Browsing by Subject "Enterohemorrhagic Escherichia coli"
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Item The Characterization of the Fucose Sensing Kinase (FUSK) and the Fucose Sensing Response Regulator (FUSR) and Their Role in Virulence Regulation in Enterohemorrhagic Escherichia Coli O157:H7(2013-01-17) Pacheco, Alline Roberta; Sperandio, Vanessa, Ph.D.EHEC causes outbreaks of bloody diarrhea worldwide, by colonizing the human large intestine, where it forms attaching and effacing (AE) lesions on the intestinal epithelium. AE lesion development requires the presence of the locus of enterocyte effacement (LEE) that encodes for a molecular syringe, a type three secretion system (T3SS), which translocates effectors to the host cell. Expression of the LEE is controlled by the AI-3/Epi/NE interkingdom signaling cascade. The two-component systems QseBC and QseEF are at the core of the AI-3/Epi/NE signaling, controlling expression of flagellar motility genes, the LEE and type 3 secreted effectors in response to AI-3 and the catecholamine hormones Epi and NE. The network of regulatory proteins that form the AI-3/Epi/NE continues to expand, as shown by recent studies from our laboratory. Microarray analyses indicate that a putative two-component system (TCS), herein named FusKR, is repressed by QseBC and QseEF. FusK is the histidine kinase and FusR is the response regulator. In this work, we started to unravel the role of FusKR in EHEC pathogenicity. We constructed isogenic knockouts of fusK and fusR, and investigated their participation in virulence gene regulation in EHEC. Microarray analysis shows that deletion of fusK and fusR alters transcription of virulence and metabolic genes. Phenotypic analyses show that fusK- and fusR- strains are hypervirulent in vitro, overexpress the LEE genes and produces higher amounts of the T3 secreted protein EspB. Nonetheless, the fusK mutant is attenuated for colonization of the mammalian intestine. Biochemical studies revealed that FusK senses fucose. Fucose is an important carbon source for commensal and pathogenic bacteria during intestinal colonization. Transcriptional analyses shows that FusKR signal transduction system regulates fucose utilization indirectly, through regulation of the predicted membrane transporter Z0461, involved in optimal fucose uptake. Gut commensal Bacteroides thetaiomicron (B.theta) degrades mucin, releasing free monosaccharides, including fucose, into the gut lumen. Co-culture of B.theta and EHEC on mucin indicates that this commensal supplies mucin-derived fucose to EHEC, reducing expression of the LEE. Our studies demonstrate that a novel TCS, FusKR, modulates intestinal colonization by EHEC, and it is involved in complex interactions with the microbiota during infection.Item The Functional Characterization of QSEC a Bacterial Adrenergic Receptor and the Luxr Homologue SDIA in EHEC(2009-09-04) Hughes, David T.; Sperandio, VanessaEnterohemorrhagic Escherichia coli (EHEC) O157:H7 is a human pathogen responsible for outbreaks of hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). The histidine sensor kinase QseC is an inner membrane adrenergic receptor which responds to the bacterial signal autoinducer-3 (AI-3) and the host signals epinephrine and norepinephrine. EHEC senses these signals in the gut in order to coordinate expression of multiple virulence factors. These factors include the locus of enterocyte effacement (LEE) genes which facilitate attachment and effacement (AE) of the gut epithelium, Shiga toxin (Stx) which causes HUS, and secreted effectors like NleA. We had previously reported that QseC is autoregulatory and regulates the flagellar genes through its cognate response regulator QseB. Here, we examined the global role of QseC in EHEC gene regulation. Microarray analysis of 뱳eC along with real time RT-PCR (qPCR) revealed QseC's regulation of Stx, NleA, and Ler, the master regulator of the LEE. Additionally, phosphotransfer studies between QseC and thirty two E. coli response regulators, revealed two new QseC phosphotransfer partners: QseF and KdpE. qPCR confirmed a role for QseC in QseF and KdpE genetic regulation. Additionally, QseC appears to regulate the LEE genes through KdpE and regulates Stx through QseF. Finally, 뱳eC and 뱳eB do not have the same phenotype. We examined this phenomenon by monitoring the flagellar response in 뱳eC and 뱳eB. It appears that, QseB plays a dual role in gene regulation based on its phosphorylation state. ?? We also studied the role of EHEC cell-cell signaling in cattle, the asymptomatic natural reservoirs of EHEC. We have shown that mutation of the LuxR homologue SdiA, decreases EHEC's ability to