PI: STARNBACH, MICHAEL N Title: Alteration of host protein stability by Legionella

Received: 10/18/2010 FOA: PA10-069 Council: 05/2011

Competition ID: ADOBE-FORMS-B FOA Title: NIH EXPLORATORY DEVELOPMENTAL RESEARCH GRANT 

PROGRAM (PARENT R21)

1 R21 AI096101-01 Dual: Accession Number: 3338682

IPF: 3212902 Organization: HARVARD UNIVERSITY (MEDICAL SCHOOL)

Former Number: Department: Microbiology

IRG/SRG: ZRG1 IDM-A (80)S AIDS: N Expedited: N

Subtotal Direct Costs 

(excludes consortium F&A) 

Year 1:

Year 2:

Animals: N 

Humans: N 

Clinical Trial: N

Current HS Code: 10

HESC: N

New Investigator: N

Early Stage Investigator: N

Senior/Key Personnel: Organization: Role Category:

Michael Starnbach President and Fellows of Harvard 

University

PD/PI 

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10/18/2010 5789461-01-5256928

President and Fellows of Harvard College

Sponsored Programs Admin

25 Shattuck St

Harvard Medical School

Boston

02115-6027

Ms. Deborah

Good

Alteration of host protein stability by Legionella

Yes No

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Dr. Michael N.

02115-0000

200 Longwood Avenue

Harvard Medical School

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Harvard Medical School

Microbiology

Professor

President and Fellows of Harvard University

MA: Massachusetts

USA: UNITED STATES

	


 		

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Evans, Kelly A. 10/18/2010

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d. Estimated Program Income

	


 		

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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

Research & Related Senior/Key Person-------------------------------------------------------------------------- 9

 Biographical Sketches for each listed Senior/Key Person---------------------------------------- 10

PHS 398 Specific Cover Page Supplement------------------------------------------------------------------------ 13

PHS 398 Specific Modular Budget------------------------------------------------------------------------------- 15

 Personnel Justification---------------------------------------- 18

PHS 398 Specific Research Plan-------------------------------------------------------------------------------- 19

 Specific Aims---------------------------------------- 20

 Research Strategy---------------------------------------- 21

 Select Agent Research---------------------------------------- 27

 Bibliography & References Cited---------------------------------------- 28

 Letters of Support---------------------------------------- 30

 Resource Sharing Plan---------------------------------------- 31

PHS 398 Checklist--------------------------------------------------------------------------------------------- 32

Table of Contents Page 3

Principal Investigator/Program Director (Last, first, middle): Starnbach, Michael, N.
County:

* ZIP / Postal Code:

* Country:

* State:

* City: 

Street2:

Organization Name:

Project/Performance Site Location(s)

Project/Performance Site Primary Location

* Street1:

Province:

President and Fellow of Harvard College

200 Longwood Avenue

Boston Suffolk

MA: Massachusetts

USA: UNITED STATES

02115

OMB Number: 4040-0010 

Expiration Date: 08/31/2011

I am submitting an application as an individual, and not on behalf of a company, state, 

local or tribal government, academia, or other type of organization. 

DUNS Number:

* Project/ Performance Site Congressional District: MA-008

Project/Performance Site Location 1

USA: UNITED STATES

* ZIP / Postal Code:

* Country:

Province:

* State:

* City: 

Street2:

* Street1:

Organization Name:

County:

DUNS Number:

* Project/ Performance Site Congressional District: 

I am submitting an application as an individual, and not on behalf of a company, state, 

local or tribal government, academia, or other type of organization. 

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Performance Sites Page 4

Principal Investigator/Program Director (Last, first, middle): Starnbach, Michael, N.
1. * Are Human Subjects Involved?

IRB Approval Date:

Human Subject Assurance Number:

2. * Are Vertebrate Animals Used?

IACUC Approval Date:

Animal Welfare Assurance Number

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?

4.d. If yes, please explain:

6. * Does this project involve activities outside of the United States or partnerships with international collaborators?

6.b. Optional Explanation:

7. * Project Summary/Abstract

11. Equipment

8. * Project Narrative

12. Other Attachments

RESEARCH & RELATED Other Project Information

Is the IACUC review Pending?

If no, is the IRB review Pending?

2.a. If YES to Vertebrate Animals

3. * Is proprietary/privileged information included in the application? 

4.a. * Does this project have an actual or potential impact on the environment? 

6.a. If yes, identify countries:

1 2 3 4 5 6

9. Bibliography & References Cited

10. Facilities & Other Resources

Yes No

1.a If YES to Human Subjects

Yes No

Yes No

If yes, check appropriate exemption number.

Yes No

Yes No

Yes No

Yes No

Yes No

Yes No

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Add Attachment Delete Attachment View Attachment

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Is the Project Exempt from Federal regulations?

5. * Is the research performance site designated, or eligible to be designated, as a historic place? Yes No

5.a. If yes, please explain:

 


  		

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Other Information Page 5

Principal Investigator/Program Director (Last, first, middle): Starnbach, Michael, N.
Project Summary

Infection with the intracellular pathogen Legionella pneumophila can lead to a severe pneumonia 

known as Legionnaires’ disease. Legionella pneumophila uses a specialized type IV secretion apparatus, also 

known as the Dot/Icm system, to secrete over 150 effector proteins directly into the host cell. The translocated 

bacterial effectors establish a vacuolar niche that supports replication of L. pneumophila in eukaryotic cells. 

While there is an extensive literature describing how several of these effectors alter host cell functions, the 

targets of most have remained elusive. A significant problem in linking a particular effector to a particular 

function is the redundant or overlapping activity of many effectors. This means that L. pneumophila mutant 

strains deficient in any one effector often have no appreciable phenotype, preventing the identification of their 

host targets. While it is well appreciated that many L. pneumophila effectors directly alter host proteins through 

functions such as E3 ubiquitin ligase activity, there have been few methods developed to monitor pathogeninduced changes in host protein stability on a large scale. Here we propose to apply a novel screening method 

called the “Global Protein Stability” (GPS) system to identify host cell proteins whose stability is altered by the 

secreted L. pneumophila effectors. Once we have identified host proteins that are stabilized or destabilized 

when a functional type IV secretion system is present, we will test whether reducing or increasing the 

prevalence of these proteins (attempting to reverse the effects of the Legionella effectors) impairs the capacity 

of L. pneumophila to replicate and survive within host cells. Once we identify which host proteins must be 

altered in order for L. pneumophila to replicate, we will take a targeted approach to identify which of the L. 

pneumophila effectors are causing these essential changes to host proteins. In addition, the GPS screen may 

also identify the targets of specific “families” of effectors that have remained elusive, such as the L. 

pneumophila E3 ubiquitin ligases. The directed approach we propose allows us to overcome the difficulties 

inherent in target identification, such as the redundancy of effectors, and identify the functions of effectors that 

have remained cryptic. Organism-induced alterations of the host are key to pathogenesis, yet it has previously 

not been possible to study alterations to individual host proteins at the scale the GPS system permits. The 

experiments described in this proposal allow, for the first time, dissection of how bacterial infection globally 

regulates host cell proteins and pathways beyond the transcriptional level. 

Project Description Page 6

Principal Investigator/Program Director (Last, first, middle): Starnbach, Michael, N.
Project Narrative

Legionella pneumophila, the causative agent of Legionnaires’ disease replicates inside host cells. To 

manipulate the host cell and replicate intracellularly, the organism injects >150 of its proteins into host cells. 

The proposed research uses a large-scale approach to identify the targets of these injected bacterial proteins –

identifying the host cell proteins that are destabilized or stabilized by the injected bacterial proteins. Once we 

identify which bacterial proteins are manipulating which host proteins, we can test methods to disrupt these 

interactions. This may lead to the development of new classes of antibiotics to treat bacterial infection.

Public Health Relevance Statement Page 7

Principal Investigator/Program Director (Last, first, middle): Starnbach, Michael, N.
RESOURCES

The PI’s laboratory consists of 1700 square feet of space in the Warren Alpert Building. There is ample 

bench, desk space and a fume hood. In addition, we have exclusive access to a tissue culture room with four 

laminar flow hoods, a CO2 incubator and microscopes. We have shared access to warm and cold rooms and 

several equipment rooms. A newly constructed SPF animal facility is located within the Warren Alpert Building. 

Space for infected mice is available in a newly constructed BL2/BL3 vivarium in an adjacent building. Apple 

Macintosh personal computers are located in both the office and laboratory space. All computers are attached 

by Ethernet to Harvard University computing facilities, the internet, software for analysis of sequence data, the 

Harvard Medical School library databases, and scientific literature databases.

