Project description:In 2011, in Germany, Escherichia coli O104:H4 caused the enterohemorrhagic E. coli (EHEC) outbreak with the highest incidence rate of hemolytic uremic syndrome. This pathogen carries an exceptionally potent combination of EHEC- and enteroaggregative E. coli (EAEC)-specific virulence factors. Here, we identified an E. coli O104:H4 isolate that carried a single nucleotide polymorphism (SNP) in the start codon (ATG>ATA) of rpoS, encoding the alternative sigma factor S. The rpoS ATG>ATA SNP was associated with enhanced EAEC-specific virulence gene expression. Deletion of rpoS in E. coli O104:H4 Dstx2 and typical EAEC resulted in a similar effect. Both rpoS ATG>ATA and DrpoS strains exhibited stronger virulence-related phenotypes in comparison to wild type. Using promoter-reporter gene fusions, we demonstrated that wild-type RpoS repressed aggR, encoding the main regulator of EAEC virulence. In summary, our work demonstrates that RpoS acts as a global repressor of E. coli O104:H4 virulence, primarily through an AggR-dependent mechanism.
Project description:We performed a high-throughput mapping of the 5’ end transcriptome of the pAA plasmid of the clinical Escherichia coli O104:H4 (E. coli O104:H4) isolate LB226692. We employed differential RNA-sequencing (dRNA-seq), a terminator exonuclease (TEX)-based RNA-seq approach allowing for the discrimination of primary and processed transcripts. This method has proven to be a powerful tool for the mapping of transcription start sites (TSS) and detection of non-coding RNAs (ncRNAs) in bacteria. We catalogued pAA-associated TSS and processing sites on a plasmid-wide scale and performed a detailed analysis of the primary transcriptome focusing on pAA virulence gene expression.
Project description:RpoS is a conserved stress regulator that plays a critical role in survival under stress conditions in Escherichia coli and other γ-proteobacteria. RpoS is also involved in virulence of many pathogens including Salmonella and Vibrio species. Though well characterized in non-pathogenic E. coli K12 strains, the effect of RpoS on transcriptome expression has not been examined in pathogenic isolates. E. coli O157:H7 is a serious human enteropathogen, possessing a genome 20% larger than that of E. coli K12, and many of the additional genes are required for virulence. The genomic difference may result in substantial changes in RpoS-regulated gene expression. To test this, we compared the transcriptional profile of wild type and rpoS mutants of the E. coli O157:H7 EDL933 type strain. The rpoS mutation had a pronounced effect on gene expression in stationary phase, and more than 1,000 genes were differentially expressed (two-fold, p<0.05). By contrast, we found 11 genes expressed differently in exponential phase. Western blot analysis revealed that, as expected, RpoS level was low in exponential phase and substantially increased in stationary phase. The defect in rpoS resulted in impaired expression of genes responsible for stress response (e.g., gadA, katE and osmY), arginine degradation (astCADBE), putrescine degradation (puuABCD), fatty acid oxidation (fadBA and fadE), and virulence (ler, espI and cesF). For EDL933-specific genes on O-islands, we found 50 genes expressed higher in wild type EDL933 and 49 genes expressed higher in the rpoS mutants. The protein levels of Tir and EspA, two LEE-encoded virulence factors, were elevated in the rpoS mutants under LEE induction conditions. Our results show that RpoS has a profound effect on global gene expression in the pathogenic strain O157:H7 EDL933, and the identified RpoS regulon, including many EDL933-specific genes, differs substantially from that of laboratory K12 strains. In this study, we characterized the RpoS regulon of E. coli O157:H7 strain EDL933 using microarray analysis.
Project description:Here, we investigated the impact of Stx2 phage carriage on Escherichia coli (E. coli) K-12 MG1655 host gene expression. Using quantitative RNA-seq analysis, we compared the transcriptome of naïve MG1655 and the lysogens carrying the Stx2 phage of the 2011 E. coli O104:H4 outbreak strain or of the E. coli O157:H7 strain PA8, which share high degree of sequence similarity.
