Mechanisms of complement lectin pathway activation and resistance by trypanosomatid parasites

Molecular Immunology 53 (2013) 328–334

Contents lists available at SciVerse ScienceDirect

Molecular Immunology journal homepage: www.elsevier.com/locate/molimm

Review

Mechanisms of complement lectin pathway activation and resistance by trypanosomatid parasites Igor Cestari a,b , Ingrid Evans-Osses b , Luregn J. Schlapbach c , Iara de Messias-Reason d , Marcel I. Ramirez b,∗ a

Seattle Biomedical Research Institute, Seattle, WA 98109, USA Laboratório de Biologia Molecular de Parasitas e Vetores, Instituto Oswaldo Cruz - Fiocruz, Rio de Janeiro 21040-900, Brazil c Pediatric Critical Care Research Group, Mater Children’s Hospital, Brisbane, Australia d Laboratório de Imunopatologia, Departamento de Patologia Médica, Universidade Federal do Paraná, Curitiba, Brazil b

a r t i c l e

i n f o

Article history: Received 7 August 2012 Accepted 15 August 2012 Available online 9 October 2012 Keywords: Complement system Lectin pathway Chagas disease Trypanosomiasis T. cruzi Parasite host cell interaction Innate immunity

a b s t r a c t Studies in the past decade have demonstrated a crucial role for the complement lectin pathway in host defence against protozoan microbes. Recognition of pathogen surface molecules by mannan-binding lectin and ficolins revealed new mechanisms of innate immune defence and a diversity of parasite strategies of immune evasion. In the present review, we will discuss the current knowledge of: (1) the molecular mechanism of lectin pathway activation by trypanosomes; (2) the mechanisms of complement evasion by trypanosomes; and (3) host genetic deficiencies of complement lectin pathway factors that contribute to infection susceptibility and disease progression. This review will focus on trypanosomatids, the parasites that cause Chagas disease, leishmaniasis and sleeping sickness (African trypanosomiasis). © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Advances in complement research have revealed important mechanisms of both host innate immune defence and pathogen immune evasion strategies, in particular: (1) the role of the lectin pathway (discovered in the late 90s) in pathogen recognition through pathogen-associated molecular patterns (PAMPs); (2) the discovery of new complement regulators and pathogen’s immune evasion strategies; and (3) the identification of genetic deficiency in genes coding for mannan-binding lectin and ficolins causing susceptibility to infection. Trypanosoma cruzi, Trypanosoma brucei and Leishmania sp. are unicellular, uniflagellated protozoan parasites responsible for causing Chagas disease, sleeping sickness and leishmaniasis, respectively. They are transmitted to human (or other mammalian hosts) by an insect vector, and together they affect more than half a million people worldwide (WHO, 2011). During the infection, these parasites migrate through the host bloodstream, where they have

Abbreviations: MAC, membrane attack complex; MASP, mannan-binding lectin-associated serine protease; MBL, mannan-binding lectin; PAMP, pathogenassociated molecular patterns. ∗ Corresponding author. Tel.: +55 21 3865 8206; fax: +55 21 2590 3495. E-mail addresses: marcelr@fiocruz.br, [email protected] (M.I. Ramirez). 0161-5890/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molimm.2012.08.015

to evade the host innate immune response and, in the case of T. cruzi and Leishmania sp., infect host cells (see Buscaglia et al., 2006 for review). The complement system is one of the first mechanisms of host defence against these parasites, and their success in infecting the host is dependent on their capacity to resist the complement attack, either by inhibiting the complement cascade or by escaping its activation. In this review, we will discuss the molecular mechanisms of complement lectin pathway activation, mechanisms of resistance to complement-mediated lysis and studies on the host genetic deficiencies of the lectin pathway associated with susceptibility to infection and disease progression focusing on trypanosomatid parasites. 2. Complement lectin pathway activation The lectin pathway represents one of the first innate immune responses to a pathogen (Lambris et al., 2008). It can be activated through the binding of mannan-binding lectin (MBL), L-ficolin, Hficolin or M-ficolin to carbohydrates on the pathogen coat (Fig. 1). MBL and ficolins are members of the collectin family of proteins. They possess collagenous and lectin domains and their main function are to recognize PAMPs on microbial surfaces (Fig. 2). Activation of the lectin pathway does not depend on a specific antibody response (such as in the classical pathway), but is

I. Cestari et al. / Molecular Immunology 53 (2013) 328–334

329

Fig. 1. Complement system activation. There are three pathways that activate the complement system: lectin, classical and alternative pathways. The lectin pathway (in 1) is activated when MBL or ficolins recognize carbohydrate-containing molecules on pathogen surface. They form a complex with MASPs (MASP2 is shown in the diagram) which cleave C2 and C4 resulting in the C3 convertase formation C4b2a (in 2). The C3 convertase can cleave C3 into C3a and C3b, the latter associates with the C3 convertase (C4b2a or C3bBb, in 3 only shown C4b2a) forming the C5 convertase C4b2a3b (in 3). The C5 convertase cleaves C5 into C5a and C5b. The fragment C5b attach to the pathogen surface forming an anchor that together with C6, C7 and C8 will hold the MAC, which is formed by several C9 molecules (in 4). The MAC is a pore that will lyse the pathogen allowing the flux of electrolytes and water. Activation of the complement system by the classical pathway is mainly dependent on an antibody response against the pathogen. It occurs by binding of the C1 complex (C1q-r2 s2 ) to antibodies that recognizes the pathogen (in 6). Once bound to the pathogen surface the C1 complex is activated and cleaves C2 and C4 to generate the C3 convertase C4b2a (similar to the lectin pathway). The alternative pathway (in 5) activation is primarily dependent on C3b, which can be formed by spontaneous hydrolysis of C3(H2 O) or by the C3b generate by activation of the classical and lectin pathways (from the C3 convertase activity). In the alternative pathway, once C3b is deposited on the pathogen surface the molecule factor B binds to C3b. The factor B is then cleaved by a soluble molecule called factor D generating the fragments Ba and Bb. The C3bBb formed on the pathogen surface is the C3 convertase of the alternative pathway (which is different from the classical pathway, C4b2a). This C3 convertase also cleaves C3 similarly to the other two pathways. Once the C3 convertase is formed all the subsequent steps are common for all the three pathways.

