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

Abstract

Helicobacter hepaticus is a gram-negative, spiral-shaped microaerophilic bacterium associated with chronic intestinal infection leading to hepatitis and 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 flavoenzymes 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 analysis and mouse infection studies. The putA mutant strain of H. hepaticus exhibited increased proline levels and resistance to oxidative stress relative to that of the wild-type strain, consistent with proline's role as an antioxidant. The significant increase in stress resistance was attributed to higher proline content, as no upregulation of antioxidant genes was observed for the putA mutant strain. The wild-type and putA mutant H. hepaticus strains displayed similar levels of infection 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.

FIG. 1.

Generation of H₂O₂ by PutA in H. hepaticus. Shown are the reactions catalyzed by the PRODH and the P5C dehydrogenase (P5CDH) domains of PutA. During the oxidation of proline, the PRODH domain of PutA can catalyze the transfer of electrons from reduced flavin adenine dinucleotide to molecular oxygen, leading to H₂O₂ formation. The intermediates in the overall conversion of proline to glutamate are P5C and glutamate-γ-semialdehyde (GSA).

FIG. 2.

RT-PCR and Western blotting analyses confirm the disruption of the putA gene in H. hepaticus. (A) RT-PCR performed with putA gene-specific primers. Lanes: 1, kb ladder; 2, wild-type H. hepaticus strain; 3, empty; 4 to 7, different colonies screened for putA deletion in H. hepaticus. (B) Western blotting analysis of protein extracts from the wild-type and putA H. hepaticus strains. Two different amounts of total protein were loaded for each cell lysate sample. Lanes: 1 and 2, putA mutant cell lysates (20 μg and 50 μg, respectively); 3 and 4, wild-type cell lysates (50 μg and 20 μg, respectively); 5 and 6, purified recombinant PutA_Hh (20 μg and 50 μg, respectively).

FIG. 3.

(A) Growth curves for the wild-type (solid curve) and the putA mutant (dashed curve) H. hepaticus strains incubated on BA plates for 0, 24, 48, 96, 144, and 192 h. Cells were resuspended in brucella medium for OD measurements at each time point. (B) Proline content for the wild-type and putA mutant H. hepaticus strains at 0, 24, 48, 96, 144, and 192 h of growth on BA plates. Approximately 3 ×10⁸ cells were used for determining proline content at each time point.

FIG. 4.

(A) Expression levels of the antioxidant genes sodF and katA were analyzed by RT-PCR in the wild-type (left panel) and the putA mutant (right panel) H. hepaticus strains in cells grown for 0, 24, 48, 96, 144, and 192 h. (B) Western blotting analysis of PutA expression in the wild-type H. hepaticus cells grown for 0, 24, 48, 96, 144, and 192 h.

FIG. 5.

Scoring results of histopathological parameters of livers of wild-type H. hepaticus-infected mice (10 animals) and those of putA mutant H. hepaticus-infected mice (9 animals). Liver tissues from mice inoculated with the wild-type H. hepaticus strain (solid bars) and the putA mutant H. hepaticus strain (open bars), stained with hematoxylin-eosin, were graded semiquantitatively for different histological parameters as described in Materials and Methods. Shown here are the percentages of animals showing portal and lobular inflammation (mild or moderate) and biliary lesions/oval changes.

FIG. 6.

Representative photomicrographs of liver sections taken on day 21 postinoculation from mice inoculated with the wild-type H. hepaticus or with the putA mutant H. hepaticus strain and from tissue from sham-inoculated control mice (hematoxylin-eosin stain; magnification, ×20). (A) Wild-type H. hepaticus strain-infected mouse tissue displays mild hepatocellular necrosis with inflammatory cell infiltration (arrow). (B) Wild-type H. hepaticus strain-infected mouse tissue exhibits mild biliary hyperplasia (arrow). (C) Mild portal inflammation and no evidence for tissue necrosis are seen in the liver of a mouse inoculated with the H. hepaticus putA mutant strain. (D) Control mouse liver shows an absence of hepatic damage or inflammation.