Expression of multidrug resistance efflux pump genes in clinical and environmental isolates of Staphylococcus aureus

Kosmidis, Chris; Schindler, Bryan D.; Jacinto, Pauline; Patel, Dhiren; Bains, Karanpreet; Seo, Susan M.; Kaatz, Glenn W.

doi:10.1016/j.ijantimicag.2012.04.014

Cited by 76 publications

(56 citation statements)

References 29 publications

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“…The 2005 strain set has been described previously, as have in vitro-derived mutants of selected strains from this set found to overexpress mepA (10,12). Another 446 unique clinical isolates were collected from centers in Boston, MA, Houston, TX, Omaha, NE, and Freiberg, Germany, in 2009 (14). Strains and plasmids used are listed in Table 1.…”

Section: Methodsmentioning

confidence: 99%

“…For all strains collected in 2009, qRT-PCR was performed using a multiplex approach exactly as described previously using the Quantitect multiplex RT-PCR kit (Qiagen, Inc., Valencia, CA) and an ABI 7500 fast real-time PCR system (Applied Biosystems, Foster City, CA) (14). The comparative threshold cycle method was used to determine the relative expression of multiple chromosomal efflux pump genes, including mepA, compared to that in S. aureus SH1000, in which expression of each gene was considered to be 1.0.…”

Section: Methodsmentioning

confidence: 99%

“…Cooperative binding of MepR dimers to each signature motif in the mepA operator may be the basis for both the greater affinity and easier substrate induction at this site, perhaps in a manner similar to the interaction of QacR with the qacA operator (9). Using quantitative reverse transcription-PCR (qRT-PCR) we have identified numerous clinical and laboratory strains of S. aureus that overexpress mepA (6,(10)(11)(12)(13)(14). Understanding the mechanism(s) of mepA overexpression may provide clues for the design of means to overcome its potential deleterious clinical consequences.…”

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confidence: 99%

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Functional Consequences of Substitution Mutations in MepR, a Repressor of the Staphylococcus aureus mepA Multidrug Efflux Pump Gene

et al. 2013

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The expression of mepA, encoding the Staphylococcus aureus MepA multidrug efflux protein, is repressed by the MarR homologue MepR. MepR dimers bind differently to operators upstream of mepR and mepA, with affinity being greatest at the mepA operator. MepR substitution mutations may result in mepA overexpression, with A103V most common in clinical strains. Evaluation of the functional consequences of this and other MepR substitutions using a lacZ reporter gene assay revealed markedly reduced repressor activity in the presence of Q18P, F27L, G97E, and A103V substitutions. Reporter data were generally supported by susceptibility and efflux assays, and electrophoretic mobility shift assays (EMSAs) confirmed compromised affinities of MepR F27L and A103V for the mepR and mepA operators. One mutant protein contained two substitutions (T94P and T132M); T132M compensated for the functional defect incurred by T94P and also rescued that of A103V but not F27L, establishing it as a limited-range suppressor. The function of another derivative with 10 substitutions was minimally affected, and this may be an extreme example of suppression involving interactions among several residues. Structural correlations for the observed functional effects were ascertained by modeling mutations onto apo-MepR. It is likely that F27L and A103V affect the protein-DNA interaction by repositioning of DNA recognition helices. Negative functional consequences of MepR substitution mutations may result from interference with structural plasticity, alteration of helical arrangements, reduced protein-cognate DNA affinity, or possibly association of MepR protomers. Structural determinations will provide further insight into the consequences of these and other mutations that affect MepR function, especially the T132M suppressor.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Functional Consequences of Substitution Mutations in MepR, a Repressor of the Staphylococcus aureus mepA Multidrug Efflux Pump Gene

et al. 2013

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“…Data from our laboratory employing clinical mepA-overexpressing strains also support S1 as the primary site (11,12). In those studies, one strain each with a mutation in the signature subsequence of S1 (GTTAG ¡ GTTAA) or S2 (GTTAG ¡ GTTGG) (where the mutations are underlined) were identified.…”

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confidence: 89%

Mutations within the mepA Operator Affect Binding of the MepR Regulatory Protein and Its Induction by MepA Substrates in Staphylococcus aureus