triggered by PAMPs composed of surface carbohydrates present on several microbes (Runza et al., 2008). Weis et al. (1992) showed that MBL has a high specificity for mannan on glycosylated proteins, and its interaction with pathogen surface carbohydrates is Ca2+ -dependent. L-ficolin and H-ficolin bind preferentially to acetylated and neutral carbohydrates such as N-acetylglucosamine (GlcNAc) and galactose, respectively (Garlatti et al., 2007; Krarup et al., 2008; Matsushita et al., 2002). Pathogen recognition by MBL

and ficolins results in activation of the complement system, which can lead to pathogen lysis through the membrane attack complex (MAC) formation. Initially, MBL or ficolins bind to the pathogen surface and associate with MBL-associated serine proteases (MASPs), thereby forming a protein complex that activates the complement cascade (Matsushita and Fujita, 1992). There are three MASP enzymes; MASP1, MASP2 and MASP3 (Matsushita and Fujita, 2001; Thiel et al., 1997), that together with a truncated version of MASP2

Fig. 2. MBL and ficolins schematic structure. (A) MBL oligomers has a “bouquet”-like structure composed by monomeric subunits of 32 kDa. It contains a cysteine-rich region at the N-terminal and a collagen-like domain that are necessary to assemble the oligomeric form. At the C-terminal there is a carbohydrate recognition domain (CRD) which binds to carbohydrates (such as mannose and GlcNAc) on pathogen surface molecules. (B) Ficolin proteins are composed of a short N-terminal region with one or two cysteine residues followed by a collagen-like domain, a short link region, and a fibrinogen-like domain. Ficolin proteins form trimeric subunits through the binding of the collagen-like domain. These subunits assemble into active oligomers through the binding of four subunits via disulfide bridges at the N-terminal regions. Ficolins recognizes acetylated carbohydrates through the C-terminal fibrinogen-like domain.

330

I. Cestari et al. / Molecular Immunology 53 (2013) 328–334

Fig. 3. Complement system activation and resistance by trypanosomatid parasites. (A) Model of complement activation by trypanosomes. Initial activation of the complement system during infection occurs mainly by the lectin pathway (and at low level by alternative pathway) since it does not depend on a specific antibody response. Once the lectin pathway is activated the C3 convertase cleaves C3 forming C3b, which boost the alternative pathway and result in simultaneous complement activation. Although the lectin and alternative pathways can be continuously activated and functional, pathogens that are able to resist them may infect the host. As the infection progress the host can mount a specific antibody response which will result in classical pathway activation. The three pathways can be continuously activated depending on the mechanism of pathogen resistance. (B) Mechanism of complement evasion by trypanosomatids. (1) Inhibition of C3 convertases. The parasites (mainly T. cruzi) express complement receptors that inhibits the C3 convertase formation or that accelerates the C3 convertase dissociation. For example, CRIT binds to C2 and inhibits its cleavage by MASP2 (and C1s), preventing C3 convertase formation. T-DAF binds to C3b and C4b and likely accelerates the dissociation of the C3 convertase. (2) Expression of a protective surface layer. Leishmania sp. expresses a surface layer formed by LPG and T. brucei express VSGs that protect the parasite from the complement attack. The molecular mechanisms are still unknown. (3) Removal of immune complexes. Leishmania sp. releases the C5b-9 complex (MAC) deposited on the parasite surface.

called MAp19 (generated by splicing of the masp2 gene) (Stover et al., 1999; Takahashi et al., 1999), associate with MBL and ficolins for lectin pathway activation (Stover et al., 1999; Vorup-Jensen et al., 2000; Matsushita et al., 2000). MASP1 and MASP3, as well as MAp44, are splicing variants of the masp1 gene (Stover et al., 2004). MASP2 is the main enzyme responsible for lectin pathway activation by cleaving the components C2 and C4. The C4b fragment (product of C4 cleavage) binds to the pathogen surface and associates with C2a forming the C3 convertase (C4b2a, similar to the C3 convertase of the classical pathway) (Fig. 1). C2a has the catalytic activity that cleaves C3. The C3 convertase has a short half-life of approximately 60 s (Hourcade et al., 1999; Kuttner-Kondo et al., 2003). During this time, it cleaves the component C3 and thereafter dissociates from the pathogen’s surface. Once C3 is cleaved, the C3b fragment can either bind to the pathogen surface to activate the alternative pathway (Fig. 1), or it can bind to the C4b2a (classical or lectin pathway C3 convertase) to form the C5 convertase (C4b2a3b). C3b can also bind to the alternative pathway C3 convertase, C3bBb, and form the C5 convertase C3bBb3b. The C5 convertase cleaves C5 into C5a and C5b. C5b then binds to the pathogen surface and associates with C6, C7 and C8 to support the formation of the MAC (C5b-9, with several C9 molecules), which results in parasite lysis. 3. Lectin pathway activation by protozoan parasites Understanding how protozoan parasites activate the lectin pathway is crucial for unravelling their mechanisms of complement evasion. Several protozoan parasites activate the lectin pathway (Cestari et al., 2009; Ambrosio and De Messias-Reason, 2005; Evans-Osses et al., 2010; Holmberg et al., 2008). MBL, for example, binds to lipophosphoglycans on the surface of Leishmania sp. (Green et al., 1994), which results in lectin pathway activation and promastigote lysis (Ambrosio and De Messias-Reason, 2005). We have

