PI: Muir, Tom Title: Peptide Autoinducers of Staphylococcal Pathogenicity 

Received: 07/05/2016 FOA: PA16-160 Council: 01/2017 

Competition ID: FORMS-D FOA Title: NIH Research Project Grant (Parent R01)

2 R01 AI042783-16A1 Dual: Accession Number: 3954056 

IPF: 6661401 Organization: PRINCETON UNIVERSITY 

Former Number: Department: 

IRG/SRG: SBCB AIDS: N Expedited: N 

Subtotal Direct Costs 

(excludes consortium F&A) 

Year 16: 

Year 17: 

Year 18: 

Year 19: 

Year 20: 

Animals: N 

Humans: N 

Clinical Trial: N 

Current HS Code: 10 

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New Investigator: N 

Early Stage Investigator: N 

Senior/Key Personnel: Organization: Role Category: 

Tom Muir PRINCETON UNIVERSITY PD/PI 

Richard Novick New York University School of Medicine Co-Investigator 

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Funding Opportunity Number: PA-16-160 . Received Date: Tracking Number: GRANT12207716 2016-07-05T12:16:58.000-04:00 

OMB Number: 4040-0001 

Expiration Date: 06/30/2016 

APPLICATION FOR FEDERAL ASSISTANCE 

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Peptide Autoinducers of Staphylococcal Pathogenicity 

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NJ-012 
Funding Opportunity Number: PA-16-160 . Received Date: Tracking Number: GRANT12207716 2016-07-05T12:16:58.000-04:00 

 

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SF 424 (R&R) APPLICATION FOR FEDERAL ASSISTANCE Page 2 

14. PROJECT DIRECTOR/PRINCIPAL INVESTIGATOR CONTACT INFORMATION 

Prefix: First Name*: Tom Middle Name: Last Name*: Muir Suffix: 

Position/Title: Professor 

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Tracking Number: GRANT12207716 Funding Opportunity Number: PA-16-160. Received Date:

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Contact PD/PI: Muir, Tom 

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Funding Opportunity Number: PA-16-160. Received Date:

2016-07-05T12:16:58.000-04:00 Tracking Number: GRANT12207716 

RESEARCH & RELATED Other ProjectInformation 

1. Are Human Subjects Involved?* ❍ Yes ● No 

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Filename 

7. Project Summary/Abstract* abstract-resubmission.pdf 

8. Project Narrative* Narrative-resubmission.pdf 

9. Bibliography & References Cited bibliography_Staph_resubmission.pdMf ime

10.Facilities & Other Resources Facilities-resubmission.pdf 

11.Equipment Equipment_Muir_resubmission.pdf 
Project Summary/Abstract Page 6 

 

 

 

 

 

 

 

 

 

Contact PD/PI: Muir, Tom 

A research program will be undertaken to study agr signal transduction in the commensal pathogen, 

Staphylococcus aureus. The accessory gene regulator (agr) locus found in all staphylococci encodes a quorum 

sensing (QS) circuit that controls the expression of virulence and other accessory genes. It consists of two 

oppositely oriented transcriptional units, of which one encodes four proteins, AgrBDCA, involved in production 

and sensing of an autoinducer peptide (AIP), and the other encodes a regulatory RNA that is the effector of 

target gene regulation. The finding that staphylococcal virulence can be inhibited through antagonism of this 

QS pathway has fueled tremendous interest in understanding the detailed mechanisms at play throughout the 

circuit. Building on recent breakthroughs that have allowed us to reconstitute much as the quorum sensing 

circuit using purified components, we propose to integrate chemical, biochemical, biophysical and genetic tools 

for the purpose of obtaining a deeper understanding into the molecular processes underlying the production 

and sensing of the autoinducer peptide (AIP) pheromone that is central to agr regulation. The program will 

move forward in three directions: Aim 1, identifying the key missing players in AIP biosynthesis; Aim 2, 

understanding how agonism and antagonism of the QS system relates to newly discovered conformational 

changes in the AIP receptor, AgrC, and; Aim 3, identifying novel modulators of agr through sophisticated 

target-based screens. These studies will lay the groundwork for the development of therapeutic strategies 

targeting agr, but also contribute to a fundamental understanding of QS systems of this type, which are 

pervasive in the low-GC bacterial phylum, Firmicutes. 
 

 

 

 

Contact PD/PI: Muir, Tom 

Staphylococcus aureus (S. aureus) is an opportunistic pathogen capable of invading mucous membranes or 

soft tissue; once invasion occurs, the bacterium deploys a diverse arsenal of virulence factors to evade the 

host immune system and to facilitate spread of the infection in the host environment. A research program will 

be undertaken to study the central quorum sensing (QS) circuit, termed agr, which regulates the onset of 

virulence as a function of bacterial population size. Building on recent breakthroughs that have allowed us to 

reconstitute much of the circuit using purified components, we propose to integrate chemical, biochemical, 

biophysical and genetic tools to gain a deeper understanding into the molecular processes underlying agr 

regulation; these studies will provide fundamental insights into how a QS circuit such as agr operates at the 

molecular level and will lay the foundation for the development of new strategies for treating Staphylococcus 

aureus infections. 

Project Narrative Page 7 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Contact PD/PI: Muir, Tom 

Laboratory: 

The Department of Chemistry at Princeton University has recently moved into the Frick Laboratory, an entirely 

new Chemical Sciences facility, totaling 265,000 square feet. The building is located in the heart of the 

sciences neighborhood that connects disciplines such as genomics, neuroscience, physics, chemical and 

biological engineering, mathematics, and molecular biology. Three entire floors of the building are dedicated 

to new research space. Professor Muir and his lab group occupy laboratory, office and group conference 

space primarily on the third floor (with addition space for spectroscopy instrumentation in the basement of the 

building). The synthetic peptide and protein chemistries described in this proposal will be performed within 

his state-of-the-art laboratory, specifically designed for modern synthetic protein chemistry and extremely well 

equipped for the chemical synthesis or protein expression, purification and characterization of peptides and 

proteins and for small molecule synthetic organic chemistry (116 feet of hood space). The laboratory also has 

dedicated cold-room, tissue culture and dark rooms for biochemistry and cell biology studies. 

Clinical: N/A 

Animal: N/A 

Computer: 

Professor Muir and his lab group have multiple high-end computers for purposes of communications, data 

processing, access to the internet, calculations, and monitoring laboratory equipment. The University Office 

of Information Technology maintains the TIGRESS High Performance Computing Center to provide resources 

meeting the broad computational requirements of the University research community. Available software 

includes statistical and database management packages, as well as programs for computer analysis of 

nucleic acid and protein sequences. 

Offices: 

The new Chemical Sciences building has three office modules on each floor. Each faculty member is allotted 

one office and one adjacent meeting room. Each module also has a shared conference room. 

Other: 

Frick Laboratory is in close proximity to the structures housing the Department of Molecular Biology and the 

University's Lewis-Sigler Institute of Integrative Genomics promoting collaborations between researchers who 

have had many overlapping research interests and scientific interactions. Core facilities include; the genomics 

center, equipped with state-of-the art next generation DNA sequencing instruments including the Ion Torrent 

Sequencer needed for some of the proposed studies; a proteomics mass spectrometry center harboring a 

range of systems tuned to proteomics applications (additional Orbitrap instruments); a macromolecular 

crystallography facility which possesses state-of-the-art robotic screening infrastructure and X-ray sources; 

and the biological imaging center equipped with a number of state-of-the-art fluorescence microscopes for 

cell biological and organismal biology applications. Lewis Library is also in close proximity and houses the 

science and technology collections of the university. Princeton University supports a glass blower, mechanical 

workshop, on-site departmental computing support personnel, and an efficient administrative infrastructure 

which provides a wide variety of services including electronic online purchasing and facility maintenance. 

Facilities & Other Resources Page 8 
 

 

 

 

 

 

 

Contact PD/PI: Muir, Tom 

Major Equipment: 

The relocation of the Muir lab from Rockefeller to Princeton University provided an excellent opportunity to 

acquire new major equipment that will greatly facilitate our research. The new MicroTOF-Q II mass 

spectrometer (Bruker) will be an essential tool for the analysis of peptides/proteins synthesized and semisynthesized in our lab. This mass spectrometer has high sensitivity and accuracy for identification of proteins 

and post-translational modifications. This instrument also allows for ms/ms analysis of peptides. Another new 

piece of equipment is the Liberty peptide synthesizer (CEM Corporation). This instrument is extensively used 

to synthesize peptides up to 40 amino acid in length for various applications in our chemical biology research. 

The Liberty synthesizer is particularly useful for the preparation of modified histone peptides that are the key 

players of this grant proposal. Analysis of the protein/peptide samples is facilitated by four analytical HPLC 

systems (Agilent) all with autosamplers and with an option of fluorescence signal detection with one of the 

instruments. Purification of peptides is facilitated by two dedicated preparative-scale HPLC systems (Waters) 

with peak-picking auto-purification capability. In addition to two standard AKTA FPLC systems (GE Healthcare) 

for protein purification, we have recently acquired a third system with a multiple angle light scattering detector 

(Wyatt Technology) that will greatly enhance our ability to characterize large protein complexes such as 

nucleosomes. Tissue culture work will be carried out in two biosafety cabinets (Baker) and mammalian and 

insect cells will be maintained in three CO2 incubators. A Zeiss microscope has also been purchased, with 

fluorescence capabilities, to monitor cells during tissue culture. The Muir lab has a designated room for work 

with radioactive substances, equipped with a MicroBeta2 automated scintillation counter (Perkin Elmer). Other 

standard major lab equipment includes two Avanti J-26XP floor centrifuges and one Optima L-80 XP 

ultracentrifuge (Beckman), several imaging systems (Odyssey (Licor), ImageQuant (GE Healthcare)), two 

freeze dryers (Labconco, Millrock), incubators/shakers (ATR), fermentor (New Brunswick Scientific), a UV-Vis 

spectrophotometer (Agilent) and an Emulsiflex-C3 cell homogenizer (Avestin). We have also acquired a plate 

reader (Molecular Devices) to monitor absorbance, luminescence and/or fluorescence of multi-well plates, 

which will be crucial for several assays currently being developed in the lab. All of the above-mentioned 

rd instruments are located on the 3 floor of the Frick Chemistry Laboratory building. Muir lab initiated the 

development of the Protein Chemistry Center as a shared research facility on the lower level of the chemistry 

department. As core equipment for the Center we have acquired a BioCore 4000 system, Chirascan CD 

spectrometer, SX20 stopped-flow Reaction Analyser (Applied Photophysics) and Fluorolog3-11 fluorimeter 

(Horiba), which will be used at crucial stages of our projects to gain further insight into biophysical properties of 

our protein/peptide samples. We have also recently installed within the Protein Chemistry Center a proteomics 

workstation containing a variety of analysis software (SCAFFOLD, Proteome Discover, MASCOT) that will be 

used to analyze, in house, the various proteomics datasets acquired in the course of this research. 

The Princeton University Screening Center (PUSC) is fully equipped to carry out all the designed experiments 

in the proposal. As for the compound collection, the Screening Center currently has 75,000 singleton 

compounds as 10 mM DMSO solutions. This collection is assembled to have maximum diversity with drug-like 

properties while being vastly differentiated from those compound collections at typical academic screening 

centers, such as the NIH MLPCN center. PUSC also has access to the 1.4 million Chiromics Maximum 

Coverage compound collection, which is custom built to be used in line with the ASMS screening modality. In 

support of all aspects of biochemical and/or cell-based functional assays, PUSC possesses fully upgraded 

Perkin-Elmer EnVision and Biotek Cytation 3 multi-mode plate readers. All small molecule dispensing is 

performed using the non-contact acoustic dispenser Labcyte ECHO 550 system which is capable of handling 

volumes as low as 2.5 nL with great accuracy. For bulk liquid and reagent handling in the preparation of the 

assay plates, PUSC uses the Agilent Bravo liquid handling system. 

For affinity selection mass spectrometry (ASMS), PUSC has a custom designed ASMS system, which consists 

of the Dionex 2-dimensional SEC-reverse phase nano flow liquid chromatography system in line with the 

Thermo QExactive orbitrap mass spectrometer capable of reaching resolution of 140000. This instrument is 

capable of screening the Maximum coverage collection of 1.4 million compounds in a one week time frame. 

The data produced from these experiments will be analyzed using the CHALIS software also accessible 

through the PUSC. All data generated from the screens at the PUSC are further analyzed by using the 

Chemaxon InstantJChem cheminformatics platform in combination with Perkin-Elmer Spotfire for data 

visualization. 

Equipment Page 9 
 

Contact PD/PI: Muir, Tom 

Other shared research facilities available for our research groups are also located on the lower level. The 

departmental NMR facility currently has seven spectrometers on site, 300-800 MHz, including three new 

Bruker Avance cryoprobe units assembled into a cluster. One of these units is 1H-optimized, one is 13C 

optimized and the third, a Cryo-QNP, has 1H, 13C, 31P and 15N capabilities. All three units have a fully robotic 

120-sample carousel allowing them to run unattended 24 hours a day, seven days a week. Importantly, a new 

Bruker 800 MHz instrument with cryoprobe was installed January 2013. This instrument will be essential to 

some of the proposed NMR studies on PHP. The mass spectrometer facility has several instruments including 

new Agilent TOF, Q-TOF, GC-MS and HPLC triple quad spectrometers and a Thermo Orbitrap instrument. 

The TOF unit is configured for walk-up high- resolution molecular mass determinations. The Q-TOF has an 

integrated chip-based nanoflow HPLC for proteomics. The triple quad is optimized for quantitativemetabolomic 

experiments, while the Orbitrap is configured for proteomic and metabolomics applications. The Chemistry 

Department retains a staff of five professionals to maintain and repair the shared instrumentation. These staff 

members collaborate with research groups on the optimal application of the equipment to a particular project, 

and instruct individual researchers on the use of the instruments. 

Equipment Page 10 
f

OMB Number: 4040-0001 

Expiration Date: 06/30/2016 

Contact PD/PI: Muir, Tom 

RESEARCH & RELATED Senior/Key Person Profile(Expanded) 

PROFILE - Project Director/Principal Investigator 

Prefix: First Name*: Tom Middle Name Last Name*: Muir Suffix: 

Position/Title*: Professor 

Organization Name*: PRINCETON UNIVERSITY 

Department: 

Division: 

Street1*: 

Street2: 

City*: 

County: 

State*: 

Province: 

Country*: 

Zip / Postal Code*: 

Phone Fax Number: E-Mail*: 

Number*: 

Credential, e.g., agency login: 

Project Role*: PD/PI Other Project Role Category: 

Degree Type: Degree Year: 

File Name 

Attach Biographical Sketch*: Muir_NIH_new_biosketch_June_20ap1p6li.cpadtiof n/pd

Attach Current & Pending Support: 

Page 11 

Funding Opportunity Number: PA-16-160 . Received Date: Tracking Number: GRANT12207716 2016-07-05T12:16:58.000-04:00 
Contact PD/PI: Muir, Tom 

PROFILE - Senior/Key Person 

Prefix: First Name*: Richard Middle Name P. Last Name*: Novick Suffix: 

Position/Title*: Professor of Microbiology and Medicine 

Organization Name*: New York University School of Medicine 

Department: 

Division: 

Street1*: 

Street2: 

City*: 

County: 

State*: 

Province: 

Country*: 

Zip / Postal Code*: 

Phone Number*: Fax Number: E-Mail*: 

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Project Role*: Co-Investigator Other Project Role Category: 

Degree Type: MD,BA Degree Year: 

File Name 

Attach Biographical Sketch*: Novick.biosketch.6-14-16.pdf 

Attach Current & Pending Support: 

Page 12 

Funding Opportunity Number: PA-16-160 . Received Date: Tracking Number: GRANT12207716 2016-07-05T12:16:58.000-04:00 
Biosketches Page 13 

Contact PD/PI: Muir, Tom 

BIOGRAPHICAL SKETCH 

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

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

NAME 

Muir, Thomas W. 

