Mutations in PA3574 ( nalD ) Lead to Increased MexAB-OprM Expression and Multidrug Resistance in Laboratory and Clinical Isolates of Pseudomonas aeruginosa

Abstract

Mutations in genes mexR and nalC have previously been shown to drive overexpression of the MexAB-OprM multidrug efflux system in Pseudomonas aeruginosa. A transposon insertion multidrug-resistant mutant of P. aeruginosa overproducing MexAB-OprM was disrupted in yet a third gene, PA3574, encoding a probable repressor of the TetR/AcrR family that we have dubbed NalD. Clinical strains overexpressing MexAB-OprM but lacking mutations in mexR or nalC were also shown to carry mutations in nalD. Moreover, the cloned nalD gene reduced the multidrug resistance and MexAB-OprM expression of the transposon mutant and clinical isolates, highlighting the significance of the nalD mutations vis-à-vis MexAB-OprM overexpression in these isolates.Pseudomonas aeruginosa is an opportunistic human pathogen characterized by an innate resistance to multiple classes of antimicrobials (11), attributable in part to a family of broadly specific, so-called multidrug efflux systems (23, 24) that work synergistically with low outer membrane permeability (9, 19) to limit antimicrobial accumulation in this organism. Several multidrug efflux systems in P. aeruginosa have been described to date (23), although the major system contributing to intrinsic multidrug resistance is encoded by the mexAB-oprM operon (10, 18, 26). MexAB-OprM exports a wide variety of antimicrobials, including most classes of antibiotics, biocides, dyes, detergents, organic solvents (i.e., aromatic hydrocarbons; reviewed in reference 23]), and homoserine lactones associated with quorum sensing (8,22). The last play a role in cell densitydependent expression of a number of virulence factors in P. aeruginosa, and thus, the activity of this efflux system can influence virulence (30). Indeed, a recent study suggests that the MexAB-OprM efflux system of P. aeruginosa promotes the release of a molecule(s) ultimately important for the virulence of this organism (12). The observation that MexAB-OprM hyperexpression in nalB strains impairs fitness and virulence (30) also suggests that this efflux system has a physiological role in P. aeruginosa independent of antimicrobial efflux and resistance. Consistent with this, mutants hyperexpressing MexABOprM were readily selected in vivo in a rat model of acute P. aeruginosa pneumonia in the absence of any antibiotic selection (16). The specific nature of the selective in vivo growth advantage provided by this efflux system is, however, unknown.Hyperproduction of MexAB-OprM has been documented in lab and clinical multidrug-resistant isolates carrying lesions in the mexR gene (4,15,20,28,35,37) (so-called nalB mutants [21]), encoding a repressor of mexAB-oprM expression (27, 35).MexAB-OprM hyperexpression also occurs independently of mutations in mexR or the mexR and mexAB-oprM promoter regions (35, 37). These so-called nalC mutants (5, 20, 35) carry a mutation in a recently identified gene (PA3721, also known as nalC) that encodes a TetR family repressor of an adjacent two-gene operon, PA3720-PA3719 (5). It is, in fact, the increased express...

Berberine inhibits the MexXY‑OprM efflux pump to reverse imipenem resistance in a clinical carbapenem‑resistant Pseudomonas aeruginosa isolate in a planktonic state

“…Furthermore, it was demonstrated that the reversion of IMP resistance by BEB may be due to inhibition of the MexXY-OprM efflux pump. Among the four efflux pumps associated with carbapenem resistance, MexAB-OprM and MexXY-OprM are constitutively expressed and their overexpression mediates intrinsic resistance in P. aeruginosa, while MexCD-OprJ and MexEF-OprN usually remain quiescent in wild-type P. aeruginosa and their overexpression results in acquired multi-drug resistant phenotypes in mutant isolates (20)(21)(22). As described previously, MexAB-OprM and MexXY-OprM are usually overexpressed in clinical P. aeruginosa isolates (4,5), and the latter was also demonstrated to be a target of BEB (14).…”

Adaptive and Mutational Resistance: Role of Porins and Efflux Pumps in Drug Resistance

“…Another characteristic of these mutants is their greater susceptibility to several ␤-lactams and aminoglycosides, which might be related to some extent to the downregulation of the mexABoprM operon observed for these mutant strains (153). Interestingly, Sobel et al (230) observed that the mutation of the mexS (PA2491) gene, which is under the transcriptional control of MexT and codes for an oxidoreductase, leads to an nfxC-like phenotype. Moreover, some studies of clinical isolates indicated that additional unidentified genes may be responsible for this phenotype (135).…”

“…More recently, it was demonstrated that the product of the armR (PA3719) gene binds to and associates with MexR in vivo, reducing its suppression of mexAB-oprM expression (44). Multidrug resistance via MexABOprM overexpression can also occur due to mutations in nalD, which encodes a TetR family repressor (230), and nalD mutations have been observed for P. aeruginosa clinical strains.…”

Antibacterial-ResistantPseudomonas aeruginosa: Clinical Impact and Complex Regulation of Chromosomally Encoded Resistance Mechanisms

“…However, the possibility that C 4 -HSL directly or indirectly influences NalDmediated regulation of mexAB-oprM to promote growth phase expression has yet to be determined experimentally. Overexpression of mexAB-oprM has been detected in nalB-, nalC-, and nalD-type multidrug-resistant mutants and selected both in vivo (2,137,181,184,239,273) and in vitro (2,24,239,245). In nalB-type strains, mutations within mexR disrupt the translation of full-length protein or compromise the repressor activity of MexR by causing a loss of dimerization, defects in (2,219).…”

“…The binding region encompasses overlapping mexR and mexA promoters, and association of MexR dimers with these sites represses transcription of the mexAB-oprM operon and negatively autoregulates its own expression (52,220). A second regulatory factor of the mexAB-oprM operon, NalD, was discovered following insertional mutagenesis of a gene, nalD, located adjacent to a putative pump gene of the major facilitator superfamily (239). NalD is a repressor of the TetR family that binds to a sequence upstream of mexAB-oprM but downstream of the mexR binding sites (160).…”