HopE and HopD Porin-Mediated Drug Influx Contributes to Intrinsic Antimicrobial Susceptibility and Inhibits Streptomycin Resistance Acquisition by Natural Transformation in Helicobacter pylori

Helicobacter pylori is a human pathogen competent for natural transformation. Intrinsic and acquired antibiotic resistance contribute to the survival and multiplication of H. pylori under antibiotics. While drug-resistance dissemination by natural transformation (NT)-mediated horizontal gene transfer remains poorly understood in H. pylori. The purpose of the study was to investigate the role of H. pylori porins (HopA, HopB, HopC, HopD, and HopE) in the intrinsic antibiotic resistance and to preliminarily reveal the potential effect of HopE and HopD porins in streptomycin resistance acquisition after NT in the presence of antibiotics. Using traditional antibiotic susceptibility tests and growth curve analysis, we found the MIC values of metronidazole, clarithromycin, levofloxacin, tetracycline, rifampin, and streptomycin in mutants lacking HopE and/or HopD were significantly elevated compare to those in wild-type strain. The quantitative analysis of the tetramethyl rhodamine isothiocyanate (TRITC)-labeled streptomycin accumulation at the single-cell level showed reduced streptomycin intracellular fluorescence in ΔhopE and ΔhopD mutant cells. Furthermore, in the presence of translation-inhibiting antibiotic streptomycin, the resistance acquisition frequency was decreased in the wild-type strain, which could be reversed by mutants lacking HopE and HopD that restored relatively high resistance acquisition frequencies. By transforming a pUC19-rpsLmut-sfgfp linear plasmid carrying a streptomycin conferring mutation, we observed that the impaired ability of rpsLmut synthesis in the wild-type strain was restored in the ΔhopE and ΔhopD mutant transformants. Our study revealed that in the presence of streptomycin, resistance acquisition at least partially relied on the deletion of the hopE and hopD genes, because their loss reduced streptomycin concentration in the cell and thus restored the expression of the resistance-conferring gene, which was inhibited by streptomycin in wild-type strain. The loss of HopE and HopD influx activity may also preserve resistance acquisition by transformation in the presence of antibiotics with other modes of action. IMPORTANCE Helicobacter pylori is constitutively competent for natural transformation (NT) and possesses an efficient system for homologous recombination, which could be utilized to study the NT-mediated horizontal gene transfer induced antibiotic resistance acquisition. Bacterial porins have drawn renewed attention because of their crucial role in antibiotic susceptibility. From the perspective of porin-mediated influx in H. pylori, our study preliminarily revealed the important role of HopE and HopD porins not only in preserving the intrinsic susceptibility to specific antibiotic but also in evading acquired antibiotic resistance by NT in the presence of translation-inhibiting antimicrobial. Therefore, the loss of HopE or HopD porin in H. pylori genomes, combined with the large number of secreted or cell-free genetic elements carrying mutations conferring antibiotic resistance, may raise the possibility that this mechanism plays a potential role in the propagation of antibiotic resistance within H. pylori communities.