1. No category

On the following pages you will find one of the Sample R01
Applications and Summary Statements indexed here:
http://www.niaid.nih.gov/ncn/grants/app/default.htm
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PI: HUSTON, CHRISTOPHER D
Title: Molecular Mechanism of Entamoeba histolytica phagocytosis
Received: 02/28/2008
FOA: PA07-070
Competition ID: VERSION-2A-FORMS
FOA Title: RESEARCH PROJECT GRANT (PARENT R01)
1 R01 AI072021-01A2
Dual:
IPF: 8738101
Organization: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
Former Number:
Department: Medicine - Infectious Diseases
IRG/SRG: ZRG1 IDM-R (02)M
AIDS: N
Expedited: N
Subtotal Direct Costs
(excludes consortium F&A)
Year 1:
200,000
Year 2:
200,000
Year 3:
200,000
Year 4:
200,000
Year 5:
200,000
Animals: N
Humans: N
Clinical Trial: N
Exemption: 10
HESC: N
New Investigator: Y
Senior/Key Personnel:
Organization:
Role Category:
Christopher Huston MD
The University of Vermont and State
Agricultural College
PD/PI
Jose Teixeira PhD
The University of Vermont and State
Agricultural College
Post Doctoral Associate
Peter Henson PhD
National Jewish Medical and Research
Center
Other (Specify)-Other Significant
Contributor
Tomoyoshi Nozaki MD, P
Gunma University Graduate School of
Medicine
Other (Specify)-Other Significant
Contributor
William Petri Jr.
University of Virginia Health System
Other (Specify)-Other Significant
Contributor
Gary Ward
The University of Vermont and State
Agricultural College
Other (Specify)-Other Significant
Contributor
Council: 10/2008
Accession Number: 3063504
Appendices
appendix_upload_2, appendix_upload_3, appendix_upload_1
Additions for Review
Accepted Publication
Entamoeba histolytica
phagocyt
06/05/2008
Supplemental Material
Supplemental Material
06/05/2008
APPLICATION FOR FEDERAL ASSISTANCE
SF 424 (R&R)
2. DATE SUBMITTED
02/28/2008
Applicant Identifier
22240
3. DATE RECEIVED BY STATE
State Application Identifier
1. * TYPE OF SUBMISSION
❍ Pre-application ❍ Application
● Changed/Corrected Application
4. Federal Identifier
AI072021
5. APPLICANT INFORMATION
* Legal Name: The University of Vermont and State Agricultural College
Department: Medicine - Infectious Diseases
Division: COLLEGE OF MEDICINE
* Street1: 85 South Prospect Street
Street2: 340 Waterman Building
* City: Burlington
County: Chittenden
Province:
* Country: USA: UNITED STATES
Person to be contacted on matters involving this application
Prefix:
* First Name:
Middle Name:
Ms.
Dayna
E.
* Phone Number: (802) 656-4067
Fax Number: (802) 656-3190
6. * EMPLOYER IDENTIFICATION NUMBER (EIN) or (TIN):
XXXXXXX
8. * TYPE OF APPLICATION:
● Resubmission
❍ Renewal
❍ New
❍ Continuation
❍ Revision
If Revision, mark appropriate box(es).
❍ A. Increase Award ❍ B. Decrease Award ❍ C. Increase Duration
❍ D. Decrease Duration ❍ E. Other (specify):
* Is this application being submitted to other agencies? ❍ Yes ● No
What other Agencies?
11. * DESCRIPTIVE TITLE OF APPLICANT'S PROJECT:
Molecular Mechanism of Entamoeba histolytica phagocytosis
12. * AREAS AFFECTED BY PROJECT (cities, counties, states, etc.)
n/a
13. PROPOSED PROJECT:
* Start Date
* Ending Date
12/01/2008
11/30/2013
* Organizational DUNS:0668111910000
* State: VT: Vermont
* ZIP / Postal Code:
05405-0160
* Last Name:
LeDuc
Email: Dayna.LeDuc@uvm.edu
Suffix:
7. * TYPE OF APPLICANT
H: Public/State Controlled Institution of Higher Education
Other (Specify):
Small Business Organization Type
❍ Women Owned
❍ Socially and Economically Disadvantaged
9. * NAME OF FEDERAL AGENCY:
National Institutes of Health/DHHS
10. CATALOG OF FEDERAL DOMESTIC ASSISTANCE NUMBER:
TITLE: Research Project Grant (Parent R01)
14. CONGRESSIONAL DISTRICTS OF:
a. * Applicant
b. * Project
VT-001
VT-001
15. PROJECT DIRECTOR/PRINCIPAL INVESTIGATOR CONTACT INFORMATION
Prefix:
* First Name:
Middle Name:
* Last Name:
Dr.
Christopher
Dwight
Huston
Position/Title: Assistant Professor
* Organization Name: The University of Vermont and State Agricultural College
Department: Medicine - Infectious Diseases
Division:
* Street1: Stafford Hall, Room 320
Street2: 95 Carrigan Drive
* City: Burlington
County: Chittenden
* State: VT: Vermont
Province:
* Phone Number: 802-656-9115
Tracking Number: GRANT00424214
* Country: USA: UNITED STATES
Fax Number: 802-847-5322
Funding Opportunity Number: PA-07-070
Suffix:
MD
* ZIP / Postal Code: 05405
* Email: christopher.huston@uvm.edu
Received Date: 2008-02-28 12:55:15.000-05:00 Time
Zone: GMT-5
OMB Number: 4040-0001
Expiration Date: 04/30/2008
SF 424 (R&R) APPLICATION FOR FEDERAL ASSISTANCE
16. ESTIMATED PROJECT FUNDING
a. * Total Estimated Project Funding
$1,505,000.00
b. * Total Federal & Non-Federal Funds $1,505,000.00
c. * Estimated Program Income
$0.00
Page 2
17. * IS APPLICATION SUBJECT TO REVIEW BY STATE EXECUTIVE ORDER 12372 PROCESS?
a. YES
❍ THIS PREAPPLICATION/APPLICATION WAS MADE AVAILABLE TO THE
STATE EXECUTIVE ORDER 12372 PROCESS FOR REVIEW ON:
DATE:
b. NO
● PROGRAM IS NOT COVERED BY E.O. 12372; OR
PROGRAM HAS NOT BEEN SELECTED BY STATE FOR REVIEW
❍
18. By signing this application, I certify (1) to the statements contained in the list of certifications* and (2) that the statements herein are true, complete
and accurate to the best of my knowledge. I also provide the required assurances * and agree to comply with any resulting terms if I accept an
award. I am aware that any false, fictitious, or fraudulent statements or claims may subject me to criminal, civil, or administrative penalties. (U.S.
Code, Title 18, Section 1001)
● * I agree
* The list of certifications and assurances, or an Internet site where you may obtain this list, is contained in the announcement or agency specific instructions.
19. Authorized Representative
Prefix:
* First Name:
Ms.
Ruth
* Position/Title: Assoc.VP for Research Admin
Department: Sponsored Programs Office
* Street1: 340 Waterman Building
* City: Burlington
Middle Name:
* Last Name:
A.
Farrell
* Organization Name: The University of Vermont and State Agricultural College
Division:
Street2: 85 South Prospect Street
County: Chittenden
* State: VT: Vermont
Province:
* Country: USA: UNITED STATES
* ZIP / Postal Code:
05405-0160
* Phone Number: 802-656-3360
Fax Number: 802-656-1326
* Email: ospuvm@uvm.edu
* Signature of Authorized Representative
* Date Signed
Ms. Ruth A. Farrell
02/28/2008
Suffix:
20. Pre-application File Name: Mime Type:
21. Attach an additional list of Project Congressional Districts if needed.
File Name: Mime Type:
Tracking Number: GRANT00424214
Funding Opportunity Number: PA-07-070
Received Date: 2008-02-28 12:55:15.000-05:00 Time
Zone: GMT-5
OMB Number: 4040-0001
Expiration Date: 04/30/2008
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
424 R&R and PHS-398 Specific
Table Of Contents
Page Numbers
SF 424 R&R Face Page------------------------------------------------------------------------------------------
1
Table of Contents---------------------------------------------------------------------------------------------
3
Performance Sites---------------------------------------------------------------------------------------------
4
Research & Related Other Project Information------------------------------------------------------------------
5
Project Summary/Abstract (Description)----------------------------------------
6
Public Health Relevance Statement (Narrative attachment)----------------------------------------
7
Facilities & Other Resources----------------------------------------
8
Equipment----------------------------------------
9
Research & Related Senior/Key Person--------------------------------------------------------------------------
10
Biographical Sketches for each listed Senior/Key Person----------------------------------------
14
PHS 398 Specific Cover Page Supplement------------------------------------------------------------------------
33
PHS 398 Specific Modular Budget-------------------------------------------------------------------------------
35
Personnel Justification----------------------------------------
39
Additional Narrative Justification----------------------------------------
40
PHS 398 Specific Research Plan--------------------------------------------------------------------------------
41
Introduction to Revised/Supplemental Application----------------------------------------
44
Specific Aims----------------------------------------
47
Significance and Related R&D----------------------------------------
49
Preliminary Studies/Phase I Final Report----------------------------------------
52
Experimental/Research Design and Methods----------------------------------------
61
Bibliography & References Cited----------------------------------------
74
Letters of Support----------------------------------------
80
PHS 398 Checklist---------------------------------------------------------------------------------------------
85
Appendix
Number of Attachments in Appendix:
3
Table of Contents
Page 3
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
RESEARCH & RELATED Project/Performance Site Location(s)
Project/Performance Site Primary Location
Organization Name: The University of Vermont and State Agricultural College
* Street1: Stafford Hall, Room 320
Street2: 95 Carrigan Drive
* City: Burlington
County: Chittenden
* State: VT: Vermont
Province:
* Country: USA: UNITED
STATES
* Zip / Postal Code: 05405
File Name
Mime Type
Additional Location(s)
Performance Sites
Tracking Number: GRANT00424214
Page 4
OMB Number: 4040-0001
Expiration Date: 04/30/2008
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
RESEARCH & RELATED Other Project Information
❍
Yes
●
No
❍
Yes
❍
No
3
4
❍
Yes
●
No
❍
Yes
❍
No
3. * Is proprietary/privileged information ❍ Yes
●
No
1. * Are Human Subjects Involved?
1.a. If YES to Human Subjects
Is the IRB review Pending?
IRB Approval Date:
Exemption Number:
1
2
5
6
Human Subject Assurance Number
2. * Are Vertebrate Animals Used?
2.a. If YES to Vertebrate Animals
Is the IACUC review Pending?
IACUC Approval Date:
Animal Welfare Assurance Number
included in the application?
4.a. * Does this project have an actual or potential impact on
❍
●
Yes
No
the environment?
4.b. If yes, please explain:
4.c. If this project has an actual or potential impact on the environment, has an exemption been authorized or an environmental assessment (EA) or
environmental impact statement (EIS) been performed?
❍
❍
Yes
No
4.d. If yes, please explain:
5.a. * Does this project involve activities outside the U.S. or
❍
Yes
●
No
partnership with International Collaborators?
5.b. If yes, identify countries:
5.c. Optional Explanation:
6. * Project Summary/Abstract
abstract.pdf
Mime Type: application/octet-stream
7. * Project Narrative
projplan.pdf
Mime Type: application/octet-stream
8. Bibliography & References Cited
ref.pdf
Mime Type: application/octet-stream
9. Facilities & Other Resources
Facilities_Upload.pdf
Mime Type: application/octet-stream
10. Equipment
Major_Equipment_Upload.pdf
Mime Type: application/octet-stream
Tracking Number: GRANT00424214
Other Information
Page 5
OMB Number: 4040-0001
Expiration Date: 04/30/2008
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
Project Summary/Abstract:
Amebiasis ranks second as a protozoan cause of death. Entamoeba histolytica, the etiologic agent, is
an intestinal ameba that acquires nutrients by phagocytosis of colonic bacteria, and phagocytosis of host cells
by E. histolytica is a prominent feature of invasive amebiasis. Despite its importance, little is known about the
mechanism of E. histolytica phagocytosis. Entamoeba histolytica induces host cell apoptosis, resulting in host
cell surface changes that engage unknown amebic phagocytosis receptors. In mammals, collectin family
members (e.g., mannose binding lectin (MBL)) and the structurally related protein C1q bind to apoptotic cells,
and initiate phagocytosis by interaction of their conserved collagenous "tails" with calreticulin. Calreticulin,
which has no transmembrane domain, serves as a bridge between collectins and the macrophage receptor
CD91. Preliminary data show that: 1) C1q, MBL, and collectin tails stimulate E. histolytica phagocytosis, 2)
C1q and MBL compete for binding to the surface of E. histolytica, 3) calreticulin is present on the amebic
surface and re-localizes to the phagocytic cup during interaction with apoptotic cells, 4) human C1q and the
collectins MBL and SP-A compete for binding to immobilized amebic calreticulin, and 5) specific E. histolytica
surface proteins interact with calreticulin. Therefore, host collectins bound to apoptotic cells and bacteria may
stimulate E. histolytica phagocytosis by interaction of their collagenous tail domain with an amebic receptor.
Calreticulin is one logical candidate, and, if calreticulin participates in amebic phagocytosis, it is hypothesized
to do so by bridging between collectins and a calreticulin receptor on the amebic surface. The specific aims
test these hypotheses. In aim 1, purified collectins, C1q, and collagenous collectin "tails" will be used to test if
E. histolytica has a receptor for the collectin tail that mediates engulfment of apoptotic cells. Except for C1q,
the collectins also opsonize bacteria in mucosal secretions. Therefore, studies will be performed to test if E.
histolytica engulfs apoptotic cells and bacteria via the same mechanism. In aim 2, RNA-mediated interference
and an alternative gene silencing method will be used to determine if calreticulin participates in E. histolytica
phagocytosis. Recombinant calreticulin will be used for binding studies to determine if E. histolytica has a
calreticulin receptor. In aim 3, either a collectin or a calreticulin receptor will be identified. The decision of
which to pursue will depend on the results from aims 1 and 2. In either case, cross-linking methods and
complementary affinity-based methods will be combined with mass spectrometry to identify interacting amebic
surface proteins. Successful completion of the proposed studies will provide a molecular understanding of E.
histolytica phagocytosis, and will substantially augment our knowledge of how E. histolytica interacts with the
host and with colonic bacteria. Furthermore, new insights into the pathogenesis of amebiasis that result from
these studies may suggest novel methods for its treatment and prevention.
Project Description
Page 6
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
Project Narrative:
Entamoeba histolytica, a single-celled intestinal parasite that causes invasive amebiasis (a disease
characterized by bloody diarrhea and liver abscesses), engulfs killed cells during invasion through host tissues.
The goal of this project is to determine the molecular mechanisms underlying E. histolytica's ability to
recognize and engulf killed cells. This will provide novel insights into how E. histolytica causes invasive
infections, possibly suggesting new methods to treat or prevent amebiasis.
Public Health Relevance Statement
Page 7
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
Facilities and Other Resources:
Laboratory: Dr. Huston has a 370 sq. ft. laboratory with other shared laboratory space in the Department of
Microbiology and Molecular Genetics, which is housed in the University of Vermont's modern Stafford Hall
research building. This space contains all of the equipment necessary to conduct any standard cellular,
biochemical, microbiological, or molecular biological procedure.
Clinical: Not applicable
Animal: Not applicable
Computer: Three Dell Pentium 4 desktop computers, a Dell Latitude D610 laptop computer with docking
station, a laser printer, and scanner are available within the laboratory for word processing, data analysis,
database searches, and figure preparation. In addition, the University of Vermont's computing facilities are
available on-line via Ethernet.
Office: Dr. Huston has a 110 sq. ft. office adjacent to his laboratory. Ample desk space is available for the
graduate student and Dr Teixeira.
Other: The core facilities of the University of Vermont include: 1) a DNA analysis facility which provides DNA
sequencing as well as quantitative RT-PCR and DNA microarray analysis using the Affymetrix system, 2) a
staffed proteomics core with both MS-MS and LC-MS capabilities, 3) a staffed flow cytometry facility, 4) a
staffed cell-imaging facility with epifluorescent microscopes, scanning and transmission electron, laser
dissecting, and confocal microscopes, and 5) a staffed structural biology/bioinformatics facility with molecular
modeling capabilities. The department provides glassware washing, drying, and autoclaving services. The
department's administrative office includes a business manager, financial assistant, and two secretaries, and
provides access to a photocopier and Fax machine.
Facilities
Page 8
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
Equipment:
Equipment important to this project within Dr. Huston's laboratory: tissue culture hood (Labconco Class II),
fume hood, CO2 incubator (Fisher Isotemp), general incubator (Precision), refrigerated table-top centrifuge
(Fisher Accuspin 3R), two microfuges (Eppendorf), gel image acquisition system (Kodak EDAS 120), inverted
tissue culture microscope (Olympus CK2), refrigerator/freezer, -20ºC upright freezer, -70ºC cabinet freezer
(Revco), a floor model environmental shaker (New Brunswick Series 25), a PCR thermocycler (MJ Research
PTC-200 DNA engine), equipment for agarose, and 1- and 2-dimensional polyacrylamide gel electrophoresis
on up to 6 samples simultaneously (Invitrogen IPGRunner (for isoelectric focusing), XCell6 MultiGel Unit (for
SDS-PAGE), and Zoom Dual Power Supply), and a computer controlled microplate spectrophotometer with
plate shaking, temperature control, and continuous wavelength capabilities (BioTek PowerWave XS).
Important departmental equipment: AKTAxpress Protein Purification system (GE Healthcare), electroporator
(BioRad GenePulser II), refrigerated floor model centrifuge (Sorvall RC5B), darkroom, cold room, icemaker,
autoclave, gel dryer, and three vacuum desiccators.
Important core equipment within the Health Sciences or Given Research Buildings (both connected to Stafford
Hall): Two laser scanning confocal microscopes (BioRad MRC 1024ES and Zeiss 510, fee-for-service), an
Olympus BX50 epifluorescent microscope equipped with an Optronics Magnafire digital camera (fee-forservice) to use for microscopy-based phagocytosis assays, two flow cytometers (a Coulter EPICS XL fourcolor flow cytometer and a Becton Dickinson LSRII 10-color flow cytometer, fee-for-service) to use for flow
cytometry-based phagocytosis assays, an ABI Prism 7700 Sequence Detector (fee-for-service) for quantitative
real time PCR, and a BioRad Molecular Imager Fx for gel image acquisition.
Equipment
Page 9
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
RESEARCH & RELATED Senior/Key Person Profile (Expanded)
PROFILE - Project Director/Principal Investigator
Prefix
* First Name
Middle Name
* Last Name
Suffix
Dr.
Christopher
Dwight
Huston
MD
Position/Title: Assistant Professor
Department: Medicine - Infectious Diseases
Organization Name: The University of Vermont and State Agricultural Col- Division: COLLEGE OF MEDICINE
lege
* Street1: Stafford Hall, Room 320
Street2: 95 Carrigan Drive
* City: Burlington
County: Chittenden
* State: VT: Vermont Province:
* Country: USA: UNITED STATES
* Zip / Postal Code: 05405
*Phone Number
Fax Number
* E-Mail
802-656-9115
802-847-5322
christopher.huston@uvm.edu
Credential, e.g., agency login: XXXXXXX
* Project Role: PD/PI
Other Project Role Category:
File Name
Bio_Dr._Christopher_Dwight_Huston_MD
_0.pdf
*Attach Biographical Sketch
Attach Current & Pending Support
Mime Type
application/octet-stream
PROFILE - Senior/Key Person
Prefix
* First Name
Middle Name
* Last Name
Suffix
Dr.
Jose
E
Teixeira
PhD
Position/Title: Post Doctoral Associate
Department: Medicine - Infectious Diseases
Organization Name: The University of Vermont and State Agricultural Col- Division:
lege
* Street1: 320 Stafford Hall
Street2: 95 Carrigan Drive
* City: Burlington
County: Chittenden
* Country: USA: UNITED STATES
* Zip / Postal Code: 05405
* State: VT: Vermont Province:
*Phone Number
Fax Number
* E-Mail
802-656-9115
802-847-5322
Jose.Teixeira@uvm.edu
Credential, e.g., agency login:
* Project Role: Post Doctoral Associate
Other Project Role Category:
File Name
Bio_Dr._Jose_E_Teixeira_PhD_1.pdf
*Attach Biographical Sketch
Mime Type
application/octet-stream
Attach Current & Pending Support
PROFILE - Senior/Key Person
Prefix
* First Name
Dr.
Peter
Middle Name
Position/Title: Margaret A. Regan Professor
Department: Pediatrics
Organization Name: National Jewish Medical and Research Center
Division:
* Street1: 1400 Jackson Street
Street2:
Tracking Number: GRANT00424214
Key Personnel
* Last Name
Suffix
Henson
PhD
Page 10
OMB Number: 4040-0001
Expiration Date: 04/30/2008
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
* City: Denver
County:
* State: CO: Colorado Province:
* Country: USA: UNITED STATES
* Zip / Postal Code: 80206
*Phone Number
Fax Number
* E-Mail
303-398-1380
303-270-2156
hensonp@njc.org
Credential, e.g., agency login:
* Project Role: Other (Specify)
Other Project Role Category: Other Significant Contributor
File Name
Bio_Dr._Peter_Henson_PhD_2.pdf
*Attach Biographical Sketch
Mime Type
application/octet-stream
Attach Current & Pending Support
PROFILE - Senior/Key Person
Prefix
* First Name
Dr.
Tomoyoshi
Middle Name
* Last Name
Suffix
Nozaki
MD, PhD
Position/Title: Professor
Department: Department of Parasitology
Organization Name: Gunma University Graduate School of Medicine
Division:
* Street1: Department of Parasitology
Street2: Gunma University Graduate School of Medicine
* City: Maebashi, Gunma 371-8511
County:
* State:
* Country: JPN: JAPAN
* Zip / Postal Code: 371 8511
Province:
*Phone Number
Fax Number
* E-Mail
81-27-220-8020
81 27 220 8025
nozaki@med.gunma-u.ac.jp
Credential, e.g., agency login:
* Project Role: Other (Specify)
Other Project Role Category: Other Significant Contributor
File Name
Bio_Dr._Tomoyoshi_Nozaki_MD,_PhD_3.
pdf
*Attach Biographical Sketch
Attach Current & Pending Support
Mime Type
application/octet-stream
PROFILE - Senior/Key Person
Prefix
* First Name
Middle Name
* Last Name
Suffix
Dr.
William
A.
Petri
Jr.
Position/Title: Wade Hampton Frost Professor of Edidemiology
Department: Department of Medicine
Organization Name: University of Virginia Health System
Division:
* Street1: University of Virginia Health System
Street2: PO Box 801340
* City: Charlottesville
County:
* State: VA: Virginia
* Country: USA: UNITED STATES
* Zip / Postal Code: 22908-1340
Province:
*Phone Number
Fax Number
* E-Mail
434-924-5621
434-924-0075
wap3g@virginia.edu
Credential, e.g., agency login:
* Project Role: Other (Specify)
Other Project Role Category: Other Significant Contributor
File Name
Bio_Dr._William_A._Petri_Jr._4.pdf
*Attach Biographical Sketch
Mime Type
application/octet-stream
Attach Current & Pending Support
Tracking Number: GRANT00424214
Key Personnel
Page 11
OMB Number: 4040-0001
Expiration Date: 04/30/2008
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
PROFILE - Senior/Key Person
Prefix
* First Name
Middle Name
* Last Name
Dr.
Gary
E
Ward
Position/Title: Associate Professor
Suffix
Department: Microbiology & Molecular Genet
Organization Name: The University of Vermont and State Agricultural Col- Division:
lege
* Street1: 316 Stafford Hall
Street2: 95 Carrigan Drive
* City: Burlington
County: Chittenden
* Country: USA: UNITED STATES
* Zip / Postal Code: 05405
* State: VT: Vermont Province:
*Phone Number
Fax Number
* E-Mail
802-656-4868
802-656-8749
Gary.Ward@uvm.edu
Credential, e.g., agency login: XXXXXXX
* Project Role: Other (Specify)
Other Project Role Category: Other Significant Contributor
File Name
Bio_Dr._Gary_E_Ward_5.pdf
*Attach Biographical Sketch
Mime Type
application/octet-stream
Attach Current & Pending Support
Tracking Number: GRANT00424214
Key Personnel
Page 12
OMB Number: 4040-0001
Expiration Date: 04/30/2008
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
RESEARCH & RELATED Senior/Key Person Profile (Expanded)
Additional Senior/Key Person Form Attachments
When submitting senior/key persons in excess of 8 individuals, please attach additional senior/key person forms here. Each additional form attached here, will provide you with the ability to identify another 8 individuals, up to a maximum of 4 attachments (32 people).
The means to obtain a supplementary form is provided here on this form, by the button below. In order to extract, fill, and attach each additional
form, simply follow these steps:
•
•
•
•
•
•
•
Select the "Select to Extract the R&R Additional Senior/Key Person Form" button, which appears below.
Save the file using a descriptive name, that will help you remember the content of the supplemental form that you are creating. When assigning a name to the file, please remember to give it the extension ".xfd" (for example, "My_Senior_Key.xfd"). If you do not name your file
with the ".xfd" extension you will be unable to open it later, using your PureEdge viewer software.
Using the "Open Form" tool on your PureEdge viewer, open the new form that you have just saved.
Enter your additional Senior/Key Person information in this supplemental form. It is essentially the same as the Senior/Key person form that
you see in the main body of your application.
When you have completed entering information in the supplemental form, save it and close it.
Return to this "Additional Senior/Key Person Form Attachments" page.
Attach the saved supplemental form, that you just filled in, to one of the blocks provided on this "attachments" form.
Important: Please attach additional Senior/Key Person forms, using the blocks below. Please remember that the files you attach must be Senior/
Key Person Pure Edge forms, which were previously extracted using the process outlined above. Attaching any other type of file may
result in the inability to submit your application to Grants.gov.
1) Please attach Attachment 1
2) Please attach Attachment 2
3) Please attach Attachment 3
4) Please attach Attachment 4
Filename
ADDITIONAL SENIOR/KEY
PERSON PROFILE(S)
MimeType
Filename
Additional Biographical
Sketch(es) (Senior/Key Person)
MimeType
Filename
Additional Current and
Pending Support(s)
Tracking Number: GRANT00424214
MimeType
Key Personnel
Page 13
OMB Number: 4040-0001
Expiration Date: 04/30/2008
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
BIOGRAPHICAL SKETCH
Provide the following information for the key personnel and other significant contributors in the order listed on Form Page 2.
Follow this format for each person. DO NOT EXCEED FOUR PAGES.
NAME
POSITION TITLE
Huston, Christopher D.
Assistant Professor
eRA COMMONS USER NAME
XXXXXXX
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)
INSTITUTION AND LOCATION
Cornell University, Ithaca, NY
Cornell University Medical College, NY, NY
DEGREE
(if applicable)
YEAR(s)
B.A.
M.D.
1990
1994
FIELD OF STUDY
Physics
Medicine
A. Positions and Honors
Positions
1994-1997 Intern/Resident, Internal Medicine, University of Vermont College of Medicine,
Burlington, VT
1997-1998 Chief Resident in Internal Medicine, University of Vermont College of Medicine,
Burlington, VT
1997-1998 Faculty Scholar, Department of Rheumatology and Immunology, University of Vermont
College of Medicine, Burlington, VT, Laboratory of Ralph C. Budd, M.D. (25% time)
1998-2001 Fellow, Division of Infectious Diseases, Department of Medicine, University of Virginia
School of Medicine, Charlottesville, VA
2001-2003 Howard Hughes Postdoctoral fellow (Parasitology), Division of Infectious Diseases,
University of Virginia School of Medicine, Charlottesville, VA, Laboratory of William A.
Petri, Jr., M.D., Ph.D.
7/2003Assistant Professor (tenure track), Division of Infectious Diseases, Department of
Medicine, University of Vermont College of Medicine, Burlington, VT
5/2005Adjunct Assistant Professor, Department of Microbiology and Molecular Genetics,
University of Vermont, Burlington VT
5/2005Adjunct Assistant Professor, Cell and Molecular Biology Program, University of
Vermont, Burlington, VT
Honors
1986-1990
1994
1994
1994
1994
1997
1999
1999
2000
2000
2001-2003
2002
20032004
Dean’s List/Distinction in All Subjects, Cornell University, Ithaca, NY
Alpha Omega Alpha Medical Honor Society, Cornell University Medical College (CUMC)
The Upjohn Achievement Award for excellence in Pharmacology, CUMC
The Jay Lawrence Award for Clinical Proficiency in Infectious Diseases, CUMC
The Good Physician Award (given by class vote), CUMC
The Frank L. Babbott, M.D. Memorial Award, University of Vermont College of Medicine
Young Investigator Award, American Society of Tropical Medicine and Hygiene
Invited Speaker: Symposium on apoptotic inflammation and enteric pathogens.
American Society for Tropical Medicine and Hygiene. Washington, D.C.
Special citation for fellows-in-training, Infectious Diseases Society of America,
Abstract #469
Invited Speaker: International symposium on Amoebiasis. Bernhard Nocht Institute for
Tropical Medicine. Hamburg, Germany.
Howard Hughes Postdoctoral Fellowship for Physicians
Poster award recipient, Woods Hole Molecular Parasitology Meeting, Abstract #268A
Mentored Clinical Scientist Development Award (K08), NIH AI053678
Invited Plenary Speaker: Brazilian Society for Protozoology 29th Annual Meeting for
Biosketches
Page 14
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
2004
2006
2007
Basic Research in Chagas' Disease. Caxambu, Minas Gerais, Brazil.
