Study On The Distribution Characteristics Of Virulence Factors And Function Of EspX1 In Avian Pathogenic Escherichia Coli

Avian pathogenic Escherichia coli(APEC)can infect poultry and cause avian colibacillosis,which brings a lot of economic losses to poultry industry.The disease often causes mixed infection with other respiratory diseases,leading to systemic infection and characteristic fibrous lesions such as pericarditis and pericarditis in poultry,resulting in septicemia and poultry death.APEC has many serotypes and no cross-protection,so it is difficult to prevent and control.The use of antibiotics leads to the emergence of multiple drug-resistant APEC strains,which greatly hinders the prevention of avian colibacillosis.APEC is similar to human Extraintestinal pathogenic Escherichia coli(Ex PEC)in serotype,phylogenetic clustering and virulence genes,and is a repository of Ex PEC virulence and drug resistance genes.Pentapeptide repetitive(PPR)protein family is widely distributed and has important functions in eukaryotes,but there are few studies on PPR in prokaryotes.This article investigated the serotype,phylogenetic clustering and distribution of virulence factors of APEC strains in different poultry farms in Jiangsu,Anhui and other places.This paper also explored the effects of PPR protein EspX1 on the biological characteristics of APEC and host inflammatory factors,so as to provide reference for elucidating the mechanism of PPR protein in prokaryotes.The main research contents are as follows:1 Molecular Epidemiological investigation on serotype,phylogenetic clustering and virulence factors of APECIn this study,210 strains of APEC were isolated and identified from different poultry farms,and serotype,phylogenetic clustering and virulence gene detection were carried out to analyze the relationship between virulence gene and serotype and phylogenetic grouping,so as to provide reference for the epidemic characteristics of APEC and formulating prevention and control strategies.The results showed that O78 was dominant serotypes,and the proportion of group B2 was the highest,accounting for 31.9%.Virulence gene test showed that the detection rate of fim C,mat and ibe B genes was more than 90%,and that of pap C,neu C,vat and ibe A was less than 22%.All isolates contained at least four virulence genes.Association analysis showed that virulence genes irp2 and ibe A were highly distributed in O2,neu C was highly distributed in O1,vat was highly distributed in O1 and O2,ETT2 was highly distributed in O78.And pap C,tsh,irp2,neu C,vat and ibe A were higher in B2 groups.This study provides a reference for understanding the dominant epidemic strains of APEC and its control.2 Distribution and prokaryotic expression of PPR protein EspX1 in APECPPR protein EspX1 was found in APEC by genome sequencing,but its distribution and function were unknown.In this experiment,the distribution of EspX1 in 111 strains of APEC was investigated,and the sequence analysis of EspX1 was carried out.The plasmid pGEX4T-1-espX1 was constructed,and the expressed protein was purified and immunized with New Zealand white rabbits.The results showed that the distribution rate of EspX1 was 41.44%,there was neither signal peptide nor transmembrane region in EspX1,and there were multiple helices in 3D structure.Fusion protein GST-EspX1 was soluble,and the immune serum with titer of1:32000 was obtained,which laid a foundation for the study of the function of EspX1.3 Construction of EspX1 deletion strain,complementary strain and detection of their biological characteristicsIn order to understand the influence of EspX1 of APEC,the Red homologous recombination system was used to construct the deletion strain ?EspX1,and the plasmid p BAD3×flag was used to construct the complementary strain C?EspX1.The biological characteristics among FJ1 B,?EspX1 and C?EspX1 were compared.The results showed that EspX1 did not affect the growth,motility and invasive ability of APEC.And EspX1 could enhance APEC's antiserum ability,antiphagocytic ability,colonization ability,virulence and resistance of some fluoroquinolone antibiotics.These suggested that EspX1 is a virulence factor of APEC.This study explored the effect of EspX1 on APEC,enriched the data on the function of PPR protein in prokaryotes,and provided reference for the follow-up study of PPR protein in prokaryotes.4 Effect of virulence factor EspX1 on the expression of inflammatory factors in host cellsPreliminary experiments have proved that EspX1 is a virulence factor of APEC.The absence of EspX1 will affect some of the biological characteristics of APEC,but the effect of EspX1 on host cells is unknow.To understand the influence of EspX1 on the host's inflammatory response,FJ1 B,?EspX1 and C?EspX1 were used to infect HD-11 cells and detected the expression of TNF-?,IL-1? and IL-8.In addition,the plasmid pEGFP-N1-espX1 was constructed and transfected into DF-1 and 293 T cells to detect the expression of these inflammatory factors.The changes of Phosphatidylserine(PS)position and the activation of Caspase 3 were also examined to verify the effect of EspX1 on apoptosis.The results showed that the expression of these inflammatory factors increased in HD-11 cells infected with ?EspX1,while decreased significantly in pEGFP-N1-espX1 transfected cells.The results showed that EspX1 could inhibit the expression of host inflammatory cytokines TNF-?,IL-1? and IL-8,but had no effect on apoptosis.