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Regulation of the PhoP-PhoQ two-component system in Pseudomonas aeruginosa and its role in virulence Gellatly, Shaan Lae

Abstract

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that can cause severe infections in individuals with underlying medical conditions. P. aeruginosa primarily infects at epithelial surfaces where it interacts initially via type IV pili, flagella and LPS. Two component regulatory systems control many aspects of pseudomonal physiology and mediate adaptation to environmental changes including those that occur in the host. This thesis outlines the contributions of these systems to the cytotoxicity to epithelial cells and sheds light on the regulation mediated by the two-component sensor PhoQ. Systems that contributed to cytotoxicity fell into several themes including motility, cyclic-di-GMP regulation, and carbon and nitrogen utilization. Several genes controlled by PhoQ were shown to be dysregulated during infection of lung epithelial cells, including upregulation of oprH-phoP-phoQ, the lipid A modification gene arnB, and downregulation of a lipid A deacylase, pagL. Consistent with this, lipid A from a phoQ mutant grown in varying magnesium concentrations displayed alterations. LPS of the phoQ mutant revealed increased inflammatory properties as demonstrated by increased secretion of the cytokines IL6, TNFalpha , and IL10 from PBMCs. The decrease in cytotoxicity of a phoQ mutant correlated with a decrease in secretion of lipases and proteases when co-incubated with cultured epithelial cells. These results suggest that the PhoP-PhoQ system might adapt the bacterium to lung epithelia and that this might contribute to and be exacerbated by the selective pressure of inhaled polymyxin therapeutics. Unlike most sensor kinases that phosphorylate their cognate response regulators, PhoQ of P. aeruginosa appears to act only as a phosphatase of its cognate regulator PhoP. Here it was demonstrated that PhoP was not activated by PhoQ in a luminescence reporter screen. The sensor kinase, RoxS, involved in regulation of the cyanide insensitive oxidase, was revealed as a candidate phosphodonor to PhoP. Since mutation of roxS was able to reduce but not eliminate expression from the oprH-phoP-phoQ operon, it is conceivable that other sensors contribute to PhoP phosphorylation. It was also demonstrated that PhoP contributed to the known polymyxin resistance of a phoQ mutant but only partially to cytotoxicity. These results emphasize the complexity of the PhoP-PhoQ regulatory system.

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