The trans-sialidase, the major Trypanosoma cruzi

Glycobiology Advance Access published August 19, 2015 Glycobiology, 2015, 1–8 doi: 10.1093/glycob/cwv057 Review

Review

The trans-sialidase, the major Trypanosoma cruzi virulence factor: Three decades of studies

2

Laboratório de Glicobiologia, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21944902, Rio de Janeiro, RJ, Brasil, and 3Vanderbilt University School of Medicine, Nashville, TN 37232, USA 1

To whom correspondence should be addressed: Tel: +55-21-3938-6646; e-mail: [email protected]

Received 2 June 2015; Revised 9 July 2015; Accepted 19 July 2015

Abstract Chagas’ disease is a potentially life-threatening disease caused by the protozoan parasite Trypanosoma cruzi. Since the description of Chagas’disease in 1909 extensive research has identified important events in the disease in order to understand the biochemical mechanism that modulates T. cruzi-host cell interactions and the ability of the parasite to ensure its survival in the infected host. Exactly 30 years ago, we presented evidence for the first time of a trans-sialidase activity in T. cruzi (T. cruzi-TS). This enzyme transfers sialic acid from the host glycoconjugates to the terminal β-galactopyranosyl residues of mucin-like molecules on the parasite’s cell surface. Thenceforth, many articles have provided convincing data showing that T. cruzi-TS is able to govern relevant mechanisms involved in the parasite’s survival in the mammalian host, such as invasion, escape from the phagolysosomal vacuole, differentiation, down-modulation of host immune responses, among others. The aim of this review is to cover the history of the discovery of T. cruzi-TS, as well as some well-documented biological effects encompassed by this parasite’s virulence factor, an enzyme with potential attributes to become a drug target against Chagas disease. Key words: chagas disease, glycoimmunology, sialic acid, trans-sialidase, Trypanosoma cruzi

Sialic acid-containing molecules Sialylated glycoconjugates decorate the surface of all mammalian cells. Given their ubiquitous presence and abundance, sialic acids (Sia) have many important biophysical effects (Crespo et al. 2009; Angata and Fukuda 2010; Varki and Gagneux 2012). Sia is a group of structurally diverse 9-carbon monosaccharides with heterocyclic ring structures (Varki and Schauer 2009). Sia bears a negative charge via a carboxylic acid group attached to the ring, as well as other chemical groups, including N-acetyl and N-glycolyl groups. The two main types of Sia found in mammals are N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc) (Traving and Schauer 1998). Neu5Ac and its hydroxylated derivative Neu5Gc differ by one oxygen atom in the N-glycolyl group (Figure 1). These usually occur as terminal structures attached to β-galactopyranosyl (βGalp) residues at the non-reducing termini of both N- and O-linked glycans (Chen and Varki 2010). The typical cell exhibits tens of millions of Sia molecules,

and it is estimated that the local concentration on the cell-surface glycocalyx can approach 100 mM (Varki and Gagneux 2012). Sia thus provides a large component of negative charge repulsion between cells, which could alter the biophysical properties of cellular interactions. Although Sia are the major cell-surface sugars in the Deuterostome lineage of animals, they are also found in other branches of life (Cohen and Varki 2010). It is important to point out that the “sialoglycophenotype” of many host cell types is exploited by numerous microorganisms, such as viruses (Neu et al. 2011), bacteria (Chang and Nizet 2014) and protozoa (Nishikawa et al. 2013). One of the most elegant mechanisms of cell-surface sialylation found in nature is the one utilized by parasite protozoan Trypanosoma cruzi (Previato et al. 1985). In T. cruzi, Sia was first described in epimastigote forms by using lectins and colorimetric methods (Pereira et al. 1980). Further, the existence of Sia in the epimastigote form was confirmed by thin-layer

© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected]

1

Downloaded from http://glycob.oxfordjournals.org/ at Universidade Federal do Rio de Janeiro on September 4, 2015

L Freire-de-Lima2, L M Fonseca2, T Oeltmann3, L Mendonça-Previato1,2, and J O Previato2

2

L Freire-de-Lima et al.

Fig. 1. Sialic acids found in mammal cells. (A) Neu5Ac and (B) Neu5Gc.

chromatography and gas-liquid chromatography coupled to mass spectrometry. Interestingly, the presence of Sia on the parasite cell surface was not detected when the epimastigote form was incubated with radioactive precursors of the biosynthetic pathway of Sia (Schauer et al. 1983), suggesting that an alternative mechanism for Sia acquisition was present in the parasite (Previato et al. 1985). Also in 1983, noting the ability of T. cruzi trypomastigotes to hydrolyze Sia from human erythrocytes and plasma glycoproteins, Pereira (1983) suggested that the parasite would express a stage-specific neuraminidase activity. Thirty years ago our group described, for the first time, the T. cruzi’s ability to incorporate Sia from exogenous sialoglycoconjugates (Figure 2A). This innovative observation suggested that the incorporation of Sia onto the parasite cell surface did not occur by the conventional route; i.e. CMP-Neu5Ac as the sugar donor, but rather by a new metabolic route involving a trans-glycosylation reaction to Sia (Previato et al. 1985). Later, Sia incorporation into trypomastigote forms was demonstrated in vitro by Zingales and collaborators (Zingales et al. 1987) and then in vivo by Previato and colleagues (Previato et al. 1990). In the Previato study, it was shown that during the acute phase of Chagas disease in mice, the parasite’s trypomastigote forms incorporate Neu5Gc onto its own cell-surface glycoconjugates. Finally, the trans-glycosylase activity for Sia was characterized as T. cruzi-TS, which is specific for alpha(α)2,3 Sia and is responsible for the transfer of Sia from the host’s sialoglycoconjugates to acceptor mucin-like

molecules expressed on the parasite cell surface (Schenkman et al. 1991; Parodi et al. 1992; Mendonça-Previato et al. 2013). Following the identification of the gene coding for both neuraminidase (Pereira et al. 1991) and T. cruzi-TS (Parodi et al. 1992; Uemura et al. 1992), it became clear that both activities were related to the same enzyme. These pioneering studies were critical to elucidate the biochemical parameters of T. cruzi-TS, as well as to understand how this Sia-dependent enzyme might be able to act as a virulence factor to ensure parasite survival in infected hosts. The next sections address the significance of this biological system, covered by both T. cruzi-TS and molecules containing α2-3-linked Sia. In addition, we will examine how the parasite exploits both normal and unusual sialoglycophenotypes of host cells.

