Characterization of TgMIC1 and TgMIC4 immunomodulatory potential: analysis of the pleiotropic effect and repercussions on immunity

• Grant number: 15/14111-4
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: August 01, 2015
• End date: July 31, 2017
• Field of knowledge: Biological Sciences - Biology
• Principal Investigator: Maria Cristina Roque Antunes Barreira
• Grantee: Camila Figueiredo Pinzan
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 13/04088-0 - Lectin from pathogens, AP.TEM
• Associated scholarship(s): 15/24726-6 - The immunomodulatory and biochemical properties of TgMIC1 and TgMIC4: the role of possible interactions with TLRs on NK cells., BE.EP.PD

Abstract

Toxoplasma gondii is an obligate intracellular protozoan able to infect a wide range of host species. The apical region of the parasite is rich in organelles that are involved in both adhesion and invasion process of the host cell. Some proteins released by micronemes (MICs) have adhesives domains which are essential to the parasite virulence; they can form complexes on the parasites surface, such as TgMIC1 / MIC4 / MIC6 and TgMIC3 / MIC8. TgMIC1 was the first microneme protein to be identified. It is a 49kDa soluble protein able to bind on host cells receptors (Fourmaux et al., 1996). TgMIC1 forms the core of the complex by simultaneously interacting with two other microneme proteins TgMIC4, TgMIC6, and host cells (Brencht et al., 2001;. Reiss et al, 2001). TgMIC4 is also a soluble adhesive protein able of interacting with host cells; It is 61kDa protein and contains six conserved apple domains exposed extracellularly (Brencht et al., 2001). Among these Apple domains, the fifth domain is essential for interaction with glycans, particularly those containing terminal galactose (Marchant et al., 2012). Although TgMIC4 weakly interacting with the surface of the host cell, that interaction is added to the strong adhesion provided by TgMIC1 (Saouros et al., 2005). Marchant and colleagues demonstrated that the fifth TgMIC4 Apple domain has lectin activity with affinity for oligosaccharides ending in galactose .We previously showed that TgMIC1 / MIC4 subcomplex binds to lactose and stimulates macrophages to secrete IL-12 (Lawrence et al., 2001). The presence of glycans associated with TLR2 and TRL4 led us to hypothesize that these receptors could be targets for the recognition by the carbohydrate recognition domain (CRD) of micromene proteins. These hypotheses was confirmed by recent studies in which recombinant forms of TgMIC1 and TgMIC4 were used to stimulate HEK293 cells transfected with TLR2 or TLR4; it was found that rTgMIC1 and rTgMIC4 activate TLR2 and TLR4 in the presence or absence of co-receptors and co-accessory molecules. In addition, bone marrow-derived macrophages from WT mice stimulated with rTgMIC1 rTgMIC4 produced high levels of proinflammatory cytokines and this production was TLR2 and TLR4 dependent. In this project we intend to evaluate the molecular mechanisms and signaling pathways involved in innate immune cell activation by microneme proteins (TgMIC1 and TgMIC4) as well studying their involvement in the experimental toxoplasmosis. We believe that these goals will be achieved, because we have essential tools for the study implementation of such as; TgMIC1 and TgMIC4 mutated proteins in their glycosylation sites, knockout parasites for the both microneme proteins (TgMIC1 and TgMIC4), double knockout mice TRL2 - / - / TRL4 - / -, MyD88 - / - and TRIF - / -. We expected that these efforts will result in better understanding of the parasitic adhesion process and the immune response generated by T. gondii infection.