Chagas Disease (CD) is a highly influential parasitic infection in 21 endemic countries of Latin America. Its impact has transcended the borders of this region and has reached regions such as North America, Japan, Europe and Australia. The large spread of CD is largely due to the migration of infected people from endemic areas to non-endemic areas . The number of infected individuals worldwide reaches 6 million, of whom around 7000 die during the course of the year. These statistics indicate that CD is the parasitic infection that causes the greatest number of deaths in Latin America. It is also a major contributor to the global burden of cardiovascular disease, as this parasitosis is the primary source of infectious cardiomyopathy around the planet . In addition to all these problems, most of the people affected by CD are of low socioeconomic resources and live in remote areas with difficult access. In addition, it must also be considered that less than 1% of chagasic individuals can be diagnosed and have chemotherapeutic treatment.
The parasite-host relationships are determining factors in the appearance of CD, as well as in the severity level of chronic symptomatic forms. Complement is the first defense barrier to prevent Trypanosoma cruzi infection from establishing, the functions of this protein complex are participation in the detection of these microorganisms and opsonization, as well as, they are involved in their elimination in order to avoid the entrance to the host cells. Activation of complement during the acute phase of CD allows controlling parasitaemia, while its proinflammatory effects favor the chronic phase. For all these reasons, this first line of defense for mammals constitutes a very strong and difficult system to violate, however, T. cruzi is a very ingenious parasite and always provided with counter-attack tactics, since it has developed different evasion mechanisms, to avoid the action of the complement cascade, which enables it to overcome the barriers imposed by the immune system and continue with the progress of CD.
The human invasion process by T. cruzi is affected by the activation of the complement system during the acute phase where it plays a key role in the control of parasitaemia, while in the chronic phase it participates in the appearance of symptomatic manifestations because it induces the production of proinflammatory molecules . The complement system is made up of around 40 circulating proteins present in the plasma/blood, which work together with regulators or receptors of the cell surface, whose activation can be carried out in three ways: Classical Pathway (CP), Alternative Pathway (AP) and Lectin Pathway (LP). These routes have some points in common: 1 the degradation of C3 in C3a and C3b and 2 the convergence in a common point, the assembly C3 convertase complex.
CP begins mainly due to C1 binding with immune complexes (antigen/antibody); as well as, it depends to a lesser degree on PAMPs (pathogen-associated molecular patterns) such as porins and lipopolysaccharides from Gram-negative bacteria; pentraxins (pentraxin 3 and C-reactive protein) are some examples . The C1 complex is structured by a C1q molecule plus two molecules of C1r and C1s, which results in a molecule of the C1q C1r2 C1s2 type. After C1q formation, its activation is mediated by its interaction with the CH3 Fc domain of IgM or CH2 Fc domain of IgG, which produces modifications in the conformation of C1q. Then C1r and C1s are activated, in addition to the serine proteases cut C4 and C2 that give rise to C3 and C5 convertases from CP .
LP is activated in the presence of mannose containing polysaccharides present on the surface of the parasite, which interact with Mannan Binding Lectin (MBL), as well as other receptors such as L-ficolin, H-ficolin or M-ficolin. This pathway contributes around 70% of parasite complement-mediated lysis during the T. cruzi invasion process [6,7].
AP is the simplest route, because it only needs to be activated due to spontaneous hydrolysis of the thiol-ester bond of C3 a-chain that produces C3 (H2O), which interacts with plasma protein FB and produces C3 (H2O) B. FD degrades FB into Ba and Bb, this last fragment binds to C3 (H2O) and forms the first C3 convertase (C3 (H2O) Bb), which has serine protease activity by acting on C3 and converting it to C3a and C3b. This latter fragment binds to FB, which allows FD to break it down and forms the second C3 convert, C3b Bb that binds to C3 to produce C3b Bb C3b, a complex structure possessing C5 convert activity [8, 9].