trans-Sialidase: a major virulence factor released to the extracellular milieu during the early stages of T. cruzi infection Several studies have shown that the trypomastigote form of T. cruzi sheds proteins which are able to modulate the host immune response during the early stages of infection (DosReis 2011). Amongst all known molecules, TS proteins have been considered as a major virulence factor, either by their capability to dampen host cell immunity or by its ability to mediate the interaction between the parasite and host cells (Frasch 1994; Chuenkova and Pereira 1995; Mendonça-Previato et al. 2010; Buschiazzo et al. 2012; Mendonça-Previato et al. 2013). It


Fig. 2. Schematic representation of T. cruzi-TS. (A) Enzymatically aTS can be bound to the T. cruzi trypomastigote through a GPI-anchor and is able to transfers sialic acid from host sialoglycoconjugates to mucin-like molecules on the parasite cell surface, or act as an adhesin independently of catalytic activity (iTS). (B) Alternatively, T. cruzi-TS can be released into the bloodstream and remodelate the host cell glycophenotype.

Trypanosoma cruzi trans-sialidase

Parasite-host cell contact Trypanosoma cruzi trypomastigotes are very promiscuous. Excluding the red blood cells, the parasite is able to invade any kind of nucleated cells, e.g., phagocytic and non-phagocytic cells. Host cell invasion involves multiple steps, one of which involves primary contact between parasite surface molecules and host cell glycoconjugates (Caradonna and Burleigh 2011; Barrias et al. 2013; de Souza and de Carvalho 2013). Although the enzymatic activity displayed by TS was proposed almost 30 years ago as a key factor for T. cruzi pathogenesis (Previato et al. 1985), it is now known that enzymatically inactive TS (iTS) may play important roles in the biology of the parasite (Todeschini et al. 2002a). In 1995, studies carried out by Cremona and colleagues demonstrated that the difference between active TS (aTS) and iTS was restricted to a single amino acid. While aTS has a tyrosine residue at position 342 (Tyr342), iTS displays a histidine residue (His342) in the same position. This naturally occurring Tyr342 → His substitution was enough to entirely abolish the T. cruzi-TS activity (Cremona et al. 1995). The importance of the adhesin property of iTS initially was demonstrated in our laboratory when we reported that iTS binds sialyl and β-Galp residues in a sequential-ordered mechanism (Todeschini et al. 2004). This finding could have important implications in the life cycle of the parasite, since inactive members of TS proteins may play key roles in T. cruzi-host cell interactions. The significance of Sia-containing glycoconjugates on cell communication and invasion by T. cruzi was proposed several years ago following the demonstration that the invasion of Sia-deficient cells (CHO-Lec 2) was reduced when compared with wild-type cells (Schenkman et al. 1993). In addition, studies from our group involving the use of a Vibrio cholerae neuraminidase suicide-type inhibitor (Hinou et al. 2005) reinforced the importance of Sia on parasite-host cell communication. Besides inhibiting both neuraminidase and TS activities, in a time- and dose-dependent fashion, the inhibitor was also able to reduce the level of infection in cultured mammalian cells (Carvalho et al. 2010). In fact, some compounds have been

synthesized and tested as specific inactivators (Buchini et al. 2008) or inhibitors (Lieke et al. 2011) of T. cruzi-TS activity, aiming towards the discovery of new agents for the cure of Chagas’ disease. Given the importance of sialidases and TS as virulence factors in several infections (Frasch 2000; Wiggins et al. 2001; Hedlund et al. 2010; Dc-Rubin and Schenkman 2012), Frasch and collaborators carried out crucial works on the 3D structure and mechanistic properties of the T. rangeli sialidase (Buschiazzo et al. 2000) and T. cruzi-TS activities (Buschiazzo et al. 2002). The comparison between the structural domains of these enzymes was fundamental to determine the catalytic mechanism of T. cruzi-TS (Watts et al. 2003; Amaya et al. 2004). At the same time, the construction and analysis of TS mutants have defined the amino acid residues relevant for the trans-sialidase activity (Paris et al. 2005). These findings are relevant to the rational design of inhibitors of T. cruzi-TS. However, to this date, an effective inhibitor is yet to be identified. Exhaustive studies carried out by PereiraPerrin’s group have shown that Sia-containing molecules may also exert control on cell signaling mediated by tyrosine kinase receptor-A (TrkA) (Chuenkova and PereiraPerrin 2004, 2005; de Melo-Jorge and PereiraPerrin 2007; Aridgides et al. 2013). In these studies it was clearly demonstrated that de-sialylation of TrKA by T. cruzi-TS leads to the rapid receptor internalization, activation and neuronal differentiation (Woronowicz et al. 2004). The authors suggested that such enzymatic activity may participate in the neural repair and neuroprotection mediated by the T. cruzi-TS, which is also known as parasite-derived neurotrophic factor (Chuenkova and Pereiraperrin 2011). On the other hand, studies published by Rubin-de-Celis and colleagues suggested that Sia content, which is incorporated by T. cruzi-TS is not a limiting factor for host cell invasion, since trypomastigote and both control and TS-transfected metacyclic forms showed equal efficiency in mammalian cell invasion (Rubin-de-Celis et al. 2006). Recently, Butler and colleagues demonstrated that the interaction of TS proteins with host sialoglycans was able to generate an “eat me” signal in epithelial cells, an event that may depend on G protein activation. Epithelium represents a natural barrier to infection, and the cells that compose them are considered not likely to be involved in phagocytosis of large bodies such as parasites. These authors speculated that the action of T. cruzi-TS on sialylated epitopes might facilitate the parasite entry into non-phagocytic cells (Butler et al. 2013). The examples described in this section strongly suggest that T. cruzi exploits the host cell sialoglycophenotype to set up parasite-host cell communication, which is essential for a successful infection.

