Browsing by Subject "Gene Expression Regulation, Bacterial"
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Item Characterization of the Activation of the FlgSR two-component system in Campylobacter jejuni(2009-06-17) Joslin, Stephanie Nicole; Hendrixson, David R.Epidemiological studies indicate that Campylobacter jejuni is the leading cause of bacterial gastroenteritis worldwide. This organism has the ability to live as a commensal or a pathogen, depending on the host with which it is associated. While colonization of the gastrointestinal tract of many avian and mammalian species results in a harmless commensal relationship, human infection can cause diarrheal disease. In both scenarios flagellar motility is crucial for promoting optimal host interactions, as non-motile C. jejuni colonize the gastrointestinal tracts of commensal hosts at levels significantly lower than motile isolates and are incapable of causing disease in humans. The means by which C. jejuni regulates flagellar gene transcription and assembly differ from the well-studied pathways in species of Salmonella, E. coli, and Vibrio. Previous studies found that C. jejuni requires the flagellar export apparatus, sigma54, and a two-component regulatory system comprised of the FlgS sensor kinase and the FlgR response regulator to activate transcription of the middle and late sigma54-dependent flagellar genes. The FlgR response regulator is an NtrC-like protein that can be divided into three domains: an N-terminal domain that is phosphorylated by FlgS, a central sigma54 interaction domain, and a C-terminal domain of unknown function. Characterization of FlgR was accomplished by generating constructs that lack the N- or C-terminal domains of the protein and the site of phosphorylation. Through genetic and biochemical analyses, we found that both the N- and C-terminal domains have suppressive functions that prevent FlgR activation of sigma54-dependent flagellar gene transcription in the absence of FlgS. Our data also indicate that unlike other NtrC-family proteins, the C-terminus of FlgR does not bind DNA and is dispensable for FlgR activity. The FlgS sensor kinase activates FlgR through phosphorylation, but little was known about its activation prior to these studies. We have identified the site of FlgS autophosphorylation and demonstrated that formation of the flagellar export apparatus and the presence of at least one other flagellum-associated protein is required for autoactivation of this protein. This study provides insight into the unusual regulation of the FlgSR two-component system and its role in activating sigma54-dependent flagellar gene transcription.Item The Role of Host Hormones and Metabolites in the Regulation of Virulence in Enterohemorrhagic Escherichia coli (EHEC)(2012-07-17) Njoroge, Jacqueline W.; Sperandio, VanessaGastrointestinal bacteria, including the enteric pathogen enterohemorrhagic Escherichia coli O157:H7 (EHEC) that causes hemorrhagic colitis, sense diverse environmental signals, and use them as cues for differential gene regulation and niche adaptation. This allows for a temporal and energy efficient up-regulation of EHEC virulence factors that is essential for successful colonization and infection of the host. These virulence factors include motility genes, Shiga toxin, and attaching and effacing (AE) lesion formation on colonic epithelial cells. AE lesion formation is primarily regulated by a pathogenicity island (PI) known as the locus of enterocyte effacement (LEE). One of the signals sensed by EHEC to activate virulence is the mammalian hormone epinephrine. We investigated the extent of epinephrine regulation in EHEC through transcriptome studies. The bacterial adrenergic kinases QseC and QseE both respond to epinephrine to regulate the LEE PI positively and negatively respectively. We also demonstrated for the first time that co-incubation with epinephrine increases the formation of AE lesions, and that QseC and QseE are the only sensors of epinephrine in EHEC. Epinephrine is not the only host hormone sensed by EHEC. We showed that another human hormone, serotonin is sensed by EHEC, Citrobacter rodentium and uropathogenic E.coli. In EHEC and C.rodentium we showed that serotonin inhibits the transcription of the LEE PI. We also determined that the mechanism of LEE PI inhibition by serotonin is through the reduction of autophosphorylation of the bacterial sensor kinase CpxA, which is itself an activator of the LEE PI. In addition to chemical signaling, nutrient availability plays an important role in bacterial gene regulation. We investigated the role that carbon nutrients play in the regulation of EHEC virulence. We showed that the LEE PI is activated under gluconeogenic conditions, which has been shown to be important for the maintenance of colonization in vivo, and inhibited under glycolytic conditions. We also identified a novel glucose concentration dependent regulator of the LEE PI, Cra. These findings enhanced our understanding of the role that epinephrine plays in virulence, and introduced two other signals, serotonin and glucose which are both important for the regulation of EHEC virulence genes. [Keywords: EHEC; bacterial pathogenesis; serotonin; epinephrine; carbon nutrition; virulence; Escherichia coli]