Receptor Heterodimerization and Co-Receptor Engagement in TLR2 Activation Induced by MIC1 and MIC4 from Toxoplasma gondii.

The microneme organelles of Toxoplasma gondii tachyzoites release protein complexes (MICs), including one composed of the transmembrane protein MIC6 plus MIC1 and MIC4. In this complex, carbohydrate recognition domains of MIC1 and MIC4 are exposed and interact with terminal sialic acid and galactose residues, respectively, of host cell glycans. Recently, we demonstrated that MIC1 and MIC4 binding to the N-glycans of Toll-like receptor (TLR) 2 and TLR4 on phagocytes triggers cell activation and pro-inflammatory cytokine production. Herein, we investigated the requirement for TLR2 heterodimerization and co-receptors in MIC-induced responses, as well as the signaling molecules involved. We used MICs to stimulate macrophages and HEK293T cells transfected with TLR2 and TLR1 or TLR6, both with or without the co-receptors CD14 and CD36. Then, the cell responses were analyzed, including nuclear factor-kappa B (NF-κB) activation and cytokine production, which showed that (1) only TLR2, among the studied factors, is crucial for MIC-induced cell activation; (2) TLR2 heterodimerization augments, but is not critical for, activation; (3) CD14 and CD36 enhance the response to MIC stimulus; and (4) MICs activate cells through a transforming growth factor beta-activated kinase 1 (TAK1)-, mammalian p38 mitogen-activated protein kinase (p38)-, and NF-κB-dependent pathway. Remarkably, among the studied factors, the interaction of MIC1 and MIC4 with TLR2 N-glycans is sufficient to induce cell activation, which promotes host protection against T. gondii infection.

MIC4 from Toxoplasma gondii: A Lectin Acting as a Toll-Like Receptor Agonist.

Tachyzoites, which are infective forms of Toxoplasma gondii, use their actinomyosin system to move over surfaces and invade host cells. Central to this process is the regulated release of micronemes organelles contents. The microneme protein 4 (MIC4) has the property to recognize galactosides residues linked to glycoproteins on the host cell surface. This property allows that MIC4 binds to TLR2- and TLR4 N-linked glycans and promote the activation of cell innate immune cells and secretion of inflammatory cytokines, acting on resistance against the parasite. Obtention of MIC4 from T. gondii requires several purification steps, is time-consuming and provides low yield. Therefore, this section details the protocol for prokaryotic expression, production, and purification of recombinant MIC4 (rMIC4) and for experimental assays to confirm its biological activity.

Production and Characterization of MIC1: A Lectin from Toxoplasma gondii.

Some lectins of pathogens interact with host cells through the recognition of specific carbohydrates displayed on the mammals' cell surface. The microneme protein 1 (MIC1) from Toxoplasma gondii has a lectin domain that specifically binds sialic acid residues, often found in the terminal positions of N-glycans of mammalian cells. The necessary studies on the MIC1 biological roles have been limited initially by the laborious purification of the protein from T. gondii tachyzoites and the low yields verified. Then Escherichia coli has been transformed with a construct containing the MIC1 gene, and the obtained recombinant MIC1 (rMIC1) has been purified from the inclusion bodies. Herein, we detail the methodology of heterologous production and purification of rMIC1 and protocols to assay the rMIC1 lectin ability.