Studies On The Sequence Characteristic And Inflammatory Mechanism Of Helicobacter Pylori CagPAI

Helicobacter pylori (H.pylori), a microaerophilic, curved-shaped and gram-negative bacterium, colonized in stomach in more than half of people in the world. It had been regarded as the class I carcinogen by the World Health Organization in 1994.The cag PAI is a 40kb locus in the chromosomal glutamate racemase gene. The cagPAI generally contains 28 genes which encode various virulence-related proteins. The cagPAI has a high correlation between virulence and genetic polymorphism. From 23 complete cagPAI sequences submitted in the NCBI database, cagPAI genes have significant genetic diversity. H.pylori exhibits a large degree of genomic and allelic diversity. Strain-specific diversity has been proposed to be involved in organism's ability to cause different diseases. There are also indications of significant geographical differences among strains. Currently, there is no complete Chinese cagPAI gene sequences in the NCBI database. However, with the high degree of polymorphism and the obviously geographical characteristics, cagPAI should have its own unique regional characteristics in China. Fully understanding of H.pylori on the genetic polymorphism and association with the disease may help to understand interaction between H.pylori and host, and which type of strain is pathogenic, then which type of strain can cause cancer and other serious gastrointestinal diseases.In this study,86 strains isolated from patients in four regions in China were selected. A total of 77 cagPAI were sequenced successfully. The cagPAI features are described, including cagPAI genes, the nucleotide length and the G+C% content. Based on the multiple sequence alignment and cluster analysis, we have found eight hypervariable regions on the cagPAI sequences. We found that the cagPAI of Chinese strains has obvious regional characteristics among the different regions in China. This study also reported that a long sequence missed in CagA C-terminal fragments in four strains. Our study also reported amino acid mutation in conserved sequence region which is the marker of the East Asian or the Western strains for the first time in China. And these mutations were found only in four similar strains in Vietnam and Thailand. In this study, the repeat sequences in the cagY were analyzed. We found that cagY genes from different strains have a high degree of heterogeneity. The identity of cagY gene is 32.9%, and the length of gene is about 3534-5394bp. Sequence comparisons revealed that the cagY sequence of Chinese strains have some differences from the ones submitted to NCBI. Most of the cagY gene contained in the Chinese strains is shorter than strains in NCBI for about lkb. The cagY repeat fragments were located in one region in 73(about 94.8%) Chinese strains while most of the Western strains have two regions where repeated fragments were found. The AGD motif in the CagL is associated with adhesion between the H.pylori and the host cell. Mutation occurred in this motif was found in some strains and this variation is not reported.In this study,26695,26695 AcagA, XA195, FJ21 and FJ15 were selected to incubate with AGS cells. The levels of IL-10, IL-4, IL-6, IL-2, IL-12, IFN-γ, TNF-α, TGF-β, IL-8 and VEGF in the cells suspension at 14 time points were detected using liquid chip technology. Finally we found that IL-8 and VEGF were high closely related to the interaction between the H.pylori and AGS cells. Basing on this phenomemon, we presumed the function of the gastric epithelial cells against the H.pylori infection in the beginning. High levels of cytokines secretion stimulated by XA195 strain withoutHP0521 gene suggests that HP0521 gene does not affect IL-8 and VEGF secretion. The capacity of IL-8 and VEGF stimulation was assessed between the 26695 and 26695ΔcagA. The conclusion is that the CagA is involved in the IL-8 and VEGF secretion. We also confirmed that the partial deletion of cagA in the 3'terminal affected on the IL-8 and VEGF stimulation basing on the analysis of the incubation of the XA195, FJ21, FJ15 and AGS cells. In addition, there is significantly difference between FJ21 and FJ15 in the capacity of IL-8 stimulation, however there is no significant difference in the VEGF stimulation. This phenomenon indicates that IL-8 and VEGF secretion stimulated by H.pylori relays on different mechanism.