Mechanism(s) of resistance of Helicobacter pylori towards Metronidazole

PhD Thesis

Nookala, Ravi 2000. Mechanism(s) of resistance of Helicobacter pylori towards Metronidazole. PhD Thesis University of East London Department of Life Sciences
AuthorsNookala, Ravi
TypePhD Thesis

Metronidazole is an essential component of the triple therapy regimen against
Helicobacter pylori infection. The development of resistance towards
metronidazole results in failure to eradicate H. pylori completely.
The main aim of the investigation was to understand further the mechanism(s)
of resistance in H. pylori. The investigation focussed upon studying the role
and function of NADH oxidase in metronidazole resistance. The NADH
oxidase levels were shown to be significantly higher in metronidazole
susceptible strains than in resistant strains. The purification and
characterisation of the enzyme responsible for the oxidation of NADH
resulted in isolation of a protein shown to be catalase. The results suggest that
NADH oxidase activity in susceptible strains is a function of a bifunctional
catalase rather than that of a distinct enzyme. This was confirmed by isolation
of catalase from E. coli cells containing cloned H. pylori catalase and
demonstration that catalase and NADH activities co-purified. The catalase
activity of the purified protein from the bacterial strains used was retained but
the oxidation of NADH was not significant in the resistant strain.
The base sequence of the catalase gene from the susceptible strain was
determined and shown to be 99% identical to that from the cloned gene. The
comparison of the derived amino acid sequence of catalase from H. pylori and
Proteus mirabilis showed that the heme-binding site is highly conserved. The
amino acids in the NAD(P)H binding site are conserved in both strain NCTC
11639 (Mtz s ) and the genomic strain ATCC 26695 (Mtz s) of H. pylori but
show significant differences compared with P. mirabilis.
A three-dimensional model of catalase from a metronidazole-susceptible H.
pylori strain showed stearic hindrance around the NAD(P)H binding site and a
substitution of an acidic for a basic residue within the phosphate binding site.
Both effects could result in NAD(P)H being less tightly bound and, hence able
to leave the catalase in exchange for NADH. Other substitutions may account
for the ability of the modified binding site to oxidise NADH.
The oxidation of NADH aids in the activation of metronidazole, which
damages DNA. The absence of NADH oxidase activity in resistant strains
results in the inability of the enzyme to activate metronidazole leading to
resistance. The finding that this NADH oxidase activity is a function of a
modified catalase in susceptible strains suggests a novel mechanism of
metronidazole resistance in H. pylori.

Publication dates
PrintAug 2000
Publication process dates
Deposited29 Jul 2014
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