Master of Science (M.Sc.)
Mark H Forsyth
The pathogen Helicobacter pylori infects up to 50% of the world’s population by colonizing the gastric mucosa, leading to gastric cancer or gastric ulcers for a fraction of infected individuals. To counteract the acidity of the stomach, H. pylori utilizes a signal transduction mechanism, a two-component signal transduction system (TCS), to adapt to local shifts in pH. This system is comprised of a sensor histidine kinase, ArsS, and a response regulator, ArsR. The sensor histidine kinase gene, arsS, encodes three different isoforms that result from slipped strand mispairing in a homopolymeric cytosine tract near the 3’ terminus. These isoforms are each functional in vitro (1), but the impact of the carboxy-terminal domain variations, if any, on subsequent gene expression has never been investigated. Given conservation of the arsS homopolymeric cytosine tract in the closely related species Helicobacter cetorum (cetacean pathogen) and Helicobacter acinonychis (large feline pathogen), we hypothesized that this poly-cytosine tract may impact the ability of H. pylori to respond to acid in one of the following ways: the ability of H. pylori to grow or survive under acidic conditions, the expression of regulon genes under acidic conditions, or the ability of H. pylori to express ArsS under acidic conditions. Isogenic mutants of H. pylori in which the hypermutability of the arsS poly-C tract was minimized to isolate each ArsS isoform were utilized in this study. ClustalW sequences and predicted protein structures demonstrated conservation of the ArsS protein among all isoforms of H. pylori, H. acinonychis, and H. cetorum up to and including the poly-proline region encoded by the 3’ poly-cytosine tract. Following the poly-proline tract, conservation between 40-100% was observed in homologous ArsS isoforms of H. pylori and H. acinonychis Growth curves at pH 7 demonstrated no significant differences in the ability of the isoforms to allow bacterial growth in vitro, while kill curves also demonstrated no significant difference in the ability of the mutants to survive under pH 4.2 acidic conditions. Western blots demonstrated no detectable difference in the protein expression of any of the three stabilized ArsS isoforms under pH 7 conditions. Cells grown under pH 5 and pH 7 conditions were each subjected to RNA-seq and genes differentially expressed identified for validation using qRT-PCR. We identified a series of regulons, including a pan-regulon of genes regulated to varying degrees among all three of the isoforms.Follow-up qRT-PCR has provided insight into variations in both induction ratios of acidic induction and basal levels at neutral conditions among the isoforms for several genes including jhp1300 (hypothetical) and fecA2 (jhp0743). These provide evidence that different isoforms of ArsS influence gene regulation and, thus, may play a larger role in the overall response to the acidic environment of the stomach.
© The Author
Polanshek, Elisabeth Huntley, "Impact Of Isoforms Of ArsS On Acid-Regulated Gene Expression In Helicobacter Pylori" (2022). Dissertations, Theses, and Masters Projects. William & Mary. Paper 1673281537.
Available for download on Monday, August 26, 2024