New Virulence Factors Invovled In K88ac+ Enterotoxigenic Escherichia Coli

Porcine enterotoxigenic Escherichia coli?ETEC?is the leading cause of diarrhea in neonatal and post-weaning piglets,which resulting in significant economic losses in swine production.K88ac+ETEC is one of the major serotypes that causing porcine post-weaning diarrhea?PWD?,which has high morbidity and mortality after infection.The primary virulence factors in ETEC includes adhesin and enterotoxin,while the role of flagella,as a new virulence factor,in the pathogenesis of K88ac+ETEC is not elucidated well.In this paper,we targeted the reference C83902 ETEC strain?08:H19:K88ac+ LT+ STa+ STb+?to study the role of flagella and K88 fimbriae played in bacterial pathogenic process.Furthermore,we sequenced the genome of strain C83902 and found some more novel virulence factors.The detailed works we did in this study is as following:1.Co-regulation of flagella and K88 fimbriae in K88ac+ enterotoxigenic Escherichia coliWhile it is known that bacterial flagella generally contribute to pathogen virulence,the role of flagella in the pathogenesis of K88ac+ETEC mediated neonatal and post-weaning diarrhea?PWD?is not currently understood.We targeted C83902 to construct isogenic mutants in the fliC?encoding the major flagellin protein?,mot A?encoding the flagella motor?,and faeG?encoding the major subunit of K88 fimbriae?genes by using X-Red recombinant system.PCR and sequencing was done to confirm the knocking out gene in the mutants.The complemented strains C83902?fliC/pfliC and C83902?faeG/pfaeG were constructed by transforming the recombined plasmid pBR322 that expressing FliC and FaeG into the mutant strain respectively.Combined assays of bacterial motility,transmission electron microscope and agglutination test were performed to test the biology function of mutants and complemented strains.The result of adhesion assay showed that the ?fliC and AfaeG mutants had a 20%and 96%reduction respectively,to adhere to porcine intestinal epithelial IPEC-J2 cells when compared with WT strain.The?fliC ofaeG double mutant showed a 97%decreased in adhesion as well while the ?motA mutant and complemented strains showed a similar adherence ability with the WT strain.qRT-PCR revealed that faeG expression was 21.5%down-regulated after deleting fliC,,while there was a 2.08-fold increase of fliC expression in AfaeG mutant.fliC and faeG expression in ?motA mutant and complemented strains were not significant different from that in WT strain.These data demonstrated that co-regulation exists between flagella and K88 fimbriae in K88ac+ ETEC.2.Flagella and K88 fimbriae in K88ac+ ETEC affect the bacterial biofilm formation and quorum sensingBacteria that form biofilms are often highly resistant to antibiotics and are capable of evading the host immune system.The reference C83902 ETEC strain and isogenic fliC and faeG mutants,as well as the complemented strains were used in this study.Biofilm formation quantitative assay showed that biofilm formation ability in ?fliC mutant decreased 47.5%when compared with WT strain,while ?faeG mutant increased 1.4-fold.As the quorum sensing?QS?-? is an important signaling pathway in E.coli and participate in the regulation of biofilm formation.We detected the AI-2 activity and confirmed the AI-2 related genes expression by using qRT-PCR.The result showed that AI-2 activity in AfliC mutant,?faeG mutant and AfliC ?faeG double mutant reduced 16.2%,19.1%and 21%,respectively,as compared with WT strain.Result of qRT-PCR revealed that transcription level of luxS and pfs gene decreased in ?fliC mutant,but increased in ?faeG mutant when compared with WT strain.This study demonstrated both flagella and K88 fimbriae playing important roles in the bacterial biofilm formation and quorum sensing in the K88ac+ETEC strain.3.Role of lipid rafts in enterotoxigenic Escherchia coli Flagellins Induced Pro-inflammatory in Intestinal Epithelial CellsLipid rafts,which refer to the cholesterol and sphingolipid-rich ordered microdomains,distributed in the cell membrane,participate in amounts of important biological processes.Likewise,to probe into the role of lipid rafts played in mediating interaction between ETEC flagellins and intestinal epithelial cell membranes,two kinds of cultured intestinal epithelial cells?Caco-2 and IPEC-J2?were targeted,firstly pretreated with methyl-?-cyclodextrin?M?CD?to efficiently deplete the membrane cholesterol and distupt the lipid rafts,and then incubated with engineered recombinant?H1/H19 serotypes?flagellins,respectively.Exgenous cholesterol was supplementd to recover the membrane cholesterol in the complemented groups.Cells cultured in the cell media throughout the experiment were regard as controls.Lastly,real-time PCR and ELIS As were performed to determined the expression level of TLR5 pathway related proinflammatoiy cytokines in cells of control groups,M?CD pretreated groups and cholesterol complemented groups.The results showed that,when removing the membrane cholesterol,recombinant flagellins induced one-third reduction of cytokines of IL-8 and TNF-a involved in TLR5 pathway in both transcription and protein level,as compared to untreated control cells.Meanwhile,IL-8 and TNF-a expression in cholesterol complemented groups were not significantly different from controls.This study demonstrated the critical role of membrane cholesterol of lipid rafts in TLR5 mediated host innate immune recognition to ETEC infection.4.Genome sequencing of the K88ac+ ETEC strain C83902In order to find out more new virulence factors than known fimbrial adhesin,flagella and enterotoxin in the K88ac+ETEC.In this study,next generation sequencing?NGS?technology was performed to sequence the genome of reference C83902 ETEC strain.The full length of bacterial chromosome is 4,920,255 bp,which contains 5,148 genes with a GC content of 50.8%.CRISPRs analysis showed the bacteria carrys both CRISPR land CRISPR 2,but Cas enzyme related genes were already abandoned during its evolution.Five complete,one incomplete and one questionable prophage were found in the choromosome.E.coli strains could be classified into five phylogroups?A,B1,B2,D and E?.The phylogenetic analysis showed that C83902 was included in the phylogenetic group A,but seems closer to strain O104:H4₂011C-3493,0103:H2₁2009,and human ETEC strian E24377.Classification and comparison of the virulence factors in C83902 revealed four novel fimbriae operon,including alpha fimbriae,stf fimbriae,sfm fimbriae and long polar fimbriae,are existing in the genome,which have not been reported in ETEC before.Otherwise,Yersinia high pathogenicity island?HPI?and Type VI secretion system?T6SS?were found as well,which would be new ETEC virulence factors,that remains to study.5.Cloning,expression and identification of alpha fimbriae in K88ac+ ETECAnalyzing the genome sequence data of K88+ ETEC strain C83902,four genes were identified which encoding the alpha fimbriae operon.By using BLAST to genomes of different E.coli strains on GenBank,the result showed that this operon majorly distributes in pathogenic E.coli.including ETEC,APEC and UPEC.We designed the primers and cloned this operon into pET42a?+?.Then the fimbriae was expressed in E.coli strain C3040,under the IPTG inducing.Transmission electron microscope was performed to obverse the morphology of fimbriae.Adhesion assay showed that alpha fimbriae don't adhere to human colon cancer cells Caco-2 in vitro.This study is the first time identifying the alpha fimbriae in an ETEC strain,which would lay the foundation for further discovering new virulence factors and elucidating pathogenesis in K88+ ETEC.