1株多杀性巴氏杆菌的全基因组序列及致病相关基因分析

通过对多杀性巴氏杆菌(Pasteurella multocida)CS株全基因组的毒力基因分析，以期从基因组角度了解P. multocida CS的致病性机理。常规方法分离细菌，并进行毒力鉴定。基于二代测序平台对P. multocida CS进行测序，应用比较基因组学方法将其与GenBank中具全基因组来源于不同地域和宿主的P. multocida基因组进行比较分析。结果显示：猪肺疫病料中分离细菌滴鼻感染小鼠，测得其LD₅₀为5×10² CFU·mL^-1，显示较强毒性。将测序后的P.multocida CS株全基因组信息提交至NCBI，获得登录号SUB11119617。通过对P.multocida CS全基因组序列的分析表明，P.multocida CS株全基因组大小为2 599 048 bp，G+C含量为39.932%，编码CDs为2 580个，占整个基因组长度的69.6%，编码CDs的平均长度为952 bp。此外还有53个tRNA基因和3个rRNA基因。有87.95% 的编码CDs可进行COGs功能分类，29个为功能未知基因。利用RaAXML的最大释然法(maximum likelihood，ML)进行系统进化分析发现，P. multocida CS株与1个猪源性、2个牛源血清A型毒株，和1个猪源性D型毒株有较近的亲缘关系。P. multocida CS株含254个毒力相关基因，分为4大类11小类。其中铁摄取系统、黏附系统、分泌系统和双组分调控系统相关的多个基因在不同的毒株(包括血清型)的分布存在多态性。通过对基因出现频率的分析发现，铁摄取系统相关的tbpA和铁复合物外膜受体蛋白基因(iron complex outer membrane receptor protein gene，irp)以及黏附的相关的ppdD和ompA基因在血清A型出现的频率较高，这是否与不同菌株的毒力差异相关有待进一步证实。P. multocida CS的分泌系统由Ⅰ型(hly基因)、Tat型、Sec-SRP型3种类型组成。其中，hlyD基因在不同的血清型的分布频率存在多态性。P. multocida CS株的双组分信号转导系统(TCSs)由16个调节相关基因，12个感应相关基因和2个有hybrid相关基因。这些基因在不同血清型分布的多态性与不同菌株毒力的关系有待进一步研究。综上所述，组成铁摄取系统、黏附系统、分泌系统和双组分调控系统的基因在不同的血清以及同一血清型内出现频率存在多态性，它们与P. multocida菌株毒力强弱的关系有待进一步研究。

Analysis on Complete Genome Sequence and Pathogenic Genes of a Pasteurella multocida Strain

This study aimed to investigate the pathogenicity of Pasteurella multocida CS strain by analyzing virulence genes of its complete genome. The bacteria strain was isolated from swine pasteurellosis by conventional methods and its virulence was tested by mice. The complete genome of P. multocida CS strain was sequenced based on the second generation sequencing platform, and analyzed its differences with others submitted to GenBank from different regions and hosts by comparative genomics methods. The LD₅₀ of P. multocida CS strain was 5×10² CFU·mL^-1, which showed strongly toxic to mice. The genomic information of CS strain was submitted to NCBI, and the accession number was SUB11119617. The analysis results of the complete genome sequence of P. multocida CS strain showed that the total genome size of CS strain was 2 599 048 bp, the content of G+C was 39.932%, the CDs encoding gene was 2 580, accounting for 69.6% of the whole genome length. The average length of CDs encoding gene was 952 bp. Besides, there were 53 tRNA and 3 rRNA genes. 87.95% of the coding genes could be assigned as COGs, and the functions of 29 genes were unknown. Phylogenetic analysis using maximum likelihood method showed that CS strain was closely related to 1 pig origin serotype A strain and 2 cattle origin serotype A strain and 1 pig origin serotype D strain. The P. multocida CS strain contained 254 virulence-related genes which were divided into 4 major classes and 11 subclasses. The distribution of several genes related to iron uptake system, adhesion system, secretion system and two-component regulatory system was different among different serotypes. By analyzing the frequency of these gene occurrence of different P. multocida serotypes, it was found that tbpA and iron complex outer membrane receptor protein gene (irp) related with iron uptake system, and ppdD and ompA genes related with adhesion appeared to be associated with bacterial toxic differention among different strains. The secretory system of P. multocida CS strain consisted of three types: type I (hly gene), Tat type and Sec-SRP type. The frequency of hlyD gene among different serotypes was different. P. multocida CS strain's two-component signal transduction system (TCSs) consisted of 16 regulatory genes, 12 induction-related genes and 2 hybrid genes. The relationship between the polymorphism of TCSS genes in different serotypes and virulence of different strains needs to be further studied. To sum up, the gene frequency of iron uptake system, adhesion system, secretion system and two-component regulatory system had polymorphism among different serotypes and different isolates of the same serotypes. These may be one of the reasons for the virulence differences among different P. multocida strains.