Cloning and expression of a high-molecular-mass major antigen of Helicobacter pylori: evidence of linkage to cytotoxin production

Abstract

A high-molecular-mass (120to 128-kDa) Helicobacter pyloni antigen has been associated with peptic ulcer disease. We created a bank of 40,000 random chromosomal fragments of H. pyloni 84-183 by using AZapII. Screening of this bank in Escherichia coli XL1-Blue with absorbed serum from an H. pyloni-infected person permitted the isolation and purification of a clone with a 3.5-kb insert. Subcloning of this insert (pMC3) permitted the expression of a recombinant H. pyloni protein that had a mass of approximately 96 kDa and that was recognized by the human serum. Sera that were obtained from H. pylori-infected persons and that recognized the native 120to 128-kDa H. pylori antigen recognized the recombinant 96-kDa pMC3 protein to a significantly greater extent than did sera that did not recognize the native H. pylori antigen. All 19 H. pyloni isolates producing the 120to 128-kDa antigen hybridized with pMC3; none of 13 nonproducers did so (P < 0.001). Because all 15 isolates producing the vacuolating cytotoxin hybridized with pMC3, we called the gene cagA (cytotoxin-associated gene). Sequence analysis of pMC3 identified an open reading frame of 859 amino acids, without a termination codon. Parallel screening of a Agtll library with human serum revealed positive plaques with identical 0.6-kb inserts and sequences matching the sequence of the downstream region of pMC3. To clone the full-length gene, we used the 0.6-kb fragment as a probe and isolated a clone with a 2.7-kb insert from the AZapII genomic library. Nucleotide sequencing of this insert (pYB2) revealed a 785-bp sequence that overlapped the downstream region of pMC3. Translation of the complete nucleotide sequence ofcagA revealed an open reading frame of 1,181 amino acids yielding a protein of 131,517 daltons. There was no significant homology with any previously reported protein sequence. These findings indicate the cloning and characterization of a high-molecular-mass H. pylori antigen potentially associated with virulence and with cytotoxin production.

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). One of these genes, picB (cagE), was recognized to encode a homologue of a protein involved in toxin export of Bordetella, which suggested that the cag PAI of Hp might play a role in secretion of yet unidentified products (Tummuru et al, 1995).…”

Reduced activation of inflammatory responses in host cells by mouse-adapted Helicobacter pylori isolates

“…cagA genotyping of isolates was performed by PCR as described previously (Jenks et al, 1998). The CagA phenotype of isolates was determined by Western blotting with a CagA-reactive hyperimmune human serum (kindly donated by Professor M. J. Blaser, New York University Medical Center, NY, USA) (Tummuru et al, 1993).…”

Helicobacter pylori

“…Antibodies to vacuolating toxin were no more prevalent in infected patients with vs. without ulcers, but antibodies to a 120-128 kDa protein were present in 100% of ulcer patients compared with about 70% of infected controls [71]. This protein has been cloned and the gene called cagA [8]. Studies indicate that strains with cagA express this and vacA whereas strains lacking cagA express neither.…”