ABSTRACT: A study on the effects of an sdiA mutant and the AHL molecule on the virulence of EHEC Comparison of wild type E. coli 8624 with and without AHL to and E. coli 8624 sdiA mutant with and without AHL to determine the effects of each factor on gene expression
Project description:A study on the effects of an sdiA mutant and the AHL molecule on the virulence of EHEC Overall design: Comparison of wild type E. coli 8624 with and without AHL to and E. coli 8624 sdiA mutant with and without AHL to determine the effects of each factor on gene expression
Project description:The goals of this experiment was to obtain a transcriptional profile of EHEC 86-24 upon deletion of fusK or fusR genes. The samples were grown in DMEM until late logaritmic phase then RNA was extracted using the TriZol method.
Project description:Escherichia coli O157:H7 is a food-borne pathogen that causes bloody diarrhea and hemolytic uremic syndrome. Hfq is an sRNA chaperone protein that is involved in post-transcriptional regulation of virulence genes in pathogenic bacteria. In EHEC strain EDL933, Hfq acts a negative regulator of the locus of enterocyte effacement (LEE) that encodes most of the proteins involved in type three secretion and attaching and effacing lesions. We deleted hfq in E. coli O157:H7 strain 86-24 and compared global transcription profiles of the hfq mutant to the wild type strain in exponential growth phase. Deletion of hfq affected transcription of genes common to nonpathogenic and pathogenic strains of E. coli as well as pathogen-specific genes. Downregulated genes in the hfq mutant included ler as well as genes encoded in LEE2-5 that encode for type three secretion and AE lesion formation. Decreased expression of the LEE genes in the hfq mutant occurred at mid-, late, and stationary growth phases in both LB and DMEM media as detected by qRT-PCR. We also confirmed decreased regulation of the LEE genes by examining secreted proteins and AE lesion formation by the hfq mutant and WT strains. Deletion of hfq also caused decreased expression of the two-component system qseBC involved in inter-kingdom signaling and virulence gene regulation in EHEC as well as an increase in stx2AB expression that encodes for the deadly Shiga toxin. Altogether, these data indicate that Hfq plays a different regulatory role in EHEC 86-24 from what has been reported for EHEC strain EDL933 and that the role of Hfq in EHEC virulence regulation extends beyond the LEE. Comparison of transcriptional regulation of the WT 86-24 isolate and the hfq mutant for the identification of regulated targets that were followed up by functional analysis.
Project description:Bacterial pathogens must be able to both recognize suitable niches within the host for colonization and successfully compete with commensal flora for nutrients in order to establish infection. Ethanolamine (EA) is a major component of mammalian and bacterial membranes and may be used by pathogens as a carbon and/or nitrogen source in the gastrointestinal tract. We examined how EA influences gene expression in the human pathogen enterohemorrhagic Escherichia coli O157:H7 (EHEC). Our results indicate EA is not only important for nitrogen metabolism, but that EA is used in cell-to-cell signaling to activate virulence gene expression. Genes encoding for the global regulatory proteins QseC, QseE, and QseA, as well as for attaching and effacement (AE) lesion formation and Shiga toxin are differentially regulated when EHEC is grown with micromolar concentrations of EA. We also constructed a deletion of eutR that encodes the regulator of the eut (EA utilization) operon and examined virulence gene expression. These results suggest that EutR is important in regulating gene expression in response to EA, but that EA signaling does not occur solely through EutR. This is the first report linking EA to cell-to-cell signaling and pathogenesis. Design of the study was to compare the transcriptional response of WT cells grown with ethanolamine to that of wild type cells grown without ethanolamine.
Project description:Enterohemorrhagic E. coli (EHEC) colonizes the large intestine and causes attaching and effacing lesions (AE). Most of the genes involved in the formation of AE lesions are encoded within a chromosomal pathogenicity island termed the Locus of Enterocyte Effacement (LEE). The LysR-like transcriptional factor QseA regulates the LEE by binding directly to the regulatory region of ler. Here, we performed transcriptome analyses comparing WT EHEC and the isogenic qseA mutant in order to elucidate the extent of QseA’s role in gene regulation in EHEC. The following results compare genes that were up-regulated and down-regulated ! 2-fold in the qseA mutant strain compared to the WT strain. At mid-exponential growth, 222 genes were up-regulated and 1874 were downregulated. At late-exponential growth, a total of 55 genes were up-regulated and 605 genes were down-regulated. During mid-exponential growth, QseA represses its own transcription, whereas during late-logarithmic growth, QseA activates expression of the LEE genes as well as non-LEE encoded effector proteins. During both growth phases, several genes carried in O-islands, were activated by QseA, whereas genes involved in cell metabolism were repressed. We also performed electrophoretic mobility shift assays, competition experiments, and DNAseI footprints, and the results suggested that QseA directly binds both the ler proximal and distal promoters, its own promoter, as well as promoters of genes encoded in EHEC-specific O-islands. Additionally, we mapped the transcriptional start site of qseA, leading to the identification of two promoter sequences. Taken together, these results indicate that QseA acts as a global regulator in EHEC, coordinating expression of virulence genes. Design of the study was to make the knockout of the qseA gene and compare the transcriptional response to that and the wild type.
