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Mapping of a domain required for protein-protein interactions and inhibitory activity of a Helicobacter pylori dominant-negative VacA mutant protein.

ABSTRACT: The Helicobacter pylori VacA toxin is an 88-kDa secreted protein that causes multiple alterations in mammalian cells and is considered an important virulence factor in the pathogenesis of peptic ulcer disease and gastric cancer. We have shown previously that a VacA mutant protein lacking amino acids 6 to 27 (Delta6-27p88 VacA) is able to inhibit many activities of wild-type VacA in a dominant-negative manner. Analysis of a panel of C-terminally truncated Delta6-27p88 VacA proteins indicated that a fragment containing amino acids 1 to 478 (Delta6-27p48) exhibited a dominant-negative phenotype similar to that of the full-length Delta6-27p88 VacA protein. In contrast, a shorter VacA fragment lacking amino acids 6 to 27 (Delta6-27p33) did not exhibit detectable inhibitory activity. The Delta6-27p48 protein physically interacted with wild-type p88 VacA, whereas the Delta6-27p33 protein did not. Mutational analysis indicated that amino acids 351 to 360 are required for VacA protein-protein interactions and for dominant-negative inhibitory activity. The C-terminal portion (p55 domain) of wild-type p88 VacA could complement either Delta6-27p33 or Delta(6-27/351-360)p48, reconstituting dominant-negative inhibitory activity. Collectively, our data provide strong evidence that the inhibitory properties of dominant-negative VacA mutant proteins are dependent on interactions between the mutant VacA proteins and wild-type VacA, and they allow mapping of a domain involved in the formation of oligomeric VacA complexes.

SUBMITTER: Torres VJ

PROVIDER: S-EPMC1418911 | biostudies-literature | 2006 Apr

REPOSITORIES: biostudies-literature

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Mapping of a domain required for protein-protein interactions and inhibitory activity of a Helicobacter pylori dominant-negative VacA mutant protein.

Torres Victor J VJ McClain Mark S MS Cover Timothy L TL

Infection and immunity 20060401 4

The Helicobacter pylori VacA toxin is an 88-kDa secreted protein that causes multiple alterations in mammalian cells and is considered an important virulence factor in the pathogenesis of peptic ulcer disease and gastric cancer. We have shown previously that a VacA mutant protein lacking amino acids 6 to 27 (Delta6-27p88 VacA) is able to inhibit many activities of wild-type VacA in a dominant-negative manner. Analysis of a panel of C-terminally truncated Delta6-27p88 VacA proteins indicated that ...[more]

PMID: 16552038

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Project description:AIM:To construct a recombinant prokaryotic expression vector inserted with Helicobacter pylori vacA gene and identify the immunity of the expressed recombinant protein, and to determine prevalence of vacA-carrying/VacA expressing H pylori isolates and seroprevalence of specific ant-VacA antibody in H pylori infected patients. METHODS:Polymerase chain reaction technique was used to amplify complete vacA gene of H pylori strain NCTC11637 and to detect vacA gene in 109 H pylori isolates. The amplification product of the complete vacA gene was sequenced after T-A cloning. A recombinant expression vector inserted with a complete vacA gene fragment, named as pET32a-vacA, was constructed. Expression of the target recombinant protein VacA (rVacA) was examined by SDS-PAGE. Western blot using commercial antibodies against whole cell of H pylori and an immunodiffusion assay using self-prepared rabbit anti-rVacA antibody were applied to determine immunoreaction and antigenicity of rVacA. Two ELISA methods were established to detect VacA expression in H pylori isolates and the specific anti-VacA antibody in sera from 125 patients infected with H pylori. RESULTS:In comparison with the reported corresponding sequences, homologies of nucleotide and putative amino acid sequences of the cloned vacA gene were 99.82% and 100%, respectively. The constructed recombinant prokaryotic expression system efficiently produced rVacA. rVacA was able to combine with the commercial antibodies against whole cell of H pylori and to induce the immunized rabbit to produce specific antibody with an immunodiffusion titer of 1:4. All tested H pylori isolates carried vacA gene, but only 66.1% expressed VacA protein. Of the serum samples tested, 42.4% were positive for specific anti-VacA antibody. CONCLUSION:A prokaryotic expression system of H pylori vacA gene was successfully constructed. The expressed rVacA can be used to detect specific anti-VacA antibody in human and to prepare antiserum in animals. The high frequency of vacA gene in H pylori isolates, but with a low frequency of VacA expression and specific anti-VacA antibody in H pylori infected patients implies that VacA is not an ideal antigen for H pylori vaccine.

Dataset Information

Mapping of a domain required for protein-protein interactions and inhibitory activity of a Helicobacter pylori dominant-negative VacA mutant protein.

Publications

Mapping of a domain required for protein-protein interactions and inhibitory activity of a Helicobacter pylori dominant-negative VacA mutant protein.

Similar Datasets

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