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

The bacterial oxidation of molecular H2 commonly occurs in nature, as hydrogen gas released by other bacteria represe

nts a useable high-energy reductant.

• 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.

• 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.

• 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

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

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

Mouse colonization assay of H. pylori SS1 and Hyd:cm (SS1)

SS1 => Normal hydrogenaseHyd:cm => Hydrogenase mutant

Inoculated by oral gavage with H. pylori culture

Exp A: 2x108 cells/doseExp B: 1x109 cells/dose

Stomachs excised, weighed, homogenized , serial dilutions were plated on BA plates

4 weeks

Incubated at 37 , 100% humidity, 5% CO℃ 2, 2% O2, balance N2 atmosphere for 5 days

Measure colonization data(CFU/gram stomach)

Mouse colonization assay of H. pylori SS1 and Hyd:cm (SS1)

1 x 103 CFU/gram stomach

Exp A: 2x108 cells/doseExp B: 1x109 cells/dose

Mouse colonization assay of H. pylori SS1 and Hyd:cm (SS1)

19 18

3

17 12

6SS1 Hyd:cm

37 of 37 9 of 38

100% 24%

A mutant H. pylori strain unable to oxidize hydrogen

is severely impaired in its ability to colonize in mice.

Hydrogen concentrations in mouse stomachs

Female C57B1 mice

Clark-type micro-electrode model H2-50Stomach mucus lining area

8 sites per mouse stomach

Anesthetized with halothane

Different days, different times

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

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

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.

Thank you

HP0631(hydA)

HP0632(hydB)