Characterization of the MexT Regulator of the mexEF-oprN Multidrug Efflux Operon of Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is resistant to many clinically-relevant antibiotics and this is partly attributable to multidrug efflux systems in this organism. One of these, MexEF-OprN, exports chloramphenicol, fluoroquinolones and trimethoprim and is positively regulated by MexT, encoded by the mexT gene that is located upstream of the efflux genes. MexT also positively regulates the mexS gene that encodes an oxidoreductase of unknown function and whose loss increases expression of mexEF-oprN. A null mutation in the mexS gene of P. aeruginosa increased expression of two 3-gene operons, PA4354-PA4355-PA4356 and PA2813-2812-2811. This increased expression, which paralleled an increase in mexEF-oprN expression in the same mutant, was, like mexEF-oprN, MexT-dependent. The PA4356 (xenB) gene product is homologous to a xenobiotic reductase in P. fluorescens shown to remove nitro groups from trinitrotoluene and nitroglycerin. As nitration is a well-known result of nitrosative stress, a role for xenB (and the co-regulated mexEF-oprN and PA2813-2812-2811) in a nitrosative stress response was hypothesized and tested. Using S-nitrosoglutathione (GSNO) as a source of nitrosative stress, expression of mexEF-oprN, xenB and PA2811 was shown to be GSNO-inducible, though in the case of xenB and PA2811 this was only seen in a mutant lacking MexEF-OprN and only for xenB, this GSNO-inducible expression was dependent upon MexT. Consistent with MexT being required for mexEF-oprN and PA4354-PA4355-PA4356 expression, MexT bound to their promoter regions. Chloramphenicol, a nitroaromatic antimicrobial that is a substrate for MexEF-OprN and resembles a nitrosated nitrosative stress product accommodated by this efflux system induced expression of mexEF-oprN, but not xenB, or PA2811, and this was dependent upon the MexT regulator. In addition to MexT positive-regulating activity of genes in response to nitrosative stress, MexT also negatively regulates the expression of mexAB-oprM through direct binding to its promoter region and the oprD gene, encoding an outer membrane porin that provides a portal of entry for basic amino acids and carbapenem, whose expression was reduced only by GSNO.