Trypanosoma cruzi-TS modulates host immune system As mentioned above, T. cruzi does not synthetize Sia. The parasite acquires Sia from host sialoglycoconjugates, through the transglycosylation reaction catalyzed by T. cruzi-TS (Previato et al. 1985). Since Sia plays essential functions on both inate and adaptative immunity (Amon et al. 2014; Chen et al. 2014) it is not surprising that TS proteins are able to dampen the host’s immune response. Over the last years, several papers have demonstrated that TS proteins might act on distant sites from the parasite as a soluble factor. Since its discovery, many laboratories have tried to elucitate how this Sia-dependent parasitic enzyme can modulate the host immunity in order to ensure the parasite’s survival in the infected host. Initial studies reported by Chuenkova and Pereira (1995) demonstrated sensitization of mice with minute amounts of the native TS prior to infection with T. cruzi led to increased parasitemia and mortality. It is important to


is a well established fact that in trypomastigote forms of T. cruzi, TS is a GPI-anchored membrane protein, which is dynamically released to the extracellular milieu. This leads to a systemic distribution of the enzyme through the bloodstream (Figure 2B). The half-life of TS proteins in the bloodstream is considerably extended due to the occurrence of a C-terminal repetitive domain termed Shed Acute Phase Antigen (SAPA) (Alvarez et al. 2004). SAPA is believed to be an evolutionary strategy adopted by the parasite to prevent the early production of antibodies against the N-terminal domain, which is responsible for its catalytic activity (Ribeirao et al. 1997). Over the last few years, many studies have shed light on the relationship between TS proteins and numerous pathologies observed during the early stages of T. cruzi infection. The administration of recombinant TS in non-infected mice is capable of inducing apoptosis in thymocytes (Mucci et al. 2002) and mature T cells (Mucci et al. 2005) and causes lowering of platelet numbers (Tribulatti et al. 2005). In addition, studies from several groups have shown that the administration of recombinant TS is able to modulate the functionality of immune cells, such as T and B lymphocytes (Chuenkova and Pereira 1995; Gao and Pereira 2001; Gao et al. 2002; Freire-de-Lima et al. 2010; Bermejo et al. 2013; Ruiz Diaz et al. 2015). Interestingly, some of these events are abrogated by the passive transfer of TS-neutralizing antibodies (Tribulatti et al. 2005; Buschiazzo et al. 2012), reinforcing the possibility that increased shedding of the enzyme correlates with the increased virulence of corresponding parasite strains (Risso et al. 2004).

3

4

glycophenotype adopted by activated T cells may favor building an effective immune response since Sia reduces the binding between CD8+ T and class MHC I. This is supported by data showing that cells from ST3GalI −/− mice show increased CD8-MHC I binding when compared with wild type (Moody et al. 2001). Besides the polyclonal activation of T cells and the massive apoptosis of immature and mature T cells, hypergammaglobulinaemia is also a typical event in the acute phase of Chagas disease (el Bouhdidi et al. 1994). In 2002, Gao and colleagues demonstrated that T. cruzi-TS, through its C-terminal (long tandem repeat) domain (SAPA), is capable of activating mouse B cells in a T-cell-independent manner. The SAPA as well as the whole enzyme is capable of inducing IL-6 (in bone marrow and splenocytes) and γ-IFN (in splenocytes). These authors were also able to correlate this T-cell-independent B-cell activation with polyclonal Ig secretion in vivo, which could explain, in part, its virulence-enhancing activity (Gao et al. 2002). In a recent study, Bermejo and colleagues were able to show that T. cruzi-TS is capable of inducing the production of IL-17 by B cells in both murine and human models. The effect was dependent on the sialylation of molecules on the cell surface, particularly CD45 (or another molecule that interacts with it), since its presence is essential for IL-17 production. It is also worth noting that TS triggers a different signaling pathway from the one classically associate with IL-17 and bypasses the activation of RORγt- and RORα. (Bermejo et al. 2013).

Sialoglycophenotypes adopted by CD8+ T cells during T. cruzi infection As cited above, the multifunctional properties of T. cruzi-TS are essential for its ability to modulate not only the sialoglycophenotype of the parasite but also of host cells. The fact that the sialylation of T. cruzi’s surface mucin-like molecules enhances its invasive capacity is no longer a question (Burleigh and Andrews 1995; de Souza et al. 2010). TS allows the protozoan to avoid immune responses through molecular mimicry, making it possible for T. cruzi to escape from the antibodies and proteins of the complement system directed against the parasite’s epitopes (Argibay et al. 2002; Gao et al. 2002). Several studies have confirmed the significance of differential sialylation for CD8+ T cells with respect to their development (Moody et al. 2001), activation via the TCR (Pappu and Shrikant 2004), and cytotoxic responses (Sadighi Akha et al. 2006). However, so far few studies have provided insights into the impact of surface sialylation of CD8+ T cells on the responses to pathogenic microorganisms. Infection with T. cruzi is of particular interest in this context because the parasite releases into the host plasma large amounts of aTS (Alcantara-Neves and Pontes-de-Carvalho 1995). Recently, we demonstrated that in a T. cruzi-TS-free system, such as the Plasmodium vivax infection, the activated CD8+ T cells predominantly exhibit the glycophenotype PNAhigh. However, in T. cruzi-infected mice, the glycophenotype PNA intermediary (PNAint) exhibited by activated CD8+ T cells can be converted into PNAlow after intravenous administration of aTS. Since the Ag-specific CD8+ T cell responses in T. cruzi-infected and TS-treated mice were impaired when compared with the T. cruzi-infected and TS-untreated group, it is reasonable to suggest that atypical acquired sialoglycophenotype (PNAlow) may contribute to increased parasite half-life in the infected host (Freire-de-Lima et al. 2010). Further studies are necessary to confirm that hypothesis, but it is plausible to speculate that the manipulation of Sia by T. cruzi-TS on CD8+ T cells from T. cruzi-infected individuals might be an evasion mechanism employed by the parasite to assure its replication in the infected host cells. Figure 3 illustrates a hypothetical molecular


