Expanding the known repertoire of C-type lectin receptors binding to Toxoplasma 2 gondii oocysts using a modified high-resolution immunofluorescence assay

Authors: FABIAN, BENEDIKT - Robert Koch Institute; LEPENIES, BERND - Hannover School Of Veterinary Medicine; SCHARES, GEREON - National Reference Center For Toxoplasmosis; Dubey, Jitender; SPANO, FURIO - Istituto Superiore Di Sanita; SEEBER, FRANK - Robert Koch Institute

Submitted to: mSphere
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/8/2021
Publication Date: 3/31/2021
Citation: Fabian, B.T., Lepenies, B., Schares, G., Dubey, J.P., Spano, F., Seeber, F. 2021. Expanding the known repertoire of C-type lectin receptors binding to Toxoplasma 2 gondii oocysts using a modified high-resolution immunofluorescence assay. mSphere. 6(2):e01341-20. https://doi.org/10.1128/mSphere.01341-20.
DOI: https://doi.org/10.1128/mSphere.01341-20

Interpretive Summary: Food safety due to microbial contamination of foods remains a public health concern worldwide. Among the parasitic contaminants, fecal contamination of foods due to protozoans such as Cyclospora, Cryptosporidium, Giardia, and Toxoplasma are of prime concern. Knowledge of oocyst biology of T.gondii or Cyclospora is limited, because of the limited availability of oocysts for experimentation. Here, the authors describe a method that allows to process minute amounts of oocysts for immunofluorescence microscopy without compromising their structural properties, compared to other published methods. These tools will allow further studies into oocyst wall composition and could also provoke experiments regarding immunological recognition of oocysts. These results will be of interest to diagnosticians, parasitologists, and public health workers. This research was completed before the closure of Toxoplasma research at ARS in 2018.

Technical Abstract: 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.