| Plague, one of the most devastating infections in human history, is a zoonotic infection that spreads to humans from natural rodent reservoirs, commonly via the bite of an infected flea. Yersinia pestis, the causative agent of plague, has killed hundreds of millions of people in the three major historical plague pandemics. In recent years, plague became somehow active in some region and WHO assigned it as a reemerge infections. On the other hand, as a typical biological warfare agent, Y. pestis might be used in the war or bioterrorism attack in future, which poses significant threats on the health and safety of human beings. It is significant to probe into the polymorphism of the genome of Y.pestis and accordingly set up database for identificating and tracking the souce of this deadly pathogen in no time.During its expansion and adaptation to new niches, Y. pestis undergoes vast genetic variations in its genome to overcome the natural selective forces, resulting in the genomic diversity in this species. Based on the principal molecular strategies for bacterial genomic polymorphisms,including single nucleotide mutation, lateral gene transfer and deletion, genome rearrangement and short repeat polymorphisms,we probe into the genomic polymorphism of Y. pestis with several molecular markers and consequently selected the efficient targets to set up database for quick identifying and tracking the origin of Y.pestis. In the same time, the great amounts of data provided us useful information to further study the adaptive microevolution and plague foci expansion of Y.pestis.To establish a framework representing the genetic relationships among different groups of Y.pestis based on SNPs analysisSingle nucleotide polymorphism (SNP) affords many advantages for analysis of phylogenetic relationships among microbial strains, especially closely related clonal organisms. Achtman et al have discovered the synonymous SNPs sSNPs in the genome of Y.pestis and genotyped the representative isolates of this homogeneous specie. Baed on their study, we used 45 sSNPs to analysis the 121 representative strains and divided them into 12 types, 3 main branches. Branch 0, considered as a more ancient group for the strains in it present less mutation in 45 SNPs compared with Y.pseudotuberculosis, including the microtus strains of type 0.PE4 and antique strains of type 0.PE5 and 0.PE6. Antiqua strains of 1.ANT grouped with orientalis strains of 1.ORI in Branch 1 and antiqua strains of type 2.ANT with medievalis strains of type 2.MED in Branch 2, suggesting the close relationships among them. The gentic relationship framework provided reliable reference for the genomic polymorphism studies in Y.pestis with other molecular markers.Improvement of the genotyping system of Y.pestis based on the different regions (DFRs)In this study, the previous described 22 DFRs and DFR23 identified by SSH (suppression subtractive hybridization) were used to investigate more Chinese Y. pestis strains for validating DFR-based genotyping system. 909 strains of Y. pestis from China were grouped into 32 genomovars. Genomovars distribution was somehow focus-specific in China, and we proposed Major and Minor genomovars for explaining their distribution and roles played in microevolution of Y. pestis. However, for the low resolution power, there were still some strains from several foci indistinguishable by the DFR profiles. By in silico DFR profiling of the sequenced genomes, we were able to compare Chinese strains and those outside China as well. Orientalis strains in China turned out to be more ancient than those aboard according to the DFR profiles, supporting the notion that the orientalis strains were originated from China. The DFR profiles of Y.pestis in Xinjiang province were more diversity, inferring this region could be an access of Y. pestis spreading between China and Central Asia. The inexpensive and easy to used DFR based method is suitable for typing amounts of strains in no time and the new identified DFRs in future will improve this typing system.The genomic polymorphism of Y.pestis based on the variable number of tandem repeat (VNTR)Y. pestis is a highly monomorphic species and there no adequate mutations were accumulated in its genome. High-diversity VNTR analysis is necessary for genomic polymorphism study of this species. In this study, we used the high diversity MLVA methods to probe into the genomic polymorphism of Y. pestis.Two popular sets of VNTR loci, including 25 loci recoverd by Vergnaud et al and 42 loci by Keim et al, were used in studying of Y. pestis. We initially analysis 500 strains from China, Former Soviet Union (FSU), Mongolia and some regions in Europe with 25 loci and genotyped them into 350 types. These high resolution power loci can differentiated the strains from foci L and M, G and H, as well as I and K1 which cannot be divided by DFR methods. For technical limitation, the 25 loci with motif average in 17bp cannot represent the loci in genome of Y. pestis.In further study, we used 94 strains to evaluate the two sets of loci and found that For the limitation of the strains amount and diversity outside China, the relationships among different strains represented by the two sets of popular loci are somehow deviated by comparing to the SNPs analysis. In this study, with the aid of the rich strain resource in China, we screened the VNTR loci in the genome of Y. pestis. After complex step by step deletion, we finally selected 18 high diversity loci as efficient targets for quickly indentifying and tracking the source of Y. pestis and in the same time, the derived great amounts data enriched our genomic polymorphism database of Y. pestis.Probe into the feasibility for genotyping Y.pestis with PCR-based IS analysis.Insertions sequences (IS) are small DNA segments capable of inserting into target DNA molecules with more or less pronounced target specificity. They play important roles in DNA translocations and other rearrangements in bacteria and also can be used as molecular tools for typing pathogenic microorganisms. In this study we analyzed 39 loci of IS in 95 representative strains and found that the electrophoresis profile of the 39 IS were bewildering. With the aid the whole sequenced genome of CO92, we revealed that the rearrangements induced by IS are very complicate. It is impracticable to describe the presence or absence of the IS in a certain loci, which will decrease the resolution power in a great degree and consequently disorder the relationships among differt groups of Y.pestis.Construct the pipeline for rapidly identifying and tracking the soure of Y. pestis in China.After systematically analysis the several molecular markes used in this study, we summarized that compared to SNPs and IS analysis, the DFR and VNTR analysis are more suitable for identifying and tracking the source of Y. pestis. In this study we chose 5 DFRs and 8 VNTRs to set up a database for quickly identifying and tracking the source of Y. pestis in China. With aid of these targets and the database we established with our previous data, we can differentiate the strains from different geographic origins, which provided us useful tools for quickly identifying and tracking the source of the strain when the plague or bioterrorism attack break out. |
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