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

doi:10.11843/j.issn.0366-6964.2022.06.019

摘要/Abstract

摘要： 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。

关键词: 多杀性巴氏杆菌CS株, 全基因组序列, 致病基因组学, 基因多态性

Abstract: 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.

Key words: Pasteurella multocida CS strain, complete genome sequence, pathogenomics, polymorphism

中图分类号:

S852.612

许文博, 吴丽梅, 刘鑫, 李升, 黄复深, 李润成. 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[J]. 畜牧兽医学报, 2022, 53(6): 1858-1869.

XU Wenbo, WU Limei, LIU Xin, LI Sheng, HUANG Fushen, LI Runcheng. Analysis on Complete Genome Sequence and Pathogenic Genes of a Pasteurella multocida Strain[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(6): 1858-1869.

参考文献 30

[1]	ZHAO X, GU Y F, GUO W N. Advance in virulence factors of Pasteurella multocida[J]. Progress in Veterinary Medicine, 2020, 41(11): 100-103. (in Chinese)赵霞, 顾有方, 郭伟娜. 多杀性巴氏杆菌毒力因子研究进展[J]. 动物医学进展, 2020, 41(11): 100-103.
[2]	HARPER M, BOYCE J D, ADLER B. Pasteurella multocida pathogenesis: 125 years after Pasteur[J]. FEMS Microbiol Lett, 2006, 265(1): 1-10.
[3]	CARTER G R. Further observations on typing Pasteurella multocida by the indirect hemagglutination test[J]. Can J Comp Med Vet Sci, 1962, 26(10): 238-240.
[4]	HEDDLESTON K L, GALLAGHER J E, REBERS P A. Fowl cholera: gel diffusion precipitin test for serotyping pasteurella multocida from avian species[J]. Avian Dis, 1972, 16(4): 925-936.
[5]	JOLLEY K A, MAIDEN M C J. BIGSdb: scalable analysis of bacterial genome variation at the population level[J]. BMC Bioinformatics, 2010, 11: 595.
[6]	HARPER M, JOHN M, TURNI C, et al. Development of a rapid multiplex PCR assay to genotype Pasteurella multocida strains by use of the lipopolysaccharide outer core biosynthesis locus[J]. J Clin Microbiol, 2015, 53(2): 477-485.
[7]	PENG Z, WANG X R, ZHOU R, et al. Pasteurella multocida: genotypes and genomics[J]. Microbiol Mol Biol Rev, 2019, 83(4): e00014-19.
[8]	LI H. Research progress on virulence factors and gene expression of Pasteurella multocida[J]. Chinese Journal of Traditional Veterinary Science, 2015, 4(9): 12-13. (in Chinese)李卉. 多杀性巴氏杆菌毒力因子及基因表达的研究进展[J]. 中兽医学杂志, 2015, 4(9): 12-13.
[9]	PORS S E, CHADFIELD M S, SØRENSEN D B, et al. The origin of Pasteurella multocida impacts pathology and inflammation when assessed in a mouse model[J]. Res Vet Sci, 2016, 105: 139-142.
[10]	PENG Z, LIANG W, WANG F, et al. Genetic and phylogenetic characteristics of Pasteurella multocida isolates from different host species[J]. Front Microbiol, 2018, 9: 1408.
[11]	DE OLIVEIRA FILHO J X, MORÉS M A Z, REBELLATO R, et al. Pathogenic variability among Pasteurella multocida type A isolates from Brazilian pig farms[J]. BMC Vet Res, 2018, 14(1): 244.
[12]	TOWNSEND K M, BOYCE J D, CHUNG J Y, et al. Genetic organization of Pasteurella multocida cap Loci and development of a multiplex capsular PCR typing system[J]. J Clin Microbiol, 2001, 39(3): 924-929.
[13]	ZHANG W J, LIU S J, ZHANG Y Y, et al. Serotype identification, virulence genes and pathogenicity detection of Klebsiella pneumoniae from foxes in some areas of Hebei Province[J]. Chinese Journal of Veterinary Science, 2020, 40(4): 735-739. (in Chinese)张文举, 刘少杰, 张艳英, 等. 河北部分地区狐狸源肺炎克雷伯菌血清型鉴定、毒力基因及致病性检测. 中国兽医学报, 2020, 40(4): 735-739.