previously shown that MBL, L-ficolins and H-ficolins bind to glycosylated molecules on the surface of T. cruzi metacyclic trypomastigotes (Cestari et al., 2009). Binding of these lectins to T. cruzi surface resulted in rapid complement activation in a Ca2+ -dependent fashion. Complement activation using human serum depleted of MBL and ficolins resulted in an approximately 70% reduction of C3b and C4b deposition on parasite surface and a significant decrease (almost 70%) of complement-mediated lysis (Cestari et al., 2009; Cestari and Ramirez, 2010). In agreement with this, lysis of T. cruzi in non-immune serum was also dependent on MASP2 and C2, both required for the activation of the lectin pathway (Cestari et al., 2009). On the other hand, depletion of C1q (a molecule required for classical pathway activation) from non-immune human serum had no significant effect in T. cruzi complement activation and lysis (Cestari et al., 2009; Cestari and Ramirez, 2010). This indicated that in the absence of an antibody response, the classical pathway may not be sufficient to fully activate the complement system against T. cruzi. Nonetheless, under these circumstances the lectin pathway can recognize the parasites and active the complement cascade. The classical pathway is important for activation of the complement system after the host develops a specific antibody response. For example, it has been shown that anti-␣-galactosyl antibodies from Chagas disease patients activate the complement system and lyse T. cruzi trypomastigotes. Since MBL also binds to IgM, IgA and IgG antibodies (Arnold et al., 2005; Malhotra et al., 1995; Roos et al., 2001), it is possible that both pathways (classical and lectin) are synergistically activated in the presence of specific antibodies and in chronic infections (Fig. 3A). The role of the alternative pathway in complement activation by trypanosomes has been studied through kinetics of complement-mediated lysis in C2-deficient serum (Cestari et al., 2009; Dominguez et al., 2002). Complement C2 is required for C3 convertase formation by the classical and lectin pathway, but not

I. Cestari et al. / Molecular Immunology 53 (2013) 328–334

331

Table 1 Complement receptors and their function in trypanosomatids. Molecule

Pathogen

Function

Complement C2 receptor inhibitor trispanning Calreticulin Complement regulatory protein GP63 Gp58/68 T-DAF

T. cruzi T. cruzi T. cruzi L. major T. cruzi T. cruzi

Binds to C2 and prevent its cleavage by C1s and MASP2–Classical and lectin pathway inhibition Binds to C1q and MBL and inhibits classical and possibly MBL-mediated lectin pathway activation Binds to C3b and C4b resulting in classical and alternative pathways C3 convertase dissociation Binds to C3 and inhibits C3 convertase formation Binds to Factor B and prevents its association with C3b inhibiting the alternative pathway Binds to C3b and C4b and inhibits complement lysis likely by dissociation of the C3 convertase

for the alternative pathway. Complement assays with C2-deficient serum resulted in slow complement activation and inefficient lysis of T. cruzi (Cestari et al., 2009; Cestari and Ramirez, 2010). Kinetics of complement-mediated lysis with serum treated with EGTA and MgCl2 (to chelate Ca2+ , classical and lectin pathway are not functional under these conditions) confirmed that the alternative pathway is slowly activated by T. cruzi and Leishmania sp. (Cestari and Ramirez, 2010; Dominguez et al., 2002; Cestari et al., 2008). The inefficient activation of the alternative pathway by T. cruzi trypomastigotes results from the inability of factor B to bind to the parasite surface (Joiner et al., 1986). Similarly, a slow activation of the complement system in serum deficient in C2 has been observed in Leishmania sp. (Dominguez et al., 2002; Moreno et al., 2007), suggesting that the alternative pathway activation can be delayed in the absence of the classical and lectin pathway. It is noteworthy that the alternative pathway can be self-activated by hydrolysis of C3; however, hydrolysed C3 represents only 5% of the amount of C3 available in serum (Pangburn and Muller-Eberhard, 1983). This supports the idea that the alternative pathway is slowly activated, although the extent of its activation can also vary between organisms. In summary, two scenarios can be postulated to explain how the complement system recognizes these parasites during infection and contributes to pathogen clearance: (1) Early activation of the complement system – Early activation of the complement system is mainly dependent on PAMP recognition by the lectin pathway (Fig. 3A). The lectin pathway also triggers the activation of the alternative pathway by generating C3b, which results in synergistic activation of the complement system. (2) Late activation of the complement system – Pathogens that escape the lectin pathway activation and succeed in infecting the host can still be detected by the classical pathway after the host mounts a specific antibody response. Since the classical pathway is effectively activated in the presence of specific antibodies, its activity is likely more pronounced later during infection (Fig. 3A). MBL binding to antibodies can also result in lectin pathway activation. Activation of the classical and lectin pathway also generates C3b, triggering the alternative pathway. 4. Mechanisms of complement evasion by protozoan parasites There are at least three distinct mechanisms of complement system evasion by trypanosomes and. The most common mechanism is the expression of complement receptors that inhibit the complement cascade preventing C3 convertase formation. In T. cruzi, there are several molecules that inhibit C3 convertase formation (Table 1). The molecule CRIT (Complement C2 receptor inhibitor trispanning) inhibits the formation of the C3 convertase by interacting with the complement component C2 (Cestari et al., 2009, 2008). CRIT is a surface molecule expressed at the metacyclic trypomastigotes stage (infectious stage). Overexpression of the crit gene in T. cruzi epimastigote stage (the insect stage, which is sensitive to complement-mediated lysis) conferred complement resistance to the parasites (Cestari et al., 2008). Trypanosome CRIT is highly conserved with Schistosoma sp. CRIT, and also shares