POSITION TITLE 

Professor of Chemistry 

eRA COMMONS USER NAME (credential, e.g., agency login) 

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

residency training if applicable.) 

INSTITUTION AND LOCATION DEGREE 

(if applicable) MM/YY FIELD OF STUDY 

University of Edinburgh, Edinburgh, UK B.Sc. 

(1 st Honors) 1985-1989 Chemistry 

University of Edinburgh, Edinburgh, UK Ph.D. 1989-1993 Organic Chemistry 

The Scripps Research Institute, San Diego, CA Postdoc 1993-1995 Bio-organic Chemistry 

A. Personal Statement 

In the Muir Lab, we have developed general protein engineering approaches that allow recombinant 

polypeptides and synthetic polypeptides (or other artificial molecules) to be ligated together through a normal 

peptide bond. This technology, which can be applied both in vitro and in vivo, opens up the world of proteins to 

the tools of organic chemistry by allowing the insertion of unnatural amino acids, posttranslational modifications 

and isotopic probes site-specifically anywhere into proteins. Our methods are now used by numerous 

laboratories worldwide, and have allowed a large number of questions to be addressed. 

My lab provides a unique and exemplary training experience for students to pursue their research. We offer 

superb research facilities and equipment for the production of proteins and chromatin via protein 

semisynthesis. Other projects include exploration of the enzymology and mechanisms of inteins using 

semisynthetic and NMR approaches, understanding the molecular mechanisms including molecular 

recognition processes underlying the Agr quorum sensing circuit controlling virulence in Staphylococci, 

investigation of the role of histidine phosphorylation in eukaryotic cells, and the use of genetic and chemical 

biology methods to study and modify, for the purposes of protein engineering, intein protein splicing elements. 

Our lab offers an extremely collaborative spirit both within as well as with other research groups. 

I have a strong history of mentoring young scientists and training graduate students and postdocs. The Muir 

lab has trained a large number of predoctoral and postdoctoral candidates, and all have gone on to successful 

independent careers. In total, there have been over 57 pre- and-postdoctoral candidates that have come 

through the Muir lab with many having assumed independent academic positions. To date, I have trained 26 

PhD students (of whom 12 are women) and 31 postdoctoral fellows (of whom 11 are women). These 

individuals have obtained positions in industry and at leading academic institutions around the world (including 

UC Berkeley, UCLA, UCSF, University of Washington, EPFL - École Polytechnique Fédérale de Lausanne, 

Scripps Institute, Penn State, Texas A&M, University of Naples, Columbia Medical School, University of 

Queensland, and many more). 

B. Positions and Honors 

Positions and Employment

1993-1995 Postdoctoral Associate, The Scripps Research Institute, San Diego, CA 

1995-1996 Senior Research Associate, The Scripps Research Institute, San Diego, CA 

1996-2000 Assistant Professor and Head of Laboratory, The Rockefeller University, New York, NY 

2000-2002 Associate Professor and Head of the Selma and Lawrence Ruben Laboratory, The Rockefeller 

University, New York, NY 
Contact PD/PI: Muir, Tom 

Biosketches Page 14 

2005-2011 Richard E. Salomon Family Professor and Head of the Selma and Lawrence RubenLaboratory, 

The Rockefeller University, New York, NY 

2005-2011 Director, Pels Family Center for Chemistry, Biochemistry and Structural Biology, The 

Rockefeller University, New York, NY 

2011-present Van Zandt Williams Jr. Class of ’65 Professor of Chemistry, Princeton University, Princeton, NJ 

2015-present Chair of Department of Chemistry, Princeton University, Princeton, NJ 

Honors and Awards 

Pew Scholar in the Biomedical Sciences (The Rockefeller University) 

Alfred P. Sloan Research Fellow (The Rockefeller University) 

Burroughs-Wellcome Fund, New Investigator in the Toxicological Sciences (The Rockefeller University) 

Irma T. Hirsch/Monique Weill-Caulier Trust Research Fellow (The Rockefeller University) 

Leonidas Zervas Award from The European Peptide Society (The Rockefeller University) 

Richard E. Salomon Family Professor (The Rockefeller University) 

Kavli Fellow (U.S. National Academy of Sciences) 

Fellow of the American Association for the Advancement of Science (AAAS) 

Vicent du Vigneaud Award in Peptide Chemistry (American Peptide Society) 

Irving Sigal Young Investigator Award (The Protein Society) 

Distinguished Teaching Award (The Rockefeller University) 

Winner of the New York Academy of Sciences Blavatnik Award for Young Scientists and Engineers (NYAS) 

Jeremy Knowles Award (Royal Society of Chemistry) 

Arthur C. Cope Scholar Award (American Chemical Society) 

MERIT Award (US National Institutes of Health) 

Fellow of The Royal Society of Edinburgh 

Breslow Award in Biomimetic Chemistry (American Chemical Society) 

C. Contributions to Science (from 150 peer-reviewed publications) 

Specific highlights from our own work include: 

Structure and Function of Inteins: Protein splicing is a remarkable posttranslational process in which an 

intervening sequence, termed an intein, becomes excised from a host protein, the extein, in an autocatalytic 

manner. In protein trans-splicing the intein is split into two pieces and splicing only occurs upon reconstitution 

of these fragments. We have for many years studied the molecular details of protein splicing that occurs in cis 

and in trans. Indeed, through our efforts, and those of others, we now have a much clearer picture of the 

nature of catalysis for all the steps in the canonical protein splicing mechanism. In addition, new technologies 

have emerged from these basic mechanistic studies and these have been used to answer a number of biology 

questions. 

• Muir TW, Sondhi D, Cole PA. Expressed protein ligation: a general method for protein engineering. 

Proc Natl Acad Sci U S A. 1998 Jun 9;95(12):6705-10. PubMed Central PMCID: PMC22605. 

• Xu R, Ayers B, Cowburn D, Muir TW. Chemical ligation of folded recombinant proteins: segmental 

isotopic labeling of domains for NMR studies. Proc Natl Acad Sci U S A. 1999 Jan 19;96(2):388-93. 

PubMed Central PMCID: PMC15146. 

• Shah NH, Dann GP, Vila-Perelló M, Liu Z, Muir TW. Ultrafast protein splicing is common among 

cyanobacterial split inteins: implications for protein engineering. J Am Chem Soc. 2012 Jul 

18;134(28):11338-41. PubMed Central PMCID: PMC3535263. 

• Liu Z, Frutos S, Bick MJ, Vila-Perelló M, Debelouchina GT, Darst SA, Muir TW. Structure of the 

branched intermediate in protein splicing. Proc Natl Acad Sci U S A. 2014 Jun 10;111(23):8422-7. 

PubMed Central PMCID: PMC4060664. 

The regulation of Chromatin Structure and Function: We have developed a suite of chemistry-driven 

methods to study how post-translational modifications of the core histone proteins in chromatin regulate the 
 

Contact PD/PI: Muir, Tom 

structure and function of the chromatin fiber. This has led to new insights into the flow and storage of 

epigenetic information in mammalian cells, information that has improved our understanding of the molecular 

basis of fundamental DNA-templated processes such as transcription and that suggests new routes for the 

treatment of human diseases, many of which have an epigenetic origin. Relevant papers are listed below. 

• McGinty RK, Kim J, Chatterjee C, Roeder RG, Muir TW. Chemically ubiquitylated histone H2B 

stimulates hDot1L-mediated intranucleosomal methylation. Nature. 2008 Jun 5;453(7196):812-6. 

PubMed Central PMCID: PMC3774535. 

• Fierz B, Chatterjee C, McGinty RK, Bar-Dagan M, Raleigh DP, Muir TW. Histone H2B ubiquitylation 

disrupts local and higher-order chromatin compaction. Nat Chem Biol. 2011 Feb;7(2):113-9. PubMed 

Central PMCID: PMC3078768. 

• Lewis PW, Müller MM, Koletsky MS, Cordero F, Lin S, Banaszynski LA, Garcia BA, Muir TW, Becher 

OJ, Allis CD. Inhibition of PRC2 activity by a gain-of-function H3 mutation found in pediatric 

glioblastoma. Science. 2013 May 17;340(6134):857-61. PubMed Central PMCID: PMC3951439. 

• Nguyen UT, Bittova L, Müller MM, Fierz B, David Y, Houck-Loomis B, Feng V, Dann GP, Muir TW. 

Accelerated chromatin biochemistry using DNA-barcoded nucleosome libraries. Nat Methods. 2014 

Aug;11(8):834-40. PubMed Central PMCID: PMC4130351. 

Protein Chemistry in Living Cells and Animals: We have for several years explored the possibility of 

performing protein chemistry inside living systems – in principle this would allow for protein structure and 

function to be controlled and manipulated in ways inaccessible to standard genetics. A number of technologies 

have emerged from this initiative – many of which have relied on insights emerging from our long-standing 

mechanistic studies of inteins, remarkable proteins which mediate protein splicing (a naturally occurring protein 

editing reaction). These include a variety of small molecule and optically controlled protein ligation reactions, 

which permit the spatial-temporal control of protein function in cells and living animals. Key papers are listed 

below. 

• Mootz HD, Muir TW. Protein splicing triggered by a small molecule. J Am Chem Soc. 2002 Aug 

7;124(31):9044-5. PubMed PMID: 12148996. 

• Giriat I, Muir TW. Protein semi-synthesis in living cells. J Am Chem Soc. 2003 Jun 18;125(24):7180-1. 

PubMed PMID: 12797783. 

• Schwartz EC, Saez L, Young MW, Muir TW. Post-translational enzyme activation in an animal via 

optimized conditional protein splicing. Nat Chem Biol. 2007 Jan;3(1):50-4. Epub 2006 Nov 26. PubMed 

PMID: 17128262. 

• David Y, Vila-Perelló M, Verma S, Muir TW. Chemical tagging and customizing of cellular chromatin 

states using ultrafast trans-splicing inteins. Nat Chem. 2015 May;7(5):394-402. PubMed PMCID: 

PMC4617616. 

Virulence Regulation in Staphyloccus aureus: In a separate area of work, we have worked for many years 

to understand the molecular details of virulence control in pathogenic Staphylococci. We have defined the 

molecular structure of a family of secreted peptides from S. aureus that control virulence in the organism 

through a conserved quorum sensing signaling pathway termed agr. Agr remains the best-characterized 

quorum sensing pathway in any Gram-positive organism and, given its biomedical importance, is now widely 

studied. Using a combination of chemistry, protein engineering and molecular genetics, we have figured out 

many aspects of the molecular mechanism of this critical process. This understanding has led to the rational 

design of global inhibitors of virulence in S. aureus that prevent infections in animal models and that thus have 

therapeutic potential. Key contributions are listed below. 

• Mayville P, Ji G, Beavis R, Yang H, Goger M, Novick RP, Muir TW. Structure-activity analysis of 

synthetic autoinducing thiolactone peptides from Staphylococcus aureus responsible for virulence. Proc 

Natl Acad Sci U S A. 1999 Feb 16;96(4):1218-23. PubMed Central PMCID: PMC15443. 

• Kee JM, Oslund RC, Perlman DH, Muir TW. A pan-specific antibody for direct detection of protein 

histidine phosphorylation. Nat Chem Biol. 2013 Jul;9(7):416-21. PubMed Central PMCID: 

PMC3686892. 

Biosketches Page 15 
 

Contact PD/PI: Muir, Tom 

• Wang B, Zhao A, Novick RP, Muir TW. Activation and inhibition of the receptor histidine kinase AgrC 

occurs through opposite helical transduction motions. Mol Cell. 2014 Mar 20;53(6):929-40. PubMed 

Central PMCID: PMC4004102. 

Published work in my bibliography: 

http://www.ncbi.nlm.nih.gov/sites/myncbi/tom.muir.1/bibliograpahy/40835818/public/?sort=date&direction=asce 

nding 

D. Research Support 

Ongoing Research Support 

NIH:NIGMS 5R37 GM086868 (PI:Muir) 8/1/03 – 7/31/17 

“Structure, Function and Applications of Inteins” 

The major goal of this project is to explore the enzymology and mechanisms of inteins using semisynthetic and 

NMR approaches. 

NIH:NIGMS 5 R01 GM107047 (PI:Muir) 9/01/13 – 4/30/17 

“Development and Applications of 'Designer Chromatin’“ 

The major goal of this project is to deepen our understanding of the molecular mechanisms underlying the 

regulation of chromatin structure and function. 

NIH:NCI P01 CA196539 (PI: Allis, Co-PI: Muir) 7/01/15 – 6/30/20 

“Oncohistones: Role of Histone H3 Mutations in the Oncogenesis of Pediatric Cancers” 

The broad goal of this project is to develop a suite of chemistry-driven tools to study the detailed mechanism 

by which histone H3 mutations, oncohistones, associated with pediatric brain and bone cancers mis-regulate 

epigenetic control of gene expression, leading to disease. 

Completed 

NIH:NIGMS 5R01 GM095880 (PI:Muir) 12/1/10-11/30/14 

“Chemistry and Biology of Protein Histidine Phosphorylation” 

The major goal of this project is to investigate the role of histidine phosphorylation in eukaryotic cells. 

NIH:NIAID 5R01 AI042783 (PI:Novick) 5/15/98 – 3/31/16 

“Peptide Autoinducers of Staphylococcal Pathogenicity” 

The major goal of this project is to understand the molecular mechanisms including molecular recognition 

processes underlying the Agr quorum sensing circuit controlling virulence in Staphylococci. 

Role: Co-PI 

Biosketches Page 16 
 

 

 

Contact PD/PI: Muir, Tom 

BIOGRAPHICAL SKETCH 

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

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

NAME: Richard P. Novick, M.D. 

eRA COMMONS USER NAME (credential, e.g., agency login): 

POSITION TITLE: Professor 

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, 

include postdoctoral training and residency training if applicable. Add/delete rows as necessary.) 

INSTITUTION AND LOCATION 

DEGREE 

(if 

applicable) 

Completion 

Date 

MM/YYYY 

FIELD OF STUDY 

Yale University, New Haven, CT B.A. 06/1954 Psychology 

New York University School of Medicine M.D. 06/1959 

A. Personal statement 

As noted below, I discovered and characterized the agr system and its autoinducing thiolactone peptide 

ligands and have spent much of the past 25 years engaged in studying it. I have published some 45 research 

papers and 8 reviews/chapters on the system. Included in my studies were mouse experiments that 

demonstrated attenuation of murine staphylococcal infections by inhibitory variants of the AIP that blocked agr 

activation. To evaluate these infection studies, I developed luciferase reporter vectors that enabled in vivo 

monitoring of the infection by IVIS imaging. My studies were greatly aided by an exceptionally productive 

collaboration with Prof Tom Muir, formerly of Rockefeller University, now at Princeton. These studies have 

been continuously supported by NIH for the past 25 years. Our joint studies have by now proceeded to the 

point where the biochemical-biophysical aspects of the work are best handled by Professor Muir, and 

constitute aims 1 & 2 of the present application, whereas my lab is best suited to evaluating the efficacy, both 

in vitro and in vivo of agr-inhibiting compounds, and constitute aim 3. 