The complement activation system is the first defense barrier against pathogenic microorganisms, whose mechanism of action depends on a proteolytic cascade that triggers a very powerful lytic effect. For these reasons, this chain of events is an ideal target for microorganisms to implement their immune evasion strategies that allow them to survive and advance the infection process until reaching the chronic phase of the disease . During the first steps of infection, T. cruzi is attacked by the AP and LP pathways of the complement, since the CP pathway is activated at later times when the production of antibodies begins.
T. cruzi is a very robust parasite, with multiple tools to escape the action of the mammalian host complement system, by inhibiting this proteolytic cascade, which allows it to survive its lytic mechanisms, as well as opsonization and chemotaxis. Epimastigotes, the non-infective form of the parasite, that live in triatomine vectors, are very susceptible to complement-mediated lysis while trypomastigotes, the infective form, present in host bloodstream, are resistant. The susceptibility of T. cruzi to complement is stage (amastigote, epimastigote, trypomastigote) and dependent strain. The morphological stages of this microorganism have different sensitivities to complementmediated lysis. In the case of epimastigotes, their presence has been determined to allow the activation of the 3 complement pathways and they are sensitive to this mechanism . In contrast, metacyclic trypomastigotes are resistant to the action of complement .
T. cruzi is a very successful microorganism because it efficiently invades and infects mammals, which allows it to multiply and perpetuate itself to guarantee its existence. Metacyclic trypomastigotes (infective forms of the host mammal) can escape complementmediated destruction, because these parasites have the surprising ability to express on their surface a variety of Complement Regulatory Proteins (CRPs), which act at different levels in the plugin path, as described in detail in the following sections:
T-DAF is a glycoprotein with a MW of 87–93 kDa , which remains attached to the plasma membrane by means of a GPI anchor . It has been determined that the blood, metacyclic and cultured trypomastigotes  express T-DAF, which manages to establish interactions with C3b and C4b, which induces the inhibition of the assembly and favors the accelerated decay of C3 and C5 convertase from AP and CP of the complement cascade [14,15]. These events inhibit parasitic lysis and allow their escape from the complement cascade of different mammalian species. From a cDNA expression library corresponding to T-DAF, it is possible to obtain a clone using monoclonal anti-DAF antibodies to carry out the immunosearch. It is possible to determine the nucleotide sequence that codes for a fragment of the protein and the high homology of T-DAF with human DAF can be evidenced [16,17].
TcCRT is a Ca++ binding protein present in T. cruzi trypomastigotes, where it is located mainly in the Endoplasmic Reticulum (ER) and then transported to the surface of the parasite, mainly through the region where the flagellum arises. According to histochemical evaluations, it has other cellular locations, such as in the Golgi apparatus, flagellar pocket, reservosomes, kinetoplast, nucleus and cytosol , but the reasons for its ubiquity are unknown . TcCRT has functions on several steps involved in the parasite/ host relationship, such as the inhibition of the formation of C3 and C5 convertase of CP due to its union by S-domain with the C1q collagen stems, which prevents the activation of C1s , as well as the degradation of C4 dependent on C1s . However, C1s inactivation has been observed to occur only when she is forming the C1 complex [C1q, (C1r, C1s) 2] . As well as, it interacts with ficolines  and MBL  and therefore, inhibits the initial steps of complement LP. The deletion of the TcCRT gene makes T. cruzi sensitive to CoML, on the other hand, when parasites are modified to induce TcCRT over expression, their resistance to complement by CP and LP increases considerably . Likewise, TcCRT has an important role as a virulence factor, whose infective potential is enhanced by binding to C1q [24,25].
TcCRP has been initially characterized by Norris et al (1989) , it is a 160 kDa glycoprotein (gp160) expressed by trypomastigotes, absent in epimastigotes . It constitutes an integral protein of the plasma membrane of metacyclic and tissue-culture trypomastigotes of T. cruzi, whose insertion occurs through a GPI anchor. According to studies of sub cellular location, it has been detected in the flagellar pocket and the flagellum .
