Mechanism Research Of Staphylococcus Aureus Induced Vascular Leakage By Activating Neutrophils

Community-associated methicillin-resistant Staphylococcus aureus(CA-MRSA)threaten human health worldwide.CA-MRSA-associated infection can be local,such as skin and soft tissue infection,and sometimes leads to serious systemic infection,sepsis,toxic shock syndrome which is caused multiple virulence factors.During severe S.aureus infection,patients always show the clinical symptom with hypotension,edema,sometimes serious to circulatory shock and multi organ failure,all of these indicates that the vascular endothelial barrier is damaged in the process of S.aureus infection.Many efforts has been made to explore the vascular endothelial barrier and S.aureus virulence factors.Previous report four possibilities:Superantigen(enterotoxin,TSST-1),α-Hemolysin,Panton–Valentine leukocidin and Cysteine proteinases.However,the mechanism underlying S.aureus infection-induced vascular leakage remains unclear we need to do more in the other mechanism.Heparin binding protein(HBP)is an early diagnostic marker for severe sepsis or septic shock caused by invasive bacterial infection.Referring to literatures,another gram-positive bacteria –Streptococcus can also induce TSS,M protein has been proved play a critical role by stimulate HBP release through the β-integrin receptor on PMN.In our previous study,Streptococcus suis can cause serious infection both in piglet and human.The HBP level of the STSS patients was higher than healthy people.We found that Sly was the main cause of HBP release.All of thease provide a useful direction for us.Firstly,we collected blood specimens with severe S.aureus infection presented sepsis shock,acute cellulitis,and respiratory failure and HA and CA-MRSA isolated from patients,to make sure the relationship between HBP and Staphylococcus aureus infection.The results showed that the CA-MRSA had more potential in stimulating HBP release.We found that the stimulus was proteinase K-sensitive and was not significantly affected by heat treatment.Combined the seperation and purification of S.aureus supernatant and activity detection with the high resolution SDS-PAGE analysis revealed that the unique component in those elution fractions that stimulated HBP release was approximately 3kDa,we then extracted peptides from the supernatant.The ethanol soluble peptides was more active in HBP-release.ESI-MS identified some activated ingredents from the ethanol soluble peptides,including 3 phenol-soluble modulin(PSM)α and δ-toxin.PSMα has recently been proposed as an important virulence factor of S.aureus.As has been proved before.PSMα1-3 can lead to PMN lysis dramatically at high concentration.However,10% serum can totally block the activation and cytolytic effects on PMNs.That means PSMα1-3 cannot effectively exert their biological functions in vivo.In our research,these five peptides were synthesized,only PSMα4 stimulated HBP release in a dose-dependent manner.Human serum had no blocking effect on PSMα4-induced HBP release.To further confirm,we constructed PSMα deletion mutant strain wild type strain which showed significant difference compared with the wild type stran in HBP release.And the culture supernatant of PSMα4 complement △α strain restored HBP release.Further study showed that PSMα4 also significantly stimulated the release of myeloperoxidase(MPO)and elastase which were all co-localized with HBP in whole blood and increased the expression of cell surface CD63 in PMNs in the presence of serum.In addition,confocal microscopy demonstrated that PSMα4 induced the translocation of CD63 from the cytoplasm to the cell surface.The fact that only PSMα4 caused PMN exocytosis in the presence of serum lipoprotein suggests that this molecule has a unique function during infection in vivo.In the investigatation of the molecular mechanism underlying PSMα4-induced HBP release,synthetic non-formyl PSMα4 failed to induce HBP release.Thus,PSMα4 amino(N)terminus formylation is required for the process,which was different from the chemotaxis and calcium ion fluxes in human neutrophils induced by PSMα4.To further investigated the FPR2 downstream signaling molecules.PI3 K specific inhibitor,wortmannin,reduced HBP release.Cytoskeleton rearrangement is often associated with degranulation and Rac2 has been recognized to regulate cytoskeleton rearrangement,the Rac specific inhibitor,NSC23766,completely abolished PSMα4-induced HBP release.Apart from that,the Ca2+chelator,EGTA,significantly reduced HBP release which was monitored by Fluo-3/AM was measured by a confocal microscope.By using alanine substitution screen,we found that some positions might be critical to HBP release induction,which significantly abolished HBP release.We are trying to further study to analyse the structure.As HBP is a potent inducer of vascular leakage,the culture supernatant of whole blood treated with PSMα4 peptide significantly increased HUVEC monolayer permeability and functional blocking antibody against HBP blocked the increased permeability significantly.PSMα4 also stimulated reactive oxygen species(ROS)production from PMNs,and 10% human serum only partially inhibited this.These all indicated that PSMα4-induced HBP release from PMNs might result in vascular leakage.Though there is no HBP in mice but it have been found to release a functional homolog of HBP.The culture supernatant of PSMα deletion mutant(△α)resulted in significantly less vascular leakage compared with the supernatant of wild type USA300 and complement strain.The synthetic PSMα4 did increase vascular leakage significantly,while the FPR2 antagonist WRW4 significantly reduced PSMα4-induced vascular leakage in mice.To further confirm the function of PMN,we depleted mouse neutrophils by injection of cyclophosphamide.In the neutropenic mice,neither the supernatant of wide type strain nor the supernatant of △α strain induced vascular leakage.Synthetic PSMα4 peptide also failed to induce vascular leakage in neutropenic mice,which means that depletion of mouse neutrophil would abolish the PSMα4-induced release of HBP functional homolog and the consequent vascular leakage.To further confirm that PSMα4-induced vascular leakage was mediated by secreted factors from PMNs,we injected the culture supernatant of mouse PMNs into the neutropenic mice.The supernatant of mouse PMNs that were stimulated with PSMα4 increased vascular leakage significantly,whereas the supernatant of mouse PMNs that were incubated with the control peptide did not.We also detected that PSMα4 4 significantly increased MPO release from mouse PMNs.These results indicate that the secreted factors from PSMα4-stimulated mouse PMNs may induce vascular leakage.In the model of bacteremia,However,compared with lung tissue of mice infected with the △α strain,the lung tissue of mice infected with the wild-type strain showed more severe plasma leakage,which was characterized by erythrocyte and plasma effusion from the capillaries into the lung tissue from the stain with hematoxylin and eosin.Evans blue effusion in the lung tissue was significantly higher in mice infected with wild-type strain than in mice infected with the Δα strain,indicating more severe plasma leakage in mice infected with the wild-type strain.In general,We found that PSMα especially PSMα4 was demonstrated to play a critical role in response to neutrophils in blood during infection because of its resistance to lipoprotein-mediated neutralization,which was confirmed by vascular leakage assay in HUVEC monolayer model and mouse bacteremia model.Our research implys PSMα4 may contribute to the research of S.aureus pathogenesis,especially in sepsis and TSS and need more attention.