Pathogenic Mechanism Of OmpA From Avian Pathogenic Escherichia Coli For Blood-brain Barrier Breakthrough

Escherichia coli meningitis is an acute fatal disease and the survivors have serious sequelae of nerve injury.It is a serious threat to the health of human beings and animals.So far,there is just antibiotic treatment and no effective vaccine prevention measures.At present,Escherichia coli(Neonatal Meningitis Escherichia coli,NMEC)is one of the main pathogens of young children meningitis.In recent years,researchers have found that APEC(Avain Pathogenic Escherichia coli,APEC)TW-XM strain can cause avian meningitis,and is similar to NMEC genome,with same susceptible animal model.Avain meningitis Escherichia coli may cause potential zoonosis.However,we still do not know the pathogenesis of APEC TW-XM.Outer membrane protein A(outer membrane protein A,OmpA)is an important functional protein of the outer membrane in Escherichia coli,which not only maintains structural stability,but also promotes bacterial adhesion and invasion,and affects bacterial pathogenicity.In view of the fact that most of the studies on OmpA focus on NMEC which OmpA plays an important role in the process of NMEC crossing the blood-brain barrier(Blood-Brain Barrier,BBB),howere,the role of OmpA in avian meningitis Escherichia coli remains to be studied.OmpA as ainitialing point in this study was explored its pathogenic mechanism in the process of APEC TW-XM pathogen infection for the host.By comparing the ompA gene sequences of the related meningitis-causing Escherichia coli strains on the NCBI GenBank,the degenerate PCR primers were designed to successfully amplify the ompA gene in APEC TW-XM strains.Then,the isogenic ompA deletion strain of APEC TW-XM strain was constructed by homologous recombination of ?-Red.Through gene cloning technique,the ompA gene was cloned into the expression plasmid vector pBR322 and transformed into the APEC TW-XM?ompA deletion strain,and the complementary strain APEC TW-XM?ompA/pompA was successfully constructed.The results of bacterial growth test showed that the loss of OmpA did not affect the normal growth ability of the bacteria.We explored OmpA effect on meningitis caused by APEC TW-XM crossing the BBB by using mouse brain microvascular endothelial cells(bEnd.3 cells)as the BBB in vitro model mice.The bEnd.3 cells adhesion and invasion test for detecting the adhesion and invasion ability of APEC TW-XM showed that the adhesion and invasion ability of deleted strains to bEnd.3 cells decreased significantly by about 37%and 43%respectively(p<0.01),compared with wild strains and complementary strains.The results of qRT-PCR detection of the infected bEnd.3 cells showed that the deletion of ompA gene significantly affected the transcriptional levels of IL-6,IL-1? and iNOS in bEnd.3 cells,which decreased by 75%,76%and 70%,respectively(p<0.01),there was no significant difference in the expression of inflammatory factors between the complementary group and the wild group(p>0.05).At the same time,qRT-PCR and Western blot were used to detect that ZO-1,Occludin and Claudins-5 tight junction proteins on the bEnd.3 cells infected with the deletion strain were significantly higher than those in the wild strain infection group at RNA(54%,50%,65%,p<0.01)and protein(36%,40%,64%,p<0.01)levels,respectively,while there was no significant difference between the supplement group and the wild group(p>0.05).It is suggested that the loss of OmpA reduces the pathogenic ability of APEC TW-XM and affects the ability to destroy the BBB.In order to further explore the mechanism that how OmpA protein promotes the adhesion and invasion of APEC TW-XM to cells,affects the production of pro-inflammatory factors and the expression of intercellular tight junction proteins,we detected its receptor protein gp96 on the cell surface by qRT-PCR and Western blot.The results showed that both the transcription and protein levels of gp96 inthe bEnd.3 cells infected by the deletion strain decreased 67%(p<0.05)and 50%(p<0.01)significantly compared with the wild group.There was no statistical difference between the complementary group and the wild group(p>0.05).The above experimental results in vitro show that OmpA can promote the adhesion and invasion of APEC TW-XM to the bEnd.3 cells by binding to the receptor gp96,and regulate the expression of pro-inflammatory factors and tight junction proteins in cells,thus affecting the damage of the BBB caused by the bacteria.In order to further explore the pathogenic mechanism of OmpA in APEC TW-XM breaking through BBB,we inoculated APEC TW-XM strain into the mouse abdominal cavity.The mouse meningitis model infected with APEC TW-XM was successfully induced by observing and scoring the clinical neurological symptoms of meningitis.The survival time of the deletion strain infection group(average 20 hours)was significantly longer than that of the wild strain infection group(average 11.4 hours).Simultaneously,the mortality rates of wild type strain,deletion strain and complementary strain infection group were 100%(10/10),40%(4/10)and 90%(9/10),respectively.The BBB permeability of the infected mice was detected by Evans Blue(EB).The results showed that the brains of the mice infected with wild strain and complementary strain were obviously blue.The quantitative results of EB showed that the content of EB in the brain of the mice in the deleted ompA strain infection group was significantly lower than that in the wild type strain infection group by 37%(p<0.01).The results showed that the BBB permeability of the mice infected with the deletion ompA strain was decreased.The results of the bacterial count in brain tissue further showed that compared with the wild strain infection group,the ability of the deletion strain to cross the BBB decreased significantly by 67%(p<0.01).The results of qRT-PCR detection of inflammatory factors in the brain tissue of mice showed that the transcription levels of IL-6,IL-1? and iNOS inflammatory factors in the brain tissue of mice in the deletion strain infection group were decreased significantly 67%,49%and 64%(p<0.01),compared with the wild strain infection group.There was no significant difference between the complementary and the wild strain infection group.The results of the tight junction proteins in the brain of mice by qRT-PCR and Western blot showed that compared with the wild strain infection group,the transcription of ZO-1,Occludin and Claudins-5 tight junction protein in the brain tissue infected with the deletion strain were increased 42%,36%and 41%(p<0.01))and protein levels were increased 34%,61%and 31%(p<0.01).There was no significant difference in the protein expression between the complementary group and the wild group(p>0.05).The expression of OmpA protein receptor gp96 in the brain of mice was detected by qRT-PCR and Western blot.The results showed that the transcription level and protein level of gp96 in the brain tissue of the deletion group were significantly decreased 68%(p<0.05)and 60%(p<0.05)compared with the wild type group,and there was no significant difference in the protein expression of gp96 between the complementary and the wild strain infection group.Consistent with the results of experiments in vitro,it is proved that OmpA affects the pathogenic ability of APEC TW-XM by breaking through the BBB resulting in meningitis.The experimental results described above in vitro and in vivo showd that OmpA can promote the adhesion and invasion of APEC TW-XM to BBB and brain colonization by effecting the expression of the receptor gp96,and regulate the expression of pro-inflammatory factors and tight junction proteins in the BBB to affect the bacterial damage of the BBB and the severe inflammatory response caused by invading the central nervous system.