Bile Resistance in Pseudomonas aeruginosa

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.

Grant Number: 1R21AI187937-01
NIH Institute/Center: NIH
Principal Investigator: Kelly Bachta