Date of Award
8-2012
Degree Name
Doctor of Philosophy
Department
Biological Sciences
First Advisor
Dr. John M. Spitsbergen
Second Advisor
Dr. Maria Scott
Third Advisor
Dr. David Huffman
Fourth Advisor
Dr. Susan Stapleton
Keywords
E. Coli, Malate, HNS, enterchemolysin
Abstract
Shiga toxin-producing Escherichia coli (STEC) are worldwide pathogens causing an estimated 200,000 infections per year in the United States. Infections with STEC sometimes progress to kidney failure ending in death. Two studies presented below describe a novel virulence factor and mechanisms required for pathogenesis of STEC seropathotype 091 :H21.
First the novel protein, YghJ, was identified and found to be conserved amongst extraintestinal and diarrheal seropathotypes of E. coli with similar proteins carried by pathogens such as Vibrio cholerae. When the yghJ gene was disrupted creating a YghJ null mutant, the defect resulted in a significant growth deficiency in minimal media with L-malate as the sole carbon source. The null mutant growth defect was complemented by replacement of the mutated copy with an intact copy of yghJ expressed from a high copy number plasmid. Structure-function analysis of YghJ revealed lipidation restricts the YghJ protein to the inner membrane. Further studies suggest an amino-terminal domain involved in carbon binding and a carboxy-terminal region with mucinase activity. Taken together, the data indicates that YghJ belongs to a new class of protein and plays an important role in carbon utilization since oxidation of L-malate to oxaloacetate is obligatory for survival of facultative anaerobes like E. coli.
The second study focused on regulation of expression of the enterohemolysin toxin gene (ehxA) carried by STEC. The role of the HNS protein, a global inhibitor, was investigated. Insertional mutation of the hns gene was constructed in STEC 091:H21and revealed that the amount of EhxA increased in the hns knockout strain. The HNS protein was found to control expression of the ehxA gene through direct binding of the promoter upstream of the enterohemolysin operon (ehxCABD). In total, the data indicates that in STEC the 88 bp DNA region upstream of ehxCABD contains a cis-acting element to which HNS binds and negatively regulates expression of enterohemolysin.
In conclusion, YghJ participates in a key carbon metabolic pathway and provides a competitive advantage in carbon-limited environments where L-malate is present. Compelling evidence suggests that HNS interferes with RNA polymerase binding blocking transcription of ehxCABD in STEC grown ex vivo.
Access Setting
Dissertation-Open Access
Recommended Citation
Rogers, Miles T., "Regulation of Expression of Enterohemolysin Toxin by the Global Repressor HNS and Characterization of a New Class of Protein Produced by Shiga Toxin-Producing Escherichia Coli" (2012). Dissertations. 73.
https://scholarworks.wmich.edu/dissertations/73