Characterization of a Helicobacter hepaticus putA mutant strain in host colonization and oxidative stress

Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588; Veterinary and Biomedical Science Department, University of Nebraska, Lincoln, Nebraska

^* To whom correspondence should be addressed. Email: dbecker3{at}unl.edu.

	Abstract

Helicobacter hepaticus is a Gram-negative, spiral shaped microaerophilic bacterium associated with chronic intestinal infection, leading to hepatitis, colonic and hepatic carcinomas in susceptible strains of mice. In the closely related human pathogen Helicobacter pylori, L-proline is a preferred respiratory substrate and is found at significantly high levels in the gastric juice of infected patients. A previous study of the proline catabolic PutA flavoenzyme from H. pylori and H. hepaticus revealed that Helicobacter PutA generates reactive oxygen species during proline oxidation by transferring electrons from reduced flavin to molecular oxygen. We further explored the preference for proline as a respiratory substrate and the potential impact of proline metabolism on the redox environment in Helicobacter species during host infection by disrupting the putA gene in H. hepaticus. The resulting putA knockout mutant strain was characterized by oxidative stress and mice infection studies. The putA mutant strain of H. hepaticus exhibited increased proline levels and resistance to oxidative stress relative to the wild-type strain consistent with proline acting as an antioxidant. The significant increase in stress resistance was attributed to higher proline content as no upregulation of antioxidant genes was observed in the putA mutant strain. The wild type and putA mutant H. hepaticus strains displayed similar infection levels in mice but in mice challenged with the putA mutant strain significantly reduced inflammation was observed suggesting a role for proline metabolism in H. hepaticus pathogenicity in vivo.