Epidemiology Investigation Of Porcine Extraintestinal Pathogenic Escherichia Coli And Functional Study Of Regulator Ydiv

Escherishia coli(E.coli),one of the model bacterium that had most variation in pathotypes,had been widely studied.According to its chromosomal characteristics and virulence gene profile,E.coli can be separated into three groups,which are commensal,intestinal pathogenic and extraintestinal pathogenic e.coli.Extraintestinal E.coli can be further divided into uropathogenic e.coli,neonatal meningitis E.coli and avian pathogenic e.coli based on infected organs.The common symptoms caused by ExPEC include meningitis,cystitis,pneumonia,pyonephritis and septicemia.Currently,most studies focus on ExPECs isolated from human and bird and the importance of ExPECs in swine is underestimated.There are only few reports about this pathogen.This is mostly due to the difference of phylogenetic origin between ExPEC isolated from human and pigs.However,In our previous study,The incidence of swine ExPEC cases was found to be increased over the years and large number of swine ExPECs possess same phylogenetic background and serogroup as human ExPECs.The molecular epidemiology characteristics,virulence genes and pathogenesis of swine ExPECs is still not clear.Therefore this study investigated porcine ExPECs collected from 10 provinces in China in 2006-2007 and analysized the genetic features of representative porcine ExPEC strain PCN033,which could cause meningitis and belongs to newly found 1687 ST type.Based on these results,we also investigate the function of potential virulence regulator gene ydiv,which would provide bases for prevention and treatment of this disease.1.Molecular epidermiology porcine ExPECsThe 81 strains of ExPECs isolated from our clinical laboratory are colleceted from 10 provinces from 2006-2007.These ExPEC strains were investigated in terms of its serogroup,phylogenetic group,MLST,and virulence genes via immunoprecipitation and PCR method.The results showed that the dominant porcine ExPEC serogroups are O11,O8,O138,O161 and O101.All ExPECs are equally distributed in phylogenetic group A,B1,B2,and D,while most human and avian ExPECs belongs to phylogenetic group B2 and D.The MLST results showed that our collection of porcine ExPECs is consist of mainly four clonal complex,namely CC10,CC1687,CC88 and CC58.The CC1687 strains are consist of strains of new sequence types found in our study.The CC1687 strains have K1 capsule and could be isolated from brain and cerebrospinal fluids,which are similar to NMEC strains.However,porcine ExPEC strains are lack of ibeA gene that is important in NMEC pathogenesis.This implicated that strains in CC1687 may have the capacity to invade the brain of diseased pigs via unique virulence profile.The CC1687 strains were isolated from different provinces in China,which indicated these strains are already endemic in these provinces.Many strains with ST types in Clonal complex 10 were also reported to be isolated from human ExPEC strains,the zoonotic potential of porcine ExPEC therefore need to be further investigated.2.Comparative genomic analysis of ExPEC from different hosts.Current research results showed that different virulence genes take part in the pathogenesis of ExPEC isolated from different hosts.We therefore speculated that porcine ExPECs make use of their unique virulence gene profile during the process of their pathogenesis.Based on the epidemiological data of porcine ExPECs,Porcine ExPEC PCN033,a hightly virulent strain that could cause meningitis in clonal complex 1687,was compared to buffalo(LS4),bird(APEC O1),and human(CFT073 and RS218)ExPEC in order to identify virulence factors that are host specific and that are shared by different hosts.We found that our swine ExPEC strain PCN033 had 692 unique genes with Blast.COG analysis of these unique genes showed that they are mostly related to organic substance catabolism,which may help porcine ExPEC PCN033 to make use of more organic substances.This may be benificial for porcine ExPEC PCN033 to adapt to different environment.The PCN033 have 15 genomic islands.Among them,G11 and G13 have type III and type VI secretion systems,which are putative virulent factors.The PCN033 is also unique in its flagellum,adhesins,and virulence genes,it is also lack of ibeA gene,which is important in the pathogenesis of human NMEC.The differences therefore indicate procine ExPECs may have unique virulent factors or mechanisms to cause meningitis in pigs.This part of study layed foundation for our further research on the pathogenesis of porcine ExPEC.3.Regulatory mechanism and biological function of ydiv in porcine ExPECBased on our previous genomic analysis data,we constructed mutant strains of regulator genes and investigate their relationship with virulence through mice infection model.The result showed that ydiv,a putative regulator with c-di-GMP domain,would significantly reduce the virulence of the PCN033 strain if deleted.We further use RNAseq to investigate its effect on transcriptome by comparing the WT and mutant strains.The result showed that ydiv could affect the transcription of 12 genes,most of them are related to flagellum.The effects of ydiv gene on flagellun were also validated by the motility assay.To investigate the mechanism of ydiv dependent regulation on flagellum,we use bacteria two-hydrid system and detected protein-protein interaction between Ydiv and flagellar regulator FlhD;We use surface plasmon resonance(SPR)technique and detected protein-DNA interaction between Ydiv and the promotor of flhDC operon as well as the promoter of ydiv itself.The functional analysis showed that ?ydiv strains' increased adherence to brain microvascular endothelial cells(BMECs)is depended on increased flagellum expression.The double mutant strain ?ydiv?fliA restore to normal adherence to BMECs.In vivo test shows ?ydiv strains have decreased amount of bacteria in all tissues except brain.The above result indicate flagellum is an important adhesin to the BMECs in porcine ExPEC and its increased expression would decrease the amount of ExPEC CFUs in tissue.The clearance assay in mice model demonstrated that the decreased amount of ?ydiv strains in different tissues is due to the increased clearance of ?ydiv strains compared to WT strains.We assume that this is because the increased flagellum expression in ?ydiv would facilitate recognition and clearance of PCN033 by the immune system.The macrophage phagocytic assay showed that increased phagocytic effect of macrophage is depend on flagellum.?ydiv strains are easier to be phagocized by macrophage while the ?ydiv?fliA strains is less likely to be phagocized when compared to WT strains.This result indicated that flagellum could mediate the recognition and phagocytosis of immune cells to ExPECs.In summary,thhis part of study showed that flagellum is an important adhesin for swine ExPEC to attach to the BMECs,Ydiv could regulate flagellum through duel mechanisms.It could suppress the expression of flagellum by both protein-protein interaction and protein-DNA interaction.The Ydiv,on one hand,functions mainly through suppressing the flagellum expression and reducing the recognition and clearance of PCN033 by the host,which would be beneficial to its survival in animals.on the other hand,Ydiv could maintain flagellum expression through negative feedback on its own promotor.Flagellum expression was kept to a level to maintain the adhesion of porcine ExPECs to BMECs.