Background: Multidrug resistant Gram-negative bacteria are a major threat to human health and new strategies to tackle these organisms are urgently needed. In Pseudomonas aeruginosa, four tripartite Mex efflux pumps (EPs) are major determinants for both intrinsic and acquired antibiotic resistance. Hence, inhibition of these EPs has been proposed as a promising strategy to potentiate the efficacy of existing antimicrobials. However, Mex EPs natural substrates and roles of P. aeruginosa are not yet fully known. In this work, we aimed at elucidating the natural substrates of Mex RNDs in P. aeruginosa to in a second phase evaluate their potential as competitive efflux pumps inhibitors to potentialize known antibiotics. To do so, we used biological, genetic and metabolomic approaches.

Results: We constructed a mutant strain of PA14 genetically deleted in the four clinically relevant Mex EPs (Δ4mex) and overexpressed in this strain each Mex multidrug EP individually. Using comparative metabolomics on culture supernatants, UHPLC-HRMS analysis identified 2567 features. Of those 210 (8.2%) were significantly more abundant in the supernatant of EP overexpressors. Most of the features were EP-specific (73.8%), while 26.2% were shared between two or more EPs. Among these we identified by-products of secondary metabolite synthesis as well as specific alkylquinolins. In comparison to the wildtype supernatant, the one of the EP deficient mutant PA14Δ4mex showed decreased accumulation of fatty acids, including long chain homoserine lactone quorum sensing signalling molecules. Our data suggested the Mex RNDs of P. aeruginosa play a membrane detoxifying role following oxidative stress. The metabolomics data were moreover supported by transposon mutagenesis findings as well as biological assays. Finally, some of the proposed natural substrates or closely related molecules showed efflux pumps inhibition effects.

Conclusion: In contrast to the well-described broad-spectrum antibiotic profile, the natural substrate profile of Mex EPs seems to be rather metabolite specific. The multidrug Mex efflux pumps in P. aeruginosa appear to transport signalling molecules, metabolic by-products, toxic metabolites, as well as oxidized fatty acids following oxidative stress. Moreover, these natural substrates are interesting sources of competitive efflux pumps inhibitors.