Helicobacter pylori causes chronic gastritis, peptic ulcer disease and is an early risk factor for gastric cancer The ecological niche of H. pylori is the human stomach, where it establishes in > 50% of the human population a long-term colonization of the mucosa. The host mounts a vigorous immune response against H. pylori, which fails to resolve the infection and may in fact contribute to the severity of the disease. The reason for the successful chronic persistence of H. pylori in its special niche is not well understood. As postulated earlier, H. pylori may evade host responses through the inhibition of antigen-specific T cell proliferation.
Data from the H. pylori mouse model indicate that CD4+ T cells are crucial for control of the H. pylori infection by the host (1). A direct correlation was reported between the level of protection against H. pylori infection and the density of T cells recruited to the gastric mucosa. In contrast to MHC class I and B cell knockout mice, MHC class II knockout mice were not protected by vaccination, indicating that control of the H. pylori infection is dependent on MHC class II-restricted, cell-mediated mechanisms.
Besides the immune system and specific genetic factors of the host (4), several studies suggest that manifestation of gastroduodenal disease is also associated with certain virulence traits of the bacteria. Bacterial factors include the presence of the cag pathogenicity island (cag-PAI) (2) or the vacuolating cytotoxin (VacA) (3). The cag-PAI is only present in type I H. pylori strains and encodes a type IV secretion system, which injects the bacterial protein CagA into the cytoplasm of gastric epithelial cells. CagA is tyrosine-phosphorylated by Src kinase (CagAP-tyr) and involved in modulating host signal transduction pathways (10,13,14). Recently, we demonstrated that injected CagA recruits the growth factor receptor binding protein 2 (Grb2) in epithelial cells and activates Ras signaling via the Raf / Mek / Erk stress kinase pathway, which results in the induction of cell proliferation and cell scattering (8).
A further H. pylori protein with an intracellular target is VacA, an AB type bacterial toxin, which is secreted from H. pylori as a 140 kDa precursor protein by an autotransporter mechanism (type V secretion) (5). Mature VacA is an oligomer of an approximately 95 kDa polypeptide. VacA may be further processed into 37 kDa and 58 kDa subunits; for the vacuolating activity the 37 kDa- and part of the 58 kDa subunit is necessary, whereas the receptor binding function is supposed to be located in the 58 kDa subunit. Variant forms of VacA have been described, which differ in their signal sequences (s1a, s1b, s2) and / or their middle regions (m1, m2). Variation in the m-region apparently correlates with the binding capacity of VacA to target cells. Several functions have been proposed for VacA (for review see (6). It has been reported to selectively inhibit the Ii-dependent pathway of antigen presentation mediated by the major histocompatibility complex (MHC) class II, or to induce apoptosis by targeting mitochondria and inducing cytochrome C release. The basic mechanism how VacA induces vacuolation and how it exerts its numerous other effects is, however, not well understood.
CagA and VacA are the only so far known virulence factors of H. pylori which have their targets within eucaryotic cells (11). CagA is not only translocated into epithelial cells, but also into professional phagocytes, such as human polymorphonuclear leucocytes (PMNs), as well as murine (J774A) and human (THP-1) macrophage cell lines (9). Thus, we reasoned that these bacterial virulence factors might have an impact on T lymphocytes, allowing H. pylori to efficiently resist the host immune system during chronic gastric H. pylori infection. We showed that the m1 form of VacA is able to block the proliferation of T cells by down-regulating expression of interleukin-2 (IL-2) and inducing cell cycle arrest. Intracellular VacA blocks the activity of calcineurin, an ubiquitously expressed cellular phosphatase, which controls the nuclear translocation and activity of the Nuclear factor of activated T cells (NFAT) by its dephosphorylation. The transcription factor NFAT is important for the transcription of a wide range of cytokines, including IL-2. Thus, H. pylori is able to induce a local immune suppression, which may favor its chronic persistence in the gastric mucosa.
Results/Project Status
Helicobacter pylori cag-Type IV secretion system facilitates corpus colonization to induce precancerous conditions in an animal model.
A number of epidemiological studies suggest that atrophic corpus-dominant gastritis is an increased risk factor for gastric carcinogenesis. We therefore studied the role of the Helicobacter pylori Type IV secretion system (T4SS) for pathogenesis in the rodent model. Mongolian gerbils were infected for 32 weeks with either H. pylori type I strain B128, or isogenic mutant strains B128#cagY or B128#cagA, defective in its T4SS, or in the production of its effector protein CagA, respectively. The bacteria were reisolated in a quantitative manner from gastric antrum and corpus separately and cytokines, such as interferon-#, (IFN-#) interleukin-1-beta (IL1##, and KC were measured by quantitative RT-PCR and gastric pH and hormones were determined. Interestingly, wild type (wt) B128-infected gerbils harbored high numbers of bacteria in the gastric antrum and corpus, whereas B128#cagY and B128#cagA were severely restricted to the antrum (12). Furthermore, all infected animals showed a strong antral inflammation and epithelial cell proliferation. B128- rather than mutant-infected gerbils, presented a severe inflammation with large lymph-aggregates, increased proliferation, significant atrophy and mucous gland metaplasia in the corpus. Only in wt-infected animals plasma gastrin levels and gastric pH values were significantly elevated. But in all infected animals the expression of pro-inflammatory cytokines IL-1#, IFN-# and KC was considerably increased in the antrum, but only in wt-infected animals an increase was seen in the corpus mucosa (12). Thus, we show that the presence of an intact T4SS allows H. pylori to colonize the gastric corpus, which results in atrophic corpus-dominant gastritis, a severe precancerous condition. These data identified the T4SS and CagA as a major risk factor for gastric cancer development in an rodent animal model.
Helicobacter pylori VacA blocks T cell activation by different mechanisms
Helicobacter pylori produces the vacuolating cytotoxin VacA, which induces cellular vacuolation in epithelial cells. We and others showed that VacA efficiently blocks proliferation of T cells (7). It specifically interferes with the T cell receptor/IL-2 signaling pathway on the level the Ca2+-calmodulin-dependent phosphatase calcineurin and induces cell cycle arrest. Nuclear translocation of Nuclear Factor of Activated T cells (NFAT), a transcription factor acting as a global regulator of immune response genes, is abrogated. In addition to IL-2 and the high affinity receptor IL-2R#. Our Affymetrix microarray data indicate that a number of chemokines are down-regulated in the Jurkat T cell line upon VacA treatment and that VacA mimics the activity of the immunosuppressive drugs cyclosporin A or FK506. Unexpectedly, we recently noticed that isolated human T cells from peripheral blood did not respond to the activity of VacA as described for the cell line. Interestingly, the treatment of these cells with certain T cell activating agents rendered these T cells fully competent for the VacA activity described above. This indicated, that a certain receptor for VacA has to be expressed before VacA can efficiently bind and be taken up by fresh T cells. Using Affymetrix microarray analysis we are now in the process to identify this putative receptor and to characterizes its function in relation to VacA treatment. Thus, we can conclude that the vacuolating cytotoxin might induce a local immune suppression in the gastric mucosa, which can explain the extraordinary chronicity of the H. pylori infection.
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