Genetic Diversity And VacA Allele Heterogeneity In Helicobacter Pylori Isolates From Different Areas In China

Objective:H.pylori (Helicobacter pylori, Hp) is a curved gram-negative bacterium that resides in the mucosa of the human stomach. It has been generally accepted that colonization of the stomach by H.pylori is associated with the risk of different gastroduodenal diseases including chronic gastritis, peptic ulcer, gastric cancer, and mucosal associated lymphoid tissue lymphoma. It is also thought to be one of the most prevalent human bacterial pathogen, infecting the stomachs of more than one-half of the human population. H. pylori show exceptional genetic variability and interspecies diversity. Moreover, the sequence diversity of H. pylori allele genes is greater than that seen in most other bacteria.Numerous previous studies for epidemiological researches for H.pylori have highlighted on vacA gene. This virulence factor gene exits in the almost all the H.pylori strains. It is responsible for the gastric epithelial damage and causes of mucosal ulceration when administered intragastrically to mice. Different allelic variants of the vacA sequence in two regions of the gene have been related to the differences in the level of cytotoxin production and to distinct clinical outcomes of the H pylori infection, and differed in the strains from distinct areas.To study the epidemiology of H.pylori infections, the method of multiple typing have been useful to differentiate the isolates under the species level. Multilocus sequence typing (MLST) was designed to provide a tool for investigating populations and population dynamics on a global scale. It applies neutral or slowly accumulating genetic variation in housekeeping genes. The evolution of these housekeeping genes is constrained by their requirement to encode functional products and is not affected by the rapid evolution that may be detected within genes encoding proteins that influence survival in a particular niche. MLST also resolves other molecular typing techniques'shortfalls, which are that their results could not be compared among different labs.Therefore housekeeping and virulence-associated genes of H. pylori were used for typing 40 isolates collected from 4 different parts of China. And the method of MLST also allows investigating the species'population and the population's evolution of H.pylori isolates from distinct regions.Methods:1. Unrelated patients accompanying dyspeptic symptoms were recorded, which were from 4 different cities, i.e. Beijing, Guangzhou, Shanghai and Chongqing between the year 2002 and 2007. Endoscopic examinations of upper part of the digestive tract were exexuted and the specimens for traumatic test were collected from antrum. Two specimens of the mucous membrane were taken from the prepyloric part and gastric corpus. Biopsies samples were cultured on Brain-heart infusion (BHI) agar containing 5% rabbit blood. The bacterial colonies identified as H.pylori were stored in brain heart infusion medium at ?70℃. Reference strains of H.pylori was obtained (in lyophilized form) from the National Collection of Type Cultures (NCTC): NCTC11637(type strain, Australia); NCTC 11638(Australia).2. Thaw and inoculated the stored H.pylori cultures. Extracted the DNA of the bacterial from the pure subculture.3. Eight fragments of the housekeeping genes'allelic loci of H.pylori were selected for the MLST scheme. There are atpA, efp, mutY, ppa, trpC, ureI, vacA and yphC.4. The eight housekeeping genes'fragments for MLST scheme were amplified respectively from each H.pylori chromosome DNA by PCR techniques. And PCR products of each isolates were purified for sequencing using QIA quick gel extraction kit. Both strands of DNA sequences were determined with an ABI PRISM 310 DNA sequencer (Applied Biosystems).5. The results of DNA sequences were assembled from the resultant chromatograms by the DNAssist software package 2.0(Gene Codes Corp). Each isolate is defined by an allelic profile consisting of the housekeeping genes integers. Each unique allelic profile is assigned as a sequence type (ST). START programme is used for analysis of MLST data, involving data summary, lineage assignment, tests for recombination and tests for selection, et al. Related STs were clustered into clonal complexes or lineages using the BURST (Based Upon Related Sequence Types) algorithm and to examine patterns of evolutionary descent.6. vacA genotypes are determined by s (signal peptide) and m (internal) region alleles of gene. Using H.pylori genomic DNA as the template as described for the MLST scheme for PCR amplification the s and m alleles of vacA gene.Results:1. The internal fragments of eight loci were amplified and sequenced for 40 cultures of collected H.pylori. All eight loci appeared to be under stabilizing selective pressure, since most of the substitutions were synonymous, as indicated by the dN/dS ratios being substantially less than 1.0. There are 38 STs and most of them were unique, and had not been described previously at the Helicobacter pylori MLST database. An UPGMA (unweighted pair group mean average) cluster analysis of distance matrix showed that the strains isolated from 4 different cities as well as 2 controlled strains could be clustered in 3 groups. The total Ia was 5.27, and mean trial variance was 0.0138 with max trials variance 0.1146. There is no evidence for linkage; therefore, genetic diversity in H.pylori is caused primarily by recombination.2. The primers on the allele s1, s2 and m1, m2 used in the study enabled the differentiation and characteristics of allele of s region and m region in vacA genotype which were isolated from examined H.pylori strains. The most frequently observed H. pylori vacA allele genotype is the s1a/m2 allele (80%), followed by s1b/m2 (17.5%). In contrast, the s1a/m1 genotype is scarcely represented (2.5%). Investigated of the 40 isolates, the distribution of STs is diverse with the vacA genotypes.Conclusion1. In this study the sequences of eight housekeeping genes'fragments from 40 strains of H. pylori, which were isolated from 4 geographical regions of China were analyzed by MLST. Sequencing and sequence analysis were done and brought up 38 STs.2. The allelic types of vacA gene of collected strains were obtained and assigned to related diseases successfully. According to our data there has showed no obvious link between STs and vacA gene subtypes.3. Although the number of isolates was relatively small in this study, the diversity of the isolates of H. pylori was established by identification of various genotypes and clonal complexes. Two weakly clonal groupings were found, superimposed on a pattern of free recombination. And our study provides an outcome that offers a basis for further population genetic analyses.