One of these families is the RND (resistance nodulation division) efflux system. RND efflux systems are tripartite efflux pumps comprised of three components: an inner membrane transporter protein (IMT) (e.g. MexB, MexY), a periplasmic membrane fusion protein (MFP) (e.g. MexA, MexX), and an outer membrane protein channel (OprM). The three components work in concert to efflux a variety of substrates including most classes of antibiotics and naturally occurring compounds such as quorum sensing molecules and bile salts. P. aeruginosa RND efflux pumps have also been implicated in invasion, adherence, and colonization of the human host. Thus, these systems make an attractive target for new antimicrobial drug discovery.
Methods: We are currently studying two RND efflux pumps in P. aeruginosa – MexAB-OprM and MexXY-OprM. Each pump has a unique substrate signature. Utilizing a DNA-barcoded small molecule library that blankets the chemical space, we are developing inhibitors targeted at MexA and MexX, the MFP protein of each system. MFPs form multimers that link the inner membrane (IM) transporter with the outer membrane (OM) channel. The goal is to discover MFP inhibitors that interfere with oligomerization and/or the interaction(s) with the IM and OM proteins.
Results: We have purified each target protein and specific mutants with well characterized functional defects. These will be used to assay for inhibitory compounds. In addition, efflux mutant strains (ΔmexA, ΔmexR, ΔmexX, ΔmexZ, and ΔoprM) and their complemented strains have been characterized with respect to sensitivity to common antibiotics.
Conclusion: Through our study of MexA and MexX, we seek to develop novel efflux inhibitors that target MDR P. aeruginosa by inhibiting efflux.