The PI’s office is 240 square feet and is attached to the laboratory space.

Administrative support, computer support, statistical consulting, bioinformatics support and secretarial 

support for this project is available in the Department of Microbiology and Molecular Genetics.

The equipment necessary to perform the research in this proposal is located either within our 

laboratory, within the Department of Microbiology & Molecular Genetics, or within the adjacent Immune 

Diseases Institute. These include centrifuges, FACScan and FACSaria flow cytometers/sorters, oligonucleotide 

synthesizers, thermal cyclers, HPLC, refrigerators, freezers, -800

 C freezer, liquid N2 storage, and 

electrophoresis equipment.

The Department of Microbiology and Molecular Genetics has just purchased an Agilent array reader 

that is available for analyzing the arrays in this project.

SCIENTIFIC AND INTELLECTUAL ENVIRONMENT

To succeed in the proposed research, we will need to combine classical and molecular genetics, 

biochemistry, immunology, cell biology, and conventional and high-resolution microscopy. The location of our 

laboratory within the Department of Microbiology and Molecular Genetics and within the wider community of 

Harvard Medical School (HMS) is ideally suited for maximizing our chances of success. The high density of 

scientific leaders and innovators with well-equipped facilities at HMS has made it possible to assemble a group 

of outstanding colleagues to provide critical support and advice for all aspects of the proposed project. The 

most important of these interactions will be with Dr. Stephen Elledge in our Department of Genetics. We also 

will formally consult on a quarterly basis with a number of Harvard Medical School colleagues including Drs. 

Dan Finley and Randy King (experts on protein turnover and the ubiquitin-proteasome pathway), Drs. Eric 

Rubin and Stephen Lory (who have expertise on genomic approaches to the study of bacterial pathogenesis), 

and Dr. Deb Hung (a chemical biologist interested in new approaches to develop antimicrobial compounds).

In addition to these outstanding colleagues, the Department of Microbiology and Molecular Genetics 

has a long tradition of collegiality, active scientific exchange, and collaboration. The week is bracketed by 

scientific and social events that bring the entire community of faculty and trainees together. Every Monday we 

have student-postdoc research presentations (two talks, 25 minutes each) and every Friday we have beer 

hour. Both events are equally well-attended and promote scientific exchange and seed new collaborations. In 

addition, the department hosts a weekly seminar series where leading researchers in the fields of microbiology, 

cell biology, and immunology are invited to present current research in these areas. Our research group also 

attends the weekly Immunology program seminar and social hour.

Facilities Page 8

Principal Investigator/Program Director (Last, first, middle): Starnbach, Michael, N.
Province:

PROFILE - Project Director/Principal Investigator

Prefix: * First Name: Middle Name:

* Last Name: Suffix:

Organization Name: Division:

Position/Title: Department:

* Street1:

Street2:

* Phone Number: Fax Number:

* E-Mail:

Credential, e.g., agency login:

* Project Role: Other Project Role Category:

* Country: * Zip / Postal Code:

* State:

* City: County/ Parish:

Attach Current & Pending Support

RESEARCH & RELATED Senior/Key Person Profile (Expanded)

*Attach Biographical Sketch

Dr. Michael N.

Starnbach

Professor Microbiology

President and Fellows of Harvard University

Harvard Medical School

Boston

Harvard Medical School

200 Longwood Avenue

MA: Massachusetts

USA: UNITED STATES 02115-0000

PD/PI

Ph.D.

1992

R21StarnbachBioOCT2010ABSFINA Add Attachment Delete Attachment View Attachment

Add Attachment Delete Attachment View Attachment

OMB Number: 4040-0001 

Expiration Date: 06/30/2011

Degree Type:

Degree Year:

Province:

PROFILE - Senior/Key Person

Prefix: * First Name: Middle Name:

* Last Name: Suffix:

Organization Name: Division:

Position/Title: Department:

* Street1:

Street2:

* Phone Number: Fax Number:

* E-Mail:

Credential, e.g., agency login:

* Country: * Zip / Postal Code:

* State:

* City: County/ Parish:

USA: UNITED STATES

* Project Role: Other Project Role Category:

1

Degree Type:

Degree Year:

*Attach Biographical Sketch Add Attachment Delete Attachment View Attachment

Attach Current & Pending Support Add Attachment Delete Attachment View Attachment

	


 		

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Key Personnel Page 9

Principal Investigator/Program Director (Last, first, middle): Starnbach, Michael, N.
BIOGRAPHICAL SKETCH 

Provide the following information for the key personnel and other significant contributors. 

Follow this format for each person. DO NOT EXCEED FOUR PAGES.

NAME 

Michael Starnbach 

eRA COMMONS USER NAME 

POSITION TITLE 

Professor 

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)

INSTITUTION AND LOCATION DEGREE 

(if applicable) YEAR(s) FIELD OF STUDY 

Vassar College, Poughkeepsie, NY A.B 1987 Biology 

Stanford University, Stanford, CA Ph.D 1992 Micro. & Immuno. 

University of Washington, Seattle, WA None Postdoctoral Research 

A. Personal Statement 

Research in my lab uses a combination of cellular and molecular approaches in the analysis of host responses 

to bacterial pathogens. Many virulence factors have been identified that allow bacteria to survive and replicate 

within the mammalian host and often within host cells. I have focused on the consequences of these survival 

strategies, specifically asking how bacterial virulence factors subvert or inhibit host responses allowing for 

chronic infection. My lab has led the efforts to dissect antigen specific T cell responses to chronic bacterial 

infection, including C. trachomatis. Approximately half the members of my research team are cellular 

immunologists focused on the T cell response to bacterial pathogens. The other half of my research team 

focuses on bacterial pathogenesis, with a particular interest in how bacterial virulence determinants modify 

host cells and how host cell innate immune pathways allow for resistance to intracellular growth. Recent work 

on cellular factors needed to constrain bacterial growth has used Legionella pneumophila as the model 

pathogen, so my lab is already prepared to conduct these studies. Working at the interface of bacterial 

pathogenesis and cellular immunology reflects my prior training, with graduate work focused on bacterial 

virulence determinates and postdoctoral work in T cell immunobiology.

B. Positions and Honors. 

1985-1987 Research Assistant; Vassar College with E. Pinina Norrod, Department of Biology 

1987-1992 Graduate student and research assistant, Stanford University School of Medicine with Stanley 

Falkow, Department of Microbiology and Immunology 

1990-1992 Research in absentia. Laboratory of Stephen Lory, University of Washington School of 

Medicine, Department of Microbiology 

1992-1995 Associate, Howard Hughes Medical Institute, and Senior Fellow, Department of Immunology; 

University of Washington School of Medicine, with Michael Bevan. 

1995-2001 Assistant Professor, Dept. of Microbiology and Molecular Genetics, Harvard Medical School. 

1997-date Tutor in Biochemical Sciences, Harvard University. 

2001-2008 Associate Professor, Dept. of Microbiology and Molecular Genetics, Harvard Medical School 

2008-date Professor, Dept. of Microbiology and Molecular Genetics, Harvard Medical School 

Academic Honors 

1987 Departmental Honors in Biology and General Honors, Vassar College 

1987-1989 Departmental USPHS Predoctoral Traineeship, Stanford University 

1989-1990 Cellular and Molecular Biology USPHS Predoctoral Traineeship, Stanford University 

1996-1999 Junior Faculty Research Award, American Cancer Society 

Biosketches Page 10

Principal Investigator/Program Director (Last, first, middle): Starnbach, Michael, N.
C. Selected peer-reviewed publications (in chronological order). 

1. Starnbach, M. N, and M. J. Bevan. 1994. Cells Infected with Yersinia Present an Epitope to Class I MHC 

Restricted Cytotoxic T-Lymphocytes. Journal of Immunology 153:1603-1612. 

2. Starnbach, M. N, M. J. Bevan, and M. F. Lampe. 1994. Protective Cytotoxic T-Lymphocytes are induced 

during Murine Infection with Chlamydia trachomatis. Journal of Immunology, 153:5183-5189. 

3. Ballard, J. D., R. J. Collier, and M. N. Starnbach. 1996. Anthrax toxin mediated delivery of a cytotoxic T-cell 

peptide epitope in vivo. Proceedings of the National Academy of Sciences, USA, 93:12531. 

4. Lampe, M. F., M. J. Bevan, C. B. Wilson, and M. N. Starnbach. 1998. Gamma interferon production by 

cytotoxic T-lymphocytes is required for resolution of Chlamydia trachomatis infection. Infection and Immunity, 

66:5457-5461. 