B. Positions and honors 

Positions and Employment 

1961-1962 Postdoctoral Fellowship with Dr. M.R. Pollock, F.R.S. National Institute forMedical 

Research, London 

1962-1963 Assistant Residency, Dept. of Medicine, Vanderbilt University Hospital, Nashville, TN. 

(Professor of Medicne, Dr. David E. Rogers) 

1963-1965 Special Postdoctoral Fellowship with Professor R.D. Hotchkiss, The Rockefeller University, 

New York. 

1970-1975 Research Associate Professor, School of Medicine, New YorkUniversity. 

1976-Present Research Professor, Dept. of Microbiology, New York University, NY. 

1969-1975 Adjunct Professor, Dept. of Microbiology, New York University, NY. 

1981-1991 Director, The Public Health Research Insititute, New York, NY 

1975-1993 Member, Chief, Dept. of Plasmid Biology, PHRI. 

1993-Present Investigator, Skirball Institute, NYUMC. 

Professor of Microbiology and Medicine, NYU School of Medicine. 

2010-Present Recanati Family Professor of Science, NYU School of Medicine 

Honors 

Phi Beta Kappa, Magna Cum Laude, Alpha Omega, M.D. with Honors, Borden Award, 

Berson Alumni Achievement Award, Member, National Academy of Sciences, Master Researcher Award, 

NYUSOM, 2009, Recanati Family Professor of Science, 2010. 

Biosketches Page 17 
 

 

 

 

Contact PD/PI: Muir, Tom 

C. Contributions to science 

1. Discovery and characterization ofplasmids in staphylococci and their carriage of staphylococcal βlactamase and other resistances 

By the early 1960’s, β-lactamase-based penicillin resistance in Staphylococcus aureus had become a 

major clinical problem, addressed by the development of methicillin. In a study of methicillin resistance in βlactamase-producing S. aureus, I observed that β-lactamase production could be lost, which led to the 

demonstration that it was plasmid-coded. This, the first demonstration of plasmids in S. aureus, led to my 

long-lasting interest in and study of plasmid biology. This work has had a major impact on clinical medicine, 

agriculture, and microbiology, as well as contributing importantly to the understanding of bacterial 

molecular genetics, and has led to the construction by my lab of a plasmid cloning vector system for S. 

aureus, now in worldwide use among staphylococcal researchers. 

a. Novick, R.P. and Richmond, M.H. (l965). Nature and interactions of the genetic elements 

governing penicillinase synthesis in Staphylococcus aureus. J. Bacteriol. 90, 467-480. 

b. Novick, R.P. and Schwesinger, M. (1976). Independence of plasmid incompatibility and replication 

control functions in Staphylococcus aureus. Nature 262, 623-626. 

c. Novick, R.P. and Hoppensteadt, F.C. (1978). On plasmid incompatibility. Plasmid 1, 421-434. 

d. Gruss, A., Ross, H.P., and Novick, R.P. (1987). Functional analysis of a palindromic sequence 

required for normal replication of several staphylococcal plasmids. Proc. Natl. Acad. Sci. USA. 

84:2165-2169. 

2. Discovery of plasmid-determined heavy metal resistance 

Given the well-known carriage and dissemination of multiple resistance genes by E. coli plasmids, 

Christine Roth, a technician and I screened the newly discovered 25 kb “penicillinase” plasmids for other 

resistances and found not only MLS resistance, but also resistance to mercury, cadmium, lead, arsenate, 

arsenite, and bismuth salts, present in various combinations. These studies led to extensive biochemical 

studies by others, notably Simon Silver, who identified the resistance mechanisms. They also 

demonstrated that bacteria were capable of developing resistance to a wide variety of environmental 

inhibitors in addition to antibiotics. And they impacted environmental microbiology significantly, as plasmidcarried and chromosomal metal resistances were soon found among a variety of microorganisms, 

especially in areas polluted by industrial wastes. Several of these resistance genes were found to be 

inducible, and their promoters have thus been useful additions to our vector system. 

a. Novick, R.P. and Roth, C. (l968). Plasmid-linked resistance to inorganic salts in Staphylococcus 

aureus. J. Bacteriol. 95, l335-l342 

b. Smith, K. and Novick, R.P. (l972). Genetic studies on plasmid-linked cadmium resistance in S. 

aureus. J. Bacteriol. ll2, 76l-772. 

c. Novick, R.P., Murphy, E., Gryczan, T.J., Baron, E. and Edelman, I. (1979) Penicillinase plasmids of 

Staphylococcus aureus: Restriction-deletion maps. Plasmid 2, 109-129 

3. Discovery that plasmid replication control requires that the plasmid replication initiator protein be 

destroyed after a single use so it cannot be re-utilized 

Control of plasmid replication had been a topic of great interest ever since Jacob, Brenner and Cuzin had 

proposed that plasmids were attached to specific sites on the inner leaf of the cell membrane and were 

replicated passively as accessories to the cell’s genome. Pritchard suggested instead that plasmids were 

autonomous and that their replication was regulated negatively, a suggestion that was confirmed by 

Nordstrom’s isolation of plasmid mutants in E. coli with increased copy numbers. We found this also to be 

true for staphylococcal plasmids and found that in S. aureus, plasmid replication was controlled indirectly 

by regulation of the rate of synthesis of the plasmid replication initiator protein. However, if this protein were 

to accumulate in active form, it would obviate the control mechanism and so I proposed that the protein 

could be used only once and had to be inactivated thereafter. Avi Rasooly, a post-doc in the lab, confirmed 

this by demonstrating that the dimeric protein was inactivated at the end of the replication cycle by the 

attachment of a short oligonucleotide to the active site tyrosine of one of the protomers. This was a key 

result in plasmid biology as it confirmed my view, previously articulated in my 1980 Scientific American 

article, that the plasmid was not a simple genome accessory but was rather a self-regulating autonomous 

endosymbiont in its own right. 

Biosketches Page 18 
 

 

 

 

 

 

Contact PD/PI: Muir, Tom 

a. Rasooly, A. and Novick, R. (1993). Replication-specific inactivation of the pT181 plasmid initiator 

protein. Science 262:1048-1050. 

b. Rasooly, A., Wang P-Z., & Novick, R. P. (1994). Replication-specific conversion of the 

Staphylococcus aureus pT181 initiator protein from an active homodimer to aninactive 

heterodimer. EMBO J., 13:5245-5251. 

c. Rasooly, A., Wang P-Z., & Novick, R. P. (1994). Replication-specific conversion of the 

Staphylococcus aureus pT181 initiator protein from an active homodimer to aninactive 

heterodimer. EMBO J., 13:5245-5251. 

4. Discovery of the agr system and its regulation by an RNA molecule and activation by thiolactonecontaining peptides 

Reports in the early 1980’s of S. aureus mutants defective in the expression of several virulence factors 

suggested that the mutations might have affected a regulatory system and so I ran a Tn551 transposon 

screen and isolated an insertion in a gene, now known as agrA, which turned out to be the response 

regulator of a two-component signal transduction system (TCS), which I named the agr system. In a very 

productive collaboration with Prof. Tom Muir, we have studied this system at great length, discovering that 

it is activated by a unique thiolactone peptide that binds to the signal receptor, AgrC, and that the TCS 

activates transcription of a divergent promoter that determines the synthesis of a 517 nt regulatory RNA, 

RNAIII, that controls translation of many virulence genes. The system occurs as 4 allelic variants that, in 

heterologous combinations, inhibit activation of the TCS. We demonstrated that this inhibition could 

attenuate or block a staphylococcal subcutaneous abscess in mice and have analyzed this effect in some 

depth, most recently finding that the inhibitory peptide could be injected at a different site from the bacteria 

and was effective after a delay of up to 8 h. 

a. Ji, G., Beavis, R., & Novick, RP (1995). Cell Density Control of Staphylococcal virulence mediated 

by an Octapeptide Pheromone. Proc. Natl. Acad. Sci. USA 92:12055 12059. 

b. G., R. Beavis, and R. P. Novick. (1997). Bacterial interference caused by autoinducing peptide 

variants. Science. 276:2027-2030. 

c. Wright JS 3rd, Jin R, Novick RP. (2005) Transient interference with staphylococcal quorum sensing 

blocks abscess formation. Proc Natl Acad Sci U S A. Feb 1;102(5):1691-6. 

5. Discovery and in depth analysis of the SaPIs – highly mobile staphylococcal pathogenicity islands 

carrying and disseminating the genes for toxic shock toxin and other superantigens 

Following the tampon-based outbreak of staphylococcal toxic shock in the late 1970’s and the identification 

by Bergdoll and Schlievert of the responsible toxin, TSST-1, I was invited by Proctor & Gamble and by 

Johnson & Johnson to clone and characterize the toxin gene. Barry Kreiswirth, a student in the lab, cloned 

the TSS gene (tst) and we found that it was flanked by 15 kb of DNA that was absent from non-TSS 

strains. This 15 kb element turned out to be a highly mobile pathogenicity island that was induced by 

certain helper phages to excise and replicate and was packaged in small infectious particles composed of 

phage virion proteins. It is the only known source of TSST-1. This element, abbreviated SaPI1 

(staphylococcal pathogenicity island 1), was the prototype of a very large family of similar elements, with 

most S. aureus strains containing one or more. We have since characterized these islands in great depth, 

aided by a very productive collaboration with Dr. José Penadés. It has become clear that these elements 

have a very important role in staphylococcal biology and pathobiology, contrbuting not only to horizontal 

gene transfer, but also to the well-being of the host organism, lagely by down-regulating the reproduction of 

infecting phages. 

a. Lindsay, J. A., A. Ruzin, H. F. Ross, N. Kurepina, and R. P. Novick. (1998). The gene for toxic 

shock toxin is carried by a family of mobile pathogenicity islands in Staphylococcus aureus. Mol. 

Microbiol.. 29:527-543. 

b. Ubeda C, Barry P, Penadés JR, Novick RP. A pathogenicity island replicon in Staphylococcus 

aureus replicates as an unstable plasmid. Proc Nat Acad Sci, US, 2007; 104: 14182-88. 

c. Ram G, Chen J, Ross HF, Novick RP. Precisely modulated pathogenicity island interference with 

late phage gene transcription. Proc Natl Acad Sci U S A. 2014 Oct 7;111(40):14536-41 

d. Chen J, Ram G, Penadés JR, Brown S, Novick RP. Pathogenicity island-directed transfer of 

unlinked chromosomal virulence genes. Mol Cell. 2015 Jan 8;57(1):138-49 

Biosketches Page 19 
Contact PD/PI: Muir, Tom 

Complete List of Published Work in MyBibliography: 

http://www.ncbi.nlm.nih.gov/sites/myncbi/richard.novick.1/bibliography/40752281/public/?sort=date&direction= 

ascending 

D. Research support 

Ongoing Research Supp ort 

Completed research support 

R01 AI22159 Novick (PI)09/01/85 - 08/31/13 

Molecular biology of TSST-1 and other superantigen toxins 

This project is a study of the novel, mobile genetic elements encoding toxic shock toxin - and other 

superantigens. 

Role: PI 

Biosketches Page 20 
Contact PD/PI: Muir, Tom 

Budget Justification – Muir Lab 

PERSONNEL 

Tom W. Muir (1 summer month) will serve as the principal investigator. In addition to 

guiding the project, he will be responsible for experimental design and for interpretation 

of all aspects of the synthetic peptide and protein chemistry, as well as structural and 

biochemical studies of agr proteins. Dr. Muir has considerable experience in the 

chemical synthesis and semisynthesis of proteins, and has worked on the study of agr 

quorum sensing for over 20 years. 

Postdoc: Stephen Xie (12 months) is an experienced protein structural biologist with 

extensive training in the area of AgrC biochemistry. Dr. Xie joined the Muir lab to study 

the agr system and has spearheaded our efforts to study AgrC by x-ray crystallography. 

He will work on the experiments outlined in Aim 2. 

Graduate Student : Aishan Zhou (12 months). Ms. Zhou is a third year graduate student 

in the Muir lab. She has received extensive training in the areas of peptide and protein 

chemistry, especially as applied to agr. Ms. Zhou has developed genetic assays and 

crosslinking approaches to study the AIP biosynthesis and AIP/AgrC binding. She will 

work on the biochemical and biophysical experiments outlined in Aim 1. 

Graduate Student: to be appointed (12 months). He/She will work on the experiments 

outlined in Aim 3. 

MATERIALS & SUPPLIES 

I. HPLC/FPLC COLUMNS 

There is an unusually large amount of analytical and semi-preparative reverse-phase 

HPLC/FPLC in the work proposed. Funds are requested for analytical and semipreparative HPLC/FPLC columns. We have budgeted for: 

• 3 analytical (4.6mm x 25cm, Vydac) columns at each 

• 3 semiPrep (1.0 x 25cm, Vydac) columns at each 

• 2 FPLC G75 size exclusion columns at each (for AgrC purification work) 

HPLC/FPLC Columns: 

II. SUPPLIES 

The following supply costs are based on three full-time (one Postdoc and two Graduate 

Students) researchers working in the laboratory. Unless otherwise stated, all projected 

consumables have been calculated based on the actual current amounts used in 

peptide/protein chemistry in the Muir laboratory. 

Budget Justification Attachment Page 36 

A. Chemicals 

Misc. Organic Chemicals 
Budget Justification Attachment Page 37 

Specialty chemicals are required on a continuing basis for the synthesis of modified 

amino acids containing stable isotopes and fluorescent probes, of dipeptide analogs, of 

unusual intermediates, and of starting resins and resin linkers. Anamount of per 

full time person per month, has been arrived at based on my own experience of the 

typical expenditures of a laboratory engaged chemical biology. This figure is in line with 

amounts used in synthetic organic research groups (adjusted for the estimated 

percentage time spent on organic non-peptide synthesis). 

Misc. organic chemicals: 

B. Peptide Synthesis 

Contact PD/PI: Muir, Tom 

We anticipate undertaking ~10 chemical syntheses of AgrD an peptides approximately 

40 residues in length per year. All amounts are based on using the in-situ 

neutralization/HBTU activation method for Boc or Fmoc-based solid-phase peptide 

synthesis. 

1). Boc-L-amino acids 

Synthesis on a 0.05-0.5 Mmole scale requires 2.20 Mmole amino acid per residue which 

gives a total of 2.20x10-3 x 40 = 0.088 mole amino acids (of all kinds) per synthesis. At 

an average formula weight of 280 grams/mole for side-chain protected Boc-amino acids 

(from Nova Biochem), this equates to around 24 grams of high purity protected amino 

acid derivatives per total synthesis. For a synthesis of a 40 residuepeptide at per 

gram of protected amino acid x 24 g the cost is per synthesis. We are budgeting 

for 30 syntheses per year. 

