Research On Construction Of Mutant E.Coli K1 With Highpathogenicity Island Gene IbeT Delection And The Invasive Capacity To HBMEC

PurposeNeonatal meningitis is a very common disease in certral neural system(CNS). E.coli K1 is one of the most common gram-negative organisms causing neonatal bacterial meningitis.The mortality and morbidity associated with E.coli meningitis remain significant,with case fatality rates above 80%of infected neonates.Most cases of E.coli meningitis develop as a result of hematogenous spread,but the mechanisms by which E.coli traverses across the blood-brain barrier are not clearly understood.The blood-brain barrier(BBB) consists mainly of specialized capillary endothelium,characterized by the presence of tight junction(TJ) which can protect the brain from pathogen.We have developed an in vitro human brain microvascular endothelialcell(HBMEC) culture with characteristics of the blood-brain barrier,which is a natural target for meningitis.Binding and invasion of E.coli have been verified in the transfected and original HEMEC model,and the results are in agreement with each other.Using comparative genetic methods,we found the E.coli K1 's highpathogenicity island genes which composed of 23 10Kb DNA.Increasing evidence suggested that ibeT,one of E.coli Kl's highpathogenicity island genes,was associated with E.coli invasion to HBMEC.In this study,we investigated the role of ibeT in E.coli invasion to HBMEC by using E44,a mutant strain of E.coli K1,to construct and characterize a mutant E44 strain with in-frame deletion of highpathogenicity island gene ibeT.After then,we detected the alteration of E.coli invasion to HBMEC.It is a significant meaning for new therapeutic approaches. Methods1.Gene recombination.Firstly,we search for the sequence of ibeT in Genedata, and design the primer of ibeT by using the Primer 5 software.Secondly,we obtained the ibeT DNA sequence by PCR.2.Using up-strain(A) and down-strain(B) of highpathogenicity island gene ibeT as the two homologous sequences,we subcloned the recombined sequence A and B into the suicide vector pCVD442.3.By homologous recombination and conjunction mobilization,we constructed the E44 mutant with deletion of the highpathogenicity island gene ibeT and selected the positive clone by using ampicillin and rifampicin.4 Concultrue HBMEC and the two kind of E.coli with different concentration,and counter the number of wild and the mutant strain of the E.coli invading into the HBMEC.5.Using immunofluorescence staining to observe the alteration of the E.coli's invasion capacity.Results1.By PCR amplification and sequence analysis we constructed a mutant E44 with the highpathogenicity island gene ibeT deleted2.Both of the two kind of the E.coli can invaded into HBMEC.the number of wild type of the E.coli is more than the mutant type obviously(p<0.05).Conclusions1.Construct a mutant E44 with highpathogenicity island gene ibeT deletion successfully。2.The existence of highpathogenicity island gene ibeT enhance the invasion capacity to the HBMEC.