Liubchenko G. Mechanisms of activation of lymphocytes with extracellular adherence protein of Staphylococcus aureus

In this work surface adhesive protein р70 (ЕАР) from Staphylococus aureus Wood-46 has been prepared and purified using a combination of biochemical, microbiological and immunological methods. For the first time molecular mechanisms of the immune response initiation and activation of lymphocyte subpopulations by EАР were examined and its immunobiological activity and immunomodulating influences were established. A protective role of EАР was demonstrated. It was determined that Eap stimulates the formation of Ca2+ channel clusters on the B lymphocytes surface in the place of contact with the microbeads coated with Eap (a model system that imitates antigen particles). For the first time it was established that EАР stimulates phosphorylation/activation of major intracellular signaling molecules such as CD19, Lyn, Erk, FcgRIIb, Zap70, pSHIP1, pSHP2. It was found, that Еap induces Ca2+-responses in intact (naive) B lymphocytes. It was shown, that p70 stimulates localized concentration of ICAM1 in a contact areas with microbeads, covered with EАР. Surface adhesive protein stimulated different levels of cell activation, as shown by the mobilization of the intracellular Ca2+ in the cells. The degree of B lymphocytes activation after the stimulation with p70 directly correlated with B cell receptor expression (BCR). Stimulation of intact murine (C57BL/6) B lymphocytes with the Eap and protein А combination provoked co-clustering of B cell receptor (sIgM) and ICAM-1 in structures that are similar to immune synapse. This effect resembled staphylococcal protein A's ability to bind sIgM and to act as B lymphocyte superantigen. Such ability of EАР to interfere with B cell receptor signaling, irrespective of antigen specificity, may suggest that Eap may assist antigen mimicry and early internalization of the sIgM signaling complexes in B cells, and, thus, helps Staphylococcus aureus to avoid B cell immune response. Eap was found to stimulate phagocytic activity of the cells in vitro as well as in vivo. Animal immunization with EАР has been accompanied with an increase in metabolic and functional activity of phygocytic system and NK cells. Significant activation of the oxygen-dependent bactericidal neutrophil activity, stimulation of IFN, TNF and interleukins production and animal protection from experimental staphylococcal infection by EАР has been observed.