Evidence of MexT-Independent Overexpression of MexEF-OprN Multidrug Efflux Pump of Pseudomonas aeruginosa in Presence of Metabolic Stress

Abstract

BackgroundThe Pseudomonas aeruginosa MexEF-OprN efflux pump confers resistance to clinically significant antibiotics. Regulation of mexEF-oprN operon expression is multifaceted with the MexT activator being one of the most prominent regulatory proteins.MethodologyWe have exploited the impaired metabolic fitness of a P. aeruginosa mutant strain lacking several efflux pump of the resistance nodulation cell division superfamily and the TolC homolog OpmH, and isolated derivatives (large colony variants) that regained fitness by incubation on nutrient-rich medium in the absence of antibiotics. Although the mexEF-oprN operon is uninducible in this mutant due to a 8-bp mexT insertion present in some P. aeruginosa PAO1 strains, the large colony variants expressed high levels of MexEF-OprN. Unlike large colony variants obtained after plating on antibiotic containing medium which expressed mexEF-oprN in a MexT-dependent fashion as evidenced by clean excision of the 8-bp insertion from mexT, mexEF-oprN expression was MexT-independent in the large colony variants obtained by plating on LB alone since the mexT gene remained inactivated. A search for possible regulators of mexEF-oprN expression using transposon mutagenesis and genomic library expression approaches yielded several candidates but proved inconclusive.SignificanceOur results show that antibiotic and metabolic stress lead to up-regulation of MexEF-OprN expression via different mechanisms and that MexEF-OprN does not only extrude antimicrobials but rather serves other important metabolic functions.

Amino Acid Substitutions Account for Most MexS Alterations in Clinical nfxC Mutants of Pseudomonas aeruginosa

“…In contrast, the PAO1 mexS allele had virtually no impact on the resistance levels of strain PA14⌬S (strain PA14⌬S PAO1 ) and failed to reverse the overexpression of mexE, whose transcripts remained 45-fold more abundant in PA14⌬S in comparison with PA14 mexS allele. Consistent with PAO1 producing inactive MexS-D 249 and MexT peptides, spontaneous excision of the extra 8-bp sequence inserted in mexT is known to trigger MexEF-OprN production in this strain, with MexT recovering its functionality in a nonfunctional MexS background (36). Confirming that MexS-N 249 (and not MexS-D 249 ) is functional, analysis of 7 drug-susceptible strains of P. aeruginosa collected from surface waters (PE1, PE1346, PE1361, PE1393, PE1423, PE1446, and PE1450) showed that the genomes of all of them encoded MexS-N 249 together with an active MexT (without any insertion in the mexT gene) (data not shown).…”

MexXY efflux pump overexpression and aminoglycoside resistance in cystic fibrosis isolates of Pseudomonas aeruginosa from chronic infections

“…MexEF-OprN, on the other hand, provides resistance to fluoroquinolones, chloramphenicol, trimethoprim, and carbapenem (Köhler et al 1997). MexEFOprN pump has been shown to be overexpressed not only in the CF isolates but also in response to metabolic stress (Wolter et al 2009;Kumar and Schweizer 2011).…”

Emergence of the P2 Phenotype in Pseudomonas aeruginosa PAO1 Strains Involves Various Mutations in mexT or mexF

“…P2 also shows increased activation of the Rhl quorumsensing subsystem, which may suggest that dysfunction of the MexEF-OprN pump mainly affects the Rhl/C 4 -HSL quorumsensing pathway of P. aeruginosa. Although the MexEF-OprN efflux pump has been extensively characterized in the context of antibiotics, it has been suggested that the main purpose of the pump may not actually be antimicrobial extrusion (33) but may rather be metabolic function (34). For example, some isolates of P. aeruginosa from a mouse model of pneumonia express the MexEF-OprN pump without any exposure to antimicrobials (35).…”