Effect Of The ClpV1 Gene Of Type ? Secretion System On The Pathogenicity Of Meningitis-associated Avian Pathogenic Escherichia Coli

Avian pathogenic Escherichia coli(APEC)is an important member of extraintestinal pathogenic E.coli(ExPEC),which is one of the main infectious pathogens of avian E.coli diseases in China.APEC has a high homology with neonatal meningitis E.coli(NMEC).With the development of intensive aquaculture,APEC has been proved as a potential zoonotic pathogen with the risk of infecting infants with meningitis,which is of great importance to public health.As a gram-negative bacterium,E.coli can release a multi-functional weapon-Type VI secretion system(T6SS),which can secret proteins as a weapon to attack target cells and participate in pathogenic process.ClpV is the component that disassembles the T6SS under the drive of adenosine triphosphate(ATP).Three T6SSs have been found in E.coli.T6SS1 is involved in the interaction with host cells,affecting the competition between bacteria and enhancing the pathogenicity to mice.T6SS2 is only involved in the interaction with brain microvascular endothelial cells of mouse,while T6SS3 does not have secretion function.It is still not clear what role T6SS1 plays in the pathogenesis of E.coli.In this study,the core component of T6SS1,ClpV1,was taken as the pointcut to explore its role in APEC meningitis,so as to better understand the pathogenic mechanism of APEC.1.Construction of clpV1 gene mutants of TW-XM strain and biological characteristics analysis of the mutant strainAccording to NCBI GenBank database,PCR primers were designed to amplify the clpV gene of APEC TW-XM(Wild-type,O2:K1).Then,the PCR amplification products were purified,cloned and sequenced.Then,we designed the primers which contain a homologies 5'terminal to the target region and a homologies 3'terminal to the chloromycin-resistance cassette for inactivating the target gene.According to the ?-Red recombination system,we used plasmid pKD3 as the template DNA to amplify the chloromycin-resistance cassette,then the purified PCR products were electroporated into TW-XM carrying a heat-sensitive plasmid pKD46.And the successful recombinant bacteria were selected.After that,the plasmid pCP20 was electroporated into the first recombination bacteria,and the chloromycin-resistance cassette was excised.Finally,the isogenic gene deletion TW-XM?clpV1 was constructed and confirmed by PCR detection and DNA sequencing data.On the base of the deleted strain,we electroporated plasmid pBR322 which could express clpV1 into the second recombination mutant to construct the complemental strain TW-XMC?clpV1.Biological characteristics analysis was conducted afterwards.Bacterial growth test indicated that there was no difference among wild type,mutants and complemental strains.Motility test showed that TW-XM?clpV1 lost its motility on semisolid medium,but the wild type and complemental strain still had strong motility.Meanwhile,biofilm formation test proved that the clp V1 gene could promote bacterial biofilm formation.Drug sensitivity test results showed that the deletion of the clpV1 gene did not affect the sensitivity to nine antibiotics of the TW-XM strain wildly used clinically.Besides,we used Real-time PCR to detect the transcriptional changes of type ? fimbriae related genes,T6SS related genes and meningitis-associated virulence factors in mutanted strains.The results showed that the transcriptional level of all genes above in clpV1 mutant decreased significantly(p<0.05 or p<0.01).The deleted strain also showed lower pathogenicity towards ducks.As a result,clpV1 could be involved in the biological function of TW-XM.In addition,the clpV1 gene is involved in the pathogenesis of TW-XM,and there are some synergistic effects existing in different virulence factors.After the clp Vl gene was deleted,the virulence of TW-XM was reduced.2.Effect of clp V1 gene on the ability of TW-XM to induce meningitisIn order to investigate the effect of clpV1 gene on the ability of TW-XM to induce meningitis in newborn mouse,a mouse infection model was established with intraperitoneal injection of TW-XM,TW-XM?clpV1 and TW-XMCAclpV1 with 107 CFU each mouse.0.1 mL PBS was intraperitoneal injectes in negative control group.At 12 hour post-infection,complete blood count,magnetic resonance imaging(MRI),cerebrospinal fluid(CSF)examination,proliferation capacity,bacterial identification,Evan's Blue(EB)method,qRT-PCR,histological and immunohistochemical analysis and other examinations were carried out.The results showed that the clinical symptoms,MRI findings and hematological indexes were abnormal in TW-XM group compared with the control group,and E.coli were observed in CSF,bacterial load in blood,brain and lung were increased(p<0.01).mRNA expression levels of IL-1?,IL-6,IL-8 and TNF-? were significantly increased in brains infected with TW-XM(p<0.01)and quantification of EB in brains was increased(p<0.01)compared with the control group.The result of histological and immunohistochemical analysis showed that the meninges was discontinuous,edematous and bleeding,and the expressions of tight junctions(TJs)proteins ZO-1,Occludin and claudin-5 were all significantly decreased(p<0.01)in TW-XM group.The results indicated that APEC TW-XM could cause meningitis and destroy BBB in mouse.However,compared to the TW-XM?clpV1 group,there were no significate difference with all indexes in the control group,which indicated that the deletion of clp V1 gene could reduce the ability of TW-XM to destroy the BBB.In summary,the deletion of the clpV1 gene in the TW XM strain weakened the virulence and the ability to induce meningitis in mice.Therefore,the clpV1 gene plays an important role in meningitis caused by TW-XM strain.