PATHOLOGICAL SIGNIFICANCE AND MOLECULAR CHARACTERIZATION OF THE VACUOLATING TOXIN GENE OF HELICOBACTER-PYLORI

Some strains of Helicobacter pylori are known to produce an extracellu lar cytotoxin that causes vacuolization in various mammalian cells. In this study, we found that concentrated culture supernatants from four Helicobacter strains isolated from patients infected with the bacteri um, but having normal gastric mucosa, lacked cytotoxic activity. We al so show that a higher percentage of strains isolated, from patients wi th polymorphonuclear leukocyte infiltration of gastric mucosa are toxi n positive (78%) versus those isolated from patients lacking such infi ltration (33%). In addition to examining the relationship between path ology and cytotoxic activity, we used the previously published N-termi nal sequence of the protein to clone and characterize vacA, the struct ural gene encoding the cytotoxin. Briefly, three oligonucleotides capa ble of encoding the first nine amino acids corresponding to the sense strand and four oligonucleotides corresponding to the noncoding strand of the last seven known amino acids of the cytotoxin protein were mad e. They were used in all 12 possible combinations in 12 different PCR reactions, with DNA from a cytotoxin-positive strain as template. In f our combinations, the expected 69-bp fragment was seen. The sequence o f this 69-bp fragment confirmed that it encoded the known N-terminal s equence of the cytotoxin. This gene is capable of encoding a 136-kDa p rotein with a 33-amino-acid signal peptide, whereas the purified cytot oxin is only 87 kDa, suggesting processing in the C-terminal region of the protein. A single copy of the vacA gene encodes the cytotoxin in H. pylori. Consequently, the insertion of a kanamycin resistance marke r in the vacA gene produced an isogenic mutant lacking the cytotoxic a ctivity. This mutant provides genetic evidence that vacA encodes the c ytotoxin. Sequence analysis of the DNA adjacent to the vacA gene demon strated that this gene is next to a putative cysteinyl tRNA synthetase gene. From the sequence arrangement, we predict that there are no oth er genes transcribed together with vacA. We also show that five of sev en cytotoxin-negative strains examined still carry the sequences encod ing it whereas the other two have suffered a deletion pf the vacA gene . We further show that in at least one cytotoxin-negative but vacA-pos itive strain (MO19), there are variations in the length of the vacA ge ne that could explain the cytotoxin-negative phenotype in this strain.