Técia Ulisses de Carvalho and Emile Barrias
Biophysics Program, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro
Email: email@example.com, firstname.lastname@example.org
Adhesion involves the recognition of molecules present in both the cells involved: the parasite and its host. Adhesion is therefore a process mediated by receptors, restricted to membrane dominions. Not every trypomastigote form that adheres to a host cell invades it or is phagocyted. In studies of interaction between host cells and T. cruzi one must take into account: (i) the strain of T. cruzi used; (ii) the evolutionary form; (iii) the form of the trypomastigote used (thin or wide); and (iv) the type of host cell. The mechanisms of recognition, signalling and invasion (or phagocytosis) of T. cruzi with host cells are complex. Various molecules in both cells have been implicated in this initial recognition, including glycoproteins, glycolipids and lectin-type proteins. Most glycoproteins that participate in this process are anchored by glycosylphosphatidylinositol (GPI), external and variable N-terminal domain is located in the glycocalyx of the parasite, facilitating connection with many components of the host cell membrane and of the extracellular matrix. One of the glycoproteins that have this function of recognition between parasite and host cell is TSSA. In addition to the adhesion process, this protein is also involved in signal triggering processes, working as an antigen (Camara et al, 2017). One of the most intensely studied molecules, present in the plasma membrane of trypomastigote forms (but also in amastigotes, albeit in a smaller amount), is a protein with neuraminidase and transialidase activity. This last enzyme removes sialic acid residues (located in position α-1-3) from glycoproteins, glycolipids and oligosaccharides present in the medium, and transfers them to acceptor molecules (called mucins) in the plasma membrane of the trypomastigote forms. It has been demonstrated that sialic acid works as a modulating molecule for the adhesion process between T. cruzi and the host cells. Molecules gp82 (present in metacyclic forms) and gp85 (present in trypomastigotes from cellular culture) are members of the superfamily gp85/sialidase. Some T. cruzi molecules are considered pleiotropic. Such is the case of the calreticulin protein of T. cruzi, as it is present not only in the parasite’s endoplasmatic reticulum, but also on its surface. This protein participates in key processes to establish infection by T. cruzi, such as inhibition of the complement system, and works as an important virulent factor. The calreticulin protein, when bound to the main components of complement, also functions as an anti-angiogenic and antitumor molecule, helping with the inhibition of tumoral growth in infected animals (Ramirez-Tolosa e Ferreira, 2017).
Depending on the evolutionary form of T. cruzi studied, different molecules have been identified as participating in the parasite-host cell adhesion process:
1) Molecules present in the membrane of metacyclic trypomastigotes that are important for the interaction process: gp82(which binds do gastric mucin); molecules similar to mucins (gp35/50); gp90 (molecule that negatively modulates the parasite’s entrance in the cell). These molecules recognize receptors, not yet identified, in the host cells. They trigger the signalling machinery in the parasite as well as in the host cell. Signalling triggered by gp35/40 or gp90 has low or no increase in cytoplasmatic calcium. Molecules gp82, gp35/50 and gp90 are anchored to the plasma membrane of the parasite through an anchor of glycosylphosphatidylinositol (GPI).
2) Molecules present in the membrane of trypomastigotes obtained from the culture of infected cells: gp85 (recognizes components of the extracellular matrix, such as fibronectin, laminin and cytokeratin 18); molecules similar to mucins (molecules with weight ranging from 70 to 200 kDa); transialidase (negatively regulates the parasite’s entrance in the cell). One antigen present in the acute phase of Chagas disease, called SAPA, has been identified as a transialidase; cysteine proteases (antigen gp57/51, cruzipain); oligopeptidases (serinea protease); penetrin, a 60 kDa-protein (affinity for proteins of the extracellular matrix, binding to heparine, heparan sulfate and collagen).
3) Molecules present in the membrane of blood trypomastigotes: molecules of the superfamily of gp85/TS, which binds to components of the extracellular matrix (fibronectin/laminin); TS; mucin.
As for the host cell (mammal), it is believed that any class of molecules exposed on its surface has the potential to be a binding receptor for T. cruzi. Most classes of receptors are characterized by carbohydrates containing residues of galactosyl, mannosyl and sialyl, in addition to proteins similar to lectins (such as galectin 3), which bind to residues of carbohydrates present on the surface of the parasite. Some lectins, such as the mannose-binding lectin, participate in processes of humoral pattern regocnition that are important for the host’s defence. In the case of Chagas disease, this lectin is involved in the regulation of host resistance and cardiac inflammation during the infection. Other molecules that work as receptors possibly involved in the pathogenesis of Chagas disease are endothelin 1 and bradykinin receptors. These are used by trypomastigotes to invade cardiovascular cells, leading to the Chagas cardiac disease. Cytokeratin 18, fibronectin, laminin and integrins are also receptor molecules, as the Tc85 present on the surface of trypomastigotes have motifs that bind to these molecules, bridging the gap between the parasite and the host cell. Below is a schematic representation of the main molecules involved in this recognition process.
