Comparative Genomics And Expression Of VasF、vasK In T6SS Of Swine-Origin Extraintestinal Pathogenic Escherichia Coli

Extraintestinal Pathogenic Escherichia coli is a kind of pathogen that cause human and animals infected severely,such as UTI,neonatal Meningitis,sepsis, pneumonia,endometritis and mastitis. In recent years, the rate of disease which caused by animal source ExPEC is rising. A growing number of studies reported animal source ExPEC and anthropogenic ExPEC virulence gene sequence is similar and high homology [1-4]. For this reason, to carry out comparative genome analysis studies of animal origin ExPEC for the differences between the virulence of its animal and human origin ExPEC source ExPEC has become essential.This study is to identify ExPEC from different hosts and test its pathogenic, and comparative genomics analysis whole genome sequences of the pig source ExPEC with 42 strains of E. coli in the NCBI, further to clone and express unknown function vasF and vasK of complete T6SS2 secretion system, in the hope of laying the foundation for molecular epidemiology and pathogenesis mechanism of ExPEC research.Methods and results are as follows:1、By the method of molecular biology,the samples from animals infected with pneumonia was separated to identify pathogen, The results showed that 5 strains ExPEC was collected, among them is the zebra(BM1), pigs(ZF3,ZF7), rabbit(TF) from the lung and the birds of the air bag(AF1). Further these ExPECs was tested LD50 with karber method, its LD50 were 1.28×107CFU、9.32×106CFU、9.97×106CFU、6.79×106CFU、1.20×108CFU。2、Blastp analysis and clustering analysis is used to compare the whole genome sequence of the pig source ExPEC(SLPE) and 42 strains of e. coli, building its evolutionary tree.The results showed that E.coli SLPE belongs to B1 populations, and the relation with APEC O78 is close, however the relation with Intestinal pathogenic Escherichia coli UMNK88 and 2.3916 from pig is far, these belong to the of different groups3、Further E.coli SLPE genome was mapped to the APEC O78 genome, by the mummer software linear relationship between E.coli SLPE and APEC O78 was compared to analysis these relationships, results show that linear of E.coli SLPE with APEC O78 is good, close evolution relations with the APEC O78 is verified.3、 Using PGAP software virulence genes of E.coli SLPE was clustered, the results showed that although evolutionary relationships of E.coli SLPE with APEC O78 is close, but E.coli SLPE has more virulence factors compared with APEC O78.Through drawing heat map of virulence genes, found that papG Ⅱ、pix pilus gene cluster adhesion,IreA and fecR iron carrier,hlyF toxin/ Invasin,pgtP exists only in ExPEC; E.coli SLPE also contains a complete T6 SS type 2 secretion system; in addition, E.coli SLPE capsule polysaccharide biosynthesis gene cluster has a different type of serotypes that is special compare with all the E.coli.4、In order to investigate the source and structure of the special polysaccharide biosynthesis gene cluster which found in the genomic of E.coli SLPE, the content of GC gene,conservative gene and non conservative gene in the group 1 E.coli K30 and Klebsiella pneumoniae CPS was analyzed and compared,The results showed that E.coli SLPE could be horizontal transferred from Klebsiella pneumoniae serotype K16 such as Klebsiella pneumoniae serotype 2069/49, UCICRE 4, MGH 46 or other K16 serotypes of CPS, whether cps is related with Pathogenic effect and extra intestinal colonization is worth further study.5、Comparing with NCBI published NMEC RS218 T6SS2 secretion system, E.coli SLPE were found to contain a complete T6 SS type 2 secretion system.Through cloning, and prokaryotic expression the vasF and vasK, fusion proteins was got,the size is about 30 kDa and 50 kDa respectively.