Molecular characterization of benzimidazole resistance in Helicobacter pylori

A family of benzimidazole derivatives (BI) was shown to possess potent and selective activity against Helicobacter pylori, although the precise cellular target of the BIs is unknown. Spontaneous H. pylori mutants were isolated as resistant to a representative BI (compound A). Genomic DNA was isolated from a BI-resistant mutant, transformed into a BI-sensitive strain, and found to be sufficient to confer BI resistance. The resistance determinant was localized to a 17-kb clone after screening a lambda-based genomic library constructed from the BI-resistant strain. Upon sequencing and mapping onto the H. pylori strain J99 genome, the 17-kb clone was shown to contain the entire nuo operon (NADH:ubiquinone oxidoreductase). Further subcloning and DNA sequencing revealed that a single point mutation in nuoD was responsible for BI resistance. The mutation resulted in a G398S amino acid change at the C terminus of NuoD. Thirty-three additional spontaneous BI-resistant mutants were characterized. Sequencing of nuoD from 32 isolated mutants revealed three classes of missense mutation resulting in amino acid changes in NuoD: G398S, F404S, and V407M. One BI-resistant isolate did not have a mutation in nuoD. Instead, a T27A amino acid change was identified in NuoB. MIC testing of the wild-type H. pylori strain and four classes of nuo mutants revealed that all NuoD mutant classes were hypersensitive to rotenone, a known inhibitor of complex I (NADH:ubiquinone oxidoreductase) suggested to bind to NuoD. Further, a nuoD knockout verified that it is essential in H. pylori and may be the target of the BI compounds.