Proteomic remodeling of outer membrane vesicles in antibiotic-resistant Helicobacter pylori reveals adaptive stress responses

Abstract

Background Antibiotic resistance in Helicobacter pylori has become a major obstacle to successful eradication therapy worldwide. Although outer membrane vesicles (OMVs) are increasingly recognized as key mediators of bacterial adaptation and host interaction, their contribution to antibiotic resistance in H. pylori remains poorly understood.Methods Metronidazole and levofloxacin-resistant derivatives of the H. pylori G27 strain were generated by stepwise antibiotic exposure. OMVs isolated from susceptible and resistant strains were characterized by transmission electron microscopy and dynamic light scattering, followed by comparative LC-MS/MS analysis of whole-cell and OMV proteomes. Functional annotation and enrichment analyses were performed to identify resistance-associated biological processes.Results Comparative proteomic analysis revealed remodeling of both cellular and OMV-associated protein profiles following acquisition of antibiotic resistance. While 236 proteins were shared among all strains, distinct resistance-associated protein signatures were identified in the levofloxacin- and metronidazole-resistant derivatives. Functional enrichment highlighted pathways associated with membrane transport, oxidative stress response, metabolic adaptation, and cellular envelope organization. OMV proteomes differed from their corresponding whole-cell proteomes, a pattern consistent with, though not proof of, selective protein packaging. Conserved vesicle-associated proteins, including UreA, UreB, GroEL, and GroES, were detected in OMVs from all strains, whereas OMVs from the levofloxacin-resistant derivative contained proteins associated with redox homeostasis and iron metabolism, including SelA and FeoB. In addition, differential representation of the virulence factors CagA and VacA was observed between susceptible and resistant OMVs.Conclusion Acquisition of antibiotic resistance in H. pylori is accompanied by coordinated remodeling of both the cellular proteome and OMV cargo. The selective representation of stress adaptation, transport, and virulence associated proteins within OMVs is consistent with the hypothesis that selective protein packaging may contribute to bacterial adaptation under antibiotic pressure and identifies OMV-associated proteins and pathways as potential targets for future therapeutic intervention.