Ectopic Expression And Membrane Complex Formation Of RPSA In Brain Microvascular Endothelial Cells Stimulated By SS2 Enolase

Streptococcus suis serotype 2(SS2)is an important zoonotic pathogen that can cause the onset of host meningitis,and it is often cured with irreversible sequelae.Enolase(ENO)is an important virulence factor for the pathogenicity of SS2.Our team has shown that ENO can act on Ribosomal protein SA(RPSA)of porcine microvascular endothelial cells(PBMECs).The interaction of the two proteins promotes cell apoptosis and inhibits the expression of tight junction protein(TJs),thereby destroying host BBB.RPSA is one of ribosomal proteins,mainly involved in protein synthesis in the intracellular.In recent years,studies have found that the abnormal distribution of RPSA on the cell surfaces mediates the invasion of host cells by pathogens.RPSA on the cell surface is mainly located in the "lipid raft",in which the inward sunken "lipid raft" is also called caveolae/rafts.Caveolae/raft-mediated endocytosis is one of the main ways for pathogens to enter the host,and it is also extremely important in the host's resistance to pathogen infection.However,the mechanism by which RPSA transfers to the cell membrane and participates in pathogenicity is still unclear.Therefore,it is necessary to reveal the location of RPSA on the cell surfaces,the interacting molecules,and the role of caveolae/rafts in RPSA-mediated SS2 infection of host cells.The following work was carried out to investigate the role of RPSA in the stimulation of human cerebral microvascular endothelial cells(HCMECs)by ENO:1.Bioinformatics analysis of the function and signal pathway of RPSA in the pathogenesis of SS2 meningitisWe first used bioinformatics methods to fully understand the role of RPSA in SS2 infection.In this study,the differences in cerebrospinal fluid and serum proteomics were comparative analysis from SS2 meningitis and health piglets,and the differentially expressed proteins(DEPs)and the signal pathways involved in the process of SS2 meningitis were systematically summarized.The results found that energy metabolism,cholesterol metabolism and exocytosis were significantly responsive in this pathology.Moreover,the proteins related to RPSA in this pathology were screened by integrating the database of protein interaction and protein co-expression.Finally,some DEPs such as vimentin(VIM)were screened to be closely related to RPSA and these DEPs were significantly enriched in signal pathways such as ribosome,energy metabolism,cell necrosis,and NOD-like receptors.2.The localization of RPSA in HCMECs stimulated by SS2Since proteins often play different functions in different subcellular locations.Western blot(WB)and immunofluorescence test have been used to study the subcellular localization of RPSA and the localization relationship between membrane RPSA and caveolae/raft during SS2 infection of HCMECs.We found that SS2 infection promoted a significant increase in the RPSA expression level.Meanwhile,SS2 infection was associated with an increased number of vacuoles inside of the cells stained with RPSA,named as "vacuolar RPSA" cells.We further found that SS2 infection promoted the intracellular transfer of RPSA to the cell surface of HCMECs.Moreover,our data revealed that aggregated RPSA and some of the CAV1 were co-localized on the cell surface under physiological conditions.SS2 infection promoted the accumulation of caveolin 1(CAV1)and the formation of membrane bulges where RPSA enveloped CAV1 on the cell surface.SS2 infection also caused dynamic changes in the localization of RPSA and CAV1 on the cell surface which could be eliminated by disruption of caveolae/rafts by using methyl-?-cyclodextrin(M?CD)to deplete the cholesterol of the cell membrane.This study for the first clarified the changes in the subcellular localization of RPSA and found that caveolae/rafts were involved in RPSA-mediated SS2 infection of HCMECs.3.RPSA-VIM-CAV1 membrane complex is involved in SS2 infection of HCMECsCombined with the possible interaction molecules of RPSA obtained by bioinformatics analysis,this research studied the composition and interrelationship of RPSA membrane complex in SS2-infected HCMECs by pull-down,mass spectrometry sequencing,co-immunoprecipitation(CO-IP)and multicolor fluorescence immunohistochemistry experiments.The results showed that the molecules of RPSA,CAV1,VIM and Junction Plakoglobin(JUP)were co-located in the plasma membrane and interacted with each other to form complex.ENO promoted the interaction between RPSA,CAV1 and VIM.However,the interaction between RPSA and JUP was weakened.These results refine the localization of RPSA on the membrane surface,indicating that RPSA is located in the desmosome/intermediate filament network.In conclusion,RPSA,CAV1,VIM and JUP proteins were co-localized on the cell surface in the form of complexes.4.The mechanism of RPSA-VIM-CAV1 complex in SS2 infection of HCMECsSince ENO promotes the interaction between RPSA,CAV1 and VIM,this study will focuses on elucidating the relationship between RPSA,VIM and caveolae/rafts in the process of ENO inhibiting the expression of TJs and inducing cell apoptosis by using immunofluorescence,RNA interference,flow cytometry and WB experiments.It was found that RPSA positively regulate VIM expression.ENO increased VIM protein levels by acting on RPSA,thereby inhibiting the tight junction protein ZO-1and promoting pro-apoptotic molecule Smac protein levels.In addition,the results also found that CAV1 on the cell surface distributed around ENO,making ENO localized in lysosomes when caveolae/rafts were normal.However,disruption of caveolae/rafts resulted in a significant accumulation of CAV1 and blocking the process of ENO into the lysosome.Disruption of caveolae/rafts significantly also elevated ENO adhesion to HCMECs,enhanced downstream signals mediated by RPSA-VIM(significant decrease of ZO-1 protein;significant increase of Smac protein),and aggravated cell death.Importantly,these effects were reversed by cholesterol supplementation.Thus,normal caveolae/rafts were essential to attenuate the ENO toxic effect on HCMECs mediated by RPSA-VIM through endocytosis.In summary,we clarified for the first time the relationship between ENO,RPSA,VIM and caveolae/rafts and the mechanism by which the interaction induced apoptosis and destroied BBB during SS2 infection,which provided a theoretical basis for the prevention and treatment of SS2 meningitis.