note that such effects are specific for the transferase activity of TS, since the results could not be replicated in mice primed with viral or bacterial sialidases. The mechanisms responsible for those effects were not determined. However, since TS injection into severe combined immunodeficiency mice did not affect parasitemia or mortality, it was proposed that TS may act on host lymphocytes which are essential components of the acquired immune system (Chuenkova and Pereira 1995). Thymocyte and T-cell apoptosis are a common feature in acute parasite infections (Begum-Haque et al. 2009; Francelin et al. 2011). In the majority of infectious diseases with thymic atrophy, the most important biological event associated with thymocyte loss is the cell death by apoptosis. This is also observed in experimental Chagas disease (Savino 2006; Morrot et al. 2012). Several articles published by Campetella’s group have pointed out the pro-apoptotic effects of aTS both on thymocytes and mature T cells (Leguizamon et al. 1999; Mucci et al. 2002; Tribulatti et al. 2007). Several hematological alterations may be the result of desialylation by the neuraminidase activity of T. cruzi-TS (Pereira 1983; de Titto and Araujo 1988; Tribulatti et al. 2005). However, further studies regarding this aspect have proposed that the pro-apoptotic effect of T. cruzi-TS on immature and mature T cells may be induced by the incorporation of sialyl residues onto the cell surface that is mediated by its trans-sialidase activity (Mucci et al. 2006). It has also been shown that there is a correlation between apoptotic cell death and an imbalance in the hypothalamuspituitary-adrenal of T. cruzi-infected mice and GH3 cells. This later effect correlates with an increased production of glucocorticoids and a reduction in prolactin levels (Lepletier et al. 2012). In 2002, our group demonstrated that T. cruzi-TS was able to activate CD4+ T cells through engagement of CD43 (Todeschini et al. 2002b), an integral membrane glycoprotein typically expressed at high levels on all leucocytes, except most resting B lymphocytes (Ostberg et al. 1998). To promote such activation, it was necessary to combine T. cruzi-TS with anti-CD3 or anti-TCR monoclonal antibodies. By itself TS was not able to induce the activation of mouse purified CD4+ T cells in vitro (Todeschini et al. 2002b). T-cell activation is accompanied by loss of Sia from core 1 O-glycans (Siaα2,3Galβ1,3GalNAc-Ser/Thr), leading to enhanced expression of asialylated Galβ1,3GalNAc glycan species which results in the induction of core 2 O-linked glycans (Galvan et al. 1998; Priatel et al. 2000). The exposed asialylated core 1 O-glycans on activated T cells may be detected with peanut agglutinin (PNA), a plant lectin that recognizes Galβ1,3GalNAc sequences on several glycoproteins, including CD43 and CD45 (Wu et al. 1996). Since glycan structures bearing terminal βGalp units are potential substrates for T. cruzi-TS, it is plausible that T. cruzi-TS would be able to re-sialylate cell-surface asialoglycoconjugates in activated T cells (showing a PNAhigh phenotype), and therefore modulate crucial physiological events, such as proliferation, cytotoxic activity and T-cell half-life. On the other hand, in T cells from non-infected mice, which express a PNAlow glycophenotype, it is more likely that aTS exerts its neuraminidase activity. In this scenario, we would expect a stronger cell-to-cell interaction between desialylated non-activated T cells, an episode able to induce cell death triggered through the Fas-FasL pathway (Azuma et al. 2000; Tringali et al. 2007; Ghrici et al. 2013). In addition, asialoglycans generated by the hydrolytic action of aTS may be a potential target for the action of pro-apoptotic proteins belonging to the galectin family (Brandt et al. 2010; Lee et al. 2013). Although it is a reasonable speculation, such hypothesis still needs confirmation. Since glycoconjugates bearing terminal Sia residues promote cell– cell repulsion, it is reasonable to suggest that the PNAhigh



5

mechanism of how T. cruzi-TS might dampen the building of an effective CD8+ T cell response.

Conclusion and perspectives In this review, we have focused on the history of the discovery of T. cruzi-TS, as well as how this Sia-dependent enzyme might be able to interfere in many biological parameters to guarantee the persistence of the parasite in the infected host. Over the last 30 years, there have been significant efforts directed towards understanding the relevance of TS on the biology of T. cruzi. However, the lack of TS-specific inhibitors and TS knockout strains has hampered progress in the area. Although hundreds of convincing works reinforce the idea that T. cruzi-TS acts as a virulence factor during the acute phase of Chagas disease, there are still open questions that need to be addressed, such as the number and the genomic organization of the genes encoding iTS and aTS in different parasite strains (Burgos et al. 2013). Another issue that requires further investigation is the impact of the altered sialoglycophenotype of cells belonging to the host immune system. Since the blood-trypomastigote releases significant quantities of TS into the host plasma, infection with T. cruzi is of particular interest in this context. Some works have proposed that in B and T lymphocytes, cellsurface glycoproteins such as CD43 and/or CD45 may act as potential acceptors for TS activity (Todeschini et al. 2002a; Freire-de-Lima et al. 2010; Muia et al. 2010; Bermejo et al. 2013). The assumption is that such a phenomenon is able to disturb the content of Sia on those cells which may enables the parasite to evade the host immune response. In vitro and in vivo experiments suggest that CD43 is a possible ligand/ acceptor molecule for T. cruzi-TS. Initial studies revealed that the parasite’s enzyme is able to costimulate host T cells through leucosialin (CD43) engagement (Todeschini et al. 2002a). Further, Freire-deLima et al. (2010) showed in vivo by using ST3Gal-I KO mice that CD43 may act as a potential Sia acceptor in activated CD8+ T cells. CD45 is another glycoprotein expressed in all leukocytes that has been identified as a possible target for the parasite’s enzyme. In

2010, Muia and colleagues demonstrated by using chemical tags in Sia to show that different isoforms of CD45 may act as possible Sia acceptors for T. cruzi-TS. More recently, Bermejo et al. (2013) showed that CD45 is essential for the activation and production of IL-17 by B cells. Although this is a very interesting hypothesis with some supporting preliminary evidence, further studies with CD43 KO and CD45 KO mice are necessary to confirm this possibility. On a different note, since T. cruzi-TS plays such an important role in the parasite’s biology, TS is a tempting target for the development of new therapeutic agents. There are several groups investigating new alternatives to replace the highly toxic drugs currently used in the treatment the Chagas disease. Over the last 10 years, many potential inhibitors of TS have been described. However, due to no specificity for the parasite’s enzyme, many of them have yet to be tested in tissue-cultured cells or animal models. Because TS is such an appealing target for drug design and therapeutic intervention in Chagas’ disease substantial efforts in this area will, without doubt, increase in the coming years.