College of Medicine New Research Initiative Award, University of Vermont
Frymoyer Scholar (one of five co-recipients), University of Vermont College of Medicine
American Society for Clinical Investigation, Young Investigator Award Recipient
Other Experience and Professional Memberships
Memberships: Infectious Diseases Society of America (1998- ), American Society of Tropical
Medicine and Hygiene (1998- ), American Society for Microbiology (2002- )
Program Committee member: 2002 International Conference on Emerging Infectious Diseases, CDC
Advisor: Influenza Vaccine Utilization Committee, Vermont Department of Health
Ad Hoc Reviewer (1998- ): Molecular Microbiology, Cellular Microbiology, Journal of Clinical
Microbiology, American Journal of Tropical Medicine and Hygiene, Mayo Clinic Proceedings,
Digestive and Liver Disease, European Journal of Clinical Microbiology and Infectious Diseases,
Coronary Artery Disease, Experimental Parasitology, Clinical and Diagnostic Laboratory
Immunology, Journal of Parasitology, PLoS Neglected Tropical Diseases
Editorial Board Member: Infection and Immunity (2002- ) (re-appointed 1/2008 until 12/31/2010)
B. Peer-reviewed publications (in chronological order)
1. Vincent MS, Roessner K, Sellati T, Huston CD, Sigal LH, Behar SM, Radolf JD, Budd RC. Lyme
arthritis synovial γδ T cells respond to Borrelia burgdorferi lipoproteins and lipidated
hexapeptides. Journal of Immunology. 1998. 161:5762-5771.
2. Huston CD, Mann BJ, Hahn CS, Petri WA. Role of host caspases in cell killing by Entamoeba
histolytica. Archives of Medical Research. 2000. 31:s216-217.
3. Huston CD, Houpt ER, Mann BJ, Hahn CS, Petri WA. Caspase 3-dependent killing of host cells
by the parasite Entamoeba histolytica. Cellular Microbiology. 2000. 2:617-625. [includes cover
photo]
4. Cheng XJ, Hughes MA, Huston CD, Loftus BB, Gilchrist CA, Lockhart LA, Ghosh S, Miller-Sims
V, Mann BJ, Petri WA, Tachibana H. The 150 kDa Gal/GalNAc lectin co-receptor of
Entamoeba histolytica is a member of a gene family containing multiple CXXC sequence
motifs. Infection and Immunity. 2001. 69:5892-5898.
5. Haque R, Huston CD, Hughes M, Houpt E, Petri WA. Current Concepts: Amebiasis. New
England Journal of Medicine. 2003. 348:1565-1573. [peer-reviewed]
6. Huston CD, Boettner DR, Miller-Sims V, Petri WA. Apoptotic killing and phagocytosis of host cells
by the parasite Entamoeba histolytica. Infection and Immunity. 2003. 71:964-972.
7. Huston CD. Parasite and host contributions to the pathogenesis of amebic colitis. Trends in
Parasitology. 2004. 20:23-26. [peer-reviewed]
8. Okada M, Huston CD, Mann BJ, Petri WA, Kita K, Nozaki T. Proteomic analysis of phagocytosis
in the enteric protozoan parasite Entamoeba histolytica. Eukaryotic Cell. 2005. 4:827-831.
9. Boettner DR, Huston CD, Sullivan JA, Petri WA. Entamoeba histolytica and Entamoeba dispar
utilize externalized phosphatidylserine for recognition and phagocytosis of erythrocytes.
Infection and Immunity. 2005. 73:3422-30.
10. Okada M, Huston CD, Oue M, Mann BJ, Petri WA, Kita K, Nozaki T. Proteomic analysis of
phagosome maturation of the enteric protozoan parasite Entamoeba histolytica. Molecular and
Biochemical Parasitology. 2006. 145:171-83.
11. Huston CD, Miller-Sims VC, Teixeira JE. Identification and characterization of EhABC A1, an
Entamoeba histolytica group A ABC transporter with similarity to Ced7. Molecular and
Biochemical Parasitology. 2006. 146:272-6.
12. Kirkpatrick BD, Huston CD, Wagner D, Alston WK, Noel F, Rouzier P, Pape JW, Bois G, Larsson
CJ, Tenney K, Powden C, O'Neill JP, Sears CL. Serum mannose-binding lectin deficiency is
associated with cryptosporidiosis in young Haitian children. Clinical Infectious Diseases. 2006.
Biosketches
Page 15
43:289-94.
13. Boettner DR, Huston CD, Linford AS, Buss SN, Houpt E, Sherman NE, Petri WA. Entamoeba
histolytica phagocytosis of human erythrocytes involves PATMK, a member of the
transmembrane kinase family. PLoS Pathogens. 2008. 4(1):122-33. PMID: 18208324.
14. Teixeira JE, Huston CD. Participation of the serine-rich Entamoeba histolytica protein in amebic
phagocytosis of apoptotic host cells. Infection and Immunity. 2008. 76:959-66. PMID:
18086807.
15. Teixeira JE, Huston CD. Evidence of a continuous endoplasmic reticulum in the protozoan
parasite Entamoeba histolytica. Eukaryotic Cell. 2008. Feb 15; [Epub ahead of print] PMID:
18281599.
C. Research Support
Committed
5 K08 AI053678-04 Huston (PI)
7/01/03-6/30/08
Funding agency: NIH/NIAID
Project title: E. histolytica phagocytosis of apoptotic host cells
This project examines the role of host cell apoptosis and associated cell surface changes in
recognition and ingestion of killed cells by Entamoeba histolytica.
1 P20 RR021905-01 Huston (PI)
8/01/06-7/31/11
Funding agency: NIH
Project title: Mechanism of Entamoeba histolytica phagocytosis
This project is one of five submitted 10/2004 as part of a COBRE grant application by the
University of Vermont. This project examines the mechanism of E. histolytica-induced host cell
surface changes that facilitate amebic phagocytosis of killed cells, and characterizes the function of
the serine-rich E. histolytica protein, a protein identified as a putative E. histolytica phagocytosis
receptor.
XXXXXXX
Completed support
XXXXXXX
Biosketches
Page 16
XXXXXXX
XXXXXXX
Biosketches
Page 17
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
BIOGRAPHICAL SKETCH
Provide the following information for the key personnel and other significant contributors in the order listed on Form Page 2.
Follow this format for each person. DO NOT EXCEED FOUR PAGES.
NAME
POSITION TITLE
Teixeira, José E.
Post-doctoral Fellow
eRA COMMONS USER NAME
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)
INSTITUTION AND LOCATION
School of Medicine of Botucatu, Paulista State
University, Brazil
School of Medicine of Ribeirão Preto, University
of São Paulo, Brazil
School of Medicine of Ribeirão Preto, University
of São Paulo, Brazil
School of Medicine of Ribeirão Preto, University
of São Paulo, Brazil
University of Virginia, Charlottesville, Virginia
DEGREE
(if applicable)
YEAR(s)
B.A.
1984-1987
Medicine (unfinished)
B.A.
1987-1990
Medical Biology
M.Sc.
1990-1994
Immunology
Ph.D.
1994-1998
Immunology
Post-doc
1998-2001
FIELD OF STUDY
Microbiology
A. Positions and Honors
Positions
1998-1999
1999-2001
2001-2003
2003-pres
Medicine
Postdoctoral Fellow, Division of Infectious Disease, Department of Medicine,
University of Virginia Health Sciences Center, Laboratory of Barbara Mann, Ph.D.
Examined Entamoeba histolytica-induced dephosphorylation in host cells and
relationship to cell killing.
Research Associate, Division of Infectious Disease, Department of Medicine,
University of Virginia Health Sciences Center, Laboratory of Barbara Mann, Ph.D.
Postdoctoral Fellow, Department of Biochemistry and Immunology, School of
Medicine of Ribeirão Preto, University of São Paulo, Brazil
Postdoctoral Fellow, Department of Medicine, University of Vermont School of
Scholarship and Fellowship Awards
1990-1994 M.Sc. Scholarship, Fundação de Amparo à Pesquisa do Estado de São Paulo
(FAPESP), Brazil
1994-1998 Doctorate Scholarship, Fundação Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior (CAPES), Brazil
1998-1999 Research Training Fellowship, International Training and Research Program in
Emerging Infectious Diseases (ITREID). John E. Fogarty International Center,
National Institutes of Health, USA
B. Peer-reviewed Publications (in chronological order)
Manço JC, Terra J, Teixeira JE, Silva GA. Avaliação Laboratorial da Distensibilidade Pulmonar e
Sua Aplicação Clínica, Jornal de Pneumologia 15: 200 - 204, 1989.
Biographical Sketches for each listed Senior/Key Person 2
Page 18
Teixeira JE, Costa RS, Lachmann PJ, Würzner R, Barbosa JE. CR1 Stump and Terminal
Complement Complex are Found in the Glomeruli of Lupus Nephritis Patients. Clin Exp Immunol.
105: 497 - 503, 1996.
Teixeira JE, Martinez R, Câmara L, Barbosa JE. Expression of Complement Receptor Type 1 on
Erythrocytes of Paracoccidioidomycosis Patients. Mycopathologia. 152: 125 - 133, 2001.
Teixeira JE, Mann BJ. Entamoeba histolytica–induced Dephosphorylation in Host Cells. Infect
Immun. 70: 1816 - 1823, 2002.
Michelin MA, Crott LSP, Assis-Pandochi AI, Coimbra TM, Teixeira JE, Barbosa JE. Influence of
the Electric Charge of the Antigen and Immune Complex (IC) Lattice on the IC Activation of
Human Complement. Int J Exp Path. 83: 105 - 110, 2002.
Yamamoto GRY, Teixeira JE, Germiani H, Boaretti AC, Marquetti PRC, Barbosa JE. In Vitro
inhibition of the Complement-Dependent Lytic Activity by Isosorbide 5-Mononitrate.
Intercontinental Cardiology. 11: 3-6, 2002
Lins CE, Crott LSP, Teixeira JE, Barbosa JE. Reduced Erythrocyte Complement Receptor Type 1
(CR1/E) in Systemic Lupus Erythematosus Is Related to a Disease Activity Index and Not to The
Presence or Severity of Renal Disease. Lupus. 13: 517-521, 2004.
Roxo PJr, Ferriani VPL, Teixeira, JE, Barbosa JE. Complement levels in Brazilian children during
and after meningococcal meningitis. Clinics. 60(2): 127-30, 2005.
Fonzar-Marana RRN, Ferriani RA, Soares SG, Cavalcante-Neto FF, Teixeira JE, Barbosa JE.
Expression of Complement System Regulatory Molecules in the Endometrium of Normal
Ovulatory and Hyperstimulated Women Correlate with Mentrual Cycle Phase. Fertil Steril. 86(3):
58-61, 2006.
Huston CD, Miller-Sims VC, Teixeira JE. Identification and characterization of EhABC A1, an
Entamoeba histolytica group A ABC transporter with similarity to Ced7. Mol Biochem Parasitol.
146(2): 272-6, 2006.
Teixeira JE, Huston CD. Participation of the serine-rich Entamoeba histolytica protein in amebic
phagocytosis of apoptotic host cells. Infect Immun. 2008. 76:959-66. PMID: 18086807.
XXXXXXX
C. Other Support: Teixeira, Jose E.
None
Biographical Sketches for each listed Senior/Key Person 2
Page 19
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
BIOGRAPHICAL SKETCH
Provide the following information for the key personnel and other significant contributors in the order listed on Form Page 2.
Follow this format for each person. DO NOT EXCEED FOUR PAGES.
NAME
POSITION TITLE
Peter M. Henson
Professor
eRA COMMONS USER NAME
XXXXXXX
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)
INSTITUTION AND LOCATION
Edinburgh University, Scotland
Edinburgh University, Scotland
University of Cambridge, England
Edinburgh University, Scotland
DEGREE
(if applicable)
BVM & S
B.Sc. Hon
Ph.D.
Hon MD
YEAR(s)
1963
1964
1967
2002
FIELD OF STUDY
Veterinary Medicine
Bacteriology
Immunopathology
Medicine
A. Positions and Honors.
Positions and Employment
1967 1969
Research Fellow, Dept Experimental Pathol, Scripps Clin & Res Foundation, La Jolla, CA
1969 1972
Associate, Dept Experimental Pathology, Scripps Clinic & Research Foundation, La Jolla, CA
1972 1977
Associate Member, Dept Immunopathology, Scripps Clin & Res Foundation, LaJolla, CA
1977 1980
Director of Research, Dept Pediatrics, National Jewish Hosp& Res Ctr, National Asthma Ctr
1980 1982
Vice-Chairman, Department of Pediatrics, National Jewish Center
1981 1982
Associate Vice-President for Professional Services, National Jewish Center
1986 1987
Co-Head, Pulmonary Division, Department of Medicine Univ Colorado Health Sci Center
1982 1996
Executive Vice President Academic Affairs, Natl Jewish Ctr Immunol & Respir Med, Den, CO
1977 present Professor of Pathology, University of Colorado School of Medicine
1977 present Senior Faculty Member, Dept of Pediatrics, National Jewish Med & Res Ctr, Denver, Colorado
1980 present Professor, Department of Medicine, University of Colorado School of Medicine
Other Experience and Professional Memberships Awards and Honors (selected)
2005
Burns Amberson Lecture, ATS Centenary Meeting
1991
Margaret A. Regan Professor of Pulmonary Inflammation
1983
Reticuloendothelial Society, Marie T. Bonazinga Award
1980
American Association of Pathologists, Parke Davis Award
July 1970 - June 1975
NIH, Research Career Development Award
July 1969 - June 1970
American Heart Association Established Investigatorship
Professional Organizations (selected)
American Association of Pathology
American Association of Immunologists
American Thoracic Society
American Society for Cell Biology
B.
Selected peer-reviewed publications (in chronological order).
(Publications selected from >400)
Fadok VA, Bratton DL, Konowal A, Freed PW, Westcott JY, Henson PM. Macrophages that have ingested
apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms
involving TGFb, PGE2, and PAF. J. Clin. Invest. 101:890-898, 1998.
Bratton DB, Fadok VA, Richter DA, Kailey JM, Frasch S C, Nakamura T, Henson PM. Polyamine regulation of
plasma membrane phospholipid flip-flop during apoptosis. J. Biol. Chem. 274:28113-28120, 1999.
Hildeman DA, Mitchell T, Teague TK, Henson P, Day BJ, Kappler J, Marrack PC. Reactive oxygen species
regulate activation induced T cell apoptosis. Immunity. 10:735-744, 1999.
Fadok VA, Bratton DL, Rose D, Pearson A, Ezekowitz A, Henson PM. A receptor for phosphatidylserinespecific clearance of apoptotic cells. Nature 405:85-90, 2000.
Biographical Sketches for each listed Senior/Key Person 3
Page 20
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
Whitlock BB, Gardai S, Fadok V, Bratton D, Henson PM. Differential roles for amb2 integrin clustering or
activation in the control of apoptosis via regulation of Akt and ERK survival mechanisms. J Cell Biol.
141:1305-1320, 2000.
Taylor PR, Carugati A, Fadok VA, Cook HT, Andrews M, Carroll MC, Savill JS, Henson PM, Botto M, Walport
MJ. A hierarchical role for classical pathway complement proteins in the clearance of apoptotic cells in vivo:
a mechanism for protection from autoimmunity. J Exp Med. 192:359-366, 2000.
Ogden CA, deCathelineau A, Hoffmann PR, Fadok VA, Bratton D, Henson PM. C1q and collectin engagement
of cell surface calreticulin initiates macropinocytosis and uptake of apoptotic cells. J. Exp. Med. 194(6):781796, 2001.
Hoffmann PR, et al, Henson PM. Phosphatidylserine (PS) induces PS receptor-mediated macropinocytosis
and promotes clearance of apoptotic cells. J Cell Biol 155(4):649-659, 2001.
Huynh, M-LH, Fadok VA, Bratton DL, Henson PM. Phosphatidylserine-dependent ingestion of apoptotic cells
promotes TGFβ1 secretion and resolution of inflammation. J Clin Invest 109:41-50, 2002.
Vandivier RW, Fadok VA, Hoffmann PR, Bratton DL, Penvari C, Brown KK, Brain JD, Accurso FJ, Henson PM.
Elastase-mediated phosphatidylserine receptor cleavage impairs apoptotic cell clearance in cystic fibrosis
and bronchiectasis. J Clin Invest. 109:661-670, 2002.
Teder P, Vandivier RW, Jiang D, Liang J, Pure E, Henson PM, Noble PW. Resolution of lung inflammation by
CD44. Science 296(5565):155-158, 2002.
Gardai SH, Xiao Y-Q, Dickinson M, Nick J, Voelker D, Greene K, Henson P. By binding SIRPα or
calreticulin/CD91, lung collectins act as dual function surveillance molecules to suppress or enhance
inflammation. Cell. 115:13-23, 2003.
Wang X, Wu Y-C, Fadok VA, Lee M-C, Gengyo-Ando K, Cheng L-C, Ledwich D, Hsu P-K, Chen J-Y, Chou BK, Henson P, Mitani S, Xue D. Cell corpse engulfment mediated by C. elegans phosphatidylserine receptor
through CED-5 and CED-12. Science. 302:2563-1566, 2003.
Gardai SJ, Hildeman DA., Frankel SK, Whitlock BB, Frasch SC, Borregaard N, Marrack P, Bratton DL, Henson
PM. Phosphorylation of Bax serine 184 by Akt regulates its activity and apoptosis in neutrophils. J Biol
Chem, 279:21085-21095, 2004.
Frasch SC, Henson PM, Nagaosa K, Fessler MB, Borregaard N, Bratton DL. Phospholipid flip-flop and
phospholipid scramblase (PLSCR 1) co-localize to uropod rafts in fMLP stimulated neutrophils. J. Biol
Chem 279:17625-17633. 2004.
Hoffmann PR, Kench JA, Vondracek A, Kruk E, Daleke DL, Jordan M, Marrack P, Henson PM, Fadok VA.
Interaction between phosphatidylserine and the phosphatidylserine receptor inhibits immune responses in
vivo. J Immunol 174:1393-1404, 2005.
Gardai SJ, McPhillips KA, Frasch C, Janssen WJ, Starefeldt A, Murphy-Ullrich JE, Bratton DL, Oldenborg PA,
Michalak M, Henson PM. Cell surface calreticulin initiates clearance of viable or apoptotic cells through
trans activation of LRP on the phagocyte. Cell. 123:321-334, 2005.
Murakawa T, Kerklo MM, Zamora MR, Wei Y, Gill RG, Henson PM, et al. Simultaneous LFA-1 and CD40
ligand antagonism prevents airway remodeling in orthotopic airway transplantation: implications for the role
of respiratory epithelium as a modulator of fibrosis. J Immunol 174:3869-3879, 2005.
Cool CD, Groshong SD, Rai PR, Henson PM, Stewart JS, Brown KK. Fibroblast Foci are Not Discrete Sites of
Lung Injury/Repair: the Fibroblast Reticulum. Am J Respir Crit Care Med 174:654-658, 2006.
Henson PM, Vandivier RW, Douglas IS. Cell death, remodeling, and repair in chronic obstructive pulmonary
disease? Proc Am Thorac Soc 3:1-5, 2006.
Morimoto K, Janssen WJ, Fessler MB, McPhillips KA, Borges VM, Bowler RP, Xiao Y-Q, Kench JA, Henson
PM, Vandivier RW. Lovastatin enhances clearance of apoptotic cells (efferocytosis) with implications for
chronic obstructive pulmonary disease. J Immunol 176, 2006, 7657-65.
McPhillips, K., Janssen, W.J., Ghosh, M., Byrne, A., Gardai, S., Remigio, L., Bratton, D.L., Kang, J.L., and
Henson, P. 2007. TNF-alpha inhibits macrophage clearance of apoptotic cells via cytosolic phospholipase
A2 and oxidant-dependent mechanisms. J Immunol 178:8117-8126.
Bianchi SM, Prince LR, McPhillips K, Allen L, Marriott HM, Taylor GW, Hellewell PG, Sabroe I, Dockrell DH,
Henson PM, Whyte MKB. Impairment of apoptotic cell engulfment by pyocyanin, a toxic metabolite of
Pseudomonas aeruginosa. Am J Respir Crit Care Med 177:35-43, 2008.
Monks J, Smith-Steinhart C, Kruk ER, Fadok VA, Henson PM. Epithelial cells remove apoptotic epithelial cells
during post-lactation involution of the mouse mammary gland. Biol Reprod. 2008
Biographical Sketches for each listed Senior/Key Person 3
Page 21
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
BIOGRAPHICAL SKETCH
Provide the following information for the key personnel and other significant contributors in the order listed on Form Page 2.
Follow this format for each person. DO NOT EXCEED FOUR PAGES.
NAME
POSITION TITLE
Tomoyoshi Nozaki
Professor of Parasitology
eRA COMMONS USER NAME
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)
INSTITUTION AND LOCATION
School of Medicine, Keio University, Tokyo
School of Medicine, Keio University, Tokyo
DEGREE
(if applicable)
YEAR(s)
M.D.
Ph.D.
1987
1997
FIELD OF STUDY
Parasitology
Parasitology
A. Positions and Honors.
Positions and Employment
April 1, 1987
Appointed as a Research Associate in the Department of Parasitology, School of
Medicine, Keio University
July 30, 1987
Licensed by the National Board for medical doctors and registered in the Ministry of
Health and Welfare, Japan (Licence No. 311355)
October 1, 1988
Appointed as a Visiting Research Associate in Laboratory of Immunopathology of Prof.
Keizo Asami, Recife, Brazil
May 18, 1989
Appointed as a Fogarty Visiting Fellow, Laboratory of Parasitic Diseases (James A.
Dvorak, Laboratory of Parasitica Diseases, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, Maryland, U. S. A.
August 1, 1992
Appointed as a Fogarty Visiting Associate at NIH
November 1, 1992
Appointed as a Research Associate, Laboratory of Molecular Parasitology (George A.
M. Cross Lab), The Rockefeller University, New York, U. S. A.
January 1, 1996
Appointed as a Research Associate in the Department of Tropical Medicine and
Parasitology, School of Medicine, Keio University, Tokyo
April 1, 1999
Appointed as Head of Laboratory of Molecular Parasitology, Department of
Parasitology, National Institute of Infectious Diseases, Tokyo
January 1, 2005
Appointed as Professor of Department of Parasitology, Gunma University Graduate
School of Medicine, Maebashi, Gunma
B. Selected peer-reviewed publications. (since 2001)
Nozaki T., Shigeta Y., Saito-Nakano Y., Imada M., and Kruger W.D. (2001) Characterization of transsulfuration
and cysteine biosynthetic pathways in the protozoan haemoflaggelate, Trypanosoma cruzi: Isolation and
molecular characterization of cystathionine beta-synthase and serine acetyltransferase from trypanosoma.
J. Biol. Chem. 276: 6516-6523.
Sanuki, J., Tokoro, M., Nozaki, T., Okuzawa, E., and Asai, T. (2001) Purification and identification of a major
soluble 40-kDa antigen from Entamoeba histolytica and its use for serodiagnosis of chronic amebiasis.
Parasitol. Int. 50, 73-80.
Saito-Nakano, Y., Nakazawa, M., Shigeta, Y., Takeuchi, T., and Nozaki, T. (2001) Identification and
characterization of genes encoding novel Rab proteins from Entameoba histolytica. Mol. Biochem.
Parasitol. 116, 219-222.
Haghighi, A., Kobayashi, S., Takeuchi, T., Masuda, G., and Nozaki, T. (2002) Remarkble genetic
polymorphism among Entamoeba histolytica isolates from a limited geographic area. J. Clin. Microbiol. 40,
4081-90.
Basombrío, M., Gómez, L., Padilla, A.M., Ciaccio, M., Nozaki, T., and Cross, G.A.M. (2002) Targeted deletion
of the Gp72 gene decreases the infectivity of Trypanosoma cruzi for mice and insect vectors. J. Parasitol.
88, 489-493.
Biographical Sketches for each listed Senior/Key Person 4
Page 22
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
Kabututu, Z., Martin, S.K., Nozaki, T., Kawazu, S., Okada, T.,Munday, C.J., Duszenko, M., Lazarus, M., Urade,
Y., and Kubata, B.K. (2002) Prostaglandin production from arachidonic acid and evidence for a 9,11endoperoxide prostaglandin H2 in Leishmania. Int. J. Parasitol. 32, 1693-1700.
Kubata, B.K., Munday, C.J., Nozaki, T., Kabututu, Z., Fukuzumi. S., Ohkubo. K., Lazarus, M., Martin, S.K.,
Duszenko, M., and Urade, Y. (2002) A key role for Trypanosoma cruzi Old Yellow Enzyme in the
metabolism of trypanocidal drugs. J. Exp. Med. 196, 1241-1251.
Haghighi, A., Kobayashi, S., Takeuchi, T., Thammapalerd, N., and Nozaki, T. (2003) Geographic diversity of
genotypes among Entamoeba histolytica field isolates. J. Clin. Microbiol., 41, 3748-3756.
Dvorak, J.A., Kobayashi, S., Nozaki, T., Takeuchi, T., and Matsubara, C. (2003) Induction of permeability
changes and death of vertebrate cells is modulated by the virulence of Entamoeba spp. Isolates. Parasitol.
Int. 52, 169-173.
Kawazu, S, Nozaki, T., Tsuboi, T., Nakano, Y., Komaki-Yasuda, K., Idenoue, N., Torii, M., and Kano, S. (2003)
Expression profiles of peroxiredoxin proteins of the rodent malaria parasite Plasmodium yoelii. Int. J.
Parasitol. 33, 1455-1461.
Ali, V., Shigeta, Y., and Nozaki, T. (2003) Molecular and structural characterization of NADPH-dependent Dglycerate dehydrogenase from the enteric parasitic protist Entamoeba histolytica. Biochem. J. 375, 729736.
Tokoro, M., Kobayashi, S., Takecuhi, T., and Nozaki, T. (2003) A novel sulfur-containing amino acid
degradation enzyme methionine gamma-lyase from the enteric protozoan parasite Entamoeba histoltyica. J.
Biol. Chem. 278, 42717-42727
Ghosh, S., Chan, J., Lea, C.R., Meints, G.A., Lewis, J.C., Tovian, Z., Flessner, R., Loftus, T.C., Bruchhaus, I.,
Fradley, K.L., Kendrick, H., Croft, S., Kemp, R., Kobayashi, S., Nozaki, T., and Oldfield, E. (2004) Effects of
Bisphosphonates on the Growth of Entamoeba and Plasmodium species in vitro and in vivo. J. Med. Chem.
47, 175-187.
Kumagai, M, Makioka, A., Takeuchi, T., and Nozaki, T. (2004) Molecular cloning and characterization of a
protein farnesyltransferase from the enteric protozoan parasite Entamoeba histolytica. J. Biol. Chem. 279,
2316-2323.
Ali, V., Shigeta, Y., Tokumoto, U., Takahashi, Y., and Nozaki, T. (2004) An intestinal parasitic protist
Entamoeba histolytica possesses a non-redundant NIF-like system for iron-sulfur cluster assembly under
anaerobic conditions. J. Biol. Chem. 279, 16863-16874.
Ali, V., Shigeta, Y., Hashimoto, T., and Nozaki, T. (2004) Molecular and biochemical characterization of Dphosphoglycerate dehydrogenase from Entamoeba histolytica: a unique enteric protozoan parasite that
possesses both phosphorylated and non-phosphorylated serine metabolic pathways. Eur. J. Biochem. 271,
2670-2681.
Saito-Nakano, Y., Yasuda, and Nozaki, T. (2004) Unique role of Rab5 and Rab7 in phagocytosis of the enteric
protozoan parasite Entamoeba histolytica. J. Biol.Chem. 279, 49497-49507.
Loftus, B., Anderson, I., Davies, R., Alismark, U. C. M., Samuelson, J., Amedeo, P., Roncaglia, P., Berriman,
M., Hirt, R. P., Mann, B. J., Nozaki, T., Suh, B., Pop, M., Duchene, M., Ackers, J., Tannich, E., Leippe, M.,
Hofer, M., Bruchhaus, I., Willhoeft, U., Bhattacharya, A., Chillingworth, T., Churcher, C., Hance, Z., Harris,
B., Harris, d., Jagels, K., Moule, S., Mungall, K., Ormond, D., Squares, R., Whitehead, S., Guillen, N.,
Gilchrist, C., Stroup, S. E., Bhattacharya, S., Lohia, A., Foster, P. G., Sicheritz-Ponten, T., Weber, C.,
Singh, U., Mukherjee, C., Petri, W. A. J., Clark, C. G., Embley, T. M., Barrell, B., Fraser, C. M., and Hall, N.
(2005). The genome of the protist parasite Entamoeba histolytica. Nature. 433. 865-868.
Okada, M., Huston, C. D., Mann, B. J., Petri, Jr., W. A., Kita, K., and Nozaki, T. (2005) Proteomic analysis of
phagocytosis in the enteric protozoan parasite Entamoeba histolytica. Eukaryot. Cell 4, 827-831.
Beck, D.L., Boettner, D., Dragulev, B., Ready, L., Mackey, A.J., Nozaki, T., Pearson, W.R., and Petri, Jr., W.A.
(2005) Identification and gene expression analysis of a large family of transmembrane kinases related to the
Gal/GalNAc lectin in Entamoeba histolytica . Eukaryot. Cell 4, 722-732.
Saito-Nakano, Y., Loftus, B. J., Hall, N., and Nozaki, T. (2005) The diversity of Rab small GTPases in
Entamoeba histolytica. Exp. Parasitol. 110, 244-252.
Mitra, B. N., Yasuda, T., Kobayashi, S., Satio-Nakano, Y., Nozaki, T. (2005) Differences in morphology of
phagosomes and kinetics of acidification and degradation in phagosomes between the pathogenic
Entamoeba histolytica and the non-pathogenic Entamoeba dispar. Cell Motil. Cytoskeleton 62, 84-99.
