Disruption of the Epithelial Apical-Junctional Complex by Helicobacter pylori CagA

Infection of the stomach by Helicobacter pylori strains containing the cag pathogenicity island, a type IV secretion system (TFSS), results in translocation of CagA protein into host epithelial cells and increases the risk of gastric diseases (1–4). H. pylori adhere to cells in the immediate vicinity of the apical-junctional complex, but the significance of this localization for pathogenesis is unclear (5, 6). The epithelial apical-junctional complex forms a network of transmembrane, scaffolding, and signaling proteins, and serves as a barrier, adhesion site, and signaling complex to control cell polarity, proliferation, and differentiation. Dysfunction of the apical-junctional complex is characteristic of many human diseases, including carcinogenesis (7–9).

To study the effects of H. pylori attachment to, and CagA translocation into, polarized epithelia, we developed a system of H. pylori infection of polarized cell monolayers Madin-Darby canine kidney (MDCK) cells (fig. S1). As observed with human infection, MDCK-adapted H. pylori (G27-MA) preferentially attached near cell-cell junctions (Fig. 1) (10). Bacteria not only preferentially targeted the junctions, but also recruited the tight-junction scaffolding protein ZO-1 to sites of attachment (Fig. 1B), and modified the distribution of ZO-1 near junctions, suggesting an interaction between bacterial factors and host proteins (Fig. 1C).

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Because CagA affects cytoskeletal organization once injected into host cells (11), we explored whether it is required to recruit ZO-1 to adherent bacteria. In MDCK cells, isogenic mutants lacking CagA (ΔCagA) adhere to the cell surface, but they adhere less frequently to junctions (fig. S1). We therefore tested wild-type and ΔcagA mutants for ZO-1 recruitment in cells that form organized tight junctions (MDCK cells), and in gastric adenocarcinoma cells (AGS) that express ZO-1 but are unable to form tight junctions. Wild-type H. pylori caused ectopic patches of ZO-1 to assemble underneath adherent bacteria in both cells (Figs. 1, B and C, and 2A). The ΔcagA strain readily attached to the cells but did not induce ZO-1 redistribution (ΔCagA in Fig. 2B and fig. S1). Genetic reconstitution of the cagA gene in the ΔcagA mutant rescued bacterial recruitment of ZO-1 (CagA* in Fig. 2B). Delivery of CagA to the host cell through the cag TFSS (12, 13) was necessary for association with ZO-1 because mutants that cannot translocate CagA [ΔvirB10 (cag7) or ΔvirB4 (cag23, cagE)], were unable to recruit ZO-1 to cell attachment sites (fig. S2). Simultaneous visualization of CagA, ZO-1, and H. pylori showed that injected CagA colocalized with ZO-1 at sites of bacterial attachment, as well as at cell-cell contacts (Fig. 2C).

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Once translocated into host cells, CagA is phosphorylated by kinases of the Src family at tyrosines within the repeated five–amino acid–motif EPIYA (14, 15, 16). H. pylori colocalization with ZO-1 was independent of this modification because a mutant form of CagA that lacks the phosphorylation domains also induced recruitment of ZO-1 [EPISA (14) in Fig. 2B] (15). Phosphorylated CagA binds the phosphotyrosine phosphatase SHP2 as well as the adaptor protein Grb2, and these signaling events correlate with cell elongation (17, 18). CagA colocalization with ZO-1 occurs independently of cell elongation, because the CagA_EPISA mutant recruited ZO-1 but had no effect on AGS cellular morphology (EPISA in Fig. 2B), and wild-type H. pylori colocalized with ZO-1 before cell elongation (Fig. 2A). Thus, CagA may mediate its effects on host cells through at least two functional domains: one that interacts with SH2 domain–containing proteins and another that interacts with components of the apical-junctional complex.

To determine whether CagA affects the barrier function of the apical-junctional complex, we used ruthenium red staining and transmission electron microscopy to examine individual junctions (Fig. 3) (10). In uninfected MDCK monolayers, tight junctions were functional as judged by the exclusion of apically applied ruthenium red from the basal-lateral space. In MDCK monolayers infected with wild-type H. pylori, individual junctions showed barrier dysfunction with leakage of ruthenium red into the basal-lateral space (Fig. 3B, right panel). Leakage of solutes (bovine serum albumin–biotin) across the whole monolayer was also detected in infected cells (Fig. 3A). Solute leakage across an epithelium has been described as a consequence of the H. pylori toxin VacA (19). Long-term infection with H. pylori mutants that lacked either CagA or VacA caused solute leakage across MDCK monolayers, but a double mutant lacking both CagA and VacA had intact barrier function, suggesting that both CagA and VacA alter tight-junction function (Fig. 3A). A clear effect of CagA on tight-junction organization was revealed in MDCK monolayers that were infected during synchronized junction formation (10), because wild-type H. pylori, not ΔCagA mutants, prevented the formation of functional tight junctions (Fig. 3C).

