S. aureus is an important human pathogen capable of causing serious, life-threatening infections and is one of the most common organisms to do so. This pathogen possesses multiple mechanisms by which it resists the killing effects of biocides and antibiotics, including overexpression of membrane-based proteins called multidrug resistance (MDR)-conferring efflux pumps (EPs). In fact, efflux is the single most important mechanism by which bacteria such as S. aureus can evade the effects of multiple structurally different antimicrobial agents simultaneously. EP activity also predisposes S. aureus to acquire target-based high level resistance-conferring mutations to some pump substrates by reducing intracellular concentrations to subinhibitory levels. EPs belong to one of five different protein families that are differentiated by structural characteristics and energy source used for substrate transport. The Multidrug and Toxic compound Extrusion (MATE) family is the most recently described and members are found not only in bacteria but also in eukaryotes including plants, yeast, and humans. Typical substrates for MATE pumps include mono- and bivalent organic cations such as biocides and disinfectants, fluoroquinolones, and anticancer agents. Acquisition of MDR S. aureus strains, including those with increased expression of MATE and other MDR efflux pump genes, can produce undesirable consequences such as prolonged hospital stays, increased healthcare costs, and most importantly increased morbidity and mortality. MepA is the first and only MATE MDR EP identified in S. aureus, and overexpression of mepA occurs in clinical strains. Point mutations in mepR, which encodes MepR, a MarR-family transcriptional repressor of mepA, that inactivate the protein frequently are the bases of mepA overexpression and are found in clinical strains and easily produced in the laboratory. However, other mechanisms of mepRA regulation also exist as mepA-overexpressing clinical strains lacking mepR mutations have been identified. This application proposes experiments designed to increase our understanding of the mepRA pump system in particular and MDR EPs of S. aureus in general. Our goals are to (1) Determine the details of MepR-DNA and MepR-inducer interactions by structural biology investigations and characterize the MepR-inducer binding site(s) using mutagenesis;(2) Determine the functional characteristics of the MepA pump and employ mutagenesis to better understand substrate/inhibitor interactions with it, which will inform the future structural biology analysis of the protein;(3) Characterize MepR-dependent and - independent mepRA regulatory mechanisms, including naturally-occurring MepR substitution and operator site mutations and trans-acting factors. MepR functional and operator site binding studies and analyses of plasmid libraries will be employed to accomplish this goal. The detailed study of MepA, combined with similar earlier work with other clinically important S. aureus MDR EPs (NorA and QacA/B), will help in the rational design of broad-spectrum EP inhibitors.
S. aureus is a major community- and nosocomially-acquired pathogen. This project will provide data increasing our understanding of Multidrug and Toxic compound Extrusion (MATE) family efflux pumps, knowledge that also may be applicable to eukaryotic MATE proteins, and will inform future work toward the design of compounds that inhibit multiple S. aureus MDR pumps simultaneously. This will be an advance in antibacterial chemotherapy resulting in an improvement in patient outcomes, which is directly relevant to the mission of the VA.
|Schindler, Bryan D; Jacinto, Pauline L; Buensalido, Joseph Adrian L et al. (2015) Clonal relatedness is a predictor of spontaneous multidrug efflux pump gene overexpression in Staphylococcus aureus. Int J Antimicrob Agents 45:464-70|
|Schindler, Bryan D; Seo, Susan M; Birukou, Ivan et al. (2015) Mutations within the mepA operator affect binding of the MepR regulatory protein and its induction by MepA substrates in Staphylococcus aureus. J Bacteriol 197:1104-14|
|Schindler, Bryan D; Frempong-Manso, Emmanuel; DeMarco, Carmen E et al. (2015) Analyses of multidrug efflux pump-like proteins encoded on the Staphylococcus aureus chromosome. Antimicrob Agents Chemother 59:747-8|
|Schindler, Bryan D; Seo, Susan M; Jacinto, Pauline L et al. (2013) Functional consequences of substitution mutations in MepR, a repressor of the Staphylococcus aureus MepA multidrug efflux pump gene. J Bacteriol 195:3651-62|
|Birukou, Ivan; Tonthat, Nam K; Seo, Susan M et al. (2013) The molecular mechanisms of allosteric mutations impairing MepR repressor function in multidrug-resistant strains of Staphylococcus aureus. MBio 4:e00528-13|
|Schindler, Bryan D; Patel, Diixa; Seo, Susan M et al. (2013) Mutagenesis and modeling to predict structural and functional characteristics of the Staphylococcus aureus MepA multidrug efflux pump. J Bacteriol 195:523-33|
|Schindler, Bryan D; Jacinto, Pauline; Kaatz, Glenn W (2013) Inhibition of drug efflux pumps in Staphylococcus aureus: current status of potentiating existing antibiotics. Future Microbiol 8:491-507|
|Kosmidis, Christos; Schindler, Bryan D; Jacinto, Pauline L et al. (2012) Expression of multidrug resistance efflux pump genes in clinical and environmental isolates of Staphylococcus aureus. Int J Antimicrob Agents 40:204-9|
|Kosmidis, Christos; DeMarco, Carmen E; Frempong-Manso, Emmanuel et al. (2010) In silico genetic correlations of multidrug efflux pump gene expression in Staphylococcus aureus. Int J Antimicrob Agents 36:222-9|
|Frempong-Manso, Emmanuel; Raygada, Jose L; DeMarco, Carmen E et al. (2009) Inability of a reserpine-based screen to identify strains overexpressing efflux pump genes in clinical isolates of Staphylococcus aureus. Int J Antimicrob Agents 33:360-3|