Project description:Staphylococcus aureus is a notorious bacterial pathogen that causes a broad range of human diseases, and isolates that are resistant to several antibiotic classes including last resort antibiotics like vancomycin and daptomycin complicate the situation. We characterized S. aureus VC40, a strain that shows full resistance to vancomycin (MIC of 64 µg/ml) and daptomycin (MIC of 4 µg/ml) as well as a decreased susceptibility to further cell wall active agents. Genome sequencing revealed mutations in genes encoding the histidine kinases WalK and VraS that control cell envelope related processes and gene expression profiling indicated the induction of the respective regulons in strain VC40. Reconstitution of the mutations in walK or vraS into the susceptible S. aureus NCTC 8325 background resulted in a considerably increased resistance to vancomycin and daptomycin with MICs surpassing the clinical breakpoints for these antibiotics, thereby generating vancomycin-intermediate S. aureus (VISA) strains. As observed for S. aureus VC40, the walKwalk and vraS mutations also led to an increased expression of the respective regulons in the NCTC 8325 background. Phenotypic studies showed that S. aureus VC40 as well as the walKwalk and vraS mutants of strain NCTC 8325 were characterized by a significantly thickened cell wall, a decreased growth rate, a reduced autolytic activity and an increased resistance to lysostaphin-induced lysis. These results demonstrate that the WalK and VraS histidine kinases act as major switches which allow S. aureus to rapidly develop vancomycin resistance up to the VISA level via mutation of one single gene locus and concomitantly contribute to cross-resistance to other antibiotics including the last resort antibiotic daptomycin.
Project description:Staphylococcus aureus is a notorious bacterial pathogen that causes a broad range of human diseases, and isolates that are resistant to several antibiotic classes including last resort antibiotics like vancomycin and daptomycin complicate the situation. We characterized S. aureus VC40, a strain that shows full resistance to vancomycin (MIC of 64 M-BM-5g/ml) and daptomycin (MIC of 4 M-BM-5g/ml) as well as a decreased susceptibility to further cell wall active agents. Genome sequencing revealed mutations in genes encoding the histidine kinases WalK and VraS that control cell envelope related processes and gene expression profiling indicated the induction of the respective regulons in strain VC40. Reconstitution of the mutations in walK or vraS into the susceptible S. aureus NCTC 8325 background resulted in a considerably increased resistance to vancomycin and daptomycin with MICs surpassing the clinical breakpoints for these antibiotics, thereby generating vancomycin-intermediate S. aureus (VISA) strains. As observed for S. aureus VC40, the walKwalk and vraS mutations also led to an increased expression of the respective regulons in the NCTC 8325 background. Phenotypic studies showed that S. aureus VC40 as well as the walKwalk and vraS mutants of strain NCTC 8325 were characterized by a significantly thickened cell wall, a decreased growth rate, a reduced autolytic activity and an increased resistance to lysostaphin-induced lysis. These results demonstrate that the WalK and VraS histidine kinases act as major switches which allow S. aureus to rapidly develop vancomycin resistance up to the VISA level via mutation of one single gene locus and concomitantly contribute to cross-resistance to other antibiotics including the last resort antibiotic daptomycin. Microarray was used to evaluate alteration in the transcriptome of mutS mutant and compared to the parental strain VC40
Project description:The GraS/GraR two-component system has been shown to control cationic antimicrobial peptide (CAMP) resistance in the major human pathogen Staphylococcus aureus. We identified a highly conserved ten base pair palindromic sequence (5’ ACAAATTTGT 3’) located upstream from GraR-regulated genes (mprF and the dltA and vraFG operons), which we show to be essential for transcriptional regulation by GraR and induction in response to colistin, a bacterial CAMP, suggesting it is the likely GraR binding site. Genome-based predictions and transcriptome analysis revealed several novel GraR target genes. Global expression changes between the wild type strain HG001 (a rsbU+ variant of strain NCTC 8325) and a DgraSR mutant, grown to mid-exponential phase in TSB with 50 µg/ml colistin, were examined. Hybridizations were performed in triplicate using RNA isolated from independent cultures.
Project description:We report the condition-dependent transcriptome of S. aureus HG001, a derivative of strain NCTC 8325, by strand-specific tiling array hybridizations. More than 40 experimental conditions were investigated ranging from optimal in vitro growth to interaction with host cells. Analyses included the systematic mapping of transcription units, annotation of non-coding RNAs, the classification of promoters according to their dependency on SigA and SigB, and the prediction of potentially new transcription factor target sites. Antisense RNAs being of particular interest because of the small number of alternative sigma factors used by S. aureus were found to be relatively rare, overlapping only 6% of the annotated coding genes.
Project description:In this dataset, we investigate the targets of degrasyn in the methicillin-sensitive S. aureus strain NCTC 8325. This includes data on enrichment studies and competition studies with conventional ABPP using a degrasyn-derived probe, competition studies with residue-specific proteomics using the isoDTB-ABPP method and global analysis of protein expression levels in response to degrasyn treatment.