5. Fling, S., A. Sutherland, L. Steele, B. Hess, S. D’Orazio, J-F. Maisonneuve, M. Lampe, P. Probst, and 

M.Starnbach. 2001. CD8+ T-cells Recognize a Novel Inclusion Membrane Associated Protein from the 

Vacuolar Pathogen Chlamydia trachomatis. Proceedings of the National Academy of Sciences, USA, 98:1160-

1165. 

6. Boyartchuk V.L., K.W. Broman, R.E. Mosher, S.E. D'Orazio, M.N. Starnbach, and W.F. Dietrich. 2001. 

Multigenic control of Listeria monocytogenes susceptibility in mice. Nature Genetics, 27:259-60. 

7. Starnbach, MN, Loomis WP, Ovendale P, Regan D, Hess B, Alderson MR, Fling SP. An inclusion 

membrane protein from Chlamydia trachomatis enters the MHC class I pathway and stimulates a CD8+

 T cell 

response. Journal of Immunology, 2003;171:4742-4749. 

8. Ho, TD, Starnbach, MN. The Salmonella-encoded type III secretion system can translocate Chlamydia 

proteins. Infection and Immunity, 2005;73:905-911. 

9. Bernstein-Hanley, I, Balsara, ZR, Ulmer, W, Coers, J, Starnbach, MN, Dietrich, WF. Genetic analysis of 

susceptibility to Chlamydia trachomatis in mouse, Genes and Immunity, 2006, 7:122-129. 

10. Misaghi, S, Balsara, ZR, Catic, A, Spooner, E, Ploegh, HL, Starnbach, MN. Chlamydia trachomatisderived deubiqitinating enzymes in mammalian cells during infection, Molecular Microbiology, 2006, 61:42-50. 

11. Bernstein-Hanley, I, Coers, J, Balsara, ZR, Taylor, GA, Starnbach, MN, Dietrich, WF. The p47 GTPases 

IGTP and Irgb10 map to the Chlamydia trachomatis susceptibility locus Ctrq-3 and mediate cellular resistance 

in mouse. Proceedings of the National Academy of Sciences, USA, 2006, September 7. 

12. Balsara, ZR, Misaghi, S, Lafave, JN, Starnbach, MN. Chlamydia trachomatis infection induces cleavage 

of the mitotic cyclin B1. Infection and Immunity, 2006, 74:5602-5608. 

13. Coers, J, Bernstein-Hanley, I, Grotsky, D, Parvanova, I, Howard, JC, Taylor, GA, Dietrich, WF, Starnbach, 

MN. Chlamydia muridarum evades growth restriction by the IFN inducible host resistance factor Irgb10. 

Journal of Immunology, 2008, 180:6237-6245. 

14. Henry, SC, Daniell, XG, Burroughs, AR, Indaram, M, Howell, DN, Coers, J, Starnbach, MN, Hunn, JP, 

Howard, JC, Feng, CG, Sher, A, Taylor, GA. Balance of Irgm protein activities determines IFNg-induced host 

defence. J Leukoc Biol. 2009 Jan 27. PMC ID: PMC2669409. 

15. Coers, J, Starnbach, MN, Howard, JC. Modeling infectious disease in mice: co-adaptation and the role of 

host-specific IFNγ responses, PLOS Pathogens, May 29, 2009. PMC ID: PMC2682201. 

Biosketches Page 11

Principal Investigator/Program Director (Last, first, middle): Starnbach, Michael, N.
D. Research Support. 

Ongoing Research Support

R01 AI039558 Starnbach (PI) 6/1/06 - 5/31/11 

NIH/NIAID 

Cytotoxic T cell Mediated Immunity to Chlamydia

The major goals of this project are to identify C. trachomatis genes encoding CD8+ T cell epitopes, test their 

efficacy in a model vaccine, and define the mechanism of CD8+ T cell protection. 

Role: PI

R01 AI062827 Starnbach (PI) 2/01/06 – 1/31/11 

NIH/NIAID 

Genetics of Innate Immunity to Chlamydia trachomatis 

The goal of this project is to identify mouse loci associated with susceptibility and/or resistance to C. 

trachomatis infection. 

Role: PI

 

R01 AI055962 Starnbach (PI) 6/1/09 – 5/31/11 

NIH/NIAID 

Inhibition of T cell Responses by Bacteria 

The major goal of this project is to identify and characterize the defect in MHC-I processing and/or presentation 

that occurs during S. flexneri infection. 

Role: PI 

Completed Research Support 

None. 

Biosketches Page 12

Principal Investigator/Program Director (Last, first, middle): Starnbach, Michael, N.
PHS 398 Cover Page Supplement

1. Project Director / Principal Investigator (PD/PI)

* Title:

* Street1:

* City:

* Country:

Street2:

County/Parish:

* State:

* Zip / Postal Code:

Prefix: * First Name:

Middle Name:

* Last Name:

Suffix:

2. Human Subjects

Clinical Trial?

* Agency-Defined Phase III Clinical Trial?

* First Name:

Middle Name:

* Last Name:

Suffix:

3. Applicant Organization Contact

Person to be contacted on matters involving this application

* Phone Number: Fax Number:

Email:

OMB Number: 0925-0001 

Province:

Prefix:

Dr. Michael

N.

Starnbach

No Yes

No Yes

Ms. Deborah

Good

Associate Director

25 Shattuck Street

Boston

USA: UNITED STATES

MA: Massachusetts

02115-6027

	


 		

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Clinical Trial & HESC Page 13

Principal Investigator/Program Director (Last, first, middle): Starnbach, Michael, N.
4. Human Embryonic Stem Cells

* Does the proposed project involve human embryonic stem cells?

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/research/registry/. 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: 

Specific stem cell line cannot be referenced at this time. One from the registry will be used.

PHS 398 Cover Page Supplement

No Yes

Cell Line(s):

	


 		

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Clinical Trial & HESC Page 14

Principal Investigator/Program Director (Last, first, middle): Starnbach, Michael, N.
PHS 398 Modular Budget, Periods 1 and 2

OMB Number: 0925-0001 

1. 

Start Date: End Date:

Budget Period: 1

* Direct Cost less Consortium F&A

A. Direct Costs

B. Indirect Costs

Consortium F&A

* Total Direct Costs

Indirect Cost Type

Indirect Cost 

Rate (%)

Indirect Cost 

Base ($) * Funds Requested ($)

Cognizant Agency (Agency Name, POC Name and Phone Number)

Indirect Cost Rate Agreement Date Total Indirect Costs

2. 

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04/28/2010

0.00

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1. 

Start Date: End Date:

Budget Period: 2

* Direct Cost less Consortium F&A

A. Direct Costs

B. Indirect Costs

Consortium F&A

* Total Direct Costs

Indirect Cost Type

Indirect Cost 

Rate (%)

Indirect Cost 

Base ($) * Funds Requested ($)

Cognizant Agency (Agency Name, POC Name and Phone Number)

Indirect Cost Rate Agreement Date Total Indirect Costs

C. Total Direct and Indirect Costs (A + B) Funds Requested ($)

2. 

3. 

4. 

* Funds Requested ($)

MTDC Organized Research (A02)

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04/28/2010

07/01/2012 06/30/2013

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PHS 398 Modular Budget, Periods 3 and 4

1. 

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* Direct Cost less Consortium F&A

A. Direct Costs

B. Indirect Costs

Consortium F&A

* Total Direct Costs

Indirect Cost Type

Indirect Cost 

Rate (%)

Indirect Cost 

Base ($) * Funds Requested ($)

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Indirect Cost Rate Agreement Date Total Indirect Costs

C. Total Direct and Indirect Costs (A + B) Funds Requested ($)

2. 

3. 

4. 

1. 

Start Date: End Date:

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* Direct Cost less Consortium F&A

A. Direct Costs

B. Indirect Costs

Consortium F&A

* Total Direct Costs

Indirect Cost Type

Indirect Cost 

Rate (%)

Indirect Cost 

Base ($) * Funds Requested ($)

Cognizant Agency (Agency Name, POC Name and Phone Number)

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Principal Investigator/Program Director (Last, first, middle): Starnbach, Michael, N.
PHS 398 Modular Budget, Periods 5 and Cumulative

1. 

