To adhere to host cells Toxoplasma gondii uses microneme proteins (MICs), making their adhesive domains essential for the parasite virulence. MICs can be found associated with each other on the tachyzoite surface, like the complex TgMIC1/MIC4/MIC6. We have previously demonstrated that pure TgMIC1/MIC4 subcomplex, obtained through adsortion to immobilized lactose, can stimulate macrophages to secrete IL-12, in a sugar-recognition dependent manner. Because TLR2 and TLR4 are N-glycosylated, we hypothesized that they could be targeted by the carbohydrate recognition domains (CRD) of microneme proteins to trigger the cytokine production. This hypothesis was confirmed by recent studies in which recombinant forms of TgMIC1 and TgMIC4 have activated TLR2- or TLR4-transfected HEK293 cells, in the presence or absence of co-receptors and accessory molecules. Alternatively, murine bone marrow-derived macrophages (BMDMs) stimulated with rTgMIC1 and rTgMIC4 were showed to produce high levels of proinflammatory cytokines, a response that was blocked in TLR4-/- macrophages and significantly inhibited in TLR2-/- macrophages. Based on these previous data, we defined the objective of this study: to characterize the interaction of TgMIC1 and TgMIC4 with TLR2 and TLR4 N-glycans in terms of identification of the triggered signaling pathways. For this, BMDMs from WT, DKO (TLR2-/-/TLR4-/-), MyD88-/-, TRIF-/- and TRAF6-/- mice will be stimulated with TgMIC1/4, rTgMIC1 and rTgMIC4 and studied concerning the release of proinflammatory cytokines. The MICs interaction with TLR2 and TLR4 will be also analysed regarding the adaptor molecules involvement in the intracellular signaling. In addition, we will certify whether this interaction is due to the recognition of N-glycans linked to TLR2 and TLR4, by using western blot and competition assay with specific sugar. Finally, we will evaluate the expression of co-receptors known to be associated with TLR2 and TLR4 in order to elucidate their role in the activation and/or regulation of signaling pathways triggered by the interaction of microneme proteins with TLR2 and TLR4.
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