Total Boc-L-amino acids: 

2). Solvents 

We will require unusually large amounts of high purity solvents for stepwise solid phase 

peptide synthesis. Based on actual laboratory records we anticipate that the synthesis of 

each polypeptide will require the following: 

• Dimethyl formamide (amine-free peptide synthesis grade), per litre (Fisher) 

at 20 litres per synthesis amounting to 600 litres in year 1 

• Dichloromethane (spectroanalysed grade), per litre (Fisher) at 4 litres per 

synthesis amounting to 120 litres in year 1 

• Trifluoroacetic acid (high purity synthesis grade), $150 per litre (Halochem) at 

30 litres in year 1 

Total solvent costs: 

3). SPPS Chemicals 

A range of high quality reagents are required for the total synthesis of peptides, namely: 

• High purity loaded PAM-resins (4g of each, Applied Biosystems), MBHA resin 

(20g, Peninsula Labs.), PEGA resin (50g,Novabiochem), Sulfonamide resin 

(Novabiochem), ninhydrin reagents, scavengers in year 1 

• Diisopropylethylamine (Applied Biosystems), 600ml year 1 

• HBTU/HATU in year 1 

Total SPPS Chemicals: 

Overall Total for Peptide Synthesis: 

III. MOLECULAR BIOLOGY/PROTEIN EXPRESSION 
Contact PD/PI: Muir, Tom 

A. Cloning 

Restriction endonucleases, high fidelity thermostable DNA polymerases, DNA ligase, 

dNTP and PCR primers are required on a continuing basis for the construction of new 

expression plasmids, PCR amplification, and DNA sequencing. 

Total Cloning: 

B. Microbiological media 

We anticipate that over the year 100 L of LB media will be used to induce 

overexpression of recombinant proteins by addition of IPTG to the final concentration of 

1 mM. Thus, at the current price of IPTG ( /5g, Sigma) we request for IPTG, 

and for media and antibiotics (from Fisher). 

Total Microbiological Media: 

C. Chromatography 

Our studies will require a range of affinity matrixes including, chitin beads from New 

England Biolabs, and Glutathione resin from Pharmacia, and Ni2+-NTA agarose (for 

affinity chromatography), and conventional sorbents for liquid chromatography (from 

Pharmacia and TosoHaas). Various ultrapure chemicals and disposable bottletop 0.2mM 

filters will be required for the preparation of buffers suitable for the gel-permeation, ionexchange and HPLC studies to be performed on the FPLC/HPLC systems. Centrifugal 

concentrators, dialysis tubing will be required for pre-, inter-, and postcolumn treatments 

of protein samples. 

• Acetonitrile (Spectranalyzed Grade, Fisher Scientific 

• Affinity chromatography media 

• Salts, buffers, filterware 

• Microconcentrators, centifugal filters 

Total Chromatography: 

D. Proteases 

Our studies require the use of specific proteases (Factor Xa, thrombin) to remove Nterminal leader sequences form expressed proteins. 

Total Proteases: 

E. Electrophoresis 

Ultrapure acrylamide and agarose, urea and SDS for standard agarose-gels and SDS 

PAGE. The cost for these general electrophoresis supplies is estimated at per 

year. 

Total Electrophoresis: 

F. Sequencing and oligo synthesis 

Total Sequencing: 

TOTAL MOLECULAR BIOLOGY COSTS YEAR 1= 

TOTAL MATERIALS & SUPPLIES COSTS YEAR 1 = 

Budget Justification Attachment Page 38 
Contact PD/PI: Muir, Tom 

TRAVEL 

A. We request per year for travel to domestic scientific meetings and 

synchrotron trips; 1 per postdoc/grad student and PI. 

Total Travel: 

PUBLICATION COSTS 

We request per year to meet the costs of publications and reprints. 

Total Publication Costs: 

TOTAL MAT. & SUPPLIES, TRAVEL, PUBLICATION YEAR 1= 

Budget Justification Attachment Page 39 
 

Contact PD/PI: Muir, Tom 

Budget justification

Personnel: Funds are requested for the salary and benefits of a postdoc (12 months calendar). The postdoc 

will be proficient in microbiological techniques and the handling of mice.First year: in salary and fringe 

benefits. 

Animals: Funds are requested for the purchase and maintenance of the mice that will be required for the 

proposed experiments. Yearly request: . 

Supplies: Funds are requested for supplies at the rate of ~ per year per researcher – which is the 

usual rate for workers in the Novick lab. First year’s request: . 

Miscellaneous: Funds are also requested for travel to one domestic meeting per year.First year: . 

Budget Justification Attachment Page 56 
OMB Number: 0925-0001 

Expiration Date: 10/31/2018 

PHS 398 Cover Page Supplement 

Contact PD/PI: Muir, Tom

1. Human Subjects Section 

Clinical Trial? ❍ Yes ● No 

*Agency-Defined Phase III Clinical Trial? ❍ Yes ❍ No 

2. Vertebrate Animals Section 

Are vertebrate animals euthanized? ● Yes ❍ No 

If "Yes" to euthanasia 

Is the method consistent with American Veterinary Medical Association (AVMA) guidelines? 

● Yes ❍ No 

If "No" to AVMA guidelines, describe method and proved scientific justification 

3. *Program Income Section 

*Is program income anticipated during the periods for which the grant support is requested? 

❍ Yes ● No 

If you checked "yes" above (indicating that program income is anticipated), then use the format below to reflect the amount and 

source(s). Otherwise, leave this section blank. 

*Budget Period *Anticipated Amount ($) *Source(s) 

Page 59 

Funding Opportunity Number: PA-16-160 . Received Date: Tracking Number: GRANT12207716 2016-07-05T12:16:58.000-04:00 
Contact PD/PI: Muir, Tom 

PHS 398 Cover Page Supplement 

4. Human Embryonic Stem Cells Section 

*Does the proposed project involve human embryonicstem cells? ❍ Yes ● No 

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://grants.nih.gov/stem_cells/registry/current.htm. 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 beused. 

Cell Line(s) (Example: 0004): 

5. Inventions and Patents Section (RENEWAL) 

*Inventions and Patents: ❍ Yes ● No 

If the answer is "Yes" then please answer the following: 

*Previously Reported: ❍ Yes ❍ No 

6. Change of Investigator / Change of Institution Section 

✔❏ Change of Project Director / Principal Investigator 

Name of former Project Director / Principal Investigator 

Prefix: 

*First Name: Richard 

Middle Name: 

*Last Name: Novick 

Suffix: 

✔❏ Change of Grantee Institution 

*Name of former institution: 

New York University School of Medicine 

Page 60 

Funding Opportunity Number: PA-16-160 . Received Date: Tracking Number: GRANT12207716 2016-07-05T12:16:58.000-04:00 
Contact PD/PI: Muir, Tom 

PHS 398 Research Plan 

OMB Number: 0925-0001 

Expiration Date: 10/31/2018 

Introduction 

1. Introduction to Application Introduction-resubmission.pdf (Resubmission and Revision) 

Research Plan Section 

2. Specific Aims Specific_Aims-resubmission.pdf 

3. Research Strategy* Research_Strategy-Final_resubmission.pdf 

4. Progress Report Publication List 

Human Subjects Section 

5. Protection of Human Subjects 

6. Data Safety Monitoring Plan 

7. Inclusion of Women and Minorities 

8. Inclusion of Children 

Other Research Plan Section 

9. Vertebrate Animals Vertebrate_animals_Novick.pdf 

10. Select Agent Research 

11. Multiple PD/PI Leadership Plan 

12. Consortium/Contractual Arrangements Signed.face.pdf 

13. Letters of Support Letters_of_Support.pdf 

14. Resource Sharing Plan(s) 

15. Authentication of Key Biologicaland/or 

Chemical Resources 

Appendix 

16. Appendix 

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Funding Opportunity Number: PA-16-160 . Received Date: Tracking Number: GRANT12207716 2016-07-05T12:16:58.000-04:00 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Contact PD/PI: Muir, Tom 

I am grateful for the opportunity to present a revised version of our application “Peptide Inducers of 

Staphylococcal Pathogenicity”, first reviewed in February 2016 at the SBCB study section (18 percentile, priority 

score 32). It seems that the reviewers found the topic of great biomedical importance and that the original proposal 

was rated very highly – as one of the reviewers put it “In all respects, it has very few weaknesses”. The reviewers 

further note “the proposed experiments are likely to have a high impact”, with the potential to provide fundamental 

insights that could lead to new therapeutic strategies. The reviewers stressed the strength of our research team, 

stating that “the investigators are peerless”, and that our work “has resulted in new mechanistic insights at virtually 

every step in the pathway.” Indeed, the reviewers seemed particularly enthusiastic about the mechanistic and 

biophysical studies the form the core of the proposal (aims 1 and 2), noting the multidisciplinary and innovative 

approaches to be taken and the body of supporting preliminary data provided. Altogether, the consensus seems to 

have been that the proposal was very strong with only minor weaknesses, which we address below. 

Perhaps the biggest critique of the grant relates to the proposed mouse infection experiments described in 

Aim 3. The reviewers unanimously characterized this line of investigation as being premature, going as far to say 

that it may be more useful to consider this an option for future experiments. Given this consensus view, we have 

completely reorganized Aim 3, replacing the mouse work (now viewed as more long term, as per the suggestion) 

with an entirely new sub-aim in which we will attempt to identify a global AIP activator of the S. aureus agr response 

employing a peptide screening technology newly developed in my group. Unlike global inhibitors of the agr 

response, which have been known for over a decade, a global activator has never been described. Such a 

compound would be of interest as a way of disrupting S. aureus biofilms (which occurs upon agr activation), with 

presumptive utility as a coating for medical devices/implants. We propose to develop such a molecule using a novel 

peptide array system. 

Critique #1 pointed out the challenges associated with identifying AIP binding sites on ArgC by performing 

crosslinking studies. We certainly appreciate the technical challenges here, however, newly added preliminary work 

strongly argues for feasibility. Specifically, we have now successfully used photocrosslinking followed by mass spec 

mapping to localize the AIP binding site to one region of the receptor, namely residues 75-90 (see revised Figure 5). 

In the future we will narrow down the site using a combination of additional mapping with strategically placed CNBr 

cleavage sites, targeted mutagenesis and genetically incorporated crosslinkers. Note that, as per the suggestion, we 

did consider using electrophilic amino acids for cysteine based crosslinking – analogous to Lei Wang’s work – 

however this approach is chemically incompatible with the AIP thiolactone. Irrespective, the proposed crosslinking 

workflow works and can allow mapping of the site as was originally hoped. 

The Critique also points out that some of the “more ambitious experiments lack preliminary data and may 

fail.” There is, of course, no guarantee that any ambitious experiment will work. Perhaps, a more relevant question 

to ask is whether the questions posed are worthy of the asking. On this level, there appears to be no pushback from 

the reviewers, rather it is a question of approach. Importantly, our newly added preliminary data strongly supports 

feasibility. For example, we have now determined the x-ray crystal structure, to 2.25 Å, of the complete cytoplasmic 

domain of AgrC (see revised Figure 6). This breakthrough underwrites a major component of specific aim 2. The 

reviewers also questioned our ability to identify the proteases responsible for degradation of AgrDC and processing 

of AgrD(1-32)-thiolactone. Our work has already shone light on this – the importance of AgrDC turnover in driving AIP 

biosynthesis came out of our published studies, and the likelihood that SpsB is not the only (even the physiologically 

relevant) protease for processing AgrD(1-32)-thiolactone is supported by our extensive preliminary data, expanded 

on in the revision. We apologize if we failed to convey our appreciation of the attendant technical challenges here, 

and regret if we gave the impression that a one-dimensional approach will be taken. In fact, we will take both a 

candidate and unbiased approach to this problem, employing various approaches (Aim 1). In terms of the former, we 

have now generated a series of S. aureus strains with candidate proteases deleted or mutated (e.g. clpPS98A), with 

many others in the works. As for the biochemical approach, we have now shown that the AgrD(1-32)-thiolactone is 

efficiently processed by the cell supernatant fraction of S. aureus cells, i.e. the protease(s) is a soluble secreted 

factor rather than a membrane protein (see revised Figure 4). This breakthrough greatly simplifies the identification 

of the protease(s) using either ‘classical’ fractionation methods, or by including photo-crosslinkers in nonhydrolysable AgrD(1-32)-thiolactone analogs. We stress that our expensive preliminary data, long track record of 

delivering the goods on this system and multifaceted and innovative approach to this problem, all argue for a 

successful outcome. 

Reviewer #1 states that the “single-molecule experiments are under-developed.” We acknowledge that our 

discussion of this experiment was a little on the brief side and devote more space to this in the revision. Lastly, 

reviewer #3 states that “innovation of the proposed experiments is not clearly described”. I respectfully disagree. 

Innovation is pervasive in the proposal, whether it is the use of a plethora of cutting-edge (and bespoke) chemical 

biology methods, new methods to generate and screen novel compounds, and the unprecedented use of a 

completely reconstituted two competent circuit in our mechanistic studies. Moreover, the proposal is laden with new 

concepts and hypotheses. For example, the link between quorum sensing and protein turnover, the proposed role of 

metabolic state in regulating the quorum sensing circuit, and the role of opposing mechanical motions in regulating 

the kinase activity of AgrC. Overall, we have tried very hard to strike a balance between asking interesting and 

biomedically relevant questions and proposing a mixture of innovative, as well as tried and tested, approaches to 

answer these. I hope and believe that the changes we have made in the revision more clearly articulate this vision. 

Key changes to the text are underlined (with the exception of Aim 3 which has been substantially reworked). 

Introduction to Application Page 62 
 

 

 

 

 

 

 

 

 

 

 

 

 

Contact PD/PI: Muir, Tom 

A research program will be undertaken to determine the molecular mechanisms underlying agr signal 

transduction in the commensal pathogen, Staphylococcus aureus. The accessory gene regulator (agr) locus 

found in all staphylococci encodes a quorum sensing (QS) circuit that controls the expression of virulence and 

other accessory genes. It consists of two oppositely oriented transcriptional units, of which one encodes four 

proteins, AgrBDCA, involved in production and sensing of an autoinducer peptide (AIP), and the other encodes 

a regulatory RNA that is the effector of target gene regulation. The finding that staphylococcal virulence can be 

inhibited through antagonism of this QS pathway has fueled tremendous interest in understanding the 

molecular mechanisms at play throughout the circuit. Such knowledge is expected to aid in the development of 

therapeutic strategies targeting agr, but also contribute to a fundamental understanding of QS systems of this 

type, which are pervasive in the low-GC bacterial phylum, Firmicutes. Significant progress has been made 

during the past funding cycle in understanding the mechanisms underlying agr signaling (see Research 

Strategy). Using reconstituted biochemical systems of defined composition, we now have a much clearer 

picture of AIP biosynthesis and secretion, as well as the molecular motions attendant to signaling through the 

two-component system. Our studies reveal many unanticipated features within the agr circuit and have 

generated a series of key biochemical questions that will form the basis of future studies, as outlined below: 

Specific Aim 1: To determine the mechanisms of AIP biosynthesis and secretion. AIPs feature a 

thiolactone macrocycle and are produced from precursor polypeptides, encoded by AgrD, through two 

proteolytic events with an intervening secretion step. Thiolactone formation is coupled to the first of these 

processing steps and involves the activity of the integral membrane protease, AgrB, which converts AgrD into 

AgrD(1-32)-thiolactone and a C-terminal proteolysis fragment, AgrDC. Our studies show that efficient 

thiolactone production requires rapid degradation of AgrDC. We will explore this newly uncovered link between 

QS and protein homeostasis in S. aureus cells. As part of this, biochemical and genetic tools will be used to 

test the hypothesis that AgrDC is degraded by dedicated AAA+ proteases such as ClpS providing the 

thermodynamic driving force for AIP production. Our preliminary studies also indicate that AgrB is not 

responsible for secretion of AgrD(1-32)-thiolactone out of cells. Thus, this key step in the biosynthesis remains 

enigmatic. We will take both an unbiased and candidate based strategy, employing various chemical biology 

and genetic approaches, to identify the secretion apparatus required. As part of this, we will test the hypothesis 

that the phenol-soluble modulin ABC transporter (Pmt) is responsible for AIP secretion. The final step in AIP 

biosynthesis involves proteolytic removal of the an N-terminal leader peptide from the newly secreted, AgrD(1-

32)-thiolactone. While it has been suggested that the general housekeeping protease, SpsB, is responsible for 

this step, our preliminary data casts considerable doubt on this assignment. Thus, we will carry out functional 

proteomic studies geared towards identifying the necessary protease for each of the four agr specificity groups. 