Start Date: End Date:

Budget Period: 5

* Direct Cost less Consortium F&A

A. Direct Costs

B. Indirect Costs

Consortium F&A

* Total Direct Costs

Indirect Cost Type

Indirect Cost 

Rate (%)

Indirect Cost 

Base ($) * Funds Requested ($)

Cognizant Agency (Agency Name, POC Name and Phone Number)

Indirect Cost Rate Agreement Date Total Indirect Costs

C. Total Direct and Indirect Costs (A + B) Funds Requested ($)

2. 

3. 

4. 

Cumulative Budget Information

* Funds Requested ($)

*Section A, Total Direct Cost less Consortium F&A for Entire Project Period

1. Total Costs, Entire Project Period

Section A, Total Consortium F&A for Entire Project Period

*Section A, Total Direct Costs for Entire Project Period

*Section B, Total Indirect Costs for Entire Project Period

*Section C, Total Direct and Indirect Costs (A+B) for Entire Project Period

2. Budget Justifications

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Principal Investigator/Program Director (Last, first, middle): Starnbach, Michael, N.
BUDGET JUSTIFICATION

PERSONNEL

Michael Starnbach (Principal Investigator, 1.2 Calendar months). Michael Starnbach will be 

responsible for the overall intellectual direction of this project. He will supervise and be 

responsible for the conduct of all the studies in this proposal, review all protocols and data, and 

supervise and train all other personnel. 

Catarina Nogueira (Post-Doc, 12 Calendar months). Catarina Nogueira will take primary 

responsibility for the GPS screens (Aim 1) and the identification of the bacterial effectors 

responsible (Aim 2). Dr. Nogueira is an expert in how bacteria modify host proteins, having 

conducted doctoral work in Craig Roy’s lab at Yale where she studied L. pneumophila’s capacity 

to manipulate host regulatory pathways to induce apoptosis. 

Hannah Ratcliffe (Research Assistant, 3 Calendar months). Hannah Ratcliffe will assist Dr. 

Nogueira with the extensive cell culture and molecular biology required for this project. She will 

also maintain tissue culture supplies and reagents. 

Personnel Justification Page 18

Principal Investigator/Program Director (Last, first, middle): Starnbach, Michael, N.
PHS 398 Research Plan

1. Application Type:

From SF 424 (R&R) Cover Page. The response provided on that page, regarding the type of application being submitted, is repeated for your 

reference, as you attach the appropriate sections of the Research Plan.

*Type of Application:

OMB Number: 0925-0001 

2. Specific Aims

3. *Research Strategy

6. Protection of Human Subjects

7. Inclusion of Women and Minorities

8. Targeted/Planned Enrollment Table

9. Inclusion of Children

10. Vertebrate Animals

13. Consortium/Contractual Arrangements

14. Letters of Support

15. Resource Sharing Plan(s)

16. Appendix

1. Introduction to Application 

(for RESUBMISSION or REVISION only)

Human Subjects Sections

2. Research Plan Attachments: 

 Please attach applicable sections of the research plan, below.

Other Research Plan Sections

5. Progress Report Publication List

11. Select Agent Research

12. Multiple PD/PI Leadership Plan

4. Inclusion Enrollment Report

New Resubmission Renewal Continuation Revision

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Principal Investigator/Program Director (Last, first, middle): Starnbach, Michael, N.
2. Specific Aims

L. pneumophila is a powerful model organism that can be used to understand the interactions between 

intracellular pathogens and the host cells they infect. Our lab has been interested in understanding, at a global 

scale, how effector proteins that are translocated into host cells during bacterial infection post-translationally 

manipulate host cell proteins and pathways. Although some host cell proteins targeted by specific L. 

pneumophila type IV secreted effectors have been identified, most have remained elusive. We have been 

working with Stephen Elledge’s lab in the Department of Genetics to adapt their recently-developed “Global 

Protein Stability” (GPS) system to analyze how translocated effector proteins impact the stability of individual 

host cell proteins. Our goal in this proposal is to globally characterize changes in host protein stability caused 

by translocated proteins and to begin identifying specific L. pneumophila effectors responsible for these 

alterations. Understanding on a large scale what host proteins are altered by specific bacterial effectors will 

allow us to uncover host pathways that are critical for L. pneumophila replication and survival inside a cell. 

Specifically, we will:

Specific Aim 1. Identify and characterize host cell proteins that are stabilized or destabilized by L. 

pneumophila translocated effectors. To better understand how L. pneumophila manipulates host cells, we 

will conduct a “Global Protein Stability” (GPS) screen to identify host proteins that are altered in stability when 

the Legionella type IV secretion system is present. We will determine the stability of >12,000 individual host 

proteins during infection with wild-type L. pneumophila and then identify which of these host proteins are more

or less stable when the cell is infected with L. pneumophila (ΔdotA) that lacks a functional type IV secretion 

system (and is unable to secrete effectors into cells). Once we identify proteins whose stability is altered by 

the L. pneumophila effectors, we will determine whether reversing these changes, by either reducing or 

increasing the prevalence of these host proteins, impairs the intracellular growth of L. pneumophila. This will 

identify host proteins whose stability must be manipulated by L. pneumophila in order for the organism to grow.

Specific Aim 2. Determine the L. pneumophila Dot/Icm-translocated substrates that are responsible for 

alterations in host protein stability. Previous loss-of-function studies using L. pneumophila mutants have 

been unable to identify the targets of many effectors due to significant redundancy in the function of the 

effectors. Once we identify changes in host protein stability mediated by the translocated effectors (in Specific 

Aim 1), we can then test which L. pneumophila effector(s) is responsible for that alteration. This allows a more 

direct approach to identify bacterial effector/host target pairs important in pathogenesis. Together these 

approaches will help us understand both the host proteins that are altered in stability and the bacterial effectors 

responsible for these changes. If these effector-mediated manipulations of host cell protein stability are 

required for growth, it will allow us to develop new classes of antibiotics that prevent these manipulations and 

prevent bacterial growth.

Through these two fundamentally linked aims we will use GPS to identify host proteins, pathways or 

networks altered by L. pneumophila translocated effectors. We then can begin to identify the specific bacterial 

virulence factors responsible for those alterations. The use of GPS is an innovative method that can be 

adapted widely to understand host-pathogen interactions.

Specific Aims Page 20

Principal Investigator/Program Director (Last, first, middle): Starnbach, Michael, N.
3. Research Strategy 

3(a). Significance. Intracellular bacterial pathogens have evolved to exploit host cells in order to survive 

within and spread from mammalian hosts. Many of these bacterial pathogens reside in vacuoles and 

translocate virulence factors into the host cell cytosol through specialized secretion systems in order to avoid 

destruction by the endocytic pathway and allow replication within the cell (12). The activities of secreted 

effectors and their impact on host cell functions is an area of intense investigation. To completely understand 

how bacteria impact, exploit, or affect host cell functions during infection, one must study multiple aspects of 

host cell protein regulation during infection, including transcriptional regulation, regulation of translation, as well 

as protein modification, localization and turnover. Much research activity over the past decade has focused on 

the opportunities available to study transcriptional regulation of host cell functions during infection. The ability 

to screen arrays of host cell genes during bacterial infection has led to an explosion in the available data 

showing which mammalian genes are induced and repressed as a result of infection. However, bacterial 

manipulations of host cell proteins (for example the activity of bacterial proteases) (18) cannot be discovered 

by monitoring transcription. It simply has not been previously possible to examine how bacterial infection 

directly affects the stability of individual host proteins at the scale afforded by transcriptional arrays. L. 

pneumophila has served as an exceptional model pathogen for dissecting complex changes in host cell biology 

that occur during infection. Especially well-characterized are the protein effectors secreted by the L. 

pneumophila type IV secretion system. Here we propose to conduct a “Global Protein Stability” (GPS) screen 

to determine the impact of Legionella type IV secretion on the stability of >12,000 individual host cell proteins. 

To accomplish this, we will identify proteins whose stability differs when cells are infected with wild-type L. 

pneumophila vs. a mutant strain (ΔdotA) that lacks a functional type IV secretion system. This approach will 

allow us to identify a significant subset of host cell proteins whose stability is altered by the L. pneumophila

type IV effectors. Once we have identified proteins whose stability is altered during infection, we will conduct 

experiments to determine whether some of these host protein alterations are necessary for intracellular growth 

of L. pneumophila. At the same time, we will also work to identify which L. pneumophila effector proteins are 

responsible for individual protein changes in the host. As a longer-term goal, we would like to target with 

experimental therapeutics the host proteins that are required for successful bacterial replication – developing 

novel classes of “host-based” antibiotics. 

The ability to identify, study and target host proteins altered by microbial infection will be compelling to 

most cellular microbiologists regardless of the microbe they study. Therefore we are confident that GPS will be 

widely adopted to study the pathogenesis of infectious disease caused by bacteria, viruses and parasites. 

