| Bacterial meningitis is the most common infectious disease of the central nervous system in newborns.Although the mortality rate has decreased in recent years due to the use of effective antibiotics,the incidence of bacterial meningitis remains relatively unchanged.In addition,nearly half of the survivors are still at high risk of lifelong neurological damage and neurological sequelae.Escherichia coli strain K1(E.coli K1)is the most common gram-negative bacterium causing neonatal meningitis.Its virulence factor FimH is the most important adhesion factor in the initial stage of E.coli K1 invasion of human microvascular endothelial cells(HBMECs).Some researchers have found that FimH also plays an important role in the invasion of HBMEC by E.coli K1;however,the underlying mechanism needs to be clarified.Further,in addition to its role as an adhesion/invasion factor,the question whether FimH regulates the recruitment and migration of PMN during the invasion of HBMECs by E.coli K1 remains to be addressed.CD48 is a FimH receptor and belongs to glycosylphosphatidylinositol(GPI)anchored protein,which is widely distributed in hematopoietic cells and vascular endothelial cells and plays an important role in pathogen recognition.GPI anchor protein exists in the"lipid rafts"in cell membrane,a structure generated by the aggregation of glycolipid,sphingomyelin,cholesterol,and other components.Because lipid rafts contain a large number of signal transduction-related signal molecules,CD48 can trigger a series of signal cascade reactions to realize signal transmission by cross-linking the signal molecules in the lipid raft through costimulatory signals.Our previous studies have shown that E.coli K1(FimH+)can induce the re-distribution of?7 nicotinic acetylcholine receptor(?7 nAChR),a regulatory molecule that mediates bacterial invasion and inflammatory pathways in lipid rafts.In addition,it has been found that E.coli K1(FimH+)can also induce the redistribution of CD48 in the lipid raft of HBMECs,and both CD48 and?7 nAChR aggregate to one of the nine components of the raft.On this basis,we hypothesized that E.coli K1 binds to HBMEC receptor CD48 through FimH to form a FimH/CD48/?7 nAChR/LRs complex.Moreover,using lipid rafts as a signal platform,the complex induces the rearrangement of HBMEC cytoskeleton protein and regulates the expression of cell adhesion molecules via a costimulatory signal,thereby enhancing the invasion of HBMECs by E.coli K1 and promoting the migration of PMN through the blood-brain barrier.A clear understanding of this mechanism provides a new strategy for clinical treatment of neonatal bacterial meningitis infection and has important guiding significance for the development of vaccines.This study will verify the above hypothesis from the following two aspects.Part one:Effect of virulence factor FimH on E.coli K1 and PMN crossing the blood brain barrieObjectives:To construct FimH gene knockout strain-?FimH;to construct the recombinant expression plasmid of pET28a-FimH1-156,and purify the recombinant fusion protein FimH1-156;to investigate the effect of the virulence factor(FimH)of E.coli K1 on the bacterial invasion of HBMECs and PMN migration.Methods:Methods:1.E.coli strain RS218(O18:Kl:H7)was isolated from cerebrospinal fluid of newborn with meningitis.The strain E44 is rifampicin resistance of E.coli strain RS218.Construction of a FimH gene knockout strain-?FimH by Red two-step homologous recombination using E44 as initial strain.The22A33 strain used in this paper was a FimB Tnpho A insertion mutant derived from E44,which causes inactivation of all type I fimbria genes,including FimH.E44 and22A33 strain are donated by professor Sheng-He Haung of Children's Hospital of Los Angeles,University of Southern California,USA.The recombinant expression plasmid of pET28a-FimH1-156was constructed and transformed into Transetta(DE3)competent cells,which was then induced by IPTG and purified to obtain the recombinant fusion protein FimH1-156.Using the HBMEC cell model and classical gentamicin protection method,compare the effects of E44?22A33 and?FimH on the invasion of HBMEC.Transwell cell migration model in vitro was used to detect the effects of E44?22A33 and?FimH,and purified FimH1-156protein on PMN migration through the blood-brain barrier.Results:1.By sequencing,the FimH gene knockout strain E.coli K1 RS218/?FimH,was successfully constructed.The FimH gene knockout strain was named?FimH.2.The expression vector pET28a-FimH1-156was successfully constructed and the puritied FimH1-156fusion protein was obtained.3.The results of bacterial invasion test showed that the invasive ability of 22A33 and?FimH on HBMECs decreased by nearly 50%compared with wild strain E44(p<0.001).4.The results of cell migration of Transwell in vitro suggested that the effect of22A33 and?FimH on PMN crossing the blood brain barrier was significantly weaker than that of wild strain E44(p<0.05).Conclusion:1.FimH gene knockout E.coli K1 RS218 strain?FimH was successfully constructed.2.The prokaryotic expression recombinant plasmid pET28a-FimH1-156was successfully constructed and the purified FimH1-156fusion protein was successfully obtained.3.The promotive effect of the virulence factor FimH of E.coli K1 on