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Efflux-mediated multidrug resistance in Bacillus subtilis: similarities and dissimilarities with the mammalian system.

ABSTRACT: Bacillus subtilis cells selected for their resistance to rhodamine 6G demonstrated a multidrug-resistance (MDR) phenotype resembling that of mammalian MDR cells. Like MDR in mammalian cells, MDR in bacteria was mediated by the efflux of the drugs from the cells. The bacterial multidrug efflux system transported similar drugs and was sensitive to similar inhibitors as the mammalian multidrug transporter, P-glycoprotein. The gene coding for the bacterial multidrug transporter, like the P-glycoprotein gene in mammalian MDR cells, was amplified in the resistant bacteria. On the other hand, the bacterial multidrug transporter showed no sequence similarity to P-glycoprotein but exhibited an obvious homology to tetracycline efflux pumps and carbohydrate-ion symporters. These results show that the transport of structurally unrelated molecules can be mediated by members of different families of membrane transporters.

SUBMITTER: Neyfakh AA

PROVIDER: S-EPMC51750 | biostudies-other | 1991 Jun

REPOSITORIES: biostudies-other

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Efflux-mediated multidrug resistance in Bacillus subtilis: similarities and dissimilarities with the mammalian system.

Neyfakh A A AA Bidnenko V E VE Chen L B LB

Proceedings of the National Academy of Sciences of the United States of America 19910601 11

Bacillus subtilis cells selected for their resistance to rhodamine 6G demonstrated a multidrug-resistance (MDR) phenotype resembling that of mammalian MDR cells. Like MDR in mammalian cells, MDR in bacteria was mediated by the efflux of the drugs from the cells. The bacterial multidrug efflux system transported similar drugs and was sensitive to similar inhibitors as the mammalian multidrug transporter, P-glycoprotein. The gene coding for the bacterial multidrug transporter, like the P-glycoprot ...[more]

PMID: 1675788

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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.

Project description:Multidrug membrane transporters can selectively extrude a wide variety of structurally and functionally unrelated substrates, and they are responsible for ineffective treatment of a wide range of diseases (e.g., infection and cancer). Their underlying molecular mechanisms remain elusive. In this study, we functionalized Ag NPs (11 nm in diameter) with two biocompatible peptides (CALNNK, CALNNE) to prepare positively and negatively charged Ag-peptide NPs (Ag-CALNNK NPs+?, Ag-CALNNE NPs-4?), respectively. We used them as photostable plasmonic imaging probes to study charge-dependent efflux kinetics of BmrA (ABC) membrane transporter of single live Bacillus (B.) subtilis cells. Two strains of the cells, normal expression of BmrA (WT) or devoid of BmrA (?BmrA), were used to study the charge-dependent efflux kinetics of single NPs upon the expression of BmrA. The NPs (1.4 nM) were stable (non-aggregated) in a PBS buffer and biocompatible to the cells. We found the high dependent accumulation of the intracellular NPs in both WT and ?BmrA upon the charge and concentration of NPs. Notably, the accumulation rates of the positively charged NPs in single live WT cells are nearly identical to those in ?BmrA cells, showing independence upon the expression of BmrA. In contrast, the accumulation rates of the negatively charged NPs in WT are much lower than in ?BmrA, showing high dependence upon the expression of BmrA and suggesting that BmrA extrude the negatively charged NPs, but not positively charged NPs, out of the WT. The accumulation of positively charged NPs in both WT and ?BmrA increases nearly proportionally to the NP concentration. The accumulation of negatively charged NPs in ?BmrA, but not in WT, also increases nearly proportionally to the NP concentration. These results suggest that both negatively and positively charged NPs enter the cells via passive diffusion driven by concentration gradients across the cellular membrane, and BmrA can only extrude the negatively charged NPs out of the WT. This study shows that single NP plasmon spectroscopy can serve as a powerful tool to identify single plasmonic NPs and to probe the charge-dependent efflux kinetics and function of single membrane transporters in single live cells in real time.

Dataset Information

Efflux-mediated multidrug resistance in Bacillus subtilis: similarities and dissimilarities with the mammalian system.

Publications

Efflux-mediated multidrug resistance in Bacillus subtilis: similarities and dissimilarities with the mammalian system.

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