Molecular Hydrogen as an Energy Source for Helicobacter pylori

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Molecular Hydrogen as an Energy Source for
Helicobacter pylori

• Jonathan W. Olson and Robert J.Maier
• 29 NOVEMBER 2002 SCIENCE
• Speaker Lai Szu Ming (???)
• Date 2002/12/24

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

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• 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
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• 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.

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• 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

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Characterize hydrogenase regulation

• The reporter gene XylE of Pseudomonas putida

• phydxylE gt hydrogenase promoter xylE gene
• pHP0630xylE gt HP0630 promoter xylE gene
  (not related to hydrogenase)
• pHelxylE gt promoterless xylE gene

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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
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Mouse colonization assay of H. pylori SS1 and
Hydcm (SS1)
SS1 gt Normal hydrogenase Hydcm gt 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)
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Mouse colonization assay of H. pylori SS1 and
Hydcm (SS1)
Exp A 2x108 cells/dose Exp B 1x109 cells/dose
1 x 103 CFU/gram stomach
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Mouse colonization assay of H. pylori SS1 and
Hydcm (SS1)
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A mutant H. pylori strain unable to oxidize
hydrogen is severely impaired in its ability to
colonize in mice.
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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
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Previously show that a whole-cell michaelis
constant (Km) For hydrogen gt 1.8µM
Average 43 µM
Under most conditions the hydrogen oxidizing
system in H. pylori would be saturated
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Discussion

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

Diet gt H2 concentration gt H. pylori controlled
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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.

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Thank you
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Merry X'mas
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HP0631(hydA)
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HP0632(hydB)