Funding This work was supported by the Fundação de Apoio à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Conflict of interest statement None declared.

Abbreviations aTS, active TS; iTS, inactive TS; NeuAc, N-acetylneuraminic acid; NeuGc, N-glycolylneuraminic acid; PNA, peanut agglutinin; SAPA, Shed Acute Phase Antigen; Sia, sialic acids; TrkA, tyrosine kinase receptor-A; βGalp, β-galactopyranosyl.


Fig. 3. Sialoglycophenotypes adopted by CD8+ T cells during T. cruzi infection. Glycan structures expressed by naïve CD8+ T cells are heavily sialylated. However, during T-cell activation, the glycan structures become poorly sialylated in response to reduced levels of expression and/or activity of sialyltransferases, which favors the mounting of an effective immune response. On the other hand, during T. cruzi infection, TS may re-sialylate the asialoglycoconjugates on T-cell surface, weakening the antigen-specific cytotoxic response.

6

References

class of mechanism-based inhibitors for Trypanosoma cruzi trans-sialidase and their influence on parasite virulence. Glycobiology. 20:1034–1045. Chang YC, Nizet V. 2014. The interplay between Siglecs and sialylated pathogens. Glycobiology. 24:818–825. Chen GY, Brown NK, Zheng P, Liu Y. 2014. Siglec-G/10 in self-nonself discrimination of innate and adaptive immunity. Glycobiology. 24:800–806. Chen X, Varki A. 2010. Advances in the biology and chemistry of sialic acids. ACS Chem Biol. 5:163–176. Chuenkova M, Pereira ME. 1995. Trypanosoma cruzi trans-sialidase: Enhancement of virulence in a murine model of Chagas’ disease. J Exp Med. 181:1693–1703. Chuenkova MV, PereiraPerrin M. 2004. Chagas’ disease parasite promotes neuron survival and differentiation through TrkA nerve growth factor receptor. J Neurochem. 91:385–394. Chuenkova MV, PereiraPerrin M. 2005. A synthetic peptide modeled on PDNF, Chagas’ disease parasite neurotrophic factor, promotes survival and differentiation of neuronal cells through TrkA receptor. Biochemistry. 44:15685–15694. Chuenkova MV, Pereiraperrin M. 2011. Neurodegeneration and neuroregeneration in Chagas disease. Adv Parasitol. 76:195–233. Cohen M, Varki A. 2010. The sialome – far more than the sum of its parts. OMICS. 14:455–464. Cremona ML, Sanchez DO, Frasch AC, Campetella O. 1995. A single tyrosine differentiates active and inactive Trypanosoma cruzi trans-sialidases. Gene. 160:123–128. Crespo HJ, Cabral MG, Teixeira AV, Lau JT, Trindade H, Videira PA. 2009. Effect of sialic acid loss on dendritic cell maturation. Immunology. 128: e621–e631. Dc-Rubin SS, Schenkman S. 2012. T rypanosoma cruzi trans-sialidase as a multifunctional enzyme in Chagas’ disease. Cell Microbiol. 14: 1522–1530. de Melo-Jorge M, PereiraPerrin M. 2007. The Chagas’ disease parasite Trypanosoma cruzi exploits nerve growth factor receptor TrkA to infect mammalian hosts. Cell Host Microbe. 1:251–261. de Souza W, de Carvalho TM. 2013. Active penetration of Trypanosoma cruzi into host cells: Historical considerations and current concepts. Front Immunol. 4:2. de Souza W, de Carvalho TM, Barrias ES. 2010. Review on Trypanosoma cruzi: Host cell interaction. Int J Cell Biol. 2010:1–18. de Titto EH, Araujo FG. 1988. Serum neuraminidase activity and hematological alterations in acute human Chagas’ disease. Clin Immunol Immunopathol. 46:157–161. DosReis GA. 2011. Evasion of immune responses by Trypanosoma cruzi, the etiological agent of Chagas disease. Braz J Med Biol Res. 44:84–90. el Bouhdidi A, Truyens C, Rivera MT, Bazin H, Carlier Y. 1994. Trypanosoma cruzi infection in mice induces a polyisotypic hypergammaglobulinaemia and parasite-specific response involving high IgG2a concentrations and highly avid IgG1 antibodies. Parasite Immunol. 16:69–76. Francelin C, Paulino LC, Gameiro J, Verinaud L. 2011. Effects of Plasmodium berghei on thymus: High levels of apoptosis and premature egress of CD4(+) CD8(+) thymocytes in experimentally infected mice. Immunobiology. 216:1148–1154. Frasch AC. 1994. Trans-sialidase, SAPA amino acid repeats and the relationship between Trypanosoma cruzi and the mammalian host. Parasitology. 108 Suppl.:S37–S44. Frasch AC. 2000. Functional diversity in the trans-sialidase and mucin families in Trypanosoma cruzi. Parasitol Today. 16:282–286. Freire-de-Lima L, Alisson-Silva F, Carvalho ST, Takiya CM, Rodrigues MM, DosReis GA, Mendonca-Previato L, Previato JO, Todeschini AR. 2010. Trypanosoma cruzi subverts host cell sialylation and may compromise antigen-specific CD8+ T cell responses. J Biol Chem. 285:13388–13396. Galvan M, Murali-Krishna K, Ming LL, Baum L, Ahmed R. 1998. Alterations in cell surface carbohydrates on T cells from virally infected mice can distinguish effector/memory CD8+ T cells from naive cells. J Immunol. 161:641–648. Gao W, Pereira MA. 2001. Trypanosoma cruzi trans-sialidase potentiates T cell activation through antigen-presenting cells: Role of IL-6 and Bruton’s tyrosine kinase. Eur J Immunol. 31:1503–1512.


