696 Whole-Genome Sequencing Detects Novel Virulence Variants of Helicobacter pylori cagA Gene and Its Relationship With Oncogenic cagA

Abstract

The H. pylori cag type 4 secretion system (T4SS) translocates CagA into host cells which can lead to NF-κβ activation. The T4SS component CagY is essential for CagA translocation. cagY harbors two repeat motifs which engender considerable genetic diversity to this locus. We used Mongolian gerbils to select for Hp derivative strains that differed in intensity of host interactions to define the biology of long-term colonization and genetic alteration of virulence factors within the gastric niche. Gerbils were initially challenged with the carcinogenic Hp strain 7.13 for 12 weeks; two derivative isolates, 7.13-1 and 7.13-2, were then obtained from 2 independent gerbils. In vitro, strain 7.13-1 induced significantly higher levels of NF-κβ activation and CagA translocation in AGS gastric epithelial cells compared to strain 7.13-2 (fold/uninfected: 100±7 vs. 26±7, respectively; p<0.001) .We next infected new populations of gerbils with parental 7.13 and the two polar 7.13 Hp derivatives for up to 16 weeks. There were no differences in colonization among the groups, although all animals that developed adenocarcinoma harbored decreased bacterial burdens. Strain 7.131 induced premalignant lesions in 25%, 50%, 62.5%, and 75% at 4, 8, 12, and 16 weeks respectively. In contrast, the incidence of dysplasia and adenocarcinoma was only 25% and 12.5% with the 7.13 parental strain or the 7.13-2 derivative 16 weeks post-infection. In vivo-readapted strains from animals infected with parental 7.13 and 7.13-1 maintained their ability to induce high levels of NFκβ activation similar to the input strains, except for strains isolated from animals that developed cancer, which were attenuated in the ability to activate NF-κβ and translocate CagA. In contrast, readapted isolates from animals infected with strain 7.13-2 induced increased levels of NFκβ activation and CagA translocation in vitro, which was concordant with increased levels of inflammation in the corresponding gerbils. To define mechanisms that mediate these phenotypes, we examined cagY rearrangements by RFLP. We identified two distinct patterns, one specific for parental 7.13 and 7.131 and the other for strain 7.13-2. Readaptation revealed that a proportion of parental 7.13 (25%) and 7.13-1 (37.5%) isolate derivatives altered their cagY pattern, developing a 7.132-type pattern in the presence of cancer which was accompanied by decreases in NFκβ activation and CagA translocation. In contrast, 62.5% of Hp readapted isolates from animals infected with strain 7.13-2 changed their cagY pattern to one that mirrored the original 7.13 parental strain and those changes were linked to increases in inflammation, NFκβactivation and CagA translocation. Collectively, these results suggest Hp can modify function of the T4SS by alternating cagY genotype in response to the neoplastic landscape within gastric mucosa.