Previous work in our laboratory and by others, has demonstrated the complexity of regulatory networks that coordinately control the expression of virulence factors in P. aeruginosa. We have used genome-wide transposon mutant analyses to define the functions of two-component regulatory systems and enzymes involved in synthesis or degradation of second messenger c-diGMP. One of the consistent features of phenotypes of strains containing mutations in these regulators is the inverse relationship between expression of genes involved in production and secretion of toxins and those responsible for biofilm formation. By analyzing the phenotypes of a set of mutants with insertions in genes encoding diguanylate cyclases and phosphodiesterases (the DDGEF and EAL domain-containing proteins) we identified a subset of six genes that control the expression of virulence factors. A detailed analysis of these novel virulence regulators will be the subject of the proposed work. This research plan will focus on establishing the role of c-diGMP production and metabolism in the expression of various virulence traits by generating active site mutants in the diguanylate cyclases and phosphodiesterases, which we have recently identified as regulators of virulence. A proteome-wide screen will be undertaken to identify c-diGMP binding proteins that serve as adapters for transmission of signals between diguanylate cyclases and effectors resulting in regulation of virulence genes. Finally, we will undertake a detailed analysis of the novel three components RocS1/RocR/ RocA1 signal transduction system in order to determine the mechanism of action of signaling domains in global transcriptional responses involving the second messenger c-diGMP. This work will be the basis of improving our understanding of the molecular circuitry, which assimilates and accurately transduces environmental stimuli, allowing pathogens to respond to and thrive in the environment of the infected host.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AI021451-20
Application #
7086992
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
Taylor, Christopher E,
Project Start
1984-08-01
Project End
2010-03-31
Budget Start
2006-04-01
Budget End
2007-03-31
Support Year
20
Fiscal Year
2006
Total Cost
$413,792
Indirect Cost
Name
Harvard University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Han, Kook; Tjaden, Brian; Lory, Stephen (2016) GRIL-seq provides a method for identifying direct targets of bacterial small regulatory RNA by in vivo proximity ligation. Nat Microbiol 2:16239
Caille, Olivier; Zincke, Diansy; Merighi, Massimo et al. (2014) Structural and functional characterization of Pseudomonas aeruginosa global regulator AmpR. J Bacteriol 196:3890-902
Cattoir, V; Narasimhan, G; Skurnik, D et al. (2013) Transcriptional response of mucoid Pseudomonas aeruginosa to human respiratory mucus. MBio 3:e00410-12
Skurnik, David; Roux, Damien; Cattoir, Vincent et al. (2013) Enhanced in vivo fitness of carbapenem-resistant oprD mutants of Pseudomonas aeruginosa revealed through high-throughput sequencing. Proc Natl Acad Sci U S A 110:20747-52
Skurnik, David; Roux, Damien; Aschard, Hugues et al. (2013) A comprehensive analysis of in vitro and in vivo genetic fitness of Pseudomonas aeruginosa using high-throughput sequencing of transposon libraries. PLoS Pathog 9:e1003582
Koh, Andrew Y; Mikkelsen, Per J; Smith, Roger S et al. (2010) Utility of in vivo transcription profiling for identifying Pseudomonas aeruginosa genes needed for gastrointestinal colonization and dissemination. PLoS One 5:e15131
Thaden, Joshua T; Lory, Stephen; Gardner, Timothy S (2010) Quorum-sensing regulation of a copper toxicity system in Pseudomonas aeruginosa. J Bacteriol 192:2557-68
Mulcahy, Lawrence R; Burns, Jane L; Lory, Stephen et al. (2010) Emergence of Pseudomonas aeruginosa strains producing high levels of persister cells in patients with cystic fibrosis. J Bacteriol 192:6191-9
Hurley, Bryan P; Goodman, Andrew L; Mumy, Karen L et al. (2010) The two-component sensor response regulator RoxS/RoxR plays a role in Pseudomonas aeruginosa interactions with airway epithelial cells. Microbes Infect 12:190-8
Lory, Stephen; Merighi, Massimo; Hyodo, Mamoru (2009) Multiple activities of c-di-GMP in Pseudomonas aeruginosa. Nucleic Acids Symp Ser (Oxf) :51-2

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