Alcantara-Neves NM, Pontes-de-Carvalho LC. 1995. Circulating transsialidase activity and trans-sialidase-inhibiting antibodies in Trypanosoma cruzi-infected mice. Parasitol Res. 81:560–564. Alvarez P, Buscaglia CA, Campetella O. 2004. Improving protein pharmacokinetics by genetic fusion to simple amino acid sequences. J Biol Chem. 279:3375–3381. Amaya MF, Watts AG, Damager I, Wehenkel A, Nguyen T, Buschiazzo A, Paris G, Frasch AC, Withers SG, Alzari PM. 2004. Structural insights into the catalytic mechanism of Trypanosoma cruzi trans-sialidase. Structure. 12:775–784. Amon R, Reuven EM, Leviatan Ben-Arye S, Padler-Karavani V. 2014. Glycans in immune recognition and response. Carbohydr Res. 389:115–122. Angata K, Fukuda M. 2010. Roles of polysialic acid in migration and differentiation of neural stem cells. Methods Enzymol. 479:25–36. Argibay PF, Di Noia JM, Hidalgo A, Mocetti E, Barbich M, Lorenti AS, Bustos D, Tambutti M, Hyon SH, Frasch AC, et al. 2002. Trypanosoma cruzi surface mucin TcMuc-e2 expressed on higher eukaryotic cells induces human T cell anergy, which is reversible. Glycobiology. 12:25–32. Aridgides D, Salvador R, PereiraPerrin M. 2013. Trypanosoma cruzi coaxes cardiac fibroblasts into preventing cardiomyocyte death by activating nerve growth factor receptor TrkA. PLoS ONE. 8:e57450. Azuma Y, Taniguchi A, Matsumoto K. 2000. Decrease in cell surface sialic acid in etoposide-treated Jurkat cells and the role of cell surface sialidase. Glycoconj J. 17:301–306. Barrias ES, de Carvalho TM, De Souza W. 2013. Trypanosoma cruzi: Entry into mammalian host cells and parasitophorous vacuole formation. Front Immunol. 4:186. Begum-Haque S, Haque A, Kasper LH. 2009. Apoptosis in Toxoplasma gondii activated T cells: The role of IFNgamma in enhanced alteration of Bcl-2 expression and mitochondrial membrane potential. Microb Pathog. 47:281–288. Bermejo DA, Jackson SW, Gorosito-Serran M, Acosta-Rodriguez EV, Amezcua-Vesely MC, Sather BD, Singh AK, Khim S, Mucci J, Liggitt D, et al. 2013. Trypanosoma cruzi trans-sialidase initiates a program independent of the transcription factors RORgammat and Ahr that leads to IL-17 production by activated B cells. Nat Immunol. 14:514–522. Brandt B, Abou-Eladab EF, Tiedge M, Walzel H. 2010. Role of the JNK/c-Jun/ AP-1 signaling pathway in galectin-1-induced T-cell death. Cell Death Dis. 1:e23. Buchini S, Buschiazzo A, Withers SG. 2008. A new generation of specific Trypanosoma cruzi trans-sialidase inhibitors. Angew Chem Int Ed Engl. 47:2700–2703. Burgos JM, Risso MG, Breniere SF, Barnabe C, Campetella O, Leguizamon MS. 2013. Differential distribution of genes encoding the virulence factor transsialidase along Trypanosoma cruzi discrete typing units. PLoS ONE. 8: e58967. Burleigh BA, Andrews NW. 1995. The mechanisms of Trypanosoma cruzi invasion of mammalian cells. Annu Rev Microbiol. 49:175–200. Buschiazzo A, Amaya MF, Cremona ML, Frasch AC, Alzari PM. 2002. The crystal structure and mode of action of trans-sialidase, a key enzyme in Trypanosoma cruzi pathogenesis. Mol Cell. 10:757–768. Buschiazzo A, Muia R, Larrieux N, Pitcovsky T, Mucci J, Campetella O. 2012. Trypanosoma cruzi trans-sialidase in complex with a neutralizing antibody: Structure/function studies towards the rational design of inhibitors. PLoS Pathog. 8:e1002474. Buschiazzo A, Tavares GA, Campetella O, Spinelli S, Cremona ML, Paris G, Amaya MF, Frasch AC, Alzari PM. 2000. Structural basis of sialyltransferase activity in trypanosomal sialidases. EMBO J. 19:16–24. Butler CE, de Carvalho TM, Grisard EC, Field RA, Tyler KM. 2013. Transsialidase stimulates eat me response from epithelial cells. Traffic. 14:853–869. Caradonna KL, Burleigh BA. 2011. Mechanisms of host cell invasion by Trypanosoma cruzi. Adv Parasitol. 76:33–61. Carvalho ST, Sola-Penna M, Oliveira IA, Pita S, Goncalves AS, Neves BC, Sousa FR, Freire-de-Lima L, Kurogochi M, Hinou H, et al. 2010. A new



