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

Abstract

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.

Keywords: Adaptation; Bacteria; IL8; Stomach.

Figure 1. CD4+ T cells are required to control H pylori colonization density and select strains with loss of T4SS function and recombination in cagY

(A) H pylori colonization density was significantly greater in Rag1−/− and T cell KO mice than in wild type mice. Adoptive transfer of WT CD4+ T cells into Rag1−/− mice markedly reduced H pylori colonization compared to Rag1−/−. Each data point represents CFU/g for an individual mouse 8 weeks PI (N=7–8 mice/group). Horizontal lines indicate mean ± standard error of the mean (SEM). (B) Single colonies recovered from WT and B cell KO mice, and mice adoptively transferred with WT CD4+ T cells, showed marked loss in the capacity to induce IL8 that was accompanied by recombination in cagY (open circles). In contrast, all colonies from Rag1−/− mice and most from T cell KO mice induced IL8 and had the same cagY RFLP (closed circles) as WT H pylori PMSS1. Each data point represents the result from a single colony (N=3-6 colonies/mouse). (C) Percent of colonies that underwent cagY recombination (open circles divided by total colonies for each group in panel B). *P≤0.05, **P≤0.01, ***P≤0.001.

Figure 2. Selection of CagY variants is mediated downstream of IFN-γ signaling

(A) H pylori colonization density was significantly higher in IfnγR−/− mice compared to WT at both 4 and 8 weeks PI. Each data point represents CFU/g from an individual mouse (N=6/group). (B) Single colonies (N=3-6/mouse) recovered from WT mice showed loss in the capacity to induce IL8 that was associated with recombination in cagY (open circles), but colonies from IfnγR−/− mice induced IL8 similarly to WT PMSS1 and had no changes in cagY (closed circles). (C) Percent of colonies that underwent cagY recombination (open circles divided by total colonies for each group in panel B). Adoptive transfer of Ifnγ−/− CD4+ T cells into Rag1−/− mice was sufficient to control bacterial load 8 weeks PI (D), but did not select H pylori variants with loss of IL8 induction or change in cagY PCR-RFLP (E,F). Horizontal lines indicate mean ± SEM. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001

Figure 3. CagY is a molecular rheostat that alters the inflammatory capacity of H pylori

Three single colonies recovered from WT mice infected with PMSS1 (Out1, with cagY PCR-RFLP equivalent to wild type cagY from PMSS1; Out2; Out3) had unique cagY PCR-RFPL patterns (A), and induced high, intermediate, or low IL8, respectively (B) (gray bars) compared to PMSS1 and its cagY deletion mutant (black bars). Complementation of ΔcagY with Out1 (ΔY [Out1]), Out2 (ΔY [Out2]), or Out3 (ΔY [Out3]) phenocopied the IL8 induction of the respective output strain (white bars). Data represent mean ± SEM of four replicates. *P≤0.05. (C) Out1, Out2 and Out3 also demonstrated decreasing translocation of phosphorylated CagA (α-PY99), which was phenocopied when PMSS1ΔcagY was complemented with the respective cagY gene. Differences in CagY (α-CagY) were also apparent by immunoblot. Arrowheads in panel A indicate unique bands.

Figure 4. Kinetics of cagY recombination and loss of T4SS function are associated with the capacity of H. pylori to induce inflammation

(A) Replicate IL8 assays (N=10) for WT H pylori strains PMSS1 and J166. Data for both strains are normalized to PMSS1. (B) Mice infected with H pylori PMSS1 showed increased inflammation in gastric tissue compared to J166, which was statistically significant at 8 and 16 weeks PI (B). (C) Colonies recovered from PMSS1-infected WT mice lost the capacity to induce IL8 and changed cagY (open symbols) more rapidly and more completely than colonies recovered from J166-infected mice. Data for each strain are normalized to their respective WT. (D) Percent of colonies that underwent cagY recombination (open circles divided by total colonies for each group in panel C. H pylori recovered 8 weeks after challenge of WT mice with ΔY[Out1], which induces high IL8, were at a lower bacterial density (E) and underwent cagY recombination more frequently (F) than H pylori from mice colonized with ΔY[Out3]. Bars represent mean ± SEM. **P≤0.01, ***P≤0.001, ****P≤0.0001.

Figure 5. Competitive advantage of CagY-mediated loss of T4SS function increases progressively over time

(A) Output colonies from mice infected with an equal mixture of isogenic H pylori PMSS1 strains bearing either the functional (PMSS1) or non-functional (SS1) cagY allele were enumerated by selective plating, and used to calculate the log10 competition index. Each data point represents a single mouse; horizontal lines=geometric mean. At early time points there was no selective advantage, but by 8 weeks PI the PMSS1 strain bearing the SS1 cagY was present at > 300-fold greater abundance. *P≤0.05, **P≤0.01. (B) Normalized IL8 induction of a sweep culture from each mouse showed a strong inverse correlation with log10 competition index.

Figure 6. CagY-mediated loss of T4SS function promotes bacterial persistence during an intense inflammatory response

(A) Il10−/− mice inoculated with H pylori PMSS1 showed significantly increased gastritis compared to WT mice 4 weeks PI. (B) H pylori bacterial burden was significantly higher in WT compared to Il10−/− mice. By 4 weeks PI, bacterial burden in Il10−/− mice was frequently near the level of detection and 3 mice were uninfected (not shown). Mice whose CFU are shown in brackets yielded the colonies whose IL8 induction is shown in brackets in panel C. (C) Loss of the capacity to induce IL8 associated with changes in cagY PCR RFLP (open circles) was more apparent in H pylori colonies recovered from Il10−/− compared to WT mice, particularly in colonies from mice that showed colonization density that resembled that in WT mice. All colonies whose IL8 induction is shown in brackets in panel C were recovered from the mice whose CFU are bracketed in panel B. (D) Average normalized IL8 induction of all colonies from each mouse showed a strong inverse correlation with bacterial burden (R²=0.78, P≤0.0001). **P≤0.01,***P≤0.001.

Figure 7. Recombination in cagY modulates T4SS function during chronic infection in humans

(A) cagY PCR-RFLP analysis of sequential A and B H pylori isolates. Arrowheads denote bands that changed in isolate A and B, which were collected from the same patient 7.4 years apart. (B) IL8 induction normalized to strain PMSS1 for sequential H pylori isolates A and B, their cagY knockouts (Δ), and strains in which their cagY genes have been exchanged. ***P≤0.001,****P≤0.0001.