一株羊源A型多杀性巴氏杆菌的全基因组测序及生物学特性分析

Whole-genome sequencing and biological characteristics analysis of a sheep-derived capsular type A Pasteurella multocida strain

【背景】多杀性巴氏杆菌(Pasteurella multocida,Pm)是一种革兰氏阴性菌,可引起动物和人类的呼吸道疾病和败血症等。本实验室前期分离鉴定一株A型Pm HN02菌株。【目的】通过对HN02菌株的全基因组测序及生物信息学分析,扩充多杀性巴氏杆菌的基因组数据库信息;通过毒力基因鉴定和系统进化树分析,明确该菌株含有的毒力基因和遗传进化关系,为临床预防和诊断提供理论依据。【方法】使用单分子实时测序(Single Molecule Real Time Sequencing,SMRT)技术对Pm HN02菌株进行全基因组测序,利用Illumina测序校正后进行基因功能注释和生物信息学分析。使用PCR鉴定菌株毒力基因,并构建进化树进行分析。【结果】Pm HN02菌株全基因组大小为2 333 292 bp,GC含量为40.15%,预测到的编码基因有2 389个,包含19个rRNA (6个23S rRNA、6个16S rRNA、7个5SrRNA)、62个tRNA基因、5个sRNA;含84个串联重复序列、66个小卫星DNA、2个微卫星DNA、9个基因岛、9个前噬菌体;分别有1 648、2 190和1 917个基因注释在GO、KEGG和COG数据库中,而且大部分富集于Pm的代谢过程;还有85个III型分泌系统效应蛋白、191个表型突变基因、165个毒力因子相关基因。根据分析结果绘制该菌株的全基因组圈图,并将基因组信息提交至NCBI后获得登录号cp037865。PCR鉴定发现该菌株含有fimA、toxA等14个毒力基因,缺失了tadD等毒力基因。系统进化树分析发现该菌株同北京的Pm3菌株(MH150895.1)进化关系最接近。【结论】研究完成了A型Pm HN02株的全基因组测序和生物学特性鉴定,揭示了其同国内外Pm分离株的进化关系,为预防Pm疾病流行和探索Pm致病机制提供了参考。

[Background] The Gram-negative Pasteurella multocida(Pm) causes respiratory diseases and septicemia in animal and human. Capsular type A Pm HN02 has been isolated and identified by our laboratory. [Objective] We sequenced the whole genome of HN02 and analyzed the bioinformation,thereby supplementing the genome information of Pm. Through identification and phylogenetic analysis,the virulence genes and genetic evolution of the strain were clarified, respectively, which laid a theoretical basis for clinical prevention and diagnosis of related diseases. [Methods] The whole genome of HN02 was sequenced by SMRT and corrected by Illumina sequencing, followed by genome annotation and bioinformatics analysis. The virulence genes were identified by PCR and the phylogenetic tree was constructed for analysis. [Results] The whole genome of HN02 was 2 333 292 bp, with GC content of 40.15%. It was predicted to have 2 389 coding genes, including 19 rRNA genes(6 23 S rRNA, 6 16 S rRNA,and 7 5 S rRNA genes), 62 tRNA genes, and 5 sRNA genes, 84 tandem repeats, 66 minisatellite DNAs,2 microsatellite DNAs, 9 genomic islands, and 9 prophages. A total of 1 648, 2 190, and 1 917 genes were annotated in Gene Ontology(GO), Kyoto Encyclopedia of Genes and Genomes(KEGG), and Cluster of Orthologous Groups of proteins(COG), respectively. Most of them were involved in the metabolic process of Pm. In addition, 85 type III secretion system effector genes, 191 phenotypic mutation genes, 165 virulence factor-related genes, and 1 important secondary metabolism gene cluster were identified. According to the analysis result, a circular map for the genome was plotted, and the genome information was submitted to NCBI to obtain the accession number cp037865. PCR identification showed that the strain had 14 virulence genes such as fimA and toxA and deleted virulence genes such as tadD. Phylogenetic analysis suggested that HN02 had the closest genetic relationship with the strain(MH150895.1) from Beijing. [Conclusion]The whole-genome sequence and biological characteristics of capsular type A Pm HN02 were elucidated and the evolutionary relationship was revealed, providing a reference for preventing the epidemic of Pm-induced disease and exploring the underlying pathogenic mechanism.