Paris G, Ratier L, Amaya MF, Nguyen T, Alzari PM, Frasch AC. 2005. A sialidase mutant displaying trans-sialidase activity. J Mol Biol. 345:923–934. Parodi AJ, Pollevick GD, Mautner M, Buschiazzo A, Sanchez DO, Frasch AC. 1992. Identification of the gene(s) coding for the trans-sialidase of Trypanosoma cruzi. EMBO J. 11:1705–1710. Pereira ME. 1983. A developmentally regulated neuraminidase activity in Trypanosoma cruzi. Science. 219:1444–1446. Pereira ME, Loures MA, Villalta F, Andrade AF. 1980. Lectin receptors as markers for Trypanosoma cruzi. Developmental stages and a study of the interaction of wheat germ agglutinin with sialic acid residues on epimastigote cells. J Exp Med. 152:1375–1392. Pereira ME, Mejia JS, Ortega-Barria E, Matzilevich D, Prioli RP. 1991. The Trypanosoma cruzi neuraminidase contains sequences similar to bacterial neuraminidases, YWTD repeats of the low density lipoprotein receptor, and type III modules of fibronectin. J Exp Med. 174:179–191. Previato JO, Andrade AF, Pessolani MC, Mendonca-Previato L. 1985. Incorporation of sialic acid into Trypanosoma cruzi macromolecules. A proposal for a new metabolic route. Mol Biochem Parasitol. 16:85–96. Previato J, Andrade A, Vermelho A, Firmino J, Mendonça-Previato L. 1990. Evidence for N-glycolylneuraminic acid incorporation by Trypanosoma cruzi from infected animal. Mem Inst Oswaldo Cruz. 85:38–39. Priatel JJ, Chui D, Hiraoka N, Simmons CJ, Richardson KB, Page DM, Fukuda M, Varki NM, Marth JD. 2000. The ST3Gal-I sialyltransferase controls CD8+ T lymphocyte homeostasis by modulating O-glycan biosynthesis. Immunity. 12:273–283. Ribeirao M, Pereira-Chioccola VL, Eichinger D, Rodrigues MM, Schenkman S. 1997. Temperature differences for trans-glycosylation and hydrolysis reaction reveal an acceptor binding site in the catalytic mechanism of Trypanosoma cruzi trans-sialidase. Glycobiology. 7:1237–1246. Risso MG, Garbarino GB, Mocetti E, Campetella O, Gonzalez Cappa SM, Buscaglia CA, Leguizamon MS. 2004. Differential expression of a virulence factor, the trans-sialidase, by the main Trypanosoma cruzi phylogenetic lineages. J Infect Dis. 189:2250–2259. Rubin-de-Celis SS, Uemura H, Yoshida N, Schenkman S. 2006. Expression of trypomastigote trans-sialidase in metacyclic forms of Trypanosoma cruzi increases parasite escape from its parasitophorous vacuole. Cell Microbiol. 8:1888–1898. Ruiz Diaz P, Mucci J, Meira MA, Bogliotti Y, Musikant D, Leguizamon MS, Campetella O. 2015. Trypanosoma cruzi trans-sialidase prevents elicitation of Th1 cell response via interleukin 10 and downregulates Th1 effector cells. Infect Immun. 83:2099–2108. Sadighi Akha AA, Berger SB, Miller RA. 2006. Enhancement of CD8 T-cell function through modifying surface glycoproteins in young and old mice. Immunology. 119:187–194. Savino W. 2006. The thymus is a common target organ in infectious diseases. PLoS Pathog. 2:e62. Schauer R, Reuter G, Muhlpfordt H, Andrade AF, Pereira ME. 1983. The occurrence of N-acetyl- and N-glycoloylneuraminic acid in Trypanosoma cruzi. Hoppe Seylers Z Physiol Chem. 364:1053–1057. Schenkman S, Jiang MS, Hart GW, Nussenzweig V. 1991. A novel cell surface trans-sialidase of Trypanosoma cruzi generates a stage-specific epitope required for invasion of mammalian cells. Cell. 65:1117–1125. Schenkman RP, Vandekerckhove F, Schenkman S. 1993. Mammalian cell sialic acid enhances invasion by Trypanosoma cruzi. Infect Immun. 61:898–902. Todeschini AR, Dias WB, Girard MF, Wieruszeski JM, Mendonça-Previato L, Previato JO. 2004. Enzymatically inactive trans-sialidase from Trypanosoma cruzi binds sialyl and beta-galactopyranosyl residues in a sequential ordered mechanism. J Biol Chem. 279:5323–5328. Todeschini AR, Girard MF, Wieruszeski JM, Nunes MP, DosReis GA, Mendonça-Previato L, Previato JO. 2002a. trans-Sialidase from Trypanosoma cruzi binds host T-lymphocytes in a lectin manner. J Biol Chem. 277:45962–45968. Todeschini AR, Nunes MP, Pires RS, Lopes MF, Previato JO, MendonçaPreviato L, DosReis GA. 2002b. Costimulation of host T lymphocytes by


