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Interaction of microneme proteins of Toxoplasma gondii with N-linked glycans of TLR4: effects on innate immunity

Grant number: 14/13324-1
Support type:Scholarships in Brazil - Doctorate
Effective date (Start): January 01, 2015
Effective date (End): September 30, 2018
Field of knowledge:Biological Sciences - Biochemistry - Molecular Biology
Cooperation agreement: Coordination of Improvement of Higher Education Personnel (CAPES)
Principal Investigator:Maria Cristina Roque Antunes Barreira
Grantee:Flávia Costa Mendonça Natividade
Home Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

Toxoplasmosis is caused by the protozoan Toxoplasma gondii, intracellular parasite able to infect any nucleated cell of warm-blooded animals. In the apical region of this parasite there are organelles that release several molecules involved in adhesion and host cell invasion process. Among these secretory products include those released by micronemes. Micronemes proteins associate with each other on the surface of tachyzoite and forms complexes as TgMIC1/MIC4/MIC6. The TgMIC1 and TgMIC4 have adhesives domains that are essential for the virulence of the parasite. In previous studies, our group isolated subcomplex TgMIC1/MIC4 by affinity to immobilized lactose and demonstrated its property of stimulating murine macrophages to secrete IL-12. The availability of recombinant forms of TgMIC1 and TgMIC4 has shown that both proteins promote the release of pro-inflammatory cytokines and modulate immunity against the parasite. Furthermore, it was found that the TgMIC1 and TgMIC4 immunomodulatory effect derives from the interaction established by their carbohydrate recognition domain with Toll-like receptors. It was also found that macrophages from mice TLR2-/- and TLR4-/- produce lower levels of TNF-±, IL-6 and IL-12(p40) and IL-1² in response to stimulation with rTgMIC1 and rTgMIC4. Finally, the use of mutants for the glycosylation sites of the receptor showed that rTgMIC1 rTgMIC4 recognize N-glycans of TLR2, especially those occupying the third and fourth position in the receptor.The aim of this project is to study the interaction of micronemes proteins with N-glycans of TLR4. Firstly, we will generate mutants for to the N-glycosylation sites of TLR4. They will be transfected into HEK293T cells, to identify the glycans involved in the TLR4 activation triggered by TgMIC1 and/or TgMIC4. Activation will be detected in HEK293T cells by measuring IL-8 levels in the cells supernatant. The relevant mutants will also be transduced in macrophages from TLR4 and TLR2 double knock-out mice; the transduced cells will be stimulated with rTgMIC1 or rTgMIC4, and assayed for the production of pro-inflammatory cytokines. The possible direct physical interaction established between the micronemes proteins and N-glycans of Toll-like receptor, whose occurrence will be indicated by the mentioned assays, will be evaluated by electrochemical impedance spectroscopy and structural facts responsible for the interaction will be assessed by analysis of crystals of complexes of micronemes proteins and TLR4, as well as by molecular modeling. (AU)

Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
MENDONCA-NATIVIDADE, FLAVIA COSTA; LOPES, CARLA DUQUE; RICCI-AZEVEDO, RAFAEL; SARDINHA-SILVA, ALINE; PINZAN, CAMILA FIGUEIREDO; PAIVA ALEGRE-MALLER, ANA CLAUDIA; NOHARA, LILIAN L.; CARNEIRO, ALAN B.; PANUNTO-CASTELO, ADEMILSON; ALMEIDA, IGOR C.; ROQUE-BARREIRA, MARIA CRISTINA. Receptor Heterodimerization and Co-Receptor Engagement in TLR2 Activation Induced by MIC1 and MIC4 from Toxoplasma gondii. INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, v. 20, n. 20 OCT 2 2019. Web of Science Citations: 0.
SARDINHA-SILVA, ALINE; MENDONCA-NATIVIDADE, FLAVIA C.; PINZAN, CAMILA F.; LOPES, CARLA D.; COSTA, DIEGO L.; JACOT, DAMIEN; FERNANDES, FABRICIO F.; ZORZETTO-FERNANDES, ANDRE L. V.; GAY, NICHOLAS J.; SHER, ALAN; JANKOVIC, DRAGANA; SOLDATI-FAVRE, DOMINIQUE; GRIGG, MICHAEL E.; ROQUE-BARREIRA, MARIA CRISTINA. The lectin-specific activity of Toxoplasma gondii microneme proteins 1 and 4 binds Toll-like receptor 2 and 4 N-glycans to regulate innate immune priming. PLOS PATHOGENS, v. 15, n. 6 JUN 2019. Web of Science Citations: 2.

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