The long-term goal of this research is to explore the molecular mechanisms that bacteria use for cell-cell communication. Here we propose an integrated structural, chemical, and biological study of recently identified quorum sensing circuits in two related bacteria, Vibrio harveyi and Vibrio cholerae. To develop a molecular understanding of how quorum sensing signals (called autoinducers) are detected, and how sensory information is transduced to control behavior on a community-wide scale, we will carry out in-depth studies that combine synthetic organic chemistry, bacterial genetics, biochemistry, and x-ray crystallography. We will identify signaling agonists and antagonists to provide lead compounds for the development of antibacterial drugs designed to modulate quorum sensing. More generally, a longstanding problem in the bacterial signaling field is to understand how extracellular information is transduced into cells. The proposed studies will further our mechanistic understanding of transmembrane signal transduction via two-component sensor kinases, of which these quorum sensing receptors represent particularly tractable examples. The proposed aims build on significant progress during the first funding period, in which extensive structure/function studies led to a specific mechanistic model for signal transduction by the quorum sensing receptor LuxPQ. This mechanism differs fundamentally from the canonical mechanism based on studies of chemotaxis receptors. In the first aim, we will use molecular genetic approaches coupled with x-ray crystallography to test and extend our model.
The second aim i s to use organic synthesis and high-throughput screening to identify novel LuxPQ agonists and antagonists. Biochemical and structural studies will be used to characterize their mode of action.
Aims 3 and 4 represent a new effort to characterize the molecular mechanisms underlying the dominant quorum sensing pathway in the human pathogen V. cholerae. In preliminary studies, we have determined the chemical structure of the relevant autoinducer, CAI-1. We have purified and crystallized the CAI-1 synthase CqsA, a pyridoxal phosphate enzyme, and in the third aim, we propose to determine its structure, identify its substrates, and characterize its enzymatic mechanism. In the fourth aim, we will combine genetic and chemical screens with x-ray crystallography to probe the molecular details of the interaction between CAI-1 and its cellular receptor.

Public Health Relevance

Quorum sensing is a process of cell-cell communication that allows bacteria to collectively control processes including biofilm formation and the secretion of virulence factors. We propose to study quorum sensing in the major human pathogen, Vibrio cholerae, and to identify molecules that target quorum sensing to inhibit virulence.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI054442-08
Application #
7760655
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Hall, Robert H
Project Start
2003-03-15
Project End
2013-02-28
Budget Start
2010-03-01
Budget End
2011-02-28
Support Year
8
Fiscal Year
2010
Total Cost
$450,657
Indirect Cost
Name
Princeton University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
002484665
City
Princeton
State
NJ
Country
United States
Zip Code
08544
van Kessel, Julia C; Rutherford, Steven T; Shao, Yi et al. (2013) Individual and combined roles of the master regulators AphA and LuxR in control of the Vibrio harveyi quorum-sensing regulon. J Bacteriol 195:436-43
Perez, Lark J; Ng, Wai-Leung; Marano, Paul et al. (2012) Role of the CAI-1 fatty acid tail in the Vibrio cholerae quorum sensing response. J Med Chem 55:9669-81
Shao, Yi; Bassler, Bonnie L (2012) Quorum-sensing non-coding small RNAs use unique pairing regions to differentially control mRNA targets. Mol Microbiol 83:599-611
Wei, Yunzhou; Ng, Wai-Leung; Cong, Jianping et al. (2012) Ligand and antagonist driven regulation of the Vibrio cholerae quorum-sensing receptor CqsS. Mol Microbiol 83:1095-108
Ng, Wai-Leung; Perez, Lark; Cong, Jianping et al. (2012) Broad spectrum pro-quorum-sensing molecules as inhibitors of virulence in vibrios. PLoS Pathog 8:e1002767
Rutherford, Steven T; Bassler, Bonnie L (2012) Bacterial quorum sensing: its role in virulence and possibilities for its control. Cold Spring Harb Perspect Med 2:
Rutherford, Steven T; van Kessel, Julia C; Shao, Yi et al. (2011) AphA and LuxR/HapR reciprocally control quorum sensing in vibrios. Genes Dev 25:397-408
Bolitho, Megan E; Perez, Lark J; Koch, Matthew J et al. (2011) Small molecule probes of the receptor binding site in the Vibrio cholerae CAI-1 quorum sensing circuit. Bioorg Med Chem 19:6906-18
Ng, Wai-Leung; Perez, Lark J; Wei, Yunzhou et al. (2011) Signal production and detection specificity in Vibrio CqsA/CqsS quorum-sensing systems. Mol Microbiol 79:1407-17
Stauff, Devin L; Bassler, Bonnie L (2011) Quorum sensing in Chromobacterium violaceum: DNA recognition and gene regulation by the CviR receptor. J Bacteriol 193:3871-8

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