Project description:Staphylococcus aureus USA300 wild type strain was cultivated in RPMI medium in 3 biological replicates and harvested at an OD500 of 0.5 before (as control) and at 30 min after exposure to 1.5 mM in RPMI HOCl stress. Cells were disrupted in 3 mM EDTA/ 200 mM NaCl lysis buffer with a Precellys24 ribolyzer followed by RNA isolation using the acid phenol extraction protocol as described. The RNA quality was approved by Trinean Xpose (Gentbrugge, Belgium) and the Agilent RNA Nano 6000 kit using an Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). Ribo-Zero rRNA Removal Kit (Bacteria) from Illumina (San Diego, CA, USA) was used to remove the rRNA. TruSeq Stranded mRNA Library Prep Kit from Illumina (San Diego, CA, USA) was applied to prepare the cDNA libraries. The cDNAs were sequenced paired end on an Illumina HiSeq 1500 (San Diego, CA, USA) using 70 and 75 bp read length and a minimum sequencing depth of 10 million reads per library. The paired end cDNA reads were mapped to the Staphylococcus aureus USA300 genome sequence (accession number FPR3757_CP255) using bowtie2 v2.2.7 (Langmead and Salzberg, 2012) with default settings for paired-end read mapping. All mapped sequence data were converted from SAM to BAM format with SAMtools v1.3 (Li et al., 2009) and imported to the software ReadXplorer v.2.2 (Hilker et al., 2016).
Project description:Staphylococcus aureus COL wild type strain was cultivated in LB medium in 3 biological replicates and harvested at an OD500 of 0.5 before (as control) and at 30 min after exposure to 1176 μM neutrophil-derived oxidant hypothiocyanous acid (HOSCN) stress. Cells were disrupted in 3 mM EDTA/ 200 mM NaCl lysis buffer with a Precellys24 ribolyzer followed by RNA isolation using the acid phenol extraction protocol as described. The RNA quality was approved by Trinean Xpose (Gentbrugge, Belgium) and the Agilent RNA Nano 6000 kit using an Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). Ribo-Zero rRNA Removal Kit (Bacteria) from Illumina (San Diego, CA, USA) was used to remove the rRNA. TruSeq Stranded mRNA Library Prep Kit from Illumina (San Diego, CA, USA) was applied to prepare the cDNA libraries. The cDNAs were sequenced paired end on an NextSeq 500 (San Diego, CA, USA) using 75 bp read length and a minimum sequencing depth of 8 million reads per library. The paired end cDNA reads were mapped to the Staphylococcus aureus COL genome sequence (accession number CP000046) using bowtie2 v2.2.7 (Langmead and Salzberg, 2012) with default settings for paired-end read mapping. All mapped sequence data were converted from SAM to BAM format with SAMtools v1.3 (Li et al., 2009) and imported to the software ReadXplorer v.2.2 (Hilker et al., 2016).
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: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.