vacA Genotypes and Genetic Diversity in Clinical Isolates of Helicobacter pylori |
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| Authors: | Shan-Rui Han Hans-Joachim Schreiber Sucharit Bhakdi Michael Loos Markus J. Maeurer |
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| Affiliation: | Department of Medical Microbiology, Johannes Gutenberg University, D-55101 Mainz, Germany |
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| Abstract: | Genetic diversity in Helicobacter pylori strains may affect the function and antigenicity of virulence factors associated with bacterial infection and, ultimately, disease outcome. In this study, DNA diversity of H. pylori isolates was examined by analysis of vacA genotypes and by restriction fragment length polymorphism (RFLP) analysis of H. pylori-associated genes (vacA, cagA, flaA, ureAB, and ureCD). Thirty-seven H. pylori isolates from 26 patients were successfully classified into distinct vacA allelic genotypes. The signal sequence allele s1 (31 of 37) predominated over the s2 allele (6 of 37) and was significantly associated with the occurrence (past or present) of gastric ulcers. A novel midregion allele, designated as m3, has been identified in two H. pylori isolates which could not be typed with midregion allele m1- or m2-specific primers. Additionally, significant nucleotide diversity yielding different amino acid sequences was demonstrated by DNA sequencing of vacA fragments from clinical isolates of H. pylori. Furthermore, RFLP analysis of 45 H. pylori isolates (including 15 paired isolates) obtained from antrum and corpus biopsy specimens from 30 individual patients showed remarkably high interhost diversity (one patient, one H. pylori strain) and intrahost identity in gene sequences coding for VacA, CagA, flagellin, and urease. Only in a single patient was a minor genotypic variation at different anatomic sites within the stomach identified. These data warrant the detailed analysis of the effect of genetic diversity on the function and antigenicity of H. pylori-associated virulence factors.Helicobacter pylori is one of the most prevalent causes of infection in human beings worldwide. H. pylori persists in the human gastric mucus layer for decades and possibly for life, even in the face of a brisk humoral antibody response (3, 26). The extent of mucosal lesions induced by H. pylori varies, with only a small number of patients developing peptic ulceration and gastric cancer (3, 19). With the increasing emergence of antibiotic-resistant H. pylori strains (27), development of an effective vaccine may represent an alternative means of controlling or even preventing H. pylori infection (5, 21).Individual H. pylori isolates demonstrate a high level of genomic diversity as defined by different techniques, including conventional or pulsed-field gel electrophoresis and restriction endonuclease digestion of genomic DNA, PCR-amplified genomic DNA, individual genes, or ribosomal DNA (1, 9, 10, 14, 24). Genomic differences may affect virulence factors, altering their function and antigenicity. Antigenic variation of certain gene products may represent an immune escape mechanism for H. pylori strains in the host organism.Vacuolating cytotoxin (VacA) is a major H. pylori-associated virulence factor (25, 29), but it may also serve as a target for induction of a strong, long-lived, and effective anti-H. pylori-directed immune response (12, 18, 25). Although almost all H. pylori strains bear the vacuolating cytotoxin gene (vacA) and through expression of this gene produce an immunoreactive protein (2), only 50 to 60% exhibit detectable cytotoxin activity (6, 24). Hence, nonfunctional but immunogenic variants of VacA exist. It was recently reported that the 95-kDa protein isolated from culture supernatants of Tox− strains (the Tox protein being a homolog of VacA) is recognized by an antiserum raised against the H. pylori cytotoxin from Tox+ strains but is not capable of inducing cell vacuolation (2). The humoral anti-VacA-directed immune response is predominantly directed against conformational epitopes of the cytotoxin (17). Therefore, the nucleotide or amino acid sequences of cytotoxin and its homolog provide the basis for the design of a genetically “detoxified” molecule which retains the structure and immunogenicity of the native protein.The level of in vitro cytotoxin activity appears to correlate with the clinical consequences of H. pylori infection. VacA functionality in turn correlates with specific vacA genotypes, defined by certain combinations of vacA signal sequences (s1a, s1b, and s2) and vacA midregion alleles (m1 and m2), including s1a-m1, s1b-m1, s1a-m2, s1b-m2, and s2-m2 (2). It is unknown whether these structural vacA differences impact on anticytotoxin-directed immunity.In this study, we employed a PCR-based method to define the vacA genotypes of 37 individual H. pylori isolates from antrum and corpus biopsy specimens obtained from 26 patients undergoing endoscopy. Genetic diversity was characterized in detail by restriction fragment length polymorphism (RFLP) analysis of H. pylori-associated genes (i.e., vacA, cagA, flaA, ureAB, and ureCD) coding for virulence factors (VacA, CagA, flagellin, and urease). This analysis was complemented by sequence analysis of individual vacA and flaA genes. |
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