3(b). Innovation. Although most investigators studying the effects of bacteria on host cells appreciate that 

gene regulation and protein turnover are both key to understanding how bacteria alter host cell functions, there 

has been a bias towards the study of gene regulation rather than protein stability. This bias is a result of the 

availability of arrays and other tools that allow the simultaneous measurement of thousands of individual host 

cell gene transcripts during a particular stage of cell infection. Although proteomics is an active area of 

investigation, the ability to monitor changes in protein abundance globally is limited. Approaches to study 

protein levels usually involve pulse-chase experiments, the administration of protease inhibitors followed by 

biochemical analysis, or biochemically trapping individual protein-protein interactions. These approaches are 

inherently limited in the number of proteins that can be monitored at one time, and are not scalable to make 

them more “global”. We are interested in understanding how L. pneumophila type IV secreted effectors 

manipulate host cell protein function and turnover at a global level, and to determine whether some of these 

manipulations are required for intracellular replication. It is known that the type IV (Dot/Icm) secretion 

apparatus is crucial for L. pneumophila survival and replication inside eukaryotic cells. Over 150 Dot/Icm 

effector proteins have been identified through a variety of genetic, biochemical and cell biological approaches, 

yet for most of these proteins the biochemical functions and specific host targets remain unknown (4, 10, 16). 

We believe that many of the L. pneumophila effector proteins secreted during bacterial infection alter host cell 

proteins and pathways and allow the bacteria to establish a replicative niche. However, we have been 

frustrated by the inability to study changes in host cell protein stability at a scale provided by transcriptional 

arrays. The GPS screens being developed by our colleagues in the Genetics department are a novel, highly 

innovative method to globally monitor protein stability in mammalian cells, and we felt it could be adapted as a 

completely new large-scale approach to identify host proteins altered during bacterial infection. As proof-ofprinciple, we have begun to use GPS to globally monitor the stability of host proteins altered by L. pneumophila

effectors during infection. GPS has the potential to profoundly increase our knowledge of the basic science of 

protein regulation during L. pneumophila infection and significantly increase our potential to target and cure this 

disease. Importantly, this method is applicable to most other classes of microbial pathogens and it is likely to 

identify significant virulence mechanisms not previously detectable using previously available methods. It isn’t 

Research Strategy Page 21

Principal Investigator/Program Director (Last, first, middle): Starnbach, Michael, N.
hard to imagine that GPS may revolutionize the study of how bacterial infection globally regulates host cell 

proteins and pathways beyond the transcriptional level. 

3(c). Approach (Specific Aim 1). Identify and characterize host cell proteins that are stabilized or 

destabilized by L. pneumophila translocated effectors.

3(c).i. Modification of host cell proteins by Dot/Icm-dependent secreted effectors is required for L. 

pneumophila intracellular replication. L. pneumophila is a facultative intracellular pathogen that is present in 

fresh-water reservoirs (5). Inhalation of aerosolized water droplets contaminated with L. pneumophila results 

in bacterial uptake by alveolar macrophages that support bacterial replication (9). Without antibiotic therapy, 

bacterial replication can lead to the development of a severe pneumonia known as Legionnaires’ disease (6). 

As the use of artificial water reservoirs, such as air conditioning units and other man-made devices capable of 

aerosolizing contaminated water have become more common, epidemics and pneumonia caused by L.

pneumophila have also become more prevalent (23). 

To establish replication in its vacuolar niche, L. pneumophila inhibits lysosomal fusion and remodels its 

membrane-bound compartment into an organelle that resembles the endoplasmic reticulum (ER) (8). The 

ability of L. pneumophila to replicate in a ER-like compartment is dependent on a type IV secretion system, 

also known as the Dot/Icm secretion apparatus (19, 22). Over 150 Dot/Icm substrates have been identified, 

but for most of these proteins, the specific effector activities remain unknown (4, 10, 16). Using GPS, we can 

determine the stability of >12,000 host proteins during infection with wild-type L. pneumophila, and then 

identify which of these host proteins are more or less stable during infection with a dotA L. pneumophila mutant 

strain that lacks a functional Dot/Icm secretion system (and therefore translocates no effectors). This 

comparison of wild-type and mutant L. pneumophila strains will provide a vastly enhanced perspective on the 

targeting of host cell proteins by L. pneumophila-translocated substrates and the extent to which this targeting 

is required for infection, replication, and dissemination. 

The “Global Protein Stability” (GPS) system is a highly innovative method developed by the Elledge lab at 

Harvard Medical School to globally monitor changes in protein stability mediated by host cell ubiquitin ligases 

(24, 25). In our lab we are adapting this method as a novel approach to understand host-pathogen 

interactions. Here we propose to apply the GPS system to understand the extent to which L. pneumophila

Dot/Icm-dependent virulence factors modulate host cellular functions by stabilizing and destabilizing host 

proteins during infection. 

3(c).ii. Global Protein Stability (GPS) profiling of L. 

pneumophila-infected mammalian cells. The Elledge lab recently 

published a description of the GPS platform as a method to identify 

the substrates of a human E3 ubiquitin ligase (24, 25), and we 

have now applied that system to identify proteins whose stability is 

altered by L. pneumophila secreted effectors. The Elledge lab 

provided to us a library of HEK 293T cells each containing a single 

copy of an expression cassette fused to a different human ORF. 

The pooled library of HEK 293T cells we obtained was assembled from an arrayed set (ORFeome v3.1) of 

12,000 human ORFs, each engineered into a unique retroviral reporter construct whose general structure is 

shown in the figure above. The reporter expression cassette contains a single pCMV promoter that with an 

internal ribosome entry site (IRES) permits the translation of two fluorescent proteins from one mRNA 

transcript. The first fluorescent protein is DsRed and the second is an enhanced GFP (EGFP) expressed as a 

fusion to one of 12,000 host cell proteins (EGFP-X). When introduced into the genome of cells, DsRed and 

EGFP-X should be produced at a constant ratio (independent of normal transcriptional regulation) because 

they are translated from the same mRNA (and regardless of where the construct integrates). However, while 

the amount of DsRed remains constant, the level EGFP becomes subject to the stability of the protein fused to 

its C-terminus. Therefore for each fusion construct in the library a baseline EGFP:DsRed ratio (reflecting 

relative stability) can be measured using fluorescent activated cell sorting (FACS). In our approach, we 

infected one copy of the library with wild-type L. pneumophila, and infected another identical copy with L. 

pneumophila ΔdotA. For any host protein whose stability is altered by a Dot/Icm-dependent secreted effector, 

there will be a dramatic difference in the EGFP:DsRed ratio in cells expressing that ORF between the two 

libraries. A differential change in EGFP:DsRed ratio will occur between corresponding ORFs in the two 

libraries anytime one or more Dot/Icm-dependent effector changes the stability of the EGFP-X fused to that 

ORF. For example, the effector may induce or deplete proteins that regulate the stability of X, or an effector 

may directly stabilize X, destabilize X, or cleave X. Controls conducted by the Elledge lab (25) have shown 

that the N-terminal EGFP fusion almost never alters the stability of the protein to which it is fused. Controls in 

our lab have shown that L. pneumophila replicates robustly in HEK 293T cells and that infection of these cells 

with L. pneumophila does not alter the inherent stability of DsRed or EGFP when expressed alone. 

Research Strategy Page 22

Principal Investigator/Program Director (Last, first, middle): Starnbach, Michael, N.
In our experiments, based on the published methods of the Elledge lab and incorporating all of the 

controls described (24, 25)), HEK 293T cells were infected for 24 h with wild-type L. pneumophila and sorted 

by high-throughput FACS into 7 bins (sorting 1 million cells into each bin). Cells were sorted into the bins 

based on their individual EGFP:DsRed ratio, with increasing EGFP:DsRed ratios corresponding to bins 1-7 

respectively. Because sorting of cells into the distinct bins was dependent on the stability of the individual 

EGFP-X they express, the stability of an individual EGFP-X can be inferred from the distribution of cells 

expressing that particular EGFP-X within the 7 bins (abundance in bin 7 indicating stability of X, abundance in 

bin 1 indicating instability of X). The ORFs (Xs) in each bin were then amplified in bulk using PCR and labeled 

with Cy3 using an in vitro transcription kit. The ORFs (Xs) in the pre-sorted library were also amplified in bulk 

using PCR and labeled with Cy5 using an in vitro transcription kit. Because the labeled material is derived 

from genomic DNA, the labels represent the presence of an ORF within the bin or overall library, and are 

completely independent of transcription. The Cy3 labeled amplified ORFs from bin 1 were then mixed 1:1 with 

the Cy5 labeled amplified ORFs from the pre-sort library and applied to a custom microarray containing probes 

for the coding region of the 12,000 host proteins comprising the library. For each of the host protein ORF 

probes represented on the microarray, the ratio of Cy3:Cy5 indicates the prevalence of cells bearing that ORF 

in that bin. The amplified ORFs from bins 2-7 were similarly prepared and applied to a total of 7 microarrays. 

