Enterohemorrhagic E. coli (EHEC) causes outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS) worldwide. EHEC colonizes the large intestine where it causes attaching and effacing (AE) lesions on intestinal epithelial cells by expression of a type three secretion system (TTSS) encoded within the locus of enterocyte effacement (LEE), and effectors encoded within and outside the LEE, such as Tir and EspFu respectively. Additionally, EHEC also produces Shiga toxins (Stx) that are responsible for HUS. We previously reported that EHEC senses three chemical signals to activate transcription of virulence genes: a bacterial aromatic autoinducer (AI-3) produced by the normal gastrointestinal flora, and two hormones (epinephrine/norepinephrine) produced by the host. These signals are detected by two membrane bound histidine sensor kinases: QseC and QseE, which subsequently relay this information to a complex regulatory cascade to activate transcription of key virulence genes. This cascade is coordinated at the transcriptional, post-transcriptional and translational/functional level. Importantly, all mutants in this signaling cascade have been shown to be attenuated in animal models of infection. Upon sensing AI-3, epinephrine and norepinephrine QseC increases its autophosphorylation, and subsequently phosphorylates its cognate response regulator (RR) QseB, leading to activation of the flagella regulon. Additionally, QseC also phosphorylates two non-cognate RRs, KdpE and QseF. KdpE directly activates transcription of the LEE genes, while QseF activates expression of EspFU and Shiga toxin. QseF and QseB coordinately activate transcription of the small RNA (sRNA) glmY that post-transcriptionally further modulate and coordinate expression of the LEE and espFu (see Progress Report). Finally QseBC activate expression of the qseEGF operon that encodes the histine sensor kinase QseE which also senses epinephrine; its cognate RR QseF (also phosphorylated by QseC, highlighting the conversion and coordination within this cascade) and the outer membrane lipoprotein QseG. QseG coordinates the change of the gating system of the LEE-encoded TTSS to promote translocation of the Tir effector into epithelial cells. Altogether, this complex signaling cascade coordinates expression of all virulence traits in EHEC ensuring their timely expression, so EHEC can swim through the mucus layer, form AE lesions on enterocytes, and express and release Shiga toxin. We have identified several members of this signaling cascade, and addressed their functional role. However, many aspects of this regulatory cascade, which are crucial for EHEC virulence, remain poorly characterized. Accordingly, the specific aims of this study are:
Specific Aim 1. To investigate the kinetics of QseC's phosphorylation of the three response regulators: QseB, QseF and KdpE.
Specific Aim 2. To elucidate the post-transcriptional regulation of the LEE and espFu genes by the glmY sRNA.
Specific Aim 3. To characterize the role of the lipoprotein QseG in attaching and effacing (AE) lesion formation.

Public Health Relevance

Enterohemorrhagic E. coli (EHEC) is a deadly human pathogen that causes bloody diarrhea and hemolytic uremic syndrome worldwide. EHEC senses the host hormones epinephrine and norepinephrine and the bacterial autoinducer AI-3 to activate virulence gene expression. These signals are sensed through the QseC receptor, and initiate a complex regulatory cascade to ensure timely expression of virulence genes. In this grant we aim to understand the intricacies of this signaling cascade and their contribution to EHEC pathogenesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI053067-15
Application #
9189584
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
Baqar, Shahida
Project Start
2003-07-01
Project End
2017-12-31
Budget Start
2017-01-01
Budget End
2017-12-31
Support Year
15
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
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