[14]	LIU H Y, YANG Y W, WANG Y W, et al. Serotype, drug resistance and pathogenicity analysis of 6 strains of Pasteurella multocida from goats[J]. Heilongjiang Animal Science and Veterinary Medicine, 2020(16): 65-68. (in Chinese)刘海燕, 杨有文, 王远微, 等. 6株山羊源多杀性巴氏杆菌血清型、耐药性及致病性分析[J]. 黑龙江畜牧兽医, 2020(16): 65-68.
[15]	HAN M L, ZHANG X X, WU T Z, et al. Isolation and Identification of Pasteurella multocida rabbits[J]. Xinjiang Agricultural Sciences, 2018, 55(7): 1333-1342. (in Chinese)韩猛立, 张星星, 吴桐忠, 等. 兔源巴氏杆菌分离与鉴定[J]. 新疆农业科学, 2018, 55(7): 1333-1342.
[16]	LIU W J, YANG M J, XU Z F, et al. Complete genome sequence of Pasteurella multocida HN06, a toxigenic strain of serogroup D[J]. J Bacteriol, 2012, 194(12): 3292-3293.
[17]	KONG C, NEOH H M, NATHAN S. Targeting Staphylococcus aureus toxins: a potential form of anti-virulence therapy[J]. Toxins, 2016, 8(3): 72.
[18]	ZHANG Y N, LI Y F, CHEN R J, et al. Resistance and virulence analysis of type A: L1 ST128 Pasteurella multocida from ducks[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(10): 2852-2863. (in Chinese)张亚楠, 李亚菲, 陈汝佳, 等. A: L1 ST128型鸭源多杀性巴氏杆菌的耐药性及毒力分析[J]. 畜牧兽医学报, 2021, 52(10): 2852-2863.
[19]	NOINAJ N, BUCHANAN S K, CORNELISSEN C N. The transferrin-iron import system from pathogenic Neisseria species[J]. Mol Microbiol, 2012, 86(2): 246-257.
[20]	OGUNNARIWO J A, SCHRYVERS A B. Characterization of a novel transferrin receptor in bovine strains of Pasteurella multocida[J]. J Bacteriol, 2001, 183(3): 890-896.
[21]	EWERS C, LUBKE-BECKER A, BETHE A, et al. Virulence genotype of Pasteurella multocida strains isolated from different hosts with various disease status[J]. Vet Microbiol, 2006, 114(3-4): 304-317.
[22]	FARAHANI M F, ESMAELIZAD M, JABBARI A R. Investigation of iron uptake and virulence gene factors (fur, tonB, exbD, exbB, hgbA, hgbB1, hgbB2 and tbpA) among isolates of Pasteurella multocida from Iran[J]. Iran J Microbiol, 2019, 11(3): 191-197.
[23]	PENG Z, LIANG W, AI W C, et al. Genotypical characteristics of swine Pasteurella multocida in China[J]. Acta Veterinaria et Zootechnica Sinica, 2019, 50(5): 1064-1072. (in Chinese)彭忠, 梁婉, 艾伟诚, 等. 我国猪群中多杀性巴氏杆菌的基因型分析[J]. 畜牧兽医学报, 2019, 50(5): 1064-1072.
[24]	HARPER M, BOYCE J D, ADLER B. The key surface components of Pasteurella multocida: capsule and lipopolysaccharide[J]. Curr Top Microbiol Immunol, 2012, 361: 39-51.
[25]	MICHAEL F S, VINOGRADOV E, LI J J, et al. Structural analysis of the lipopolysaccharide from Pasteurella multocida genome strain Pm70 and identification of the putative lipopolysaccharide glycosyltransferases[J]. Glycobiology, 2005, 15(4): 323-333.
[26]	YIN L, QI K Z, SONG X J, et al. Type Ⅲ secretion system 2 pathogenicity islands of Escherichia coli[J]. Microbiology China, 2017, 44(12): 3031-3037. (in Chinese)尹磊, 祁克宗, 宋祥军, 等. 大肠杆菌Ⅲ型分泌系统2毒力岛研究进展[J]. 微生物学通报, 2017, 44(12): 3031-3037.
[27]	WILLIAMS R H, WHITWORTH D E. The genetic organisation of prokaryotic two-component system signalling pathways[J]. BMC Genomics, 2010, 11: 720.
[28]	WILLETT J W, CROSSON S. Atypical modes of bacterial histidine kinase signaling[J]. Mol Microbiol, 2017, 103(2): 197-202.
[29]	LAUB M T, GOULIAN M. Specificity in Two-component signal transduction pathways[J]. Annu Rev Genet, 2007, 41: 121-145.
[30]	BEIER D, GROSS R. Regulation of bacterial virulence by two-component systems[J]. Curr Opin Microbiol, 2006, 9(2): 143-152.