similarities with CRIT of human monocytic cells (Inal et al., 2005). CRIT is a trans-membrane protein that presents an N-terminal extracellular domain called ed1. This domain shares similarities with the C4␤-chain, which is involved in C2 binding (Cestari et al., 2009; Inal and Schifferli, 2002). The ed1 domain binds to C2 and inhibits its cleavage by C1s and MASP2, thereby preventing formation of the classical and lectin pathway C3 convertase (Cestari et al., 2009; Inal et al., 2005; Inal and Schifferli, 2002). Another molecule involved in complement inhibition by T. cruzi is calreticulin (CRT) (Ferreira et al., 2004a). CRT localizes at the surface of trypomastigotes (Ferreira et al., 2004b). It binds to collagenous tails of C1q and MBL, resulting in inhibition of the classical pathway and interference of MBL binding to mannan (Ferreira et al., 2004b; Ramirez et al., 2011; Valck et al., 2010). GP160, also called complement regulatory protein (CRP), is a molecule expressed in T. cruzi trypomastigotes that binds to C3b and C4b and dissociates the classical and alternative pathways C3 convertase (Norris, 1998; Norris et al., 1989). Over-expression of the gene coding for CRP in the complement sensitive epimastigotes rendered parasites resistant to lysis (Norris, 1998). Besides having a similar mechanism of action to the human molecule decay accelerating factor (DAF), CRP is also anchored by glycosylphosphatidylinositol and shares DNA sequence similarities with DAF (Beucher and Norris, 2008; Norris et al., 1991; Beucher et al., 2003). Similarly, an 87–93 kDa protein identified on the surface of T. cruzi trypomastigotes, called trypomastigotes-decay accelerating factor (T-DAF), was shown to share cDNA similarity to human DAF and inhibit parasite lysis (Tambourgi et al., 1993). Another molecule shown to inhibit C3 convertase formation in T. cruzi is gp58/68 (Fischer et al., 1988). Purified gp58/68 inhibited formation of cell-bound and fluid-phase alternative pathway C3 convertase in a dosedependent fashion. Unlike DAF, gp58/68 was unable to dissociate the C3 convertase. However, its inhibition of the C3 convertase seems to depend on its association with factor B, rather than C3b (Fischer et al., 1988). Another complement evasion strategy employed by T. cruzi relies on stabilization and inhibition of C3 convertase by host factors. Trypomastigotes parasites induce host cells to release plasma membrane-derived microvesicles that bind to C4b2a and inhibit its activity on the parasite surface (Cestari et al., 2012). Infection of mouse with T. cruzi trypomastigotes resulted in higher levels of microvesicles circulating in plasma. In addition, infection of mouse with T. cruzi in presence of exogenous microvesicles resulted in higher parasitemia according to its role in complement evasion (Cestari et al., 2012). It is thought that complement receptors from host cells present on the microvesicles (such as CR1 and DAF) could be involved in C3 convertase inhibition; however, it is still to be determined. Unlike T. cruzi, the mechanism of complement evasion by Leishmania sp. is not based on C3 convertase inhibition. The surface glycoprotein GP63 is the only molecule reported to interfere with the C3 convertase (Brittingham et al., 1995) (Table 1). GP63, also known as major surface protease, is the major C3b acceptor (Russell, 1987). C3b binding to GP63 results in its conversion to iC3b (inactive C3b), thereby preventing C3 convertase formation on the parasite surface (Brittingham et al., 1995). Furthermore,

332

I. Cestari et al. / Molecular Immunology 53 (2013) 328–334

surface deposited iC3b is recognized by the complement receptor 3 (also known as Mac-1), resulting in parasite phagocytosis by macrophages (Brittingham et al., 1995), in which the parasites can multiply and further develop. Lipophosphoglycan (LPG) has been shown to protect Leishmania sp. from the complement system, likely by forming a protective layer on the parasite surface (Spath et al., 2003). Deletion of the gene coding for the Golgi GDP-mannose transporter LPG2, required for the synthesis of surface lipophosphoglycan, rendered parasites highly susceptible to complement-mediated lysis (Spath et al., 2000). Furthermore, LPG2 null L. major parasites were incapable of establishing macrophage infection and were less effective at infecting mice. L. major also releases the membrane attack complex (C5b-9) deposited on its surface during complement activation (Puentes et al., 1990). The majority of C5b-9 formed on metacyclic promastigotes was spontaneously released into the serum as soluble C5b-9. Although all the components of the cascade were active and C5b-7 was stably bound to the parasite surface, most of the C5b-9 was unstable and released from the surface, indicating that these parasites employ different strategies to avoid destruction by the complement system. For the African trypanosome T. brucei, evasion of the complement system depends on the expression of a single variant surface glycoprotein (VSG) that forms a coat on the parasite surface (Russo et al., 1994; Engstler et al., 2007). This parasite has a repertoire of more than 1000 genes (and pseudogenes) coding for VSGs, which are anchored to the parasite surface by glycosylphosphatidylinositol [see Rudenko, 2011 for a comprehensive review]. Once the host develops an antibody response against a specific surface VSG, parasites that have already switched to a different vsg gene can escape the host antibody response until the host develops an antibody response to the new surface VSG, resulting in waves of parasitemia (Rudenko, 2011). Furthermore, removal of immune complexes deposited on the parasite surface has also been shown to depend on VSGs (Russo et al., 1994; Engstler et al., 2007). Host immunoglobulins form immune complexes with VSG on the cell surface and are rapidly removed by a hydrodynamic force generated by parasite motility, resulting in the transfer of the immune complexes to the posterior end of the cell, where they are endocytosed (Engstler et al., 2007). Overall, the mechanisms of complement evasion by trypanosomes include: (1) the inhibition of C3 convertase (Fig. 3B). This seems to be the most common mechanism in T. cruzi. C3 is a key molecule in complement activation and is also responsible for activation of the host cellular immune response. The expression of complement inhibitors of C3 convertase has also been shown in many other non-protozoa parasites and it seems to be one of the major mechanisms of complement evasion (Lambris et al., 2008); (2) expression of a protective layer on the cell surface (Fig. 3B). For Leishmania sp., a protective layer on the parasite surface formed by LPG also contributes to its immune evasion, although the specific mechanism of how it blocks complement is still unknown. VSGs, on the surface of African trypanosomes, are another example of a surface layer that protects the parasites against complementmediated lysis; (3) removal of immune complexes from the parasite surface (Fig. 3B). Shedding of the membrane attack complex allows Leishmania sp. to evade lysis. It has also been shown that T. cruzi sheds membrane-derived vesicles (Frevert et al., 1992); although there is no evidence for C5b-9 release, it is possible that a similar mechanism could also happen in T. cruzi. On the other hand, T. brucei evades lysis by rapid endocytosis of immune complexes deposited on the surface VSGs. Although complement resistance in this parasite seems to be primarily dependent on antigenic variation, rapid removal of complement factors and antibodies likely contribute to evasion of both the complement system and phagocytosis mediated by immunoglobulin and C3 receptors.

