Kavanaugh, L. G. | bioRxiv Science

bioRxiv2024

Determination of Pseudomonas aeruginosa MexXY-OprM specificity in a major efflux knockout system reveals distinct antibiotic substrate classes

Defining the substrates of Resistance-Nodulation-Division (RND) systems is often complicated by the presence of multiple systems in a single bacterial species. Using a major efflux knockout strain, we developed a Pseudomonas aeruginosa system that enables controlled expression of MexXY-OprM in the absence of background efflux complexes with overlapping substrate profiles. This system identified three groups of potential substrates: substrates, partial substrates, and nonsubstrates, and defined trimethoprim as a new substrate for MexXY-OprM.

microbiology↗

bioRxiv2023

Berberine dimers as chemical probes of Pseudomonas aeruginosa MexXY-OprM efflux function and inhibition

The Resistance-Nodulation-Division (RND) efflux pump superfamily is pervasive among Gram-negative pathogens and contributes extensively to clinical antibiotic resistance. The opportunistic pathogen Pseudomonas aeruginosa contains 12 RND-type efflux systems, with four contributing to resistance including MexXY-OprM which is uniquely able to export aminoglycosides. At the site of initial substrate recognition, small molecule probes of the inner membrane transporter (e.g., MexY) have potential as important functional tools to understand substrate selectivity and a foundation for developing adjuvant efflux pump inhibitors (EPIs). Here, we optimized the scaffold of berberine, a known but weak MexY EPI, using an in-silico high-throughput screen to identify di-berberine conjugates with enhanced synergistic action with aminoglycosides. Further, docking and molecular dynamics simulations of di-berberine conjugates reveal unique contact residues and thus sensitivities of MexY from distinct P. aeruginosa strains. This work thereby reveals di-berberine conjugates to be useful probes of MexY transporter function and potential leads for EPI development.

microbiology↗