Single-step PCR amplification and enzyme restriction analysis of the entire Helicobacter pylori cytotoxin vacA gene for genetic variability studies

Centro de Investigación sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública, Cuernavaca, Mor, Mexico.
FEMS Microbiology Letters (Impact Factor: 2.12). 09/1999; 178(1):55-62. DOI: 10.1111/j.1574-6968.1999.tb13759.x
Source: PubMed


To monitor changes along the entire Helicobacter pylori vac A gene we carried out full-length single-step PCR amplification in 21 gastritis and gastric cancer isolates. HindIII restriction analysis led us to detect a > 400-bp internal insertion in vacA subsequently shown to be a direct 451-bp gene duplication. We found HindIII profiles for 16 genes that allowed their grouping into two restriction patterns that were related to theoretical profiles for previously sequenced Western genes. Comparisons with theoretical HindIII patterns for Japanese isolates appear suggestive of geographical H. pylori clonality. Full-length single-step PCR amplification seems suitable for quick restriction pattern assignment and detection of gene size changes.

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Available from: Roberto Herrera-Goepfert, Apr 30, 2015
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    • "For example, the vacA gene encoding the vacuolating cytotoxin (VacA) exhibits a remarkable degree of genotypic and phenotypic variation [9,21-23]. The vacuolating activity of VacA varies approximately 30-fold across different isolates due to the presence of at least five different vacA alleles [24]. Two families of the vacA alleles, type m1 and type m2, are only about 70% identical [25]. "
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    ABSTRACT: Clinical isolates of the gastric pathogen Helicobacter pylori display a high level of genetic macro- and microheterogeneity, featuring a panmictic, rather than clonal structure. The ability of H. pylori to survive the stomach acid is due, in part, to the arginase-urease enzyme system. Arginase (RocF) hydrolyzes L-arginine to L-ornithine and urea, and urease hydrolyzes urea to carbon dioxide and ammonium, which can neutralize acid. The degree of variation in arginase was explored at the DNA sequence, enzyme activity and protein expression levels. To this end, arginase activity was measured from 73 minimally-passaged clinical isolates and six laboratory-adapted strains of H. pylori. The rocF gene from 21 of the strains was cloned into genetically stable E. coli and the enzyme activities measured. Arginase activity was found to substantially vary (>100-fold) in both different H. pylori strains and in the E. coli model. Western blot analysis revealed a positive correlation between activity and amount of protein expressed in most H. pylori strains. Several H. pylori strains featured altered arginase activity upon in vitro passage. Pairwise alignments of the 21 rocF genes plus strain J99 revealed extensive microheterogeneity in the promoter region and 3' end of the rocF coding region. Amino acid S232, which was I232 in the arginase-negative clinical strain A2, was critical for arginase activity. These studies demonstrated that H. pylori arginase exhibits extensive genotypic and phenotypic variation which may be used to understand mechanisms of microheterogeneity in H. pylori.
    Full-text · Article · Apr 2007 · BMC Microbiology
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    • "To analyze how VacA cytotoxin affects AGS cells, we used two strains: the VacA1 2074-Cd (s1/m1) strain and the 4767-C (s1/m1) strain, which presents a duplication of 451 base pairs from the codon 459 through to 594 (Perales et al., 1999). This duplication creates a stop codon nine base pairs downstream from the point of insertion and due to that, a truncated protein of 606 amino acids would be produced. "
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    ABSTRACT: The Helicobacter pylori infection of gastric mucosa is one of the most common infectious diseases and is associated with a variety of clinical outcomes, including peptic ulcer disease and gastric cancer. Helicobacter pylori-induced damage to gastric mucosal cells is controlled by bacterial virulence factors, which include VacA and CagA. Outer membrane vesicles are constantly shed by the bacteria and can provide an additional mechanism for pathogenicity by releasing non-secretable factors which can then interact with epithelial cells. The present report shows that external membrane vesicles are able to induce apoptosis not mediated by mitochondrial pathway in gastric (AGS) epithelial cells, as demonstrated by the lack of cytochrome c release with an activation of caspase 8 and 3. Apoptosis induced by these vesicles does not require a classic VacA+ phenotype, as a negative strain with a truncated and therefore non-secretable form of this protein can also induce cell death. These results should be taken into account in future studies of H. pylori pathogenicity in strains apparently VacA-.
    Preview · Article · Aug 2006 · FEMS Microbiology Letters
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    ABSTRACT: Helicobacter pylori has acquired great importance during the last two decades, after being recognized as an important pathogen that infects a great portion of the human population. This microorganism is recognized as the main causal agent of chronic gastritis and duodenal ulcers, and it is associated with the subsequent development of gastric carcinoma. The pathogenic mechanisms of H. pylori and their relation to gastric ailments have not been clearly defined. However, at present it is well established that urease, vacuolating cytotoxin VacA, and the pathogenicity island (cag PAI) gene products, are the main factors of virulence of this organism. Thus, individuals infected with strains that express these virulence factors probably develop a severe local inflammation that may induce the development of peptic ulcer and gastric cancer. The way the infection spreads throughout the world suggests the possibility that there are multiple pathways of transmission. Due to the importance that H. pylori has acquired as a human pathogen, laboratories worldwide are attempting to develop a vaccine that confers long-term immunological protection against infection by this microorganism. Hence, the objective of this review is to present the most relevant findings of the biology of H. Pylori and its interaction with the human host.
    Preview · Article · Jan 2001 · Salud publica de Mexico
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