colonize the bovine intestine. The LuxR proteins are transcription factors that are activated or repressed by the quorum sensing molecules, autoinducer-1 (AI-1) which are N-acyl homoserine lactones (AHL). Generally, in these systems, LuxI synthesizes the AHL that LuxR senses. EHEC does not encode a LuxI homologue, indicating that it can respond to AHLs through SdiA, but cannot produce them. EHEC uses SdiA to sense its environment through other AHL-producing bacteria. ?? Microarray analysis and qPCR confirmed that, in response to AHL, SdiA represses the transcription of the LEE genes, which encode bovine colonization and human virulence factors. Additionally, electrophoretic mobility shift assays have indicated that SdiA binds the promoter of ler.?? Previous reports have indicated that glutamate-dependent acid resistance (AR2) is required for EHEC to survive in cattle. qPCR comparing WT EHEC to 볤iA showed a decreased expression of AR2 genes. When AHL was added to WT EHEC an increase was seen in AR2 gene expression. This effect was absent in 볤iA. Functional acid resistance tests have confirmed that SdiA is essential in facilitating acid resistance specifically through the AR2. ?? Finally, previous reports have indicated that sdiA is required for EHEC to survive in cattle. To this end, we have confirmed the presence of AHLs in the bovine rumen and have shown that hydrophobic rumen extracts containing AHLs can decrease LEE gene expression and increase AR2 gene expression. This effect is enhanced in the presence of SdiA. ?? These findings have led us to compose a more complete picture of adrenergic signaling in EHEC and given us a greater understanding of the role of cell-cell signaling in cattle, the natural reservoir of EHEC.??Item The Functional Characterization of the LysR-Type Transcriptional Regulator QseD and the SorC-Type Transcriptional Regulator LsrR in Enterohemorrhagic Escherichia coli(2010-07-12T18:53:55Z) Habdas, Benjamin J.; Sperandio, VanessaEnterohemorrhagic Escherichia coli (EHEC) O157:H7 is a human pathogen responsible for numerous outbreaks of hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) throughout the world. EHEC is able to sense and respond to biotic cues from its environment, such as the human host produced catecholamines epinephrine and norepinephrine, through two two-component systems QseBC and QseEF, and abiotic environmental cues, such as phosphate and sulfate levels through QseEF [1-2]. Additionally, quorum sensing (QS) signaling cascades have evolved to sense microbial population density and diversity through the recognition of bacterially produced autoinducers (AI) AI-2, and 3 by LsrR, and QseBC respectively [1, 3]. Through the interpretation and integration of these multiple regulatory signaling networks that often involve intracellular regulatory proteins, such as the lysine regulator (LysR) type transcriptional (LTTR) family member QseA, EHEC is able to coordinate the expression of its multiple virulence factors [4]. These factors include the production of flagella that confer bacterial motility, the locus of enterocyte effacement (LEE) encoded type three secretion system (TTSS) that facilitates formation of attaching and effacing (AE) lesions on gut epithelium, and is positively regulated by QseA, and Shiga toxin (Stx), which causes cellular damage and HUS. // Here, we show that yjiE, renamed Quorum Sensing E. coli Regulator D (QseD), which was predicted to encode a transcriptional regulator of the LTTR family, functions in a QS-dependent manner to regulate gene expression in both pathogenic and commensal strains of E. coli. LTTRs, the largest known family of prokaryotic DNA binding proteins, contain two functional domains, an N-terminal helix-turn-helix (HTH) and a C-terminal co-factor binding domain which allows for oligomerization [5]. We have demonstrated that QseD indirectly represses transcription of the LEE in EHEC and represses the flagella regulon expression in K-12 E. coli. Additionally QseD regulates the expression of iraD, which has recently been demonstrated to prevent degradation of RpoS by RssB sequestration, leading to an altered bacterial stress-response [6-7]. However, what is most intriguing is that while qseD is prevalent in many enterobacteria it seemingly exists almost exclusively in EHEC O157:H7 isolates as a helix-turn-helix truncated "short" isoform (sQseD). Due to the inability of the sQseD to bind to DNA and the predicted in silico ability of LTTR family members to form hetero-dimers in order to bind DNA, a targeted yeast-two-hybrid (Y2H) approach was used to exclude the known LTTR regulators of LEE transcription QseA and LrhA, as QseD interaction partners. Taken