Maintenance of Type IV Secretion Function During Helicobacter pylori Infection in Mice.

The Helicobacter pylori type IV secretion system (T4SS) encoded on the cag pathogenicity island (cagPAI) secretes the CagA oncoprotein and other effectors into the gastric epithelium. During murine infection, T4SS function is lost in an immune-dependent manner, typically as a result of in-frame recombination in the middle repeat region of cagY, though single nucleotide polymorphisms (SNPs) in cagY or in other essential genes may also occur. Loss of T4SS function also occurs in gerbils, nonhuman primates, and humans, suggesting that it is biologically relevant and not simply an artifact of the murine model. Here, we sought to identify physiologically relevant conditions under which T4SS function is maintained in the murine model. We found that loss of H. pylori T4SS function in mice was blunted by systemic Salmonella coinfection and completely eliminated by dietary iron restriction. Both have epidemiologic parallels in humans, since H. pylori strains from individuals in developing countries, where iron deficiency and systemic infections are common, are also more often cagPAI+ than strains from developed countries. These results have implications for our fundamental understanding of the cagPAI and also provide experimental tools that permit the study of T4SS function in the murine model.IMPORTANCE The type IV secretion system (T4SS) is the major Helicobacter pylori virulence factor, though its function is lost during murine infection. Loss of function also occurs in gerbils and in humans, suggesting that it is biologically relevant, but the conditions under which T4SS regulation occurs are unknown. Here, we found that systemic coinfection with Salmonella and iron deprivation each promote retention of T4SS function. These results improve our understanding of the cag pathogenicity island (cagPAI) and provide experimental tools that permit the study of T4SS function in the murine model.

CagY-Dependent Regulation of Type IV Secretion in Helicobacter pylori Is Associated with Alterations in Integrin Binding.

Strains of Helicobacter pylori that cause ulcer or gastric cancer typically express a type IV secretion system (T4SS) encoded by the cag pathogenicity island (cagPAI). CagY is an ortholog of VirB10 that, unlike other VirB10 orthologs, has a large middle repeat region (MRR) with extensive repetitive sequence motifs, which undergo CD4+ T cell-dependent recombination during infection of mice. Recombination in the CagY MRR reduces T4SS function, diminishes the host inflammatory response, and enables the bacteria to colonize at a higher density. Since CagY is known to bind human α5ß1 integrin, we tested the hypothesis that recombination in the CagY MRR regulates T4SS function by modulating binding to α5ß1 integrin. Using a cell-free microfluidic assay, we found that H. pylori binding to α5ß1 integrin under shear flow is dependent on the CagY MRR, but independent of the presence of the T4SS pili, which are only formed when H. pylori is in contact with host cells. Similarly, expression of CagY in the absence of other T4SS genes was necessary and sufficient for whole bacterial cell binding to α5ß1 integrin. Bacteria with variant cagY alleles that reduced T4SS function showed comparable reduction in binding to α5ß1 integrin, although CagY was still expressed on the bacterial surface. We speculate that cagY-dependent modulation of H. pylori T4SS function is mediated by alterations in binding to α5ß1 integrin, which in turn regulates the host inflammatory response so as to maximize persistent infection.IMPORTANCE Infection with H. pylori can cause peptic ulcers and is the most important risk factor for gastric cancer, the third most common cause of cancer death worldwide. The major H. pylori virulence factor that determines whether infection causes disease or asymptomatic colonization is the type IV secretion system (T4SS), a sort of molecular syringe that injects bacterial products into gastric epithelial cells and alters host cell physiology. We previously showed that recombination in CagY, an essential T4SS component, modulates the function of the T4SS. Here we found that these recombination events produce parallel changes in specific binding to α5ß1 integrin, a host cell receptor that is essential for T4SS-dependent translocation of bacterial effectors. We propose that CagY-dependent binding to α5ß1 integrin acts like a molecular rheostat that alters T4SS function and modulates the host immune response to promote persistent infection.

Host Determinants of Expression of the Helicobacter pylori BabA Adhesin.

Expression of the Helicobacter pylori blood group antigen binding adhesin A (BabA) is more common in strains isolated from patients with peptic ulcer disease or gastric cancer, rather than asymptomatic colonization. Here we used mouse models to examine host determinants that affect H. pylori BabA expression. BabA expression was lost by phase variation as frequently in WT mice as in RAG2-/- mice that do not have functional B or T cells, and in MyD88-/-, TLR2-/- and TLR4-/- mice that are defective in toll like receptor signaling. The presence of other bacteria had no effect on BabA expression as shown by infection of germ free mice. Moreover, loss of BabA expression was not dependent on Leb expression or the capacity of BabA to bind Leb. Surprisingly, gender was the host determinant most associated with loss of BabA expression, which was maintained to a greater extent in male mice and was associated with greater bacterial load. These results suggest the possibility that loss of BabA expression is not driven by adaptive immunity or toll-like receptor signaling, and that BabA may have other, unrecognized functions in addition to serving as an adhesin that binds Leb.

CagY Is an Immune-Sensitive Regulator of the Helicobacter pylori Type IV Secretion System.

BACKGROUND & AIMS: Peptic ulcer disease and gastric cancer are caused most often by Helicobacter pylori strains that harbor the cag pathogenicity island, which encodes a type IV secretion system (T4SS) that injects the CagA oncoprotein into host cells. cagY is an essential gene in the T4SS and has an unusual DNA repeat structure that predicts in-frame insertions and deletions. These cagY recombination events typically lead to a reduction in T4SS function in mouse and primate models. We examined the role of the immune response in cagY-dependent modulation of T4SS function. METHODS: H pylori T4SS function was assessed by measuring CagA translocation and the capacity to induce interleukin (IL)8 in gastric epithelial cells. cagY recombination was determined by changes in polymerase chain reaction restriction fragment-length polymorphisms. T4SS function and cagY in H pylori from C57BL/6 mice were compared with strains recovered from Rag1-/- mice, T- and B-cell-deficient mice, mice with deletion of the interferon gamma receptor (IFNGR) or IL10, and Rag1-/- mice that received adoptive transfer of control or Ifng-/- CD4+ T cells. To assess relevance to human beings, T4SS function and cagY recombination were assessed in strains obtained sequentially from a patient after 7.4 years of infection. RESULTS: H pylori infection of T-cell-deficient and Ifngr1-/- mice, and transfer of CD4+ T cells to Rag1-/- mice, showed that cagY-mediated loss of T4SS function requires a T-helper 1-mediated immune response. Loss of T4SS function and cagY recombination were more pronounced in Il10-/- mice, and in control mice infected with H pylori that expressed a more inflammatory form of cagY. Complementation analysis of H pylori strains isolated from a patient over time showed changes in T4SS function that were dependent on recombination in cagY. CONCLUSIONS: Analysis of H pylori strains from mice and from a chronically infected patient showed that CagY functions as an immune-sensitive regulator of T4SS function. We propose that this is a bacterial adaptation to maximize persistent infection and transmission to a new host under conditions of a robust inflammatory response.