Conventional therapies have proven to be inadequate in the treatment of many if not most chronic biofilm infections due to the extraordinary tolerance of biofilms to antimicrobial treatments. We have reason to believe that we have identified a biofilm-specific regulatory circuit required to induce the transition of biofilm bacteria from a susceptible to a drug resistant state. This pathway relies on the membrane-bound two-component sensor SagS and the MerR-like, c-di-GMP responsive transcriptional regulator BrlR (PA4878). BrlR activates the expression of several genes encoding proteins involved in multidrug efflux, thus, affecting MIC, susceptibility, and recalcitrance of P. aeruginosa biofilms o killing by bactericidal antimicrobial agents. SagS contributes to brlR expression and BrlR DNA binding in a manner dependent on c-di-GMP levels. Induction of brlR expression is linked to biofilm development, with transition to the irreversible attachment stage, regulated by SagS, marking the timing of biofilm cells gaining their heightened tolerance to antimicrobial agents. Our findings thus, suggested SagS to act as a molecular switch to control biofilm formation and tolerance. However, little is known about how this is accomplished by SagS. Understanding the mechanism underlying this switch, however, will enable us to manipulate the biofilm tolerance response, with the potential to impair the transition of biofilm bacteria from a susceptible to a resistant phenotype likely resulting in enhanced treatment options in fighting biofilm infections. The goal of this project is to elucidate SagS-dependent signaling and regulatory events contributing to biofilm cells transitioning to an antimicrobial tolerant state in a BrlR-dependent manner. The project is founded on the hypotheses that BrlR and SagS are linked via a signaling cascade, requiring in a sequential manner the action of SagS, in particular its periplasmic sensory HmsP domain, SagS-signaling to activate DGC PA3177, with PA3177 contributing to the biofilm c-di-GMP level which in turn is required to activate BrlR via c-di-GMP binding and thus, biofilm tolerance. Experimentally, we will determine in Aim 1 the contribution of the HmsP domain of SagS to its function as a molecular switch by determining residues or extended sequences within the HmsP domain, responsible for SagS contributing to in vitro and in vivo biofilm tolerance and biofilm formation, and interaction with BfiSR.
In Aim 2, we will determine how SagS, in a manner dependent on input and signaling, contributes to DGC PA3177 to modulate biofilm c-di-GMP levels.
In Aim 3, we will determine how PA3177 and c-di-GMP contribute to BrlR function, identify BrlR amino acid residues contributing to c-di-GMP binding, and determine whether impaired c-di-GMP binding by BrlR results in biofilm cells impaired in transitioning to an antimicrobial tolerant state.

Public Health Relevance

The bacterium Pseudomonas aeruginosa is the cause of many serious human infections, particularly in hospitalized patients, and/or those with weak immune systems, due to the ability of P. aeruginosa to form biofilms, or highly structured, surface-associated communities that are highly refractory to antimicrobial treatment. This proposed research is aimed at understanding the mechanism of biofilm tolerance by elucidating SagS signaling and regulatory events contributing to biofilm cells transitioning to an antimicrobial tolerant state. In the long-term, SagS and BrlR could be a target for the design of new therapeutic strategies in the treatment and management of biofilm infections in humans

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Bacterial Pathogenesis Study Section (BACP)
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Lu, Kristina
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State University of NY, Binghamton
Schools of Arts and Sciences
United States
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Poudyal, Bandita; Sauer, Karin (2018) The ABC of Biofilm Drug Tolerance: the MerR-Like Regulator BrlR Is an Activator of ABC Transport Systems, with PA1874-77 Contributing to the Tolerance of Pseudomonas aeruginosa Biofilms to Tobramycin. Antimicrob Agents Chemother 62:
Dingemans, Jozef; Poudyal, Bandita; Sondermann, Holger et al. (2018) The Yin and Yang of SagS: Distinct Residues in the HmsP Domain of SagS Independently Regulate Biofilm Formation and Biofilm Drug Tolerance. mSphere 3:
Poudyal, Bandita; Sauer, Karin (2018) The PA3177 Gene Encodes an Active Diguanylate Cyclase That Contributes to Biofilm Antimicrobial Tolerance but Not Biofilm Formation by Pseudomonas aeruginosa. Antimicrob Agents Chemother 62:
Petrova, Olga E; Gupta, Kajal; Liao, Julie et al. (2017) Divide and conquer: the Pseudomonas aeruginosa two-component hybrid SagS enables biofilm formation and recalcitrance of biofilm cells to antimicrobial agents via distinct regulatory circuits. Environ Microbiol 19:2005-2024
Chambers, Jacob R; Cherny, Kathryn E; Sauer, Karin (2017) Susceptibility of Pseudomonas aeruginosa Dispersed Cells to Antimicrobial Agents Is Dependent on the Dispersion Cue and Class of the Antimicrobial Agent Used. Antimicrob Agents Chemother 61:
Petrova, Olga E; Sauer, Karin (2017) High-Performance Liquid Chromatography (HPLC)-Based Detection and Quantitation of Cellular c-di-GMP. Methods Mol Biol 1657:33-43
Chambers, Jacob R; Sauer, Karin (2017) Detection of c-di-GMP-Responsive DNA Binding. Methods Mol Biol 1657:293-302
Chambers, Jacob R; Sauer, Karin (2017) Detection of Cyclic di-GMP Binding Proteins Utilizing a Biotinylated Cyclic di-GMP Pull-Down Assay. Methods Mol Biol 1657:317-329
Petrova, Olga E; Sauer, Karin (2016) Escaping the biofilm in more than one way: desorption, detachment or dispersion. Curr Opin Microbiol 30:67-78
Gupta, Kajal; Liao, Julie; Petrova, Olga E et al. (2014) Elevated levels of the second messenger c-di-GMP contribute to antimicrobial resistance of Pseudomonas aeruginosa. Mol Microbiol 92:488-506

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