The Gram-negative bacterium Helicobacter pylori is a causative agent of gastritis and peptic ulcer disease in humans. Strains producing the CagA antigen (cagA(+)) induce strong gastric inflammation and are strongly associated with gastric adenocarcinoma and MALT lymphoma. We show here that such strains translocate the bacterial protein CagA into gastric epithelial cells by a type IV secretion system, encoded by the cag pathogenicity island. CagA is tyrosine-phosphorylated and induces changes in the tyrosine phosphorylation state of distinct cellular proteins. Modulation of host cells by bacterial protein translocation adds a new dimension to the chronic Helicobacter infection with yet unknown consequences.
Helicobacter pylori (Hp) carries a type IV secretion system encoded by the cag pathogenicity island (cag‐PAI), which is used to: (i) translocate the bacterial effector protein CagA into different types of eukaryotic cells; and (ii) induce the synthesis and secretion of chemokines, such as interleukin‐8 (IL‐8). The cag‐PAI in Hp 26695 consists of 27 putative genes, six of which were identified as homologues to the basic type IV secretion system represented by the Agrobacterium tumefaciens virB operon. To define the role and contribution of each of the 27 genes, we applied a precise deletion/insertion mutagenesis procedure to knock out each individual gene without causing polar effects on the expression of downstream genes. Seventeen out of 27 genes were found to be absolutely essential for translocation of CagA into host cells and 14 out of 27 for the ability of Hp fully to induce transcription of IL‐8. The products of hp0524 (virD4 homologue), hp0526 and hp0540 are absolutely essential for the translocation of CagA, but not for the induction of IL‐8. In contrast, the products of hp0520, hp0521, hp0534, hp0535, hp0536 and hp0543 are not necessary for either translocation of CagA or for IL‐8 induction. Our data argue against a translocated IL‐8‐inducing effector protein encoded by the cag‐PAI. We isolated a variant of Hp 26695, which spontaneously switched off its capacity for IL‐8 induction and translocation of CagA, but retained the complete cag‐PAI. We identified a point mutation in gene hp0532, causing a premature translational stop in the corresponding polypeptide chain, providing a putative explanation for the defect in the type IV secretion system of the spontaneous mutant.
SummaryType I strains of Helicobacter pylori ( Hp ) use a type IV secretion system (T4SS), encoded by the cag pathogenicity island ( cag -PAI), to deliver the bacterial protein CagA into eukaryotic cells and to induce interleukin-8 secretion. Translocated CagA is activated by tyrosine phosphorylation involving Src-family kinases. The mechanism and structural basis for type IV protein secretion is not well understood. We describe here, by confocal laser scanning microscopy and field emission scanning electron microscopy, a novel filamentous surface organelle which is part of the Hp T4SS. The organelle is often located at one bacterial pole but can be induced by cell contact also along the lateral side of the bacteria. It consists of a rigid needle, covered focally or completely by HP0527 (Cag7 or CagY), a VirB10-homologous protein. HP0527 is also clustered in the outer membrane. The VirB7-homologous protein HP0532 is found at the base of this organelle. These observations demonstrate for the first time by microscopic techniques a complex T4SS-associated, sheathed surface organelle reminiscent to the needle structures of bacterial type III secretion systems.
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