Distribution Of Type V Secretion System In Avian Pathogenic E.coli And Functional Analysis Of ApaC And YejO

Bacterial pathogens utilize various mechanisms.to invade mammalian hosts and cause diseases.One of these strategies for many bacterial pathogens is the secretion system,which play various roles in bacterial virulence.Type V secretion system(T5SS)is one of the most simple secretion system,which is widely distributed in Escherichia coli.Privious studies have shown that T5SS plays an important role in the pathogenic mechanism including adhesion and invasion of host cells,bacterial motility,aggregation,biofilm formation,and resistance in bactericidal of serum.This study investigated the distribution of T5SS in avain.pathogenic E.coli(APEC)and determine the roles of T5SS ApaC,Yej O in the APEC and E.coli.1.Distribution and epidemiological analysis of type V secretion system(T5SS)in avian pathogenic Escherichia coliThis study was carried out to analyze the distribution of T5SS in APEC and its correlation with E.coli phylogenetic group and other virulence factors.15 E.coli T5SS were selected for primers designing,and the distribution of these T5SS in APEC clinical isolates were detected by PCR.The phylogenetic group and virulence factor of APEC strains were determined.The correlations between T5SS distribution and phylogenetic group and other virulence factors of APEC were analyzed.Results of this study revealed that T5SS was widely distributed in APEC isolates.T5SS ydeK and pplfP was present in 98.55%and 92.03%of the APEC isolates,respectively.However,the distribution rates of T5SS upaC and pic in APEC were less than 10.00%.Most APEC isolates belonged to A,B1 and D phylogenetic groups,following by B2.The combinations analysis showed that T5SS ehaA,ehaB,pic and vat were predominated present in phylogenetic group D APEC isolates.While,the phylogenetic groups A and B1 were the predominated groups that harbor the ehaG,ag43/flu and apaC.However,no significant correlation between the distribution of T5SS and other virulence genes in APEC were found.In general,we can conclude T5SS is widely distributed in APEC,and some T5SS was correlated with the phylogenetic group of APEC.2.Construction and biological characteristics analysis of APEC apaC mutant strainThe apaC gene mutant and complemented strains were constructed using Red homologous recombination method and complementary plasmid.Then,the differences in growth characteristics,motility,biofilm formation ability,adhesion and invasion ability,intracellular survival and virulence of wild-tye,mutant and complemented strains were compared to determine the effect of ApaC on the biological characteristics and virulence of APEC and E.coli.It was found that ApaC had no effect on the growth characteristics and motility of the E.coli.Whereas,ApaC was shown to enhance the biofilm formation ability,agglutination,and survival ability in serum.Compared with the wild-type strain,the cell adhesion ability and intracellular survival ability were significantly decreased for the mutant strain.However,the invasion ability of mutant strain was increased.The animal infeciton experiments showed that ApaC was necessary for the colonization and virulence in vivo.These data indicated that T5SS ApaC was involved in the regulation of bacterial biological characteristics and pathogenicity of E.coli,which will help us to understand the pathogenic mechanism of APEC.3.Construction and biological characteristics analysis of APEC yjeO gene mutant strainThis study constructed the yejO mutant and complemented strains usingby Red homologous recombinationand complementary plasmid.Then,the growth characteristics,motility,biofilm formation ability,adhesion and invasion ability,intracellular survival ability and pathogenicity of bacterial strains of wild-ype,mutant and complemented strains were compared.The results showed that the deletion of the yejO gene did not affect the growth rate,motility and survival ability in serum.,However,the mutant strain showed significantly decreased biofilm formation ability..YejO was found to promote the adhesion of E.coli to DF-1 cells,but had no effect on the invasion ability.The results of animal infection experiments showed that Yej O plays an important role in colonization and virulence of APEC.These data indicated that T5SS Yej O was involved in the biofilm formation,adhesion,colonization and virulence of APEC.