Pseudomonas aeruginosaMipA-MipB envelope proteins act as new sensors of polymyxin

Abstract

AbstractDue to the rising incidence of antibiotic resistant infections, the last-line antibiotics polymyxins have resurged in the clinics in parallel with new bacterial strategies of escape. The Gram-negative opportunistic pathogenPseudomonas aeruginosadevelops resistance to colistin/polymyxin by distinct molecular mechanisms, mostly through modification of the lipid A component of the LPS by proteins encoded within thearnBCDATEF-ugd(arn) operon. In this work, we characterized a polymyxin-induced operon, namedmipBA, notably present inP. aeruginosastrains devoid of thearnoperon. We showed thatmipBAis activated by ParR/ParS two-component regulatory system in response to polymyxin. MipA and MipB localize to bacterial outer membrane and form a complexin vitro. Structural modeling revealed that the lipoprotein MipB adopts a β-lactamase fold with two additional C-terminal domains,while MipA folds as an outer-membrane β-barrel, harboring an internal negatively charged channel, able to host a polymyxin molecule. Nano differential scanning fluorimetry (DSF) showed that polymyxin stabilized MipA proteinin vitro. Mass spectrometry-based quantitative proteomics on whole bacterial membranes demonstrated that the ΔmipBAmutant synthesized less MexXY-OprA proteins in response to polymyxin compared to the wild-type strain, as a consequence of impaired transcriptional activation of themexoperon. We propose MipA/MipB to act as membrane (co)sensors working in concert to activate ParS histidine kinase and help the bacterium to cope with polymyxin-mediated envelope stress through synthesis of the efflux pomp, MexXY-OprA.