Characterization of and human serologic response to proteins in Helicobacter pylori broth culture supernatants with vacuolizing cytotoxin activity

Abstract

Helicobacter pylori infection is strongly associated with histologic gastritis and peptic ulcer disease. Broth culture supernatants from a subset of H. pyloni strains induce vacuolization in cultured cells, a phenomenon that has been attributed to cytotoxin activity. Concentrated culture supernatants from 15 of 28 (53.6%) H. pylori strains tested induced vacuolization in HeLa cells in titers ranging from 1:10 to 1:180. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of supernatants from these 28 strains and 2 control strains demonstrated an 82-kilodalton (kDa) protein band in 3 of 16 supernatants with vacuolizing activity, but in none of 14 supernatants without vacuolizing activity. By immunoblotting with human sera, a 128-kDa band was recognized in all 16 supernatants with vacuolizing activity, compared with 9 of 14 (64%) supernatants without vacuolizing activity (P = 0.014). Serologic recognition of the 128-kDa band in H. pyloni culture supernatants was more prevalent among persons infected with vacuolizing H. pylori strains than among persons infected with nonvacuolizing strains, but the difference was not statistically significant (80 versus 45%; P = 0.079); human serologic recognition of the 82-kDa band was less common. The 128-kDa band was recognized by 100% of 31 serum samples from H. pyloni-infected patients with duodenal ulcer disease, compared with 60.8% of 74 serum samples from H. pyloni-infected persons without peptic ulcer disease (P = 0.0001). These data indicate that antigenic 128-and 82-kDa proteins are present in H. pyloni broth culture supernatants with vacuolizing activity and that serologic responses to the 128-kDa protein are more prevalent among H. pyloni-infected persons with duodenal ulceration than among infected persons without peptic ulceration.

Role of type IV secretion inHelicobacter pyloripathogenesis

“…More than 18 years ago, Cover and co-workers reported a strong association between serologic responses to CagA and peptic ulcer disease (Cover et al, 1990). Independent observations led to cloning of the cagA gene (Tummuru et al, 1993;Covacci et al, 1993) and identification of the role of adjacent cag PAI genes in inflammation (Tummuru et al, 1995).…”

“…Much of the research interest worldwide is focused on the CagA effector protein, because CagA-positive but not CagAnegative Hp strains are associated with the development of severe gastric diseases. CagA was discovered in the early 1990s by independent pioneering work in the labs of Martin Blaser, Jean Crabtree and Antonello Covacci (Cover et al, 1990;Crabtree et al, 1991;Covacci et al, 1993). Recent functional studies in animal and cell culture models provided compelling evidence for the importance of CagA and cag PAI in Hp pathogenesis (Covacci and Rappuoli, 2000;Ogura et al, 2000;Rieder et al, 2005;Backert and Meyer, 2006;Hatakeyama, 2008).…”

Conservation of the cytotoxin-associated (cag A) gene of Helicobacter pylori and investigation of association with vacuolating-cytotoxin activity and gastroduodenal disease

“…To investigate the association between presence of the cagA gene and the development of HaeIII restriction site variation in and around the cagA gene, and to estimate cagA gene copy number. As there was no HaeIII restriction site within the amplified region of cagA according to the published sequences [15,17], it was assumed that one band in the genomic Southern blot was indicative of a single cagA copy. The results on cagA RFLP's obtained for both sets of H. pylori are given in Table 2 (cytotoxin set) and Table 4 (London Hospital isolates).…”

“…immune evasion, inflammatory mediators, cytotoxins and urease) [13], but to date only two potential virulence factors have been found more frequently in duodenal ulcer disease compared with individuals who are histologically normal. These are the 87 kDa vacuolating cytotoxin and a 120-128 kDa protein [14,15] coded by the cytotoxin-associated (cagA) gene [16,17]. Antibodies to this protein in either serum or mucosal secretions have been shown to be associated with peptic ulcer [15,18].…”

“…These are the 87 kDa vacuolating cytotoxin and a 120-128 kDa protein [14,15] coded by the cytotoxin-associated (cagA) gene [16,17]. Antibodies to this protein in either serum or mucosal secretions have been shown to be associated with peptic ulcer [15,18].…”