Transfection assays of epimastigotes with TcCRP indicate that these forms become more resistant to CoML, which strongly indicates that this protein is the protagonist in complement evasion by T. cruzi [29,30]. TcCRP is an inactive trans-sialidase that forms covalent bonds with C3b and C4b, which prevents the formation of functional C3 convertase from AP and CP, thus blocking the lysis of parasites mediated by the complement cascade [27,31].
TcCRIT is a protein of 32 kDa with an N-terminal extracellular domain of 27 aa called ed1, with much similarity to the C4β chain that participates in the interaction with C2 [32,33]. Trypomastigotes express it on its surface , where the binding of the ed1 domain with C2 occurs and prevents its degradation caused by MASP2 and C1s, which blocks the formation of C3 convertase [33,6,32] and ultimately inhibits the activation of LP and CP. TcCRIT is a C2 receptor present on T. cruzi (competes for C4 for binding to C2) and inhibits C2 cleavage by C1s, that is, it participates in the modulation of CP [34,35,19]. Cestari Idos et al (2009) , found that transgenic parasites that overexpress TcCRIT are highly resistant to Complement- Mediated Lysis (CoML). MBL, Ficolins and the MASP-2 enzyme have been shown to bind to the surface of metacyclic trypomastigotes and LP activates, but the mechanism of these processes is still unknown . But this morphological stage is resistant to CoML because it inhibits LP, due to the expression of TcCRIT . This protein have homology with ShCRIT of Schistosoma haematobium and hCRIT of humans.
This protein has a MW of 58 kDa in its unreduced form and 68 kDa in a reduced condition . Gp58/68 and an 80-85 kDa protein form a collagen/fibronectin receptor in trypomastigotes , which plays a fundamental role in the binding of trypomastigotes to mammalian cells [38,39,36]. This glycoprotein has complement regulatory functions, because it prevents the formation of C3 convertase from AP on the surface of T. cruzi and in the fluid medium. Its mechanism of action depends on blocking the binding of complement Factor B (FB) with C3b.
A detailed review of the mechanisms of complement evasion by T. cruzi shows its high efficiency and great ability to escape the action of this protein cascade. This microorganism produces a diversity of complement regulatory proteins, this function is to exert its action at various levels of the complement cascade and thus ensure the inhibition of the different routes of this prodigious mechanism belonging to the innate immune system.
- Jackson Y, Pinto A and Pett S. Chagas disease in Australia and New Zealand: risks and needs for public health interventions. Trop Med Int Health 2014; 19: 212-218.
- Cucunuba ZM, Okuwoga O, Basanez MG and Nouvellet P. Increased mortality Attibuted to Chagas Disease: A Systematic Review and Meta- Analysis. Parasit Vectors. 2016; 9: 42.
- Luz PR, Miyazaki MI, Neto NC, Padeski MC, Barros ACM and Boldt ABW, et al. Genetically Determined MBL Deficiency is Associated with Protection against Chronic Cardiomyopathy in Chagas Disease. PLoS Negl Trop 2016; 10: e0004257.
- Kishore U, Ghai R, Greenhough TJ, Shrive AK, Bonifati DM and Gadjeva MG, et al. Structural and functional anatomy of the Globular Domain of Complement Protein C1q. Immunol. Lett. 2004; 95: 113-128.
- Ricklin D, Hajishengallis G, Yang K and Lambris J. Complement: a key system for immune surveillance and homeostasis. Nat. Immunol. 2020; 11: 785–797.
- Cestari I, Evans-Osses I, Schlapbach L, de Messias-Reason I and Ramirez M. Mechanisms of Complement Lectin Pathway Activation and Resistance by Trypanosomatid Parasites. Mol Immunol. 2012; 53: 328–34.
- Cestari I, Ramirez M. Inefficient Complement System Clearance of Trypanosoma cruzi Metacyclic Trypomastigotes Enables Resistant Strains to Invade Eukaryotic Cells. PLoS One. 2010; 5: e9721.
- Lambris JD, Ricklin D and Geisbrecht BV. Complement Evasion by Human Pathogens. Nature Reviews Microbiology. 2008; 6: 132-142.