Nakada-Tsukui, K., Saito-Nakano, Y., Ali, V., and Nozaki, T. (2005) A retromer-like complex is a novel Rab7
effector that is involved in the transport of the virulence factor cysteine protease in the enteric protozoan
parasite Entamoeba histolytica. Mol. Biol. Cell 16, 5294-5303.
Biographical Sketches for each listed Senior/Key Person 4
Page 23
Ali, V. and Nozaki, T. (2006) Biochemical and functional characterization of phosphoserine aminotransferase
from Entameba histolytica, which possesses both phosphorylated and non-phosphorylated serine metabolic
pathways. Mol. Biochem. Parasitol. 145, 71-83.
Okada, M., Huston, C. D., Oue, M., Mann, B. J., Petri, Jr., W. A., Kita, K., and Nozaki, T. (2006) Kinetics and
strain variation of phagosome proteins of Entamoeba histolytica by proteomic analysis. Mol. Biochem.
Parasitol. 145, 171-83.
Makioka, A., Kumagai, M., Takeuchi, T., and Nozaki, T. (2006) Characterization of protein geranylgeranyltransferase I from the enteric protist Entamoeba histolytica. Mol. Biochem. Parasitol. 145, 216-25.
Mitra, B.N., Kobayashi, S., Saito-Nakano, Y., Nozaki, T. (2006) Entamoeba histolytica: differences in
phagosome acidification and degradation between attenuated and virulent strains. Exp. Parasitol. 114, 5761.
Gilchrist, C.A., Houpt, E., Trapaidze, N., Fei, Z., Crasta, O., Ascharpour, A., Evans, C., Martino-Catt, S., Baba,
D.J., Stroup, S., Hamano, S., Ehrenkaufer, G., Okada, M., Singh, U., Nozaki, T., Mann, B.J., Petri, W.A.
(2006) Impact of intestinal colonization and invasion on the Entamoeba histolytica transcriptome. Mol.
Biochem. Parasitol. 147, 163-76.
Sato, D., Nakada-Tsukui, K., Okada, M., Nozaki, T. (2006) Two cysteine protease inhibitors, EhICP1 and 2,
localized in distinct compartments, negatively regulate secretion in Entamoeba histolytica. FEBS Lett. 580,
5306-12.
Razmjou, E., Haghighi, A., Rezaian, M., Kobayashi, S., Nozaki, T. (2006) Genetic diversity of glucose
phosphate isomerase from Entamoeba histolytica. Parasitol. Int. 55, 307-11.
Sato, D., Yamagata, W., Kamei, K., Nozaki, T., Harada, S. (2006) Expression, purification and crystallization of
L-methionine gamma-lyase 2 from Entamoeba histolytica. Acta. Crystallograph Sect F Struct Biol Cryst
Commun. 62, 1034-6.
Saito-Nakano, Y., Mitra, B.N., Nakada-Tsukui, K., Sato, D., Nozaki, T. (2007) Two Rab7 isotypes, EhRab7A
and EhRab7B, play distinct roles in biogenesis of lysosomes and phagosomes in the enteric protozoan
parasite Entamoeba histolytica. Cell. Microbiol. 9, 1796-808.
Mitra, B.N., Saito-Nakano, Y., Nakada-Tsukui, K., Nozaki, T. (2007) Rab11B small GTPase regulates secretion
of cysteine proteases in the enteric protozoan parasite Entamoeba histolytica. Cell. Microbiol. 9, 2112-25.
Clark, C.G., Alsmark, U.C., Tarzeiter, M., Saito-Nakano, Y., Ali, V., Marion, S., Weber, C., Mukherjee, C.,
Bruchhaus, I., Tannich, E., Leippe, M., Sicheritz-Ponten, T., Foster, P.G., Samuelson, J., Noel, C.J., Hirt,
R.P., Embley, T.M., Gilchrist, C.A., Mann, B.J., Singh, U., Ackers, J.P., Bhattacharya, S., Bhattacharya, A.,
Lohia, A., Guillen, N., Duchene, M., Nozaki, T., Hall, N. (2007) Structure and content of the Entamoeba
histolytica genome. Adv. Parasitol. 65, 51-190.
Picazarri, K., Nakada-Tsukui, K., Nozaki, T. (2008) Autophagy during proliferation and encystation in the
protozoan parasite Entamoeba invadens. Inf. Immun. 76, 278-88.
Sato, D., Yamagata, W., Harada, S., Nozaki, T. (2008) Kinetic characterization of methionine gamma-lyases
from the enteric protozoan parasite Entamoeba histolytica against physiological substrates and
trifluoromethionine, a promising lead compound against amebiasis. FEBS J. 275, 548-60.
C. Research Support.
XXXXXXX
15590378
T. Nozaki (PI)
Project 01/01/04-12/31/08
Ministry of Education, Culture, Sports, Science and Technology of Japan
Title: Phagosome of Entamoeba histolytica
This project identifies the proteins in the phagosome of Entamoeba histolytica by proteomics.
Biographical Sketches for each listed Senior/Key Person 4
Page 24
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
BIOGRAPHICAL SKETCH
Provide the following information for the key personnel in the order listed for Form Page 2.
Follow this format for each person. DO NOT EXCEED FOUR PAGES.
NAME
POSITION TITLE
William A. Petri, Jr., M.D., Ph.D.
Wade Hampton Frost Professor of Epidemiology
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)
INSTITUTION AND LOCATION
University of Wisconsin-Madison
University of Virginia
University of Virginia
Case Western Reserve University (Univ. Hosp.)
University of Virginia
DEGREE
(if applicable)
Ph.D.
M.D.
YEAR(s)
1980
1982
1985
1988
FIELD OF STUDY
Chemistry
Microbiology
Medicine
Internal Medicine
Infectious Diseases
A. Positions and Honors.
Assistant (1988-92), Associate (1992-6), Professor (1996-present), Chief (2001-present), and Wade Hampton
Frost Professor of Epidemiology (2002 – present), Division of Infectious Diseases & International Health,
Departments of Medicine, Pathology, and Microbiology, University of Virginia
Editor, Infection & Immunity (1999-2009)
Member, NIH Pathogenic Eukaryotic (PTHE) Study Section (2005-2009)
Member, NIAID Microbiology & Infectious Diseases Research Committee, (MIDRC) NIH (2001-2005).
Member, NIH Tropical Medicine and Parasitology Study Section (1993-1997)
Chair, NIH DBBD Minority & Disabil. Predoc. Fellowship Review Committee (2003- 2007)
Parasitic Diseases Panel, U.S.-Japan Cooperative Medical Science Program (1998-2008)
Past President, American Society of Tropical Medicine & Hygiene (2003)
Inventor of the Year Award, University of Virginia Patents Foundation (2003; shared with Dr. Barbara Mann)
NIAID Blue Ribbon Panel on Bioterrorism and its Implications for Biomedical Research (2002)
Board of Directors American Type Culture Collection (1993-2001)
Squibb Award, Infectious Diseases Society of America (1999)
Burroughs Wellcome Fund Scholar & New Investigator Awards in Molecular Parasitology (1992-2003)
Lucille P. Markey Scholar in Biomedical Research (1985-1993)
Member of the Association of American Physicians, American Society for Clinical Investigation, Phi Beta
Kappa (Wisconsin), and Alpha Omega Alpha (Virginia). Appointed Life Member of the American Society
of Tropical Medicine & Hygiene, and elected Fellow of the American Academy of Microbiology, the
Infectious Diseases Society of America, and the American College of Physicians.
B. Selected Peer-reviewed Publications.
Schaenman JM, Gilchrist CA, Mann BJ, Petri WA Jr. Identification of two Entamoeba histolytica sequencespecific hgl5 enhancer-binding proteins with homology to the RNA-binding motif RRM. J. Biol. Chem.
2001, 276: 1602-1609
Haque R, Ali IKM, Sack RB, Ramakrishnan G, Farr BM, and Petri, WA Jr. Amebiasis and Mucosal IgA
Antibody against the E. histolytica Adherence Lectin in Bangladeshi Children. J. Infect. Dis. 2001,
183:1787-93.
Cheng X-J, Hughes MA, Huston CD, Loftus B, Gilchrist CA, Lockhart LA, Ghosh S, Miller-Sims V, Mann BJ,
Petri WA Jr., Tachibana H. The 150 kDa Gal/GalNAc lectin co-receptor of Entamoeba histolytica is a
member of a gene family containing multiple CXXC sequence motifs. Infect. Immun. 2001; 69:5892-8.
Ayeh-Kumi P, Ali IKM, Lockhart L, Petri WA Jr, Haque R. E. histolytica: Genetic diversity of clinical
isolates from Bangladesh as demonstrated by polymorphisms in serine-rich gene. Exp Parasitol 2001;
99:80-88.
Biographical Sketches for each listed Senior/Key Person 5
Page 25
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
Singh U, Gilchrist CA, Schaenman JM, Rogers JB, Hockensmith JW, Mann BJ and Petri WA Jr. Contextdependent roles of the Entamoeba histolytica core promoter element GAAC in transcriptional activation and
protein complex assembly. Molec Biochem Parasitol 2002; 120:107-116.
Haque R, Duggal P, Ali IM, Hossain MB, Mondal D, Sack RB, Farr BF, Beaty TH, Petri WA Jr. Innate and
Acquired Resistance to Amebiasis in Bangladeshi Children. J Infect Dis 2002; 186:547-552.
Houpt ER, Glembocki DJ, Obrig TG, Moskaluk CA, Lockhart LA, Wright RL, Seaner RM, Keepers T,
Wilkins TD, Petri WA Jr. The mouse model of amebic colitis reveals mouse strain susceptibility to
infection and exacerbation of disease by CD4 + T cells. J. Immunology 2002, 169:4496-503.
Barwick R, Uzicanin A, Lareau S, Malakmadze N, Imnadze P, Iosava M, Ninashvili N, Wilson M, Hightower
AW, Johnston S, Bishiop H, Petri WA Jr., and Juranek DD. Outbreak of amebiasis in Tbilisi, Republic of
Georgia, 1998. J Am Soc Trop Med Hyg 2002; 67:623-31.
McCarthy JS, Peacock D, Trown KP, Bade P, Petri WA Jr, Currie BJ. Endemic invasive amoebiasis in
northern Australia. Med J Australia 2002; 177:570.
Beck DL, Tanyuksel M, Mackey A, Pearson W, Loftus B, Haque R, Petri WA Jr. Sequence conservation of
the Gal/GalNAc lectin from clinical isolates. Exp Parasitol 2002; 101:157-63.
Petri WA Jr, Mann BJ, Haque R. The bittersweet interface of parasite and host: Lectin-carbohydrate
interactions during human invasion by the parasite Entamoeba histolytica. Ann Rev Micro 2002, 56:39-64.
Medina-Bolivar F, Wright R, Funk V, Sentz D, Barroso L, Wilkins TD, Petri WA Jr., and Cramer CL A nontoxic lectin for antigen delivery of plant-based mucosal vaccines. Vaccine 2003, 21: 997-1005.
Gilchrist CA, Leo M, Line CG, Mann BJ, Petri WA Jr. Calcium modulates promoter occupancy by the
Entamoeba histolytica Ca2+-binding transcription factor URE3-BP. J Biol Chem. 2003; 278: 4646 - 4653.
Huston CD, Miller-Sims V, Petri WA Jr. Apoptotic killing and phagocytosis of host cells by the parasite
Entamoeba histolytica. Infect Immun 2003, 71:964-72.
Ali, I.K.M., Hossain, M.B., Roy, S., Ayeh-Kumi, P.F., Petri WA Jr., Haque, R., Clark, C.G. 2003 Entamoeba
moshkovskii infections in children, Bangladesh. Emerg Infect Dis 2003, 9: 580-584.
Haque R, Huston CD, Hughes M, Houpt E, Petri WA Jr. Current Concepts: Amebiasis. New Engl J Med 2003,
348:1565-73.
Tachibana H, Watanabe K, Cheng X-J, Tsukamoto H, Kaneda Y, Takeuchi T, Ihara S, Petri WA Jr.
Neutralizing human antibodies specific for the Entamoeba histolytica Gal/GalNAc lectin heavy subunit.
Infect Immun 2003, 71:4313-9.
Petri WA Jr. Therapy of intestinal protozoa. Trends Parasitol 2003, 19:523-6.
Haque, R, Ali IM, Hossain MB, Mondal D, Sack RB, Farr BF, Beaty TH, Petri WA Jr. Epidemiologic and
clinical characteristics of acute diarrhea and endemic E. histolytica infections in preschool children in an
urban slum of Dhaka, Bangladesh. Am J Trop Med Hyg 2003, 69:398-405.
Houpt E, Barroso L, Lockhart L, Wright R, Cramer C, Lyerly D, Petri WA Jr. Prevention of intestinal amebiasis
by vaccination with the Entamoeba histolytica Gal/GalNac lectin. Vaccine 2004, 22:611-617.
Duggal P, Haque, R, Ali IM, Hossain MB, Mondal D, Sack RB, Farr BF, Beaty TH, Petri WA Jr. HLA Class II
alleles influencing susceptibility to E. histolytica infection in Bangladeshi Children. J Infect Dis 2004,
189:520-6
Pacheco-Yépez J, Shibayama M, Campos-Rodríguez R, Beck D, Houpt E, Petri WA Jr, Tsutsumi V. In vitro
and in vivo interaction of Entamoeba histolytica Gal/GalNAc lectin with various target cells: An
immunocytochemical analysis. Parasitol International 2004, 53:35-47.
Ramakrishnan G, Gilchrist CA, Musa H, Torok M, Grant PA, Mann BJ, Petri WA Jr. Histone
acetyltransferases and deacetylase in Entamoeba histolytica. Molec Biochem Parasitol 2004, 138:205-216.
Loftus B, et al. The genome of the protist parasite Entamoeba histolytica, Nature 2005, 433:865-868.
Okada M, Huston CD, Mann BJ, Petri WA Jr., Kita K, Nozaki T. Comprehensive proteomics analysis of
phagocytosis by the enteric protozoan parasite Entamoeba histolytica. Eukaryotic Cell 2005, 4:827-31.
Beck DL, Boettner DR, Dragulev B, Ready K, Nozaki T, Petri WA Jr. Identification and gene expression
analysis of a large family of transmembrane kinases related to the Gal/GalNAc lectin in Entamoeba histolytica.
Eukaryotic Cell 2005, 4:722-732.
Boettner DR, Huston CD, Sullivan JA, Petri WA Jr. Entamoeba histolytica and Entamoeba dispar utilize
Biographical Sketches for each listed Senior/Key Person 5
Page 26
externalized phosphatidylserine for recognition and phagocytosis of erythrocytes. Infect Immun 2005, 73:
3422-3430.
Roy S, Kabir M, Mondal D, Petri WA Jr, Haque R. Real-time PCR assay for the diagnosis of Entamoeba
histolytica infection. J Clin Microbiol 2005, 43:2168-72.
Tanyuksel M, Yilmaz H, Ulukanligil M, Araz E, Cicek M, Koru O, Tas Z, Petri WA Jr. Modern diagnostic
techniques to avoid misdiagnosis of amebiasis in Turkey. Experimental Parasitol 110:322-326, 2005.
Simonishvili S, Tsanava S, Sanadze K, Chlikadze R, Miskalishvili A, Lomkatsi N, Imnadze P, Petri WA Jr,
Trapaidze N. Entamoeba histolytica: The serine-rich gene polymorphism-based genetic variability of
clinical isolates from Georgia. Experimental Parasitol 110:313-317, 2005.
Haque R, Mondal D, Duggal P, Kabir M, Roy S, Farr BM, Sack RB, Petri WA Jr. Entamoeba histolytica
Infection in Children and Protection from Subsequent Amebiasis. Infect Immun 74:904-909, 2006.
Okada M, Huston CD, Oue M, Mann BJ, Petri WA Jr, Kita K, Nozaki T. Kinetics and strain variation of
phagosome proteins of Entamoeba histolytica by proteomic analysis. Molec Biochem Parasitol 145:171183, 2006.
Solaymani-Mohammadi S, Rezaian M, Babaei Z, Rajabpour A, Meamar AR, Pourbabai AA, and Petri WA Jr.
Comparison of a Stool Antigen Detection Kit and PCR for Diagnosis of Entamoeba histolytica and
Entamoeba dispar Infections in Asymptomatic Cyst-Passers in Iran. J Clin Microbiol 2006, 44:2258-61.
Tarleton JL, Haque R, Mondal D, Shu J, Farr BM, Sack RB, Petri WA Jr. The cognitive effects of diarrhea,
malnutrition, and Entamoeba histolytica infection on school-age children in Dhaka Bangladesh. Am J Trop
Med Hyg 74: 475 – 481, 2006.
Gilchrist CA, Houpt E, Trapaidze N, Fei Z, Crasta O, Asgharpour A, Evans C, Martino-Catt S, Baba DJ, Stroup
S, Hamano S, Ehrenkaufer G, Okada M, Singh U, Nozaki T, Mann BJ, Petri WA Jr. Impact of intestinal
colonization and invasion on the E. histolytica transcriptome. Molec Biochem Parasitol 2006, 147:163-176.
Mehra A, Frederick J, Petri WA Jr, Bhattacharya S, Bhattacharya A. Expression and Function of a Family of
Transmembrane Kinases from the Protozoan Parasite Entamoeba histolytica. Infect Immun 2006 74: 53415351.
Mondal D, Petri WA Jr, Sack RB, Kirkpatrick BD, Haque R,. Entamoeba histolytica - associated diarrheal
illness is negatively associated with the growth of preschool children: Evidence from a prospective study.
Trans R Soc Trop Med Hyg 2006; 100:1032-8.
Leo M, Haque R, Kabir M, Roy S, Lahlou RM, Mondal D, Tannich E, Petri WA Jr. Point-Of-Care Tests for the
Rapid Diagnosis of Amebiasis J Clin Microbiol 2006; 44: 4569-4571.
Solaymani-Mohammadi S, Lam M, Zunt JR, and Petri WA Jr. Entamoeba histolytica Encephalitis Diagnosed
by Polymerase Chain Reaction of Cerebrospinal Fluid. Trans R Soc Trop Med Hyg 2007; 101:311-313.
Ali IKM, Mondal D, Roy S, Haque R, Petri WA Jr, Clark CG. Evidence for a link between parasite genotype
and outcome of infection with Entamoeba histolytica. J. Clin. Microbiol. 2007; 45:285-289.
Haque R, Mondal D, Shu J, Roy S, Kabir M, Davis AN, Duggal P, Petri WA Jr. IFN- Production by
Peripheral Blood Mononuclear Cells is Associated with Childhood Resistance to Amebiasis. Am J Trop
Med Hyg 2007; 76:340-344.
Haque R, Roy S, Siddique A, Mondal U, Rahman SMM, Mondal D, Petri WA Jr. Multiplex Real-Time PCR
Assay for Detection of Entamoeba histolytica, Giardia lamblia and Cryptosporidium spp. Am J Trop Med
Hyg 2007, 76:713-717.
Tanyuksel M, Ulukanligil M, Guclu Z, Araz E, Koru O, Petri WA Jr. Two cases of rarely recognized infection
by Entamoeba moshkovskii. Am J Trop Med Hyg 2007;76 723-724.
Petri WA Jr., Kirkpatrick BD, Haque R, Duggal P. Genes influencing susceptibility to infection. J Infect Dis
2008; 197:4-6.
Boettner DR, Huston CD, Linford AS, Buss SN, Houpt E, Sherman NE, Petri Jr WA. Entamoeba histolytica
Phagocytosis of Human Erythrocytes Involves PATMK, a Member of the Transmembrane Kinase Family.
PloS Pathogens, January 18, 2008.
XXXXXXXX
Biographical Sketches for each listed Senior/Key Person 5
Page 27
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
C. Relevant Research Support
2 R01 AI026649-18 Petri (PI)
12/01/06-11/30/11
Percent Effort: 10%
NIH/NIAID
Annual Direct Costs: $250,000
“Structure and Function of E. histolytica Lectin”
This project will focus on the role of transmembrane kinase 96 in the endocytosis of apoptotic corpses as it has
been identified as part of the phagosome proteome.
R01 AI-37941-10 Petri (PI)
2/1/96 - 2/28/09
Percent Effort: 10%
NIH/NIAID
Annual Direct Costs: $200,000
“Gene Expression in E. histolytica”
The major goals of this project are to analyze the amebic lectin hgl5 promoter.
Biographical Sketches for each listed Senior/Key Person 5
Page 28
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
BIOGRAPHICAL SKETCH
Provide the following information for the key personnel and other significant contributors in the order listed on Form Page 2.
Follow this format for each person. DO NOT EXCEED FOUR PAGES.
NAME
POSITION TITLE
Ward, Gary E
eRA COMMONS USER NAME
Associate Professor
XXXXXXXX
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)
INSTITUTION AND LOCATION
University of New Brunswick, Canada
University of California at San Diego, CA
University of California at San Francisco, CA
DEGREE
(if applicable)
B.Sc.
Ph.D.
Postdoc
YEAR(s)
1979
1985
1985-1989
FIELD OF STUDY
Biophysics
Marine Biology
Cell Biology
A. Positions and Honors
Positions and employment
1979-1985
Graduate Research Assistant (Supervisor: Dr. Victor .D. Vacquier)
Marine Biology Research Division, University of California, San Diego CA
1985-1989
Postdoctoral Fellow (Supervisor: Dr. Marc W. Kirschner)
Dept. of Biochemistry and Biophysics, University of California, San Francisco CA
1989-1996
Senior Staff Fellow (Lab Chief: Dr. Louis H. Miller)
Laboratory of Parasitic Diseases, National Institutes of Health, Bethesda MD
1996-2002
Assistant Professor (1996-2002)
Dept. of Microbiology and Molecular Genetics, University of Vermont, Burlington VT
2002Associate Professor
Dept. of Microbiology and Molecular Genetics, University of Vermont, Burlington VT
Other Experience and Professional Memberships
1980Member, American Society for Cell Biology
1996Finance Committee, American Society for Cell Biology (chair 2002-present)
1998-2000
Finance Committee, Federation of American Societies for Experimental Biology
2000
Member, NIH CSR Special Emphasis Review Panel ZRG1 AARR-1
2002Treasurer and Member of the Executive Committee, American Society for Cell Biology
2003Member, American Society for Microbiology
2005National Library of Medicine, Public Access Working Group
2006
NIH Pathogenic Eukaryotes (PTHE) Study Section (June 2006)
2006
NIH CSR Special Emphasis Review Panel ZRG1 BCMB-Q (90) (S)
2007
National Library of Medicine, PubMed Central Advisory Board
Honors
1979
1979-1983
1986
1985-1988
2001
Governor General of Canada Gold Medal (University of New Brunswick)
Postgraduate Fellow, Natural Sciences and Engineering Research Council of Canada
(University of California, San Diego)
Martin D. Kamen Prize for Graduate Research in Biological Chemistry (University of California,
San Diego)
Jane Coffin Childs Postdoctoral Fellow (University of California, San Francisco)
Burroughs Wellcome Fund New Investigator in Molecular Parasitology
B. Selected peer-reviewed publications (in chronological order)
Ward, G.E. and Vacquier, V.D. (1983). Dephosphorylation of a Major Sperm Membrane Protein is Induced by
Egg Jelly During Sea Urchin Fertilization Proc. Nat. Acad. Sci. USA 80, 5578-5582.
Ward, G.E., Garbers, D.L., and Vacquier, V.D. (1984). Effects of Extracellular Egg Factors on Sperm
Guanylate Cyclase. Science 227, 768-770.
Biographical Sketches for each listed Senior/Key Person 6
Page 29
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
Ward, G. E., Brokaw, C.B., Garbers, D.L., and Vacquier, V.D. (1985). Chemotaxis of Arbacia punctulata
Spermatozoa to Resact, a Peptide from the Egg Jelly Layer. J. Cell Biol. 101, 2324-2329.
Ward, G.E., Moy, G.W., and Vacquier, V.D. (1986). Phosphorylation of Membrane-bound Guanylate Cyclase
of Sea Urchin Spermatozoa. J. Cell Biol. 103, 95- 101.
Ward, G.E. and Kirschner, M.W. (1990). Identification of Cell Cycle-Regulated Phosphorylation Sites on
Nuclear Lamin C. Cell 61, 561-577.
Adams, J. H., Hudson, D. E., Torii, M., Ward, G. E., Wellems, T. E., Aikawa, M., and Miller, L.H. (1990). The
Duffy Receptor Family of Plasmodium knowlesi is Located within the Micronemes of Invasive Malaria
Merozoites. Cell 63, 141-153.
Ward, G. E., Miller, L. H., and Dvorak, J. A. (1993). The Origin of Parasitophorous Vacuole Membrane Lipids
in Malaria-infected Erythrocytes. J. Cell Science 106, 237-248.
Ward, G. E., Fujioka, H., Aikawa, M. and Miller, L. H. (1994). Staurosporine Inhibits Invasion of Erythrocytes
by Malarial Merozoites. Exp. Parasitol., 79, 480-487.
Suss-Toby, E., Zimmerberg, J., and Ward, G.E. (1996) Toxoplasma Invasion: The Parasitophorous Vacuole is
Formed from Host Cell Plasma Membrane and Pinches Off via a Fission Pore. Proc. Nat. Acad. Sci. USA
93, 8413-8418.
Ward, G. E., Tilney, L. G. and Langsley, G. (1997). Rab GTPases and the Unusual Secretory Pathway of
Plasmodium. Parasitology Today 13, 57-62.
Tardieux, I., Baines, I., Mossakowska, M. and Ward, G.E. (1998) Actin-binding Proteins of Invasive Malaria
Parasites: Regulation of Actin Polymerization by a Complex of 32/34-kDa Proteins Associated with Heat
Shock Protein-70kDa. Mol. Biochem. Parasitol. 93, 295-308.
Carey, K.L. and Ward, G.E. (1999) 96-well Plates Providing High Optical Resolution for High-throughput
Immunofluorescence-based Screening of Monoclonal Antibodies against Toxoplasma gondii. J. Immunol.
Meth. 230, 11-18.
Carey, K.L., Donahue, C.G., and Ward, G.E. (2000) Identification and Molecular Characterization of GRA8, a
Proline-rich Dense Granule Protein of T. gondii. Mol. Biochem. Parasitol. 105, 25-37.
Donahue, C.G., Carruthers, V., Gilk, S.D. and Ward, G.E. (2000) The Toxoplasma Homolog of Plasmodium
Apical Membrane Antigen-1 (AMA-1) is a Microneme Protein Secreted in Response to Elevated
Intracellular Calcium Levels. Mol. Biochem. Parasitol. 111, 15-30.
Wichroski, M.J., Melton, J., Donahue, C.G., Tweten, R.K., and Ward, G.E. (2002) Clostridium septicum AlphaToxin is Active against the Parasitic Protozoan Toxoplasma gondii and Targets Members of the SAG
Family of GPI-anchored Surface Proteins. Infect.Immun. 70, 4353-4361.
Ward, G.E., Carey, K.L. and Westwood, N.J. (2002) Using Small Molecules to Study Big Questions in Cellular
Microbiology. Cell. Microbiol. 4, 471-482.
Wichroski, M.J. and Ward, G.E. (2003) Biosynthesis of Glycosylphosphatidylinositol is Essential to the Survival
of the Protozoan Parasite Toxoplasma gondii. Euk. Cell 2, 1132-1136.
Gaskins, E., Gilk, S., DeVore, N.. Mann, T., Ward, G. and Beckers, C.J. (2004) Identification of the Membrane
Receptor of a Class XIV Myosin in Toxoplasma gondii. J. Cell Biol. 165, 383-393
Carey, K.L., Westwood, N.J., Mitchison, T.J., and Ward, G.E. (2004). A Small-Molecule Approach to
Studying Invasive Mechanisms of Toxoplasma gondii. Proc. Nat. Acad. Sci. USA 101, 7433-7438.
Carey, K.L., Jongco, A., Kim, K. and Ward, G.E. (2004). The T. gondii rhoptry protein, ROP4, is secreted into
the parasitophorous vacuole and becomes phosphorylated in infected cells. Euk. Cell, 3, 1320-30.
Mital, J, Meissner, M, Soldati, D. and Ward, G.E. (2005). Conditional expression of Toxoplasma gondii Apical
Membrane Antigen-1 (TgAMA1) demonstrates that TgAMA1 plays a critical role in host cell invasion. Mol.
Biol. Cell 16: 4341-4349.
Alexander, D.L., Mital, J., Ward, G.E., Bradley, P., and Boothroyd, J. (2005). Identification of the Moving
Junction Complex of the Apicomplexan parasite, Toxoplasma gondii: a Collaboration Between Distinct
Secretory Organelles. PLoS Pathogens 1: 137-149.
Mital, J., Schwarz, J. Taatjes, D.J. and Ward, G.E. (2005). Laser Scanning Cytometer-based Assays for
Measuring Host Cell Attachment and Invasion by the Human Pathogen, Toxoplasma gondii. Cytometry
Part A 69A:13–19 (2005).
Gilk, S.D., Raviv, Y., Hu, K., Murray, J.M., Beckers, C.J.M., and Ward, G.E. (2006). Identification of PhIL1, a
novel cytoskeletal protein of the Toxoplasma gondii pellicle, through photosensitized labeling with [125I]-5Iodonapthalene-1-azide (INA). Euk. Cell 5: 1622-1634.
Biographical Sketches for each listed Senior/Key Person 6
Page 30
Brydges, S.D., Zhou, X.W., Hunyh, M., Harper, J.M., Mital, J., Ajoble, D., Daubener, W., Ward, G.E., and
Carruthers, V.B. (2006). Targeted Deletion of MIC5 Enhances Trimming Proteolysis of Toxoplasma
Invasion Proteins. Euk. Cell 5: 2174-2183.
