Targeting intrinsic resistance to combat multi-drug resistant P. aeruginosa

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 DESCRIPTION (provided by applicant): Antibiotic resistance is often thought of as a trait acquired by previously susceptible bacteria, either by horizontal gene transfer or by de novo mutations in chromosomal genes, and selected for by antibiotic exposure. However, the spectrum of antibiotic activity is even more limited by intrinsic antibiotic resistance, a phenotyp mediated by many gene products that limit access of antibiotics to their bacterial targets. I propose to identify intrinsic resistance mechanisms that limit utility of multiple antibiotic classs against the human pathogen Pseudomonas aeruginosa. This study will increase our basic understanding of mechanisms that contribute to P. aeruginosa cell envelope homeostasis under stress conditions, which are often distinct from those used by Enterobacteriaceae. These studies are significant, as intrinsic resistance mechanisms represent new targets for the development of antimicrobials against a clinically important and often-multidrug resistant pathogen. Two independent and complementary approaches will be employed to achieve this goal. First, I will use the novel tool of intrabodies. These heavy-chain-only IgGs of camelid origi will be expressed in the bacterial cytoplasm and periplasm, assayed for their ability to attenuate intrinsic antibiotic resistance or cell wall integrity, and then used to identify and purify their bacterial targets. In parallel, I will construct an InSeq library in P. aeruginosa and use this resource to identify and map transposon insertions that diminish bacterial fitness in the presence of antibiotics. Information obtained through these two separate approaches will be combined to generate a comprehensive list of P. aeruginosa gene products that contribute to intrinsic resistance. Bacterial proteins that play critical roles in maintaining intrinsic antibiotc resistance have not been heavily exploited in antibiotic development to date, though this approach has many advantages. Intrinsic resistance mechanisms are conserved among all members of a species, not just the subset that have acquired antibiotic resistance through mutation or acquisition of new genetic information; they promote bacterial homeostasis and survival in response to multiple antibiotic- or immune-mediated stresses, not just a single antimicrobial; they are often the substrate for acquired resistance, and their derepression or overexpression can lead to high level acquired antibiotic resistance. Thus, identifying and disarming mechanisms of intrinsic resistance is likely to have a broad impact on antimicrobial susceptibility of P. aeruginosa, making this course of study highly significant.