By compiling Cy3:Cy5 ratio data from all 7 arrays, we were able to characterize the prevalence of cells bearing 

a particular ORF in each of the seven bins. The presence of a cell bearing a particular ORF in higher number 

bins indicated that that protein is stable in the cells; the presence of a cell bearing a particular ORF in lower 

number bins indicated that that protein is unstable in the cells. To represent the protein stability information 

from the arrays quantitatively we calculated a protein stability index (PSI) for each of the host proteins in the 

library. PSI values for each host protein range from 1 to 7 with a higher PSI indicating relative protein stability. 

The calculation of PSI is described in the published report (25), but essentially identifies in which bin the 

population of cells bearing a particular ORF “peaks”.

In parallel to the experiment described above, we 

infected the library with the L. pneumophila dotA mutant strain. 

We then sorted this library into 7 bins as above, amplified the 

ORFs from each bin, and hybridized them to 7 microarrays. We 

calculated a PSI for each ORF for the ΔdotA infection. We then 

compared the PSI value we obtained for each host protein in the 

library during infection with wild-type with the PSI value we 

obtained during infection with the dotA mutant. The subtraction 

of the “wild-type” PSI value from the “ΔdotA” PSI value gives us 

a ΔPSI. ΔPSI expresses the impact of Dot/Icm-secreted 

effector/s on any particular ORF. The theoretical range of PSI 

values varies from -6 (the theoretical limit of proteins most 

“stabilized” as a result of the dotA mutation) to +6 (the theoretical limit of proteins most “destabilized” as a 

result of the dotA mutation). The figure above is a graph representing the PSI values from our preliminary 

run. 

3(c).iii. Notes on the arrays and hybridization controls used in the Global Protein Stability (GPS) 

profiling of L. pneumophila-infected mammalian cells. The custom microarrays used in the GPS system were 

designed by the Elledge lab and are manufactured by Agilent. The arrays contain over 44,000 different 

oligonucleotide probes of uniform length. The probes correspond to the coding region of the 12,000 host cell 

ORFs in the ORFeome v3.1 library. The number of probes per ORF ranges from 1 to 5 and the number of 

probes was designed to reflect the length of the ORF. Because most ORFs will hybridize to more than one 

probe, we can determine the consistency of the arrays and our hybridization procedures within any single 

array. These internal hybridization controls have proven remarkably consistent. Additionally, we can control 

across arrays by comparing the hybridization of the Cy5 labeled pre-sort ORFs between arrays. This has also 

been remarkably consistent. As an additional control across arrays we always spike the ORF mix with control 

sequences from Agilent labeled with Cy3 and Cy5 at predetermined ratios. The spike-in control sequences 

correspond to control probes incorporated into all Agilent microarrays. 

Even though the GPS system in its current form has yielded very exciting results, the dependence of 

the system on multiple microarrays may produce some noise and limit resolution. We are now working in 

collaboration with scientists at the Harvard/MIT Broad Institute to develop Illumina based sequencing methods. 

Once developed, this will provide us with deep sequence-based quantitation for every bin in our GPS runs, 

enhancing the resolution of each screen greatly. Although a similar method of deep sequencing has been 

used to identify essential Haemophilus genes in vivo using “transposon capture and sequencing” (TraCS, 

which inspired us to explore the use of deep sequencing for our work) (7), this will be the first example of deep 

sequencing being used to identify the impact of bacterial virulence factors on host proteins. 

Research Strategy Page 23

Principal Investigator/Program Director (Last, first, middle): Starnbach, Michael, N.
3(c).iv. Small-scale validation of host proteins that are predicted by GPS to be stabilized by L. 

pneumophila Dot/Icm secreted effectors. As a first assessment of our initial GPS screen, we focused on host 

proteins that were stabilized in the presence of a functional Dot/Icm system. To optimize our initial selection, 

we generated graphs for several hundred of the proteins predicted to be most stabilized (most negative PSI) 

from our GPS runs. The graphs represent the distribution of cells bearing that ORF over bins 1-7 as 

determined by the microarray hybridization - showing the results when cells bearing that ORF were either 

infected with wild-type L. pneumophila (blue) or 

the ΔdotA strain (red). Several examples are 

shown in the figure. For each of these graphs, 

a clear shift is observed in the ”peak” bin when 

a functional Dot/Icm system is present. Again, 

the examples in the figure reflect preliminary 

GPS screen results where we examined host proteins stabilized in the presence of a functional Dot/Icm system 

(those at the extreme low end of PSI values), but we are just as interested in candidates destabilized in the 

presence of a functional Dot/Icm system (those at the extreme high end of PSI values). 

We are also fortunate that the ORFeome v3.1 library is available to us as an arrayed set of ORFs in a 

Gateway entry vector. Therefore, we will confirm all of our GPS “hits” by picking out ORFs based on graphs 

where the peak representing the distribution of wild-type L. pneumophila-infected cells was clearly in a different 

bin than the peak representing the distribution of the ΔdotA-infected cells (as shown graphically with 5 

examples in the figure above). We will then clone each ORF into the original retroviral GPS destination vector 

described in section 3(c).ii above. These constructs will be individually packaged into retrovirus and then used 

to transduce HEK 293T cells. Each transduced cell line will be infected with either wild-type L. pneumophila or 

the dotA mutant strain. The cells containing the individual ORFs will be then subjected to flow cytometry and 

changes to EGFP:DsRed ratio will be identified will be compared to the original GPS “hit”. 

The GPS system is ultimately a screening tool where “hits” must be validated under endogenous 

promoters and expression levels in cells. We will examine the stability of several candidate “hit” proteins by 

comparing immunoblots of these host proteins in wild-type L. pneumophila and ΔdotA infected cells (using 

commercially available antibodies when available). After identifying and validating candidates, we will have a 

new “catalog” of host proteins whose stability are altered by L. pneumophila Dot/Icm-secreted effectors during 

infection. 

3(c).v. Additional experiments and plans to exploit the information gained from these GPS screens.

1. We strongly believe that GPS can be used to identify host protein targets as well as the protein 

networks affected by infection with pathogens such as L. pneumophila. A key goal for this project is to 

carefully determine the depth and validity of the data in our screen. The only previously conducted GPS 

screen (24) identified targets of a ubiquitin ligase, comparing untreated cells with cells expressing a dominant 

negative inhibitor of that ubiquitin ligase. This screen yielded >350 candidate substrates of the SCF ubiquitin 

ligase. A screen examining the impact of >150 L. pneumophila specific translocated effectors on host protein 

stability is likely to cause even greater perturbations than the activity of a single ubiquitin ligase. We imagine 

being able to mine from our data significant pathways in protein turnover that are mediated by infection that 

have been previously undiscovered. It also should be noted that the ubiquitin ligase GPS screen used the 

ORFeome v1.1 library (~8,000 ORFS) whereas our use of the ORFeome v3.1 library (12,000 ORFs) should 

allow even deeper protein stability network analysis. In any new GPS screens, we will employ the justconstructed ORFeome v5.1 library (17,000 ORFs, almost half the human genome) and corresponding 

microarrays or sequence-based quantitation. Complete coverage of the human genome is a near term goal of 

the groups assembling the ORFeome libraries. In addition to identifying individual host cell targets, a number 

of bioinformatics tools are available that will also assist us in revealing regulatory networks of proteins 

impacted by L. pneumophila infection. Together, this information will significantly increase our understanding 

of L. pneumophila infection and the basic cell biology of host-pathogen interactions in general. 

2. Little is known about how the majority of L. pneumophila Dot/Icm effector proteins are temporally 

regulated and translocated to host cells, therefore we will conduct identical GPS screens at several other 

timepoints after infection (besides the 24 h point used above) to gain a more complete understanding of how 

host cell protein stability changes throughout different stages of L. pneumophila infection. By understanding 

how individual host proteins are altered over the complete L. pneumophila replication cycle we can gain insight 

into L. pneumophila growth regulation and work towards interventions that block the progression of early 

intracellular replication and survival. 

