Expansion of cagA Copy Number in Helicobacter pylori During Co-Infection in a Mouse Model

Abstract

Introduction The virulence factor CagA is critical in mediating inflammation, and Helicobacter pylori PMSS1 dynamically modulates cagA copy number in response to host immune pressure. Co-infection with Salmonella enterica serotype Typhimurium has been shown to heighten systemic inflammation and reduce H. pylori colonization while maintaining type IV secretion system functionality. This suggests that the inflammatory environments induced by co-infection may influence H. pylori virulence mechanisms. This study investigates whether H. pylori PMSS1 regulates its cagA copy number during systemic co-infection in a mouse model.Methods Across three independent mouse experiments, H. pylori isolated from control and co-infected mice was analyzed for cagA gene copy number using quantitative real-time polymerase chain reaction. Three isolates with the highest cagA copy numbers observed to date were further characterized for their virulence phenotypes and subjected to whole-genome sequencing to identify the mutations associated with co-infection.Results H. pylori isolates recovered from the stomachs of co-infected mice exhibited significantly higher cagA copy numbers than those from control mice, with the mean copy number increasing from 2.64 (+/- 1.03) in the control group to 3.22 (+/- 1.63) in the co-infected group. Three H. pylori isolates with high cagA copy numbers (6.1, 9.1, and 11.0 copies) were assessed for virulence features. Notably, two of these isolates maintained elevated cagA phosphorylation and cell elongation, suggesting their potential relevance as a model for H. pylori infection studies in mice. Whole-genome sequencing further revealed co-infection-associated nonsynonymous mutations, notably in the fur gene and membrane transport-related genes such as an MFS transporter and a corrinoid ABC transporter substrate-binding protein.Conclusions These findings suggest that co-infection with S. promotes an increase in the cagA copy number, thereby enhancing the virulence potential of H. pylori. The study highlights the complex interplay between H. pylori and co-infecting pathogens and their combined impact on H. pylori-mediated disease, and the utility of high-cagA copy isolates as valuable models for in vivo studies addressing pathogenesis.