Expanding the known repertoire of C-type lectin receptors binding to Toxoplasma gondii oocysts using a modified high-resolution immunofluorescence assay
The environmental stage of the apicomplexan Toxoplasma gondii, the oocyst, is vital to its life cycle but largely understudied. Because oocysts are excreted only by infected felids, their availability for research is limited. We report the adaptation of an agarose- based method to immobilize minute amounts of oocysts to perform immunofluorescence assays (IFA). Agarose-embedding allows high-resolution confocal microscopy imaging of antibodies binding to the oocyst surface as well as unprecedented imaging of intracellular sporocyst structures with a lectin after on-slide permeabilization of the immobilized oocysts. To identify new possible molecules binding to the oocyst surface, we used this method to screen a library of C-type lectin receptor (CLR)-human IgG constant region (hFc) fusion proteins from the group of related CLRs called the Dectin-1 cluster against oocysts. In addition to CLEC7A previously reported to decorate T. gondii oocysts, we present experimental evidence for specific binding of three additional CLRs to the surface of this stage. We discuss how these CLRs, known to be expressed on neutrophils, dendritic cells or macrophages, could be involved in the early immune response by the host, such as oocyst antigen uptake in the intestine. In conclusion, we present a modified IFA technique that allows material-saving immunofluorescence microscopy with T. gondii oocysts in higher resolution than previously published, which allowed us to describe three additional CLRs binding specifically to the oocyst surface.
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