Inhibition of the multidrug efflux pump LmrS from Staphylococcus aureus by cumin spice Cuminum cyminum.

Staphylococcus aureus is a serious causative agent of infectious disease. Multidrug-resistant strains like methicillin-resistant S. aureus compromise treatment efficacy, causing significant morbidity and mortality. Active efflux represents a major antimicrobial resistance mechanism. The proton-driven multidrug efflux pump, LmrS, actively exports structurally distinct antimicrobials. To circumvent resistance and restore clinical efficacy of antibiotics, efflux pump inhibitors are necessary, and natural edible spices like cumin are potential candidates. The mode of cumin antibacterial action and underlying mechanisms behind drug resistance inhibition, however, are unclear. We tested the hypothesis that cumin inhibits LmrS drug transport. We found that cumin inhibited bacterial growth and LmrS ethidium transport in a dosage-dependent manner. We demonstrate that cumin is antibacterial toward a multidrug-resistant host and that resistance modulation involves multidrug efflux inhibition.

Modulation of the multidrug efflux pump EmrD-3 from Vibrio cholerae by Allium sativum extract and the bioactive agent allyl sulfide plus synergistic enhancement of antimicrobial susceptibility by A. sativum extract.

The causative agent of cholera, Vibrio cholerae, is a public health concern. Multidrug-resistant V. cholerae variants may reduce chemotherapeutic efficacies of severe cholera. We previously reported that the multidrug efflux pump EmrD-3 from V. cholerae confers resistance to multiple structurally distinct antimicrobials. Medicinal plant compounds are potential candidates for EmrD-3 efflux pump modulation. The antibacterial activities of garlic Allium sativum, although poorly understood, predicts that a main bioactive component, allyl sulfide, modulates EmrD-3 efflux. Thus, we tested whether A. sativum extract acts in synergy with antimicrobials and that a main bioactive component allyl sulfide inhibits EmrD-3 efflux. We found that A. sativum extract and allyl sulfide inhibited ethidium bromide efflux in cells harboring EmrD-3 and that A. sativum lowered the MICs of multiple antibacterials. We conclude that A. sativum and allyl sulfide inhibit EmrD-3 and that A. sativum extract synergistically enhances antibacterial agents.

LmrS is a multidrug efflux pump of the major facilitator superfamily from Staphylococcus aureus.

A multidrug efflux pump designated LmrS (lincomycin resistance protein of Staphylococcus aureus), belonging to the major facilitator superfamily (MFS) of transporters, was cloned, and the role of LmrS in antimicrobial efflux was evaluated. The highest relative increase in MIC, 16-fold, was observed for linezolid and tetraphenylphosphonium chloride (TPCL), followed by an 8-fold increase for sodium dodecyl sulfate (SDS), trimethoprim, and chloramphenicol. LmrS has 14 predicted membrane-spanning domains and is homologous to putative lincomycin resistance proteins of Bacillus spp., Lactobacillus spp., and Listeria spp.

MOLECULAR MODELLING, 3D-QSAR, AND DRUG DOCKING STUDIES ON THE ROLE OF NATURAL ANTICOAGULANT COMPOUNDS IN ANTITHROMBOTIC THERAPY.

Thromboembolic disorders are the leading cause of human mortality. Therefore, development of effective anticoagulant therapy is critical. Factor XIIIA (FXIIIA) protein is a crucial factor in the blood coagulation cascade, and hence it is a vital target for evolution of new antithrombotic agents. Structure-function studies of clotting factor active sites, clot formation, and thrombus structure have gained prominence in the efforts to develop novel anticoagulants. Factor XIIIA was homology modelled with the human transglutaminase-2 crystal structure as a base template for BLAST analysis. Docking and comparative binding site analysis revealed active site residue conservation and inhibitor-protein interactions. Nineteen small molecules possessing suspected anticoagulant properties were successfully docked into the FXIIIA active site following the best CoMFA and CoMSIA prediction values. Dabigatran etexilate was anticipated to be the best FXIIIA inhibitor among the nineteen anticoagulants with the highest binding affinity for the FXIIIA protein and the highest FlexX dock score of -29.8 KJ/mol. Structural properties of FXIIIA inhibitors with increased antithrombotic activity were predicted by this docking study.

Comparative genome analysis of non-toxigenic non-O1 versus toxigenic O1 Vibrio cholerae.

Pathogenic strains of Vibrio cholerae are responsible for endemic and pandemic outbreaks of the disease cholera. The complete toxigenic mechanisms underlying virulence in Vibrio strains are poorly understood. The hypothesis of this work was that virulent versus non-virulent strains of V. cholerae harbor distinctive genomic elements that encode virulence. The purpose of this study was to elucidate genomic differences between the O1 serotypes and non-O1 V. cholerae PS15, a non-toxigenic strain, in order to identify novel genes potentially responsible for virulence. In this study, we compared the whole genome of the non-O1 PS15 strain to the whole genomes of toxigenic serotypes at the phylogenetic level, and found that the PS15 genome was distantly related to those of toxigenic V. cholerae. Thus we focused on a detailed gene comparison between PS15 and the distantly related O1 V. cholerae N16961. Based on sequence alignment we tentatively assigned chromosome numbers 1 and 2 to elements within the genome of non-O1 V. cholerae PS15. Further, we found that PS15 and O1 V. cholerae N16961 shared 98% identity and 766 genes, but of the genes present in N16961 that were missing in the non-O1 V. cholerae PS15 genome, 56 were predicted to encode not only for virulence-related genes (colonization, antimicrobial resistance, and regulation of persister cells) but also genes involved in the metabolic biosynthesis of lipids, nucleosides and sulfur compounds. Additionally, we found 113 genes unique to PS15 that were predicted to encode other properties related to virulence, disease, defense, membrane transport, and DNA metabolism. Here, we identified distinctive and novel genomic elements between O1 and non-O1 V. cholerae genomes as potential virulence factors and, thus, targets for future therapeutics. Modulation of such novel targets may eventually enhance eradication efforts of endemic and pandemic disease cholera in afflicted nations.

Genome Sequence of Non-O1 Vibrio cholerae PS15.

The draft genome sequence of a non-O1 Vibrio cholerae strain, PS15, organized into 3,512 open reading frames within a 3.9-Mb genome, was determined. The PS15 genome sequence will allow for the study of the evolution of virulence and environmental adaptation in V. cholerae.

Molecular cloning and characterization of mannitol-1-phosphate dehydrogenase from Vibrio cholerae.

Vibrio cholerae utilizes mannitol through an operon of the phosphoenolpyruvate-dependent phosphotransferase (PTS) type. A gene, mtlD, encoding mannitol-1-phosphate dehydrogenase was identified within the 3.9 kb mannitol operon of V. cholerae. The mtlD gene was cloned from V. cholerae O395, and the recombinant enzyme was functionally expressed in E. coli as a 6×His-tagged protein and purified to homogeneity. The recombinant protein is a monomer with a molecular mass of 42.35 kDa. The purified recombinant MtlD reduced fructose 6-phosphate (F6P) using NADH as a cofactor with a K(m) of 1.54 +/- 0.1 mM and V(max) of 320.8 +/- 7.81 micronmol/min/mg protein. The pH and temperature optima for F6P reduction were determined to be 7.5 and 37°C, respectively. Using quantitative real-time PCR analysis, mtlD was found to be constitutively expressed in V. cholerae, but the expression was up-regulated when grown in the presence of mannitol. The MtlD expression levels were not significantly different between V. cholerae O1 and non-O1 strains.