Molecular Hydrogen as an Energy Source for Helicobacter pylori

1. Molecular Hydrogen as an Energy Source for Helicobacter pylori Jonathan W. Olson and Robert J.Maier 29 NOVEMBER 2002 SCIENCE Speaker: Lai Szu Ming (賴思明) Date: 2002/12/24

2. The bacterial oxidation of molecular H2 commonly occurs in nature, as hydrogen gas released by other bacteria represents a useable high-energy reductant.

6. Once H2 is bound and “split” by a membrane-associated hydrogenase , further oxidation-reduction and energy-generating steps are facilitated by a series of membrane-bound heme-containing electron carriers.

7. Hydrogen is a by-product of colonic fermentation , and hydrogen has been reported to be produced (measured as excreted gas) in the gastrointestinal tract of both rodents and humans . • H2 levels were determined in the termite hind-gut and recently from the cockroach midgut , but H2 levels in tissues of vertebrate animal hosts has not been assessed. • Molecular hydrogen is used as an energy reservoir for pathogenic bacteria residing in animals is not known.

8. Previously reported that lab-grown H. pylori can express a membrane bound “uptake-type” hydrogenase .(NiFe hydorgenase) FEMS Microbiology Letters 141 (1996) 71-76 Hydorgen uptake hydrogenase in Helicobacter pylori

9. Characterize hydrogenase regulation • The reporter gene XylE of Pseudomonas putida • phyd:xylE => hydrogenase promoter + xylE gene • pHP0630:xylE => HP0630 promoter + xylE gene (not related to hydrogenase) • pHel:xylE => promoterless xylE gene

10. The reporter gene XylE XylE gene 2,3-dioxygenase catechol 2-hydroxymuconic semialdehyde Measure at 375nm absorbance spectrum 1 unit of catechol 2,3-dioxygenase activity oxidizes 1mMole catechol/min, Activities are expressed as units/min/108 cells

11. Mouse colonization assay of H. pyloriSS1 and Hyd:cm (SS1) SS1 => Normal hydrogenase Hyd:cm => Hydrogenase mutant Inoculated by oral gavage with H. pylori culture Exp A: 2x108 cells/dose Exp B: 1x109 cells/dose 4 weeks Stomachs excised, weighed, homogenized , serial dilutions were plated on BA plates Incubated at 37℃, 100% humidity, 5% CO2, 2% O2, balance N2 atmosphere for 5 days Measure colonization data (CFU/gram stomach)

12. Mouse colonization assay of H. pyloriSS1 and Hyd:cm (SS1) Exp A: 2x108 cells/dose Exp B: 1x109 cells/dose 1 x 103 CFU/gram stomach

13. Mouse colonization assay of H. pyloriSS1 and Hyd:cm (SS1) 18 19 6 3 17 12 A mutant H. pylori strain unable to oxidize hydrogen is severely impaired in its ability to colonize in mice.

14. Hydrogen concentrations in mouse stomachs Female C57B1 mice Anesthetized with halothane Clark-type micro-electrode model H2-50 Stomach mucus lining area Different days, different times 8 sites per mouse stomach

15. Previously show that a whole-cell michaelis constant (Km) For hydrogen => 1.8μM Average 43 μM Under most conditions the hydrogen oxidizing system in H. pylori would be saturated

16. Discussion • H. pylori infection <=> hydrogen & hydrogenase • Colonic H2 <=> move into other tissue • H. pylori is very limited in its use of oxidizable carbon substrates <=> H2 as a high energy reductant produced by colonic fermentations from other host-residing bacteria. • H2 concentration <=> Diet Diet => H2 concentration => H. pylori controlled

17. Conclusion • H2 use must represent a large energy boost for a bacterium living in an energy-poor environment (such as gastric mucosa). • H2 is an energy substrate not used by the host, so competition for this high-energy substrate in the gastric environment is not a factor. • Other human pathogens contain uptake-type hydrogenases, so H2 utilization within animal hosts may extend beyond just H. pylori and gastric infections.

18. Thank you

19. Merry X'mas

21. HP0631(hydA)

22. HP0632(hydB)