together, these results show that QseD regulates alternate targets in EHEC and K- 12 E. coli, and that EHEC O157:H7 has evolved to encode a truncated form of this protein. // We also studied the role of the LsrR regulon in EHEC pathogenesis and environmental persistence through biofilm formation. LsrR, a negative regulator of lsrK and of the lsrACDBFG operon, has been shown to regulate the uptake and removal of AI-2, the cell-to-cell signaling product of LuxS, from the environment through regulation of the LsrACDB AI-2 uptake pump [8-9]. LsrK, an AI-2 kinase, has been shown to alleviate lsrACDBFG operon repression by generating the inhibitory ligand of LsrR DNA binding, phospho-AI-2 [10]. In E. coli, LsrR has been implicated along with LsrK in AI-2 dependent regulation of biofilm architecture and small-RNA (sRNA) expression [11]. However, while it has been suggested that AI-2 signaling can affect pathogenesis in EHEC, the direct effects of LsrR and LsrK have never been examined [12]. // Here we show that in EHEC both LsrR and LsrK regulate virulence expression, and that this regulation is altered in the absence of a functioning LuxS enzyme. In EHEC, while lsrR and lsrK both positively regulate motility in the presence of luxS, in its absence they both repress motility in a temperature dependent manner. Additionally, in the presence of luxS, lsrR increases biofilm formation. In microarray studies, LsrR was also shown to down-regulate the LEE, and differentially regulate non-LEE effectors (Nle's). Taken together, these results show that both LsrR and LsrK have regulatory roles in the pathogenesis of EHEC and that their effects are altered by the absence of luxS. // These findings have given us a more complete and greater understanding of the genetic regulatory networks and their signaling and integration in EHECItem Regulation of EHEC Lee Pathogenicity Island by Bacterial and Host Signaling(2006-08-11) Walters, Matthew S.; Sperandio, VanessaEnterohemorrhagic E. coli O157:H7 (EHEC) causes outbreaks of bloody diarrhea and hemolytic-uremic syndrome throughout the world. The locus of enterocyte effacement (LEE) consists of five major operons (LEE1 - LEE5) and is required for formation of attaching and effacing (AE) lesions that disrupt intestinal epithelial microvilli. We have previously reported that expression of EHEC LEE genes is regulated by the luxS quorum sensing system. The luxS gene in EHEC affects the production of autoinducer-3 (AI-3), which activates the LEE. Epinephrine and norepinephrine also activate the LEE in a manner similar to AI-3. The luxS mutant had diminished transcription from the LEE promoters during mid-exponential growth phase, decreased levels of the LEE-encoded proteins EscJ, Tir, and EspA, and reduced secretion of EspA and EspB, encoded by LEE4. Epinephrine enhanced LEE expression in both wildtype (WT) and the luxS mutant, but WT still exhibited greater LEE activation. The results suggest a possible synergistic relationship between AI-3 and epinephrine. The combined effects of these two signaling molecules may lead to greater LEE expression and a more efficient infection. Given the virulence defects resulting from the luxS mutation, we next examined pathways which may be affected that lead to reduced AI-3 synthesis. We show that several species of bacteria synthesize AI-3, suggesting a possible role for AI-3 in inter-species bacterial communication. The LuxS enzyme produces the autoinducer-2 (AI-2) precursor 4,5-dihydroxy-2,3-pentanedione (DPD) and homocysteine. Homocysteine is required for the de novo synthesis of methionine in the cell. The luxS mutation leaves the cell with only one pathway for the synthesis of homocysteine, involving the use of oxaloacetate and Lglutamate. The exclusive use of this pathway appears to alter metabolism in the luxS mutant, leading to decreased production of AI-3. Addition of aspartate and increasing the cellular concentration of aromatic amino acids, such as tyrosine, restored AI-3-dependent phenotypes in a luxS mutant. The defect in AI-3 production, but not in AI-2 production, was also restored by expressing the P. aeruginosa S-adenosylhomocysteine hydrolase, which produces homocysteine directly from S-adenosylhomocysteine, in the luxS mutant. Furthermore, Phenotype MicroArrays (Biolog) revealed that the luxS mutation caused numerous metabolic deficiencies, while AI-3 signaling had little effect on metabolism. These studies examine the effects of the luxS mutation on LEE expression, how AI-3 production is affected by mutation of luxS, and explores the roles of the LuxS / AI-2 system in metabolism and QS.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]