Over-Expression of Hypochlorite Inducible Major Facilitator Superfamily (MFS) Pumps Reduces Antimicrobial Drug Susceptibility by Increasing the Production of MexXY Mediated by ArmZ in Pseudomonas aeruginosa.

Nisanart Charoenlap,Punyawee Dulyayangkul,Skorn Mongkolsuk,Naphat Satapoomin,Paiboon Vattanaviboon,Matthew B Avison

doi:10.3389/fmicb.2020.592153

Abstract

Pseudomonas aeruginosa, a well-known cause of nosocomial infection, is frequently antibiotic resistant and this complicates treatment. Links between oxidative stress responses inducing antibiotic resistance through over-production of RND-type efflux pumps have been reported in P. aeruginosa, but this has not previously been associated with MFS-type efflux pumps. Two MFS efflux pumps encoded by mfs1 and mfs2 were selected for study because they were found to be sodium hypochlorite (NaOCl) inducible. Antibiotic susceptibility testing was used to define the importance of these MFS pumps in antibiotic resistance and proteomics was used to characterize the resistance mechanisms involved. The results revealed that mfs1 is NaOCl inducible whereas mfs2 is NaOCl, N-Ethylmaleimide and t-butyl hydroperoxide inducible. Deletion of mfs1 or mfs2 did not affect antibiotic or paraquat susceptibility. However, over-production of Mfs1 and Mfs2 reduced susceptibility to aminoglycosides, quinolones, and paraquat. Proteomics, gene expression analysis and targeted mutagenesis showed that over-production of the MexXY RND-type efflux pump in a manner dependent upon armZ, but not amgRS, is the cause of reduced antibiotic susceptibility upon over-production of Mfs1 and Mfs2. mexXY operon expression analysis in strains carrying various lengths of mfs1 and mfs2 revealed that at least three transmembrane domains are necessary for mexXY over-expression and decreased antibiotic susceptibility. Over-expression of the MFS-type efflux pump gene tetA(C) did not give the same effect. Changes in paraquat susceptibility were independent of mexXY and armZ suggesting that it is a substrate of Mfs1 and Mfs2. Altogether, this is the first evidence of cascade effects where the over-production of an MFS pump causes over-production of an RND pump, in this case MexXY via increased armZ expression.

Highlights

MATERIALS AND METHODSPseudomonas aeruginosa is Gram-negative opportunistic human pathogen causing nosocomial infections
Our secondary analysis of microarray data (Small et al, 2007) for P. aeruginosa undergoing hypochlorite stress indicated that mfs1 and mfs2 expression was increased by 10-fold and 5-fold, respectively
Apart from the increased expression of mfs1 and mfs2 in response to hypochlorite stress, secondary analysis of the microarray data showed that the expressions of other known antibiotic resistance-associated genes mexAB-oprM, mexXY, mexZ, armZ, and amgRS were 4, 3, 2, 10, and 4-fold higher than in untreated cells (Small et al, 2007)

Summary

Program in Applied Biological Sciences

Links between oxidative stress responses inducing antibiotic resistance through over-production of RND-type efflux pumps have been reported in P. aeruginosa, but this has not previously been associated with MFS-type efflux pumps. Proteomics, gene expression analysis and targeted mutagenesis showed that over-production of the MexXY RND-type efflux pump in a manner dependent upon armZ, but not amgRS, is the cause of reduced antibiotic susceptibility upon overproduction of Mfs and Mfs. Changes in paraquat susceptibility were independent of mexXY and armZ suggesting that it is a substrate of Mfs and Mfs. Changes in paraquat susceptibility were independent of mexXY and armZ suggesting that it is a substrate of Mfs and Mfs2 This is the first evidence of cascade effects where the over-production of an MFS pump causes over-production of an RND pump, in this case MexXY via increased armZ expression

MATERIALS AND METHODS

RESULTS AND DISCUSSION

CONCLUSION

DATA AVAILABILITY STATEMENT