Morgan, R.E., Evans, K.M., Patterson, S., Catti, F. Ward, G.E., and Westwood, N.J. (2007). Targeting
Invasion and Egress: From tools to drugs? Curr Drug Targets 8: 61-74.
Evans, K.M., Haraldsen, J.D., Pearson, R.J., Slawin, A.M.Z, Ward, G.E. and Westwood, N.J. (2007) Synthesis
and chemical characterization of target identification reagents based on an inhibitor of human cell invasion
by the parasite Toxoplasma gondii. Org. Bioorg. Chem. 5, 2063 – 2069.
XXXXXXX
C. Research Support
Ongoing Research Support
NIH, R01 AI054961
Small molecule approaches to studying T. gondii invasion
03/01/03-02/28/09
Goal: To use the small molecule inhibitors and enhancers of Toxoplasma invasion recently
identified by high-throughput screening to elucidate the mechanisms of host cell invasion and
to identify parasite proteins involved in the process
Role: P.I.
NIH, R01 AI063276
Functional Studies of Toxoplasma gondii AMA1 and AMA2
07/01/05-03/31/10
Goal: To characterize the biological function of the parasite proteins AMA1 and AMA2, and to
elucidate the function of cell-surface processing of AMA1.
Role: P.I.
NIH, P20 RR021905
Vermont Immunobiology/Infectious Diseases Center
08/01/06-06/30/11
Goal: To establish a Center of Biomedical Research Excellence (COBRE) in Immune Responses to
Infectious Pathogens at the University of Vermont.
Role: Co-PI, Co-Director
Completed Research Support
XXXXXXX
NIH, K02 AI01719
Characterization of Novel Toxoplasma Surface Proteins
04/01/00-03/31/06
Goal: To identify novel proteins on the surface of the T. gondii tachyzoite and to establish the
biological function of these proteins and their potential utility as diagnostic reagents
Role: P.I.
NIH, R29 AI42355
Surface Proteins of Toxoplasma gondii
Biographical Sketches for each listed Senior/Key Person 6
02/01/98-01/31/03
Page 31
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
Goal: To use identify novel proteins on the surface of the T. gondii tachyzoite and to establish the
biological function of these proteins
Role: P.I.
Biographical Sketches for each listed Senior/Key Person 6
Page 32
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
PHS 398 Cover Page Supplement
OMB Number: 0925-0001
Expiration Date: 9/30/2007
1. Project Director / Principal Investigator (PD/PI)
Prefix:
* First Name: Christopher
Dr.
Middle Name: Dwight
* Last Name:
Suffix:
Huston
MD
❍ No
* New Investigator?
Degrees:
● Yes
MD
2. Human Subjects
Clinical Trial?
● No
❍Yes
* Agency-Defined Phase III Clinical Trial?
● No
❍Yes
3. Applicant Organization Contact
Person to be contacted on matters involving this application
Prefix:
* First Name: Dayna
Ms.
Middle Name: E.
* Last Name:
LeDuc
Suffix:
* Phone Number: (802) 656-4067
Fax Number: (802) 656-3190
Email: Dayna.LeDuc@uvm.edu
* Title:
Research Administrator
* Street1:
Street2:
* City:
County:
* State:
OSP, 85 South Prospect Street
340 Waterman Building
Burlington
Chittenden
VT: Vermont
Province:
* Country:
USA:
* Zip / Postal Code:
Clinical Trial & HESC
Tracking Number: GRANT00424214
05405
Page 33
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
PHS 398 Cover Page Supplement
OMB Number: 0925-0001
Expiration Date: 9/30/2007
4. Human Embryonic Stem Cells
* Does the proposed project involve human embryonic stem cells?
●No
❍Yes
If the proposed project involves human embryonic stem cells, list below the registration number of the
specific cell line(s) from the following list: http://stemcells.nih.gov/registry/index.asp . Or, if a specific
stem cell line cannot be referenced at this time, please check the box indicating that one from the registry will be used:
Cell Line(s):
Specific stem cell line cannot be referenced at this time. One from the registry will be used.
Clinical Trial & HESC
Tracking Number: GRANT00424214
Page 34
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
PHS 398 Modular Budget, Periods 1 and 2
OMB Number: 0925-0001
Expiration Date: 9/30/2007
Budget Period: 1
Start Date: 12/01/2008
End Date: 11/30/2009
A. Direct Costs
Funds Requested ($)
* Direct Cost less Consortium F&A
Consortium F&A
0.00
* Total Direct Costs
200,000.00
B. Indirect Costs
Indirect Cost Type
1. Rsrch On Campus MTDC
200,000.00
Indirect Cost
Base ($)
Indirect Cost
Rate (%)
50.50
* Funds Requested ($)
200,000.00
101,000.00
Total Indirect Costs
101,000.00
Funds Requested ($)
301,000.00
2.
3.
4.
Cognizant Agency (Agency Name, POC Name and Phone Number) DHHS, Louis Martillotti, 212-264-2069
Indirect Cost Rate Agreement Date
05/16/2007
C. Total Direct and Indirect Costs (A + B)
Budget Period: 2
Start Date: 12/01/2009
End Date: 11/30/2010
Funds Requested ($)
A. Direct Costs
200,000.00
* Direct Cost less Consortium F&A
B. Indirect Costs
Indirect Cost Type
1.
Rsrch On Campus MTDC
Consortium F&A
0.00
* Total Direct Costs
200,000.00
Indirect Cost
Rate (%)
Indirect Cost
Base ($)
50.50
* Funds Requested ($)
200,000.00
101,000.00
2.
3.
4.
Cognizant Agency (Agency Name, POC Name and Phone Number) DHHS, Louis Martillotti, 212-264-2069
Indirect Cost Rate Agreement Date
05/16/2007
C. Total Direct and Indirect Costs (A + B)
Modular Budget
Tracking Number: GRANT00424214
Total Indirect Costs
Funds Requested ($)
Page 35
101,000.00
301,000.00
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
PHS 398 Modular Budget, Periods 3 and 4
OMB Number: 0925-0001
Expiration Date: 9/30/2007
Budget Period: 3
Start Date: 12/01/2010
End Date: 11/30/2011
A. Direct Costs
Funds Requested ($)
* Direct Cost less Consortium F&A
200,000.00
Consortium F&A
0.00
* Total Direct Costs
200,000.00
B. Indirect Costs
Indirect Cost Type
1. Rsrch On Campus MTDC
Indirect Cost
Rate (%)
50.50
Indirect Cost
Base ($)
* Funds Requested ($)
200,000.00
101,000.00
Total Indirect Costs
101,000.00
Funds Requested ($)
301,000.00
2.
3.
4.
Cognizant Agency (Agency Name, POC Name and Phone Number) DHHS, Louis Martillotti, 212-264-2069
Indirect Cost Rate Agreement Date
05/16/2007
C. Total Direct and Indirect Costs (A + B)
Budget Period: 4
Start Date: 12/01/2011
End Date: 11/30/2012
Funds Requested ($)
A. Direct Costs
* Direct Cost less Consortium F&A
B. Indirect Costs
Indirect Cost Type
1.
Rsrch On Campus MTDC
200,000.00
Consortium F&A
0.00
* Total Direct Costs
200,000.00
Indirect Cost
Rate (%)
Indirect Cost
Base ($)
50.50
* Funds Requested ($)
200,000.00
101,000.00
2.
3.
4.
Cognizant Agency (Agency Name, POC Name and Phone Number) DHHS, Louis Martillotti, 212-264-2069
Indirect Cost Rate Agreement Date
05/16/2007
C. Total Direct and Indirect Costs (A + B)
Modular Budget
Tracking Number: GRANT00424214
Total Indirect Costs
Funds Requested ($)
Page 36
101,000.00
301,000.00
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
PHS 398 Modular Budget, Period 5 and Cumulative
OMB Number: 0925-0001
Expiration Date: 9/30/2007
Budget Period: 5
Start Date: 12/01/2012
End Date: 11/30/2013
A. Direct Costs
Funds Requested ($)
* Direct Cost less Consortium F&A
200,000.00
Consortium F&A
0.00
* Total Direct Costs
200,000.00
B. Indirect Costs
Indirect Cost
Rate (%)
Indirect Cost Type
1.
Rsrch On Campus MTDC
Indirect Cost
Base ($)
50.50
* Funds Requested ($)
200,000.00
101,000.00
Total Indirect Costs
101,000.00
Funds Requested ($)
301,000.00
2.
3.
4.
Cognizant Agency (Agency Name, POC Name and Phone Number) DHHS, Louis Martillotti, 212-264-2069
Indirect Cost Rate Agreement Date
05/16/2007
C. Total Direct and Indirect Costs (A + B)
Cumulative Budget Information
1. Total Costs, Entire Project Period
* Section A, Total Direct Cost less Consortium F&A for Entire Project Period
$
Section A, Total Consortium F&A for Entire Project Period
$
* Section A, Total Direct Costs for Entire Project Period
$
1,000,000.00
* Section B, Total Indirect Costs for Entire Project Period
$
505,000.00
* Section C, Total Direct and Indirect Costs (A+B) for Entire Project Period
$
1,505,000.00
1,000,000.00
2. Budget Justifications
Personnel Justification
PersonnelJustification.pdf
Consortium Justification
Additional Narrative Justification NarrativeJustification.pdf
Modular Budget
Tracking Number: GRANT00424214
Page 37
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
Attachments
PersonnelJustification_attDataGroup0
File Name
PersonnelJustification.pdf
Mime Type
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Modular Budget
Tracking Number: GRANT00424214
Page 38
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
Personnel Justification:
Christopher D. Huston, M.D., Principal Investigator (3.6 calendar months), will be responsible for the
overall administrative duties and direction of the project. Dr. Huston will supervise and direct the work of Dr.
Teixeira and the graduate assistant, and will conduct some of the experiments outlined in this proposal. At the
bench, his focus will be on conducting phenotypic assays relevant to all three aims.
Jose E. Teixeira, Ph.D., Post-doctoral Associate (12 calendar months), will have primary responsibility for
conducting many of the experiments outlined in aims 1 and 3 of this proposal. Dr. Teixeira has a Ph.D. in
immunology, and has several years of experience doing E. histolytica research. He is skilled in molecular
biology, and in culture and molecular manipulation of E. histolytica. He will complete the studies on the
specificity of the host collectin-E. histolytica interaction, and focus on the more challenging aim of identifying
and characterizing cell surface proteins that interact with the collagenous collectin tail and/or calreticulin.
Bradley Heron, B.S., Graduate Assistant (12 calendar months), will work in cooperation with Dr. Teixeira.
His primary focus will be on the experiments outlined in aim 2 of this proposal with an emphasis on silencing
calreticulin expression by RNAi and on mapping the functional domains of E. histolytica calreticulin. He will
also participate in characterizing the candidate collectin or calreticulin receptor(s) identified by Dr. Teixeira.
Peter Henson, Ph.D., Other Significant Contributor (<5% effort, no funds requested), is a world leader in
studying macrophage phagocytosis of apoptotic cells, and the role of collectins and calreticulin in this process.
He will continue to provide us with purified collectins and collagenous collectin tails, provide the calreticulin
knockout and wild-type mouse embryonic fibroblast cell lines, and assist if necessary with macrophage
experiments.
Tomoyoshi Nozaki, M.D., Ph.D., Other Significant Contributor (<5% effort, no funds requested), a
leading Entamoeba researcher with expertise in phagocytosis and membrane trafficking, will assist with the
project's overall experimental design and with data interpretation through discussions with Dr. Huston, and will
provide reagents including antibodies and vectors as they become available.
William A. Petri, M.D., Ph.D., Other Significant Contributor (<5% effort, no funds requested), is a leading
E. histolytica researcher with expertise in amebic adherence and molecular manipulation of amebae. He will
assist with our RNA-mediated interference experiments to knock-down expression of calreticulin and the
calreticulin-interacting proteins we identify. Dr. Petri's laboratory has developed a system to express short
hairpin RNAs in E. histolytica that has been used to silence a lectin subunit and a transmembrane kinase.
Gary Ward, Ph.D., Other Significant Contributor (<5% effort, no funds requested), will provide expertise
on biochemical methods for protein-protein cross-linking and purification, two-dimensional electrophoresis, and
protein identification by mass spectrometry. These are methods with which he has extensive experience.
Personnel Justification
Page 39
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
Additional Narrative Justification:
Equipment:
No major (>$5000) equipment is requested.
Materials and Supplies:
Funds are requested for the purchase of consumable goods for tissue culture and molecular biology including:
plasticware, pipettes, media and serum for culture of mammalian cells and amebae, media for culture of
bacteria, restriction enzymes, enzymes for PCR, antibodies, fluorescent succinimidyl esters for use in
phagocytosis assays, biotinylation reagents and cross-linking reagents, C1q and mannose binding lectin,
columns for purification of recombinant proteins, and standard laboratory chemicals ($27,293 in year one)
Travel:
Funds are requested for travel one scientific meeting each year for Drs. Huston and Teixeira ($2000 annually).
Publication costs:
Funds are requested to cover publication costs ($1500 annually).
Equipment or Facility Rental/User Fees:
Funds are requested to cover user fees for: 1) confocal microscopy for candidate receptor localization and
confirmation of flow cytometry phagocytosis assay results ($65/hour x 1 hour/week x 52 weeks = $3380
annually), 2) flow cytometry for phagocytosis assays ($40/hour x 4 hours/week x 52 weeks = $8320 annually),
3) fee-for-service mass spectrometry for candidate receptor identification ($3000 annually; note that in the first
year we do not anticipate incurring this expense but expect the cost to be greater than this in years two, three,
and four) and 4) use of the qRT-PCR machine for assessing mRNA levels in RNAi experiments ($250
annually).
Additional Justification
Page 40
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
OMB Number: 0925-0001
Expiration Date: 9/30/2007
PHS 398 Research Plan
1. Application Type:
From SF 424 (R&R) Cover Page and PHS398 Checklist. The responses provided on these pages, regarding the type of application being submitted, are repeated for your reference, as you attach the appropriate sections of the research plan.
*Type of Application:
❍ New
● Resubmission
❍ Renewal
❍ Continuation
❍ Revision
2. Research Plan Attachments:
Please attach applicable sections of the research plan, below.
1. Introduction to Application
intro_ra.pdf
(for RESUBMISSION or REVISION only)
2. Specific Aims
rplan_nar.pdf
3. Background and Significance
rplan_bas.pdf
4. Preliminary Studies / Progress Report
rplan_prs.pdf
5. Research Design and Methods
rplan_rdm.pdf
6. Inclusion Enrollment Report
7. Progress Report Publication List
Human Subjects Sections
Attachments 8-11 apply only when you have answered "yes" to the question "are human subjects involved" on the R&R Other Project Information
Form. In this case, attachments 8-11 may be required, and you are encouraged to consult the Application guide instructions and/or the specific
Funding Opportunity Announcement to determine which sections must be submitted with this application.
8. Protection of Human Subjects
9. Inclusion of Women and Minorities
10. Targeted/Planned Enrollment Table
11. Inclusion of Children
Other Research Plan Sections
12. Vertebrate Animals
13. Select Agent Research
14. Multiple PI Leadership
15. Consortium/Contractual Arrangements
16. Letters of Support
rplan_con.pdf
17. Resource Sharing Plan(s)
18. Appendix
List of Research Plan Attachments
Tracking Number: GRANT00424214
Page 41
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
Attachments
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intro_ra.pdf
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Tracking Number: GRANT00424214
Page 42
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
Appendix
File Name
Appendix_Upload_1.pdf
Appendix_Upload_2.pdf
Appendix_Upload_3.pdf
Mime Type
application/octet-stream
application/octet-stream
application/octet-stream
List of Research Plan Attachments
Tracking Number: GRANT00424214
Page 43
Introduction to the Revised Application:
I am grateful to the reviewers for their enthusiasm and insightful comments. The reviewers' major
concern regarding the original application "[was] that it [was] heavily dependent on untested and/or not in hand
reagents". This prompted us to focus on developing all of the reagents necessary to conduct the proposed
work. The summary statement for the A1 application noted "[the] revised application [was] improved over the
previous submission and there [was] a great deal of interest in the proposed hypothesis. The project [was]
nicely supported by preliminary data and the investigator now has several key reagents in hand, including
monoclonal antibodies and recombinant proteins." Reviewer 1 noted that the "mechanisms of phagocytosis by
[E. histolytica] are not well understood [and that] these studies should lead to a better understanding of amebic
pathogenesis and may lead to a new anti-amebic vaccine target." Reviewer 2 noted that "the investigator has
done an excellent job at responding to the previous critiques…[and that]…all tools are in hand." The major
concern regarding the A1 application was "that the project could fail if the RNAi experiments show that amebae
phagocytose properly when calreticulin is not expressed." In revising the application, I have taken this and all
other concerns very seriously. The proposal has been revised substantially, and the A2 application is much
stronger as a result. Changes in the revised application are highlighted throughout in italics.
I would like to begin with a comment on my productivity, which reviewer 1 noted had been modest. I
am very pleased to report that we have published three papers since the last application (two exclusively from
my laboratory), and a fourth manuscript is under review:
1.
Teixeira JE, Huston CD. Participation of the serine-rich Entamoeba histolytica protein in amebic
phagocytosis of apoptotic host cells. Infection and Immunity. 2008. 76:959-66. PMID: 18086807
(see attached PDF).
2.
XXXXXXX
3.
Teixeira JE, Heron B, Huston CD. C1q- and collectin-dependent phagocytosis of apoptotic host
cells by the intestinal protozoan Entamoeba histolytica. 2008. Submitted.
4.
Boettner DR, Huston CD, Linford AS, Buss SN, Houpt E, Sherman NE, Petri WA. Entamoeba
histolytica phagocytosis of human erythrocytes involves PATMK, a member of the
transmembrane kinase family. PLoS Pathogens. 2008. 4:122-133. PMID: 18208324.
I hope that this work, in conjunction with another first author paper published in 2006, will mollify any concerns
regarding my ability to conduct independent, top quality research on E. histolytica.
As noted above, the primary concern of each reviewer was that the project could fail if no phenotype is
observed when calreticulin expression is silenced by RNA-mediated interference (RNAi). Major changes have
been made to the application, which speak directly to this concern. A new Specific Aim (Aim 3) has been
added, and our hypothesis/model (Figure 1), which is that host collectins bound to apoptotic cells and bacteria
stimulate phagocytosis by interaction of their conserved collagenous tail domain with an amebic receptor, has
been altered to address the possibility that calreticulin may not be the E. histolytica collectin receptor. Text on
the Specific Aims page and elsewhere has been re-written to reflect more clearly that our emphasis is on
identification of an E. histolytica collectin receptor. Calreticulin remains our leading candidate, since it is the
macrophage collectin receptor and is present in purified E. histolytica phagosomes, on the amebic surface, and
in the phagocytic cup (see PMID: 18208324, and Figures 9 and 13). Furthermore, if calreticulin participates,
we hypothesize that it triggers phagocytosis via a calreticulin receptor, analogous to interaction of calreticulin
with CD91 on macrophages. Calreticulin's involvement, however, is simply a hypothesis.
The new Aim, Aim 3, is to identify either a collectin receptor or a receptor for calreticulin. The decision of
which to pursue will be based on the results of Aims 1 and 2, and the predictions of our model. For example, if
we find that inhibition of calreticulin expression by RNAi reduces E. histolytica phagocytosis, then identifying
amebic surface proteins that interact with calreticulin will take priority. On the other hand, if RNAi knockdown
of calreticulin expression causes no phagocytosis defect, then we will identify amebic surface proteins that
interact with the collectins. In either case, we will use a cross-linking method included in the A1 application
(previously Aim 2b: Identification and characterization of E. histolytica surface proteins that bind calreticulin) as
our primary method (see Figure 17). Either C1q tails or recombinant calreticulin will be used as the bait
protein, and parallel samples using free collectins and/or calreticulin to inhibit binding will control for specificity.
Complementary affinity methods are also included. These methods and the proposed methods for candidate
prioritization and characterization are virtually identical to those proposed previously to identify surface proteins
Introduction
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Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
that bind calreticulin. Furthermore, all of the necessary reagents are in hand, since we have now purified the
N-terminal collagenous C1q tail and shown it is a relevant ligand (see new Figure 6C) and we made
biologically active recombinant calreticulin previously (see Figure 14 and new Figure 15). In the summary
statement, reviewer 1 noted that "attempts to find the calreticulin binding partner [were] well described" and
reviewer 2 similarly wrote "these experiments [were] well-described." The experiments in Aim 3 represent an
unbiased approach for identification of a collectin and/or a calreticulin receptor, and the data showing that
collectins stimulate E. histolytica phagocytosis are very strong (see Figures 5, 6, and 7); therefore, these
experiments will yield important results regardless of whether calreticulin participates.
Substantial new preliminary data has also been added to the revised application. Figure 5C shows C1q
binding to apoptotic blebs, as demonstrated by confocal microscopy. Figure 6B includes new data showing
that stimulation of phagocytosis by C1q depends on ligand density, and the method used suggests a way to
ensure equal quantities of different ligands are presented to E. histolytica (see below). Figure 6C summarizes
new results demonstrating stimulation of phagocytosis by single-ligand particles coated with mannose binding
lectin (MBL), and collagenous collectin tails purified from C1q and surfactant protein A (SP-A). These data
provide further evidence that collectins and the collectin tail stimulate amebic phagocytosis, and also document
successful purification of biologically active C1q tails in my laboratory. Finally, Figure 15 shows new flow
cytometry data demonstrating collectin-independent binding of recombinant E. histolytica calreticulin to
apoptotic but not to viable lymphocytes. Collectively, these data demonstrate significant interim progress. A
number of additional changes have been made to address the specific concerns of each reviewer.
Response to the critiques of each specific aim:
Aim 1 is to test the hypothesis that E. histolytica has a receptor for the collagenous tail of C1q and the
collectins. The approach in the A1 proposal was to determine if collectins or collagenous collectin tails
stimulate phagocytosis using collectin- or collectin tail-coated beads, to examine the specificity of the
interaction using soluble collectins (and collectin tails) and N-glycanase to determine if E. histolytica recognizes
N-linked sugars or the protein core of the ligands, and to extend the current studies by examining the role of
collectins in phagocytosis of bacteria.
Reviewer 1 noted no specific experimental concerns, but felt aim 1 was now "partially complete". As noted
above, a manuscript reporting this work has been submitted. However, important work remains to be done. A
true understanding of the molecular mechanism underlying engulfment of collectin-opsonized apoptotic cells
and particles will require detailed studies of specificity as proposed in sections 1.1-1.3. These experiments will
also lay the groundwork for specificity controls to be used during receptor identification. Finally, studies to
determine if E. histolytica phagocytoses bacteria via the same mechanism will help to define the role collectindependent phagocytosis plays in E. histolytica biology.
Reviewer 2 correctly pointed out that we must ensure that equal amounts of the collectin/C1q and Nglycanase-treated collectin/C1q are coupled to the beads used in phagocytosis assays to determine if E.
histolytica recognizes N-linked sugars or the protein core of the collectin ligands (Aim 1.3). I agree. We have
two methods to address this concern. The first is to treat the ligands with N-glycanase after they are bound to
beads, enabling direct comparison of beads prepared as a single batch. Un-coated beads will be treated to
control for unanticipated effects of N-glycanase on the beads themselves, and the proteins can be removed by
boiling to enable SDS-PAGE to confirm deglycosylation. The alternative method is more complicated but has
the advantage of enabling comparison of different ligands (e.g., C1q vs. MBL) as well as the effect of enzyme
treatment on a given ligand. By varying the concentration of C1q-biotin used to prepare C1q-coated particles
while holding the number of particles fixed, we have shown that particle uptake depends on ligand density and
that the streptavidin-coated particles become saturated with ligand as all of the biotin-binding sites are taken.
At low concentrations, addition of more C1q-biotin to the coupling reaction results in higher ligand density and
greater stimulation of phagocytosis. Once enough ligand is used to saturate the beads, however, inclusion of
more C1q-biotin during bead preparation has no effect (see new Figure 6B). By using beads fully saturated
with different ligands, it is possible to directly compare different ligands or to determine the effect of Nglycanase-treatment. To ensure the beads are fully saturated, a set of beads prepared with each ligand can
be made using progressively higher concentrations of ligand and uptake of beads from each set can be
assayed. Beads found to be fully saturated with C1q can then be compared directly to beads fully saturated
with N-glycanase-treated C1q. These new approaches are included in section 1.3.
Aim 2a (now Aim 2) was to determine if calreticulin functions as an E. histolytica phagocytosis receptor.
Preliminary experiments demonstrated calreticulin on the cell surface, interaction of E. histolytica calreticulin
with human C1q, and enhanced phagocytosis by trophozoites over-expressing FLAG-calreticulin. A genetic
Introduction
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Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
approach using RNAi to inhibit calreticulin expression, and immunological and biochemical approaches using
recombinant scFv anti-calreticulin antibodies and recombinant calreticulin were proposed.
Reviewers 1 and 2 both felt that the RNAi experiments to disrupt calreticulin expression were critical,
raising the general concern that the project could fail if trophozoites not expressing calreticulin phagocytose
properly. As described above, we have incorporated a new Aim, Aim 3, to ensure identification of an E.
histolytica collectin receptor regardless of whether calreticulin participates. Aim 2a, now called Aim 2, has
been retained, since calreticulin is a strong candidate based on our preliminary data. Both reviewers 1 and 2
also suggested that genetic approaches other than RNAi be included to ensure that calreticulin silencing is
achieved. Section 2.1 now includes an alternate silencing method developed by David Mirelman and
colleagues that we will employ. This method will also be employed as an alternative to RNAi for silencing
candidate receptors identified in Aim 3. Dr. Mirelman has already been kind enough to provide us with the
necessary plasmids.
Reviewer 1 noted that calreticulin silencing may cause "a problem with secretion…, although the
phenotype is slight in Trypanosomes." We have added a control for this possibility to section 2.1 in which
recombinant E. histolytica calreticulin is added during phagocytosis assays performed with calreticulin knockdown trophozoites. We already have active recombinant calreticulin (see Figures 14 and 15), and, if
calreticulin functions during phagocytosis as a bridge between targets and an E. histolytica surface protein,
then addition of exogenous calreticulin should complement any defect specifically due to absence of
calreticulin from the cell surface. Reviewer 1 felt that "the reasons for using host cells deficient in calreticulin
[were] less compelling." These experiments have been removed.
Reviewer 2 felt that experiments using anti-calreticulin antibodies to inhibit phagocytosis were not feasible,
because of the need to stimulate calreticulin exposure by "priming" the cells at 37º C during which time the
antibodies may be endocytosed. This is a good point, and these experiments have been removed.
Aim 2b in the A1 proposal was to identify amebic surface proteins that interact with calreticulin.
Calreticulin has no transmembrane domain, so we hypothesized that it is retained on the cell surface by
another protein that participates in cell signaling. Two complementary approaches were proposed: a method
in which cell surface proteins are biotinylated and then biotinylated proteins that co-precipitate with
endogenously expressed FLAG-calreticulin are analyzed, and a method using a chemical cross-linking reagent
and recombinant calreticulin as bait. As noted above, this subaim has been replaced in the new proposal with
Aim 3, in which we will identify either a collectin receptor or a calreticulin receptor, with the choice based on the
results from Aims 1 and 2. However, the methodology in either case will be largely unchanged.
Reviewer 1 felt that "Attempts to find the calreticulin binding partner are well described but are very likely
going to produce disulfide isomerase". I agree that the chaperone function of calreticulin could complicate
identification of the relevant cell surface binding partner, and these anticipated challenges are the basis for our
proposed use of two complementary approaches and for the prioritization scheme detailed in Aim 3. It should
be noted, however, that FLAG-PDI is not detected on the amebic surface while FLAG-calreticulin is present on
the surface following stimulation (see Figure 9). Furthermore, both proposed methods favor identification of
cell surface proteins that interact with calreticulin; in the first method, the analysis would be limited to cell
surface proteins that are biotinylated using a membrane impermeant reagent, and, in the second method, the
cross-linking bait would be recombinant calreticulin added to the exterior of intact amebic trophozoites.
Reviewer 2 felt "these experiments [were] well-described."
Budget: The committee recommended reducing the budget to 7 modules from 10 modules. We have
carefully reviewed the budget and reduced it to 8 modules. These cuts were achieved by eliminating one
graduate student, by reducing the primary investigator's effort from 4.8 to 3.6 calendar months, and by
minimizing the supply budget. Based on user costs for flow cytometry and confocal microscopy, and costs for
protein identification by mass spectrometry (all of which are essential for the project), we feel that this is the
minimum budget necessary for successful completion of the Specific Aims (please see budget justification).
In revising this application, I have made every effort to address all of the reviewers' concerns. New
methods have been added to ensure equal amounts of N-glycanase-treated ligands are presented to E.
histolytica, and to provide an alternative method for gene silencing. Most importantly, success of the project no
longer depends on involvement of calreticulin in phagocytosis. Whether we pursue a collectin or a calreticulin
receptor, the well characterized ligands and methods we have developed will make receptor identification
possible, and all of the necessary reagents are in hand to begin immediately. I again thank the reviewers for
their time and insightful comments.
Introduction
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Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
A. Specific Aims:
Amebiasis is the second leading protozoan cause of death worldwide. Entamoeba histolytica, the
causative agent, is an intestinal ameba that acquires nutrients by phagocytosis of colonic bacteria.
Phagocytosis of host cells during tissue invasion is a defining pathologic feature of invasive amebiasis.
Despite the importance of phagocytosis to E. histolytica biology and its presumed role in pathogenesis, the
molecular mechanisms underlying E. histolytica phagocytosis are virtually unknown.
