Pseudomonas aeruginosa is an opportunistic pathogen that causes a multitude of infections, which are difficult to treat because of this bacterium's intrinsic and acquired antibiotic resistance. This antibiotic resistance can be attributed to synergy between a low-permeability outer membrane (OM) and active efflux from the cell. The genome of P. aeruginosa encodes 35 proposed drug efflux systems belonging to five different families, but our working hypothesis is that the clinically relevant efflux pumps contributing to intrinsic and/or acquired drug resistance belong to the resistance nodulation division (RND) family which contains 12 members. Genome sequence and expression analysis of these systems poses several questions that remain unanswered. First, because only a subset of the RND operons contains genes for OM channel proteins, the question is whether these systems must recruit other OM channels from elsewhere on the genome to function as tripartite efflux systems for drug efflux across the entire cell envelope, as current dogma says, or whether they can function as two-component systems for other substrates. Second, with the exception of a single pump, the other RND pumps are tightly regulated but the regulatory networks governing their expression and the inducing substrates remain unknown. Our other working hypotheses therefore are: (i) efflux pumps not encoding their own OM channels may either recruit hitherto unidentified proteins for function with certain substrates but may also function as two-component efflux systems for other substrates; (ii) Efflux pump expression is governed by specific and probably also global transcriptional regulators. We will test these hypotheses using the recently discovered MexJK efflux pump that is normally silent and does not encode its own OM channel. We also propose to amend these studies to probe whether any global transcriptional regulators may be involved in RND efflux pump expression. Specifically we propose to:
Aim 1 -Establish the regulation of MexJK by its cognate regulator MexL using biochemical and molecular studies; identify an inducer that may be present in certain growth media; identify a putative activating factor present in P. aeruginosa but absent in E. coil.
Aim 2 : Establish the molecular architecture of MexJK, specifically its OM membrane channel requirement, if any, using genetic and biochemical methods.
Aim 3 : Probe other, perhaps global regulators of efflux operon expression, specifically MexS and PA4878.
