Expression And Functional Analysis Of The Protein YijP Contributes To Escherichia Coli Invasion Of Brain Microvascular Endothlial Cells

IntroductionEscherichia coli is the most common microorganism causing meningitis in the neonatal period. Most cases of ? coli meningitis occur as a result of hematogenous spread, but it is not clear how circulating E. coli cross the blood - brain barrier. Several bacterial genes had been reported, including ompA, ibeA, ibeB , and yijP relating to invasion of BBB.The yijP gene also calledf577 and ibe23 , has a 1,734 - nucle-otide ORF. Sequence analysis indicates that the gene encodes a 66.6 - KDa membrane protein, we expressed and purified the re-combinant 6 + His.tag - yijP protein to analyze its function.Methods and Materials1. Preparation of the fusion 6+His.tag - yijP protein (1)Expressed the fusion 6 +His.tag - yijP protein in M15(PREP4, pQE-yijP).(2)Purified the fusion protein by Ni - NTA Agarose affinity chro-matography.(3)Refblded the fusion protein by dialysis way.2. Function analysis of the fusion .protein(1)MTT assay was used to demonstrate the proliferatous inhibition of HBMEC by different concentration yijP protein.(2)Cell counting method was used to detect the viability inhibition by yijP protein in different time.(3)lncubated the HBMEC with 3g/ml yijP for 16h. Both phase contrast microscopy and fluorescent microscopy were used to observe the morphological changes in the HBMEC.(4)Sub - diploid peak was analyzed with a FACScan flow eytome-ter.(5)The DNA ladder was studied by agarose gel electrophoresis.(6) The active caspase - 3 was detected by Western blotting.(7) Statistical analysis was performed using t - test.Results1. Expressed and purified the recombinant 6 +His.tag - yijP proteinM15(pREP4, pQE - yijP ) was induced by IPTG for 5 hours. The 6+ His.tag - yijP fusion protein was expressed in bacterial cells and was observed as a band of 41 KD on SDS -PAGE gel.2. Function analysis of the fusion protein(1) MTT Assay: cells began to die at yijP 3g/ml medium ( P < 0.05) ,and cells died in a dose -depending way.(2) Cell Counting: We incubated the cells with yijP 3g /ml for 44 h. Cells began to die after incubated 16 h. After 44 h incubation or high dose ( over 6g / ml) of yijP protein, we observed more necrotic or detached cells.(3) Incubated the HBMEC with 3g/ml yijP for 16 h. We observed cell shrinkage, membrane blebbing under phase contrast microscopy , implying the occurrence of apoptosis. And using fluorescentmicroscopy, we observed that nuclear condensation and fragmentation.(4) Sub - diploid peak was founded with a FACScan flow cytome-ter.(5) A stronger DNA laddering pattern consistent with inter - nu-cleosomal DNA cleavage was observed after 16 h incubation with yijP protein.(6) Cleavage of caspase-3, a hallmark of apoptosis was evident. All evidence supported that yijP protein mediated apoptotic phenomenon.Conclusion1. The fusion protein, yijP genes coding produce, contributes to Escherichia coli invasion of brain microvaseular endothlial cells was successfully expressed and purified.2. yijP protein is toxic to HBMEC in vitro, indicting that yijP genes coding produce is likely a toxin.3. yijP protein is a toxin in which it can induce HBMEC apoptosis.