Our published studies have shown that E. histolytica induces host cell apoptosis, causing surface
changes on the dying cell that stimulate amebic phagocytosis. Phagocytosis of host cells has no obvious
evolutionary benefit for E. histolytica, so it is logical that phagocytosis of apoptotic cells may be due to surface
similarities between apoptotic cells and bacteria. Host collectins are pattern recognition molecules of the
innate immune system that opsonize apoptotic cells and bacteria via a C-terminal lectin domain, are present in
colonic mucous, and have a conserved N-terminal collagenous tail domain that stimulates macrophage
phagocytosis. The related serum protein C1q has no lectin domain but shares the collectin tail, and also
opsonizes apoptotic cells. Calreticulin is the macrophage receptor for the collectin tail. Human calreticulin,
which has no transmembrane domain, binds to the collectin tail as
well as directly to apoptotic cells, and stimulates macrophage
phagocytosis by interaction with CD91.
We identified calreticulin in E. histolytica phagosomes. We
now present preliminary evidence that: 1) opsonization of apoptotic
lymphocytes or particles with human C1q, collectins, and purified
collagenous collectin tails stimulates E. histolytica phagocytosis, 2)
human C1q interacts with E. histolytica calreticulin, 3) calreticulin is
present on the amebic surface and within the phagocytic cup, 4) E.
histolytica calreticulin binds to apoptotic cells independently of
collectins, and 5) specific E. histolytica surface proteins coprecipitate with calreticulin. Based on these data, we hypothesize
that host collectins bound to apoptotic cells or bacteria trigger
phagocytosis via an E. histolytica collectin receptor.
Calreticulin is a candidate collectin receptor that may also
bind apoptotic cells and bacteria directly, and may trigger
phagocytosis by interaction with an amebic calreticulin
Figure 1: Model and predictions.
receptor (Figure 1). The specific aims test these hypotheses.
Aim 1: Test the hypothesis that E. histolytica has a phagocytosis receptor specific for the collagenous
collectin tail. We will assay binding of biotinylated C1q and C1q tails to amebic trophozoites using flow
cytometry to determine if binding is saturable and inhibited by free collectins/collectin tails. Using Nglycanase to deglycosylate C1q and C1q-tails, we will determine if E. histolytica recognizes sugars on C1q
or its protein core. Finally, we will use purified collectins (which bind apoptotic cells and bacteria), C1q,
and C1q-tails in combination to determine if E. histolytica uses a collectin receptor to phagocytose bacteria.
Aim 2: Test the hypothesis that cell surface calreticulin is an E. histolytica phagocytosis receptor that
stimulates phagocytosis by interaction with a transmembrane signaling partner. Amebic calreticulin
may be a collectin receptor, and new preliminary data show that it binds to apoptotic cells independently of
the collectins. We will use RNA-mediated interference and an alternative gene silencing method to test if
calreticulin participates in amebic phagocytosis. We will assay phagocytosis of apoptotic cells and
particles coated with recombinant calreticulin, and conduct binding studies using biotinylated calreticulin to
determine if E. histolytica has a calreticulin receptor. If so, we will functionally map calreticulin.
Aim 3: Identify the E. histolytica collectin or calreticulin receptor. We will decide whether to pursue an
amebic collectin or calreticulin receptor based on the results from Aims 1 and 2, and the predictions of our
model (see Figure 1). In either case, the methods will be similar. We will use a multifunctional crosslinker
and either C1q tails or recombinant calreticulin as bait to purify interacting E. histolytica surface proteins.
Inhibition using free collectins and/or calreticulin will ensure specificity. Alternatively, we will biotinylate E.
histolytica surface proteins and use affinity methods to isolate collectin and/or calreticulin interacting
surface proteins. Interaction of candidate receptors with the bait protein will be confirmed in vitro, the
candidates will be localized during phagocytosis by confocal microscopy, and their function will be
examined using RNAi, an alternative gene silencing method, and/or a dominant negative approach.
Specific Aims
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Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
Significance: By providing a molecular understanding of amebic phagocytosis, these studies will substantially
increase knowledge of how E. histolytica interacts with the host and with colonic bacteria. This will provide
novel insights into the pathogenesis of amebiasis, and may suggest new methods of treatment and prevention.
Specific Aims
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Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
B. Background and Significance:
Amebiasis: Entamoeba histolytica, the intestinal protist that causes amebic colitis and liver abscess, causes
an estimated 50 million symptomatic infections annually, and is the second leading protozoan cause of death
worldwide [1, 2]. Approximately 50% of children in Dhaka, Bangladesh are infected with E. histolytica by age
five, highlighting the seriousness of amebiasis as a health problem in the developing world [3]. In the United
States, water-borne pathogens are of growing concern because of the risk of bioterrorism (CDC Category B)
[4]. A recent outbreak of amebiasis due to disruption of the city water supply in Tblisi, Republic of Georgia,
demonstrates the susceptibility of more developed nations to re-emergence of E. histolytica [5].
Entamoeba histolytica is spread by fecal-oral transmission (reviewed in [6]). After ingestion of the
infectious cyst form of the organism, excystation occurs in the terminal ileum or colon. The motile trophozoites
then colonize the colon by adhering to intestinal mucous glycoproteins, in large part via an amebic Dgalactose/N-acetyl-D-galactosamine (Gal/GalNAc) specific adherence lectin [7, 8]. In a minority of infections,
trophozoites penetrate the intestinal epithelium causing amebic colitis, and extra-colonic spread may result in
amebic liver abscess. Acute inflammation, tissue destruction (probably due directly to amebic proteinases and
cytotoxic ability, and to the host’s acute inflammatory response), and amebic phagocytosis of host erythrocytes
and immune cells are the major pathologic features of early invasive disease [9-15]. Interestingly,
inflammation is often minimal despite ongoing tissue destruction in well established disease [9, 10, 16, 17].
Amebic Phagocytosis: Phagocytosis of colonic bacteria is essential for E. histolytica nutrient acquisition and
growth [18-20], and, as noted above, amebic phagocytosis of host cells is a
defining pathologic feature of invasive amebiasis (Figure 2) [6, 15]. Indeed, light
microscopic examination of clinical stool samples can only distinguish E.
histolytica from infection with the intestinal commensal Entamoeba dispar if
amebae that have ingested host erythrocytes are observed [21]. The best
experimental data which establish phagocytosis as a virulence trait is the inability
of phagocytosis deficient E. histolytica mutants to cause liver abscesses in an
Figure 2: Colonic biopsy
animal model [22, 23]. However, each of the mutants used in these studies was
showing phagocytic E.
defective in at least one other virulence trait. Therefore, though phagocytosis is
histolytica trophozoites
clearly a critical aspect of E. histolytica biology, concrete conclusions on the
(arrow). (H&E stain,
Photo: Harrison Juniper)
function of phagocytosis in virulence remain impossible.
The mechanism of E. histolytica phagocytosis is poorly defined. Most data indicate that the signaling
mechanisms mediating particle uptake, vesicle trafficking, and phagosome maturation in Entamoebae are
similar to those of other phagocytes [24-29]. However, other studies describe unique vesicle trafficking events
following phagocytosis [30-34]. Several receptors have also been suggested including: 1) an as yet
unidentified mannose-containing amebic surface molecule that interacts with bacterial mannose binding
proteins [35], 2) a Gal/GalNAc lectin that is implicated in adherence and cell killing [36], 3) an unusual 112 kDa
adhesin that appears to be comprised of two proteins and also possesses proteinase activity [37], and 4) an
immunogenic serine-rich E. histolytica surface protein (the SREHP) that we recently implicated through a
monoclonal antibody screen (PMID: 18086807) [38].
We showed that E. histolytica induces caspase 3-dependent apoptosis of lymphocytes, using a mechanism
that requires ameba-host cell contact via the Gal/GalNAc specific adherence lectin (PMID: 11207613) [39]. In
this study, we also observed frequent ingestion of apoptotic host cells during cecal invasion in mice. Our study
on cell killing led us to hypothesize that apoptotic cell killing facilitates amebic phagocytosis, since the final step
in apoptosis is phagocytic clearance of dying cells [40]. This is indeed the case. We found that host cell
caspase 3 activation precedes amebic phagocytosis, and that E. histolytica ingests apoptotic cells more
efficiently than healthy and necrotic cells (PMID: 12540579) [41]. We also found that E. histolytica
preferentially ingests Ca2+ ionophore-treated red blood cells (RBCs) (PMID: 15908370 ) [42], which is not
surprising since Ca2+ ionophore treatment causes RBC membrane changes reminiscent of apoptosis [43, 44].
Phosphatidylserine (PS) is exposed on apoptotic cells [45], and appears to be a ligand for an amebic
receptor that triggers ingestion of both apoptotic lymphocytes and ionophore-treated RBCs. We showed that
insertion of PS into the outer membrane leaflet of healthy cells stimulates amebic phagocytosis [41], and pretreatment of ionophore-treated erythrocytes with Annexin V (which binds to PS) blocks phagocytosis [42].
These results are consistent with those of a previous study that showed that liposomes containing
phosphatidylserine or a synthetic negatively charged phospholipid, dicetyl phosphate, stimulate E. histolytica
Background & Significance
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Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
actin polymerization [46]. However, the increase in phagocytosis that can be attributed to recognition of PS is
modest, and Annexin V had no effect on phagocytosis of apoptotic lymphocytes [41]. This suggests that E.
histolytica uses multiple ligands and receptors for engulfment of apoptotic cells, the identity of which is an
important unanswered question. Since phagocytosis of bacteria has likely been a major evolutionary pressure
on E. histolytica, ligands present on both apoptotic cells and bacteria are logical candidates to consider.
The Role of the Collectins and C1q in Phagocytosis: The collectins are a family of pattern recognition
molecules of the innate immune system that bind to surface blebs on apoptotic cells and to microbe-associated
sugars, and facilitate macrophage phagocytosis [47-54]. In humans, collectin family members include the
mannose binding lectin (MBL), and the surfactant proteins A and D (SP-A and SP-D respectively) [54].
Though SP-A and SP-D were named for their initial identification in lung secretions, it is now clear that the
collectins are broadly distributed at mucosal surfaces where they opsonize bacteria and facilitate macrophage
phagocytosis [55-60]. Consistent with a role in phagocytosis of bacteria in the lumen of the gastrointestinal
tract, MBL, SP-A, and SP-D have each been identified in intestinal secretions [55, 56, 61-64].
The collectins are multimeric proteins whose basic functional unit is a trimer of three identical or nearly
identical polypeptides, with the number of trimeric subunits varying among different family members [54]. The
monomeric polypeptides are modified with N-linked oligosaccharides [65-68], and have four recognized
structural domains [54]. From N-terminal to C-terminal, these are: a short cysteine-rich region, a collagen-like
region with repeating motifs of Gly-X-Y (where X and Y are often proline or hydroxyproline), an α-helical coiledcoil "neck region", and a C-terminal globular "head" that contains a Ca2+-dependent C-type lectin domain. The
C-terminal lectin domain mediates binding to polysaccharide ligands on the surface of microorganisms and
apoptotic cells [69-71]. The hydrophobic neck region is required for formation of the trimeric subunits, and is
believed to align the collagen-like chains facilitating their assembly into a collagen helix [72-74]. Cysteine
residues within the short N-terminal region then form disulfide bridges between monomers, stabilizing the
trimeric subunits. Together, the assembled cysteine-rich regions and collagen-helices form a collagenous "tail"
that is recognized by the collectin receptor [50, 75-77].
C1q, a subunit of the first component of the classical complement pathway, has no lectin domain, but
shares the conserved collagenous tail [50]. C1q is a serum protein that activates complement after binding to
IgG [78]. C1q also binds directly to apoptotic cells, and facilitates macrophage phagocytosis via the collectin
receptor [50, 79]. Unlike the collectins, however, C1q does not opsonize bacteria [50, 52]. Thus, C1q
participates in macrophage phagocytosis of apoptotic cells, but the collectins (i.e., MBL, SP-A, and SP-D)
participate in both phagocytosis of apoptotic cells and, within mucosal secretions, in phagocytosis of bacteria.
Calreticulin and its Role in Phagocytosis: Calreticulin is a soluble endoplasmic reticulum (ER) chaperone
protein that binds to oligosaccharides on incompletely folded proteins, and retains them in the ER for continued
maturation ([80], reviewed in [81]). It is well established that calreticulin is also present on the surface of many
types of cells, where it is a receptor (also called cC1qR) for the collagenous collectin tail [82-87].
Several lines of evidence suggest that calreticulin plays an evolutionarily conserved role in phagocytosis.
A Dictyostelium discoideum double knock-out mutant of calreticulin and its membrane bound homologue
calnexin (not present in the E. histolytica genome databases) is severely defective in phagocytosis [88]. The
critical role calreticulin plays as an ER chaperone, however, complicates interpretation of studies done using
knock-out lines. Direct evidence that cell surface calreticulin functions in phagocytosis comes from a
macrophage study in which anti-calreticulin antibodies blocked phagocytosis, apparently by inhibiting
interaction of calreticulin with the collagenous tail of C1q or MBL bound to the surface of apoptotic cells [50].
Calreticulin lacks a transmembrane domain, but is retained on the macrophage surface by CD91 [89], and
ligation of calreticulin signals particle uptake by macrophages [50]. Furthermore, calreticulin may participate in
phagocytosis independently of its role as a collectin receptor. Calreticulin itself has lectin activity and binds
directly to apoptotic cells, where it can function as a ligand that stimulates CD91-dependent phagocytosis [90].
Highly conserved calreticulin homologues have been identified in multiple invertebrates, vertebrates, and
plants [88, 91-96]. All have an N-terminal signal sequence that guides translocation into the ER/secretory
pathway and a C-terminal ER retrieval signal (i.e., a "-KDEL" peptide), and all are further divided into three
domains based on analysis of their primary amino acid sequence. In human calreticulin, the N-terminal 180
amino acids are predicted to form a globular ß-sheet, residues 181-290 comprise a proline-rich domain (the Pdomain), and the carboxy-terminal 110 amino acids (the C-domain) are rich in aspartate and glutamate. The
N-terminal domain is believed to mediate protein-protein interactions [81]. Both the P-domain and the Cdomain possess Ca2+ binding ability, and the P-domain possesses calreticulin’s lectin activity [81].
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Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
A calreticulin homologue with 53% identity and 70% similarity to human calreticulin over its entire length is
present in the E. histolytica genome databases of both the Sanger Institute and The Institute for Genome
Research (TIGR) (TIGR locus #: 33.m00213, BLASTP E value=6 e-88 vs. the GenBank NR database). In both
databases, a nearly identical sequence corresponding to the N-terminal 140 amino acids is present, but in
each case the sequence is located at the end of an assembled contig and is incomplete. There is no obvious
CD91 homologue in either database. Similarly, despite the apparent role calreticulin plays in D. discoideum
phagocytosis [88], no obvious CD91 homologue is present in the genome of this organism either.
In recent proteomics studies, we and others identified calreticulin in purified E. histolytica phagosomes,
suggesting it may participate in E. histolytica phagocytosis (PMID: 18208324) [97, 98]. In no case, however,
have confirmatory data or functional studies of amebic calreticulin been presented.
Summary of Rationale and Significance: Entamoeba histolytica’s ability to engulf colonic bacteria is
essential for nutrient acquisition [18-20], and its ability to engulf host cells is a defining pathologic feature of
invasive amebiasis [15, 21]. However, the mechanisms underlying E. histolytica phagocytosis are poorly
understood, and the role of phagocytosis in virulence remains undetermined. Our published studies of
apoptotic cell killing and phagocytosis have defined a model in which E. histolytica induces contact-dependent
host cell apoptosis, resulting in host cell surface changes that engage as yet unknown amebic phagocytosis
receptors (PMIDs: 11207613, 12540579, and 15908370)[39, 41, 42]. The ability of the collectin family of host
pattern recognition molecules to opsonize both apoptotic cells and, within intestinal secretions, bacteria makes
them candidate ligands for an amebic phagocytosis receptor [47-54, 62-64]. Furthermore, identification of
calreticulin (the receptor for the collagenous collectin tail) within purified amebic phagosomes (PMID:
18208324) [97], suggests that E. histolytica calreticulin may trigger amebic phagocytosis by interaction with a
transmembrane signaling partner, analogous to interaction of macrophage calreticulin with CD91 [50]. Given
this and the preliminary data presented below, we hypothesize that host collectins bound to apoptotic
cells or bacteria trigger phagocytosis via an unknown E. histolytica receptor. Calreticulin, which has
no transmembrane domain, is a leading receptor candidate that we hypothesize serves as a bridge
between collectins/apoptotic cells and an E. histolytica calreticulin receptor (see Figure 1). Aim 1, to
determine if E. histolytica has a phagocytosis receptor specific for the collagenous collectin tail, has been
partially completed. The remaining specificity studies will address mechanism and will aid in receptor
identification. The studies on phagocytosis of bacteria will help to define the role of collectin-dependent
phagocytosis in E. histolytica biology. Aim 2 focuses on calreticulin, which may function as a phagocytosis
receptor either by binding collectins, or by binding directly to host cells and/or bacteria. The results of Aims 1
and 2 will define our priorities in Aim 3, which is identification of either a collectin or a calreticulin receptor. For
example, if RNAi silencing of calreticulin expression reduces E. histolytica phagocytosis and recombinant
calreticulin binding to the amebic surface is saturable, then calreticulin is very likely the E. histolytica collectin
receptor and it will be most important to identify calreticulin's downstream signaling partner. However, if
calreticulin silencing produces no phenotype, then we will directly pursue a collectin receptor. Successful
completion of the proposed studies will result in a detailed understanding of the molecular mechanism
underlying E. histolytica phagocytosis, substantially augmenting our knowledge of how E. histolytica interacts
with the host and with commensal bacteria in the gut lumen, and may lead to identification of new vaccine
candidates. Furthermore, amebae with a defined phagocytosis defect will be made in this work, enabling in
vivo studies of the role phagocytosis plays during pathogenesis of amebiasis (beyond the current aims).
Background & Significance
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Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
C. Preliminary Studies:
1. Preliminary studies establishing a system to study amebic phagocytosis and linking apoptotic
killing to phagocytosis of host cells by E. histolytica: We found
that E. histolytica apoptotic killing precedes phagocytosis using
confocal microscopy and FITC conjugated antibodies against active
caspase 3 [41]. To delineate further phagocytosis from adherence
and cytolysis, we then compared amebic ingestion of healthy and
already apoptotic Jurkat T lymphocytes (killed by pre-incubation with
actinomycin D) using confocal microscopy. Phagocytosis was
isolated from cell killing by treating amebae with 10 mM NH4Cl,
which partially blocks cytotoxicity [99]. Healthy cells were found
Apoptotic
Healthy
adherent to, but rarely ingested by NH4Cl-treated amebae, while
% amebae
30 ± 9
62 ± 7*
positive
apoptotic cells were predominantly phagocytosed (Figure 3A and B).
Phagocytic
39 ± 11
142 ± 30*
Twice as many amebae ingested apoptotic than healthy cells, and
index
the phagocytic index (the percentage of amebae with ingested cells
Figure 3: Confocal microscopy analysis
multiplied by the average number of cells per ameba) was more than
of the interaction of healthy (A) and
threefold higher for apoptotic cells (Figure 3C).
apoptotic (B) Jurkat cells with NH4Cl-
treated amebae. TAMRA-labeled cells
We developed a rapid and quantitative flow cytometry-based
(red) were centrifuged onto HM-1:IMSS
phagocytosis assay to independently test whether E. histolytica
strain E. histolytica trophozoites, and
ingested apoptotic cells more efficiently than healthy cells, and to
incubated for 10 min (37ºC). Unbound
facilitate our continued studies of phagocytosis (PMID:
cells were then washed away, and
12540579)[41]. This method measured E. histolytica ingestion of
remaining cells were fixed and stained
apoptotic and healthy Jurkat cells labeled red or green with the
for the E. histolytica Gal/GalNAc lectin
fluorescent succinimidyl esters TAMRA and CFSE respectively. The
with anti-lectin polyclonal antibodies and
labeled cell populations were mixed in a 1:1 ratio, and incubated with
a FITC conjugated secondary antibody
NH4Cl-treated amebae in the presence or absence of D-galactose,
(green). Original magnification ×400.
which blocks Gal/GalNAc lectin-mediated adherence and nearly
(C) Mean phagocytosis of Jurkat cells
incubated as above (n=3, mean and SD,
completely inhibits amebic cell killing [100]. Following incubation,
* indicates p ≤ 0.008).
adherent Jurkat cells were washed off, and the ameba-Jurkat
mixture was analyzed using two-color flow cytometry. Amebae and
Jurkat cells were distinguished by forward and side scatter characteristics, and phagocytosis was quantitated
as the percent of
E. histolytica fluorescence
Figure 4: FACS analysis of phagocytosis of healthy and apoptotic
(+Jurkats + D-galactose)
amebae with
Jurkat cells by E. histolytica. Shown is a two-color dot plot of
*
increased red or green
11.9±0.9%*
0.5±0.1%
amebic fluorescence following incubation of NH4Cl-treated E.
fluorescence.
histolytica trophozoites with CFSE-labeled healthy (green) and
The labeled
TAMRA-labeled apoptotic (red) Jurkat cells in the presence of 50
mM D-galactose (E. histolytica:healthy Jurkat:apoptotic Jurkat cell
apoptotic and healthy
ratio=1:1:1, 37ºC, 10 min). Values are percentages of amebae
Jurkat cell populations
positive for phagocytosis of apoptotic (upper left), healthy (lower
were easily separated
1.7±0.5%
right), or both apoptotic and healthy (upper right) cells (mean and
by their red and green
SD, n=3, * indicates p < 0.006 vs. ingestion of healthy cells).
fluorescence, and the
large size and vesicularity of E. histolytica enabled analysis of greater than 90% of amebae, while excluding
greater than 95% of uningested cells (data not shown). Consistent with the confocal microscopy data, twice as
many amebae ingested apoptotic cells than ingested healthy cells [41]. In the presence of D-galactose, six
times as many amebae ingested apoptotic cells as ingested healthy cells (Figure 4).
These data stress the role of receptors in addition to the Gal/GalNAc lectin in recognition of surface
changes on dying cells. Furthermore, we have established two complementary assays to study amebic
phagocytosis with these studies. Examining phagocytosis by confocal microscopy sectioning (as opposed to
conventional microscopy) eliminates artifacts due to adherence of uningested cells to the outside of amebae.
As defined above, the phagocytic index provides a combined estimate of both the number of amebae active in
phagocytosis and the efficiency of phagocytosis per ameba. The advantage of microscopy is direct
visualization of phagocytosis. The major disadvantages of microscopy are the limited number of cells that can
be examined and that it is time consuming. The FACS assay for phagocytosis that we developed overcomes
these difficulties. Using flow cytometry to measure uptake of fluorescent host cells by amebae, it is possible to
measure phagocytosis by thousands of amebae very quickly, enabling assays under a variety of conditions
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Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
and providing statistical power. The major disadvantage is that it is more prone to artifact than microscopy.
For this reason, results obtained by FACS will be confirmed independently by microscopy.
2. Preliminary studies in support of specific aim 1: Collectin-dependent phagocytosis: The collectins
and C1q bind to apoptotic cells and bacteria, and facilitate macrophage phagocytosis [47, 50-52]. C1q is
restricted to serum, but the collectins are present in serum and in mucosal secretions [55-60, 62-64].
Consistent with the possibility that the collectins or C1q facilitate E. histolytica phagocytosis, we found that
20% more E. histolytica trophozoites engulfed serum-treated apoptotic lymphocytes than lymphocytes treated
with bovine serum albumin (BSA) (negative control) (n=3, p=0.0006 (flow cytometry data, not shown)). We
then compared phagocytosis of apoptotic lymphocytes that had been pre-treated with either purified human
C1q (Quidel Corp.), or with bovine serum albumin (BSA) (negative control) to determine if opsonization of
apoptotic cells with C1q facilitates amebic phagocytosis. Significantly more trophozoites engulfed cells pretreated with C1q than control cells, and the effect was dose dependent (Figure 5A and B). C1q-biotin bound to
both apoptotic and healthy cells as determined by flow cytometry using a streptavidin-Alexa 488 conjugate for
detection, but C1q had no effect on phagocytosis of healthy cells (data not shown). Confocal microscopy
showed that C1q-biotin bound diffusely to healthy cells, but localized to blebs on apoptotic cells (Figure 5C).
Figure 5: C1q facilitates clearance of apoptotic
lymphocytes by HM-1:IMSS E. histolytica trophozoites.
Apoptotic (UV-treated) lymphocytes were CFSE-labeled,
treated with purified human C1q or BSA (negative
control)(30 min, 37ºC), and incubated with amebic
trophozoites (30 min, 37ºC, ameba:host cell=1:4) in the
presence of 100 mM D-galactose to inhibit the amebic
Gal/GalNAc lectin. (A) Representative FACS histograms
showing amebic fluorescence. M1 gate indicates
phagocytic amebae. (B) Dose effect of C1q on amebic
phagocytosis (mean and SD of phagocytic index, n=3, *
indicates p < 0.02 vs. BSA control). (C) Confocal
microscopy showing different patterns of C1q-biotin
binding to healthy and apoptotic cells. Scale bar is 10 µm.
To determine if C1q alone is adequate to trigger amebic phagocytosis, we measured engulfment of
latex beads coupled to either C1q or BSA (negative control). For this, C1q and BSA were biotinylated using
the amine-reactive reagent Sulfo-NHS-LC-Biotin (Pierce Biotechnology), and then used to coat streptavidinmodified 2 µm fluorescent latex beads (Polysciences, Inc.). Bead phagocytosis was assayed using flow
cytometry. The phagocytic index (i.e., the product of the percent amebae positive and the mean fluorescence
of positive amebae) for uptake of C1q-coated beads was nearly ten times that for control beads (Figure 6A and
C). Bead ingestion was assayed independently by microscopy with similar results (phagocytic index of 15.5 ±
7.5 vs. 4.9 ± 2.7, mean and SD, n=5, p=0.02 vs. BSA control). Given the different effects C1q opsonization
had on phagocytosis of apoptotic and healthy cells, we next examined the effect of ligand density by varying
Figure 6: E. histolytica HM-1:IMSS strain trophozoites ingest C1q-, collectin-, and collectin tail-coated single-ligand
particles, and particle uptake depends on ligand density. Streptavidin-modified 2 µm fluorescent latex beads were
coated with biotinylated C1q, MBL, C1q tails, SP-A tails, or BSA (negative control). (A) Representative FACS
histograms of amebic fluorescence following incubation with C1q-coated beads (45 min, 37ºC, ameba:bead=1:10) in
75 mM D-galactose. M1 gate indicates phagocytic amebae. (B) Dependence on C1q-density. C1q-coated particles
with increasing ligand density were prepared by incubating a fixed number of particles with increasing concentrations
of C1q-biotin. Phagocytosis was assayed as above, except using a 90 min time point. (C) Summary of results for
different ligands normalized to the BSA control (mean and SD, n=3, p ≤ 0.005 for b and < 0.05 for c vs. BSA control).
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Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
the concentration of C1q-biotin used to prepare single-ligand particles while holding the number of particles
fixed. The particles were expected to become saturated as the available streptavidin binding sites became
occupied. As shown in Figure 6B, the phagocytic index appeared to be directly related to the amount of C1q
on the beads. This result suggests that increased ligand density on apoptotic blebs may explain the different
effects C1q opsonization had on amebic phagocytosis of apoptotic versus viable cells.
We have assayed E. histolytica phagocytosis of single-ligand beads coated with MBL, C1q tails, and
SP-A tails to determine if the effect of C1q is also seen with collectins and related to the collagenous collectin
tail that members of this protein family share. MBL was purchased (US Biological), and our collaborator, Dr.
Peter Henson (University of Colorado School of Medicine, Denver, CO)(see attached letter and biosketch),
provided us with purified human SP-A tails. We purified the collagenous C1q tail by pepsin digestion and gel
filtration chromatography according to a published protocol [101]. The C1q tails were greater than 90% pure
as judged by SDS-PAGE and silver staining (data not shown). As summarized in Figure 6C, these ligands all
significantly stimulated E. histolytica phagocytosis compared to control beads. Though the largest effect was
observed with intact C1q, these beads were all prepared with non-saturating concentrations of ligand, so it is
impossible to determine if the observed differences in particle uptake were due to functional differences
between the ligands or due to differences in ligand density. We have also conducted transwell migration
assays using these ligands as chemoattractants and a standard method [102]; we found that C1q, MBL, and
purified C1q tails all increased E. histolytica migration by approximately 10-fold when compared to BSA
(negative control; also purified from serum) (p ≤ 0.005 for each vs. BSA control)(data not shown). Collectively,
these data strongly suggest recognition of the collagenous C1q/collectin tail by an amebic receptor.
We have begun using
Figure 7: C1q-biotin binds to the amebic
the C1q-biotin used to make
surface. HM-1:IMSS trophozoites were
these single-ligand particles to incubated at 4º C with C1q-biotin plus either
assess further the basis of the
unlabeled MBL or BSA (negative
C1q/ameba interaction. For
control)(C1q:MBL=1:2.5 (molar ratio)), washed,
this, C1q-biotin was incubated
fixed, blocked with a streptavidin-biotin
with E. histolytica trophozoites
blocking reagent, and stained with streptavidinAlexa 488. Representative FACS histograms
on ice in the presence of
of amebic fluorescence are shown (n=3).
unlabeled MBL or BSA in
molar excess, followed by
detection of bound C1q-biotin with streptavidin-Alexa 488 and flow cytometry (Figure 7). C1q-biotin bound the
E. histolytica surface, and binding was specifically inhibited by free MBL. Experiments using multiple doses of
C1q to determine saturability of binding, and inhibition with a variety of collectins and collectin tails are needed
to verify this result. These data and specific engulfment of the C1q-biotin beads strongly suggest that
biotinylation of C1q does not interfere with its biological activity. In addition, inhibition of C1q-biotin binding by
MBL further supports the hypothesis that the collagenous tail common to C1q and MBL mediates C1q binding.
