Project description:Staphylococcus aureus prefers the human anterior nares as its habitat, but nothing is known about the nutritional situation in this ecological niche. Analysis of nasal secretions showed a complex mixture of nutrients at low concentration. Based on these findings a synthetic nasal medium (SNM) was composed, mimicking nasal secretions. We used microarrays in order to investigate pathways and expression patterns important in a synthetic medium mimicking nasal secretions compared to standard laboratory complex medium
Project description:Staphylococcus aureus prefers the human anterior nares as its habitat, but nothing is known about the nutritional situation in this ecological niche. Analysis of nasal secretions showed a complex mixture of nutrients at low concentration. Based on these findings a synthetic nasal medium (SNM) was composed, mimicking nasal secretions. We used microarrays in order to investigate pathways and expression patterns important in a synthetic medium mimicking nasal secretions compared to standard laboratory complex medium Staphylococcus aureus USA300 was inoculated in complex medium (BM) or synthetic nasal medium (SNM) to an OD578nm of 0.005 and grown under aerobic conditions until OD578nm of 0.02. RNA was stabilized and extracted at this early growth phase and hybridization was made on Affymetrix microarrays. The aim was to identify genes which are important during growth under the limited conditions present during colonization of human nares
Project description:Staphylococcus aureus is a common human and animal opportunistic pathogen. In humans nasal carriage of S. aureus is a risk factor for various infections. Methicillin-resistant S. aureus ST398 is highly prevalent in pigs in Europe and North America. The mechanism of successful pig colonization by MRSA ST398 is poorly understood. Previously, we developed a nasal colonization model of porcine nasal mucosa explants to identify molecular traits involved in nasal MRSA colonization of pigs. Here, we report the analysis of the transcriptome of MRSA ST398 strain S0462 during colonization on the explant epithelium. Major regulated genes were encoding metabolic processes and regulation of these genes represents metabolic adaptation to nasal mucosa explants. Colonization was not accompanied by significant changes in transcripts of main virulence associated genes or known human colonization factors. Here, we document regulation of two genes which have potential influence on S. aureus colonization; cysteine extracellular proteinase (scpA) and von Willebrand factor-binding protein (vwbp, located on SaPIbov5). Colonization with isogenic-deletion strains (Δvwbp and ΔscpA) did not alter the nasal S. aureus colonization compared to wild type. Our results suggest that nasal colonization with MRSA ST398 is a complex event that is accompanied with changes in bacterial gene expression regulation and metabolic adaptation.
Project description:Staphylococcus aureus is a common human and animal opportunistic pathogen. In humans nasal carriage of S. aureus is a risk factor for various infections. Methicillin-resistant S. aureus ST398 is highly prevalent in pigs in Europe and North America. The mechanism of successful pig colonization by MRSA ST398 is poorly understood. Previously, we developed a nasal colonization model of porcine nasal mucosa explants to identify molecular traits involved in nasal MRSA colonization of pigs. Here, we report the analysis of the transcriptome of MRSA ST398 strain S0462 during colonization on the explant epithelium. Major regulated genes were encoding metabolic processes and regulation of these genes represents metabolic adaptation to nasal mucosa explants. Colonization was not accompanied by significant changes in transcripts of main virulence associated genes or known human colonization factors. Here, we document regulation of two genes which have potential influence on S. aureus colonization; cysteine extracellular proteinase (scpA) and von Willebrand factor-binding protein (vwbp, located on SaPIbov5). Colonization with isogenic-deletion strains (Î?vwbp and Î?scpA) did not alter the nasal S. aureus colonization compared to wild type. Our results suggest that nasal colonization with MRSA ST398 is a complex event that is accompanied with changes in bacterial gene expression regulation and metabolic adaptation. Number of the samples: 5 (timepoint 0 min, 30 min, 60 min, 90 min and 180 min) in 4 replicates. 4 control samples
Project description:Type I toxin-antitoxin (TA) systems are widespread genetic modules in bacterial genomes. They express toxic peptides whose overexpression leads to growth arrest or cell death, whereas antitoxins regulate the expression of toxins, acting as labile antisense RNAs. The Staphylococcus aureus (S. aureus) genome contains and expresses several functional type I TA systems, but their biological functions remain unclear. Here we addressed and challenged experimentally, by proteomics, if a type I TA system, the SprF1/SprG1 pair, influences the overall gene expression in S. aureus. Deleted and complemented S. aureus strains were analyzed for their proteomes, both intracellular and extracellular, during growth. Comparison of intracellular proteomes between the strains points on SprF1 antitoxin as an agent downregulating protein expression. In the strain naturally expressing the SprG1 toxin, cytoplasmic proteins are excreted into the medium, but this is not due to unspecific cell leakages. Such toxin-driven release of the cytoplasmic proteins may modulate the host inflammatory response that, in turn, may amplify the S. aureus infection spread.