Specific Aim 2: To understand the mechanism of activation and inhibition of the agr two component 

system. AIPs are recognized by the membrane-bound receptor histidine kinase, AgrC. Some AIPs are 

agonists of this receptor, while other are antagonists. Using AgrC reconstituted into nanometer-scale lipid 

bilayer discs, we have shown that ligand-induced activation and inhibition of the receptor occurs through 

opposite helical twisting motions that result in rheostat-like control over kinase activity. This breakthrough sets 

the stage for the next phase of the program where we will use photo-crosslinking strategies to pinpoint the 

ligand-binding pocket in AgrC and how this site couples to mechanical transduction. We will also perform 

structural studies on the soluble intracellular domain of AgrC engineered to be trapped in either the active or 

inactive state. Also proposed are single molecule biophysical studies designed to test the hypothesis that the 

rheostat-like behavior of AgrC activity reflects a continuum of dynamic states within the intracellular domain. As 

part of this aim, we will also initiate structural studies on the sensor domain of AgrC bound to 

agonist/antagonist ligands. Finally, we will explore the hypothesis, generated by our preliminary results, that 

agr activation is regulated by the metabolic state of the cell leading to down-regulation in times of stress. 

Specific Aim 3: To identify new pharmacological modulators of the agr system. In this aim, we will 

identify novel inhibitors or activators of agr by performing high-throughput screens against reconstituted AgrC 

using newly established chemistries and biochemical assays. These studies will serve as starting point for 

exploring the viability of targeting agr for biomedical applications, a long-term goal of this program. 

Collectively, these investigations will provide fundamental insights into how a QS system such as agr operates 

at the molecular level and will lay the foundation for the development of new strategies for treating S. aureus 

infections. 

Specific Aims Page 63 
Contact PD/PI: Muir, Tom 

SIGNIFICANCE: Staphylococcus aureus (S. aureus) is part of the commensal microbial flora of ~30% of the 

adult population. In spite of its normal, beneficial nature, S. aureus is an opportunistic pathogen capable of 

invading mucous membranes or soft tissue [1]. Once invasion occurs, S. aureus is a remarkable, dynamic 

pathogen that is known to cause both acute and chronic illnesses such as bacteremia, sepsis, endocarditis and 

toxic shock syndrome [1]. As a consequence, S. aureus is a major health threat worldwide. Although the 

immune system and treatment with antibiotics can clear S. aureus infections, there are several risk factors that 

include a weakened immune system, surgery, and/or implanted medical devices that can lead to fatal 

infections [2]. Notably, these risk factors often persist in a hospital environment, where virulent S. aureus 

strains can thrive (nosocomial infections) and infect vulnerable patients being treated for an unrelated problem 

[3]. Such infections have only become more lethal with the emergence of antibiotic resistant, highly virulent 

strains of S. aureus (e.g. MRSA and VRSA) that can strike down healthy individuals in addition to the elderly 

and very young [4]. As an illustration, in 2005, there were over 278,000 MRSA-related hospitalizations, and 

estimates place MRSA-related deaths of at least 18,000 per year in the United States, which is nearly as many 

deaths as AIDS, tuberculosis and viral hepatitis combined [5]. Public concern over these ‘superbugs’ has risen 

dramatically in recent years as reflected by wide-spread coverage in the popular media (for a good example, 

see http://www.pbs.org/wgbh/pages/frontline/hunting-the-nightmare-bacteria/) and certainly there is a general 

consensus among microbiologists, physicians and public health experts alike that new therapies, including 

entirely new classes of antibiotics, are desperately needed to address this problem. 

Figure 1: Virulence regulation in S. aureus. 

Schematic of the agr circuit. Activation of the agr 

response results from interaction of a cognate AIP-AgrC 

pair (i.e. within a specificity group) while non-cognate 

interactions are inhibitory. Inset: structure of AIPs I-IV. 

S. aureus is an opportunistic pathogen that 

deploys a diverse arsenal of virulence factors to 

evade the host immune system and to facilitate 

spread of the infection in the correct host 

environment. The bacterium utilizes two main 

classes of virulence factors, each associated with 

different phases of population growth [6]. During 

the lag and early exponential phases, virulent S. 

aureus cells produce cell wall-associated factors 

that facilitate tissue attachment and evasion of the 

host immune system, allowing the bacteria to accumulate and possibly form a biofilm [7, 8]. For example, 

microbial surface components, recognizing adhesive matrix molecules, adhere to the extracellular matrix to 

give the bacteria an attachment point in the host. Protein A on the bacterial surface binds IgG antibodies to 

form a protective coat and evade the host immune system [7]. Once the bacterial population achieves a 

threshold density by late exponential phase, monitored by a quorum sensing (QS) system (see below), cell 

wall-associated factors are down-regulated, allowing for detachment from the original colonization site and 

establishment of an invasive infection [9]. At the same time, the bacterium secretes enzymes and toxins, 

termed exoproteins, to degrade host tissue and to promote spread of the infection. These include degradative 

enzymes such as proteases, hemolysins, as well as enterotoxins that are the causative agents of S. aureus 

food poisoning and contribute to toxic shock syndrome and other diseases by stimulating T-cells to produce 

proinflammatory cytokines in excess amounts [10]. Importantly, the coordinated expression of virulence factors 

is not only conserved within the Staphylococci [11], but also throughout the phylum Firmicutes, which includes 

pathogenic bacteria such as Enterococcus faecalis [12], Listeria monocytogenes [13], Clostridium perfringens 

[14], and Clostridium botulinum [15]. The onset of virulence in these organisms is regulated by a signaling 

network, including one QS system termed the accessory gene regulator (agr) [16]. 

Overview of the agr QS system: The agr locus consists of two oppositely oriented transcription units, 

of which one encodes four proteins, AgrBDCA, involved in producing or sensing of the autoinducer peptide, 

AIP, and the other encodes a regulatory RNA that is the effector of target gene regulation (Figure 1). Agr is 

universally conserved among the Staphylococci and its variation is thought to be the driving force behind 

speciation within the genus [11]. Indeed, there are 4 allelic variants of agr in S. aureus that differ in their ligandreceptor specificities, such that heterologous receptor-ligand interactions are in general inhibitory [17, 18]. All 

S. aureus AIPs contain a thiolactone macrocycle (Figure 1) and are produced through sequential processing of 

a ribosomally generated polypeptide encoded by the agrD gene product [19]. Sensing of AIP is achieved 

Research Strategy Page 64 
 

 

Contact PD/PI: Muir, Tom 

through the AgrCA two-component system (TCS) in which AgrC is a membrane-bound receptor-histidine 

kinase (RHK) that undergoes autophosphorylation following cognate AIP binding and then transfers the 

phosphoryl group to a conserved response regulator (RR), AgrA. Phosphorylated AgrA dimerizes and binds to 

the genomic region between the agr promoters, activating both, thus completing a strong positive feedback 

loop [16, 20]. The P3 transcript encodes a regulatory RNA, RNAIII, that is the ultimate effector molecule of the 

agr response – at 514 nucleotides in length, RNAIII is a multifunctional molecule that acts as both a messenger 

(encoding δ-hemolysin) and an antisense RNA that regulates the expression of numerous genes [21-23]. 

Considerable effort has gone into defining the structure-activity relationships within the AIPs and AgrC that 

govern agonism and antagonism [19, 24-31]. These studies have mostly relied on cell-based assays involving 

coupled transcriptional readouts although, as discussed below, we have recently succeeded in reconstituting 

the TCS using purified components, thereby providing a platform for studying the detailed molecular 

mechanisms involved in signaling. 

Figure 2: AIP biosynthesis and the agr TCS. (a) Schematics of AIP biosynthesis (a) and AgrC activation/inhibition (b). 

Outstanding questions are highlighted with green arrows. ECM, extracellular matrix. 

AIP Biosynthesis: In the prevailing view of AIP biosynthesis (Figure 2A), the peptide is translated as a 

precursor, AgrD, in which the mature AIP sequence is embedded [24]. An N-terminal leader sequence forms 

an amphipathic helix that drives AgrD to the inner leaflet of the cell membrane where the first step in the 

maturation process occurs, namely processing of AgrD by a membrane-integrated cysteine peptidase, AgrB. 

Processing gives rise to two cleavage products, AgrD(1-32)-thiolactone and AgrDC [32, 33]. At face value, this 

step is expected to be thermodynamically unfavorable (⊗Go = ~10 kcal/mol based on model reactions), due to 

the high energy nature of the thioester bond in AgrD(1-32)-thiolactone [34]. In a key breakthrough, we have 

recently shown that this process is driven by two critical features of the system: (i) membrane association of 

AgrD(1-32)-thiolactone, which stabilizes the macrocycle, and (ii) rapid degradation of AgrDC, which affects the 

reaction equilibrium position, favoring product formation [34]. This work exposes a hitherto unappreciated 

connection between QS and protein homeostasis in S. aureus cells. The precise mechanism by which AgrDC is 

recognized and then degraded remains unknown, however our studies indicated that this must be an active 

process. Elucidation of this process will be a focus of Specific Aim 1. 

The second step in AIP biosynthesis involves transport of the AgrD(1-32)-thiolactone intermediate to 

the outside of the cell where subsequent processing can then take place (Figure 2A). That translocation occurs 

before the second processing step is supported by the observation that large quantities of AgrD(1-32)-

thiolactone can be detected in the extracellular matrix [35, 36]. AgrD lacks any signal peptide seen in proteins 

meant for secretion via the common Sec, Tat or Com secretion pathways [37, 38]. Consequently, it has been 

suggested that the AgrB protease is also responsible for the secretion step – i.e. processing and transport are 

somehow coupled [39]. However, AgrB (22 kDa) lacks any homology to known bacterial transporters (i.e. an 

ABC cassette) [40]. In preliminary data, we have engineered S. aureus cells to generate AgrD(1-32)-

thiolactone independent of AgrB and used this system to show that AIPs are still efficiently generated and 

secreted even when agrB is genetically deleted (vide infra). This strongly suggests that AgrB is not the 

transporter for AgrD(1-32)-thiolactone. We imagine two possibilities; either AgrD(1-32)-thiolactone passively 

crosses the cell membrane, or another transporter system is involved. This key issue will be tackled in Aim 1. 

The final step in AIP biosynthesis involves removal of the amphiphathic leader from AgrD(1-32)-

thiolactone (Figure 2A). We have used a defined biochemical system to show that AgrB does not perform this 

step [34]. Indeed, the prevailing view in the literature is that this is carried out by the housekeeping serine 

protease, SpsB, which is attached to the outer leaflet of the bacterial membrane [41]. However, it is unclear 

how this single protease can correctly process the AgrD(1-32)-thiolactones from the four different agr groups in 

S. aureus, all of which have different sequences around the processing site (Figure 3C) . Moreover, AIPs from 

Research Strategy Page 65 
groups I-IV all have different lengths, and there appears to be no room for sloppiness in the processing since 

addition or removal of residues from the mature AIP can convert an agonist into an antagonist of the response 

[19]. Indeed, in preliminary studies employing purified components, we have shown that processing of AgrD(1-

32)-thiolactones by SpsB is extremely inefficient (see later). Based on this, we hypothesize that other (or 

additional) proteases are involved in this step and will conduct experiments to test this in Aim 1. 

Contact PD/PI: Muir, Tom 

AgrC Activation: AgrC is a ~430-residue integral membrane protein that adopts a modular 

architecture shared among all members of the RHK family, with an N-terminal sensor domain, that spans the 

membrane 6 or 7 times, connected to a C-terminal histidine kinase domain that possesses all enzymatic 

activities required for the TCS and that is tightly regulated by AIP binding to the sensor domain [42-44]. 

Genetic and biochemical studies indicate that AgrC is an obligate dimer and that autophosphorylation between 

the protomer subunits can occur in trans [45]. We have succeeded in reconstituting purified recombinant AgrCI (i.e. from group I S. aureus) into nanometer scale lipid bilayer discs (nanodiscs) [46]. This breakthrough has 

revealed several surprising features of the system. For example, we discovered the activity of AgrC is highly 

dependent on the presence of anionic lipids, which are present in S. aureus (and indeed most Firmicutes). We 

speculate that this represents an evolutionary adaption and creates a biochemical barrier to horizontal gene 

transfer into bacteria with lower levels of these lipids (i.e. Gram negatives where RHKs are absent). We also 

discovered that AgrC has a dramatically lower affinity for ATP (Km = ~2 mM) compared other HKs (Km typically 

in the 100s μM). We hypothesize that the atypically low affinity allows the agr response to be down-regulated 

in times of stress [47] – the cellular ATP concentration drops to sub-millimolar levels during stress or stationary 

phase [48]. Our bioinformatics analysis points to amino acid substitutions in the G1 box of the catalytic domain 

of AgrC as the origin of this low ATP affinity. Using this as a guide, we propose to engineer AgrC to confer 

higher ATP affinity on the receptor. By introducing this variant into S. aureus cells we will test, as part of 

Specific Aim 2, our hypothesis that the agr response is down-regulated by lowered ATP levels duringstress. 

Perhaps the central question for understanding agr regulation, and indeed any analogous QS circuit, is 

how ligand binding is transduced by the RHK into an intracellular output. For agr this is even more fascinating 

since, depending on the AIP, AgrC can either by ac tivated or inhibited. Using our reconstituted system we have 

shown that the inhibitory interaction can either be neutral (i.e. does not affect the basal activity of AgrC) or 

involve inverse agonism (i.e. leads to a reduction of basal activity) [46]. Our studies also reveal the kinase and 

sensor domains in AgrC are connected by a helical linker whose conformational state exercises rheostat-like 

control over kinase activity (Figure 2B). Binding of AIP results in twisting of the linker in different directions 

(anti-clockwise in the case of agonism, clockwise in the case of inverse agonism). We hypothesize that this 

twisting motion alters the dynamic behavior within the kinase domain and will conduct a series of biophysical 

and structural studies to test this as part of Specific Aim 2. 

Potential of agr as a Therapeutic Target: Interfering with the agr response has long been suggested 

as a route to combating S. aureus infections [25]. Since agr is not required for survival but for virulence, it has 

been argued that resistance development against agr-targeting reagents would be lower since they impose 

milder selection pressure versus classic, bactericidal or bacteristatic antibiotics [49]. AIP inhibitors have been 

shown to attenuate the spread of S. aureus infections in mice, phenocopying the use of agr-null S. aureus 

strains as infective agents [25]. Despite this result there has been considerable debate over whether inhibition 

or activation of agr would be the better option, with arguments in favor of the latter revolving around the ability 

to disperse persistent S. aureus biofilms through activation of the response [50]. At the very least, this strategy 

has potential in preventing the adhesion of S. aureus biofilms to medical implants. Regardless of which 

strategy, activation of inhibition, would be the more beneficial for treating an infection, a major limitation of all 

medicinal chemistry efforts in this area to date has been the reliance on peptide-based modulators, all of which 

have highly labile thiolactone linkages [51]. The instability of AIP peptides in vivo along with their 

immunogenicity make them poor candidates as drugs [52, 53], no doubt stymying progress in this area. In 

preliminary studies, we have exploited our ability to reconstitute AgrC in nanodiscs to perform high-throughput 

small molecule screens leading to the identification of several molecules that are competitive with AIPs. This 

work will form the basis of additional screening and chemistry efforts, as part of Specific Aim 3, designed to 

furnish the community with the first potent small molecule modulators of AgrC. In addition, we will develop a 

novel peptide array methodology in order to identify the first global activators of the agr response. Such agents 

will have utility both as research tools and as therapeutic leads. 

