The assembly of R-bodies in Pseudomonas aeruginosa and their contribution to toxicity in phagocytes

About This Grant

PROJECT SUMMARY Pseudomonas aeruginosa is a leading cause of bacterial infections, including chronic wound and lung infections that burden healthcare systems worldwide. It is also one of the six ESKAPE bacteria, a group of opportunistic pathogens that contribute most significantly to the antimicrobial resistance crisis and the challenge of treating recalcitrant infections. In such pathogens, traits that confer survival in the environment are co-opted to support host colonization and enable prolonged infection; disrupting these traits is a promising strategy for weakening pathogen resilience and aiding bacterial clearance. We recently discovered that intriguing structures called R-bodies, which are bacterial weapons that kill grazing protists, contribute to host colonization and damage and to virulence in P. aeruginosa. Our overarching objectives are to elucidate how R-bodies are made, how they function, and how a putative R-body-associated toxin harms host cells so that (i) R-bodies can be exploited in biotechnological applications and (ii) molecular mechanisms of R-body-mediated virulence can be inhibited to fight P. aeruginosa infections. R-bodies are coiled protein ribbons that extend into needle-like structures inside host cells and that are hypothesized to deliver bacterial toxins that kill the eukaryotic cell, ultimately protecting kin bacteria from phagocytic attack. We have shown that R-body production is conferred by the reb cluster, a genomic locus that is recognized for its strong association with virulence in P. aeruginosa clinical isolates. In addition to apparent R-body structural genes, the reb cluster contains other uncharacterized genes that contribute to host damage, including a reduction in host RNA and host translational inhibition. However, the mechanisms whereby reb cluster products orchestrate R-body synthesis and deleterious effects on the host are not understood. The goal of the proposed work is to test the model that the P. aeruginosa R-body supports delivery of an effector that disables predatory and phagocytic cells, ultimately protecting the bacterial population. We will use genetic, biochemical, structural-biology, microscopic-analysis, and infection-model approaches to investigate the factors enabling R-body assembly and function (Aim 1); characterize the roles of R-bodies in determining outcomes when bacterial populations are attacked by amoebae or human macrophages (Aim 2); and determine the contributions of another reb-cluster product, PA14_27675, to the molecular pathology of infection (Aim 3). Elucidating the R-body-mediated mechanisms that confer virulence in P. aeruginosa will help us to design strategies for treating the devastating infections caused by this bacterium.