Molecular Characterization of Benzimidazole Resistance in Helicobacter pylori

Abstract

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.

Inhibition of Biofilm Formation by Esomeprazole in Pseudomonas aeruginosa and Staphylococcus aureus

“…roton pump inhibitors (PPI) have been shown to have novel antibacterial properties (9,15). Against Helicobacter pylori, benzimadazole PPI have been shown to have direct antimicrobial effects (15).…”

Benzoxazoles, Phthalazinones, and Arylurea-Based Compounds with IMP Dehydrogenase-Independent Antibacterial Activity against Francisella tularensis

“…4). The quinone site is the binding site for rotenone and other nonselective inhibitors of bacterial and mitochondrial complex I. Helicobacter pylori strains resistant to benzimidazoles contain mutations in nuoB and nuoD that also change the residues near the NuoB-NuoD interface, suggesting that this region may be the site of benzimidazole binding (48). Given the sequence variation between F. tularensis and T. thermophilus, it is not possible to dock the inhibitor into NuoB with confidence.…”

Structure–function relationships in mitochondrial complex I of the strictly aerobic yeast Yarrowia lipolytica

“…When the effects of mutations in the 49 kDa and PSST subunits of Y. lipolytica complex I [22,[24][25][26][27] and other model organisms like Rhodobacter capsulatus [28], Neurospora crassa [29], Escherichia coli [30] and Helicobacter pylori [31], are compared with their map positions in the hydrogenase model, a striking picture emerges ( Figure 2). Mutations of the cysteine ligands of cluster N2 in the PSST subunit, generated in N. crassa or E. coli lead to inactivity and the absence of the cluster N2 In summary, the observed pattern of effects is fully consistent with the following hypotheses [22,23]: (i) The proximal cluster in the hydrogenase small subunit has evolved into cluster N2 that resides in the PSST subunit, close to the interface with the 49 kDa subunit; and (ii) domains involved in the ligation of the [NiFe] centre in the hydrogenase large subunit have evolved to form a significant part of the ubiquinone reducing catalytic core in complex I.…”