Browsing by Subject "Neisseria gonorrhoeae"

As bacterial resistance to multiple antibiotics continues to become a growing problem across the globe, the imperativeness for understanding mechanisms of antibiotic and multidrug resistance is increasingly apparent. Currently, the gram-negative bacteria Neisseria gonorrhoeae and Escherichia coli are considered urgent public health threats due to the rise in multidrug resistant strains. Mechanisms by which these bacteria become resistant to antibiotics include the overexpression of multidrug efflux systems. Prior to the introduction of antibiotics, the primary purpose of these multidrug efflux systems was to protect the bacteria from cytotoxins in the environment including innate host defense molecules or toxic molecules produced by the bacteria. Overtime, the bacteria have adapted these efflux systems to protect against clinically relevant antibiotics used to clear these bacterial infections. These multidrug efflux systems are energetically expensive to synthesize; thus, they are often tightly regulated at the transcription level by transcription repressors or activators. Many multidrug efflux systems are regulated by transcriptional regulators with proximal genes that specifically regulate a single multidrug efflux system. However, the expression of a few multidrug efflux systems is controlled by unique repressors that act as global regulators, which have a larger role in regulating complex virulence and stress response systems. Specifically, examples of multidrug efflux regulators in N. gonorrhoeae and E. coli that act as global regulators within their respective genomes include N. gonorrhoeae MtrR and E. coli MprA. Understanding the global regulatory activities of these two transcription regulators will broaden our understanding of the regulatory mechanisms that enable bacterial survival during host infection, the mechanisms that contribute to antibiotic resistance, as well as the fundamentals of bacterial transcription regulation. To provide novel insight into the global regulatory activities and function of N. gonorrhoeae MtrR, this dissertation expounds a series of original structural, biochemical, and in vivo studies identifying cytotoxin and DNA recognition mechanisms of MtrR. Previous work showed that MtrR represses directly the mtrCDE efflux transporter genes by binding an operator between the mtrR and mtrC genes; additionally, MtrR represses directly the rpoH oxidative stress response sigma factor. MtrR-mediated repression of the mtrCDE genes had been shown to be relieved upon exposure of gonococci to toxic hydrophobic agents and detergents (i. e. MtrR is “induced” by these toxic molecules). However, physiologically relevant innate host molecules recognized by MtrR had not been identified. In this work, we identify bile salts present at extra-urogenital gonococcal infection sites that MtrR directly binds, to result in derepression of the mtrCDE genes in vitro and in vivo. Furthermore, we use x-ray crystallography to solve structures of MtrR in its induced form and bound to the mtrCDE and rpoH operators. With these structures, we determined the structural mechanism of induction of MtrR. In addition, the MtrR-operator structures reveal a degenerate consensus sequence to which MtrR binds within the mtrCDE and rpoH operators. Mechanisms for cytotoxin and DNA recognition were confirmed by structure-guided site-directed mutagenesis studies and a combination of biochemical binding assays utilizing isothermal titration calorimetry (ITC) or fluorescence polarization (FP). Importantly, this structural and biochemical work also reveals the mechanisms by which common mutations in multidrug resistant strains of N. gonorrhoeae confer resistance. To elucidate the function of E. coli MprA and realize its potential as a drug target, this dissertation also includes research describing the ligand-binding mechanisms of MprA. MprA (formerly EmrR) represses directly the EmrAB efflux pump in E. coli. Previously published work identified MprA as the molecular target of a small molecule inhibitor (DU011) of the biosynthesis of an important virulence factor in E. coli, the polysaccharide capsule. This lead molecule has the potential for optimization for drug development and reveals a novel function of MprA as a regulator of polysaccharide capsule synthesis. We characterized the interaction between MprA and DU011 and compared this to the binding between MprA and other previously identified ligands including salicylate and 2,4-dinitrophenol (DNP) utilizing ITC assays. Through these studies, we revealed a novel binding mode for MprA and laid the groundwork for future structural studies and drug optimization. Collectively, this work provides important insight into the breadth of regulatory functions of N. gonorrhoeae MtrR and E. coli MprA, two key global regulators from highly prevalent multidrug resistant pathogens. Specifically, the original research presented here provides a biochemical evaluation of the bacterial stress response mechanisms controlled by MtrR and MprA and their contribution to antibiotic resistance. Indeed, the biochemical and structural characterization of these two regulators will inform future work to combat multidrug resistance.

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