New approaches to antimicrobial drug discovery are urgently needed to combat untreatable infections caused by antibiotic resistant or persistent, antibiotic tolerant, bacteria. The long-term goal of this proposal is to develop novel therapy options that may prevent or reduce the complications of human bacterial acute and chronic or relapsing infections, and that could serve as alternatives or adjuncts to antibiotics. To achieve a paradigm shift in antimicrobial therapy, we propose to develop inhibitors of bacterial signaling pathways that control bacterial virulence and antibiotic tolerance mechanisms. Population density-dependent signaling, generally referred to as quorum sensing (QS), is one such mechanism. QS regulates multiple aspects of virulence. It is important for the development of acute infections and as recently discovered for the formation of antibiotic-tolerant cell populations, a process underlying pathogen persistence in chronic infections. This renewal application will test the hypothesis that novel inhibitors of QS, previously identified by us, can lead to the development of highly potent compounds with anti-infective activity in vivo. We will test this hypothesis by employing Pseudomonas aeruginosa, a recalcitrant Gram-negative bacterium that defies eradication by antibiotics and exemplifies current problematic pathogens in hospitals and intensive care units. In the last funding cycle, we demonstrated that P. aeruginosa pathogenesis can be disrupted in vivo by pharmacologically interfering with the multiple virulence factor regulator (MvfR) regulon, a component of QS circuitry that controls virulence and, as we recently discovered, the formation of antibiotic tolerant cells. We elucidated the mechanism of MvfR regulon activation and identified several small chemical compounds that inhibit the MvfR transcription factor and/or interfere with MvfR regulon activity in vivo. Using these chemical compounds, we will test our hypothesis through three Specific Aims: 1) To study the mechanisms of action of the candidate compounds using biochemical, mass spectrometric, and molecular genetic analyses. 2) To improve the most potent QS inhibitors through structure-activity relationship (SAR) studies. 3) To validate the in vivo efficacy of SAR-improved inhibitors in attenuating P. aeruginosa infection in suitable animal models. QS signaling circuits are evolutionarily conserved and play central roles in modulating virulence mechanisms in many different human pathogens. Therefore, by selectively interfering with QS, our data should yield paradigmatic insights that will be generally relevant for the development of new classes of anti-infectives that could limit development of multi-drug resistance and antibiotic tolerance in bacterial pathogens, while preserving beneficial commensal bacteria. Moreover, the study and inhibition of mechanisms involved in antibiotic tolerance could have a major impact on elucidating this unresolved phenomenon, as well as enabling the discovery of the first probe compounds targeted against antibiotic tolerant cells.

Public Health Relevance

Enter the text here that is the new public health relevance information for your application. Using no more than two or three sentences, describe the relevance of this research to public health. Antibiotic resistant and tolerant microbes are responsible for a substantial portion of human infections and are a growing threat to human health worldwide. The goal of this project is to develop novel classes of antimicrobial agents that prevent the formation of multi-antibiotic resistance and antibiotic-tolerant bacteria and enable their eradication by selectively interfering with pathways that mediate virulence. Data derived from the innovative approach proposed will reveal the importance of anti-virulence therapy, the mechanisms that mediate virulence and antibiotic tolerance, and the classes of antimicrobial agents that can target such mechanisms.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
High Priority, Short Term Project Award (R56)
Project #
2R56AI063433-06A1
Application #
8528902
Study Section
Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
Program Officer
Taylor, Christopher E,
Project Start
2012-08-21
Project End
2013-07-31
Budget Start
2012-08-21
Budget End
2013-07-31
Support Year
6
Fiscal Year
2012
Total Cost
$474,950
Indirect Cost
$192,041
Name
Massachusetts General Hospital
Department
Type
DUNS #
073130411
City
Boston
State
MA
Country
United States
Zip Code
02199
Maura, Damien; Hazan, Ronen; Kitao, Tomoe et al. (2016) Evidence for Direct Control of Virulence and Defense Gene Circuits by the Pseudomonas aeruginosa Quorum Sensing Regulator, MvfR. Sci Rep 6:34083
Hazan, Ronen; Que, Yok Ai; Maura, Damien et al. (2016) Auto Poisoning of the Respiratory Chain by a Quorum-Sensing-Regulated Molecule Favors Biofilm Formation and Antibiotic Tolerance. Curr Biol 26:195-206
Maura, Damien; Ballok, Alicia E; Rahme, Laurence G (2016) Considerations and caveats in anti-virulence drug development. Curr Opin Microbiol 33:41-46
Hazan, Ronen; Maura, Damien; Que, Yok Ai et al. (2014) Assessing Pseudomonas aeruginosa Persister/antibiotic tolerant cells. Methods Mol Biol 1149:699-707
Baldini, Regina L; Starkey, Melissa; Rahme, Laurence G (2014) Assessing Pseudomonas virulence with the nonmammalian host model: Arabidopsis thaliana. Methods Mol Biol 1149:689-97
Starkey, Melissa; Lepine, Francois; Maura, Damien et al. (2014) Identification of anti-virulence compounds that disrupt quorum-sensing regulated acute and persistent pathogenicity. PLoS Pathog 10:e1004321
Dulcey, Carlos Eduardo; Dekimpe, Valérie; Fauvelle, David-Alexandre et al. (2013) The end of an old hypothesis: the pseudomonas signaling molecules 4-hydroxy-2-alkylquinolines derive from fatty acids, not 3-ketofatty acids. Chem Biol 20:1481-91
Que, Yok-Ai; Hazan, Ronen; Strobel, Benjamin et al. (2013) A quorum sensing small volatile molecule promotes antibiotic tolerance in bacteria. PLoS One 8:e80140
Kefala, Katerina; Kotsifaki, Dina; Providaki, Mary et al. (2012) Purification, crystallization and preliminary X-ray diffraction analysis of the C-terminal fragment of the MvfR protein from Pseudomonas aeruginosa. Acta Crystallogr Sect F Struct Biol Cryst Commun 68:695-7
Hazan, Ronen; Que, Yok-Ai; Maura, Damien et al. (2012) A method for high throughput determination of viable bacteria cell counts in 96-well plates. BMC Microbiol 12:259

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