Objective The total protein expression of human gastric mucosal epithelial cells (GES-1) infected with Helicobacter pylori strains of varying virulence was analyzed in vitro to elucidate the pathogenic mechanisms associated with virulence and identify potential protein markers.

Methods  The virulence of 35 strains of Helicobacter pylori isolated from different clinical diseases was determined by cell infection experiment in vitro. Five strains of high virulence and 5 strains of low virulence were co-cultured with GES-1 adherent cells for a duration of 24 hours. The infected cells were analyzed by high performance liquid chromatogramy-mass spectrometry system and label-free technology.

Results A total of 361 proteins exhibited differential expression in the high virulence group, with a higher number of differentially expressed proteins observed between the high virulence group and the blank control group compared to the low virulence group. Eighteen potential virulence-related marker proteins were identified. The impact of highly virulent strains on GES-1 cells primarily manifests in various cellular compartments including the nucleus, cytoplasm, mitochondria and ribosomes. The specific functions are predominantly involved in apoptosis, cell proliferation, cell decay, cell adhesion, amino acid metabolism, RNA degradation, protein processing, oxidative phosphorylation, ATP synthesis and metabolism, respiratory chain electron transfer and chemical carcinogenesis–reactive oxygen species pathway. Comprehensive biological analysis revealed that high virulent Helicobacter pylori can induce an upregulation of endogenous superoxide anion radic (O₂^●―) in gastric mucosal epithelial cells, thereby augmenting intracellular H₂O₂ accumulation and leading to cellular damage as well as increased levels of reactive oxygen species (ROS), thus increasing the risk of gastric cancer.

Conclusion The present study unveiled the molecular determinants associated with Helicobacter pylori virulence at the level of whole protein expression, offering molecular markers for the development of a screening method to assess Helicobacter pylori virulence and provided molecular insights into unraveling the pathogenic mechanism of this bacterium.