Dataset Information

Contribution of resistance-nodulation-division efflux pump operon smeU1-V-W-U2-X to multidrug resistance of Stenotrophomonas maltophilia.

ABSTRACT: KJ09C, a multidrug-resistant mutant of Stenotrophomonas maltophilia KJ, was generated by in vitro selection with chloramphenicol. The multidrug-resistant phenotype of KJ09C was attributed to overexpression of a resistance nodulation division (RND)-type efflux system encoded by an operon consisting of five genes: smeU1, smeV, smeW, smeU2, and smeX. Proteins encoded by smeV, smeW, and smeX were similar to the membrane fusion protein, RND transporter, and outer membrane protein, respectively, of known RND-type systems. The proteins encoded by smeU1 and smeU2 were found to belong to the family of short-chain dehydrogenases/reductases. Mutant KJ09C exhibited increased resistance to chloramphenicol, quinolones, and tetracyclines and susceptibility to aminoglycosides; susceptibility to ?-lactams and erythromycin was not affected. The expression of the smeU1-V-W-U2-X operon was regulated by the divergently transcribed LysR-type regulator gene smeRv. Overexpression of the SmeVWX pump contributed to the acquired resistance to chloramphenicol, quinolones, and tetracyclines. Inactivation of smeV and smeW completely abolished the activity of the SmeVWX pump, whereas inactivation of smeX alone decreased the activity of the SmeVWX pump. The enhanced aminoglycoside susceptibility observed in KJ09C resulted from SmeX overexpression.

SUBMITTER: Chen CH

PROVIDER: S-EPMC3232770 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Contribution of resistance-nodulation-division efflux pump operon smeU1-V-W-U2-X to multidrug resistance of Stenotrophomonas maltophilia.

Chen Chao-Hsien CH Huang Chiang-Ching CC Chung Tsao-Chuen TC Hu Rouh-Mei RM Huang Yi-Wei YW Yang Tsuey-Ching TC

Antimicrobial agents and chemotherapy 20110919 12

KJ09C, a multidrug-resistant mutant of Stenotrophomonas maltophilia KJ, was generated by in vitro selection with chloramphenicol. The multidrug-resistant phenotype of KJ09C was attributed to overexpression of a resistance nodulation division (RND)-type efflux system encoded by an operon consisting of five genes: smeU1, smeV, smeW, smeU2, and smeX. Proteins encoded by smeV, smeW, and smeX were similar to the membrane fusion protein, RND transporter, and outer membrane protein, respectively, of kn ...[more]

PMID: 21930878

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Project description:Bacterial efflux pumps in the resistance-nodulation-cell division (RND) family of Gram-negative bacteria contribute significantly to the development of antimicrobial resistance by many pathogens. In this study, we selected the MtrD transporter protein of Neisseria gonorrhoeae as it is the sole RND pump possessed by this strictly human pathogen and can export multiple antimicrobials, including antibiotics, bile salts, detergents, dyes, and antimicrobial peptides. Using knowledge from our previously published structures of MtrD in the presence or absence of bound antibiotics as a model and the known ability of MtrCDE to export cationic antimicrobial peptides, we hypothesized that cationic peptides could be accommodated within MtrD binding sites. Furthermore, we thought that MtrD-bound peptides lacking antibacterial action could sensitize bacteria to an antibiotic normally exported by the MtrCDE efflux pump or other similar RND-type pumps possessed by different Gram-negative bacteria. We now report the identification of a novel nonantimicrobial cyclic cationic antimicrobial peptide, which we termed CASP (cationic antibiotic-sensitizing peptide). By single-particle cryo-electron microscopy, we found that CASP binds within the periplasmic cleft region of MtrD using overlapping and distinct amino acid contact sites that interact with another cyclic peptide (colistin) or a linear human cationic antimicrobial peptide derived from human LL-37. While CASP could not sensitize Neisseria gonorrhoeae to an antibiotic (novobiocin) that is a substrate for RND pumps, it could do so against multiple Gram-negative, rod-shaped bacteria. We propose that CASP (or future derivatives) could serve as an adjuvant for the antibiotic treatment of certain Gram-negative infections previously thwarted by RND transporters. IMPORTANCE RND efflux pumps can export numerous antimicrobials that enter Gram-negative bacteria, and their action can reduce the efficacy of antibiotics and provide decreased susceptibility to various host antimicrobials. Here, we identified a cationic antibiotic-sensitizing peptide (CASP) that binds within the periplasmic cleft of an RND transporter protein (MtrD) produced by Neisseria gonorrhoeae. Surprisingly, CASP was able to render rod-shaped Gram-negative bacteria, but not gonococci, susceptible to an antibiotic that is a substrate for the gonococcal MtrCDE efflux pump. CASP (or its future derivatives) could be used as an adjuvant to treat infections for which RND efflux contributes to multidrug resistance.

Project description:Clinical strains of Stenotrophomonas maltophilia are often highly resistant to multiple antibiotics, although the mechanisms of resistance are generally poorly understood. Multidrug resistant (MDR) strains were readily selected by plating a sensitive reference strain of the organism individually onto a variety of antibiotics, including tetracycline, chloramphenicol, ciprofloxacin, and norfloxacin. Tetracycline-selected MDR strains typically showed cross-resistance to erythromycin and fluoroquinolones and, in some instances, aminoglycosides. MDR mutants selected with the other agents generally displayed resistance to chloramphenicol and fluoroquinolones only, although two MDR strains (e.g., K1385) were also resistant to erythromycin and hypersusceptible to aminoglycosides. Many of the MDR strains expressed either moderate or high levels of a novel outer membrane protein (OMP) of ca. 50 kDa molecular mass, a phenotype typical of MDR strains of Pseudomonas aeruginosa hyperexpressing drug efflux systems. Indeed, the 50-kDa OMP of these S. maltophilia MDR strains reacted with antibody to OprM, the outer membrane component of the MexAB-OprM MDR efflux system of P. aeruginosa. Similarly, a ca. 110-kDa cytoplasmic membrane protein of these MDR strains also reacted with antibody to the MexB component of the P. aeruginosa pump. The outer and cytoplasmic membranes of several clinical S. maltophilia strains also reacted with the anti-OprM and anti-MexB antibodies. N-terminal amino acid sequencing of a cyanogen bromide-generated peptide of the 50-kDa OMP of MDR strain K1385, dubbed SmeM (Stenotrophomonas multidrug efflux), revealed it to be very similar to a number of outer membrane multidrug efflux components of P. aeruginosa and Pseudomonas putida. Deletion of the L1 and L2 beta-lactamase genes confirmed that these enzymes were responsible for the bulk of the beta-lactam resistance of K1385 and its parent. Still, overexpression of the MDR efflux mechanism in an L1- and L2-deficient derivative of K1385 did yield a modest increase in resistance to a few beta-lactams. These data are consistent with the MDR efflux mechanism(s) playing a role in the multidrug resistance of S. maltophilia.

Dataset Information

Contribution of resistance-nodulation-division efflux pump operon smeU1-V-W-U2-X to multidrug resistance of Stenotrophomonas maltophilia.

Publications

Contribution of resistance-nodulation-division efflux pump operon smeU1-V-W-U2-X to multidrug resistance of Stenotrophomonas maltophilia.

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