Genetic analysis of the Helicobacter pylori vacuoiating cytotoxin: structural similarities with the IgA protease type of exported protein

Abstract

The human gastric bacterial pathogen Helicobacter pylori has been implicated in type B gastritis, peptic ulceration and gastric adenocarcinoma. Here we report on the cloning and genetic characterization of an H. pylori gene named vacA, which encodes the vacuolating cytotoxin VacA, a novel type of antigenic bacterial toxin that induces the formation of intracellular vacuoles in epithelial cells. The vacuolating cytotoxin activity is expressed by a subset of clinical isolates (Vac+), all of which produce the 87 kDa cytotoxin antigen, but strains which produce neither the activity nor the cytotoxin protein (Vac-) also carry the gene. Isogenic H. pylori mutants in vacA generated by transposon shuttle mutagenesis produce neither the VacA antigen nor a vacuolating activity in a cell culture model. The vacA gene itself encodes a precursor protein of 139.6 kDa consisting of a 33-amino acid signal sequence, the 87 kDa cytotoxin and a 50 kDa C-terminal domain with features typical of a bacterial outer membrane protein. The VacA precursor shows no significant primary sequence homology with any previously reported protein, but its structural organization closely resembles the IgA protease-type of exoprotein produced by pathogenic Neisseriae and Haemophilus species. Our current data support a model for secretion of the cytotoxin through the two bacterial membranes which involves the 50 kDa domain for outer membrane translocation with subsequent proteolytic cleavage and release of the mature 87 kDa cytotoxin into the extracellular environment.

Phosphorylation of Helicobacter pylori CagA by c-Abl leads to cell motility

“…Cells were seeded in tissue culture plates for 48 h before infection and serum-starved for 16 h. Phase-contrast microscopy was performed using an inverted microscope (model TS 100, INTAS). H. pylori strains P1, P12 and Hp26695 and their isogenic mutants DCagA and DPAI have been described elsewhere (Schmitt and Haas 1994;Corthesy-Theulaz et al, 1996;Tomb et al, 1997;Odenbreit et al, 2000;Wessler et al, 2000). Bacteria were cultured on agar plates containing 10% horse serum under microaerophilic conditions at 371C for 48 h. For infection, bacteria were harvested in phosphate-buffered saline (PBS), pH 7.4 and added to the host cells at a multiplicity of infection (MOI) of 100 for the indicated periods of time.…”

Helicobacter pylori Activates the Histidine Decarboxylase Promoter through a Mitogen-activated Protein Kinase Pathway Independent of Pathogenicity Island-encoded Virulence Factors

“…AGS-B cells express the cholecystokinin-B/gastrin receptor through stable transfection and were described before (30 (50), and PAI (missing the cag pathogenicity island (PAI); and G27 (wild type) and the isogenic cagI strain (8). For the construction of the PAI strain two approximately 2-kb DNA fragments upstream of the cag-PAI (region 545254 -547164) and downstream of the PAI (584570 -586563) (51) were amplified by polymerase chain reaction and cloned into pBluescript separated by a kanamycin resistance gene.…”

A Dominant Negative Mutant of Helicobacter pyloriVacuolating Toxin (VacA) Inhibits VacA-induced Cell Vacuolation

“…The H. pylori vacA gene is translated as a 140-kDa protoxin, which undergoes amino-and carboxyl-terminal processing to yield a mature secreted toxin of about 87 kDa (13)(14)(15)(16). Secretion of VacA probably occurs via a mechanism analogous to that used for secretion of Neisseria gonorrhoeae IgA protease (14 -15).…”