Project description:In the present study we analyzed the response of S. aureus to mupirocin, the drug of choice for nasal decolonization of S. aureus. Mupirocin selectively inhibits the bacterial isoleucyl-tRNA synthetase (IleRSs) leading to the accumulation of uncharged isoleucyl-tRNA and hence (p)ppGpp. The latter is a signal for the induction of the stringent response, an important global transcriptional and translational control mechanism that allows bacteria to adapt to nutritional deprivation. To identify proteins with an altered synthesis pattern in response to mupirocin treatment we used the highly sensitive 2-dimensional gel electrophoresis technique in combination with mass spectrometry. Obtained results were complemented by DNA-microarray, Northern blot and metabolome analysis. Whereas expression of genes involved in nucleotide biosynthesis, DNA metabolism, energy metabolism and translation was significantly down-regulated, expression of the isoleucyl-tRNA synthetase, the branched chain amino acids pathway, genes with functions in oxidative stress resistance (ahpC, katA), putative roles in stress protection (SACOL1759, SACOL2131, SACOL0815) and transport processes was increased. Of particular interest were the differences in the transcription of genes encoding virulence associated regulators (i.e. arlRS, saeRS, sarA, sarR, sarS) as well as genes directly involved in the virulence of S. aureus (i.e. fnbA, epiE, epiG, seb). In the present study we analyzed the response of S. aureus to mupirocin, the drug of choice for nasal decolonization of S. aureus. Mupirocin selectively inhibits the bacterial isoleucyl-tRNA synthetase (IleRSs) leading to the accumulation of uncharged isoleucyl-tRNA and hence (p)ppGpp. The latter is a signal for the induction of the stringent response, an important global transcriptional and translational control mechanism that allows bacteria to adapt to nutritional deprivation.
Project description:In the present study we analyzed the response of S. aureus to mupirocin, the drug of choice for nasal decolonization of S. aureus. Mupirocin selectively inhibits the bacterial isoleucyl-tRNA synthetase (IleRSs) leading to the accumulation of uncharged isoleucyl-tRNA and hence (p)ppGpp. The latter is a signal for the induction of the stringent response, an important global transcriptional and translational control mechanism that allows bacteria to adapt to nutritional deprivation. To identify proteins with an altered synthesis pattern in response to mupirocin treatment we used the highly sensitive 2-dimensional gel electrophoresis technique in combination with mass spectrometry. Obtained results were complemented by DNA-microarray, Northern blot and metabolome analysis. Whereas expression of genes involved in nucleotide biosynthesis, DNA metabolism, energy metabolism and translation was significantly down-regulated, expression of the isoleucyl-tRNA synthetase, the branched chain amino acids pathway, genes with functions in oxidative stress resistance (ahpC, katA), putative roles in stress protection (SACOL1759, SACOL2131, SACOL0815) and transport processes was increased. Of particular interest were the differences in the transcription of genes encoding virulence associated regulators (i.e. arlRS, saeRS, sarA, sarR, sarS) as well as genes directly involved in the virulence of S. aureus (i.e. fnbA, epiE, epiG, seb). In the present study we analyzed the response of S. aureus to mupirocin, the drug of choice for nasal decolonization of S. aureus. Mupirocin selectively inhibits the bacterial isoleucyl-tRNA synthetase (IleRSs) leading to the accumulation of uncharged isoleucyl-tRNA and hence (p)ppGpp. The latter is a signal for the induction of the stringent response, an important global transcriptional and translational control mechanism that allows bacteria to adapt to nutritional deprivation. In total four independent hybridization experiments with each representing a biological replicate including a control and a treated sample were carried out. To account for the dye bias two of the four replicates were dye swapped.
Project description:Defects in innate immunity affect many different physiologic systems and several studies of patients with primary immunodeficiency disorders demonstrated the importance of innate immune system components in disease prevention or colonization of bacterial pathogens. To assess the role of the innate immune system on nasal colonization with Staphylococcus aureus, innate immune responses in pediatric S. aureus nasal persistent carriers (n=3) and non-carriers (n=3) were profiled by RNAseq. We stimulated previously frozen peripheral blood mononuclear cells (PBMCs) from these subjects with i) live S. aureus (a mixture of all carriage isolates), or ii) heat-killed S. aureus.PBMC gene expression profiles differed between persistent and non-S. aureus carriers following stimulation with either live or dead S. aureus. These observations suggest that individuals susceptible to persistent carriage with S. aureus may possess differences in their live/dead bacteria recognition pathway and that innate pathway signaling is different between persistent and non-carriers of S. aureus.
Project description:CodY is a conserved regulator in gram-positive organisms described to repress metabolic genes mainly involved in nitrogen metabolism but also to control the expression of virulence genes in pathogens. We constructed codY gene-replacement mutants in three unrelated S. aureus strains (Newman, UAMS-1, RN1HG). codY mutants grew slower in a chemically defined medium compared to the wild type strains. However, only codY mutants were able to grow in medium lacking threonine. Excess of isoleucine resulted in growth inhibition in the wild type but not in the codY mutants indicating a role of isoleucine for CodY dependent repression of metabolic genes. The prototypic CodY-repressed operon ilvDBCleuABCilvA is preceded by a CodY-binding motif and repressed after up-shift with isoleucine and to a lesser extent guanidine. Transcription of the quorum sensing system agr followed a similar expression pattern. The codY dependent repression of agr is in line with the concomitant influence of CodY on typical agr regulated genes such as cap, spa fnbA and coa. However, transcriptional analysis revealed that most of these virulence genes (e.g. cap, fnbA, spa hla) are also regulated by CodY in an agr negative background. Microarray analysis revealed the large majority of codY-repressed genes are involved in amino-acid transport and metabolism, genes showing codY dependent activation were mainly involved in nucleotide transport and metabolism or virulence. In summary, CodY in S. aureus not only acts as repressor for genes involved in nitrogen metabolism but also contributes to virulence gene regulation by supporting as well as substituting agr function.