5. Host lectin pathway deficiencies and susceptibility to parasitic infections Genetic deficiency of components of the complement lectin pathway is a critical factor contributing to infection susceptibility (Schlapbach et al., 2010a; Santos et al., 2001; Boldt et al., 2011; Luz et al., 2010). Infection by intracellular organisms, such as Leishmania sp., may be enhanced by opsonisation (Brittingham et al., 1995). Therefore, while most studies investigating MBL in bacterial infections have focused on MBL deficiency, several studies of parasitic infections have also looked at high MBL-producing genotypes. Santos et al. (2001) performed a case-control study in a Brazilian area where visceral leishmaniasis is epidemic. In their study, the probability of developing visceral leishmaniasis correlated with increasing MBL serum levels. Similarly, wild-type MBL genotype was found more frequently in patients with leishmaniasis, whereas mutant alleles associated with low MBL levels seemed to be protective. A subsequent study from the same region reported that highproducing MBL genotypes were more frequent in patients with visceral leishmaniasis and the MBL levels were higher in these patients when compared to asymptomatic patients (Alonso et al., 2007). In addition, high MBL-producing genotypes were associated with an increased risk of severe visceral leishmaniasis. These findings were reproduced by a recent study in an Azerbaijan population where alleles associated with high MBL levels were found more frequently in patients with visceral leishmaniasis compared to healthy controls (Asgharzadeh et al., 2007). These studies suggest that besides having evolved mechanisms to evade the activation of the complement lectin pathway, Leishmania sp. also takes advantages of these complement factors to infect the host. One hypothesis is that the binding of MBL to the surface of Leishmania parasites could lead to increased macrophage phagocytosis mediated by MBL receptors, thereby favouring parasite invasion and disease progression. High levels of MBL were associated with increased risk of severe cardiomyopathy in chronic Chagas disease, which may be due to the complement-mediated pro-inflammatory role of MBL in tissue injury (Luz et al., 2010). In addition, a recent study by Boldt et al. (2011) compared MASP2 genotypes in patients with Chagas-associated cardiomyopathy. This study analyzed six MASP2 polymorphisms in 208 patients with chronic Chagas disease and 300 healthy individuals from southern Brazil. This study found that MASP2*CD genotypes, which mostly resulted in low MASP2 levels, were associated with a high risk of chagasic cardiomyopathy, suggesting that MASP2 genotype could be related to the development of the disease. Low levels of MASP2 could also result in failure (or inefficiency) of lectin pathway activation, which could favour parasite infection and disease progression. It is noteworthy that MASP2 genotype may be useful in predicting symptomatic Chagas disease. H-ficolin merits particular attention among the lectin pathway proteins, since it is only present in humans and, in contrast to MBL, genetically determined deficiency is extremely rare. The first reports of H-ficolin deficiency caused by the FCN3 + 1637delC frame-shift mutation concerned a patient suffering from repeated infections (Munthe-Fog et al., 2009) and a neonate with severe necrotizing enterocolitis (Schlapbach et al., 2010a). In addition, a higher rate of gram-positive infections has been reported in neonates with lower H-ficolin concentrations (Schlapbach et al., 2010b). Similarly, only limited clinical data are available on L-ficolin. Lower L-ficolin concentrations were reported in children with recurrent respiratory infections (Atkinson et al., 2004; Cedzynski and Atkinson, 2009). Although the role of ficolin deficiency in trypanosomatids infections is still unknown, it is possible that ficolins also play an important role in controlling infection, as observed for bacterial infections. Altogether, this suggests that abnormal levels of serum lectins impact infection and serum lectins

