Analysis Of Genetic Diversity And Adaptive Colonization Of Helicobacter Pylori

Helicobacter pylori (H.pylori), 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. H.pylori is also thought to be one of the most prevalent human bacterial pathogen. It shows exceptional genetic variability and interspecies diversity, which makes the infection heterogeneity among different people, regions, etc. Moreover, the sequence diversity of H.pylori allelic genes is greater than that seen in most other bacteria.H.pylori has co-evolved with human beings at least since they migrated out of Africa approximately 58,000 years ago and probably throughout the whole process of human evolution. Consequently, it is believed that the bacterial factors present play an important role in the pathogenicity of the bacterium, in addition to host-related factors, and environmental factors.In this research, the diversity of H.pylori virulence factors (the gene cagA,vacA,babA,dupA and iceA) and their contribution to the different geographic distribution were discussed. And the method of MLST was taken to investigate the species'population and the population's evolution of isolates from distinct regions. Furthermore, using animal models of Mongolian gerbil, the current research also studied the genomic variation during the adaptive micro-evolution of H.pylori.Results:1. The diversity analysis of H.pylori virulence factors.The related virulence factors cagA, vacA, babA, dupA and iceA of 122 clinical isolates were detected. The positive rates for the iceA1, dupA, babA2, vacA s1a, vacA s1b, vacA s1c, vacA m1 and m2 genes were 73.9%, 83.7% and 40.5%, 97.5%, 2.5%, 88.5%, 49.2% and 50.8%, respectively. The vacA m1 type was more prevalent in the gastric cancer group (73.3%). Analyses for the C-terminal region of CagA protein, of 66.3% H.pylori presented 3 EPIYA motifs, and the East Asian type was prevalent, for 89.5%.The prevalent genotype of the Chinese H.pylori strains in the current research was vacA s1a/s1c +, babA2+, iceA1+ and CagA-ABD. And the CagA isoforms were related to the different isolated geographic areas. In Tibet, there were 66.7% of the H.pylori strains with 4 and above EPIYA motifs, as well as of 90.5% Tibetan strains. Possess of 4/5 EPIYA motif may be the marker of the H.pylori from Tibetan. However, 60% of the Mongolian strain presented Western CagA type.2. The relationship among virulence factors.In this study, the vacA m1 genotype has been strongly associated with iceA1 gene (76.8% vacA m1-positive). The H.pylori with vacA m2 genotype presented iceA2 gene, except with Tibetan strains. The synergistical interaction among H.pylori virulence factors should take the geographic variation into consideration. And the H.pylori strains in Tibet should be paid special attention to, which differed from others.Strains with Western CagA-type, had been reported with 90% vacA m2 and 50% iceA2gene. While strains carrying 4/5 EPIYA motifs, were presentation 96.7% of vacA m1 and babA2 gene. There's an association among virulence factors, which would induce serious disease.3. The analysis of MLSTSeven fragments of the housekeeping genes'(atpA, efp, mutY, ppa, trpC, ureI and yphC) allelic loci of H.pylori were selected for the MLST scheme. The results of STs were all new and had not been described previously at the Helicobacter pylori MLST database. BURST cluster analysis of distance matrix showed that the strains as well as 2 controlled strains could be clustered in 3 groups, and the rest were the singletons. To the Tibetans, the ST2237 may be the ancestor strains. And the H.pylori isolated from Inner Mongolia, were associated with western strains.4. Examination the SNPs of the H.pylori adaptive strains.Used the animal model of Mongolian Gerbil, by means of whole genome sequencing and SOAPsnp analysis, 61 SNPs loci were detected from the H.pylori adaptive strains. The SNPs were examined with PCR and sequencing analysis. The SNPA was detected in the Asn130-Gly150 domain of AlpB protein, which was strongly correlated to the H.pylori adaptive conolization. Clinical H.pylori strains from different sources, could be clustered into 2 groups based on this domain. At this locus, not only adaptive H.pylori, but also the clinical isolates showed greater variability. This may be due to different microhabitats of the host-populations, which may lead to the variation of the AlpB protein. And the polymorphism in the Asn130-Gly150 domain could be a new classification mark of bacteria.Conclusions:In this study, the diversity of H.pylori clinical isolates was analyzed by 5 primary virulence factors. The genotype of Chinese H.pylori has usually been with vacA s1a/s1c/m2 +; babA2+; iceA1+; CagA-ABD. To strains of Tibetan, the CagA protein always carried 4/5 EPIYA motif, and the positive rate of vacA s1a/s1c/m1 gene was much higher than other regions of China. And the isolates from Mongolian were associated with vacA m2,dupA+, and Western CagA-type frequently. The results of MLST were all new sequence types, which could be clustered into 3 groups. There is no evidence for linkage; therefore, genetic diversity in H.pylori is caused primarily by recombination. Phylogenetic analysis by MST detected that Mongolian strains had genetic link to the western H.pylori. Research on the microevolution of adaptive H.pylori strains found that Asn130-Gly150 domain of AlpB protein was strongly related to the H.pylori conolization. The variation of this domain was also detected among clinical isolates, which may contribute to the heterogeneous of H.pylori adhesion and attachment.This report provides new insight into the molecular epidemiological characteristic and systematic data on the population genetic evolution of H.pylori in China. The establishment of a good and reliable platform for epidemiological analysis will be invaluable for further investigations of H.pylori infections and prevention. And the investigation of bacteria microevolution will help to establish the evolutionary model of H.pylori.