Upload
hortense-caldwell
View
214
Download
2
Embed Size (px)
Citation preview
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)