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 H 2 commonly occurs in nature, as hydrogen gas released by other bacteria represents a useable high-energy reductant.

6. Once H 2 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. H 2 levels were determined in the termite hind-gut and recently from the cockroach midgut, but H 2 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 phyd:xylE=> hydrogenase promoter + xylE gene pHP0630:xylE=> HP0630 promoter + xylE gene (not related to hydrogenase) pHel:xylE=> promoterless xylE gene The reporter gene XylE of Pseudomonas putida

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/10 8 cells

11. Mouse colonization assay of H. pylori SS1 and Hyd:cm (SS1) SS1=> Normal hydrogenase Hyd:cm=> Hydrogenase mutant Inoculated by oral gavage with H. pylori culture Exp A: 2x10 8 cells/dose Exp B: 1x10 9 cells/dose Stomachs excised, weighed, homogenized, serial dilutions were plated on BA plates 4 weeks Incubated at 37 ℃, 100% humidity, 5% CO 2, 2% O 2, balance N 2 atmosphere for 5 days Measure colonization data (CFU/gram stomach)

12. Mouse colonization assay of H. pylori SS1 and Hyd:cm (SS1) 1 x 10 3 CFU/gram stomach Exp A: 2x10 8 cells/dose Exp B: 1x10 9 cells/dose

13. Mouse colonization assay of H. pylori SS1 and Hyd:cm (SS1) 19 18 3 17 12 6 SS1Hyd:cm 37 of 379 of 38 100%24% 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 Clark-type micro-electrode model H2-50 Stomach mucus lining area 8 sites per mouse stomach Anesthetized with halothane Different days, different times

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

16. Discussion H. pylori infection hydrogen & hydrogenase Colonic H 2 move into other tissue H. pylori is very limited in its use of oxidizable carbon substrates H 2 as a high energy reductant produced by colonic fermentations from other host-residing bacteria. H 2 concentration Diet Diet => H 2 concentration => H. pylori controlled

17. Conclusion H 2 use must represent a large energy boost for a bacterium living in an energy-poor environment (such as gastric mucosa). H 2 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 H 2 utilization within animal hosts may extend beyond just H. pylori and gastric infections.

18. Thank you

21. HP0631(hydA)

22. HP0632(hydB)