Two critical hallmarks of chronic Pseudomonas aeruginosa infection in patients with cystic fibrosis (CF) are the appearance of alginate over-producing mucoid colonies and the development of antibiotic resistance. A rapid emergence of antibiotic resistance in P. aeruginosa isolates in CF patients due to in vivo selection of strains that overproduce AmpC ?-lactamase, an enzyme that hydrolyzes the ?-lactams, contributes to both morbidity and mortality. Previous studies investigating the regulation of the amp genes in P. aeruginosa using genetic and molecular biological techniques led to the identification of a second ?-lactamase, PoxB oxacillinase. We showed that the expression of both ampC and poxB are under the control of AmpR, a member of the LysR family of transcriptional regulators. We have demonstrated that AmpR is a global transcriptional regulator which regulates expression of many virulent genes that are important for establishing chronic infections. Our long-term goal is to establish the regulatory circuit that exists between the amp and pox genes and their role in the development of resistance to ?-lactams and the establishment of chronic infection. To date, we know very little about the molecular mechanisms that govern the expression of pox operon. The experiments proposed in this grant will extend our understanding as they will focus on (1) characterizing the poxAB operon and determining if AmpR regulation is by transcriptional derepression, and (2) determining if expression of the poxAB operon is regulated by a previously uncharacterized two-component system. These analyses will provide invaluable insight into the bacterial adaptation to the onslaught of antibiotics and the immune response. It will likely lead to novel therapeutic approaches in combating the persistent P. aeruginosa infection that ultimately leads to the untimely death of CF patients.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Enhancement Award (SC1)
Project #
5SC1AI081376-04
Application #
8120231
Study Section
Special Emphasis Panel (ZGM1-MBRS-8 (MV))
Program Officer
Taylor, Christopher E,
Project Start
2008-08-01
Project End
2013-07-31
Budget Start
2011-08-01
Budget End
2013-07-31
Support Year
4
Fiscal Year
2011
Total Cost
$308,732
Indirect Cost
Name
Florida International University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
071298814
City
Miami
State
FL
Country
United States
Zip Code
33199
Zincke, Diansy; Balasubramanian, Deepak; Silver, Lynn L et al. (2016) Characterization of a Carbapenem-Hydrolyzing Enzyme, PoxB, in Pseudomonas aeruginosa PAO1. Antimicrob Agents Chemother 60:936-45
Balasubramanian, Deepak; Kumari, Hansi; Mathee, Kalai (2015) Pseudomonas aeruginosa AmpR: an acute-chronic switch regulator. Pathog Dis 73:1-14
Balasubramanian, Deepak; Kumari, Hansi; Jaric, Melita et al. (2014) Deep sequencing analyses expands the Pseudomonas aeruginosa AmpR regulon to include small RNA-mediated regulation of iron acquisition, heat shock and oxidative stress response. Nucleic Acids Res 42:979-98
Caille, Olivier; Zincke, Diansy; Merighi, Massimo et al. (2014) Structural and functional characterization of Pseudomonas aeruginosa global regulator AmpR. J Bacteriol 196:3890-902
Kumari, Hansi; Murugapiran, Senthil K; Balasubramanian, Deepak et al. (2014) LTQ-XL mass spectrometry proteome analysis expands the Pseudomonas aeruginosa AmpR regulon to include cyclic di-GMP phosphodiesterases and phosphoproteins, and identifies novel open reading frames. J Proteomics 96:328-342
Balasubramanian, Deepak; Schneper, Lisa; Kumari, Hansi et al. (2013) A dynamic and intricate regulatory network determines Pseudomonas aeruginosa virulence. Nucleic Acids Res 41:1-20
Balasubramanian, Deepak; Schneper, Lisa; Merighi, Massimo et al. (2012) The regulatory repertoire of Pseudomonas aeruginosa AmpC ýý-lactamase regulator AmpR includes virulence genes. PLoS One 7:e34067
Balasubramanian, Deepak; Kong, Kok-Fai; Jayawardena, Suriya Ravi et al. (2011) Co-regulation of {beta}-lactam resistance, alginate production and quorum sensing in Pseudomonas aeruginosa. J Med Microbiol 60:147-56
Kong, Kok-Fai; Schneper, Lisa; Mathee, Kalai (2010) Beta-lactam antibiotics: from antibiosis to resistance and bacteriology. APMIS 118:1-36
Kong, Kok-Fai; Aguila, Alian; Schneper, Lisa et al. (2010) Pseudomonas aeruginosa ?-lactamase induction requires two permeases, AmpG and AmpP. BMC Microbiol 10:328

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