Toxicity of Toxoplasma gondii proteins MIC1 and MIC4 on human T cell lines

Author(s)

Igor Emiliano Lemos de Souza

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2023-04-28

Examining board members

Maria Cristina Roque Antunes Barreira; Marcelo Dias Baruffi; Larissa Dias da Cunha; Maria Aparecida de Souza

• Advisor: Maria Cristina Roque Antunes Barreira

Abstract

Toxoplasma gondii is an obligate intracellular parasite that causes toxoplasmosis, a cosmopolitan zoonosis that has a high incidence worldwide. The disease can range from mild or asymptomatic in immunocompetent individuals to severe disease in immunodeficient patients or fetuses of mothers with acute toxoplasmosis. One of the most interesting characteristics of T. gondii is its ability to possibly infect all nucleated cells of homeothermic hosts. This invasion process is mediated by a series of molecules, including those secreted by specialized organelles - rhoptry, microneme, and dense granule. Such proteins help in the interaction between protozoan and host cells, the creation, and maintenance of the parasitophorous vacuole, and the egress of the parasite from the infected cell. Our group has studied two lectins secreted by micronemes, MIC1, and MIC4, which form a complex with the MIC6 protein. When the MIC1/MIC4/MIC6 complex is exposed on the parasite\'s plasma membrane, the MIC1 and MIC4 proteins recognize glycosylated receptors on the host cell, allowing a stronger interaction between the parasite and the host cell. The scarcity of studies involving the interaction between T. gondii and human lymphocytes led us to evaluate the effect of the recombinant proteins MIC1 and MIC4 on Jurkat human T lymphocyte cells. It was observed that these MICs interact better with Jurkat cells at 37oC, this binding being dependent on their carbohydrate recognition domains. Several lectins have been shown to be cytotoxic to lineage cells. Thus, Jurkat cells were stimulated with different concentrations of MIC1r or MIC4r, resulting in the loss of viability of these cells. Using annexin V and propidium iodide labeling and assessment of chromatin cleavage, rMIC1 and rMIC4 were determined to cause apoptosis in Jurkat cells. It is noteworthy that MIC1r and MIC4r increase caspases 3/7/8 labeling, and the cytotoxic effects triggered by these MICs are reversed after incubation with the pan-caspase inhibitor Emricasan. Stimulation of Jurkat cells with MIC1r or MIC4r also induced a reduction in mitochondrial potential. Taken together, these data suggest that MIC1r and MIC4r stimulate cell death by activating the extrinsic and intrinsic pathways of apoptosis. Additional data showed that the pathways used by these MICs to cause cell death in Jurkat cells depend on MAPK JNK and p38, intracellular signaling molecules involved in the cellular response to stress. In fact, MIC1r and MIC4r induce stress signaling to cells, which can be observed by the release of ROS through the NADPH oxidase complex. Corroborating these data, it was discovered that the reducing agent NAC considerably reduced the labelling of caspases 3/7 and propidium iodide in groups stimulated with MIC1 or MIC4. Taken together, our data indicate that microneme proteins interact with receptors present in human lymphocytic cells in a manner dependent on carbohydrate recognition and cause cell death dependent on ROS release and MAPK activation. (AU)

• FAPESP's process: 19/07411-2 - Evaluation of the role of micronucleus 1 and 4 proteins in the immunopathology of Toxoplasmosis
• Grantee: Igor Emiliano Lemos de Souza
• Support Opportunities: Scholarships in Brazil - Doctorate