Date Thesis Awarded

5-2024

Access Type

Honors Thesis -- Access Restricted On-Campus Only

Degree Name

Bachelors of Science (BS)

Department

Biology

Advisor

Mark Forsyth

Committee Members

Matthew Wawersik

Randolph Coleman

Abstract

Helicobacter pylori is a gram-negative pathogenic bacterium that infects the human stomach and is the leading cause of peptic ulcers and gastric cancer. This infection requires utilisation of two component systems to sense and respond to changes in the environment. Our lab established a novel regulon controlled by the CrdRS two component system (Allen, Bate, et al., 2023). Of these novel genes identified, we chose acxA to research further. acxA is part of the operon acxABC encoding the subunits of the enzyme acetone carboxylase which converts acetone into acetoacetate which is eventually converted to acetyl-coA for entry into the TCA cycle. The operon acxABC therefore may be essential for acetone metabolism and generating energy for H. pylori. Acetone, a ketone body, may be present in the human stomach especially during starvation conditions. Due to the ancient association between H. pylori and the human stomach, and the frequency of starvation during human evolution, we speculate that the ability of H. pylori to metabolize acetone as a source of carbon provided an advantage to the survival of this bacterium. We analysed the distribution of this operon across Helicobacter species using BLASTP and found acetone carboxylases to be mostly present in gastric species and less prevalent within enterohepatic species suggesting acetone metabolism may be adaptive in the gastric niche. The extensive regulation of acxA and increased colonization of murine stomachs due to acetone carboxylase, all documented in the literature, suggests the critical importance of acetone carboxylase to H. pylori. We evaluated the effect of nitric oxide (NO) on acxA mRNA expression and using RT-qPCR on H. pylori 26695 CrdR D53A and CrdS H173A phospho-incompetent mutants, we demonstrated that acxA mRNA expression is decreased by NO in a CrdRS dependent fashion. We next investigated the effect of acetone on acxA mRNA expression using various concentrations of acetone. The limited increase in acxA mRNA expression led us to culturing H. pylori in F-12 medium, a defined, minimal medium. We then investigated the effect of acetone supplementation on the growth of H. pylori 26695 in F-12 medium. Our data suggests that H. pylori derives a growth advantage in the presence of acetone, perhaps as an alternative source of carbon. To delineate the role of acetone carboxylase in this growth advantage, we created a H. pylori 26695 ΔacxA mutant which had decreased growth compared to the control strain in the presence of acetone but no defect in growth in the absence of acetone. This suggests a crucial role for acetone carboxylase in H. pylori metabolism. Definition of key energy sources such as acetone for this gastric pathogen are critical to the study of H. pylori colonization and may aid in developing novel therapeutics.

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