Mechanism(s) of resistance of Helicobacter pylori towards metronidazole
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.