The Study On The Function Of PSM? On The Traversal Of Staphylococcus Aureus Across The Blood Brain Barrier

Staphylococcus aureus?S.aureus?is an important pathogen widely distributed in nature.The morbidity caused by S.aureus is only less than Escherichia coli.The overuse of antibiotics directly leads to the prevalence of MRSA,making it increasingly difficult to cure the S.aureus infections.For S.auerus,CA-MRSA has been an alarming epidemic,which can cause many serious infections such as toxic shock,sepsis,meningitis etc.The mortality of S.aureus meningitis is over 50%,affecting human healthy severely.Usually,bacterial meningitis is the complex result of the interaction between pathogen and host.The main processes include bacterial survival in blood,colonization,invasion of vascular space,and penetration of blood-brain barrier?BBB?,then leading to the occurrence of meningitis.Among these processes,penetrating BBB is a key to cause meningitis.At present,there are few reports about the mechanism of S.aureus causing meningitis.In 2007,S.auerus phenol-soluble modulins?PSM?were discovered and proved to be key virulence factor of CA-MRSA.PSM?is the most virulent and studied in PSM family,composed of four similar structurally amphiphilic peptides about 22 amino acids and encoded by the same operon.In the research of artificially synthesized PSM?,people found that it played a variety of functions such as biofilm development,cell lysis and pro-inflammatory activity.It is reported that PSM?can increase the permeability of vascular endothelial cells,suggesting that PSM?may be related to the of S.aureus meningitis.In our study,we focused on the mechanism of PSM?affect S.aureus penetrating the BBB by a series of experiments in vitro and in vivo.Besides,we also did some work about structure-function relationship in PSM?using an alanine screen.The first part of our studies demonstrated that PSM?could increase the permeability of BBB and enhance the expression of pro-inflammatory cytokines.The stains used here were USA300-a typical strain of CA-MRSA and its isogenic?PSM?deletion mutant.In order to know the bacteria concentration used in the following experiments and whether the knockout of PSM?genes will affect the growth rate of S.auerus,the growth curves of the two strains were firstly tested and the result showed that the growth curve of USA300 was coincidence with?PSM?.The survival curve of mice showed that survival rate of mice infected with?PSM?was greatly improved compared with USA300.It convinced us that PSM?is an important virulence factor of S.auerus in our experiments.Subsequently,Balb/C mice were infected via tail vein injection,and then the bacterial loads were counted in blood and brain of mice to evalute the ability of USA300 and?PSM?penetrating BBB in mice.Before this,the cardiac perfusion experiment showed that there was no significant difference on bacterial loads in brain between perfused mice and non perfused mice.For the convenience,we did not make heart perfusion in the following experiments of counting bacterial loads.The results in vivo showed that bacterial loads were stable and there was no significant difference in blood between USA300 infected mice and?PSM?infected mice,but the bacterial loads in the brain with a rising trend.At the time of 48h and 72h after infection,bacterial loads in brain of USA300 group is more than that in?PSM?group.Meanwhile,we observed the histopathology change in brain tissue of the infected mice with the two strains,and found that the brain tissue was more severe injured in USA300 group than that of?PSM infected mice.In order to observe the overall damage of the brain tissue more directly,we injected with Evans Blue through tail vein.After heart perfusion,we found that the penetration of Evans Blue in brain of USA300 infected mice is more visible than that of?PSM infected mice at 72h post infection,which was coincidence with the result of quantitative evaluation of extravasated Evans Blue.These experiments showed that PSM?plays an important role in the process of S.aueus penetrating BBB of mice.Next,we adopted the in vitro model of BBB and evaluate the ability of S.aureus penetrating hCMEC/D3 monolayer on the basis of the traversal rate.We added 5?10⁴hCMEC/D3 into a Milicell well.By detecting transendothelial electrical resistance,we found that it need about 5 days for these cells to grow into monolayer.At this time,we added logarithmic growth S.auerus to Milicell well and counted the CFU of the cell medium in the lower chamber.After a period of time,only hundreds of S.auerus can be found penetrated the monolayer cells.In addition,we detected the adhesion rate and invasion rate of USA300 and?PSM?.The result indicated that USA300 and?PSM?had low rate for adherence and invade.These reminded us of the S.aureus could not crossing BBB directly.Then we added the S.auerus supernatant into the upper chamber of Milicell well and utilized Lucifer Yellow as indicator.According to the fluorescence intensity in the cell medium of the lower chamber and transendothelial electrical resistance as evaluation criterion.The results showed that the supernatant of USA300 and?PSM?could both increase the permeability of hCMEC/D3 monolayer,and the cell permeability caused by USA300 supernatant was more obviously than that of?PSM?.At the same time,we utilized the supernatant of USA300 and?PSM?interacting with hCMEC/D3respectively.The cytotoxic effect was evaluated by LDH kit.We can see that the supernatant of two strains can lead to the lysis of hCMEC/D3,and the degree of cell lysis caused by USA300 was more serious.The above results told us that S.aureus supernatant could destroy the integrity of hCMEC/D3 monolayer,and PSM?played an important role in the process.Based on the above experimental results,the expression of claudin5,a protein related to tight junction of cells,was detected by western bolt after the interaction between S.auerus supernatant and hCMEC/D3.The results showed that there was no significant change in the expression of cluadin5 protein compared with control at 1h.Besides,the expression of other proteins such as occludin and JAM also needed to be detected.There were some studies showed that PSM?can elicit inflammatory responses and cause a specific release of cytokines.The mice were infected with logarithmic growth phase of USA300 and?PSM?through tail vein injection,and the release of meningitis related cytokines were detected after a period of time.According to the results,when mice were infected with S.aureus,the expression of TNF-?,IL-8,MIP-1?,IFN-?were significantly increased compared with control.In addition,the cytokines associated with meningitis such as IL-1,IL-6,MCP etc,still needed to be detected.In the second part of the study,we took insight into structure-function relationship in phenol-soluble modulins?4 using an alanine screen.An alanine substitution peptides library of PSM?4 was designed and synthesized.The hemolysis was measured by colorimetric OD₅₄₀?nm?and cytolytic on hCMEC/D3 and PMN were measured by LDH kit.The CD measurements with the selected PSM?4 derivatives to determine whether the structural change correlates with PSM-mediated phenotypes.The 3D structure of PSM?4 was modeled by homology modeling.We found the key amino acid positions of PSM?4 were I3A,V4A,I15A and I17A,which contributed significantly to cytolytic and chemotaxis.The degree of?-helicity showed positive correlation to PSM?4derivatives'cytolytic.In conclusion,we preliminarily proved that S.auerus crossing BBB was related to the destruction of cerebral microvascular endothelial cells and the release of cytokines,and PSM?plays an important role in this process.We also found the key amino acid position contributed to the cytotoxicity and chemotaxis of PSM?4.What we did will provide some help for further related research.