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BackgroundMetronidazole (MTZ) is among the first-line drugs against the human gastric pathogen Helicobacter pylori (H. pylori). MTZ is used as a prodrug that is activated by an oxygen-insensitive enzyme NADPH nitroreductase (RdxA). Loss-of-function mutations in rdxA make H. pylori MTZ resistant; however, experimental proof is lacking.MethodsWe collected 139 gastric biopsy samples from patients suspected of H. pylori infection in Shanghai, and amplified Hp-specific rdxA gene from 134 samples. All these rdxA genes were sequenced and phylogenetically compared. The effect of mutations on RdxA function was measured by expressing them in Escherichia coli DH5α by using the MTZ sensitivity test.ResultsIn total, 134 gastric biopsy samples were identified as H. pylori positive. Of the 134 samples, 74 and 6 had point mutations at the various sites or promoter region of rdxA, 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 H. pylori, with 49 clustering with hpEastAsia, 3 with hpEurope, and 2 with hpNEAfrica. All 134 rdxA were expressed in E. coli 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.ConclusionThe occurrence of MTZ resistance induced by site-mutation of RdxA in patients with H. pylori 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 H. pylori to MTZ. Thus, the functional mutants involved in MTZ resistance facilitate clinical diagnosis and medication based on sequence analysis.

Podophyllotoxin (PTOX) is naturally produced by the plant Podophyllum species. Some of its derivatives are anticancer drugs, which are produced mainly by using chemical semi-synthesis methods. Recombinant bacteria have great potential in large-scale production of the derivatives of PTOX. In addition to introducing the correct enzymes, the transportation of PTOX into the cells is an important factor, which limits its modification in the bacteria. Here, we improved the cellular uptake of PTOX into Escherichia coli with the help of the zero-valent sulfur transporter YedE1E2 in the presence of cetyltrimethylammonium bromide (CTAB). CTAB promoted the uptake of PTOX, but induced the production of reactive oxygen species. A protein complex (YedE1E2) of YedE1 and YedE2 enabled E. coli cells to resist CTAB by reducing reactive oxygen species, and YedE1E2 was a hypothetical transporter. Further investigation showed that YedE1E2 facilitated the uptake of extracellular zero-valent sulfur across the cytoplasmic membrane and the formation of glutathione persulfide (GSSH) inside the cells. The increased GSSH minimized oxidative stress. Our results indicate that YedE1E2 is a zero-valent sulfur transporter and it also facilitates CTAB-assisted uptake of PTOX by recombinant bacteria.