The Mechanism Study Of Staphylococcal Efb Protein Causing Infection Through Inhibiting Platelet Aggregation

The Gram-positive bacterium Staphylococcus aureus (S.aureus) is an important pathogen, which can cause varieties of infections ranging from superficial skin lesions to endocarditis, septic arthritis and even septicemia causing death in the past 15 years.Platelets are a group of cells with important function and involved in hemostasis, wound healing, and in?ammation. The activated platelets can phagocytize microorganisms and secret several kinds of antimicrobial peptides. Moreover, varieties of cytokines and chemotatic factors secreted by activated platelets could recruit other components of innate defense system such as macrophage and neutraphiles to resist infection indirectly. The activation and aggregation of platelets is closely relative with IE( infective endocarditis).There are two goups of molecules that interact with platelets with different mechanism expressed by Staphylococcus aurues. The molecules in group 1 can promote platelets aggregation; the molecule in group 2 can function as inhibiting platelets aggregation. The reason why Staphylococcus aureus expresses two kinds of molecules with opposite function is not clear. At the same time, the relationship between platelet and Staphylococcal infection has not been discovered..Efb(extracellular fibrinogen-binding protein) is the only protein which belongs to group 2. Efb is a bifunctional protein, with full-length of 131 amino acids. The two repeat region located at N terminal of Efb could bind to fibrinogenαchain, and thus inhibits platelet aggregation; the C terminal of Efb inhibits complement activation through binding to C3(d). The pathogenicity of efb mutant strain decreases on animal model of wound healing.Efb was chosen as the target in our study. efb mutant strain was constructed. Functional recombinant Efb and its specific antibodies were prepared. The aim of our study is to investigate the relationship between the inhibiting platelets aggregation function of Efb and Staphylococcal infection. Our work is divided into two parts:1,The construction of efb mutant strain and the biological function study of recombinant Efb. efb mutant strain was constructed by gene homologous recombination in S.aureus 8325-4. The resultsof animal model showed that the virulence and lethality of efb mutant decreased remarkably. Recombinatant Efb and its N,C terminal protein was dividually expressed and purified. The complement activation assay demonstrated that Efb and EfbC could inhibit the classical complement activation pathway; the inhibition ELISA assay showed that Efb and EfbC could inhibit the alternative complement activation pathway. Furthermore, Efband EfbN could inhibit platelets aggregation by interacting with fibrinogen, suggesting that Efb functions through binding to fibrinogen. The sepecific polyclonal antibodies against to Efb could block its complement inhibition function.2,The study of the mechanism by which Efb initiates the Staphylococcal infection through inhibiting platelets aggregation. The whole blood survival assay in vitro showed that the exsitence of platelets and status of platelets aggregation is closely relative with the survival of S.aureus. The expression profile result showed that Efb was expressed at the lag phase of S.aureus growth. On the acute peritonitis animal model, the infection caused by S.aureus were aggravated significantly by pre-treated with Efb(N) or inhibiting platelets aggregation medicine. Compared with control group, the number of platelets and ratio of neutrphile both performed remarkably difference in the treatment group through monitoring the hemogram of experimental animals. Furthermore, the level of TNFαin blood increased significantly in the treatment group. The number of survival bacteria from treatment group abdominal cavity was notably larger than that from the control group. The RT-PCR results suggested that the main virulence genes'expression level of agr from lavage increased significantly compared to control group. All these results indicated that EfbN and anti-platelets aggregation medicines could increase the survival of S.aureus in vivo and then aggravate infection caused by S.aureus.by inhibiting platelets aggregation.In summary, our study revealed that inhibiting platelets aggregation could aggravate infection caused by S.aureus in the early stage of infection. The conclusion of our work can not only improve the study of anti-infection caused by S.arueus, but also indicate that the potential risk of anti-platelets aggregation medicines.