Based on these data, we concluded that C1q and the collectins are strong stimuli for amebic
phagocytosis. The relatively small effect seen when C1q is used to opsonize apoptotic cells may reflect use of
multiple ligands for phagocytosis, or opsonization of the control cells with collectins and C1q in the growth
medium during tissue culture. It was possible that phagocytosis of C1q-tail coated particles was an artifact,
due to non-specific tethering of the particles to amebae via C1q's "sticky" globular head domain. Uptake of
C1q tail-coated particles (which lack the "sticky" globular head) and migration of amebae towards purified C1q
tails, however, both strongly suggest that the effect is mediated at least in part by a specific E. histolytica
receptor. Using single-ligand particles and flow cytometry as described above, it is possible to rapidly
determine the adequacy of any protein ligand to stimulate E. histolytica phagocytosis. The experiments
proposed in aim 1 use this method, purified collectin tails, and purified collectins to examine if enhanced
clearance of C1q opsonized cells by E. histolytica occurs via a specific receptor for the conserved collectin tail.
3. Preliminary studies in support of specific aim 2: Calreticulin-dependent phagocytosis: Cell surface
calreticulin is the macrophage receptor for the collagenous tail of C1q and the collectins [85-87], and has been
implicated as a phagocytosis receptor in evolutionarily diverse species [50, 51, 84, 88]. We and others found
calreticulin in purified E. histolytica phagosomes (PMID: 18208324)[97, 98]. Therefore, to begin testing the
possibility that cell surface calreticulin mediates E. histolytica phagocytosis, we cloned calreticulin in frame with
the amino-terminal E. histolytica signal sequence from the Gal/GalNAc lectin heavy subunit followed by a
FLAG epitope tag for stable expression in E. histolytica under regulation of the lectin promoter. We cloned
protein disulfide isomerase (PDI), a representative ER protein, in the same way to use as a control (Figure 8A).
Preliminary Studies/Progress
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Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
Figure 8: Stable expression and ER localization of epitopetagged proteins in E. histolytica HM-1:IMSS strain trophozoites.
(A) FLAG-tagged fusion proteins stably expressed in amebic
trophozoites. Constructs were comprised of either PDI or
calreticulin cloned in frame with an amino-terminal signal peptide
and FLAG epitope tag into the E. histolytica expression vector
pGIR235 [140]. (B) Anti-FLAG immunoblot showing expression
of the full-length recombinant proteins. Whole amebic lysates
were separated by SDS-PAGE, transferred to PVDF membrane,
and blotted with anti-FLAG monoclonal antibodies. Lane 1, no
vector control. Lane 2, FLAG-PDI. Lane 3, FLAG-Calreticulin.
The predicted molecular weights are 37.5 and 45.1 kDa for PDI
and Calreticulin respectively. (C) Confocal microscopy showing
co-localization of the expressed fusion proteins with the ER
chaperone BiP, and (D) virtually no co-localization with the early
golgi marker Arf-1. Amebic trophozoites expressing recombinant
proteins were permeabilized, and stained with an anti-FLAG
mouse monoclonal antibody and polyclonal rabbit anti-BiP serum
OR rabbit anti-human Arf-1 serum followed by goat anti-mouse
Alexa 488 (green) and goat anti-rabbit Alexa 568 (red) secondary
antibodies. Slides were examined with a confocal microscope.
No staining was seen with the secondary antibodies alone (data
not shown). 10 µm scale bars are shown.
Immunoblots using an anti-FLAG mouse monoclonal antibody (M2 clone, Stratagene) confirmed stable
expression of the full length proteins in E. histolytica (Figure 8B). Both fusion proteins localized to the amebic
ER, as verified with confocal microscopy by immunofluorescent staining and co-localization with the ER
chaperone BiP (HSP70) using anti-BiP rabbit polyclonal antiserum (a gift from our collaborator, T. Nozaki,
Gunma University, Gunma, Japan)(see attached letter and biosketch) (Figure 8C). Consistent with what had
been the prevailing view of the ER in Entamoeba species [103-105], the ER-targeted fusion proteins and BiP
co-localized in what appeared to be a subset of vesicles in fixed trophozoites. It should be noted, however,
that we recently published evidence that E. histolytica has a continuous ER compartment, as determined using
an ER-targeted green fluorescent protein (GFP)-fusion protein to visualize the ER in living trophozoites and the
method fluorescence loss in photobleaching (FLIP)(paper in press attached; PMID: 18281599)[106]. This ERtargeted GFP-fusion also co-localized with BiP and, like the FLAG-calreticulin and FLAG-PDI proteins, had a
vesicular staining pattern in fixed cells. It is not currently possible to knock-out the endogenous copies of
calreticulin and PDI in E. histolytica and express fusion proteins under their own promoters. Therefore, to
confirm that the calreticulin- and PDI-fusion proteins were properly localized to the ER, we compared their
distribution to that of ADP-ribosylation factor-1 (Arf-1) using anti-human Arf-1 rabbit polyclonal antiserum (a gift
of V. Hsu, Brigham and Women’s Hospital, Boston, MA). Arf-1 is a GTP-binding protein that exists in
equilibrium between golgi-bound and cytosolic pools [107], and this antiserum was previously reported to label
a subset of vesicles corresponding to the amebic golgi apparatus [104]. Consistent with exclusion of the ERtargeted fusion proteins from the E. histolytica golgi apparatus, the anti-Arf-1 serum specifically stained a
subset of vesicles, which conspicuously did not co-localize with the fusion proteins (Figure 8D).
We next used these amebae and surface immunostaining followed by flow cytometry to determine if
calreticulin is present on the amebic surface under some conditions. The baseline fluorescence histograms for
parasites expressing FLAG-calreticulin and FLAG-PDI were no different than histograms of parasites stained
with an isotype control antibody (data not shown). However, FLAG-calreticulin was readily detected on the
surface of a subset of amebic trophozoites following incubation with lymphocytes (Figure 9A (bottom
histogram) and B). No surface staining was observed with amebae expressing the irrelevant protein PDI
(Figure 9A, top histogram). Furthermore, both recombinant proteins were expressed and stained at equal
levels in permeabilized trophozoites (data not shown), and the calreticulin surface staining observed after
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Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
Figure 9: Calreticulin becomes exposed on
the surface of stimulated E. histolytica HM1:IMSS strain trophozoites. Amebae
expressing FLAG-tagged calreticulin or the
irrelevant ER protein PDI were incubated
alone or with Jurkat lymphocytes (45 min,
37ºC, ameba:host cell=1:2), and stained for
flow cytometry with an anti-FLAG monoclonal
antibody and FITC conjugated anti-mouse
IgG. (A) Representative FACS histograms
showing amebic fluorescence. R2 gate
indicates surface stained amebae. (B)
Percent of amebae positive relative to PDI
control without Jurkat cells (mean and SD,
n=3, * indicates p < 0.003 vs. control).
A.
R2
B.
R2
incubation with lymphocytes was specific and not simply an artifact of membrane damage during incubation or
sample preparation, since no FLAG-PDI staining occurred without permeabilization.
To determine if cell surface calreticulin might enable phagocytosis by interaction with C1q and other
collectins bound to apoptotic cells, we first did co-immunoprecipitation experiments using our FLAG-tagged
constructs. Anti-FLAG immunoprecipitation was performed after incubating E. histolytica trophozoites with
apoptotic lymphocytes that had been pre-treated with purified human C1q. Roughly equal quantities of FLAGcalreticulin and FLAG-PDI were recovered (Figure 10, bottom). The same quantity of C1q was recovered with
immunoprecipitation from the untransfected and FLAG-PDI expressing control amebas. In contrast, a large
amount of C1q co-precipitated with FLAG-calreticulin (Figure 10, top), suggesting either direct interaction
between human C1q and the recombinant protein or the presence of both proteins within a larger complex.
Figure 10: Human C1q co-immunoprecipitates with E.
histolytica calreticulin. Apoptotic Jurkat cells were pretreated with purified human C1q (30 min, 37ºC), and then
incubated with untransfected E. histolytica (HM-1:IMSS) or
E. histolytica expressing FLAG-tagged PDI or calreticulin
(30 min, 37ºC, ameba:host cell=1.3:1) before lysis and
immunoprecipitation with an anti-FLAG monoclonal
antibody. (Top) Anti-C1q immunoblot of
immunoprecipitated proteins. (Bottom) The same
membrane stripped and re-probed with an anti-FLAG
monoclonal antibody. The precipitant from an equal
number of amebae was run in each lane.
Phagocytosis assays comparing engulfment of apoptotic lymphocytes by amebae constitutively expressing
FLAG-calreticulin or FLAG-PDI further suggest that calreticulin may be a phagocytosis receptor. Twenty
percent more amebae over-expressing calreticulin ingested apoptotic lymphocytes compared to wild-type
amebae after 20 min of co-incubation. Furthermore, more than twice as many amebae over-expressing
calreticulin were phagocytic compared to those carrying the PDI vector (% phagocytic amebae: FLAGcalretcilin=48.2 ± 2.5, FLAG-PDI=22.2 ±
Figure 11: Calreticulin over3.1, and wild-type=40.1 ± 1.3 (mean and
expression enhances phagocytosis
SD, n=3, p=0.007 for FLAG-calreticulin vs.
of apoptotic lymphocytes. Apoptotic
(UV-treated) lymphocytes were
HM-1:IMSS control)) (Figure 11). The
CFSE-labeled and incubated with
possibility that over-expression of
untransfected E. histolytica (HMcalreticulin enhances phagocytic ability
1:IMSS
control) or E. histolytica
indirectly cannot currently be excluded. For
constitutively expressing either
example, over-expression of calreticulin
FLAG-calreticulin or FLAG-PDI (20
may enhance phagocytosis by increasing
min, 37ºC, ameba:host cell=1:4).
surface expression of other molecules
Representative FACS histograms of
involved in phagocytosis due to the
amebic fluorescence are shown. R2
chaperone function of calreticulin in the ER.
gate indicates phagocytic amebae.
Encouraged by these data, we sought anti-calreticulin antibodies to facilitate our continued studies of E.
histolytica calreticulin and its possible involvement in amebic phagocytosis. Preliminary immunoblots and
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Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
immunofluores
Figure 12: Anti-calreticulin scFv characterization. (A)
cent
Immunoblots showing specific binding of scFv reagents
staining/flow
to an amebic protein the size of FLAG-calreticulin. HM1:IMSS lysates were probed with monoclonal scFv
cytometry
peptides, followed by a mouse anti-myc IgG and antiusing several
mouse IgG-HRP conjugate. (B) Confocal microscopy
commercially
showing
co-localization of calreticulin with the ER
available
chaperone BiP. Permeabilized amebic trophozoites were
antibodies
stained with anti-calreticulin scFv F11A and polyclonal
demonstrated
rabbit anti-BiP serum followed by mouse anti-myc, goat
no crossanti-mouse Alexa 488 (green), and goat anti-rabbit Alexa
reactivity (data
568 (red) antibodies. No staining was seen with the
not shown).
secondary antibodies alone, and the anti-myc/anti-mouse
Given this, we
Alexa 488 combination did not stain the anti-BiP rabbit
antibodies (not shown). Scale bar is 10 µm.
chose to make
recombinant
monoclonal single-chain Fv (scFv) antibodies using phage display and two phagemid libraries (Tomlinson I and
J) developed by the MRC Centre for Protein Engineering (Cambridge, UK) [108-110]. These phagemid
libraries each include over 100 million different scFv peptides comprised of VH and VL domains derived from a
single human antibody framework, and connected by a flexible linker. Antigen-specific phage are selected by
standard phage display and panning against the antigen of interest. For scFv production, selected phage are
then used to infect an Escherichia coli strain (HB2151) that is incapable of suppressing termination at an
amber stop codon located 3' to the scFv gene in the phagemid vector. Recombinant scFv antibodies produced
with these libraries are monovalent like Fab
Figure 13: Confocal microscopy showing calreticulin in the
fragments and can be used for the same
phagocytic cup during amebic engulfment of lymphocytes and
applications as conventional monoclonal
particles. Amebae were allowed to interact with either apoptotic
antibodies. Furthermore, they can be purified or
Jurkat cells or 2 µm red fluorescent latex beads (10 min, 37ºC),
detected using Protein A-based reagents
fixed, and stained with anti-calreticulin F11A scFv followed by a
(because they are derived from human VH3
mouse anti-myc monoclonal IgG and goat anti-mouse IgG
family variable domains that bind protein A
Alexa 488 (green) conjugate. At right is control staining with the
independently of the Fc domain [111]), and
anti-myc IgG and anti-mouse IgG Alexa 488. (A) Interaction
reagents that bind to incorporated myc or
with apoptotic lymphocytes. 10 µm scale bars are shown. (B)
Interaction with beads. Orthogonal views of four representative
6×histidine (His) tags.
We have used this method and purified
recombinant 6xHis-tagged E. histolytica
calreticulin expressed in E. coli as antigen to
select and produce four monoclonal scFv
reagents that specifically bind to E. histolytica
calreticulin. All four monoclonal scFv peptides
specifically bound an amebic protein that ran
alongside FLAG-calreticulin when used as
primary detection reagents for immunoblots
(Figure 12A). All four reagents also bound E.
histolytica as determined by immunofluorescent
staining and flow cytometry, while no binding
was detected with an irrelevant monoclonal scFv
reagent (data not shown). Finally, confocal
microscopy was performed with one of the
reagents (scFv F11A) and demonstrated
excellent co-localization with the ER marker BiP,
further validating its specificity (Figure 12B).
z-sections and two control sections with DIC images are shown.
Arrows indicate partially engulfed cells or beads.
We used these scFv reagents for
immunofluorescent confocal microscopy to
localize native calreticulin during E. histolytica
phagocytosis. Calreticulin dramatically relocalized to the phagocytic cup at early time
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Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
points (≤ 10 minutes) during phagocytosis (Figure 13A). Consistent with our proteomic analysis of the E.
histolytica phagosome and with macrophage data [97, 112], calreticulin was only rarely detected in the
phagosome at 40 minutes (data not shown). The F11A scFv reagent stained permeabilized Jurkat cells faintly,
leading us to question whether it detected E. histolytica calreticulin, host calreticulin, or both. To
unambiguously localize amebic calreticulin, we performed a similar experiment using red-fluorescent latex
beads. Again, calreticulin was present in the phagocytic cup at early time points, but was detected less often
surrounding completely engulfed particles (Figure 13B).
These scFv reagents and the recombinant calreticulin used to produce them also enabled us to
determine if amebic calreticulin interacts directly with human C1q. For this, we established an ELISA-based
method to measure binding of recombinant calreticulin to immobilized C1q. Specific, saturable binding of
amebic calreticulin to C1q was readily detected (Figure 14A), and, consistent with published studies on
calreticulin from other species [113], calreticulin binding was Ca2+-dependent (data not shown). The basis for
this interaction was explored by examining the effect of free collectins on calreticulin binding, including human
SP-A and SP-A tails provided by our collaborator, Dr. Henson. We did not yet have C1q tails when this
experiment was done. Free C1q, MBL, SP-A, and, to a lesser extent, purified SP-A tails specifically inhibited
calreticulin binding (Figure 14B). Together, these data indicate that recombinant E. histolytica calreticulin
produced in E. coli is biologically active, and that E. histolytica calreticulin interacts directly with human C1q.
Furthermore, the ability of free C1q and collectins to specifically inhibit the C1q-calreticulin interaction further
demonstrates that we have these reagents in hand, that they are biologically active, and that the calreticulinC1q interaction likely occurs via the conserved collagenous tail these molecules have in common.
Figure 14: Amebic calreticulin binds directly to human C1q.
Free collectins inhibit calreticulin binding. (A) Saturable binding
of calreticulin to C1q. Recombinant E. histolytica calreticulin was
incubated with immobilized human C1q or BSA (control), washed,
and bound calreticulin was detected using biotinylated F11A scFv
and streptavidin-HRP. Graph shows optical density vs.
[calreticulin] (mean and SD, n=3). (B) Effect of free collectins on
calreticulin-C1q binding. Calreticulin was interacted with
immobilized C1q in the presence of excess free C1q, MBL, SP-A,
or SP-A tails (1:3 molar ratio). Bound calreticulin was detected
as above. Graph shows specific binding normalized to the PBS
control (mean and SD, n=3, * indicates p<0.05 vs. BSA control).
Human calreticulin has been found on the surface
of apoptotic cells, where it acts in trans to trigger
CD91-dependent macrophage phagocytosis that is
collectin-independent [90]. Given this, we wondered if
E. histolytica calreticulin also binds directly to
apoptotic cells, independently of C1q and the
collectins. To test this, we biotinylated recombinant E.
histolytica calreticulin, and assayed calreticulin-biotin
binding to healthy and apoptotic lymphocytes that
were grown in serum-free (i.e., collectin-free) media.
Bound calreticulin-biotin was detected with
streptavidin-Alexa 488 and flow cytometry. As shown
in Figure 15, E. histolytica calreticulin specifically
bound to a subset of apoptotic cells, but did not bind to
healthy cells. These data are consistent with the
possibility that calreticulin functions in phagocytosis
both as a collectin receptor and, independently of the
collectins, by binding directly to apoptotic cells.
Figure 15: E. histolytica calreticulin binds directly to
apoptotic host cells, but not healthy cells. Healthy (A)
and apoptotic (B) Jurkat lymphocytes grown in serumfree media were washed, fixed, blocked with a
streptavidin/biotin blocking reagent, and incubated with
biotinylated recombinant E. histolytica calreticulin (30
µg/ml, 1 hr., room temperature). Cells were then
washed, and bound calreticulin-biotin was detected with
streptavidin-Alexa 488 and flow cytometry.
With these experiments we have shown calreticulin to be on the amebic surface and within the phagocytic
cup during interaction with beads and apoptotic lymphocytes, established a positive correlation between
calreticulin expression and phagocytic ability, and demonstrated the ability of E. histolytica calreticulin to
interact directly with human C1q. Inhibition of calreticulin binding by free collectins supports the notion that the
calreticulin-C1q interaction is via the conserved collagenous tail. Amebic calreticulin also bound apoptotic cells
directly, suggesting it could function in phagocytosis either in conjunction with and/or independently of the
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Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
collectins. Given these data and the described role of calreticulin in macrophage phagocytosis [50, 51], we
hypothesize that calreticulin is an amebic phagocytosis receptor. The studies in aim 2 will directly test the
function of cell surface calreticulin.
4. Preliminary studies in support of specific aim 3: Receptor identification: Since calreticulin is our
leading candidate for an E. histolytica collectin receptor, we biotinylated E. histolytica surface proteins and then
performed co-immunoprecipitation experiments to determine if calreticulin might interact with amebic cell
membrane proteins that conduct a phagocytosis signal, and to begin developing a method to identify candidate
proteins. Biotinylation of amebic surface proteins was accomplished using the membrane impermeable aminereactive reagent Sulfo-NHS-LC-Biotin (Pierce) with a method that was recently demonstrated to label amebic
surface proteins selectively [114, 115]. After surface labeling, amebae were incubated with Jurkat
lymphocytes, and FLAG-calreticulin was then immunoprecipitated. Lysates from wild-type HM-1:IMSS strain
amebas and amebas expressing recombinant FLAG-PDI served as controls for the specificity of coprecipitating proteins. The precipitated surface proteins were detected by blotting with a streptavidinHorseradish Peroxidase (HRP) conjugate and chemiluminescence following separation by SDS-PAGE and
transfer to PVDF membrane (Figure 16). Failure to detect any Jurkat proteins (lane 1) verified the specificity of
the streptavidin-HRP conjugate for biotinylated proteins, and probing whole amebic lysates (lanes 2, 3, and 4)
verified successful biotinylation of surface proteins on the control amebas and those expressing FLAG-tagged
calreticulin. At least one biotinylated protein was detected in the FLAG-calreticulin precipitant (arrow, lane 7)
that was not present in either of the untransfected or FLAG-PDI controls (lanes 5 and 6, respectively). If the
results from aim 2 confirm calreticulin's function as an E. histolytica phagocytosis receptor, we will scale-up this
approach and use it and a complementary cross-linking method for identification of calreticulin-interacting E.
histolytica surface proteins in aim 3. We hypothesize that calreticulin interacts with at least one
transmembrane signaling protein that initiates phagocytosis, but calreticulin could act as part of a larger
signaling complex. For example, calreticulin may be retained on the amebic surface by interaction with a lipid
anchored protein. A recent report demonstrating raft-like membrane domains in E. histolytica pinocytosis and
adhesion supports this possibility [116]. Of course, as noted earlier, calreticulin may not be the E. histolytica
receptor for phagocytosis of C1q-/collectin-coated cells and particles. If the results from Aim 2 indicate that
calreticulin is not an E. histolytica phagocytosis receptor, the collagenous collectin tail will be used as the bait
protein for the cross-linking method and an affinity method (which utilizes the method used here to biotinylate
E. histolytica surface proteins) for identification of the collectin receptor (see Figure 17).
Figure 16: At least one biotinylated amebic surface protein
specifically co-immunoprecipitates with FLAG-calreticulin. Surface
proteins of untransfected E. histolytica or E. histolytica constitutively
expressing FLAG-calreticulin or FLAG-PDI were biotinylated with the
amine-reactive reagent Sulfo-NHS-LC-Biotin. Labeled amebae were
then stimulated by incubation with Jurkat lymphocytes (30 min, 37ºC),
lysed, and immunoprecipitation was done using an anti-FLAG
monoclonal antibody. Shown is a streptavidin-HRP blot of lysates or
immunoprecipitant from an equal number of amebic trophozoites.
Lanes: 1=Jurkat lysate, 2=HM-1:IMSS lysate, 3=FLAG-PDI lysate,
4=FLAG-calreticulin lysate, 5=HM-1:IMSS precipitant, 6=FLAG-PDI
precipitant, 7=FLAG-calreticulin precipitant. The arrow indicates a
band specific to the FLAG-calreticulin precipitant.
6. Summary of preliminary results: In these experiments, we have developed methods and made or
acquired reagents to study E. histolytica phagocytosis that include: 1) quantitative and rapid assays for
phagocytosis, 2) a method for construction of single-ligand particles, 3) a method to label and isolate amebic
surface proteins that interact with calreticulin, 4) E. histolytica trophozoites that stably express epitope-tagged
calreticulin and control amebae, 5) biologically active biotinylated-C1q, 6) biologically active C1q, MBL, SP-A,
purified collagenous C1q tails, and purified collagenous SP-A tails, 7) biologically active recombinant amebic
calreticulin, and 8) a series of purified monoclonal scFv peptides that bind to E. histolytica calreticulin. Using
these methods and reagents, we have: 1) delineated E. histolytica phagocytosis from cell killing and
established that apoptotic cell killing facilitates phagocytosis; 2) shown that unrecognized receptor-ligand pairs
mediate phagocytosis; 3) demonstrated that opsonization of apoptotic cells with human C1q increases E.
histolytica phagocytosis, as does opsonization of latex particles with C1q, MBL, and C1q tails; and 4)
demonstrated that over-expression of calreticulin increases E. histolytica's phagocytic ability, calreticulin is
recruited to the amebic surface and the phagocytic cup during incubation with lymphocytes, and that E.
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Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
histolytica calreticulin interacts directly with human C1q and also binds directly to apoptotic cells.
Many interesting and important questions remain including: 1) Is the conserved collagenous collectin
tail a ligand for a specific amebic phagocytosis receptor? 2) If the collagenous tail is a specific ligand for
amebic phagocytosis, does E. histolytica engulf both apoptotic host cells and colonic bacteria via a common
mechanism? 3) Does cell surface calreticulin function as an amebic phagocytosis receptor, and, if so, is it a
receptor for the collagenous collectin tail, does it bind directly to target particles (e.g., by virtue of its own lectin
activity), or both? And 4) what is the identity of the receptor responsible for uptake of C1q/collectin opsonized
apoptotic cells and particles, be it calreticulin or another amebic surface protein? If calreticulin (which has no
transmembrane domain) is a phagocytosis receptor, what protein(s) does calreticulin interact with to stimulate
amebic phagocytosis? These questions are the basis for the current specific aims.
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D. Research Design and Methods:
General methods to be used throughout these studies: Unless otherwise indicated, experiments will be
conducted using HM-1:IMSS strain E. histolytica trophozoites (American Type Culture Collection (ATCC))
grown in TYI-S-33 medium (trypticase yeast extract, iron, and serum) supplemented with 100 U of penicillin/ml
and 100 µg/ml of streptomycin [117]. We will use Jurkat (Clone E6-1) T lymphocytes (ATCC) as host target
cells [118]. These cells will be cultured in serum-free medium (AIM V Medium (Gibco, Invitrogen)
supplemented with 50 µg/ml streptomycin and 10 µg/ml gentamicin) to avoid potential confounding effects due
to residual serum components (including C1q and the collectins) [52]. Apoptosis will be induced by ultraviolet
(UV) irradiation for 15 minutes, followed by a 3.5 hour incubation at 37ºC prior to use. Phagocytosis assays
and preparation of single-ligand particles will be performed as described above (Figures 3 and 4 (phagocytosis
assays), and Figure 6 (single-ligand particles)).
Aim 1: Test the hypothesis that E. histolytica has a phagocytosis receptor specific for the collagenous
collectin tail.
Rationale: C1q and the collectins both facilitate macrophage phagocytosis by binding to apoptotic blebs with
their globular head domains, and then to macrophage calreticulin via their conserved collagenous tail [50-52].
The collectins, but not C1q, also bind to bacteria [47, 48], and are present in intestinal secretions [55, 56, 6164]. The ability of E. histolytica to ingest both apoptotic cells and colonic bacteria suggests they may have
features in common that facilitate E. histolytica phagocytosis. One possible feature is the ability of collectins to
bind to both. Consistent with this, E. histolytica ingested apoptotic cells opsonized with human C1q better than
control cells, and latex beads coated with C1q, MBL, and C1q tails better than control beads. To understand
the significance of these data, it is critical to determine the mechanism of the C1q-ameba interaction. We
hypothesize that C1q and the collectins facilitate E. histolytica phagocytosis by interaction with an amebic
receptor specific for their conserved collagenous tail, providing a mechanism for engulfment of both host cells
and bacteria. Because of confounding effects due to C1q’s ability to bind IgG, inhibitory anti-C1q antibodies
are of limited use. We will address this hypothesis with experiments using purified collectins and collectin tails.
The critical questions include: 1) whether soluble collectins inhibit phagocytosis of C1q tail-coated beads, and
2) whether C1q/C1q-tail binding to the amebic surface is saturable and inhibited by free collectins.
Method:
1.1: Determine if C1q tails specifically stimulate amebic phagocytosis: This subaim is now partially
complete. Our collaborator, Dr. Henson, has provided us with purified SP-A collagenous tails that partially
inhibited binding of amebic calreticulin to human C1q in our hands (see Figure 14). Furthermore, we have
purified the collagenous C1q tail using a published protocol [50, 101, 119]. These C1q tails stimulated E.
histolytica phagocytosis when immobilized on latex beads and were also chemoattractants for E. histolytica
(Figure 6C and data not shown). Therefore, the collagenous C1q tail can trigger E. histolytica phagocytosis.
Additional studies are needed to determine if C1q tail-, C1q-, and MBL-coated particles are phagocytosed by a
common collectin receptor. Two experiments are planned. In the first, we will use our flow cytometry and
confocal microscopy phagocytosis assays (see Figures 3 and 4) to determine if increasing concentrations of
soluble C1q, C1q tails, SP-A tails, and MBL inhibit E. histolytica phagocytosis of C1q tail-coated single-ligand
particles. Inhibition with a commercially available polyclonal rabbit anti-human C1q antibody (Quidel) or a
rabbit control antibody will serve as an additional control. BSA-coated and C1q-coated beads will be included
as negative and positive controls for phagocytosis. In the second experiment, we will determine if the
conserved collagenous tail is the basis for recognition of intact C1q by E. histolytica. For this, we will use flow
cytometry and confocal microscopy to measure the effect of increasing concentrations of soluble C1q, MBL,
and C1q tails on engulfment of beads coated with intact C1q and of C1q-opsonized apoptotic lymphocytes.
Here, BSA will again serve as a negative control. As noted above, the ability of C1q's globular head to bind to
IgG limits the use of inhibitory anti-C1q antibodies in experiments that use intact C1q.
Interpretation, potential pitfalls, and alternative approaches: We anticipate that soluble collectins, SP-A
tails, and C1q tails present in excess will inhibit phagocytosis of C1q tail-coated single-ligand particles and
apoptotic cells coated with intact C1q. Inhibition with soluble collectin tails and, in the case of the C1q tailcoated particles, with antibodies directed against the C1q tail will suggest existence of a specific amebic
receptor. Given the complexity of the surface of apoptotic lymphocytes and the high likelihood that multiple
ligand-receptor pairs participate in phagocytosis, the experiments using apoptotic cells will yield important
information on the relative contribution of an amebic collectin receptor.
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An obvious potential pitfall is that E. histolytica may not have a receptor for the collectin tail. This potential
pitfall is common to all of the experiments in aim 1, and the alternative approaches for identification of ligands
that trigger amebic phagocytosis that we will use in this case are discussed in detail after section 1.4. An
additional potential pitfall of our approach is that due to redundant mechanisms for target recognition it may be
difficult to detect a significant role for recognition of the collagenous collectin tail in amebic uptake of cells.