3. As we screen, identify, and validate proteins whose stability is altered by L. pneumophilatranslocated substrates we will also conduct experiments to determine the effect of “preventing”/”counteracting” 

these individual changes from occurring. For example, with the stabilized proteins where a L. pneumophila

effector might “increase” the amount of a particular host protein within the cell, we will use siRNA to knockdown that protein within the cell. We then can examine whether this attempt to “counteract” infection-induced 

Research Strategy Page 24

Principal Investigator/Program Director (Last, first, middle): Starnbach, Michael, N.
stabilization of a host cell protein makes the cell more resistant to (or perhaps more susceptible to) infection 

with L. pneumophila. For the siRNA experiments, we are fortunate that the groups involved in creating the 

ORFeome libraries also have produced corresponding siRNA libraries to which we have complete access. In 

parallel we plan to also overexpress each “destabilized” protein candidate in host cells to see the effect on 

bacterial replication and development. Again we are fortunate to have access to the arrayed ORFeome 

libraries in Gateway entry vector. This will greatly facilitate making the mammalian cell overexpression 

constructs. 

Certainly some of the knock-downs or overexpression constructs may be deleterious to the cell or will 

not be appropriately “tuned” in expression level for us to observe differences in bacterial infectivity or growth. 

And we certainly expect that with most of our candidates, changing the level of a single protein won’t have any 

effect on infection. But we are optimistic that, either through individual interventions, through “pooled” 

interventions (such as using multiple RNAis or overexpressing multiple genes), or through insights into 

regulatory networks during infection, we will be able to use information about changes in host cell protein 

stability to intervene in the intracellular replication and survival of L. pneumophila. 

4. As we identify candidate proteins and networks altered during L. pneumophila infection, we will use 

any available small molecule compounds known to affect specific targets – treating cells with the compounds 

and looking for an effect on L. pneumophila replication. Our experiments knocking-down or overexpressing the 

proteins may also identify host proteins or regulatory pathways that would be exciting to directly target in a 

chemical screen. Small molecule screening platforms and expertise are available to us at both the Harvard 

Institute of Chemistry and Cell Biology (http://iccb.med.harvard.edu/) and the Broad Institute of Harvard and 

MIT (http://www.broadinstitute.org/science/platforms/chemical-biology-platform/chemical-biology-platform).

5. We are confident that our proposed screen examining changes in host protein stability that are 

dependent on a functional type IV secretion system is the most direct application for the GPS platform with L. 

pneumophila. We have considered conducting a GPS screen that compares changes in protein stability 

between uninfected and infected cells. However, the scale of this perturbation is so drastic that it would be 

difficult to tease apart what alterations are actively caused by a L. pneumophila infection and which are a 

response of the cell being infected. While this might be a minor concern in our current screen we can examine 

individual hits using the L. pneumophila thyA mutant strain that has a functional Dot/Icm system (so substrates 

are secreted initially) but fails to replicate due its thymidine auxotrophy (1, 13). This will allow us to exclude 

protein alterations that are simply due to L. pneumophila intracellular replication and the “stress” caused by the 

presence of a large vacuole containing replicating bacteria and not from the activity of the Dot/Icm 

translocation machinery. Furthermore, in normal human cells the TLR pathways would also be activated after 

L. pneumophila infection and proteins in those pathways would certainly be altered. However, HEK 293T do 

not express the major TLRs therefore limiting the number of hits that result from innate immune responses 

rather than direct effects of L. pneumophila Dot/Icm-secreted substrates. Overall the GPS platform is 

amenable to many novel screens that explore several different aspects of pathogenesis and protein stability. 

Certainly many pathogens that affect or manipulate the host cell could also be explored by GPS in this manner 

and we anticipate the GPS methodology will be broadly applied. 

 

3(c). Approach (Specific Aim 2). Determine the L. pneumophila Dot/Icm-translocated substrates that 

are responsible for alterations in host protein stability.

Over the last decade many investigators have been interested in identifying the function of individual 

secreted effectors in several different bacterial pathogens. However, with a pathogen like L. pneumophila

where there are over 150 translocated substrates, many effectors have overlapping functions with other 

effectors. This has limited progress in determining the role of individual effectors since mutant strains of L. 

pneumophila that are deficient in only one translocated substrate rarely have severe phenotypes (14, 15, 17, 

20, 21). Multiple mutations often need to be made in order for an appreciable phenotype to be seen. 

Furthermore, even in cases where the biochemical activity of a specific effector (e.g. a cysteine protease) is 

known, determining the host targets remains a daunting task. With our list of host proteins that are altered in 

stability only when the Dot/Icm system is functional (from Specific Aim 1), we are more strongly equipped to 

pursue individual effector/host target interactions through gain-of-function analyses that side-step the problem 

of redundancy. 

3(c) vi. Using a gain-of-function screen to identify L. pneumophila-translocated effectors that alter the 

stability of proteins. Since the host proteins we identified in Specific Aim 1 using the GPS platform are 

stabilized or destabilized only in the presence of Dot/Icm-translocated substrates, we will seek to identify 

specific L. pneumophila effectors that are responsible for these changes. To do this, we will first create stable 

cell lines transfected with the GPS retroviral construct (described in section 3(c).vi above) where EGFP is 

fused to specific host proteins whose alterations are essential for L. pneumophila infection to progress 

normally. We will then take these “GPS reporter” cell lines and transfect them individually with the genes 

encoding the 150 known and putative Dot/Icm-translocated effectors also known as the L. pneumophila 

Research Strategy Page 25

Principal Investigator/Program Director (Last, first, middle): Starnbach, Michael, N.
“secretome”. Craig Roy at Yale University has agreed to provide us with their existing set of the 150 effectors. 

Once we have the transfected cells, we can use flow cytometry as a high-throughput read-out to determine 

whether the expression of individual Dot/Icm-translocated effectors recapitulates the shift in the EGFP:DsRed 

ratio observed when the cells were infected with wild-type L. pneumophila compared to the dotA mutant. In 

these experiments, we will avoid possible cytotoxic effects of overexpressing effectors in host cells by using 

Gateway cloning to shuttle these genes into the destination vector pT-Rex-DEST31 that allows high-level 

tetracycline-regulated expression of each gene of interest in mammalian cells expressing the Tet repressor. 

This will allow for temporal regulation of each effector protein and allow us to examine the impact of the various 

effectors at various time points after expression. 

Candidate bacterial effectors that are able to alter stabilization of a specific GPS construct will then be 

further characterized. Taking advantage of the well-established genetic tools in L. pneumophila, we will make 

clean in-frame deletions of these genes. These mutant strains will then be assayed for their ability to alter the 

specific host protein identified above. If more than one candidate effector is identified for a single host protein, 

we will make a L. pneumophila strain in which all candidate effectors are deleted. Given the functional 

redundancy among effectors, we may not be able to identify all the effectors involved in altering the stability of 

a certain host protein. Nevertheless, we believe that this approach will allow us to identify novel bacterial 

effector/host protein target interactions by focusing on host protein changes that are dependent on Dot/Icm 

secreted effectors. 

3(c) vii. Determining the role of L. pneumophila Dot/Icm-translocated substrates that possess E3 

ubiquitin ligase domains. As discussed above, redundancy among L. pneumophila effectors has made it 

difficult to determine the specific host function targeted by any particular effector. Effectors have been grouped 

into “families” whose members share particular functions or functional motifs. Among these families implicated 

in modulation of host protein stability are the 6 effector proteins carrying domains that are normally associated 

with E3 ubiquitin ligase activity (3, 11). LegU2/LubX (Legionella U-box protein) (2, 11) functions to ubiquitinate 

the host cell cycle protein Clk1, while Lpp2082, LegU1, LegAU13, LicA and Lpg2160 are associated with 

multiple components of the host ubiquitination machinery but further functional description has been elusive 

(3). We will create a L. pneumophila strain bearing clean deletions in all six E3 ligase domain effectors. As 

time allows, we will conduct a new GPS screen comparing this strain to wild-type - allowing us to assess how 

deletion of a single family of effectors (all E3 ligase domain containing proteins) in an otherwise wild-type L. 

pneumophila alters the stability of the host proteome. Since the GPS platform was developed to identify 

substrates of mammalian E3 ubiquitin ligases we are confident that this screen will allow us to identify the 

targets of the E3 ligase domain effectors, and serve as an additional proof-of-principle for the use of GPS. This 

approach not only will help us dissect how all E3 ligase domain containing effectors in L. pneumophila co-opt 

the eukaryotic ubiquitination machinery to target other host proteins for degradation, but it is another approach 

that allows us to circumvent functional redundancy with all other “families” of effectors that have similar 

activities or protein domains. 

