An Analysis of Outer Inflammatory Protein A in cag Pathogenicity Island Negative and Positive Strains of Helicobacter pylori

OipA, outer inflammatory protein A, is an outer membrane protein virulence factor of the gastric pathogen Helicobacter pylori. oipA expression is regulated by phase variation at a CT dinucleotide repeat located within the 5’ end of the gene, such that the gene is alternatively inframe (Phase On) or out-of-frame (Phase Off). H. pylori isolates lacking the cag Pathogenicity Island (cagPAI negative), the primary virulence determinant of H. pylori, induce less host inflammation and almost uniformly possess an oipA allele that is phase off. Meanwhile, cagPAI positive H. pylori isolates almost always possess phase on oipA alleles. The cagPAI encodes a type IV secretion system (T4SS) that elicits the secretion of the pro-inflammatory cytokine, Interleukin 8 (IL-8), by infected gastric epithelial cells. OipA has been shown to play a role in inflammation and as an adhesin that assists in the attachment of H. pylori to host cells. Based on the current study, we hypothesize that the highly conserved, albeit apparently non-expressed, oipA in cagPAI negative strains can affect the adherence character of these isolates of reduced virulence when altered to a phase on status; however OipA’s role in inflammation may be dependent upon the presence of the cagPAI. We sought to determine the role of OipA in cagPAI positive and negative H. pylori infection of human gastric adenocarcinoma (AGS) cells using the cagPAI positive strain, 26695, and cagPAI negative strains, J68 and J75. The oipA CT repeat length was experimentally altered in H. pylori 26695 in order to turn oipA phase off, and turn oipA phase on in J68 and J75. Real time quantitative PCR (RT-qPCR) was used to determine the relative quantity of oipA mRNA. Experimentally turning oipA phase off in strain 26695 resulted in a >5-fold decrease in oipA transcript levels compared to the wild type. The novel phase on oipA J68 mutant resulted in a >6-fold increase in oipA transcription, and Western blotting using antibodies to a FLAG epitope tagged inserted within the J68 oipA allele revealed protein expression at the whole cell level. AGS attachment assays revealed that H. pylori adherence was significantly increased in both the cagPAI positive and negative strain when oipA was phase on compared to isogenic oipA phase off strains. Quantification of IL-8 by enzyme-linked immunosorbent assay showed that this variation in H. pylori attachment was only linked to changes in IL-8 production by AGS cells when infected by the cagPAI positive strain, not cagPAI negative. Taken together, these results indicate that oipA, although phase off in cagPAI negative isolates of H.pylori, can still serve as an adhesin independent of a cag T4SS. However, both OipA and the cagPAI are necessary to induce the host pro-inflammatory cytokine response. Using RT-qPCR, we quantified oipA transcription in response to attachment of H. pylori to AGS cells. oipA transcript levels were at least 7-fold higher in the H. pylori cells attached to AGS cells compared to non-adherent H. pylori cells from the same experiments. This apparent attachment selection favoring bacteria expressing higher levels of oipA was found in both phase on and phase off mutants of the cagPAI positive strain 26695. This finding further adds to the role OipA plays in attachment to host cells.