- Fromell K. Adler A, Aman A, Maniel VA, Huang S and Duhrop C. et al. Assessment of the role of C3 (H2O) in the Alternative Pathway. Front Immunol. 2020.
- Tapia V, Galdames P and Ramírez G. Mecanismos de Evasion Del Sistema del Complemento Utilizados Por Trypanosoma cruzi. Avances en Ciencias Veterinarias. 2012; 27: 10-19.
- Osorio L, Rios I, Gutiérrez B and González J. Virulence factors of Trypanosoma cruzi: who is who? Microbes Infect. 2012: 14 1390-1402.
- Davitz MA, Low MG and Nussenzweig V. Release of Decay-Accelerating Factor (DAF) from the Cell Membrane by Phosphatidylinositol Specific C (PIPLC). Selective Modification of A Complement Regulatory Protein. J Exp Med. 1986; 163: 1150-1161.
- Tambourgi DV, Cavinato RA, De Abreu CM, Peres BA and Kipnis TL. Detection of Trypanosoma-Decay Accelerating Factor Antibodies in Mice and Humans Infected with Trypanosoma cruzi. Am J Trop Med Hyg. 1995; 52: 516-520.
- Kinoshita T, Medof ME and Nussenzweig V. Endogenous Association of Decay Accelerating Factor (DAF) with C4b and C3b on Cell Membranes. J Immunol. 1986; 136: 3390-3395.
- Solana ME, Ferrer MF, Novoa MM, Song WC, Gomez RM. Decay- Accelerating Factor 1 Deficiency Exacerbates Trypanosoma cruzi-Induced Murine Chronic Myositis. Muscle Nerve. 2012; 46: 582-587.
- Fenton B and Joiner K. Developmentally Regulated Virulence Factors of Trypanosoma cruzi and Their Relationship to Evasion of Host Defenses. The Journal of Eukaryotic Microbiology, 1993; 40: 207-213.
- Tambourgi DV, Kipnis TL, da Silva WD, Joiner KA, Sher A and Heath S, et al. A partial cDNA clone of Trypomastigote Decay-Accelerating Factor (T-DAF), a Developmentally Regulated Complement Inhibitor of Trypanosoma cruzi, has Genetic and Functional Similarities to The Human Complement Inhibitor DAF. Infect Immun. 1993; 61: 3656-3663.
- Gonzalez A, Valck C, Sanchez G, Hartel S, Mansilla J and Ramirez G, et al. Trypanosoma cruzi Calreticulin Topographical Variations in Parasites Infecting Murine Macrophages. Am J Trop Med Hyg. 2015; 92: 887–897.
- Ramírez-Toloza G and Ferreira A. Trypanosoma cruzi Evades the Complement System as an Efficient Strategy to Survive in the Mammalian host: The Specific Roles of Host/Parasite Molecules and Trypanosoma cruzi calreticulin. Front Microbiol. 2017; 8: 1667.
- Ferreira V, Valck C, Sanchez G, Gingras A, Tzima S and Molina MC, et al. The Classical Activation Pathway of the Human Complement System is Specifically Inhibited by Calreticulin from Trypanosoma cruzi. J Immunol. 2004; 172: 3042–3050.
- Valck C, Ramírez G, Lopez N, Ribeiro CH, Maldonado I and Sánchez G, et al. Molecular mechanisms involved in the inactivation of the first component of human complement by Trypanosoma cruzi calreticulin. Mol Immunol. 2010; 47: 1516-1521.
- Sosoniuk E, Vallejos G, Kenawy H, Gaboriaud C, Thielens N and Fujita T, et al. Trypanosoma cruzi Calreticulin Inhibits the Complement Lectin Pathway Activation by Direct Interaction with L-Ficolin. Mol Immunol. 2014; 60: 80–85.
- Sanchez-Valdez FJ, Brandan CP, Ramirez G, Uncos AD, Zago MP and Cimino RO, et al. A Monoallelic Deletion of the TcCRT Gene Increases the Attenuation of a Cultured Trypanosoma cruzi Strain, Protecting against an in vivo Virulent Challenge. PLoS Negl Trop Dis. 2014; 8: e2696.