Gao W, Wortis HH, Pereira MA. 2002. The Trypanosoma cruzi trans-sialidase is a T cell-independent B cell mitogen and an inducer of non-specific Ig secretion. Int Immunol. 14:299–308. Ghrici M, El Zowalaty M, Omar AR, Ideris A. 2013. Induction of apoptosis in MCF-7 cells by the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus Malaysian strain AF2240. Oncol Rep. 30: 1035–1044. Hedlund M, Aschenbrenner LM, Jensen K, Larson JL, Fang F. 2010. Sialidasebased anti-influenza virus therapy protects against secondary pneumococcal infection. J Infect Dis. 201:1007–1015. Hinou H, Kurogochi M, Shimizu H, Nishimura S. 2005. Characterization of Vibrio cholerae neuraminidase by a novel mechanism-based fluorescent labeling reagent. Biochemistry. 44:11669–11675. Lee YK, Lin TH, Chang CF, Lo YL. 2013. Galectin-3 silencing inhibits epirubicin-induced ATP binding cassette transporters and activates the mitochondrial apoptosis pathway via beta-catenin/GSK-3beta modulation in colorectal carcinoma. PLoS ONE. 8:e82478. Leguizamon MS, Mocetti E, Garcia Rivello H, Argibay P, Campetella O. 1999. Trans-sialidase from Trypanosoma cruzi induces apoptosis in cells from the immune system in vivo. J Infect Dis. 180:1398–1402. Lepletier A, de Frias Carvalho V, Morrot A, Savino W. 2012. Thymic atrophy in acute experimental Chagas disease is associated with an imbalance of stress hormones. Ann N Y Acad Sci. 1262:45–50. Lieke T, Grobe D, Blanchard V, Grunow D, Tauber R, ZimmermannKordmann M, Jacobs T, Reutter W. 2011. Invasion of Trypanosoma cruzi into host cells is impaired by N-propionylmannosamine and other N-acylmannosamines. Glycoconj J. 28:31–37. Mendonça-Previato L, Penha L, Garcez TC, Jones C, Previato JO. 2013. Addition of alpha-O-GlcNAc to threonine residues define the post-translational modification of mucin-like molecules in Trypanosoma cruzi. Glycoconj J. 30:659–666. Mendonça-Previato L, Todeschini AR, de Lima LF, Previato JO. 2010. The trans-sialidase from Trypanosoma cruzi a putative target for trypanocidal agents. Open Parasitol J. 4:111–115. Moody AM, Chui D, Reche PA, Priatel JJ, Marth JD, Reinherz EL. 2001. Developmentally regulated glycosylation of the CD8alphabeta coreceptor stalk modulates ligand binding. Cell. 107:501–512. Morrot A, Barreto de Albuquerque J, Berbert LR, de Carvalho Pinto CE, de Meis J, Savino W. 2012. Dynamics of lymphocyte populations during Trypanosoma cruzi infection: From thymocyte depletion to differential cell expansion/contraction in peripheral lymphoid organs. J Trop Med. 2012:747185. Mucci J, Hidalgo A, Mocetti E, Argibay PF, Leguizamon MS, Campetella O. 2002. Thymocyte depletion in Trypanosoma cruzi infection is mediated by trans-sialidase-induced apoptosis on nurse cells complex. Proc Natl Acad Sci USA. 99:3896–3901. Mucci J, Mocetti E, Leguizamon MS, Campetella O. 2005. A sexual dimorphism in intrathymic sialylation survey is revealed by the trans-sialidase from Trypanosoma cruzi. J Immunol. 174:4545–4550. Mucci J, Risso MG, Leguizamon MS, Frasch AC, Campetella O. 2006. The trans-sialidase from Trypanosoma cruzi triggers apoptosis by target cell sialylation. Cell Microbiol. 8:1086–1095. Muia RP, Yu H, Prescher JA, Hellman U, Chen X, Bertozzi CR, Campetella O. 2010. Identification of glycoproteins targeted by Trypanosoma cruzi transsialidase, a virulence factor that disturbs lymphocyte glycosylation. Glycobiology. 20:833–842. Neu U, Bauer J, Stehle T. 2011. Viruses and sialic acids: Rules of engagement. Curr Opin Struct Biol. 21:610–618. Nishikawa Y, Ogiso A, Kameyama K, Nishimura M, Xuan X, Ikehara Y. 2013. Alpha2–3 sialic acid glycoconjugate loss and its effect on infection with Toxoplasma parasites. Exp Parasitol. 135:479–485. Ostberg JR, Barth RK, Frelinger JG. 1998. The Roman god Janus: A paradigm for the function of CD43. Immunol Today. 19:546–550. Pappu BP, Shrikant PA. 2004. Alteration of cell surface sialylation regulates antigen-induced naive CD8+ T cell responses. J Immunol. 173: 275–284.

7

8

Varki A, Schauer R. 2009. Sialic acids. In: Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, Hart GW, Etzler ME, editors. Essentials of Glycobiology. Cold Spring Harbor (NY). Watts AG, Damager I, Amaya ML, Buschiazzo A, Alzari P, Frasch AC, Withers SG. 2003. Trypanosoma cruzi trans-sialidase operates through a covalent sialyl-enzyme intermediate: Tyrosine is the catalytic nucleophile. J Am Chem Soc. 125:7532–7533. Wiggins R, Hicks SJ, Soothill PW, Millar MR, Corfield AP. 2001. Mucinases and sialidases: Their role in the pathogenesis of sexually transmitted infections in the female genital tract. Sex Transm Infect. 77:402–408. Woronowicz A, De Vusser K, Laroy W, Contreras R, Meakin SO, Ross GM, Szewczuk MR. 2004. Trypanosome trans-sialidase targets TrkA tyrosine kinase receptor and induces receptor internalization and activation. Glycobiology. 14:987–998. Wu W, Harley PH, Punt JA, Sharrow SO, Kearse KP. 1996. Identification of CD8 as a peanut agglutinin (PNA) receptor molecule on immature thymocytes. J Exp Med. 184:759–764. Zingales B, Carniol C, de Lederkremer RM, Colli W. 1987. Direct sialic acid transfer from a protein donor to glycolipids of trypomastigote forms of Trypanosoma cruzi. Mol Biochem Parasitol. 26:135–144.


a trypanosomal trans-sialidase: Involvement of CD43 signaling. J Immunol. 168:5192–5198. Traving C, Schauer R. 1998. Structure, function and metabolism of sialic acids. Cell Mol Life Sci. 54:1330–1349. Tribulatti MV, Mucci J, Cattaneo V, Aguero F, Gilmartin T, Head SR, Campetella O. 2007. Galectin-8 induces apoptosis in the CD4(high) CD8(high) thymocyte subpopulation. Glycobiology. 17:1404–1412. Tribulatti MV, Mucci J, Van Rooijen N, Leguizamon MS, Campetella O. 2005. The trans-sialidase from Trypanosoma cruzi induces thrombocytopenia during acute Chagas’ disease by reducing the platelet sialic acid contents. Infect Immun. 73:201–207. Tringali C, Lupo B, Anastasia L, Papini N, Monti E, Bresciani R, Tettamanti G, Venerando B. 2007. Expression of sialidase Neu2 in leukemic K562 cells induces apoptosis by impairing Bcr-Abl/Src kinases signaling. J Biol Chem. 282:14364–14372. Uemura H, Schenkman S, Nussenzweig V, Eichinger D. 1992. Only some members of a gene family in Trypanosoma cruzi encode proteins that express both trans-sialidase and neuraminidase activities. EMBO J. 11:3837–3844. Varki A, Gagneux P. 2012. Multifarious roles of sialic acids in immunity. Ann N Y Acad Sci. 1253:16–36.