INNOVATION: Uncovering the quorum-sensing dependence of exo-protein regulation represents a major 

advance in understanding S. aureus physiology and pathology [16]. The Muir/Novick team has made many of 

Research Strategy Page 66 
 

 

 

Contact PD/PI: Muir, Tom 

the key contributions to our understanding of the agr response over the last 25+ years. Highlights include: 

• Pioneering studies leading to the isolation, cloning and sequencing of the agr locus [21, 54, 55]. 

• Discovery of allelic variation within agr and subsequent characterization of bacterial interference 

between S. aureus specificity groups - studies that exposed the therapeutic potential of agr [17, 25, 47]. 

• Elucidation of the AIP structure and the development of the first synthetic routes to the molecule which, 

along which the establishment of robust cell-based assays, have allowed numerous structure-activity 

relationship studies to be performed on the system by many groups [19, 25, 26, 30, 31]. 

• The first demonstration that pharmacological inhibition of the agr response can attenuate the spread of a 

S. aureus infection in an animal [25]. Parenthetically, this work is, to our knowledge, the first example of 

targeting any QS system for therapeutic purposes. 

While the pioneering studies summarized above have helped provide a roadmap for understanding the 

agr system [16], the reliance on cell-based transcriptional reporter assays has greatly hindered detailed 

mechanistic studies on the QS circuitry. With this in mind, we have over the last few years made a significant 

investment in establishing defined, biochemical systems for studying the biosynthesis of the AIP, as well as the 

engagement of this molecule with the AgrC receptor [46]. This successful reconstitution of an entire TCS 

module from highly purified components is without precedent and, as noted in the preceding section, has 

already answered a number of longstanding questions in the agr field, whilst at the same time revealing 

several hitherto unknown aspects of the system [46]. Likewise, reconstitution of the critical first step in AIP 

biosynthesis represents an important technical breakthrough that has provided quantitative insights into the 

molecular biosynthesis that would not have been possible otherwise [34]. In addition to these biochemical 

advances, we have also developed powerful chemical biology tools to study the agr circuit in S. aureus cells, 

including an orthogonal route to the AIP biosynthesis that allows us to uncouple AIP maturation from AIP 

secretion. Collectively, these new tools put our team in a unique position to tackle the various mechanistic 

questions outlined in this proposal. We anticipate this work will yield molecular insights into QS that will extend 

beyond the S. aureus agr system. Moreover, the proposed work is expected to lead to the identification of the 

first small molecule inhibitors of S. aureus virulence, as well as the first global activators of the agr QS system. 

APPROACH: A research program will be undertaken to study the agr QS circuit responsible for virulence 

regulation in S. aureus. We will use chemical biology methods in conjunction with structural and genetic 

approaches to deduce the molecular mechanisms underlying the production and sensing of the AIP 

pheromone central to the QS circuit. These investigations will lay the foundation for the development of new 

strategies for treating S. aureus infections. 

Aim 1. To determine the mechanisms of AIP biosynthesis and secretion. 

AIP biosynthesis is a multistep process in which the AgrD precursor polypeptide is sequentially processed, 

leading to the secretion of the mature thiolactone-containing peptide into the extracellular milieu. Whilst there 

has been important progress in understanding AIP maturation, including our own work during the current 

funding cycle, several key aspects of the process remain mysterious. In particular, we know little of how 

AgrD(1-32)-thiolactone is secreted out of S. aureus cells and then further processed into the AIP, nor do we 

know the nature of the apparatus required for degradation of AgrDC which we have shown is intimately tied to 

the first step in the process. The goal of this aim is therefore to shed light on these remaining questions such 

that we might be able to reconstitute the entire AIP biosynthesis from purified components. 

1.1 : How is AgrDC rapidly degraded in S. aureus cells? The first step in AIP biosynthesis, involving 

AgrB-mediated processing of AgrD into AgrD(1-32)-thiolactone and AgrDC, represents, to the best of our 

knowledge, the only known proteolytic event where an S-nucleophile serves as the final acceptor of the 

peptidyl group [34]. In principle, this step poses a significant thermodynamic challenge for the organism, given 

the high-energy nature of a thioester relative to an amide. Indeed, using a fully reconstituted system, we have 

shown that this step, which we refer to as proteolytic cyclization, is fully reversible and that the equilibrium lies 

far to the left (K -5

eq = 1 x10 ). This finding raises the question of how enough AgrD(1-32)-thiolactone is produced 

in vivo to ultimately generate sufficient AIP to trigger the agr response. The concentration of AIP in culture 

medium reaches 5 μM within two hours of the onset of autoinduction [56]. Since every AIP produced also 

generates an equivalent of AgrDC, simple calculations reveal that, all else being equal, the intracellular AgrDC 

concentration would be in low millimolar range [57]. This is hard to reconcile with the equilibrium behavior of 

proteolytic cyclization noted above - such a high AgrDC concentration would completely inhibit AIP production, 

Research Strategy Page 67 
Contact PD/PI: Muir, Tom 

by lowering AgrD(1-32)-thiolactone levels. This analysis indicates that AgrDC must be rapidly degraded in S. 

aureus cells (we estimate a half-life on the order of 10 s, [34]) in order that the reverse reaction be sufficiently 

suppressed to allow adequate flux of AgrD(1-32)-thiolactone through the pathway. We have experimentally 

validated this idea by over-expressing AgrDC in S. aureus cells – as predicted this increases full-length AgrD 

levels whilst suppressing AIP production [34]. The focus of this sub-aim is to identify the proteolytic apparatus 

responsible for AgrDC degradation. Both candidate-based and unbiased approaches will be taken, as follows. 

Figure 3: Identification of the proteolytic apparatus targeting AgrDC. (a) Predicted effect of ClpP inactive mutation on 

AIP biosynthesis. (b). Identification of the AgrDC protease using activity-guided fractionation employing a fluorescent 

substrate analog. (c) Alignment of S. aureus AgrD with a gap placed between AIP and flanking sequences. Residues 

involved in AgrB recognition and conserved residues without known function are highlighted in green and blue, 

respectively. (d) Does release of AgrDC from the membrane, after proteolytic cyclization, trigger degradation? We 

hypothesize that artificially anchoring AgrDC via an amphipathic helix (blue) to the membrane will prevent degradation. 

There is considerable evidence that the ClpP-based AAA+ protease machinery (ClpCP or ClpXP) is 

required for virulence regulation in S. aureus. Both genetic deletion and chemical inhibition of ClpP, the 

catalytic subunit, lead to down regulation of the agr response [58, 59]. The molecular basis of this effect is 

unknown [60]. We hypothesize that this multimeric complex is directly responsible for degradation of AgrDC. 

Several experiments are proposed to test this. Firstly, we will directly look at the levels of full-length AgrD, 

AgrDC and AIP in an available ClpP-inactivated (clpPS98A) S. aureus line [61] using a combination of 

immunoblotting (using tagged versions of AgrD and AgrDC), mass spectrometry (for the AIP) and bioassays 

(for the AIP). We predict an increase in AgrD and AgrDC levels, but a decrease in the amount of AIP (Figure 

3A). Importantly, all of the tools required for this experiment are already available within the Muir laboratory 

[34]. We will also perform in vitro proteolysis assays using recombinant S. aureus ClpCP or ClpXP, generated 

using established methods [62, 63] and using synthetically generated AgrDC from the four specificity groups as 

substrates. Importantly, the necessary materials for this work are also already in hand. We will employ an 

established RP-HPLC based assay to test whether the peptides are degraded in an ATP-dependent manner. 

While the AAA+ protease system is a strong candidate for the AgrDC protease, we will nonetheless also take 

an unbiased approach to this problem. For this will we employ an activity-guided fractionation strategy to 

identify the protease responsible for degrading AgrDC. Accordingly, we will use a fluorescence-based assay to 

follow the biochemical enrichment of protease activity in cellular lysates from S. aureus cells in late exponential 

phase (i.e. when agr is most active). We will follow standard multi-step fractionation schemes for identifying 

host enzymes. Key to this effort will be the development of an AgrDC substrate peptide modified with a 

fluorescein-dabsyl donor-quencher FRET pair - degradation of the peptide will lead to a fluorescent signal 

(Figure 3B). Once the protease activity has been suitably enriched, we will use mass spectrometry to identify 

the proteins in the active fraction. Candidates will then be generated by recombinant expression in a suitable 

host and assayed for proteolytic activity as described above for ClpP-based AAA+ proteases. We will also 

validate the protease in vivo by making genetic deletions in S. aureus cells. 

Once the protease that degrades AgrDC is identified, experiments will be designed to pinpoint at AgrDC 

sequence features recognized by the protease. Intriguingly, previous studies [64], as well as our own 

preliminary data, have shown that the most highly conserved residues within AgrDC are not required for 

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Contact PD/PI: Muir, Tom 

substrate recognition by AgrB (Figure 3C). We speculate that these conserved motifs are recognized by the 

protease and will perform in vitro biochemical studies to test this, initially by performing an alanine-scan 

through the peptide sequence. Both the proteolysis activity by the wild-type protease and the physical 

interaction to a catalytically dead protease will be used as readouts. If key residues required for AgrDC 

degradation identified in vitro are not involved in AgrB recognition, we will transfect the corresponding mutant 

AgrD sequences into S. aureus and gauge the effect on proteolytic cyclization and on AIP production using 

established assays [34]. Alterations in the levels of starting material would provide strong in vivo evidence for a 

direct role of the protease in AgrDC degradation. In other studies, we will explore how the degradation 

machinery discriminates between full-length AgrD and AgrDC – the latter is a fragment of the former but is 

preferentially degraded in order for the agr circuit to function. We imagine two non-mutually exclusive 

possibilities. The first relates to the differential localization of full-length AgrD and AgrDC – the former is 

membrane associated whereas the latter is not [32]. Conceivable, this could affect substrate recognition by the 

protease for steric and/or electrostatic reasons. This will be tested by fusing an amphiphilic membrane 

localization sequence to either terminus of AgrDC and testing whether this remains a viable substrate for the 

degradative machinery using both cell-based and in vitro assays systems, the latter employing a liposomebased system (Figure 3D). Substrate specificity may also simply relate to peptide length, AgrDC is ~14 

residues long vs. ~46 for full-length AgrD. This idea will be explored by systematically extending the length of 

AgrDC through the addition of extra residues to the N-terminus, in this case the in vitro assay will be used. 

1.2 : How is AgrD(1-32)-thiolactone secreted from S. aureus cells? Following proteolytic cyclization, 

AgrD(1-32)-thiolactone is somehow transported to the outside of the cell, whereupon the final step in AIP 

biosynthesis can take place (Figure 2A). As noted earlier, AgrD lacks a canonical Sec, Tat or Com signal 

sequence, ruling out the involvement of these common secretion pathways in the transport step. In preliminary 

studies, we have explored whether AgrB is involved in AgrD(1-32)-thiolactone secretion, as some have 

suggested [39]. To this end, we have developed an intein-fusion strategy for producing AgrD(1-32)-thiolactone 

that is completely independent of AgrB (Figure 4A). This has allowed us to uncouple any role AgrB might have 

in transport from its known role in proteolytic cyclization. Remarkably, we find that AIP is still produced in the 

absence of AgrB, ruling out a role for this protease in the transport step. Given this result, we imagine two 

possibilities; either AgrD(1-32)-thiolactone can passively partition across the membrane, or an as yet unknown 

active transport system is involved. Both these possibilities will be explored. In the case of passive diffusion, 

we note that AgrD(1-32)-thiolactone is quite lipophilic (LogPoct/water = 0.6) - it can interact with the membrane 

both through the N-terminal amphipathic helical region [32] and through the conserved hydrophobic motif 

within the thiolactone macrocycle (Figure 3B) [34]. Thus, removal of the highly acidic AgrDC region during the 

first step might trigger an innate ability within the remainder of the AgrD sequence to partition across the 

membrane. To test this idea, we will exploit the reactivity of thioesters with nucleophiles such as aminooxycontaining reagents. Thus, we will generate liposomes containing an aminooxy-PEG, a reagent that cannot 

itself diffuse across membranes. These liposomes will then by incubated with a mixture of AgrD(1-32)-

thiolactone and a highly charged peptide-thioester (poly-Glu), the latter serving as an internal control for assay 

integrity. Generation of a PEG adduct of AgrD, but not the control peptide, as gauged by SDS-PAGE and 

LCMS, would indicate that AgrD(1-32)-thiolactone can partition the membrane (Figure 4B). 

Although a passive transport mechanism is worth testing, particularly given the lipophilicity of AgrD(1-

32)-thiolactone, such a process would be unusual for a secreted signaling peptide [65]. Thus, the involvement 

of a hitherto unknown protein transporter system will also be explored. We will again take both candidatebased and unbiased approaches to this problem. Beginning with the former, we will test the hypothesis that the 

phenol soluble modulin ABC transporter (Pmt) is responsible for AgrD(1-32)-thiolactone secretion. Phenol 

soluble modulins (PSMs) are a class of secreted peptides produced by S. aureus during infections [66]. The 

surfactin-like properties of PSMs give them cytolytic activity towards a variety of cell types including 

neutrophils, red blood cells and even other bacteria [66]. Most PSMs are induced by the agr system; RNAIII 

actually encodes one PSM, namely δ-hemolysin, whereas the other two main classes of PSMs, PSMα and 

PSMβ, are directly regulated by phosphorylated AgrA [67]. Similar to AgrD, all PSMs contain an amphipathic 

helical region whose sequence varies considerably across the family (Figure 4C). All PSMs are actively 

secreted by the four-component Pmt complex [68], which we note is the only known transporter system upregulated by agr [69]. Given this, along with the obvious similarity between PSMs and AgrD(1-32)-thiolactone 

(i.e. both are dominated by an amphipathic domain), we propose that Pmt i s responsible for secretion of both. 

This will be tested by constructing an S. aureus strain lacking the genes for both Pmt and for PSMs – needed 

to ensure cell viability, as previously shown [68]. We expect this ⊗pmt strain to have a severely impaired ability 

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to generate AIPs as determined by bioassays and mass spectrometry analysis of supernatants using 

established protocols [30, 70]. We also propose an experiment designed to sterically trap an AgrD(1-32)/Pmt 

complex. In this case we will express in S. aureus, a AgrD(1-32) fused though its C-terminus to a bulky protein 

such as ubiquitin or GFP (Figure 4D). We expect this fusion peptide to recognized by Pmt via the amphipathic 

region, but then get stuck in the pore during transport, thereby providing us the opportunity to 

immunoprecipitate the complex. In a variation of this assay, we will make membrane preparations of S. aureus 

protoplasts using established methods [71] and incubate these with synthetic AgrD(1-32)-thiolactone peptides 

containing a diazirine photocrosslinker (photo-leucine) placed within the amphiphilic region (Figure 4D). 

Protocols for preparing and analyzing such peptides are well-established in the Muir group [72, 73]. 

Importantly, these assay systems can also be used in an unbiased manner, via proteomic analysis, to identify 

other putative transporters in the event that Pmt proves not to be the transporter. 

