The Functional Characterization of QSEC a Bacterial Adrenergic Receptor and the LuxR Homologue SDIA in EHEC

dc.contributor.advisorSperandio, Vanessaen
dc.creatorHughes, David T.en
dc.description.abstractEnterohemorrhagic 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 ΔqseC 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, ΔqseC and ΔqseB do not have the same phenotype. We examined this phenomenon by monitoring the flagellar response in ΔqseC and ΔqseB. 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 ΔsdiA 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 ΔsdiA. 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.en
dc.format.digitalOriginborn digitalen
dc.subjectEnterohemorrhagic Escherichia colien
dc.subjectEscherichia coli Proteinsen
dc.titleThe Functional Characterization of QSEC a Bacterial Adrenergic Receptor and the LuxR Homologue SDIA in EHECen
dc.type.materialTexten School of Biomedical Sciencesen Microbiologyen Southwestern Medical Centeren of Philosophyen


Original bundle

Now showing 1 - 1 of 1
Thumbnail Image
2.9 MB
Adobe Portable Document Format

License bundle

Now showing 1 - 1 of 1
No Thumbnail Available
941 B
Item-specific license agreed upon to submission