Bile Resistance in Pseudomonas aeruginosa

About This Grant

PROJECT SUMMARY Pseudomonas aeruginosa (Pa), a ubiquitous, environmental Gram-negative bacterium, is a leading cause of healthcare-associated infections worldwide resulting in >400,000 deaths annually. Treatment is challenging because Pa harbors a wide array of intrinsic and acquired antibiotic resistance mechanisms. Of all invasive infection types, Pa bloodstream infections (PABSI) have very poor patient outcomes and bloodstream infections secondary to Pa are more lethal than those of any other bacterium. The reasons for this are unclear. Recently, we were the first to show that during bloodstream infection, Pa traffics to the liver, expands in the gallbladder, and is excreted in the gastrointestinal (GI) tract. Additionally, we showed that the gallbladder was the critical organ that facilitated high level Pa excretion and promoted transmission. To survive in the liver and gallbladder and establish a niche in the GI tract, Pa must be adapted to thrive in bile, a complex fluid produced by the liver, concentrated in the gallbladder and excreted into the GI tract. Bile is composed of bile acids and salts, cholesterol, proteins, and lipids that are inherently antimicrobial. Resisting bile exposure is universally critical for pathogen success in the GI tract and the GI-resident bacterial population serves as a reservoir for invasive Pa infections. To dissect the mechanisms by which Pa resists bile exposure, this project will explore an exciting, newly identified bile resistance sodium-hydrogen antiporter, and, by expanding our bile resistance analysis of multiple Pa strains, identify shared (common) and strain-specific bile resistance pathways. The approach of Aim 1 is two-fold. First, we will investigate the role of the sodium-hydrogen antiporter, shaA-F, in bile resistance. This operon was identified by an early comparative transposon-insertion (INSeq) experiment as critical for bile resistance. Additionally, we will characterize the role of each protein in the sha operon in GI carriage and pathogenesis. Second, to complement genetic approaches, RNA sequencing will be utilized to profile global bacterial transcriptional responses to bile, with an emphasis on understanding if and how sha is regulated by bile exposure. Aim 2 will deploy comparative transposon insertion sequencing (INSeq) across additional representative Pa strains to identify additional shared and strain-specific genes necessary for bile resistance. Following gene deletion, both shared and strain-specific targets will be assayed in vitro and in vivo to determine their impact on bile resistance and Pa pathogenesis in a model of PABSI. The proposed work will produce the first multi-strain comparative analysis of Pa responses to bile exposure by deploying a powerful combination of genetic and transcriptomic approaches. As bile resistance is critical for the establishment of pathogens in the human GI tract and bile exposure increases antimicrobial resistance in many pathogens, we believe that pathways identified by this proposal present exciting new targets for the development of antimicrobial agents to treat Pa infections.