Figure 4: Studying the final steps in AIP biosynthesis. (a) Intein-fusion strategy used to show that AgrB is not involved 

in AgrD(1-32)-thiolactone transport. (b) Liposome-based assay to probe passive AgrD(1-32)-thiolactone membrane 

crossing. (c). Sequence alignment between PSMs and AgrDs. (d) Schematic of strategies proposed to trap a complex 

between AgrD(1-32)-thiolactone and a transporter protein, e.g. Pmt. (e) SpsB liposomes can process group I AgrD(1-32)-

thiolactone to give active AIP as detected by a cell reporter bioassay. (f) Results of AgrD chimera experiments. (g) 

Addition of synthetic AgrDI(1-32)-thiolactone (+) to different cell fractions, followed by a bioassay for AIP production. 

1.3 : How is AgrD(1-32)-thiolactone processed into the mature AIP? The four AIPs produced by the 

S. aureus specificity groups differ in both sequence and in length (Figure 3C). All are generated from the 

corresponding AgrD(1-32)-thiolactones through processing within a linker region that connects the amphipathic 

helix and the macrocycle. This linker is 8 residues long in all four groups and is of variable sequence - the 

exception is a conserved glycine residue at its N-terminus, which likely defines the end of the preceding 

amphipathic helix. What is remarkable is that the precise cleavage point within this linker varies from group to 

group (Figure 3C). Thus, the AIPs from groups I-IV all have different lengths. MS analyses of culture 

supernatants indicate that processing is precise in each group (data not shown). Moreover, we have previously 

shown that addition or removal of even a single residue from a native AIP can convert an agonist into an 

antagonist of the agr response [19]. To date, the only known protease implicated in the processing of AgrD(1-

32)-thiolactone is SpsB, a type I signal peptidase that is attached to the outer leaflet of the bacterial membrane 

[41]. The catalytic domain of SpsB has been shown to cleave a short peptide corresponding to the linker region 

of a group I AgrD [41]. In preliminary results, we have extended this work by demonstrating that purified 

recombinant full-length SpsB embedded in a liposomal system can process the group I AgrD(1-32)-thiolactone 

to give detectable amounts of the native AIP (Figure 4E). However, the efficiency of cleavage is poor and, 

perhaps even more importantly, we have repeated this experiment with the group II AgrD(1-32)-thiolactone and 

in this case found no evidence of AIP production based on MS analysis or bioassays (data not shown). This 

result raises questions over whether SpsB is the only (or even the physiologically relevant) protease involved 

in the final step of AIP maturation. Indeed, it would be nothing short of remarkable if a single protease were 

capable of precisely processing all four groups, given the differences in AIP length. In support of this assertion, 

we have generated a series of S. aureus strains (based on an agr null background), each expressing a unique 

AgrD chimera (by swapping different regions from the 4 groups). The culture supernatants from these cells 

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Contact PD/PI: Muir, Tom 

were isolated and the AIPs generated characterized by mass spectrometry (a segment of this data is 

summarized in Figure 4F). This experiment reveals that the cleavage site (and hence length of the mature AIP) 

is defined by the sequence of the linker region. This strongly argues for the involvement of more than one 

protease in the final maturation step. 

We will identify the protease(s) responsible for processing AgrD(1-32)-thiolactones using two 

orthogonal and unbiased approaches, namely (i) activity-guided fractionation employing synthetic AgrD(1-32)-

thiolactone substrates and a combination of reporter cell assays and ms to identify active fractions, and (ii) 

affinity enrichment employing analogs of AgrD(1-32)-thiolactones containing non-hydrolysable amide isosteres 

(e.g. hydroxyethylene, phosphinates, reduced amides,) at the scissile peptide bond and if necessary 

photocrosslinkers to trap enzyme-substrate complexes for ms analysis. The extensive SAR analysis we have 

performed on the system (e.g. Figure 4F) will guide placement of these crosslinkers. In important preliminary 

studies, we have shown that the activity responsible for processing AgrDI(1-32)-thiolactone is present in the 

supernatant of S. aureus cells, i.e. it is a secreted soluble factor(s) rather than a membrane associated protein 

(Figure 4G). This breakthrough greatly simplifies the proposed biochemical studies, by allowing the use of 

standard chromatographic separations. Once candidate proteases are identified, we will verify using both 

biochemical methods, employing recombinant proteins, and using appropriate genetic deletions in S. aureus. 

AIM 2. To understand the mechanism of activation and inhibition of the agr two component system. 

Perhaps the most remarkable aspect of the agr system is the bacterial interference phenomenon whereby 

AIPs from a different specificity group can actually inhibit the response [17]. This intra-group activation vs. 

inter-group inhibition operates through the receptor histidine kinase, AgrC. We have recently shown that AIPinduced agonism and antagonism of AgrC occurs through opposite helical twisting motions that result in 

rheostat-like control over autokinase activity [46] (Figure 2B). However, several key aspects of receptor 

regulation remain unresolved at the molecular level. The goal of this aim is to provide a detailed understanding 

of how ligand binding differentially affects signaling through changes in the structure and dynamics ofAgrC. 

2.1 : Where is the AIP binding site on AgrC? While it has long been appreciated that AIPs bind the 

sensor domain of AgrC [27], the exact AIP binding site within this multi-pass membrane domain remains 

unknown. Without this information, it is difficult to formulate a testable hypothesis for how AIP binding leads to 

conformational changes in the helical linker that ultimately propagate to altered phosphohistidine levels. We 

will take a multi-pronged approach to this problem. Firstly, we will pursue a comprehensive photocrosslinking 

strategy to identify key contact residues within the sensor domain. In preliminary results, we have generated 

biotinylated analogs of AIP-I in which key residues within the macrocycle and the tail are replaced with 

diazirine or benzophenone containing amino acids (Figure 5A). These peptides not only retain the ability to 

activate AgrC nanodiscs, but become specifically crosslinked to the sensor domain following irradiation (Figure 

5A). Using this in vitro system, we will map the AIP binding site by chemically digesting the crosslinked sensor 

domain followed by SDS-PAGE and MS analysis of the peptides, a process in which we have considerable 

experience [72, 73]. For this, we will use a CNBr digestion protocol employing engineered versions of the 

sensor domain with Met residues strategically incorporated in the sequence. Importantly, we have already 

generated several Met-engineered versions of AgrC-I (retaining full activity) for this purpose (data not shown). 

Note, the presence of the biotin handle on the AIP provides a useful enrichment handle for crosslinked 

peptides. Indeed, in new preliminary data, we have successfully used this crosslinking workflow to show that 

the macrocyclic region of AIP-I (employing M8pM-AIP-I, Figure 5A) interacts with a specific motif in AgrC-I 

encompassing residues 75-90 (Figure 5B). We will use this strategy to further refine the binding site of the 

thiolactone ring by moving the position of the crosslinker. By extension, by placing the crosslinker on the AIP 

tail, we expect to get additional information on the extent of the binding pocket in terms of primary sequence 

coverage. As a further extension of these studies, we will also incorporate a genetically encoded crosslinker, 

benzoylphenylalanine (BPA), into AgrC using amber suppression methods [74]. This powerful approach allows 

residue-level resolution from crosslinking studies without the need for MS analysis [75]. Importantly, in 

extensive preliminary studies we have shown that it is possible to generate active versions of AgrC-I nanodiscs 

containing site-specifically installed BPA (Figure 5C). Ultimately, we imagine that the bi-directionality of the two 

crosslinking strategies, i.e. from the ligand and from the receptor, will allow us to pinpoint the binding site of 

AIP-1 on its receptor, AgrC-I. The crosslinking studies will then be used to guide double mutant cycle 

experiments on the receptor and the ligand in order to confirm a direct interaction [76]. These studies will 

exploit our ability to determine the dissociation constant for the AIP-AgrC interaction using a fluorescencebased assay [46]. We will then go on to perform a similar series of studies to map the binding site of AIP-II on 

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AgrC-I. Notably, AIP-II acts as an inverse agonist of this receptor [77] and so it is conceivable that different 

residues on the receptor are involved in the interaction. 

Currently, there is no high-resolution structural information available for the sensor domain of AgrC, or 

indeed any related sensor. Indeed, as note earlier, there is still some controversy over whether the sensor 

contains 6 or 7 transmembrane (TM) segments [42, 43]. Thus, depending on where the AIP binding site is 

mapped in the primary sequence from the above studies, it may be difficult to deduce how binding translates to 

mechanical motions in the linker helix region without some structural information on the sensor. An important 

exception to this situation would be if the AIP were found to engage residues in the last TM helix of the sensor. 

In this case, we would hypothesize that contacts would lead to conformational changes in the TM segment that 

would directly propagate into the linker helix given that we believe this to be one contiguous secondary 

structure element [46]. Unfortunately, the small size of the AgrC receptor (~45 kDa) places it well below the 

current size threshold for single particle cryo-EM studies on membrane protein preparations, in using 

nanodiscs [80-82]. Given this, we will attempt to identify an AgrC protein that is active and well behaved in a 

detergent-solubilized system suitable for crystallography – ligand affinity, autophosphorylation and 

monodispersity by SEC-MALS will be used to assess favorable properties. AgrC homologs from a number of 

Staphylococci species will be screened. In preliminary studies, we have already successfully over-expressed 

(in E. coli) and purified fully active AgrC homologs from all four S. aureus sub-groups as well as several other 

species including thermophiles (Syntrophothermus lipocalidus, data not shown and manuscript submitted). 

Importantly, these studies will encompass a series of constructs ranging from the full-length receptors down to 

the isolated sensor domains. Once a suitable candidate system is identified, we will initiate crystallization trials 

using dedicated sparse matrix screens optimized for membrane proteins (e.g. MembFac, MiTeGen etc). We do 

not underestimate the technical challenges associated with this undertaking (see Bio for previous structural 

work on membrane proteins in Muir lab). Indeed, this effort is likely to require multiple rounds of optimization, 

both at the level of construct engineering and crystallization conditions. Nonetheless, we are committed to this 

program and believe that the biomedical importance of AgrC coupled with the availability of a range of angonist 

and antagonist ligands make it a superior model system for understanding how ligand binding to an RHK 

sensor leads to transduction of a signal to the HK domain. 

Figure 5: 

Understanding 

the mechanism 

of AgrC 

regulation. (a) 

Crosslinking AIP 

analogs to AgrC 

nanodiscs. (b) 

Results of 

crosslinking 

experiments using 

AIP-1 

M8pM. Right: 

schematic of 

AgrC-I membrane 

topology with 

crosslink site 

shown in green. 

Left: example of 

ms data used to 

define the site of 

crosslinking. (c) Generation of AgrC nanodiscs containing Bpa using amber suppression methodology. (d) Use of GCN4-

AgrC chimera proteins reveals helical twisting controls autokinase activity. (e) Model for AgrC activation based on 

unleashing of the CA domain from the bound state on the DHp domain. 

2.2 : How do ligand-induced helical-twisting motions in AgrC control kinase activity? Our 

assertion that twisting motions in the linker helix leads to changes in autokinase activity is based on two key 

experiments [46]. Firstly, we generated a series of soluble chimeric proteins (i.e. lacking the sensor domain) in 

which the leucine zipper protein, GCN4, was fused to the cytoplasmic domain of AgrC via the linker helix. By 

sequentially moving the fusion junction one residue at a time, we were able to turn the autokinase activity on 

and off in a periodic fashion in the absence of any AIP (Figure 5D). That the periodicity was exactly 3.6 ± 0.1 

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Contact PD/PI: Muir, Tom 

residues strongly argues for helical twisting. This remarkable result was then validated in the context for the 

full-length receptor by using a cysteine crosslinking strategy and employing a range of AIP ligands [46]. The 

assay not only confirmed that AIP binding induces twisting of the linker, but revealed this occurs in different 

directions from the basal state when an agonist binds compared to an inverse agonist (Figure 2B). This 

breakthrough raises the question of how exactly these twisting motions control autokinase activity. The linker 

helix connects the last TM segment of the sensor domain to the so-called dimerization and histidine 

phosphotransder (DHp) subdomain (Figure 2B), a helical hairpin region involved in homodimerization and 

containing the histidine phosphoacceptor residue. The DHp region is then connected via a flexible linker to the 

ATP-binding catalytic (CA) domain. We hypothesize that in the fully off state of the receptor, the CA domain 

engages an exposed surface on the DHp such that phosphorylation of the His is not permitted. In this model, 

binding of agonist leads to an occlusion of this DHp surface and hence unleashing of the CA domain, which is 

now able to engage the exposed histidine (Figure 5E). This type of repositioning has been proposed for other 

systems [83, 84]. We will employ structural and biophysical approaches to test this in the context of AgrC. 

We propose to solve the x-ray structures of a series of GCN4-AgrC chimeras encompassing the “offstate”, “basal-state” and “on-state” of activity. Such a structural series will provide numerous insights into the 

details of AgrC regulation. Note, the design of these proteins will be guided by our previous work, as discussed 

above [46]. In practice, two series of constructs will be used. The first series will encompass the entire 

cytoplasmic domain of AgrC (i.e. DHp + CA) fused to GCN4. In preliminary studies, we have succeeded in 

crystallizing the fusion construct corresponding to the “off-state”. Using a combination of molecular 

replacement (using the structures of GCN4 coiled coil [85] and the isolated CA domain of AgrC [86] as search 

models) and SAD-phasing (employing selenomethionine containing constructs) we have now solved this 

structure at 2.25 Å resolution (Figure 6A). Consistent with our model (Figure 5E), this structure reveals that the 

CA domain is sequestered away from the active histidine through engagement with a surface at the base of the 

DHp domain (Figure 6A, inset). While we will continue to pursue structural studies of the other states, we are 

cognizant that the expected increase in dynamics (i.e. of the unleashed CA domain) associated with these 

constructs could make crystallization challenging. Thus, we will in parallel pursue structural studies on the 

corresponding GCN4-DHp constructs. These proteins are expected to be less dynamic, but should still provide 

insights into which surfaces within the DHp become exposed and occluded on going from the off- to on-state. 

Encouragingly, we have now 

obtained diffraction quality crystals of 

one of these GCN4-DHp constructs 

trapped in the ‘on-state’ (Figure 6B). 

Figure 6: Effect of linker helix 

conformation on CA domain 

dynamics (a) Structure of AgrC-I 

cytoplasmic domain trapped in the ‘offstate’. Inset: close-up of the DHp-CA 

interface. (b) Crystals of GCN4-DHp 

construct trapped in the active state. (c) 

Schematic of single-molecule 

experiment design to probe AgrC 

dynamics as a function of activity state. 

(d) Close-up of the CA of AgrC (pdb: 

4BXI) and the CA of TM853, highlighting 

the difference in the G1 box residues. 

In addition to high-resolution 

structural studies, we will also study 

the dynamic behavior of AgrC using 

single molecule fluorescence 

resonance energy transfer (FRET) 

experiments employing a total internal reflection (TIRF) microscope set-up (Figure 6B). These studies will be 

performed in collaboration Professor Haw Yang (Princeton University, letter attached), an expert in single 

molecule biophysics whose laboratory contains the necessary instrumentation for the proposed experiments. 

For convenience, we will employ our GCN4-AgrC chimeric constructs in these studies, again comparing the 

behavior of constructs that mimic a various activity states (Figure 5D). In practice, we will exploit the dimeric 

nature of these constructs and the fact that autophosphorylation occurs between protomers (i.e. in trans) within 

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Contact PD/PI: Muir, Tom 

AgrC [45]. Thus, an ALEXA 555 donor fluorophore will be incorporated into the DHp of one protomer using 

cysteine chemistry and an ALEXA 647 acceptor will be introduced into the CA domain of the other (Figure 6C). 

