Genome Sequencing,Transcriptome Analysis And Identification Virulence Genes Of Avian Pasteurella Multocida

Pasteurella multocida（Pm）belongs to Pasteurella genus which is a member of the Pasteurellaceae family.Pm is classified into five serogroups（A、B、D、E and F）based on capsule specificity and 16somatic serotypes based on LPS antigens.A serotype is designated by its capsular type followed by the somatic type,such as A:1、B:2 as so on.Pm is the etiologic agent of avian cholera,haemorrhagic septicaemia in ungulates.Avian cholera is mainly caused by capsular serotype A、F and D.This disease is distributed around the word and is harmful to poultry and wild poultry,causing huge economic losses.At present,many virulence factors of Pm were found,such as capsule、lipopolysaccharide、outer membrane proteins、sialidases、iron uptake proteins and so on.But using comparative study of virulent and attenuated strains to find out virulent genes is necessary to study.The aim of this research was to find out virulence-associated genes of virulent Pm strain through genome sequencing technology、RNA-seq technology and homologous recombination to construct mutant technology,and to lay the foundation for ecluidating the pathogenic mechanism and vaccine development of avian cholera.In this study,two virulent strains PmC48-1 and Pm72-4 and an attenuated strain Pm731 were sequenced by genome-wide scanning,and comparative genomics analysis four strains（PmC48-1、Pm72-4、Pm731and one published strain Pm70）to find out different genes among them.The result showed that the bases in the genome of PmC48-1、Pm72-4 and Pm731 were 2,304,775 bp、2,259,533 bp and 2,260,636 bp respectively,and the G+C%content were 40.377%、40.278%and 40.42%respectively,and the number of genes were 2131、2028 and 2068,respectively.A unique gene cluster of the virulent strain PmC48-1was found.The gene cluster of PmC48-1 was 44,468 bp in length by compare with the genome of Pm70,and was flanked by an 20 bp direct repeat sequence which has high degree of homology with the 3’-terminal sequence of gene PM_RS03305（rpL25）,suggesting the gene cluster inserted into the gene PM_RS03305.Using the PHAST software,it was found that the gene cluster contained a complete phage sequence and an incomplete phage sequence.It was a prophage associated genomic island harboring 67 putative coding sequences（CDS）.Then the three strains PmC48-1、Pm72-4 and Pm731 were sequenced by RNA-seq,then find out common differently expression genes（DEGs）between virulent strains（PmC48-1、Pm72-4）and attenuated strain Pm731.The up-regulated and down-regulated genes were analyzed by GO and KEGG Pathway enrichment and some of the DEGs were verified using Real-time RT-PCR.Transcriptome analysis showed that there were 190 differential expression genes between the virulent strains（PmC48-1、Pm72-4）and the attenuated strain Pm731,of which 75 were up-regulated and 115 were down-regulated.GO significant enrichment analysis showed that these DEGs mainly involved in catalytic activity、binding、transporter activity in terms of molecular function,and membrane and cell in terms of cellular component,and metabolic process、single-organism process、cellular process、localization in terms of biological process.KEGG Pathway enrichment analyisis indicate that the DEGs involved in carbohydrate metabolism、energy metabolism、amino acid metabolism、metabolism of cofactors and vitamins、translation、membrane transport、signal transduction、cell motility.4up-regulated genes and 8 down-regulated genes were selected to be verified via Real-time RT-PCR,and showed that they were consistent with the RNA-seq analysis results.In order to further verify the virulence of DGEs,10 different genes which contain six candidate genes（（PP₀0004、PP₀0009、PP₀0019、PP₀0053、PP₀0060、PP₀0068））from genomics screening and 4 candidate genes（dppA、fbpA、rsgA-2、PM0472）from transcriptome were selected to construct mutants strains of PmC48-1.Double-crossover mutants was identified by PCR,and study the genetic stability、growth characteristics and pathogenicity of double-crossover mutants strains.The study showed that only four double-crossover mutants of four genes（PP₀0004、PP₀0009、PP₀0019、dppA）were successfully obtained through homologous recombination respectively,named PmC48-1Δ4、PmC48-1Δ9、PmC48-1Δ19 and PmC48-1ΔdppA.The four mutants has good genetic stability after 20 generations and has basically same growth ability compared with the wild strain PmC48-1.The results of the pathogenicity test of mutants in BALB/c mice showed that the mice infected with 10 CFU wild type strain PmC48-1 were all died within 24 h,and the LD₅₀ is less than 10CFU.The mice infected with 5×10³ CFU of each four mutants（PmC48-1Δ4、PmC48-1Δ9、PmC48-1Δ19 and PmC48-1ΔdppA）though intraperitoneal injection were all health live within 10 days,and the LD₅₀ of four mutant strains were more than 5×10³ CFU.The result indicated that the four mutants insignificantly attenuated compared to the wild strain PmC48-1 in virulence.According to the gene annotation,PP₀0004 gene encodes a putative phage tail protein,PP₀0009 gene encodes a tail lengh tape measure protein,PP₀0019 gene encodes a XRE family transcriptional regulator and dppA gene encodes a peptide transporter,and dppA gene involved membrane transport and cell motility pathway and has transporter activity.In this study,we found four virulence associated genes（PP₀0004、PP₀0009、PP₀0019、dppA）of Pm through genome sequencing、RNA-seq and construction mutant technology,and the three gene（PP₀0004、PP₀0009、PP₀0019）are located in a prophage associated genomic island.These findings lay the foundation for the study pathogenic mechanism of avian cholera and provide ideas for development of safe and effective new vaccines of Pm.