I. Cestari et al. / Molecular Immunology 53 (2013) 328–334

are a determinant factor in the development of leishmaniasis and Chagas disease. 6. Conclusion Understanding pathogen and host interaction is a key aspect in the development of new therapeutics against infectious disease. Although much remains to be investigated on the molecular basis of parasitic infection, the current knowledge of the complement system highlights the importance of the lectin pathway as a key mediator of host defence. The studies on host deficiencies in components of the lectin pathway also demonstrate its association with infection susceptibility and disease progression. This opens a new opportunity to investigate the role of the lectin pathway as disease modifiers, which highlight its applicability for diagnostic and evaluation of disease progression at the individual and population level. Acknowledgments We are very grateful to Dr Lindsay Carpp for revision of the manuscript. This work was supported by CNPq (476737/20044) and for “Programa de Parasitologia Basica/CAPES”. I.C. was a recipient of CNPq (141757/2006-0) and CAPES (PDEE4261/06-2) scholarships. I.E.O. is a recipient of CAPES fellowship and M.I.R. is a CNPq fellow. References Alonso, D.P., Ferreira, A.F., Ribolla, P.E., de Miranda Santos, I.K., do Socorro Pires e Cruz, M., Aecio de Carvalho, F., Abatepaulo, A.R., Lamounier Costa, D., Werneck, G.L., Farias, T.J., et al., 2007. Genotypes of the mannan-binding lectin gene and susceptibility to visceral leishmaniasis and clinical complications. Journal of Infectious Diseases 195 (8), 1212–1217. Ambrosio, A.R., De Messias-Reason, I.J., 2005. Leishmania (Viannia) braziliensis: interaction of mannose-binding lectin with surface glycoconjugates and complement activation. An antibody-independent defence mechanism. Parasite Immunology 27 (9), 333–340. Arnold, J.N., Wormald, M.R., Suter, D.M., Radcliffe, C.M., Harvey, D.J., Dwek, R.A., Rudd, P.M., Sim, R.B., 2005. Human serum IgM glycosylation: identification of glycoforms that can bind to mannan-binding lectin. Journal of Biological Chemistry 280 (32), 29080–29087. Asgharzadeh, M., Mazloumi, A., Kafil, H.S., Ghazanchaei, A., 2007. Mannose-binding lectin gene and promoter polymorphism in visceral leishmaniasis caused by Leishmania infantum. Pakistan Journal of Biological Sciences 10 (11), 1850–1854. Atkinson, A.P., Cedzynski, M., Szemraj, J., St Swierzko, A., Bak-Romaniszyn, L., Banasik, M., Zeman, K., Matsushita, M., Turner, M.L., Kilpatrick, D.C., 2004. L-ficolin in children with recurrent respiratory infections. Clinical and Experimental Immunology 138 (3), 517–520. Beucher, M., Norris, K.A., 2008. Sequence diversity of the Trypanosoma cruzi complement regulatory protein family. Infection and Immunity 76 (2), 750–758. Beucher, M., Meira, W.S., Zegarra, V., Galvao, L.M., Chiari, E., Norris, K.A., 2003. Expression and purification of functional, recombinant Trypanosoma cruzi complement regulatory protein. Protein Expression and Purification 27 (1), 19–26. Boldt, A.B., Luz, P.R., Messias-Reason, I.J., 2011. MASP2 haplotypes are associated with high risk of cardiomyopathy in chronic Chagas disease. Clinical Immunology 140 (1), 63–70. Brittingham, A., Morrison, C.J., McMaster, W.R., McGwire, B.S., Chang, K.P., Mosser, D.M., 1995. Role of the Leishmania surface protease gp63 in complement fixation, cell adhesion, and resistance to complement-mediated lysis. Journal of Immunology 155 (6), 3102–3111. Buscaglia, C.A., Campo, V.A., Frasch, A.C., Di Noia, J.M., 2006. Trypanosoma cruzi surface mucins: host-dependent coat diversity. Nature Reviews Microbiology 4 (3), 229–236. Cedzynski, M., Atkinson, A.P., St Swierzko, A., MacDonald, S.L., Szala, A., Zeman, K., Buczylko, K., Bak-Romaniszyn, L., Wiszniewska, M., Matsushita, M., et al., 2009. L-ficolin (ficolin-2) insufficiency is associated with combined allergic and infectious respiratory disease in children. Molecular Immunology 47 (2–3), 415–419. Cestari, I., Ramirez, M.I., 2010. Inefficient complement system clearance of Trypanosoma cruzi metacyclic trypomastigotes enables resistant strains to invade eukaryotic cells. PLoS ONE 5, e9721. Cestari, I., Evans-Osses, I., Freitas, J.C., Inal, J.M., Ramirez, M.I., 2008. Complement C2 receptor inhibitor trispanning confers an increased ability to resist complementmediated lysis in Trypanosoma cruzi. Journal of Infectious Diseases 198 (9), 1276–1283.

