Genomic Characterization And Population Structure Of Helicobacter Pylori In China

Helicobacter pylori(H.pylori)is a gram negative bacteria that colonizes in human stomach.The pathogen can cause several digestive diseases ranged from mild infection like gastritis to more serious clinical outcomes like digestive ulcer and gastric carcinoma.H.pylori can establish persistent infection without regular treatment,it is generally used as a stable biomarker that reflects human population structure and historic migrations.It is estimated that H.pylori has coevolved with human beings for about 60,000 years since their migrations out of Africa.Accordingly,seven populations have been found in globally collected H.pylori isolates based on sequence diversity of the seven house keeping genes.Despite MLST analysis has been widely used to infer H.pylori populations in different geographies,this method shows very low discrimination power for Chinese isolates.In this study we selected isolates collected from eight provinces of China for whole genome sequencing.Besides,these strains are isolated from patients with different digestive diseases,and also,they are from different minorities in China.In combination with public available genomes,we perform a comprehensive bioinformatic analysis on sixty-five Chinese H.pylori genomes.Moreover,totally 107 genomes of East Asian and 491 genomes of global isolates are used to explore the genetic flux between Chinese strains and other groups.A fine population structure is inferred based on co-ancestry matrix analysis of these isolates.Finally we focus on analyzing genomic and population structure of prophages in these isolates.Genomes of seven isolates derived from one patient with gastritis are sequenced to identify possible prophages.We found a parallel co-evolution pattern between phages and H.pylori genomes.Since only very limited information has been known about characterization of phage in H.pylori,our analysis will shed new insight into potential role of phage in the diversification and evoluation of this bacteria.Part 1:comparative genomic analysis of H.pylori isolates in ChinaTotally we identify 3072 pan genes and 1147 core genes in 65 genomes of China.The number of accessory genes is 1925.0-47 strain specific genes that mainly encodes hypothetical proteins are found among different individual isolates.Corrected by reads sequences,237,250 core genome SNPs are identified based on these assembled genomes,which reflects high genetic heterogeneity in different isolates.These data demonstrate that H.pylori has evolved a highly diversified gene pool to adapt to different gastric micro-niche in such population.Global alignment identify two distinct type IV secretion systems that related to horizontal gene fragment transfer,which partially contribute to the genetic diversity among isolates of China.We extracted 85 virulence genes from genomes of Chinese H.pylori,of which 76 gene sequences are quite conservative.They mainly encode urease proteins that responsible for protecting H.pylori from high acid disruption,flagella proteins that responsible for motility and a type IV secretion system(CagPAI)that responsible for secretion of CagA into epithelial cells.Six outer membrane genes show high genetic diversity,of which sabA and sabB devide Chinese H.pylori strains into two genetic distant clades,this finding suggest a potential distinct outer membrane antigen recognition mechanism to evade from host immune systems in different populations in China.None of these virulence genes shows any discrimination power between sequence diversity and clinical outcomes,which further emphasize the complex interactions among H.pylori,human beings and outer environment.Only toxin related genes like cagA,cagPAI and vacA like protein encoding genes show discrimination power between isolates of China and western countries.Finally we analyzed 491 H.pylori genomes that represent whole population level dataset,the results show that H.pylori has an open pan gene size and a core gene set including 697 genes,which would provide preliminary data for screening species specific genes.We estimate H.pylori hold a relatively stable core gene pool in different populations or subpopulations.The results of this section indicate that it is difficult and inexplicable to get genes or genomic characterizations involving serious clinical outcomes by simply comparing H.pylori genomes without considering the complex and quite long term interations between bacteria,human host and environment.Tight combination with different host genetic background and related genomic data would be necessary to explore the mechanism of H.pylori induced serious diseases.Part 2:Population structure analysis of H.pylori in ChinaIn this section we use Chromopainter and fineSTURCTURE,in combination with phylogenetic tree to explore population structure of H.pylori in China,as well as in East Asia and all over the world.We find geography specific linages exist in China H.pylori populations.Besides,we also find ethnicity specific clades in Southwest populations.HspEAsia population are divided into 16 subgroups including 10 subgroups for China.Co-ancestry matrix indicate that hspEAsia,hspAmerind and hpSouthindia each has a relatively specific gene pool that have minor inter population genetic exchanges compared to that of intra populations.More intimate genetic flux are found among subgroups of H.pylori from Chinese,Japanese,Korean and Malaysia Chinese.The formation of these geography specific subgroups maybe due to the genetic drift and bottleneck.Empirically mapping this population structure to human historic migrations in China,we preliminarily find that the population structure of H.pylori in China may be related to some large scale human migrations in the past several hundreds of years.From this section,we accurately identify the population structure of H.pylori and their human host with different ethnicities in China,this will be helpful for learning about the relationship between H.pylori of China and gastric cancer by substracting possible bias generated from host genetic difference in large scale GWAS analysis.Part3:Genomic characterization and distribution of prophage in H.pylori of ChinaFew studies report the phage of H.pylori during the past thirty years.In this study we find an intact prophage YN4-84P in a strain isolated from Naxi ethnicity.This prophage is genetically close to KHP30,a template phage which was purified from an isolate of Japan in 2013.Unexpectedly,the portal protein gene of YN4-84P is inserted by IS605,which may impact phage assembly.In the global H.pylori population,we find 8.2%strains carrying prophage,whearas in Chinese population the infection rate is 13.07%,which is lower than that of hpEurope.Genetic diversity of these prophages also show geography specific clusters.Thus we infer that co-evolution may exist among prophage,H.pylori and human host.Some outer membrane genes related to adhesion and colonization are found in prophage genomes or near the prophage insertion site,which indicate that the genetic diversity in these genes maybe due to the infection of phages.Prophage analysis of seven complete genomes derived from one patient with gastritis show a consistent diversification pattern between phage and their host genomes.The results indicate that phage may play an important role in strain diversification and evolution of H.pylori.