the invasion of HBMEC by E.coli K1 was confirmed.4.The promotive effect of the virulence factor FimH of E.coli K1 on PMN migration across the blood-brain barrier was confirmed.Part two:Molecular mechanisms of E.coli K1 and PMN crossing the blood-brain barrier mediated by the virulence factor FimHObjectives:To detect whether the calcium signal is involved in the migration process of bacteria and PMNs regulated by virulence factor FimH of E.coli K1 strain;to determine whether the virulence factor FimH of E.coli K1 regulates the expression of HBMEC endothelial adhesion molecule ICAM-1 and dephosphorylation of actin binding protein cofilin;and to determine whether there is an interaction between CD48 and?7 nAChR on HBMECs.Methods:To detect the effects of E44 and 22A33 on intracellular calcium concentration[Ca2+]iin HBMEC,and the effects of TFP pretreatment with calmodulin inhibitor trifluoperazine on HBMEC invasion and PMN migration induced by E44 and 22A33;Immunofluorescence and western blotting were used to detect the effects of wild E44 and FimH knockout strain?FimH on the expression of ICAM-1 on HBMEC;HBMEC lipid raft was separated by density gradient centrifugation,and the distribution of CD48 and?7 nAChR on lipid raft was observed before and after FimH+E44 treatment.The effects of E44?22A33 and purified FimH protein on the expression and co-localization of CD48 and?7 nAChR on HBMEC.The effects of E44 and FimH on the dephosphorylation of HBMEC actin binding protein cofilin were detected by western blotting.The interaction between exogenous and endogenous CD48 and?7 nAChR was detected by immunoprecipitation.Results:1.E44 treatment significantly increased HBMEC[Ca2+]i,but 22A33treatment had no obvious effect on HBMEC[Ca2+]i.After pretreatment with calmodulin inhibitor TFP,the invasiveness of E44 and 22A33 to HBMEC decreased by 48%and 13%respectively(P<0.05).TFP pretreatment could also decrease the mobility of PMN induced by E44 and 22A33.Compared with 22A33 group,the decrease of E44 induced PMN mobility after TFP pretreatment was more severe than that of 22A33 group(P<0.05).2.The results of immunofluorescence and western blotting showed that compared with?FimH group,E44 treatment could significantly up-regulate the expression of ICAM-1,and the average immunofluorescence density was 19.546 vs 14.689(E44 vs?FimH)(P<0.05).The average gray scale of western blotting was 1.323 vs 0.793(E44 vs?FimH)(P<0.05).3.Separation results of lipid raft:the lipid raft can be divided into 9 components by density gradient centrifugation.In the control group,CD48 was distributed in 5-9components of lipid raft.After treatment with E44(FimH+),CD48 was partially distributed in 1-4 components of lipid raft,especially in the second component.Based on the results of previous studies on the redistribution of?7 nAChR before and after E44(FimH+)treatment,it was suggested that both?7 nAChR and CD48 appeared in the second component of lipid raft after E44 treatment,indicating that?7 nAChR and CD48 may interact with each other.4.Results of immunofluorescence double staining:the expression of CD48 and?7nAChR in E44 treatment group was significantly higher than that in 22A33 treatment group,and the co-localization of CD48 and?7 nAChR in HBMEC was obvious in E44 treatment group.In addition,the expression and co-localization of CD48 and?7nAChR induced by purified FimH protein in HBMEC were consistent with those of E44 treatment group.5.The results of western blotting showed that compared with?FimH treatment group,the cofilin phosphate level of E44 treatment group was significantly lower than that of?FimH treatment group,that is,the dephosphorylation level was increased in E44treatment group.The results suggest that E.coli K1 virulence factor FimH can induce the dephosphorylation of cofilin.6.Exogenous and endogenous Co-IP results.The results of exogenous Co-IP showed that anti-Myc Protein G-Agarose could precipitate Flag-?7 nAChR protein in 293T cells which co-transfected with Myc-CD48 and Flag-?7 nAChR eukaryotic expression vector.Anti-Flag Protein G-Agarose can also precipitate Myc-CD48.However,the control group could not precipitate the target protein,indicating the interaction between exogenous?7 nAChR and CD48 protein.The results of endogenous Co-IP showed that anti-?7 nAChR antibody not only precipitated?7 nAChR protein,but also precipitated CD48 protein at the same time.Similarly,anti-CD48 antibody can precipitate CD48 protein as well as?7 nAChR protein,indicating that endogenous?7 nAChR and CD48 also interact in HBMEC.Conclusion:1.Calcium signal is involved in the promotive role of the virulence factor FimH in the invasion of E.coli K1 and the migration of PMN across the blood-brain barrier.2.E.coli K1 virulence factor FimH can up-regulate the expression of ICAM-1,an important adhesion molecule of HBMECs,and thus promote the migration of PMN across the blood-brain barrier.3.E.coli K1 virulence factor FimH induces the dephosphorylation activation of actin binding protein cofilinin HBMECs.4.In HBMECs,CD48 and?7 nAChR interact with each other and form a FimH/CD48/?7 nAChR complex.Lipid rafts act as a signal platform for costimulatory signal transmission. |
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