Project description:Escherichia coli is a diverse species of bacteria. Some isolates of E. coli can cause hemorrhagic uremic syndrome in humans through consumption of contaminated food. We hypothesize that secreted and surface-associated proteins in E. coli may contribute to bacterial persistence in food products and to pathogenicity in humans. Here we report the proteomic analyses of secretomes from two serotypes of E. coli, O157:H7 and O104:H4, that cause human foodborne illness. The secreted proteins were isolated from the liquid cultures and analyzed by liquid chromatography-tandem mass spectrometry. We report 57 secreted proteins for O157:H7 and 67 for O104:H4 with increased abundance in minimal medium. As signature proteins, O157:H7 expressed intimin and translocated intimin receptor. By contrast, O104:H4 expressed enteroaggregative fimbriae, serine protease autotransporters, Shiga toxin, and beta-lactamase. The E. coli O104:H4 released more cytosolic proteins into the culture media than the O157:H7 serotype, indicating that it might be a leaky strain. The differential abundance of two transpeptidases YbiS and YnhG, along with other lipoproteins in O157:H7 indicates that this serotype secretes several peptidol-lipoprotein components of biofilm. In addition, we observed differential abundance of several ABC transporters, proteases and lipoproteins, which give each strain a unique survival strategy and drug resistance. The identification of secreted proteins will enable us understand better how these bacteria persist in food products and cause human disease.
Project description:RpoS is a conserved stress regulator that plays a critical role in survival under stress conditions in Escherichia coli and other γ-proteobacteria. RpoS is also involved in virulence of many pathogens including Salmonella and Vibrio species. Though well characterized in non-pathogenic E. coli K12 strains, the effect of RpoS on transcriptome expression has not been examined in pathogenic isolates. E. coli O157:H7 is a serious human enteropathogen, possessing a genome 20% larger than that of E. coli K12, and many of the additional genes are required for virulence. The genomic difference may result in substantial changes in RpoS-regulated gene expression. To test this, we compared the transcriptional profile of wild type and rpoS mutants of the E. coli O157:H7 EDL933 type strain. The rpoS mutation had a pronounced effect on gene expression in stationary phase, and more than 1,000 genes were differentially expressed (two-fold, p<0.05). By contrast, we found 11 genes expressed differently in exponential phase. Western blot analysis revealed that, as expected, RpoS level was low in exponential phase and substantially increased in stationary phase. The defect in rpoS resulted in impaired expression of genes responsible for stress response (e.g., gadA, katE and osmY), arginine degradation (astCADBE), putrescine degradation (puuABCD), fatty acid oxidation (fadBA and fadE), and virulence (ler, espI and cesF). For EDL933-specific genes on O-islands, we found 50 genes expressed higher in wild type EDL933 and 49 genes expressed higher in the rpoS mutants. The protein levels of Tir and EspA, two LEE-encoded virulence factors, were elevated in the rpoS mutants under LEE induction conditions. Our results show that RpoS has a profound effect on global gene expression in the pathogenic strain O157:H7 EDL933, and the identified RpoS regulon, including many EDL933-specific genes, differs substantially from that of laboratory K12 strains. In this study, we characterized the RpoS regulon of E. coli O157:H7 strain EDL933 using microarray analysis. A precise rpoS deletion mutant of EDL933 was constructed and employed in this study. EDL933 wild type and rpoS mutants were inoculated in triplicate into LB media at a starting OD of 0.0001 and grown aerobically at 37C. Cultures were harvested at OD600 = 0.3 in exponential phase and OD600=1.5 in stationary phase. For RNA extraction, cultures were mixed directly with a boiling lysis buffer containing SDS and EDTA followed by acidic hot phenol to minimize RNA degradation. RNA samples were hybridized to Affymetrix E. coli Genome 2.0 Array according to Affymetrix's standard protocols.