Inhibition of known amebic receptors (e.g., with D-galactose, D-mannose, and Annexin V) would be helpful in
this case, by isolating the ligand of interest. Also, our system for measuring ingestion of single-ligand beads
would be extremely helpful in determining if a negative result is truly negative. Inactivation of the C1q tails by
purification or linkage to streptavidin beads is a third potential pitfall. This is unlikely, since the C1q tails we
purified stimulated both phagocytosis and chemotaxis. The C1q tail-coated beads, however, were not
phagocytosed as well as beads coated with intact C1q. While the most likely explanation for this is that the
ligand was present at lower density, it is possible that the tails lost much of their specific activity during
purification. Therefore, if we find that the soluble C1q tails have no effect on phagocytosis of C1q-coated
apoptotic cells and particles, we will prepare C1q tail-coated particles using saturating concentrations of ligand
(see Figure 6B) to enable accurate comparison of the tails to intact C1q. If the tails appear inactive, the
proposed experiments using MBL and the binding experiments outlined in section 1.2 should still enable us to
determine if the collagenous tail is the basis by which E. histolytica recognizes C1q-coated particles.
1.2: Examine the binding specificity of purified C1q, and determine if E. histolytica binds to the
conserved collectin tail and C1q via a common receptor: We will conduct binding experiments using
biotin-labeled C1q and flow cytometry to examine the interaction of C1q with the amebic surface and determine
if E. histolytica possesses a specific receptor for the collectin tail. Biotinylation of C1q enables detection of
binding without the use of secondary antibodies, eliminating the confounding effect of C1q’s ability to bind to
IgG. This reagent was prepared using commercially available human C1q (Quidel Corp.) and the amine
reactive reagent Sulfo-NHS-LC-biotin (Pierce) according to the manufacturer’s protocol. Importantly,
biotinylation does not appear to interfere with the ability of C1q to bind the amebic surface and partial inhibition
by unlabeled MBL suggests the binding is specific for the conserved collectin tail (see Figure 7); however,
additional work is necessary to confirm the specificity of the observed C1q-biotin binding. This will include
comparison of the relative staining of the amebic population in the presence of increasing concentrations of
biotin-C1q to determine if binding is saturable, and measurement of BSA-biotin binding as a control for
specificity. We will then examine the ability of increasing concentrations of unlabeled purified C1q, MBL, SP-A,
and purified C1q tails and SP-A tails to inhibit C1q-biotin binding. C1q and MBL are commercially available
(C1q (Quidel), MBL (US Biologicals)), we have purified the C1q tails, and Dr. Henson has provided us with the
SP-A and purified SP-A tails. The method to detect binding in all of these experiments will be similar to that
used previously (i.e., E. histolytica trophozoites will be incubated at 4ºC with biotin-C1q, washed, fixed, blocked
with a streptavidin-biotin blocking reagent (Vector Laboratories), and bound biotin-C1q will be detected using a
streptavidin-Alexa 488 conjugate and flow cytometry). MBL and SP-A (both of which specifically inhibited the
calreticulin-C1q interaction in preliminary experiments (see Figure 14)) will also be biotinylated, and binding to
the surface of E. histolytica trophozoites will again be measured with flow cytometry. We will determine if
binding is saturable, and examine the specificity of binding by measuring the effect of increasing doses of
unlabeled C1q, MBL, SP-A, SP-A tails, C1q tails, and BSA (negative control). Finally, we will examine if
binding of biotin-labeled C1q tails is saturable and inhibited by free collectins, C1q, and SP-A and C1q tails.
Interpretation, potential pitfalls, and alternative approaches: Saturable binding of C1q to the amebic
surface that is specifically inhibited by unlabeled C1q, collectins, C1q tails, and SP-A tails will provide strong
evidence for an E. histolytica receptor specific for the collagenous collectin tail. Saturable binding of biotinlabeled MBL and SP-A to the amebic surface that is inhibited by unlabeled C1q would provide additional
evidence that the same receptor also recognizes the collectins. Binding studies using the collagenous C1q
tails our important because they may establish the C1q tail as the optimal "bait" protein for purification of a
collectin receptor in Aim 3. A potential pitfall of our approach is that while biotin-C1q appears to be biologically
active, biotinylation could interfere with binding of MBL, SP-A, or the C1q tail. Unlabeled MBL and SP-A
specifically blocked calreticulin-C1q binding and immobilized biotin-C1q tails and MBL stimulated phagocytosis
in our preliminary work (see Figures 14 and 6C), but it will be necessary to validate the biotinylated reagents if
no specific binding to amebae is observed. This would be done using flow cytometry by examining binding via
their globular head domains to apoptotic lymphocytes [52] and via their tail domains to macrophages [50]. Our
alternative approach if biotinylation interferes with binding in these assays will be to measure binding of
unlabeled MBL, SP-A, and C1q tails using commercially available monoclonal antibodies while competing with
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Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
other collectins. This is possible, since, unlike intact C1q, MBL, SP-A, and the C1q tail do not bind IgG.
1.3: Determine if E. histolytica recognizes sugars or proteins on C1q and the collectins: The collectins
are modified by N-linked glycosylation [65-67, 120]. Calreticulin is a chaperone and has lectin activity [81].
Therefore, we will determine if E. histolytica binds C1q and the collectins via sugar residues or their protein
cores by determining the effect N-glycanase (PNGase)(Roche) treatment has on phagocytosis of C1q-, and
C1q tail-coated single-ligand particles. To isolate the effect of PNGase treatment and ensure that the ligands
are presented to E. histolytica at equal density, we will biotinylate and couple each ligand to the beads prior to
N-glycanase treatment. This will enable comparison of treated beads to untreated beads that were prepared
as part of the same batch. PNGase treatment will be done using a standard protocol which has been used for
SP-A [65]. PNGase cleaves the amide bond in the asparagine residue of N-linked glycopeptides, releasing the
oligosaccharide and generating an aspartic acid residue on the protein chain at the site of hydrolysis [121,
122]. Successful PNGase treatment is reflected by an increase in mobility on SDS-PAGE. To confirm success
of the PNGase treatment, therefore, proteins bound to the beads will be removed by boiling, and separated by
SDS-PAGE. Amebic phagocytosis of PNGase treated beads will be compared to phagocytosis of untreated
beads incubated under the same conditions, but without PNGase. Beads coated with the following proteins
and commercially available neoglycoproteins will be used: C1q, C1q tails, BSA (not glycosylated),
Glucose(Glc)-BSA (does not bind to E. histolytica, but is modified by N-linked glycosylation), and GalNAc-BSA
(binds E. histolytica) [123]. Streptavidin is not glycosylated [124]; however, PNGase treatment of unmodified
streptavidin-coated particles will be an additional control.
Interpretation, potential pitfalls, and alternative approaches: Reduced phagocytosis of C1q- and C1q tailcoated beads following PNGase treatment would indicate that these beads are recognized via a lectin activity,
whereas no difference in phagocytosis would indicate they are recognized based on the protein
sequence/conformation. A potential pitfall of this approach is that the negative charge associated with the
aspartate residues generated by PNGase treatment may alter phagocytosis. A charge effect would be evident
if PNGase-treatment increases phagocytosis of Glc-BSA-coated beads. Our alternative approach in this case
would be to express non-glycosylated recombinant proteins in E. coli and make beads using these proteins. A
second potential pitfall of this approach is that PNGase treatment of already immobilized proteins may be
ineffective, due to an inability of the enzyme to access tightly packed proteins on the surface of beads. Failure
of PNGase treatment would be evident if it does not reduce phagocytosis of GalNAc-BSA-coated beads and
from the SDS-PAGE analysis of proteins removed from the treated beads. In this case, we would treat
unbound biotinylated C1q, C1q tails, BSA, Glc-BSA, and GalNAc-BSA with PNGase, and use these proteins at
saturating concentrations to prepare single-ligand particles (see Figure 6B). This would require preparation of
beads using increasing concentrations of each ligand; comparison of beads prepared from a given ligand used
at different concentrations, would then enable selection of those for which the biotin-binding sites of the beads
have been saturated. Ignoring effects on ligand packing due to removal of N-linked sugars, beads prepared
with saturating concentrations of a given ligand should have that ligand present at nearly equal densities.
1.4: Determine if E. histolytica uses a collectin-dependent mechanism for engulfment of both
apoptotic cells and colonic bacteria: Unlike C1q, the collectins are present in intestinal secretions and
opsonize bacteria at mucosal surfaces [55-64]. Therefore, we will conduct phagocytosis experiments using
MBL and SP-A as opsonins to determine if collectin-dependent phagocytosis is an important mechanism for E.
histolytica phagocytosis of both apoptotic cells and bacteria. We have already demonstrated phagocytosis of
MBL-coated single-ligand particles (Figure 6C). We will next assay ingestion of apoptotic lymphocytes
opsonized with either MBL or SP-A. This experiment will be conducted using the same methods we have
already used for C1q (see Figure 5). The controls will include cells treated with BSA (negative control), cells
treated with C1q (positive control for phagocytosis), and competition with increasing doses of purified C1q, C1q
tails, and SP-A tails. As noted above, due to the likelihood that multiple receptor-ligand pairs are important, it
may be necessary to isolate the ligand of interest with known phagocytosis inhibitors (i.e., D-galactose, and
Annexin V). Since both MBL and SP-A bind to mannose, we would not include it here.
After determining if the collectins facilitate E. histolytica phagocytosis of apoptotic cells, we will determine if
they facilitate ingestion of bacteria. For this, E. coli expressing green fluorescent protein (GFP)(strain DH5-α, a
gift from U. Singh, Stanford University, Palo Alto, CA) will be pre-incubated with MBL or SP-A to allow binding,
and E. histolytica’s ability to ingest the opsonized bacteria will be measured using flow cytometry and confocal
microscopy. Several controls will assist with interpretation. First, phagocytosis of collectin-coated E. coli will
be compared to phagocytosis of E. coli treated with BSA and C1q (negative controls). Second, the ability of
purified C1q tails, SP-A tails, or intact C1q to inhibit ingestion of collectin opsonized E. coli will be measured.
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These should block phagocytosis if E. histolytica recognizes collectins on the surface of bacteria by interaction
with the collectin tail. Finally, if the collectins have no effect on amebic phagocytosis of bacteria, collectin
binding to the GFP-expressing bacteria will be verified using two-color flow cytometry. To do this, collectin
opsonized GFP-expressing E. coli will be immunostained with commercially available primary antibodies (an
anti-human MBL mouse monoclonal antibody (clone 131-10, Staten Serum Institute), or an anti-human SP-A
mouse monoclonal antibody (clone HYB 238, Santa Cruz Biotechnology) and an anti-mouse IgGphycoerythrin-Cy5 (PE-Cy5) conjugate (BD Biosciences) for secondary detection. Samples stained with
isotype control antibodies will be included. These antibodies are known to work for this application [52, 125].
Interpretation, potential pitfalls, and alternative approaches: We anticipate that MBL and SP-A will
facilitate amebic phagocytosis of apoptotic cells, and that C1q tails and SP-A tails will specifically inhibit
phagocytosis of collectin-opsonized targets. It is currently unknown if E. histolytica uses distinct mechanisms
for phagocytosis of host cells and bacteria, and, if we find that the collectins also facilitate engulfment of E. coli
that can be inhibited with collectin tails, it will indicate that E. histolytica can ingest both apoptotic cells and
colonic bacteria via the same mechanism.
Two potential pitfalls are common to all of the experiments in aim 1. First, it is possible that E. histolytica
has no receptor for the collectin tail. For example, an unidentified amebic lectin may mediate uptake of C1qcoated particles. A novel lectin would be evident from the experiments in section 1.3, and would be a
phagocytosis receptor that could be identified in aim 3 using intact C1q as the "bait" protein even if it is not
specific for C1q's tail domain. Another alternate approach would be to seek additional ligands that trigger
amebic phagocytosis by evaluating the role of other features unique to the surface of apoptotic cells. Important
molecules to consider would include intracellular adhesion molecules (e.g., ICAM3, thrombospondin, oxidized
low-density lipoproteins, carbohydrates, and calreticulin (reviewed in [40])). For these studies, our flow
cytometry-based phagocytosis assay and the method we have developed to construct single-ligand particles
would be invaluable tools by enabling screening of a large number of candidate ligands. A second potential
pitfall is that E. histolytica may produce its own “collectin-like” molecules. This is a very interesting possibility
that our experimental design is not capable of excluding. However, opsonization of targets with amebic
“collectin-like” molecules should not prevent us from accurately determining if host collectins participate, since
we easily detected an effect using purified C1q in our preliminary experiments (see Figures 5 and 6).
Aim 2: Test the hypothesis that cell surface calreticulin is an E. histolytica phagocytosis receptor that
stimulates phagocytosis by interaction with a transmembrane signaling partner.
Rationale: Studies in evolutionarily diverse species indicate that calreticulin plays an important and conserved
role in phagocytosis [50, 51, 84, 88]. Furthermore, though calreticulin is a chaperone in the ER, it is known to
be on the surface of many cell types where it is the receptor for the collagenous tail of C1q and the collectins
(cC1qR) [85-87]. Preliminary experiments demonstrated calreticulin on the E. histolytica surface and within the
phagocytic cup, interaction of E. histolytica calreticulin with human C1q, collectin-independent binding of
amebic calreticulin to apoptotic but not healthy cells, and a positive correlation between calreticulin expression
and E. histolytica's phagocytic ability. Therefore, we hypothesize that amebic cell surface calreticulin is an E.
histolytica phagocytosis receptor. Obvious potential mechanisms for participation of cell surface calreticulin in
phagocytosis include binding to C1q and collectins deposited on apoptotic cells and microbes, and/or binding
to target cells and microbes independently of the collectins. In either case, we hypothesize that a calreticulin
receptor analogous to the macrophage receptor CD91 transmits the signal that initiates particle uptake (see
Figure 1). We will determine if calreticulin participates in E. histolytica phagocytosis by using RNA-mediated
interference and/or a method for transcriptional gene silencing. We will determine if E. histolytica has a
calreticulin receptor by assaying phagocytosis of cells or particles coated with recombinant calreticulin, and by
determining if binding of recombinant calreticulin to the amebic surface is saturable and specific.
Method:
2.1: Silencing calreticulin expression: Despite the important role of calreticulin as an ER chaperone, the D.
discoideum calreticulin/calnexin double mutant had a specific defect in phagocytosis [88], suggesting a similar
approach may be useful to evaluate calreticulin's function in E. histolytica phagocytosis. Molecular methods
for disruption of protein expression or function that have been used successfully in E. histolytica include
expression of dominant negative mutants [13], anti-sense strategies using anti-sense RNA expression [12, 30,
36, 126, 127] or peptide nucleic acid (PNA) oligomers [128], RNA-mediated interference (RNAi) [97, 129, 130],
and a recently developed method for silencing gene transcription [131, 132]. To ensure success in silencing
calreticulin expression, we have two approaches: RNAi and the method for transcriptional gene silencing.
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Because of its relative simplicity and speed, we will attempt RNAi first. For this, we will use a system
recently developed by our collaborator, Dr. William Petri (University of Virginia, Charlottesville, VA)(see
attached letter and biosketch), to express short hairpin RNAs under control of the E. histolytica U6 promoter
(Genbank accession number U43841). This system has been used successfully to silence expression of one
of the Gal/GalNAc lectin subunits and, in a paper on which I am second author, to silence expression of a
transmembrane kinase (PMID: 18208324) [97]. To express short hairpin RNAs constitutively in E. histolytica,
four sequences (plus a scrambled negative control) based on the gene of interest (i.e., calreticulin) are
designed corresponding to a 29 base sense strand, a 9 base loop, a 29 base antisense (complementary)
strand, and the RNA pol III terminator sequence (6 thymidines). Note that 29 base dsRNAs are used rather
than the usual 21-23 dsRNA length because of data indicating that longer hairpins are more effective in
reducing gene expression [133]. Using a published method, chimeric DNA products containing the U6
promoter and the short hairpins are generated by two rounds of PCR using E. histolytica genomic DNA as
template and primers to amplify the U6 promoter that incorporate the sense, anti-sense, and loop sequences
[134]. These PCR products are then cloned into an amebic vector which has a hygromycin selection cassette.
Following stable transfection of amebae, efficacy of the RNAi constructs will be assessed using quantitative
real time PCR and Western blots (with our scFv antibodies), and simultaneous measurement of actin mRNA
and protein levels will control for mRNA and protein quantity/gel loading.
Should RNAi fail to silence calreticulin expression, we will use an alternative method for transcriptional
silencing developed by Dr. David Mirelman [131, 132]. Dr. Mirelman has already sent us the necessary
plasmids. Though this method appears to be useful for silencing genes in E. histolytica, it remains poorly
understood and has the disadvantage that it does not work in HM-1:IMSS strain trophozoites; rather, G3
trophozoites, a clone derived from the HM-1:IMSS strain, must be used. It has the advantage that it has been
used to achieve complete silencing of multiple E. histolytica genes. To silence calreticulin gene expression,
the open reading frame of calreticulin would be cloned under control of the 5' upstream region (473 bp) of the
E. histolytica amoebapore-a (Ehap-a) gene into a plasmid that has a neomycin selection cassette. This
upstream Ehap-a region contains a short interspersed nuclear element (SINE) sequence, which mediates
epigenetic silencing via an unclear mechanism [132]. Entamoeba histolytica G3 trophozoites would then be
transfected with this silencing vector and an empty control vector, and, after selection with neomycin,
calreticulin mRNA and protein levels would be assessed as described above. Again, simultaneous
measurement of actin mRNA and protein levels would control for mRNA and protein quantity/gel loading.
Once calreticulin silencing is achieved by either of these methods, the effect of inhibiting expression of
calreticulin on phagocytosis of apoptotic cells, C1q-coated beads, and GFP-expressing E. coli will be
measured. To control for effects on phenotype related to disruption of calreticulin's chaperone function rather
than absence of calreticulin from the cell surface, we will conduct these assays with and without addition of
recombinant calreticulin, which we reason should reverse any defect due to absence of cell surface calreticulin.
We already have recombinant calreticulin and have confirmed its biological activity (see Figures 14 and 15).
Specificity will also be determined by assessing the effect of scrambled short hairpin RNAs (RNAi control) or
the empty vector control (transcriptional silencing method) on gene expression and phenotype. Finally,
standard assays for growth, cytotoxic ability (Chromium51 release assay), and motility (chemotaxis) will be
conducted to examine the specificity of an observed defect in phagocytosis [39, 102, 135].
Interpretation, potential pitfalls, and alternative approaches: Given our substantial preliminary data
suggesting that amebic calreticulin functions in phagocytosis, we anticipate that inhibition of calreticulin
expression will reduce amebic phagocytosis. If addition of recombinant calreticulin reverses the phagocytosis
defect, furthermore, it will strongly implicate calreticulin on the amebic surface. On the other hand, a
phagocytosis defect that is not reversed by addition of recombinant calreticulin may actually represent a defect
in secretion. Observation of a specific phagocytosis defect would provide additional evidence supporting our
model, and pursuit of a calreticulin receptor in aim 3. Though calreticulin is a strong collectin receptor
candidate, it is possible that calreticulin does not participate in E. histolytica phagocytosis. This would be the
case if no phagocytosis defect is observed despite successful silencing of calreticulin expression. In this case,
we would pursue a collectin receptor in aim 3.
It should be possible to interfere with calreticulin expression using these complementary silencing
methods. A potential pitfall is that calreticulin may be essential, thereby limiting the ability to silence its
expression. If this is the case, RNAi may still work, since dsRNAs targeting different regions of the gene would
be expected to silence gene expression with variable efficiency. An alternative approach should RNAi fail
would be to express calreticulin domains to obtain a dominant negative phenotype. Expression of the N, P,
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and C domains independently is the obvious starting point, and this method would be further informed by the
mapping experiments outlined in section 2.4.
2.2: Experiments using recombinant calreticulin: Another method we will use to determine if cell surface
calreticulin plays a direct role in amebic phagocytosis will use soluble, recombinant calreticulin. We have
already expressed and purified this reagent, and have demonstrated that it is biologically active (see Figures
14 and 15). Three experiments are planned. First, we will examine the effect of free calreticulin present in
excess on phagocytosis by assaying phagocytosis of unopsonized apoptotic cells, unopsonized bacteria, C1qor collectin-coated apoptotic cells, C1q- or collectin-coated single-ligand beads, and collectin-coated bacteria in
the presence of increasing concentrations of soluble calreticulin. In all assays an irrelevant His-tagged protein
(E3 kinase, negative control)(this vector is available from Invitrogen) produced and purified in the same way as
the recombinant calreticulin will be included as a control. Second, to determine if calreticulin participates in
adherence, we will assay adherence of E. histolytica to collectin-coated and uncoated viable and apoptotic
cells, and GFP-expressing E. coli in the presence or absence of excess free calreticulin. The assay for
adherence to host cells is standard [100], and an epifluorescent microscope will be used to assay adherence to
GFP-E. coli. Finally, we will determine if E. histolytica phagocytoses calreticulin-coated apoptotic cells and
single-ligand particles. We have already shown that recombinant calreticulin binds to apoptotic lymphocytes,
but not to healthy lymphocytes. To determine if calreticulin can function as ligand for phagocytosis, we will
assay phagocytosis of calreticulin-opsonized apoptotic cells (grown in serum-free media; see Figure 15) and
calreticulin-coated particles prepared as in Figure 6. In each case, cells or particles treated with an irrelevant
His-tagged protein (E3-kinase) will again serve as a negative control, and C1q-coated apoptotic cells or
particles will serve as a positive control for phagocytosis. An additional control for specificity will be to assay
uptake of calreticulin-coated cells and particles in the presence of excess free calreticulin.
Interpretation, potential pitfalls, and alternative approaches: These experiments will help to determine if
E. histolytica has a receptor for calreticulin that stimulates phagocytosis, and, if it does, will also help to
determine the mechanism by which calreticulin participates in phagocytosis (i.e., if it functions as a collectin
receptor, stimulates phagocytosis by binding directly to apoptotic cells and/or bacteria, or both). Interpretation
of them, however, will be more complicated than interpretation of the calreticulin silencing studies, because the
results would depend on the degree of saturation of a calreticulin receptor when it reaches the cell surface. If
the calreticulin receptor is completely saturated with endogenously produced calreticulin, then pre-treatment of
targets with exogenous calreticulin would inhibit phagocytosis, and calreticulin-coated cells and particles would
not be phagocytosed. If the receptor is not saturated with endogenous calreticulin, then free calreticulin would
likely increase phagocytosis at low concentrations but would inhibit phagocytosis if left in at high
concentrations, and E. histolytica would phagocytose calreticulin-coated beads. Our alternative approach if the
results of these experiments are difficult to interpret will be to conduct the same experiments using the
calreticulin-deficient trophozoites produced in section 2.1 (whose calreticulin receptor, if one exists, will
obviously not be saturated with endogenous calreticulin).
2.3: Experiments to determine if amebic surface calreticulin is a collectin/C1q receptor, and if amebic
calreticulin binds directly to target cells and bacteria: These experiments will examine binding of biotinlabeled reagents (either C1q and collectins, or calreticulin) to the amebic surface, and to apoptotic cells and
bacteria. The method will be similar to that described above in section 1.2, and use the biotinylated C1q,
collectins, and calreticulin used previously (see Figures 7 and 15). The following experiments will be
performed. First, the ability of increasing concentrations of our anti-calreticulin scFv antibodies to block binding
of biotin-C1q to amebic trophozoites will be measured using flow cytometry in order to determine if E.
histolytica interacts with C1q and the collectins via cell surface calreticulin. Note that though FLAG-calreticulin
was only detected on the amebic surface after priming trophozoites with Jurkat lymphocytes (Figure 9), biotinC1q bound to the surface of unstimulated trophozoites at 4ºC and these experiments will help to determine if
calreticulin is the receptor. The effects of a scFv antibody we have made against an unrelated membrane
antigen (a GP63 homologue) and of a non-binding scFv antibody will also be assayed (negative controls). Of
course, these experiments are contingent upon evidence from aim 1 of a specific amebic collectin receptor.
Second, we will use flow cytometry to measure binding of increasing concentrations of biotin-labeled
recombinant calreticulin to E. coli in order to determine if E. histolytica calreticulin interacts directly with
bacteria in addition to apoptotic cells. The unrelated His-tagged protein E3 kinase expressed and purified in
the same way will again serve as a control for specificity. It should be possible to determine the relative
importance of direct target-binding vs. collectin-binding by calreticulin by addition of varying concentrations of
purified C1q and collectins. Finally, we will determine if biotin-calreticulin binds to the amebic surface, and, if
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so, if binding to amebae is saturable and specific. For this, binding of increasing concentrations of biotincalreticulin will be assayed by flow cytometry. To control for specificity, inhibition of calreticulin-to-ameba
binding by pre-incubation of calreticulin-biotin with anti-calreticulin scFv antibodies (and the anti-GP63 and
non-binding control scFv reagents) will be assessed, as will the ability to inhibit binding with excess nonbiotinylated calreticulin or His-E3-kinase (negative control).
Interpretation, potential pitfalls, and alternative approaches: If the anti-calreticulin scFv antibodies inhibit
binding of biotin-labeled C1q and collectins to the surface of the amebic trophozoites, it will directly implicate
calreticulin as an amebic collectin receptor. The relevant collectin/C1q domains will be evident from the
collectin/C1q studies in aim 1, and the relevant calreticulin domain(s) will become evident by correlating the
effect of different monoclonal scFv antibodies on collectin/C1q binding with the in vitro mapping studies
planned in section 2.4. We have already shown that biotinylated recombinant calreticulin binds directly to
apoptotic cells (Figure 15), and we think it is likely that amebic calreticulin also binds directly to bacteria.
Finally, if biotin-calreticulin binds to the amebic surface in a saturable manner, it would provide additional
evidence of a specific calreticulin receptor. In the context of the mapping studies proposed in section 2.4, the
scFv inhibition studies will determine the domain(s) required for interaction of calreticulin with this protein, and
the calreticulin receptor would be identified in Aim 3. A potential pitfall of our approach is that calreticulin-biotin
may not bind to the amebic surface despite existence of a calreticulin receptor if the receptor is delivered to the
cell surface already saturated with endogenously produced calreticulin. Such a false negative result would be
suggested if we observe no specific binding despite evidence from the calreticulin silencing experiments that
calreticulin participates in phagocytosis. Our alternative approach in this case would be to conduct binding
studies using the calreticulin-deficient trophozoites produced in section 2.1.
2.4: In vitro mapping of the basis for collectin/C1q-calreticulin and calreticulin-ameba interactions:
The goal of this sub-aim is to map the calreticulin domains recognized by the scFv monoclonal antibodies used
in Figures 12-14. This will enable correlation of the results of our assays of collectin-calreticulin binding and
calreticulin-ameba binding described above (section 2.3) with the calreticulin domains involved, providing new
information on the structure of calreticulin, and possibly suggesting methods beyond those already included in
aim 3 to identify interacting proteins. These studies may also suggest how best to interfere with calreticulin
function by expressing dominant negative transgenes in E. histolytica (an alternate approach for section 2.1).
The approach for mapping will be to clone fragments of the E. histolytica calreticulin gene using PCR into the
same His-tagged expression vector we used to express the whole protein (pCR T7/NT-TOPO, Invitrogen),
express the proteins in E. coli, and purify them using nickel columns as before. Protein fragments
corresponding to the N-, P-, and C-domains will be made (these are easily identified by sequence alignment
with human calreticulin (data not shown)). After expressing the desired calreticulin fragments, the monoclonal
anti-calreticulin scFv antibodies will be screened for binding to the calreticulin fragments using Western blots.
In the context of the binding assays described above that use these scFv antibodies, this will provide a basic
map of the functional domains of E. histolytica calreticulin. Experiments to refine this map will depend on the
initial results. For example, if we find that the P- and C-domains are critical for retention of calreticulin on the
amebic surface (i.e., for interaction with the proposed transmembrane signaling partner), then inverse PCRbased mutagenesis will be used to delete or mutate one or both of calreticulin's Ca2+ binding domains.
Interpretation, potential pitfalls, and alternative approaches: Specific recognition of a given recombinant
calreticulin fragment would localize the binding epitope within approximately 150 amino acids, and enable
correlation of the phenotype obtained with the antibody and the calreticulin domain involved. The chosen initial
approach has the advantage of simplicity, and the ability to make numerous monoclonal reagents using phage
display should enable a logical approach to more sophisticated mapping studies.
A potential pitfall is that many of the scFv reagents may bind to the same epitope. This could happen if
one clone overgrew our cultures early during panning for anti-calreticulin antibody production. Two alternative
approaches can be used if necessary. First, phenotypic experiments can be conducted using the recombinant
calreticulin fragments. Second, it would be possible to generate scFv monoclonals specific for the different
domains by re-panning, and then to screen these reagents in phenotypic assays.
Aim 3: Identify the E. histolytica collectin or calreticulin receptor.
Rationale and prioritization scheme: C1q and the collectins bind to apoptotic cells and stimulate
macrophage phagocytosis via a conserved collagenous tail domain [48-54]. Calreticulin is the macrophage
receptor for the collagenous collectin tail, and serves as a bridge between collectins on apoptotic cells/bacteria
and CD91 on the macrophage surface [50, 51, 85-87]. In our preliminary studies, we have demonstrated that
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C1q stimulates E. histolytica uptake of apoptotic cells, as do C1q-, MBL, and C1q tail-coated particles.
