Pathological significance and molecular characterization of the vacuolating toxin gene of Helicobacter pylori
| Autor: | T U Westblom, L Janzon, D Ilver, Staffan Normark, Suhas H. Phadnis |
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| Rok vydání: | 1994 |
| Předmět: |
Sequence analysis
Bacterial Toxins Molecular Sequence Data Immunology Mutant Sequence alignment Biology Polymerase Chain Reaction Microbiology Homology (biology) Bacterial Proteins Amino Acid Sequence Cloning Molecular Gene Peptide sequence Base Sequence Helicobacter pylori Structural gene Nucleic acid sequence bacterial infections and mycoses Molecular biology digestive system diseases Infectious Diseases Genes Bacterial bacteria Parasitology Research Article |
| Zdroj: | Infection and Immunity. 62:1557-1565 |
| ISSN: | 1098-5522 0019-9567 |
| DOI: | 10.1128/iai.62.5.1557-1565.1994 |
| Popis: | Some strains of Helicobacter pylori are known to produce an extracellular 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 bacterium, but having normal gastric mucosa, lacked cytotoxic activity. We also show that a higher percentage of strains isolated from patients with polymorphonuclear leukocyte infiltration of gastric mucosa are toxin positive (78%) versus those isolated from patients lacking such infiltration (33%). In addition to examining the relationship between pathology and cytotoxic activity, we used the previously published N-terminal sequence of the protein to clone and characterize vacA, the structural gene encoding the cytotoxin. Briefly, three oligonucleotides capable 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 made. They were used in all 12 possible combinations in 12 different PCR reactions, with DNA from a cytotoxin-positive strain as template. In four combinations, the expected 69-bp fragment was seen. The sequence of this 69-bp fragment confirmed that it encoded the known N-terminal sequence of the cytotoxin. This gene is capable of encoding a 136-kDa protein with a 33-amino-acid signal peptide, whereas the purified cytotoxin 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 marker in the vacA gene produced an isogenic mutant lacking the cytotoxic activity. This mutant provides genetic evidence that vacA encodes the cytotoxin. Sequence analysis of the DNA adjacent to the vacA gene demonstrated that this gene is next to a putative cysteinyl tRNA synthetase gene. From the sequence arrangement, we predict that there are no other genes transcribed together with vacA. We also show that five of seven cytotoxin-negative strains examined still carry the sequences encoding it whereas the other two have suffered a deletion of the vacA gene. We further show that in at least one cytotoxin-negative but vacA-positive strain (MO19), there are variations in the length of the vacA gene that could explain the cytotoxin-negative phenotype in this strain. |
| Databáze: | OpenAIRE |
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