The Role Of Cryptococcus Neoformans Affecting S100A10 For Further Affecting MMP9 In Its Invasion To Host Blood-Brain Barrier

Background Cryptococcus,as a kind of environmental saprophytic microorganism,can cause Cryptococcosis-an opportunistic invasive fungal infection which is well described and easily recognised when it occurs as meningitis in HIV-infected persons and immunocompromised people.Central nervous system infection is the most important clinical manifestation of cryptococcal disease,and the most important cause of high mortality is regarded to Cryptococcal meningitis / meningoencephalitis.As Cryptococcus demonstrates predilection for invasion of the brain,it is reported that more than 90% of cryptococcal disease infesting the central nervous system,but the mechanism by which Cryptococcus crosses the blood–brain barrier(BBB)to cause brain invasion is largely unknown.In order for Cryptococcus to cross the BBB,there must be a way to either cross human brain microvascular endothelial cells,which are the main constitute of the BBB,or go in between tight junctions.In current hypothesis,Cryptococcus might cross the BloodBrain Barrier transcellularly,paracellularly and through infected phagocytes(the Trojan horse mechanism).S100A10 protein,a member of the S100 protein family in the calcium-binding protein family,is present in the cytoplasm and nucleus of many cells,It can bind to annexin A2 to form heterotetramer(S100A10)2-(annexinA2)2.S100A10 is present as a membrane protein on a cell membrane of a variety of cells,and with which plasminogen is activated by tPA(tissue plasminogen activator),uPA(urokinase-type plasminogen activator)that degrade the cell matrix and activate the matrix metalloproteinase(MMP),which is closely related to cell exocytosis and substance transport.MMP9(Matrix metalloproteinase 9),is described as a member of highly homologous secretory or membrane-associated calcium-dependentzinc endopeptidases MMPs,including the active form of MMP9 and 82 kDa in an inactive form of 92 kDa.Most of the cells do not reserve and synthesis MMPs,until signal need for MMPs transmiss to the cells.Subsequently,there are inactive form of zymogen,secretion of extracellular,then activated,and waterfall effect.MMPs are associated with the opening of the Blood-Brain Barrier,and MMP-9 breaks down IV,V,VII,X,XI,elastin,microfibrin,laminin,osteonectin.In this study,we try to illustrate how Cryptococcusneoformans travel across the BloodBrain Barrier through affecting S100A10,and then discuss whether the subsequently affected MMP9 would also play a role in Cryptococca meningitis / meningoencephalitis.In addition,the effect of GM6001 on the protection of Blood-Brain Barrier new method.ObjectiveFirstly,to explore that after Cryptococcus neoformans infected mouse brain microvascular endothelial cells(b.End3),whether the down-regulated expression of S100A10 affects the expression of matrix metalloproteinase 9(MMP9)at m RNA level and intracellular and extracellular protein levels.Secondly,to detect down-expression of MMP9 with silencing MMP9 gene after invasion of Cryptococcus neoformans;further more,we are supposed to explore the integrity of BBB established by LV-musMMP9-shRNA b.End3 and CFU counts of Cryptococcus neoformans in vitro.Thirdly,we explore that whether the matrix metalloproteinase inhibitor GM6001 could do good to the blood brain barrier when invading by Cryptococcus neoformans,and in the meanwhile,lay the experimental fundation to further study for clinical application in vitro.MethodsPart one By transfecting LV-musMMP9-shRNA?LV-musS100A10-shRNA into b.End3 respectively,the very cell lines were screened by puromycin.The obtained differential b.End3 cell lines were observed by fluorescence microscope and varified by RTqPCR.Analysis of mRNAand protein level of the MMP9 expression from Blood-Brain Barrier model constructed by LV-musS100A10-shRNA b.End3 and NC b.End3 in vitro which arerespectively infected by Cryptococcusneoformans.Part two LV-musMMP9-shRNA b.End3 and NC b.End3 were used to construct Blood-Brain Barrier models in vitro basing on transwell.Then both models were infected by Cryptococcusneoformans B3501,and their integrality was detected by TEER as well as colony forming unit at transwell.Part three Blood-Brain Barrier model,infected by Cryptococcusneoformans,was constructed by b.End3 within different concentrations of GM6001.Transwell assays was then performed to access the migration and invasion of Cryptococcus neoformans.Colony forming units were counted to detect its invasiveness as well as its integrality by TEER.ResultsPart one The study had successfully constructed stable transfected cell lines which could effectively interference S100A10,MMP-9 expression respectively.The difference of intracellular and extracellular levels of matrix metalloproteinase 9 from NC b.End3 group and LV-musS100A10-shRNA b.End3 group were demonstrated at mRNA levels.The expression of MMP9 were down-regulated while silencing S100A10 of C.neoformans transversal.Part two The concentration of intracellular and extracellular MMP9 were indeed decreased at mRNA levels when the constructed BBB model of LV-musMMP9-shRNA b.End3 infected by C.neoformans.Furthermore,CFUs of lower chambers in BBB model constructed by LV-musMMP9-shRNA b.End3 were much fewer than that of NC b.End3 group as well as the variation of TEER in two groups.Part three In an in vitro assay of BBB integrity,comparing with control group without GM6001,the group with GM6001 showed less decrease in TEER and fewer CFUs in lower chambers.ConclusionS100A10 is essential and sufficient for Cryptococcus neoformans migration across Blood-Brain Barrier.It can up-regulate MMP9 and consequently decrease the integrity of BBB.MMP9 may be the key or direct molecule which leading to BBB breakdown.Inhibition of MMP9 with GM6001 resulting in a reduction in BBB permeability provides a potential and brand new therapeutic idea for cryptococcal encephalitis / meningitis.