Helicobacter pylori causes clinical disease primarily in those individuals infected with a strain that carries the cytotoxin associated gene pathogenicity island (cagPAI). The cagPAI encodes a type IV secretion system (T4SS) that injects the CagA oncoprotein into epithelial cells and is required for induction of the pro-inflammatory cytokine, interleukin-8 (IL-8). CagY is an essential component of the H. pylori T4SS that has an unusual sequence structure, in which an extraordinary number of direct DNA repeats is predicted to cause rearrangements that invariably yield in-frame insertions or deletions. Here we demonstrate in murine and non-human primate models that immune-driven host selection of rearrangements in CagY is sufficient to cause gain or loss of function in the H. pylori T4SS. We propose that CagY functions as a sort of molecular switch or perhaps a rheostat that alters the function of the T4SS and “tunes” the host inflammatory response so as to maximize persistent infection.
Background & Aims Peptic ulcer disease and gastric cancer are most often caused by Helicobacter pylori strains that harbor the cag pathogenicity island (cagPAI), 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-8 (IL8) in gastric epithelial cells. cagY recombination was determined by changes in PCR restriction fragment-length polymorphisms. T4SS function and cagY in H pylori from C57BL/6 mice were compared to strains recovered from Rag1−/− mice, T and B cell deficient mice, mice with deletion of IFNGR or IL10, and Rag1−/− mice that received adoptive transfer of control or Ifng−/− CD4+ T cells. To assess relevance to humans, 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, demonstrated 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 demonstrated 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.
Helicobacter pylori uses natural competence and homologous recombination to adapt to the dynamic environment of the stomach mucosa and maintain chronic colonization. Although H. pylori competence is constitutive, its rate of transformation is variable, and little is known about factors that influence it. To examine this, we first determined the transformation efficiency of H. pylori strains under low O 2 (5% O 2 , 7.6% CO 2 , 7.6% H 2 ) and high O 2 (15% O 2 , 2.9% CO 2 , 2.9% H 2 ) conditions using DNA containing an antibiotic resistance marker. H. pylori transformation efficiency was 6-to 32-fold greater under high O 2 tension, which was robust across different H. pylori strains, genetic loci, and bacterial growth phases. Since changing the O 2 concentration for these initial experiments also changed the concentrations of CO 2 and H 2 , transformations were repeated under conditions where O 2 , CO 2 , and H 2 were each varied individually. The results showed that the increase in transformation efficiency under high O 2 was largely due to a decrease in CO 2 . An increase in pH similar to that caused by low CO 2 was also sufficient to increase transformation efficiency. These results have implications for the physiology of H. pylori in the gastric environment, and they provide optimized conditions for the laboratory construction of H. pylori mutants using natural transformation.
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