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Because the apical-junctional complex controls cell polarity (7), we asked whether H. pylori infection of MDCK cells disrupts epithelial morphology. At short times after the infection of polarized monolayers (24 hours), overt changes in MDCK cell morphology or behavior were not observed. However, after several days of infection, MDCK cells elongated and extended processes between adjacent cells, resulting in dysplastic cell shapes (Fig. 4, C and D). Intra-epithelial dysplasia depended on the injection of CagA and was reversible if monolayers were treated with antibiotics (20). Infection with an isogenic ΔcagA mutant over the same period caused some cellular enlargement but not dramatic effects on cell shape (Fig. 4B).

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We next examined whether CagA affects the molecular organization of the apical-junctional complex. Membranes from infected MDCK cells were isolated and fractionated in iodixanol density gradients, which resolved distinct membrane fractions containing intercellular junctions, apical and basal-lateral membranes, and cytosolic proteins (10, 21). In uninfected cells and cells infected with a ΔCagA mutant, ZO-1 sediments with membranes in a narrow peak between fractions 10 and 12 (1.09 to 1.108 g/ml) (Fig. 4E). However, membranes containing ZO-1 from cells infected with wild-type H. pylori have a broader distribution between fractions 8 and 18 (1.071 to 1.176 g/ml), and also contained CagA (Fig. 4E). The cytoplasmic phosphatase SHP2 was weakly present or not present in membrane fractions containing apical-junctional proteins in control and ΔcagA-infected MDCK monolayers. In contrast, infection with wild-type H. pylori caused a shift in SHP2 distribution into membrane fractions containing CagA and ZO-1 (Fig. 4E). Not all tight-junction protein distributions were changed; the sedimentation profiles of the tight-junction membrane proteins occludin and claudin-1 (22) were similar in ΔcagA and wild-type infected cells (Fig. 4E).

This analytical method also revealed the involvement of the transmembrane protein junctional adhesion molecule (JAM) in the tight-junction protein complexes induced by CagA. In wild-type–infected MDCK mono-layers, JAM distribution shifted from the lower density fractions (1.19 to 1.23 g/ml) to the fractions containing CagA (Fig. 4E). We confirmed an association between adherent bacteria, ZO-1, and JAM by confocal immunofluorescence microscopy of infected AGS cells (Fig. 4F).

Although AGS cells rapidly elongated in response to infection, polarized MDCK cells did not show morphological changes for several days. We hypothesized that the slower effect of CagA on polarized MDCK cells occurs because ZO-1 is sequestered in stable multiprotein complexes in the tight junctions, whereas in AGS cells ZO-1 is not assembled into tight junctions. To test this, we infected MDCK cells during the development of the apical-junctional complex, when individual proteins are available before their assembly into complexes (10). CagA interfered with the assembly of functional tight junctions (Fig. 3C) and altered the composition of junctional protein complexes. As early as 6 hours after infection with wild-type H. pylori, membranes containing ZO-1 cofractionated with phosphorylated CagA in two distinct density peaks (Fig. 4G; 6 hours, represented by the solid and dashed lines). The peak of lighter membranes (solid line) contained less ZO-1 than that from cells infected with ΔCagA H. pylori (Fig. 4F; 36 versus 53%, respectively, after 6 hours). After 20 hours of infection, the CagA-dependent difference in ZO-1 distribution was exacerbated. Whereas in ΔCagA-infected cells, most of the ZO-1 was found in the lighter membrane peak, the majority of ZO-1 in wild-type–infected MDCK cells fractionated in a denser peak (dashed line). Furthermore, in wild-type–infected MDCK cells, but not in uninfected or ΔCagA infected cells, a broader third peak containing phosphorylated CagA and ZO-1 appeared at a higher density. The pattern of ZO-1 fractionation in cells infected with ΔCagA H. pylori was similar to that of uninfected controls. The shifts in ZO-1 density fractionation seen with wild-type infection are likely the result of aberrant formation of the apical-junction protein complex, induced by CagA delivery, and could be responsible for the observed defects in barrier function and cell polarity.

H. pylori infection and CagA translocation into a tight polarized epithelium target the apical-junctional complex and are linked directly to changes in the structure, function, and morphology of these cells. CagA-mediated recruitment of the scaffolding protein ZO-1 and the tight-junction protein JAM to sites of bacteria attachment on host cell membranes may help to target and retain H. pylori at epithelial cell-cell junctions. Localization of signaling molecules, such as SHP2 and Src to CagA in close proximity to the tight junction may alter apical-junctional complex function (23, 24). In addition, we have previously found CagA translocation–dependent changes in gene expression of tight-junction genes in H. pylori–infected AGS cells (25). One consequence of long-term H. pylori infection with CagA+ strains, but not CagA–strains, is a greatly increased likelihood of serious noninfectious sequelae, particularly peptic ulcer disease and gastric cancer. Learning how the long-term disruption of normal apical-junctional complex signaling by CagA could be responsible for these subsequent cellular changes can teach us as much about human biology as it does about the pathogenesis of H. pylori infection.