et al. 2015

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The expression of mepA, encoding the Staphylococcus aureus MepA multidrug efflux protein, is repressed by the MarR homologue MepR. Repression occurs through binding of two MepR dimers to an operator with two homologous and closely approximated pseudopalindromic binding sites (site 1 [S1] and site 2 [S2]). MepR binding is impeded in the presence of pentamidine, a MepA substrate. The effects of various mepA operator mutations on MepR binding were determined using electrophoretic mobility shift assays and isothermal titration calorimetry, and an in vivo confirmation of the effects observed was established for a fully palindromic operator mutant. Altering the S1-S2 spacing by 1 to 4 bp severely impaired S2 binding, likely due to a physical collision between adjacent MepR dimers. Extension of the spacing to 9 bp eliminated the S1 binding-mediated DNA allostery required for efficient S2 binding, consistent with positive cooperative binding of MepR dimers. Binding of a single dimer to S1 was maintained when S2 was disrupted, whereas disruption of S1 eliminated any significant binding to S2, also consistent with positive cooperativity. Palindromization of binding sites, especially S2, enhanced MepR affinity for the mepA operator and reduced MepA substrate-mediated MepR induction. As a result, the on-off equilibrium between MepR and its binding sites was shifted toward the on state, resulting in less free MepR being available for interaction with inducing ligand. The selective pressure(s) under which mepA expression is advantageous likely contributed to the accumulation of mutations in the mepA operator, resulting in the current sequence from which MepR is readily induced by MepA substrates. E fflux of antimicrobial agents and biocides is an important bacterial resistance mechanism (1). The ability of selected efflux proteins to recognize multiple structurally diverse substrates amplifies the problem, resulting in a multidrug resistance (MDR) phenotype. Several MDR-conferring efflux proteins, here referred to as MDR-EPs, have been studied in Staphylococcus aureus, including NorA-B-C, MdeA, and QacA (2). All of these proteins are members of the major facilitator superfamily and are secondary transporters that are dependent on the proton motive force for substrate efflux. More recently, a novel multidrug and toxic compound extrusion (MATE)-family protein named MepA was identified (3, 4). This protein also is an MDR-EP and has select fluoroquinolones, biocides, and tigecycline as substrates.Expression of mepA is regulated by MepR, a winged helix-turnhelix repressor belonging to the MarR family, that is encoded by a sequence immediately upstream of mepA (3). The mepR and mepA genes each have their own promoter elements and operator sequences to which MepR binds (5). The interactions of MepR with operator sequences upstream of mepR and mepA differ considerably, in that the protein binds as a single dimer to the operator of mepR but as a pair of dimers to that of mepA (6). Binding of MepR to the mepA operator is readily preven...

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“…Bloodstream isolates of S. aureus (one per patient) collected from various locations in 2005 (n ϭ 232; Detroit area) and again in 2009 (n ϭ 563; Boston, Detroit, Houston, Omaha, and Freiburg, Germany) were analyzed using quantitative reverse transcription-PCR (qRT-PCR) for increased expression of several MDR efflux pump genes, including mepA (17)(18)(19)). An additional 126 unique nonbloodstream isolates collected in 2009 from San Francisco were also evaluated in the same manner.…”

Section: Methodsmentioning

confidence: 99%

Mutagenesis and Modeling To Predict Structural and Functional Characteristics of the Staphylococcus aureus MepA Multidrug Efflux Pump

Schindler

Patel

Seo

et al. 2012

Journal of Bacteriology

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MepA is a multidrug and toxin extrusion (MATE) family protein and the only MATE protein encoded within the Staphylococcus aureus genome. Structural data for MATE proteins are limited to a single high-resolution example, NorM of Vibrio cholerae. Substitution mutations were created in MepA using gradient plates containing both a substrate and reserpine as an efflux pump inhibitor. Site-directed mutagenesis of plasmid-based mepA was used to reproduce these mutations, as well as unique or low-frequency mutations identified in mepA-overexpressing clinical strains, and to mutagenize conserved acidic residues. The effect of these changes on protein function was quantitated in a norA-disrupted host strain by susceptibility testing with and without inhibitors and by determining the proficiency of ethidium efflux. Up-function substitutions clustered in the carboxy half of MepA, near the cytoplasmic face of the protein. Repeated application of the same gradient plate conditions frequently reproduced identical substitution mutations, suggesting that individual residues are required for interaction with specific substrates. Acidic residues critical to protein function were identified in helices 4 and 5. In silico modeling revealed an outward-facing molecule, with helices 1, 2, 4, 7, 8, and 10 having contact with a central cavity that may represent a substrate translocation pathway. Functionally important residues within this cavity included S81, A161, M291, and A302. These data provide a critical starting point for understanding how MATE multidrug efflux proteins function and will be useful in refining crystallographic data when they are available.

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Expression of multidrug resistance efflux pump genes in clinical and environmental isolates of Staphylococcus aureus

Cited by 76 publications

References 29 publications

Functional Consequences of Substitution Mutations in MepR, a Repressor of the Staphylococcus aureus mepA Multidrug Efflux Pump Gene

Functional Consequences of Substitution Mutations in MepR, a Repressor of the Staphylococcus aureus mepA Multidrug Efflux Pump Gene

Mutations within the mepA Operator Affect Binding of the MepR Regulatory Protein and Its Induction by MepA Substrates in Staphylococcus aureus

Mutagenesis and Modeling To Predict Structural and Functional Characteristics of the Staphylococcus aureus MepA Multidrug Efflux Pump

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