- Johnson CA, Kleshchenko YY, Ikejiani AO, Udoko AN, Cardenas TC and Pratap S, et al. Thrombospondin-1 interacts with Trypanosoma cruzi surface calreticulin to enhance cellular infection. PLoS One. 2012; 7: e40614.
- Ramirez G, Valck C, Aguilar L, Kemmerling U, Munoz RL and Morello C, et al. Roles of Trypanosoma cruzi calreticulin in parasite-host interactions and in tumor growth. Mol Immunol. 2012; 52: 133-140.
- Norris KA, Harth G and So M. Purification of a Trypanosoma cruzi Membrane Glycoprotein which Elicits lytic Antibodies. Infect. Immun 1989; 57: 2372– 2377.
- Norris KA, Bradt B, Cooper NR and So M. Characterization of a Trypanosoma cruzi C3 Binding Protein with Functional and Genetic Similarities to the Human Complement Regulatory Protein, Decay-Accelerating Factor. J Immunol. 1991; 147: 2240-2247.
- Van Voorhis WC, Barrett L, Koelling R and Farr AG. FL-160 proteins of Trypanosoma cruzi are expressed from a multigene family and contain two distinct epitopes that mimic nervous tissues. J Exp Med. 1993; 178: 681-694.
- Norris KA, Schrimpf JE and Szabo MJ. Identification of the gene family encoding the 160-kilodalton Trypanosoma cruzi complement regulatory protein. Infect Immun. 1997; 65: 349–357.
- Norris KA. Stable Transfection of Trypanosoma cruzi Epimastigotes with the Trypomastigote-Specific Complement Regulatory Protein cDNA confers Complement Resistance. Infect Immun. 1998; 66: 2460–2465.
- Beucher, M, Meira WS, Zegarra V, Galvao LMC, Chiari E and Norris KA. Expression and Purification of Functional, Recombinant Trypanosoma cruzi Complement Regulatory Protein. Protein Expr Purif. 2003; 27: 19-26.
- Inal JM. Complement C2 receptor Inhibitor Trispanning: from Man to Schistosome. Springer Semin Immunopathol. 2005; 27: 320–331.
- Cestari Idos S, Krarup A, Sim RB, Inal JM and Ramirez MI. Role of early lectin pathway activation in the complement-mediated killing of Trypanosoma cruzi. Mol Immunol. 2009; 47: 426-437.
- Cestari Idos S, Evans-Osses I, Freitas JC, Inal JM and Ramirez MI. Complement C2 Receptor Inhibitor Trispanning Confers an Increased Ability to Resist Complement-Mediated Lysis in Trypanosoma cruzi. J Infect Dis. 2008; 198: 1276-1283.
- Inal JM and Schifferli JA. Complement C2 Receptor Inhibitor Trispanning and the Betachain of C4 Share a Binding site for Complement C2. J Immunol. 2002; 168: 5213-5221.
- Velge P, Ouaissi MA, Cornette J, Afchain D and Capron A. Identification and Isolation of Trypanosoma cruzi Trypomastigote Collagen Binding Proteins: Possible Role in Cell-Parasite Interaction. Parasitology. 1988; 97: 255–268.
- Fischer E, Ouaissi MA, Velge P, Cornette J and Kazatchkine MD. gp 58/68, a Parasite Component that Contributes to the Escape of the Trypomastigote form of T. cruzi from Damage by the Human Alternative Complement Pathway. Immunology. 1988; 65: 299-303.
- Ouaissi, MA, Afchain D, Capron A and Grimaud JA. Fibronectin receptors on Trypanosoma cruzi trypomastigotes and their biological function. Nature. 1984; 308: 380–382.
- Ouaissi MA, Cornette J qnd Capron A. Identification and isolation of Trypanosoma cruzi trypomastigote cell surface protein with properties expected of a fibronectin receptor. Mol Biochem Parasitol. 1986; 19: 201– 211.