3(c) viii. Summary. The two aims described here use the highly innovative Global Protein Stability 

screening platform to identify host proteins that are altered in stability due to the secretion of effectors from the 

L. pneumophila type IV secretion system. Through a directed ORFeome screen as well as a specific gain-offunction study, we will for the first time have an understanding of how secreted bacterial effectors have a global 

impact on the stability of the human proteome. These approaches open new avenues to identify the specific 

effectors that are responsible for host protein changes at a level not previously possible. We believe that the 

ability to catalog how pathogens manipulate host cell proteins at a global scale is likely to bring about a shift in 

the way host-pathogen interactions are studied in the future. 

Research Strategy Page 26

Principal Investigator/Program Director (Last, first, middle): Starnbach, Michael, N.
11. Select Agent Research

No select agents will be used in this project. The BL-2 work proposed in this application 

have been approved by Harvard’s Committee on Microbiological Safety. All safety 

procedures for use with BL-2 level pathogens will be followed. 

Select Agent Research Page 27

Principal Investigator/Program Director (Last, first, middle): Starnbach, Michael, N.
Literature Cited

1. Berger, K. H., and R. R. Isberg. 1993. Two distinct defects in intracellular growth complemented by a 

single genetic locus in Legionella pneumophila. Mol Microbiol 7:7-19 

2. de Felipe, K. S., S. Pampou, O. S. Jovanovic, C. D. Pericone, S. F. Ye, S. Kalachikov, and H. A. 

Shuman. 2005. Evidence for acquisition of Legionella type IV secretion substrates via interdomain 

horizontal gene transfer. Journal of bacteriology 187:7716-7726 

3. Ensminger, A. W., and R. R. Isberg. E3 ubiquitin ligase activity and targeting of BAT3 by multiple 

Legionella pneumophila translocated substrates. Infect Immun 78:3905-3919 PMC2937443

4. Ensminger, A. W., and R. R. Isberg. 2009. Legionella pneumophila Dot/Icm translocated substrates: a

sum of parts. Current opinion in microbiology 12:67-73 PMC2741304

5. Fields, B. S. 1996. The molecular ecology of legionellae. Trends Microbiol 4:286-290 

6. Fraser, D. W., T. R. Tsai, W. Orenstein, W. E. Parkin, H. J. Beecham, R. G. Sharrar, J. Harris, G. F. 

Mallison, S. M. Martin, J. E. McDade, C. C. Shepard, and P. S. Brachman. 1977. Legionnaires' 

disease: description of an epidemic of pneumonia. N Engl J Med 297:1189-1197 

7. Gawronski, J. D., S. M. Wong, G. Giannoukos, D. V. Ward, and B. J. Akerley. 2009. Tracking 

insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus 

genes required in the lung. Proc Natl Acad Sci U S A 106:16422-16427 PMC2752563

8. Horwitz, M. A. 1983. Formation of a novel phagosome by the Legionnaires' disease bacterium 

(Legionella pneumophila) in human monocytes. J Exp Med 158:1319-1331 

9. Horwitz, M. A., and S. C. Silverstein. 1981. Interaction of the legionnaires' disease bacterium 

(Legionella pneumophila) with human phagocytes. II. Antibody promotes binding of L. pneumophila to 

monocytes but does not inhibit intracellular multiplication. J Exp Med 153:398-406 

10. Isberg, R. R., T. J. O'Connor, and M. Heidtman. 2009. The Legionella pneumophila replication 

vacuole: making a cosy niche inside host cells. Nat Rev Microbiol 7:13-24 PMC2631402

11. Kubori, T., A. Hyakutake, and H. Nagai. 2008. Legionella translocates an E3 ubiquitin ligase that has 

multiple U-boxes with distinct functions. Mol Microbiol 67:1307-1319 

12. Meresse, S., O. Steele-Mortimer, E. Moreno, M. Desjardins, B. Finlay, and J. P. Gorvel. 1999. 

Controlling the maturation of pathogen-containing vacuoles: a matter of life and death. Nat Cell Biol 

1:E183-188 

13. Mintz, C. S., J. X. Chen, and H. A. Shuman. 1988. Isolation and characterization of auxotrophic 

mutants of Legionella pneumophila that fail to multiply in human monocytes. Infect Immun 56:1449-

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14. Murata, T., A. Delprato, A. Ingmundson, D. K. Toomre, D. G. Lambright, and C. R. Roy. 2006. The 

Legionella pneumophila effector protein DrrA is a Rab1 guanine nucleotide-exchange factor. Nat Cell 

Biol 8:971-977 

15. Nagai, H., J. C. Kagan, X. Zhu, R. A. Kahn, and C. R. Roy. 2002. A bacterial guanine nucleotide 

exchange factor activates ARF on Legionella phagosomes. Science 295:679-682 

16. Ninio, S., and C. R. Roy. 2007. Effector proteins translocated by Legionella pneumophila: strength in 

numbers. Trends Microbiol 15:372-380 

17. Ninio, S., D. M. Zuckman-Cholon, E. D. Cambronne, and C. R. Roy. 2005. The Legionella IcmSIcmW protein complex is important for Dot/Icm-mediated protein translocation. Mol Microbiol 55:912-

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18. Pirbhai, M., F. Dong, Y. Zhong, K. Z. Pan, and G. Zhong. 2006. The secreted protease factor CPAF 

is responsible for degrading pro-apoptotic BH3-only proteins in Chlamydia trachomatis-infected cells. J 

Biol Chem 281:31495-31501 

19. Segal, G., M. Purcell, and H. A. Shuman. 1998. Host cell killing and bacterial conjugation require 

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20. Shen, X., S. Banga, Y. Liu, L. Xu, P. Gao, I. Shamovsky, E. Nudler, and Z. Q. Luo. 2009. Targeting 

eEF1A by a Legionella pneumophila effector leads to inhibition of protein synthesis and induction of 

host stress response. Cell Microbiol 11:911-926 

21. VanRheenen, S. M., Z. Q. Luo, T. O'Connor, and R. R. Isberg. 2006. Members of a Legionella 

pneumophila family of proteins with ExoU (phospholipase A) active sites are translocated to target 

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22. Vogel, J. P., H. L. Andrews, S. K. Wong, and R. R. Isberg. 1998. Conjugative transfer by the 

virulence system of Legionella pneumophila. Science 279:873-876 

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24. Yen, H. C., and S. J. Elledge. 2008. Identification of SCF ubiquitin ligase substrates by global protein 

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Principal Investigator/Program Director (Last, first, middle): Starnbach, Michael, N.
Harvard Medical School, Department of Genetics

77 Avenue Louis Pasteur, NRB Room #158D, Boston Massachusetts 02115

(617) 525-4510 • Fax (617) 525-4500

selledge@genetics.med.harvard.edu

 

Michael Starnbach, Ph.D.

Department of Microbiology and Molecular Genetics

Harvard Medical School

Oct.12, 2010

Dear Michael:

I’m writing to indicate my support for your application for funding to NIH. I am pleased that we are

working together to conduct Global Protein Stability (GPS) screens to identify proteins whose stability

is altered during infection with Legionella pneumophila. Screening cells infected with microbial

pathogens is an application where I feel GPS could make a genuine contribution to our understanding

of disease. The members of my lab and I are happy to continue providing advice and access to the

libraries for these studies.

Sincerely,

Stephen J. Elledge, Ph.D. 

Letters of Support Page 30

Principal Investigator/Program Director (Last, first, middle): Starnbach, Michael, N.
15. Resource Sharing Plan(s)

Research resources generated with funds from this grant will include DNA constructs 

and transfected cell lines. These resources, as available, will be freely distributed upon 

request to qualified academic investigators for non-commercial research. My institution 

and I will adhere to the NIH Grants Policy on Sharing of Unique Research Resources 

including the “Sharing of Biomedical Research Resources: Principles and Guidelines for 

Recipients of NIH Grants and Contracts” issued in December, 1999. 

http://ott.od.nih.gov/NewPages/Rtguide_final.html. Specifically, material transfers would 

be made with no more restrictive terms than in the Simple Letter Agreement or the 

UBMTA and without reach through requirements. Should any intellectual property arise 

which requires a patent, we would ensure that the technology remains widely available 

to the research community in accordance with the NIH Principles and Guidelines 

document. 

Resource Sharing Plan Page 31

Principal Investigator/Program Director (Last, first, middle): Starnbach, Michael, N.
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