333

Cestari, I., Krarup, A., Sim, R.B., Inal, J.M., Ramirez, M.I., 2009. Role of early lectin pathway activation in the complement-mediated killing of Trypanosoma cruzi. Molecular Immunology 47 (2-3), 426–437. Cestari, I., Ansa-Addo, E., Deolindo, P., Inal, J.M., Ramirez, M.I., 2012. Trypanosoma cruzi immune evasion mediated by host cell-derived microvesicles. Journal of Immunology 188 (4), 1942–1952. Dominguez, M., Moreno, I., Lopez-Trascasa, M., Torano, A., 2002. Complement interaction with trypanosomatid promastigotes in normal human serum. Journal of Experimental Medicine 195 (4), 451–459. Engstler, M., Pfohl, T., Herminghaus, S., Boshart, M., Wiegertjes, G., Heddergott, N., Overath, P., 2007. Hydrodynamic flow-mediated protein sorting on the cell surface of trypanosomes. Cell 131 (3), 505–515. Evans-Osses, I., Ansa-Addo, E.A., Inal, J.M., Ramirez, M.I., 2010. Involvement of lectin pathway activation in the complement killing of Giardia intestinalis. Biochemical and Biophysical Research Communications 395 (3), 382–386. Ferreira, V., Molina, M.C., Valck, C., Rojas, A., Aguilar, L., Ramirez, G., Schwaeble, W., Ferreira, A., 2004a. Role of calreticulin from parasites in its interaction with vertebrate hosts. Molecular Immunology 40 (17), 1279–1291. Ferreira, V., Valck, C., Sanchez, G., Gingras, A., Tzima, S., Molina, M.C., Sim, R., Schwaeble, W., Ferreira, A., 2004b. The classical activation pathway of the human complement system is specifically inhibited by calreticulin from Trypanosoma cruzi. Journal of Immunology 172 (5), 3042–3050. Fischer, E., Ouaissi, M.A., Velge, P., Cornette, J., 1988. 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 65 (2), 299–303. Frevert, U., Schenkman, S., Nussenzweig, V., 1992. Stage-specific expression and intracellular shedding of the cell surface trans-sialidase of Trypanosoma cruzi. Infection and Immunity 60 (6), 2349–2360. Garlatti, V., Belloy, N., Martin, L., Lacroix, M., Matsushita, M., Endo, Y., Fujita, T., Fontecilla-Camps, J.C., Arlaud, G.J., Thielens, N.M., et al., 2007. Structural insights into the innate immune recognition specificities of L- and H-ficolins. EMBO Journal 26 (2), 623–633. Green, P.J., Feizi, T., Stoll, M.S., Thiel, S., Prescott, A., McConville, M.J., 1994. Recognition of the major cell surface glycoconjugates of Leishmania parasites by the human serum mannan-binding protein. Molecular and Biochemical Parasitology 66 (2), 319–328. Holmberg, V., Schuster, F., Dietz, E., Sagarriga Visconti, J.C., Anemana, S.D., Bienzle, U., Mockenhaupt, F.P., 2008. Mannose-binding lectin variant associated with severe malaria in young African children. Microbes and Infection 10 (4), 342–348. Hourcade, D.E., Mitchell, L.M., Medof, M.E., 1999. Decay acceleration of the complement alternative pathway C3 convertase. Immunopharmacology 42 (1-3), 167–173. Inal, J.M., Schifferli, J.A., 2002. Complement C2 receptor inhibitor trispanning and the beta-chain of C4 share a binding site for complement C2. Journal of Immunology 168 (10), 5213–5221. Inal, J.M., Hui, K.M., Miot, S., Lange, S., Ramirez, M.I., Schneider, B., Krueger, G., Schifferli, J.A., 2005. Complement C2 receptor inhibitor trispanning: a novel human complement inhibitory receptor. Journal of Immunology 174 (1), 356–366. Joiner, K., Sher, A., Gaither, T., Hammer, C., 1986. Evasion of alternative complement pathway by Trypanosoma cruzi results from inefficient binding of factor B. Proceedings of the National Academy of Sciences of the United States of America 83 (17), 6593–6597. Krarup, A., Mitchell, D.A., Sim, R.B., 2008. Recognition of acetylated oligosaccharides by human L-ficolin. Immunology Letters 118 (2), 152–156. Kuttner-Kondo, L.A., Dybvig, M.P., Mitchell, L.M., Muqim, N., Atkinson, J.P., Medof, M.E., Hourcade, D.E., 2003. A corresponding tyrosine residue in the C2/factor B type A domain is a hot spot in the decay acceleration of the complement C3 convertases. Journal of Biological Chemistry 278 (52), 52386–52391. Lambris, J.D., Ricklin, D., Geisbrecht, B.V., 2008. Complement evasion by human pathogens. Nature Reviews Microbiology 6 (2), 132–142. Luz, P.R., Miyazaki, M.I., Neto, N.C., Nisihara, R.M., Messias-Reason, I.J., 2010. High levels of mannose-binding lectin are associated with the risk of severe cardiomyopathy in chronic Chagas Disease. International Journal of Cardiology 143 (3), 448–450. Malhotra, R., Wormald, M.R., Rudd, P.M., Fischer, P.B., Dwek, R.A., Sim, R.B., 1995. Glycosylation changes of IgG associated with rheumatoid arthritis can activate complement via the mannose-binding protein. Nature Medicine 1 (3), 237–243. Matsushita, M., Fujita, T., 1992. Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine protease. Journal of Experimental Medicine 176 (6), 1497–1502. Matsushita, M., Fujita, T., 2001. Ficolins and the lectin complement pathway. Immunological Reviews 180, 78–85. Matsushita, M., Endo, Y., Fujita, T., 2000. Cutting edge: complement-activating complex of ficolin and mannose-binding lectin-associated serine protease. Journal of Immunology 164 (5), 2281–2284. Matsushita, M., Kuraya, M., Hamasaki, N., Tsujimura, M., Shiraki, H., Fujita, T., 2002. Activation of the lectin complement pathway by H-ficolin (Hakata antigen). Journal of Immunology 168 (7), 3502–3506. Moreno, I., Molina, R., Torano, A., Laurin, E., Garcia, E., Dominguez, M., 2007. Comparative real-time kinetic analysis of human complement killing of Leishmania infantum promastigotes derived from axenic culture or from Phlebotomus perniciosus. Microbes and Infection 9 (14–15), 1574–1580. Munthe-Fog, L., Hummelshoj, T., Honore, C., Madsen, H.O., Permin, H., Garred, P., 2009. Immunodeficiency associated with FCN3 mutation and ficolin-3 deficiency. New England Journal of Medicine 360 (25), 2637–2644.

