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: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: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:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goal of this study is to investigate the significantly different pathways and genes between ST398 and ST239. Methods: mRNA profiles of ST398 and ST239 at mid-logarithmic growth phase (4h) were generated by deep sequencing, respectively in quadruplicate and duplicate samples, using the Hiseq2000 (Illumina, CA) sequencer. The four samples of ST398 are J-92 (Sample1), W-604 (Sample2), R-1025 (Sample3) and R-1089 (Sample4) and grouped to G1, while the two samples of ST239 are J-95 (Sample5) and J-99 (Sample6) and grouped to G2. The sequence reads of ST398 and ST239 that passed quality filters were respectively aligned to S. aureus subsp. aureus ST398 (RefSeq accession number AM990992) and S. aureus subsp. aureus TW20 (RefSeq accession number NC _017331) using the Burrows-Wheeler Alignment tool (BWA) followed by ANOVA (ANOVA). Only the consistent data between the four ST398 samples and two ST239 samples were reserved for further analysis. qRT–PCR validation was performed using SYBR Green assays. Results: Using an optimized data analysis workflow, RNA-seq analyses revealed four types of significantly differentially expressed genes between ST398 and ST239 (G1 only, G2 only, G1/G2>2, G2/G1>2), and only the type of G1/G2>2 was included in this study. The type of G1/G2>2 included 164 genes in total, in which there are 14 top genes showing G1/G2>5 including essB gene. Conclusions: Our data provide new information to the signicantly different genes between ST239 and ST398, especially the highly expressed genes in ST398 compared to ST239 which might be closely related to the high virulence of ST398.
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:Livestock-associated (LA) methicillin-resistant Staphylococcus aureus (MRSA) and strains of sequence type 398 (ST398), which first became known for its widespread colonization of pigs but are now also rapidly emerging in the number of human colonization and infections. The ability of broad host adaption in combination with a consciously evolves by acquisition of virulence gene or mobile genetic elements (MGE) have been increasingly addressed ST398 lineage a serious threat to public health. The present study was aimed to track out how the diverse ST398 lineage, which colonized or infected in a broad range of reservoirs and various geographic regions, is actually reflected in the course of virulence evolution. We therefore profiled the extracellular proteome, representing the main reservoir of virulence factors, of 30 representative clinical isolates using label-free quantitative mass spectrometry. The results show that these isolates can be divided into five distinct clusters based on their exoproteome identities and abundance signatures. The majority of proteins identified were predicted as cytoplasmic proteins showing substantial heterogeneity among our 30 investigated isolates. Only 50% of isolates their exoproteome clustering of isolates can be correlated the clustering based on genome sequences suggested that the large-scale extend of genotype changes over time. To assess the virulence and cytotoxicity of the 30 investigated isolates, we employed infection models based on Galleria mellonella and HeLa cells. The results uncovered the grouping of clinical isolates based on their virulence or cytotoxicity have apparently distinctive exoproteome signatures and particular exoproteins could play decisive roles in pathogenicity of this specific S. aureus lineage. Altogether, the combination of exoproteome and virulence analysis contribute to the comprehensive insights for the impact of genome diversity on the global production of virulence factors of this zoonotic lineage, and more importantly, our outcomes as well as our approach provided an effective pipeline to define proteomic signatures of S. aureus virulence.
Project description:Staphylococcus aureus (S. aureus) has already to be one of the most commonly identified bacteria that cause food poisoning. S. aureus colonization in humans can cause serious infections, toxinoses and life threatening diseases. The bacteriocin nisin has been extensively used as potential natural preservative in the food industry, but the overall transcriptional response mechanisms of S. aureus to nisin are still poorly understood. To detect the possible molecular mechanism of nisin against S. aureus, Affymetrix GeneChips were used to determine the global comparative transcription of S. aureus cells triggered by treatment with sub-inhibitory concentrations of nisin. Staphylococcus aureus planktonic cells were exposed for 60 minutes to nisin at concentration of 4 M-NM-<g/ml (1/2M-CM-^W MIC). 2 samples including 2 control samples are analyzed.
Project description:Staphylococcus aureus (S. aureus) has already to be one of the most commonly identified bacteria that cause food poisoning. S. aureus colonization in humans can cause serious infections, toxinoses and life threatening diseases. The bacteriocin nisin has been extensively used as potential natural preservative in the food industry, but the overall transcriptional response mechanisms of S. aureus to nisin are still poorly understood. To detect the possible molecular mechanism of nisin against S. aureus, Affymetrix GeneChips were used to determine the global comparative transcription of S. aureus cells triggered by treatment with sub-inhibitory concentrations of nisin.