AUTHOR=Huang Jia , Li Zhiyu , Ge Fulin , Sun Chao , Deng Zixin , Yao Weiyan , He Xinyi 

TITLE=Functional determination of site-mutations in rdxA involved in metronidazole resistance of Helicobacter pylori

JOURNAL=Frontiers in Cell and Developmental Biology

VOLUME=12

YEAR=2024

URL=https://www.frontiersin.org/journals/cell-and-developmental-biology/articles/10.3389/fcell.2024.1435064

DOI=10.3389/fcell.2024.1435064

ISSN=2296-634X

ABSTRACT=<sec><title>Background</title><p>Metronidazole (MTZ) is among the first-line drugs against the human gastric pathogen <italic>Helicobacter pylori</italic> (<italic>H. pylori</italic>). MTZ is used as a prodrug that is activated by an oxygen-insensitive enzyme NADPH nitroreductase (RdxA). Loss-of-function mutations in <italic>rdxA</italic> make <italic>H. pylori</italic> MTZ resistant; however, experimental proof is lacking.</p></sec><sec><title>Methods</title><p>We collected 139 gastric biopsy samples from patients suspected of <italic>H. pylori</italic> infection in Shanghai, and amplified <italic>Hp</italic>-specific <italic>rdxA</italic> gene from 134 samples. All these <italic>rdxA</italic> genes were sequenced and phylogenetically compared. The effect of mutations on RdxA function was measured by expressing them in <italic>Escherichia coli</italic> DH5α by using the MTZ sensitivity test.</p></sec><sec><title>Results</title><p>In total, 134 gastric biopsy samples were identified as <italic>H. pylori</italic> positive. Of the 134 samples, 74 and 6 had point mutations at the various sites or promoter region of <italic>rdxA</italic>, generating truncated and extended fused proteins, respectively. The remaining 54 were full-length with single nucleotide variation (SNV) compared with the wild-type RdxA from <italic>H. pylori</italic>, with 49 clustering with hpEastAsia, 3 with hpEurope, and 2 with hpNEAfrica. All 134 <italic>rdxA</italic> were expressed in <italic>E. coli</italic> DH5α; 22 and 112 resultant strains showed MTZ-sensitive and MTZ-resistant phenotypes, respectively. Comparative analysis of single nucleotide polymorphisms (SNPs) in the functional and inactivated RdxA revealed 14 novel mutations in RdxA, 5 of which conferred MTZ resistance: S18F, D59S, L62I, S79N, and A187V.</p></sec><sec><title>Conclusion</title><p>The occurrence of MTZ resistance induced by site-mutation of RdxA in patients with <italic>H. pylori</italic> infection was 83.6% (112/134) in the Shanghai region. The major form of loss-of-function mutation was truncation of RdxA translation at a rate of 58/112 (51.8%). Molecular detection reliably determined the resistance of <italic>H. pylori</italic> to MTZ. Thus, the functional mutants involved in MTZ resistance facilitate clinical diagnosis and medication based on sequence analysis.</p></sec>