Antibiotic Inducibility of the MexXY Multidrug Efflux System of Pseudomonas aeruginosa : Involvement of the Antibiotic-Inducible PA5471 Gene Product

Abstract

The MexXY components of the MexXY-OprM multidrug efflux system of Pseudomonas aeruginosa are encoded by a MexZ repressor-regulated operon that is inducible by antibiotics that target the ribosome. Mutant strains disrupted in a gene, PA5471, were shown to be compromised for drug-inducible mexXY expression and, therefore, MexXY-OprM-mediated antimicrobial resistance. The PA5471 gene was inducible by the same ribosometargeting agents that induce mexXY expression. Moreover, vector-driven expression of cloned PA5471 was sufficient to promote mexXY expression and MexXY-mediated resistance in the absence of antibiotic exposure, consistent with PA5471 directly or indirectly activating mexXY expression following its own upregulation in response to antibiotics. The requirement for PA5471 for mexXY expression and antimicrobial resistance was, however, obviated in mutants lacking the MexZ repressor of mexXY expression, suggesting that PA5471 directly or indirectly modulates MexZ activity in effecting mexXY expression. While the recruitment of PA5471 and MexXY in response to ribosome disruption by antimicrobials is consistent with their genes playing a role in protecting cells from the adverse consequences of disrupting the translation process, reminiscent of transtranslation, these genes appear to operate independently in their contribution to resistance: mutants defective in trans-translation showed a much more modest (twofold) decrease in resistance to ribosome-targeting agents than those lacking PA5471 or MexXY, and this decrease was observed whether functional PA5471/MexXY was present or not.Multidrug efflux systems of the resistance-nodulation-division (RND) family contribute significantly to intrinsic and acquired resistance to antimicrobials in a number of gram-negative bacteria (43, 45). Despite their significance as determinants of antibiotic resistance, however, RND-type multidrug exporters also, in many instances, accommodate biocides (42, 45), organic solvents (48), detergents (43), including bile salts (9,18,46,60), toxic fatty acids/lipids (54), and in some instances, plant-derived antimicrobials (phytoalexins and isoflavonoids) (7, 39), metabolic inhibitors (52), organometallic compounds (tributyltin) (25), quorum-sensing effector molecules (13,26,40), and, possibly, virulence factors (21) in addition to antibiotics. Clearly, RND pumps can and do function as other than antibiotic exporters.Pseudomonas aeruginosa expresses several three-component RND-type multidrug efflux systems, among which four, MexAB-

Primary mechanisms mediating aminoglycoside resistance in the multidrug-resistant Pseudomonas aeruginosa clinical isolate PA7

“…Involvement of the MexXY-OprA/OprM in aminoglycoside resistance of P. aeruginosa PA7 Aminoglycoside resistance in P. aeruginosa PA7 was compared with the two aminoglycoside-sensitive strains PAO1, the most well-known strain (Sobel et al, 2003;Morita et al, 2006), and DSM 1128, a taxonomically related PA7 strain (Martin & Beveridge, 1986;Kiewitz & Tümmler, 2000;Roy et al, 2010). The MICs of the antipseudomonal aminoglycosides (amikacin, arbekacin, gentamicin and tobramycin) for P. aeruginosa PA7, PAO1 and DSM1128 are shown in Table 1.…”

“…The mexXY genes from PAO1 (Mine et al, 1999) were released via the digestion of pSportI : : mexXY (Morita et al, 2006) with EcoRI and cloned into pSTV28 and pYM101, digested with EcoRI, and dephosphorylated to yield pYM115 and pYM120, respectively.…”

“…The plasmids pEX18Tc (Hoang et al, 1998), pYM101 (Morita et al, 2010), and their derivatives were maintained and selected using medium supplemented with 2.5210 mg tetracycline ml 21 for E. coli or 50-150 mg tetracycline ml 21 for P. aeruginosa. The plasmids pSportI (Morita et al, 2006), pBR322 (Morita et al, 1998), pFLP2 (Hoang et al, 1998) and their derivatives were maintained and selected using medium supplemented with 100 mg ampicillin ml 21 for E. coli or 200 mg carbenicillin ml 21 for P. aeruginosa. The plasmids pSTV28 (Masaoka et al, 2000), pKOV [identical to pKO3 (Link et al, 1997), except for the addition of a 3 kb stuffer sequence in the multiple cloning site (http://arep.med.harvard.edu/labgc/pko3.html)], and their derivatives were maintained and selected using medium supplemented with 2-10 mg chloramphenicol ml 21 for E. coli.…”

“…In-frame deletion mutants of the oprA (PSPA7_3271), mexXY (PSPA7_3269-3270), mexXY-oprA (PSPA7_3269-3270-3271), oprM (PSPA7_0527), mexAB-oprM (PSPA7_0525-0526-0527) and/or aacA4 (PSPA7_3724.1) genes from P. aeruginosa PA7 and their derivatives were constructed using the previously described sacB-based strategy (Morita et al, 2006(Morita et al, , 2010. The plasmids and P. aeruginosa mutants are listed in Supplementary Table S1, while the primer pairs are listed in Supplementary Table S2.…”

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

“…However, it is becoming increasingly clear that the transcriptional regulation of RND pumps in organisms like P. aeruginosa is quite complex (Li et al 2015) and that RND pump overexpression can occur in response to the presence of not only antimicrobials but also various environmental stresses (Poole 2012;Alvarez-Ortega et al 2013). For example, in CF isolates of P. aeruginosa, overexpression of the MexXY pump has been linked to environmental conditions that the organism encounters in CF lungs (Morita et al 2006). To gain more insights into the interplay between the antibiotic susceptibility, the expression of RND pumps, and the underlying mechanisms of RND pump expression, we analyzed the expression of RND pumps in antibiotic-resistant CF isolates of P. aeruginosa and sequenced their regulatory genes that may be responsible for their overexpression.…”