Project description:The ability to respond to stress is at the core of an organism’s survival. The hormones epinephrine and norepinephrine play a central role in stress responses in mammals, which require the synchronized interaction of the whole neuroendocrine system. Bacteria also sense and respond to epinephrine and norepinephrine as a means to gauge the metabolic and immune state of the host. Mammalian adrenergic receptors are G-coupled protein receptors (GPCRs), bacteria, however, sense these hormones through histidine sensor kinases (HKs). HKs autophosphorylate in response to multiple signals and transfer this phosphate to response regulators (RRs). Two bacterial adrenergic receptors have been identified in EHEC, QseC and QseE, with QseE being downstream of QseC in this signaling cascade. We mapped the QseC signaling cascade in the deadly pathogen enterohemorrhagic E. coli (EHEC), which exploits this signaling system to promote disease. Through QseC, EHEC activates expression of metabolic, virulence and stress response genes, synchronizing the cell response to these stress hormones. Coordination of these responses is achieved by QseC phosphorylating three of the thirty two EHEC RRs. The QseB RR, which is QseC’s cognate RR, activates the flagella regulon which controls bacteria motility and chemotaxis. The QseF RR, which is phosphorylated by the QseE adrenergic sensor, coordinates expression of virulence genes involved in formation of lesions in the intestinal epithelia by EHEC, and the bacterial SOS stress response. The third RR, KdpE, controls potassium uptake, osmolarity response, and also the formation of lesions in the intestine. Adrenergic regulation of bacterial gene expression shares several parallels with mammalian adrenergic signaling having profound effects in the whole organism. Understanding adrenergic regulation of a bacterial cell is a powerful approach to study the underlying mechanisms of stress and cellular survival. Experiment Overall Design: Microarray comparisons reveal some trends with respect to signaling cascades. Comparative methods were used to identify networks.
Project description:Adherence of pathogenic Escherichia coli strains to intestinal epithelia is essential for infection. For enterohemorrhagic E. coli (EHEC) serotype O157:H7, we have previously demonstrated that multiple factors govern this pathogen’s adherence to HeLa cells (39). One of these factors is CadA, a lysine decarboxylase, and this protein has been proposed to negatively regulate virulence in several enteric pathogens. In the case of EHEC strains, CadA modulates expression of the intimin, an outer membrane adhesin involved in pathogenesis. Here, we experimentally inactivated cadA in O157:H7 strain 86-24 to investigate the role of this gene in EHEC adhesion to tissue culture monolayers, global gene expression patterns, and colonization of the infant rabbit intestine. As expected, the cadA mutant did not possess lysine decarboxylation activity and was hyper-adherent to tissue-culture cells. Adherence of the cadA mutant was nearly 2-fold greater than that of the wt and complementation of the cadA defect reduced adherence back to wt levels. Furthermore, the cadA mutant affected the expression of intimin protein. Disruption of the eae gene (encoding the intimin protein) in the cadA mutant significantly reduced its adherence to tissue-culture cells. However, adherence of the cadA eae double mutant was greater than that of an 86-24 eae mutant, suggesting that the enhanced adherence of the cadA mutant is not entirely attributable to enhanced expression of intimin in this background. Gene array analysis revealed that the cadA mutation significantly altered EHEC gene expression patterns; expression of 1332 genes was down-regulated and 132 genes up-regulated in the mutant compared to the wild type strain. Interestingly, the gene expression variation shows an EHEC-biased gene alteration including intergenic regions. Two putative adhesins: flagella and F9 fimbriae were up-regulated in the cadA mutant, suggestive of their association with adherence in absence of the Cad regulatory mechanism. Remarkably, in the infant rabbit model, the cadA mutant out-competed the wild type strain in the ileum but not in the cecum or mid-colon, raising the possibility that CadA negatively regulates EHEC pathogenicity in a tissue-specific fashion. Experiment Overall Design: Arrays used as a starting point for further examination of the effects of the cadA deficiency. A limited number of biologically significant phenotypes and gene expression profiles were examined using qRT-PCR
Project description:Escherichia coli 8624 and the isogenic mutants in qseE, qseF and qseG are compared to determine the role that each of the genes play in regulation of the transcriptome. These results are verified by qRT-PCR and reveal the important role of this three-component signaling system. Escherichia coli 8624 and the isogenic mutants in qseE, qseF and qseG are compared to determine the role that each of the genes play in regulation of the transcriptome. Single Affymetrix E. coli 2.0 Arrays are used as a starting point for this study and these results are further verified by qRT-PCR.