334

I. Cestari et al. / Molecular Immunology 53 (2013) 328–334

Norris, K.A., 1998. Stable transfection of Trypanosoma cruzi epimastigotes with the trypomastigote-specific complement regulatory protein cDNA confers complement resistance. Infection and Immunity 66 (6), 2460–2465. Norris, K.A., Harth, G., So, M., 1989. Purification of a Trypanosoma cruzi membrane glycoprotein which elicits lytic antibodies. Infection and Immunity 57 (8), 2372–2377. Norris, K.A., Bradt, B., Cooper, N.R., So, M., 1991. Characterization of a Trypanosoma cruzi C3 binding protein with functional and genetic similarities to the human complement regulatory protein, decay-accelerating factor. Journal of Immunology 147 (7), 2240–2247. Pangburn, M.K., Muller-Eberhard, H.J., 1983. Initiation of the alternative complement pathway due to spontaneous hydrolysis of the thioester of C3. Annals of the New York Academy of Sciences 421, 291–298. Puentes, S.M., Da Silva, R.P., Sacks, D.L., Hammer, C.H., Joiner, K.A., 1990. Serum resistance of metacyclic stage Leishmania major promastigotes is due to release of C5b-9. Journal of Immunology 145 (12), 4311–4316. Ramirez, G., Valck, C., Molina, M.C., Ribeiro, C.H., Lopez, N., Sanchez, G., Ferreira, V.P., Billetta, R., Aguilar, L., Maldonado, I., et al., 2011. Trypanosoma cruzi calreticulin: a novel virulence factor that binds complement C1 on the parasite surface and promotes infectivity. Immunobiology 216 (1–2), 265–273. Roos, A., Bouwman, L.H., van Gijlswijk-Janssen, D.J., Faber-Krol, M.C., Stahl, G.L., Daha, M.R., 2001. Human IgA activates the complement system via the mannanbinding lectin pathway. Journal of Immunology 167 (5), 2861–2868. Rudenko, G., 2011. African trypanosomes: the genome and adaptations for immune evasion. Essays in Biochemistry 51 (2011), 47–62. Runza, V.L., Schwaeble, W., Mannel, D.N., 2008. Ficolins: novel pattern recognition molecules of the innate immune response. Immunobiology 213 (3–4), 297–306. Russell, D.G., 1987. The macrophage-attachment glycoprotein gp63 is the predominant C3-acceptor site on Leishmania mexicana promastigotes. European Journal of Biochemistry 164 (1), 213–221. Russo, D.C., Williams, D.J., Grab, D.J., 1994. Mechanisms for the elimination of potentially lytic complement-fixing variable surface glycoprotein antibody-complexes in Trypanosoma brucei. Parasitology Research 80 (6), 487–492. Santos, I.K., Costa, C.H., Krieger, H., Feitosa, M.F., Zurakowski, D., Fardin, B., Gomes, R.B., Weiner, D.L., Harn, D.A., Ezekowitz, R.A., et al., 2001. Mannan-binding lectin enhances susceptibility to visceral leishmaniasis. Infection and Immunity 69 (8), 5212–5215. Schlapbach, L.J., Thiel, S., Kessler, U., Ammann, R.A., Aebi, C., Jensenius, J.C., 2010a. Congenital H-ficolin deficiency in premature infants with severe necrotising enterocolitis. Gut 60 (10), 1438–1439. Schlapbach, L.J., Mattmann, M., Thiel, S., Boillat, C., Otth, M., Nelle, M., Wagner, B., Jensenius, J.C., Aebi, C., 2010b. Differential role of the lectin pathway of

complement activation in susceptibility to neonatal sepsis. Clinical Infectious Diseases 51 (2), 153–162. Spath, G.F., Epstein, L., Leader, B., Singer, S.M., Avila, H.A., Turco, S.J., Beverley, S.M., 2000. Lipophosphoglycan is a virulence factor distinct from related glycoconjugates in the protozoan parasite Leishmania major. Proceedings of the National Academy of Sciences of the United States of America 97 (16), 9258–9263. Spath, G.F., Garraway, L.A., Turco, S.J., Beverley, S.M., 2003. The role(s) of lipophosphoglycan (LPG) in the establishment of Leishmania major infections in mammalian hosts. Proceedings of the National Academy of Sciences of the United States of America 100 (16), 9536–9541. Stover, C.M., Thiel, S., Thelen, M., Lynch, N.J., Vorup-Jensen, T., Jensenius, J.C., Schwaeble, W.J., 1999. Two constituents of the initiation complex of the mannan-binding lectin activation pathway of complement are encoded by a single structural gene. Journal of Immunology 162 (6), 3481–3490. Stover, C.M., Lynch, N.J., Hanson, S.J., Windbichler, M., Gregory, S.G., Schwaeble, W.J., 2004. Organization of the MASP2 locus and its expression profile in mouse and rat. Mammalian Genome 15 (11), 887–900. Takahashi, M., Endo, Y., Fujita, T., Matsushita, M., 1999. A truncated form of mannosebinding lectin-associated serine protease (MASP)-2 expressed by alternative polyadenylation is a component of the lectin complement pathway. International Immunology 11 (5), 859–863. Tambourgi, D.V., Kipnis, T.L., da Silva, W.D., Joiner, K.A., Sher, A., Heath, S., Hall, B.F., Ogden, G.B., 1993. A partial cDNA clone of trypomastigote decayaccelerating factor (T-DAF), a developmentally regulated complement inhibitor of Trypanosoma cruzi, has genetic and functional similarities to the human complement inhibitor DAF. Infection and Immunity 61 (9), 3656–3663. Thiel, S., Vorup-Jensen, T., Stover, C.M., Schwaeble, W., Laursen, S.B., Poulsen, K., Willis, A.C., Eggleton, P., Hansen, S., Holmskov, U., et al., 1997. A second serine protease associated with mannan-binding lectin that activates complement. Nature 386 (6624), 506–510. Valck, C., Ramirez, G., Lopez, N., Ribeiro, C.H., Maldonado, I., Sanchez, G., Ferreira, V.P., Schwaeble, W., Ferreira, A., 2010. Molecular mechanisms involved in the inactivation of the first component of human complement by Trypanosoma cruzi calreticulin. Molecular Immunology 47 (7–8), 1516–1521. Vorup-Jensen, T., Petersen, S.V., Hansen, A.G., Poulsen, K., Schwaeble, W., Sim, R.B., Reid, K.B., Davis, S.J., Thiel, S., Jensenius, J.C., 2000. Distinct pathways of mannan-binding lectin (MBL)- and C1-complex autoactivation revealed by reconstitution of MBL with recombinant MBL-associated serine protease-2. Journal of Immunology 165 (4), 2093–2100. Weis, W.I., Drickamer, K., Hendrickson, W.A., 1992. Structure of a C-type mannosebinding protein complexed with an oligosaccharide. Nature 360 (6400), 127–134. WHO: http://wwwwhoint/en/2011.