Furthermore, E. histolytica calreticulin interacts with human C1q and is present in purified E. histolytica
phagosomes, on the amebic surface, and in the phagocytic cup. Based on these data and analogy with the
mechanism of macrophage phagocytosis, we hypothesize that host collectins bound to apoptotic cells or
bacteria trigger phagocytosis via an E. histolytica collectin receptor. Calreticulin is a candidate
collectin receptor that may also bind apoptotic cells and bacteria directly, and may trigger
phagocytosis by interaction with an amebic calreticulin receptor (see Figure 1). In this aim, we will
identify and partially characterize either a collectin or a calreticulin receptor. The decision of which to pursue
will be based on the predictions of our model and the results from aims 1 and 2. Specifically, if we find that
silencing calreticulin has no effect on phagocytosis and the collectin binding/inhibition studies in sections 1.1
and 1.2 demonstrate specific collectin binding and phagocytosis due to recognition of the collagenous tail, then
we will pursue a collectin receptor using purified C1q tails as the bait protein and the methods outlined below.
On the other hand, if calreticulin silencing by RNAi reduces E. histolytica phagocytosis and the binding and
phagocytosis studies using recombinant calreticulin that are proposed in sections 2.2 and 2.3 indicate the
presence of an E. histolytica calreticulin receptor, then this would provide strong evidence that calreticulin is
the collectin receptor and its downstream signaling partner (i.e., the calreticulin receptor) would be identified
using recombinant calreticulin as the bait protein. A potential pitfall is that E. histolytica has no receptor
specific for the collagenous collectin tail and that calreticulin also does not participate in E. histolytica
phagocytosis. For example, it is possible that the phagocytosis of C1q-, C1q tail-, and MBL-coated particles
we observed is not specific to the collagenous tail domain, but is related to sugar modifications and mediated
by a novel amebic lectin (the Gal/GalNAc specific lectin is excluded, since our preliminary experiments have all
been performed in the presence of D-galactose). This would be evident by interpreting the deglycosylation
experiments in section 1.3 in the context of the C1q tail-binding studies. Identification of C1q interacting
membrane proteins would still be important and would be possible using the methods described below.
Method:
3.1: Identification of candidate cell surface signaling partners: After deciding whether to pursue a
collectin receptor or a calreticulin receptor using the prioritization scheme outlined above, we will use
complementary cross-linking and affinity-based methods to identify candidate E. histolytica cell membrane
receptors. These experiments will be done in collaboration with Dr. Gary Ward (University of Vermont,
Burlington, VT)(see attached biosketch and letter of support), who has extensive experience using biochemical
cross-linkers and 2-dimensional electrophoresis (2DE) for identification of cell membrane proteins in
Toxoplasma gondii [136].
The first approach, which is summarized in Figure 17A, will be to use the multi-functional cross-linker
Sulfo-SBED (Pierce) to covalently label either a collectin or a calreticulin receptor on the amebic surface. In
either case, the method will be similar, with differences limited to the "bait" protein used and the controls for
specificity. This “re-tagging” method was used successfully to identify an H. pylori adhesin ([137], reviewed in
[138]). Sulfo-SBED has an amine-reactive group linked via a disulfide bond to a photoreactive aryl azide group
and a biotin label (Figure 17A, 1). The “bait” protein (i.e., purified C1q tails or recombinant calreticulin) is
conjugated to the cross-linker by reaction with the amine-reactive group (Figure 17A, 2), and then allowed to
bind to the “prey” (i.e., E. histolytica membrane proteins that interact with the bait). Upon UV irradiation, the
aryl azide group forms a covalent bond with any protein in its immediate vicinity (i.e., the prey). Exposure to
reducing conditions then cleaves the disulfide bond, resulting in transfer of the biotin label to the prey, and the
biotin label is used to purify the prey using streptavidin conjugated agarose beads (Figure 17A, 3). The
purified biotinylated protein(s) will be separated by 1-dimensional electrophoresis (1DE) and proteins of
interest will be identified using in-gel trypsin digestion and mass spectrometry. This will be conducted at the
W.M. Keck Biomedical Mass Spectrometry Laboratory at the University of Virginia, a fee-for-service facility that
has provided us with excellent results on other projects (PMIDs: 15821141, 16290089, 18086807, and
18208324)[29, 38, 97, 139]. For identification of a C1q/collectin receptor, a Sulfo-SBED-C1q tail conjugate will
be used as bait, and binding and UV cross-linking will be performed in parallel in the presence or absence of
excess free MBL, C1q, and C1q tails to enable identification of proteins that interact specifically. Similarly, for
identification of calreticulin interacting surface proteins, Sulfo-SBED-calreticulin will be used as bait, and crosslinking will be performed with and without competition by excess free calreticulin. Samples prepared using a
Sulfo-SBED-BSA conjugate will serve as an additional control for specificity in all experiments.
The second approach is summarized in Figure 17B, and is specific to identification of amebic surface
proteins that interact with calreticulin. This approach is very similar to the method we developed to
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Figure 17: Schemes for identification of an E. histolytica collectin or calreticulin receptor. In approach (A), the
multifunctional cross-linker Sulfo-SBED (1) is conjugated to either purified C1q tails or recombinant calreticulin, which will
serve as the "bait" protein (2). After allowing binding to E. histolytica surface proteins, the cross-linker’s photoreactive
aryl azide group binds covalently to proteins in its immediate vicinity following UV irradiation. Exposure to reducing
conditions then cleaves the disulfide bond resulting in transfer of the biotin label to the interacting protein, enabling
purification of the biotinylated protein using streptavidin conjugated beads. Cross-linking in the presence of either C1q,
MBL, and C1q tails in excess (collectin receptor) or calreticulin in excess (calreticulin receptor) will control for specificity.
This approach is well suited for identification of transient or weakly interacting proteins, and can be used to identify
receptors for any protein ligand. Approach (B) is an affinity-based method for identification of amebic membrane proteins
that interact with FLAG-calreticulin. Here, surface proteins are biotinylated using Sulfo-NHS-LC-Biotin, and FLAGcalreticulin interacting proteins are isolated by immunoaffinity purification with anti-FLAG antibodies. Experiments
conducted in tandem with FLAG-PDI and FLAG-Lgl expressing amebae control for specificity. Approach (C) is an
affinity-based method for identification of amebic membrane proteins that interact with the collagenous tail of C1q. Here,
surface proteins are biotinylated, and purified C1q tails are allowed to bind. Amebic membrane proteins that interact with
the C1q tail are isolated by immunoaffinity purification using anti-C1q antibodies and detected using streptavidin-HRP.
Experiments conducted in tandem in the presence of excess MBL and/or SP-A tails control for specificity. If lysis
conditions disrupt the C1q-tail/receptor interaction, amebae can be lysed with a dialyzable detergent such as noctylglucoside, dialyzed, and the membrane fraction can be probed with C1q tails. (part A adapted from ref. 137)
demonstrate co-precipitation of specific surface proteins with FLAG-calreticulin (also see Figure 16). Briefly,
the strategy is to 1) biotinylate the E. histolytica surface proteins in trophozoites expressing FLAG-calreticulin,
2) immunoaffinity purify FLAG-calreticulin and calreticulin interacting proteins using anti-FLAG antibodies, 3)
separate the purified proteins using 1DE, or, if necessary, 2DE, and 4) identify the biotinylated proteins that copurify with FLAG-calreticulin using streptavidin-HRP blots. Two controls will be used to assess the specificity
of co-purifying proteins. First, we will use samples prepared using our FLAG-PDI expressing trophozoites.
Second, we will use trophozoites expressing a FLAG-Gal/GalNAc lectin light subunit (FLAG-Lgl) fusion cloned
into the same plasmid we used to express FLAG-calreticulin. This plasmid has already been provided by Dr.
Petri, and the FLAG-Lgl fusion localizes to the amebic surface [140]. Comparison of blots from samples
prepared and run in tandem using FLAG-PDI and FLAG-Lgl expressing trophozoites (negative controls) will
enable identification of biotinylated surface proteins that specifically co-purify with calreticulin. Proteins of
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interest will be identified using mass spectrometry as described above.
The third approach, which is an affinity method for identification of membrane proteins that interact with
the collagenous C1q tail, is similar in many respects to the affinity method described above for identification of
amebic surface proteins that interact with calreticulin (see Figure 17C). Use of the purified collagenous C1q
tail as the bait protein instead of intact C1q will reduce non-specific binding, by eliminating the ability of C1q to
bind to many proteins through its C-terminal globular head; therefore, our first choice here and for the crosslinking method described above will be to use purified C1q tails as the bait. We already have this reagent (see
Figure 6C). To identify amebic surface proteins that interact with the collagenous C1q tail, we will biotinylate
the amebic surface proteins using Sulfo-NHS-LC-biotin, incubate the cells or cell lysates with purified C1q tails,
and then use a commercially available anti-C1q antisera (Quidel) for immunopurification of the C1q tail.
Samples will be prepared in tandem in the presence of excess MBL and SP-A tails to control for specificity,
and biotinylated (i.e., cell surface) amebic proteins that specifically co-purify with the C1q tails will then be
identified by mass spectrometry. It should be noted that despite amebic phagocytosis of C1q tail-coated
particles (Figure 6C), we have not yet proven that C1q tails bind specifically to the amebic surface. If the tail
purification process disrupts the its specific binding activity, it would be evident from the experiments using
biotinylated C1q tails that are outlined in section 1.2, and our alternative approach in this case would be to use
intact C1q as the bait protein (which we have already shown binds specifically (Figure 7)).
Each of these approaches has advantages and disadvantages. The cross-linking method can be
adapted to identification of receptors for any protein ligand. In addition, it is more suitable for identification of
transient or weak protein-protein interactions, because it results in covalent-labeling of the protein(s) of
interest. The re-tagging method may also be more selective, since C1q tail- or calreticulin-protein interactions
that occur after amebic lysis will not affect the results. The second method, which applies to identification of a
calreticulin receptor, has the advantages that we have already used it with some success, and that it will work
even if the calreticulin interacting proteins on the cell surface are already saturated with endogenous
calreticulin. This method and the affinity-based method for collectin receptor identification also do not require
direct interaction of all proteins of interest with the collectin or calreticulin bait, which may be beneficial if the
receptor is a member of a larger signaling complex, such as a lipid anchored protein present in a lipid raft. A
recent report demonstrating involvement of raft-like membrane domains in E. histolytica pinocytosis and
adhesion supports this possibility [116]. Both the second and third methods are most well suited for
identification of strong protein-protein interactions, but have the advantage that they should be relatively easy
to scale-up, simplifying acquisition of protein quantities suitable for identification by mass spectrometry. A
potential pitfall in either case is that cell lysis and membrane protein solubilization may disrupt interaction of the
bait with its receptor. Complementary use of the cross-linking method controls for this difficulty, and alternative
lysis conditions such as use of a dialyzable detergent like N-octylglucoside may be helpful.
Reproducible production of large quantities of the bait interacting proteins will be a difficult problem to
overcome in conducting these studies. Entamoeba histolytica’s large size and the ease of axenic culture are
advantages here, since they enable production of grams of starting material. Variation in the amebic cultures
(e.g., density or time-to-harvest) with resultant differences in protein expression may be a source of prep-toprep variability, so cultures will be carefully standardized and consistently harvested during log-phase growth.
In addition, a cocktail of protease inhibitors (AEBSF, EDTA, bestatin, E-64, leupeptin, and aprotinin (Sigma, St.
Louis, MO.)) will be used and all protein purification will be done on ice to limit the activity of the amebic
proteinases. Prep-to-prep reproducibility will be carefully evaluated by visualizing proteins with silver stain
prior to proceeding with preparative gels and protein identification.
For protein separation, our first approach will be to use simple 1DE SDS-PAGE. The purification steps
should simplify the protein mixtures adequately to make excision of the relevant bands and protein
identification by mass spectrometry possible with this method, and the method has the advantage that it
reliably separates membrane proteins. We recognize, however, that the protein mixtures may be complex,
necessitating 2DE for protein separation. One potential pitfall of 2DE is its limited ability to separate
membrane proteins. However, Dr. Ward's laboratory has recently published a paper describing methods using
the detergents ASB-14 and octylglucoside for 2DE sample preparation [141]. This method dramatically
improved solubility of transmembrane proteins for 2DE, and enabled the 2DE purification of several
transmembrane T. gondii proteins. Therefore, it is unlikely that we will encounter insurmountable difficulties in
resolving the E. histolytica proteins of interest even if 2DE is necessary. A second challenge, whether using
1DE or 2DE, will be to accurately pick protein bands or spots of interest for mass spectrometry from parallel
streptavidin-blots and preparative gels. For this, we will use a previously described method in which proteins
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on a PVDF membrane are stained with colloidal gold prior to blotting with streptavidin-HRP [142]. This method
makes it simple to identify biotinylated proteins of interest within the overall pattern of proteins separated by
1DE or 2DE, since the immunoblot and colloidal gold staining are done on the same membrane. Once the
landmarks are clear, it is relatively simple to identify the same landmarks and proteins of interest on parallel
Coomassie blue-stained preparative gels.
It will be very important to prioritize the candidate proteins we identify, so that we can take a logical
approach to characterizing them (see 3.2 and 3.3 below). If a protein is consistently identified using both
methods, it will obviously be given higher priority. The proteins will be further prioritized based on analysis of
the primary protein sequence with specific attention to the following questions: 1) Is it predicted to be in the
secretory pathway and/or to have a transmembrane domain?, 2) Does it have recognized signaling motifs?, 3)
Are there known homologues with roles in adhesion, endocytosis, and phagocytosis by other organisms?, and
4) Is it a known E. histolytica cell surface protein, either of unknown function (e.g., one of a large family of
recently identified transmembrane kinases [143, 144]) or with a proposed role in adherence and/or
phagocytosis (e.g., the Gal/GalNAc specific lectin [36], or the 112 kDa adhesin [37])?
Interpretation, potential pitfalls, and alternative approaches: We believe that it will be possible to
overcome the technical challenges these experiments pose, and to identify appropriate candidate receptors for
further evaluation. Our ability to visualize biotinylated E. histolytica proteins that specifically coimmunoprecipitate with FLAG-calreticulin on streptavidin-HRP blots supports this. Controls for specificity
included in our experimental design (e.g., competition with free collectins or free calreticulin, and, for the
method in Figure 17B, co-purification of FLAG-PDI and FLAG-Lgl interacting proteins) will limit the possibility
that irrelevant proteins will be isolated, and prioritization of the identified proteins as described above will be
critical. In the case of calreticulin interacting proteins, it is very likely that PDI would co-purify with FLAGcalreticulin, since in other species the two interact in the ER. However, the cross-linking method would only
label proteins on the surface of intact trophozoites, and analysis of proteins isolated using the affinity-based
methods will be restricted to surface proteins that are biotinylated.
The major potential pitfall of this aim is that E. histolytica may not have a specific receptor for the
collagenous C1q tail, and also may not have a receptor for calreticulin that mediates phagocytosis. We believe
this is unlikely given the strength of our preliminary data. However, if we find in aim 1 evidence that E.
histolytica has no receptor for the collagenous collectin tail and in aim 2 evidence that calreticulin does not
participate in E. histolytica phagocytosis, our alternative approach will be to first identify the true ligands that
stimulate E. histolytica phagocytosis. Since C1q-coated beads were rapidly phagocytosed by E. histolytica,
the PNGase studies outlined in section 1.3 might provide a critical lead. For example, if we find that N-linked
sugars on C1q are the basis for recognition of these particles, then the relevant receptor might be a novel
amebic lectin, and we would identify this lectin using intact C1q as the bait protein and PNGase-treated C1q to
control for specificity. Published studies on membrane changes that occur during apoptosis would provide
additional potential ligands. Potential ligands known to be exposed during apoptosis that we would screen
include ICAM3, thrombospondin, and oxidized low-density lipoproteins [40]. The re-tagging method described
above could then be adapted for receptor identification.
3.2: Confirmation of the bait-candidate interaction: To verify that the proteins identified in 3.1 interact with
the bait protein, we will begin by expressing and purifying the candidate’s predicted extracellular domain in E.
coli using the vector pET101/D-TOPO (Invitrogen), which incorporates a carboxy-terminal V5 epitope in
addition to a 6×His tag for protein detection and purification. Whether the recombinant candidate protein
interacts with the bait in vitro will then be assessed using co-immunoprecipitation experiments, either by
immunoprecipitating with an anti-V5 antibody or with an anti-bait antibody (i.e., anti-calreticulin scFv or antiC1q), and then probing immunoblots with both anti-V5 antibody and the anti-bait antibody. In addition, the
recombinant protein and phage display will be used to generate monoclonal scFv antibodies directed against
the candidate protein. These monoclonal scFv antibodies will be used to examine localization of the candidate
protein in E. histolytica trophozoites at rest, and during phagocytosis of apoptotic cells and bacteria. We will
then conduct co-localization studies. In the case of a calreticulin receptor, we will use our FLAG-calreticulin
expressing E. histolytica trophozoites, using anti-FLAG antibodies to stain calreticulin and scFv antibodies
against the novel protein. In the case of a collectin receptor, we will use biotin-C1q and/or biotin-C1q tails with
streptavidin-Alexa 488 for detection, and the scFv antibodies to detect the candidate protein. Finally, the new
scFv antibodies will be used in excess for phenotypic studies (i.e., adherence and phagocytosis assays). The
anti-GP63 and our non-binding scFv reagents will be used as negative controls.
Research Design & Methods
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Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
Interpretation, potential pitfalls, and alternative approaches: While confocal microscopy is not adequate
to confirm protein-protein interaction, a combination of co-localization of the novel proteins with calreticulin or
biotin-C1q, and confirmation that they interact in vitro will provide strong supportive evidence, and inhibition of
phagocytosis by antibodies against the candidate protein would provide additional support. Actual confirmation
that the proteins interact in vivo would require experiments using fluorescence resonance energy transfer
(FRET) or protein complementation assays (e.g., by producing fusions to complementary fragments of GFP)
[145], both of which are unlikely to work without a detailed knowledge of the mechanism of protein-protein
interaction, and are beyond the current aims. It is noteworthy, however, that the chosen method may provide
information to facilitate the future use of these methods.
The major potential pitfall of the chosen approach is the possibility that the recombinant proteins will be
dysfunctional, resulting in a false negative result. This could result from placement of the epitope tags, or from
altered maturation of the proteins in E. coli. Our alternative approach if no interaction is observed will be to
express the candidate protein on the surface of Chinese Hamster Ovary (CHO) cells, and then to quantitate
binding of the bait protein (either calreticulin or the C1q tail) using an ELISA-based method similar to the one
used for the experiment in Figure 14. For this, the predicted extracellular domain of the candidate protein will
be cloned in frame using PCR into the vector pDisplay (Invitrogen), which results in a fusion of the protein of
interest with a mammalian N-terminal signal sequence and C-terminal transmembrane anchoring domain.
Surface expression can be verified using a confocal microscope by probing for incorporated hemagglutinin and
myc epitope tags. Binding of calreticulin or the C1q tail to wild-type cells may complicate these experiments.
However, comparison of calreticulin and/or C1q tail binding to cells displaying the candidate receptor and cells
transfected with the empty control vector should enable interpretation.
3.3: Interference with expression and/or activity of the candidate proteins, and functional studies:
Specific interference with the expression or function of the candidate signaling proteins identified in 3.1 will
allow us to experimentally test whether they participate in phagocytosis, and will generate amebae with
sustained defects for future in vivo experiments. Our method of choice to disrupt protein expression will again
be to induce RNAi by expressing short hairpin RNAs as in section 2.1. If RNAi fails, the transcriptional gene
silencing method described in section 2.1 would again be used. If the candidate protein’s sequence suggests
obvious mutations that may have a dominant negative phenotype, we will also express these mutant proteins.
As in section 2.1, chimeric DNA molecules linking the E. histolytica U6 promoter to 29 base short hairpin RNAs
targeting four sequences (plus a scrambled negative control) in the gene of interest will be generated by two
rounds of PCR using E. histolytica genomic DNA as template and primers directed to the U6 promoter
sequence that incorporate the sense, anti-sense, and DNA loop sequences, as well as the RNA pol III
terminator sequence (6 thymidines) [134]. These PCR products will be cloned into an E. histolytica expression
vector containing a hygromycin selection cassette. Following stable transfection of amebae, efficacy of the
RNAi constructs will be assessed using quantitative real time PCR and Western blots (with the scFv antibodies
generated in section 3.2) and the effect on phagocytosis of inhibiting expression of the candidate protein will be
measured. In each case, specificity will be determined by assessing the effect of scrambled short hairpin
RNAs on gene expression and phenotype. Standard assays for growth, cytotoxic ability (Chromium51 release
assay), and motility (chemotaxis) will also be conducted to examine the specificity of an observed defect in
phagocytosis [39, 102, 135].
Interpretation, potential pitfalls, and alternative approaches: If interference with the expression (RNAi or
transcriptional silencing) or with the function (dominant negative approach) of a protein that interacts with
calreticulin reduces amebic phagocytosis, it will provide additional evidence that calreticulin functions as a
bridge between apoptotic cells/target particles and a calreticulin receptor. If, on the other hand, silencing
expression of another C1q interacting surface protein interferes with phagocytosis, it would provide evidence
that that protein is the collectin receptor. An important potential pitfall is that the identified protein may be one
member of a protein family whose members have redundant functions (e.g., one of a large family of
transmembrane kinases identified in the E. histolytica genome database [143, 144]). In this case, interfering
with expression of a single family member may have no observable phenotype despite a role in phagocytosis.
RNA-mediated interference is particularly well suited for silencing entire gene families [146, 147], and, in the
case that the target gene belongs to a gene family, the most highly conserved sequences can be targeted.
Timeline:
Research Design & Methods
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Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
Specific Aim
Specific Aim 1-Collectin-dependent phagocytosis
1.1-C1q tail expts.
1.2-C1q/collectin binding expts.
1.3-N-glycanase treatment of collectins
1.4-collectin-dependent phagocytosis of bacteria
Specific Aim 2-Functional studies of calreticulin
2.1-calreticulin silencing
2.2-recombinant calreticulin expts.
2.3-expts. to test if E. his. calreticulin is a collectin
receptor
2.4-calreticulin mapping expts.
Specific Aim 3-Collectin or calreticulin receptor
identification
3.1-candidate identification
3.2-confirmation of candidate-bait interaction
3.3-functional evaluation of candidates
Year 1
Year 2
Year 3
Year 4
Year 5
Expected results and future directions: Successful completion of the proposed studies will help to define
the molecular mechanisms underlying amebic phagocytosis, and will deepen our understanding of how E.
histolytica interacts with the host and with commensal bacteria in the gut lumen. In addition to further
characterizing the ligands that stimulate phagocytosis, we anticipate identifying at least one novel amebic
phagocytosis receptor that is important for signaling particle uptake (i.e., either a collectin receptor or the
calreticulin/calreticulin receptor complex). There are a number of obvious future directions. We believe that
aim 1 will confirm that the collagenous collectin tail is a ligand for an amebic phagocytosis receptor. This
would suggest several additional possibilities. Though no clear collectin homologues are present in the E.
histolytica genome databases, it would be important to determine if E. histolytica produces its own “collectinlike” molecules that opsonize bacteria in the gut. C1q is a known chemoattractant for human leukocytes [148],
and we have already shown that C1q, MBL, and the collagenous C1q tail are chemoattractants for E.
histolytica. Entamoeba histolytica also migrates towards amebic lysates [149], and, if migration towards
amebic lysates is dependent on the amebic collectin receptor, it would suggest both the existence of amebic
collectin-like molecules and a method to identify them using a chemotaxis assay and biochemical fractionation.
The experiments proposed in aims 2 and 3 also lead to important future work. First, structure/function studies
of the collectin or calreticulin receptor identified in aim 3 will be necessary to elucidate the cell signaling
pathways that initiate E. histolytica phagocytosis. Though genes encoding proteins with limited similarity to
regions of CD91’s extracellular domain can be identified in the E. histolytica genome databases, no genes
encoding proteins with CD91’s intracellular signaling motif (an NPXY motif [150]) are present, and the cytosolic
adapter protein Ced-6/Gulp that interacts with this motif in macrophages is also absent. These observations
suggest unique aspects to the intracellular signaling events that mediate E. histolytica phagocytosis, and
understanding these events may lead to novel anti-parasitic drugs or insight into unidentified signaling
pathways in other phagocytes. Second, regardless of the role (or lack thereof) of calreticulin in amebic
phagocytosis, the methods we have developed to study E. histolytica phagocytosis should enable successful
identification of a collectin receptor that initiates uptake of apoptotic cells and bacteria. This will enable
generation of an E. histolytica strain with a defined defect in phagocytosis, and will provide the reagent
necessary for future in vivo studies to delineate the specific contribution of phagocytosis to invasive amebiasis.
Since removal of apoptotic cells by macrophages limits inflammation [40, 151], it is interesting to speculate that
apoptotic cell killing and phagocytosis by E. histolytica may reduce inflammation during amebic invasion. This
would be consistent with the paucity of inflammation that is typical of well established amebiasis in humans [9,
10, 16, 17], and this possibility would be examined in future animal studies using histology, immunostaining,
cytokine assays, and Affymetrix gene microarrays. Finally, it is possible that the surface receptor identified in
aim 3 will be a novel candidate for inclusion in a vaccine to prevent amebiasis. Towards this end, it will be
important to determine if the receptor is immunogenic, both in animals infected with E. histolytica and in
people. Both could be easily determined by ELISA and/or immunoblots using serum samples and recombinant
antigen, and, if immunogenic, an obvious next step would be to determine if anti-receptor antibodies prevent
disease in passively immunized animals.
Research Design & Methods
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Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
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Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
1400 Jackson Street
Denver, CO 80206
303 388 4461
800 423 8891
http://www.nationaljewish.org
THE NUMBER 1 HOSPITAL IN THE U.S. FOR RESPIRATORY DISEASES
U.S. NEWS & WORLD REPORT, 1998 - 1999
February 5, 2008
Christopher D. Huston, M.D.
Assistant Professor
Department of Medicine
University of Vermont College of Medicine
Rm 320 Stafford Hall
95 Carrigan Drive
Burlington, VT 05405
Dear Chris,
Of course we will be happy to help in any way we can with your project, titled
“Molecular mechanism of Entamoeba histolytica phagocytosis”. In light of our own
findings on macrophage phagocytosis of apoptotic cells, I find your preliminary data
indicating that E. histolytica may engulf apoptotic host cells and bacteria using a collectinand calreticulin-dependent mechanism very interesting.
As you know, we have considerable experience working with macrophages that may
be of benefit to you. In particular, we have successfully purified surfactant protein A and
collagenous collectin tails, both of which you have already used in your experiments. The
new data you generated using the collectins and collectin tails we sent you suggest that your
approach to these studies should work well. We can provide more of these reagents for
your experiments. We can also provide target cells from calreticulin knock-out mice for use
in your assays.
I look forward to working with you on this project. Good luck with your proposal.
Yours sincerely
Peter M. Henson, Ph.D.
Margaret A. Regan Professor of Pulmonary Inflammation
Letters of Support
Page 80
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
Letters of Support
Page 81
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
Letters of Support
Page 82
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
William A. Petri, M.D., Ph.D.
Professor of Internal Medicine,
Microbiology, and Pathology
Chief, Division of Infectious Diseases
and International Health
February 1, 2008
Christopher D. Huston, M.D.
Assistant Professor of Medicine
Division of Infectious Diseases
University of Vermont College of Medicine
Rm 320 Stafford Hall
95 Carrigan Drive
Burlington, VT 05405
Dear Chris,
I am writing to offer my enthusiastic support for your experiments to knock-down expression
of calreticulin and calreticulin interacting proteins hypothesized to participate in Entamoeba
histolytica phagocytosis. I find your work to be highly original and of great significance for
what it will reveal about the mechanisms by which amebic phagocytosis contributes to
virulence.
As you know, we have developed an shRNA expression system for E. histolytica that has
been used to knock down expression of a transmembrane kinase and a lectin subunit. The
transmembrane kinase work has just been published (Boettner et al. PLoS Pathogens. 2008.
4(1):e8.). You are welcome to the use of the shRNA system for your experiments. Please
also feel free to ask for any of our specialized reagents, animal model systems, or gene
expression profiling arrays that might prove useful for your work.
Sincerely,
William A. Petri, Jr.
Wade Hampton Frost Professor of Epidemiology
University of Virginia Health System, PO Box 801340 • Charlottesville, VA 22908-1340
Office: 434-924-5621 • Fax: 434-924-0075 • E-mail: wap3g@virginia.edu
Letters of Support
Page 83
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
Christopher D. Huston, M.D.
Department of Medicine
University of Vermont College of Medicine
Rm 320 Stafford Hall, 95 Carrigan Drive
Burlington, VT 05405
February 1, 2008
Dear Chris,
I am writing to confirm my willingness to serve as a collaborator on your NIAID grant proposal
titled "Molecular mechanism of Entamoeba histolytica phagocytosis". Your preliminary data
suggesting that E. histolytica calreticulin functions as a phagocytosis receptor are very intriguing.
The data indicating the host collectins are ligands are also very strong, and, as we have discussed,
these methods could readily be adapted to identification of E. histolytica collectin-binding proteins.
As you know, my lab has considerable experience using biochemical cross-linkers and twodimensional electrophoresis to identify novel Toxoplasma gondii membrane proteins. We recently
published a manuscript describing the use of the detergents ASB-14 and octylglucoside in the 2D
gel purification of several T. gondii transmembrane proteins (Gilk et al. Eukaryotic Cell. 2006.
5:1622-34.); this method may be very helpful in your studies to identify transmembrane E.
histolytica proteins that initiate phagocytosis. I can be available to provide technical assistance on
use of biochemical cross-linkers, 2D gel electrophoresis, and protein identification by mass
spectrometry. In addition, should you need greater loading capacity or resolution than are possible
with your 2D gel equipment, you are welcome to use the equipment in my laboratory.
Good luck with your application, and I look forward to working with you on this exciting project.
Sincerely,
Gary Ward, Ph.D.
Professor
Department of Microbiology and Molecular Genetics
Letters of Support
Page 84
Principal Investigator/Program Director (Last, first, middle): Huston, Christopher, Dwight
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