Several different pairwise combinations will be tested for an optimal FRET response – the newly available 

crystal structure of the AgrC cytoplasmic domain (see above) will also help guide the placement of 

fluorophores. These two species will then be mixed and subjected to a refolding protocol, thereby giving a 

statistical mixture, 50% of which will contain the desired donor-acceptor pair (note, the remaining components, 

containing one or the other probes, will be silent in the assay). In preliminary results, we have shown that the 

GCN4-AgrC chimeras can be denatured and then efficiently refolded into mono-disperse dimers with retention 

of full autokinase activity (data not shown). Our hypothesis predicts that constructs in different activity states 

will have different time-dependent FRET behavior (Figure 6c). Our recent structural data on the “off-state” 

construct (Figure 6A) suggests that it will primarily exhibit a single FRET state, with only occasional excursions 

to other states reflecting transient unleashing of the CA domain from its DHp docking site. By contrast, we 

predict the more active GCN4-AgrC constructs will exhibit a more complex FRET behavior reflecting disruption 

of this autoinhibitory CA-DHp interaction. This could be manifest in several ways including alteration in the 

dwell time in any given state (on or off) or a change in the number or distribution of FRET states observed 

(schematized in Figure 6c). A unique power of our system is the availability of GCN4 -AgrC constructs spanning 

a range of activities. By c omparing the single molecule FRET behavior of this activity series, it should be 

possible to rigorously model the physical characteristics of the active state of the kinase. 

2.3 : Is AgrC activity tuned to the metabolic state of S. aureus cells? In the final part of this aim, we 

will test our hypothesis that agr activation is internally regulated by the metabolic state of the cell, leading to 

down-regulation in times of stress. This idea derives from our discovery that the Km-ATP of AgrC is in a range 

(low millimolar) that makes it sensitive to changes in ATP/ADP levels that accompany stress [46]. Bioinformatic 

studies point to amino acid substitutions in the ATP-binding pocket of the CA domain as the origin of this low 

ATP affinity. Specifically, AgrC has a non-canonical G1 box compared to kinases that bind to ATP with higher 

affinity [46, 87-89]. Specifically, it lacks two conserved glycine residues and has an Asn residue in place of a 

conserved Asp residue (which engages the adenine ring of ATP) (Figure 6D). Using this information, we 

propose to engineer the CA domain of AgrC to have a higher ATP affinity. We will systematically mutate the 

G1 box, and if necessary second-shell residues, in AgrC-I with the view to lowering Km-ATP of AgrC into a range 

that would make the kinase less sensitive to falling ATP levels during times of stress. If necessary, we will 

employ the ROSETTA design suite developed by Baker and coworkers in these studies [90]. Our existing 

AgrC-nanodisc system will serve as the workhorse for these protein-engineering studies. Once a suitably 

engineered version of AgrC-I is developed, this will then be introduced into an appr opriate S. aureus genetic 

background using established methods. Our hypothesis predicts that cells containing an altered agr circuit 

based on this engineered AgrC protein will have a dramatically different behavior during late-exponential and 

stationary phase, namely the agr response will not be down-regulated as in wild-type cells. 

AIM 3. To identify new pharmacological modulators of the agr system (Completely NEW) 

In this final aim, we will develop new chemistries, and exploit the availability of the reconstituted AgrC/AgrA 

assay system, to identify new chemical probes of agr that will lay the foundation for new therapeutic strategies. 

3.2: Identification of a global activator of agr. Unlike global inhibitors of the agr response, which 

have been known for some time, a global activator (i.e. one that activates all four groups) has never been 

described. Such a compound would disrupt S. aureus biofilms (which occurs upon agr activation [9]), with 

presumptive utility as a coating for medical devices/implants. We propose to identify such a molecule using a 

peptide array system (Figure 7A). In preliminary studies, we have shown that AIP-I can be immobilized to a 

surface through its N-terminus via click chemistry. Importantly, the immobilized pheromone retains the ability to 

active the agr response in S. aureus reporter cells (Figure 7A, inset). With this as a launch pad, we will 

generate spatially defined arrays of AIP analogs using a novel chain inversion strategy. Briefly, we will employ 

standard SPOT peptide synthesis on a nitrocellulose surface functionalized with a bifunctional linker 

possessing an aryl hydrazine and an azide. A spatially defined library of AIP analogs will be synthesized off the 

hydrazine handle using standard Fmoc-SPPS and each peptide will be outfitted with an N-terminal PEG spacer 

followed by a propargyl group. Click chemistry will then be performed (thereby anchoring the peptide to the 

surface through both its termini) followed by protecting group removal and C-terminal cleavage. Subsequent 

oxidation of the C-terminal hydrazide (and spontaneous thiolactone formation [31]) will directly afford the 

immobilized library of thiolactone-containing AIPs. This strategy will allow us to quickly generate hundreds of 

AIP analogs, allowing unprecedented chemical diversity to be explored. As a convenient high-throughput 

readout of activity, we will employ our AgrC nanodiscs in conjunction with our available anti-pHis antibodies – 

activation of AgrC by an immobilized peptide will lead to autophosphorylation which can be readout in an 

Research Strategy Page 74 
 

 

 

 

 

 

 

 

Contact PD/PI: Muir, Tom 

ELISA format. Hits will then be resynthesized and activity verified using established in vitro and cell-based 

assays. Identification of a global activator will open up many exciting new possibilities for exploring agr biology 

(including with our ongoing mechanistic work) and for future biomedical applications. 

Figure 7: Identification of small molecule 

modulators of AgrC. (a) Proposed peptide array 

technology used to identify global agr activators. 

Inset: AIP-I immobilized through click chemistry to a 

passivated glass surface can still activate agr in S. 

aureus reporter cells (GFP readout). (b) Schematic 

of primary screen based on ASMS. SEC: sizeexclusion chromatography. Bottom: hit molecules 

identified in proof-of-principle screen. 

3.2: Identification of small molecule 

modulators or agr. The availability of AgrCnanodiscs provides a powerful system for small 

molecule screens. In proof-of-principle studies, 

we have successfully applied an affinity-based 

small molecule screening (ASMS) strategy to 

discover AIP competitive binders of AgrC-I. 

These studies were performed in the Princeton 

Small Molecule Screening Center. The Center 

possesses both propriety (one million 

compounds) and commercial (~100,000 

compounds) compound collections and is fully equipped to perform both phenotypic (i.e. cell-based) and 

targeted (i.e. using purified components) screens. In ASMS, a series of pooled compound mixtures (grouped 

based on mass considerations) are mixed with the target of interest, in this case AgrC-I-nanodiscs, and then 

resolved by size exclusion chromatography (SEC) [91]. Target-associated compounds are then identified by 

LC-MS/MS using a UPLC-Orbitrap system. In a pilot study, we have performed this analysis on ~50,000 

compounds in the presence and absence of saturating amount of AIP-I. This has led to the identification of 

several AIP-competitive binders of AgrC-I (Figure 7B). Encouraged by these results, we will repeat the ASMS 

screen using the full compound collection available in the Center (see letter of collaboration from Center 

Director, Han Kim). Validation of the hits from the screen will involve multiple phases. Firstly, we will employ a 

newly developed high-throughput fluorescence-based assay involving displacement of a solvatochromic 4-

dimethylamino-phthalimide labeled AIP from AgrC-nanodiscs [92]. We have shown that this system allows 

quantitative measurement of specific binding of ligands to the AgrC sensor domain (data not shown), making it 

an ideal secondary screening assay to measure sensor affinity of initial hits from SEC-based ASMS. 

Compounds that advance from this stage will then be assayed for their ability to activate or inhibit AgrC 

autokinase activity using our established biochemical [31, 46, 93, 94] and cell-based assays [95]. These lead 

molecules will serve as starting points for medicinal chemistry efforts designed to improve both potency and 

efficacy, as moni tored by the in vitro and cell-based assays described above. The Muir laboratory is well 

equipped, both practically and intellectually, to conduct studies of this type (e.g. [95]). 

We stress that the goal of this aim is to identify and then optimize tool compounds (global activators 

and inhibitors of agr) using various biochemical and cell-based assays. We are, however, cognizant that some 

of these modulators could have value as therapeutic leads. While beyond the scope of the current proposal, 

we do note that animal (murine) models of infection are available and have been used successfully to test our 

AIP inhibitors in the past [25, 47]. We envision this as being an exciting line of enquiry for the future. 

SUMMARY: The accessory gene regulator (agr) locus found in all staphylococci encodes a quorum sensing 

(QS) circuit that controls the expression of virulence and other accessory genes. A research program will be 

undertaken to study agr signal transduction in the pathogen, Staphylococcus aureus. Building on recent 

breakthroughs that have allowed us to reconstitute much of the quorum sensing circuit using purified 

components, we will integrate chemical, biochemical, biophysical and genetic tools for the purpose of obtaining 

a deeper understanding into the molecular mechanisms underlying the production and sensing of the 

autoinducer peptide (AIP) pheromone that is central to agr regulation. The program will move forward in three 

directions, namely (i) identifying the key missing players in AIP biosynthesis, (ii) understanding how agonism 

and antagonism of the QS system relates to newly discovered conformational changes in the AIP receptor, 

AgrC, and (iii) identifying small molecule modulators of agr through sophisticated target-based screens. These 

studies will provide fundamental insights into how a QS circuit such as agr operates at the molecular level and 

will lay the foundation for the development of new strategies for treating S. aureus infections. 

Research Strategy Page 75 
Contact PD/PI: Muir, Tom 

Vertebrate animals 

1) Use of animals. The experiments to be conducted here are noted in the experimental approach section 

under Specific Aim 3. 

Mice will be used as described below and tagged with luciferase as described in the proposal. 

Animals to be used will be 8 or 12 week-old outbred Swiss-Webster mice of either sex. 

General Methods 

i) Mouse deep quadriceps (DQ) model. Three infecting doses, 107, 108, 109 bacteria + cytodex beads will 

be delivered in 25 μl PBS. Animals will be imaged daily to evaluate the persistence and localization of the 

infection. Twenty-seven mice will be used for this validation study. 

ii) Mouse kidney abscess model. A reproducible kidney abscess-inducing dose will be determined by IV 

injection of graded doses of bacteria, 107, 108, 109 - 3 mice for each dose, evaluated by IVIS imaging daily for 

5 days. Nine mice will be used for this validation study. 

iii) Toxicity and pharmacokinetics. We propose to test the 4 most effective small molecules (SM1-4) for IV 

toxicity and to measure IV pharmacokinetics. 

a) IV Toxicity: 500 μg/mouse (20 mg/kg). Mice will be monitored for signs of toxicity, including ataxic 

gate, decrease in activity, ruffled fur, weight loss, etc. 3 6-8 week old mice will be used per test, 12 

total. 

b) Pharmacokinetics: Three 12 week-old Swiss-Webster mice of either sex will be used for each 

experiment. Mice will be anesthetized and the femoral vein cannulated. The SM to be tested will be 

injected in 100 μl phosphate buffered normal saline (PBS) and 20 μl samples will be withdrawn for 

processing at 15 min intervals for 90 min. 

iv) Testing for agr inhibition 

a) Correlation between agr inhibition and attenuation of infection (DQ model). It is proposed to 

use 50 μg/mouse of the 4 most effective SMs in this test, 5 mice each for agr+ and agr-bacteria, and 5 

untreated controls. If reproducible attenuation is not observed, we will use 4x this dosage (200 

μg/mouse). Imaging will be performed at 24 h intervals for 5 days, after which mice will be euthanized 

and viable bacteria enumerated in tissue homogenates. 5 mice per group. Mice will be euthanized and 

viable counts compared with luciferase signals. Maximum number of mice - 90. 

b) Efficacy of compound (kidney model). With the most effective SM and dose, we wouldverify 

efficacy in the kidney model, using USA300. This test would use 10 mice – 5 with the SM and 5 

untreated. 

c) Repeated dosage. With the most effective SM and dose, we will test for the effects of repeated 

dosage, administering the molecule at 0, 3 and 6 h after the bacteria. For this test, we will use USA300 

in the DQ model and 10 mice. 

d) Delayed administration. We will test for the effects of delayed administration of the best SM, using 

intervals of 0, 24 or 48 h between injection of bacteria and peptide. These tests will use the most 

effective SM with USA300 in the kidney model and will require 20 mice – 5 for each interval and 5 

untreated. 

Vertebrate Animals Page 76 

v) Synergism with antibiotics. If the hypothesis that inhibiting agr attenuates an infection is not substantiated, 

we will test for SM synergy with vancomycin (vnc) in the DQ model. Each test would use 20 mice – 5 with SM, 

2 mg/kg, 5 with vnc, 33 mg/kg, 5 with both, and 5 untreated, with treatment at 0, 24, or 48 h post infection. If 

the attenuation hypothesis is substantiated, the most effective agr inhibitor would again be tested for synergy 

with vnc. The SM and antibiotic will each be used alone at the same single doses as above, and in 

combination, using the same therapeutic regimen as above. The proposed experiments would utilize 40 mice. 

Testing of additional SMs would require 20 mice each. In the event that we are unable to identify a suitably 

effective small molecule by high-throughput screening, we will do essentially the same experiments as those 

proposed above with 4 different AIP derivatives: AIP-II, AIP-II N3A mutant, and the lactone derivatives of each. 

The maximum number of mice for both of the above sets of experiments would be 440. 

2) Justification for the use of animals. The proposed studies are pre-clinical trials of therapeutic agentsand 
4) Minimization of pain and distress. Animals will be anesthetized with isoflurane for injections and for 

imaging. Infected animals will receive ibuprofen a dosage of 7.5 mg/kg, mixing the liquid form of Children's 

ibuprofen in the drinking water at that dosage. If an animal remains in discomfort despite the presence of 

ibuprofen in the drinking water, Buprenorphine at 0.05-0.1 mg/kg will be administered subcutaneously every 12 

hours. 

Contact PD/PI: Muir, Tom 

therefore can be done only with live animals. Although invertebrate models for studying staphylococcal 

infections are available, we do not believe that results obtained with such models could satisfactorily translate 

to the vertebrate organism. We generally use mice, since these are the “lowest” form of vertebrate suitable for 

such studies. It is noted, parenthetically, that our imaging system enables a 5-10-fold reduction in the numbers 

of animals required for our experiments, in comparison with methods that require point-by-point enumeration of 

bacteria. 

3) Veterinary care. Animals are housed in micro isolator cages, 5 mice to a cage, in an animal room with 

proper ventilation, temperature control, and light-dark cycle, and are given a standard diet with water ad 

libitum. 

Cages are changed weekly by the Skirball animal facility staff. Dead animals are autoclaved before discarding, 

or are dissected for the enumeration of viable bacteria in tissues and organs. In general, intravenous (IV) or 

intraperitoneal (IP) injections will be of 100 μl in PBS. Intramuscular (IM) injections will be of 25 μl of PBS. 

Moribund animals (ruffled fur, ataxic gait) will be euthanized by CO2 narcosis. For imaging, the animal will be 

anesthetized with isoflurane. 

5) Euthanasia. Animals will be euthanized by CO2 narcosis, either at the end of an experiment or during an 

experiment if they show signs of severe morbidity. This is the standard method recommended by the AVMA. 

Vertebrate Animals Page 77 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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