Trypanosoma cruzi e o Sistema Complemento: Mecanismos de ativação e o papel do gene CRIT (Complement C2 Receptor Inhibitor Trispanning) na resistência à lise em cepas de classe I e II / Trypanosoma cruzi E O SISTEMA COMPLEMENTO: MECANISMOS DE ATIVAÇÃO E O PAPEL DO GENE CRIT (COMPLEMENT C2 RECEPTOR INHIBITOR TRISPANNING) NA RESISTÊNCIA À LISE EM CEPAS DE CLASSE I E II / Trypanosoma cruzi e o Sistema Complemento: Mecanismos de ativação e o papel do gene CRIT (Complement C2 Receptor Inhibitor Trispanning) na resistência à lise em cepas de classe I e II / Trypanosoma cruzi E O SISTEMA COMPLEMENTO: MECANISMOS DE ATIVAÇÃO E O PAPEL DO GENE CRIT (COMPLEMENT C2 RECEPTOR INHIBITOR TRISPANNING) NA RESISTÊNCIA À LISE EM CEPAS DE CLASSE I E II

Igor dos Santos Cestari

Trypanosoma cruzi, the agent of Chagas disease, infects 18 million people in Latin America. T. cruzi has an heteroxicenous life cycle infecting vertebrates and invertebrate hosts. Two classes of T. cruzi have been proposed based on molecular markers, the class I with a sylvatic life cycle infecting mostly marsupials, while class II parasites have a domestic life cycle infecting placental mammals. T. cruzi needs to evade the host innate immunity to infect cells and establish infection. The most important innate immunity mechanism of vertebrate hosts is the complement system, which is constituted of several proteins activated in cascade that culminates with parasite lysis. Three pathways may activate complement: i) classical pathway, activated by immunoglobulins binding to the pathogen surface, ii) lectin, activated by MBL (Mannan-Binding Lectin) and Ficolins binding to carbohydrate found in pathogen surface; and iii) alternative, by C3b binding to surface molecules of the pathogen. Some surface molecules in T. cruzi have been described for inhibing lysis by complement, such as CRP (Complement Regulatory Protein) and DAF (Decay Accelerating Factor) that bind to C3b and C4b and inhibit the C3 convertase. The prevailing concept about complement activation by T. cruzi is that it activates mainly the alternative pathway, which was shown through deposition of C3b on parasite surface, and the absence of lyses with factor B-depleted serum. However, the experiments were done with long time incubation of parasite and serum, and without considering the lectin pathway. Here, we have characterized the mechanisms of complement pathway activation of epimastigote forms of T. cruzi class I (Colombiana strain) and class II (Y strain); and determined the functional role of CRIT (Complement C2 Receptor Inhibitor Trispanning) gene in the resistance to complement mediated lyses. Assays of Lethal Serum Concentration-50 showed that Colombiana strain was more lysed by complement than Y strain, 50% of lysis was detected with serum concentration between 6,25% and 12,5% in Colombiana, and between 12,5% and 25% in Y strain. Kinetics of complement activation with NHS 25% showed that Y strain and Colombiana strain differ in the velocity of complement activation. At 5 minutes of incubation with NHS25% the parasite survival was 2,6% for Colombiana and 44,6% for Y strain, both strains being totally lysed at 30 minutes. When the NHS 25% was treated with EGTA (that blocks classical and lectin pathways) the lysis by alternative pathway was slow and similar between the strains, and the parasites survived with 30 minutes of incubtation. Activation of the complement in the absence of classical pathway showed that the quick complement activation by T. cruzi is mediated by lectin pathway, with 27% of parasite survival after 5 minutes. Incubation of the NHS 25% with increasing concentrations of mannose inhibited the parasite lysis, with survival of 26% at 1mM and 72% at 40mM, indicating that T. cruzi activate the lectin pathway through the binding of MBL to parasite surface mannoses. We identified the gene CRIT, a complement C2 receptor described in schistosome by Inal, J.M. (2000), in T. cruzi Y, Colombiana, Cl brener and Dm28c strains. Tc-CRIT of Y strain is 97% similar to S. mansoni CRIT. Tc-CRIT expression was detected in the infective forms of T. cruzi Y strain through the monoclonal antibody anti-CRIT-ed1. The over-expression of Tc-CRIT in epimastigote forms of Y strain resulted in 70% increase of resistance to complement-mediated lyses. Assays with NHS25% treated with EGTA showed that CRIT does not inhibit the alternative pathway, acting specifically as a classical and lectin pathway inhibitor. The over-expression of Y Tc-CRIT strain in the Colombiana strain restored the resistance to lysis in 40%. Our results showed that T. cruzi activate quickly the classical and lectin pathway of complement, and the infective forms of the parasite express a C2 receptor capable of inhibiting specifically the complement lyses mediated by classical and lectin pathways.

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