After the recognition (adhesion) of the parasite to the surface of the host cell, a series of cellular signalling occur, culminating in the parasite’s internalization in a host cell. There are many mechanisms used for this process. One entrance mechanism is phagocytosis/macropinotysosis, in which cells emit pseudopodes and there is participatin of actin filaments. Although the phagocytosis process is described as being restricted to specialized cells, the macropinocytosis process can be observed in professional and non-professional phagocytes. In these processes there is not just the participation of actin filamnets, but also activation of thyrosin kinases, phosphatidylinositol kinases and PAK kinases (De Souza et al, 2013). The main difference between phagocytosis and macropinocytosis is the emission of pseudopodes or membrane ruffles, as well as the internalization of extracellular fluids concomitant to the parasite’s entrance in the cell. The process known as autophagy can also be triggered by the entrance (and formation of autofagosome) of T. cruzi. This process is marked by the activation of the mTor signalling pathway and by the recruiting of protein LC3b. Endocytosis mechanisms, in which no emission of pseudopodes occurs but there is involvement of actin filaments, are involved in the entrance process of T. cruzi, as receptors present in clathrin-mediated endocytosis sites, as well as in proteins present in regions known as membrane macro domains (flotillins and caveolins), have been described as important for the entrance of this parasite. The regions of membrane micro domains are also classically known as signalling hotspots, as many proteins that trigger different signalling pathways are related to this region. Trypomastigotes can also enter through membrane invagination, without the participation of actin filaments. This last process has been considered an active mechanism for the parasite’s entry with energy waste.
In the early moments of T. cruzi recognition with the cell host, there is a transient increase in the cytoplasmatic levels of calcium (in the parasite as well as in the host cell). This calcium increase is important for the parasite’s entrance: if this transient increase in cytoplasmatic calcium is blocked, using thapsigargina, for instance, this entry is reduced. It has also been demonstrated that a recruitment of lysosomes to the parasite’s invasion site occurs, although this phenomenon does not take place for all invading parasites (it happens in about 20% of them). In addition to starting the acidification process of the future parasitophorous vacuole, lysosomes have the function of donating membrane for the formation of the vacuole. There have also been descriptions of the participation of the plasma membrane regions and early endosomes of guest cells in this entry mechanism. The fusion of vesicles of the endocytic pathway (early endosome, late endosome, and recycling endosome) is described as crucial for the maturation of the vacuole containingT. cruzi. Regardless of the entrance mechanism used by the parasite, it ends up in a transient endocytic vacuole. This vacuole will always be fused to lysosomes (regardless of whether at the entrance site or somewhere along the endocytic pathway), forming a phagolysosome. This vacuole is considered transient because after two hours the parasite destroys its membrane and escapes into the cytoplasm, where it concludes the differentiation process into amastigote. It has been demonstrated that transient calcium increases in the cytoplasm of the host cell after interaction with trypomastigote forms of T. cruzi causes a reorganization of the actin cytoskeleton. It seems that actin is depolymerized at the entrance site, facilitating the invasion by the parasite. Several works using cytochalasin D (agent that depolymerizes actin filaments) to treat host cells before the interaction process show very controversial data: some authors describe the treatment as inhibiting trypomastigote forms, while others describe a marked increase in this entrance. Other authors say no effects are observed in the entrance of trypomastigotes after this treatment. However, when all these data are analyzed, we can see that treatment time, interaction times with the trypomastigotes, the host cells used and the strains of T. cruzi are all different.
The actin cytoskeleton has been shown to be very important in the retention of trypomastigote cells in the cytoplasm of the host cell. During the formation of the parasitophorous vacuole, we can see the presence of a belt of filamentous actin around this vacuole. It is believed that this belt is related to the phagolysosome fragmentation process. The entrance of trypomastigote forms triggers, in the host cells, a signalling process that leads to the parasite’s invasion of the cell. In professional phagocytic cells, this activates tyrosine kinases, recruits PI-3 kinase and actin for the parasite’s entrance site. These data show that the main entrance mechanism into macrophages is phagocytosis. In professional non-phagocytic cells, there is no activation of tyrosine kinases, as shown in works in which inhibitors of these enzymes are used, resulting in no reduction of the invasion process. However, activation of PI-3 kinase does occur, and this activation seems to be the regulator of phagocytosis, with the participation of lysosomes of the host cell. Tyrosine phosphatases have also been demonstrated to participate in this process. More recently, the invasion process of T. cruzi has also been related to the microvesicles released by these parasites. Microvesicles can be secreted naturally or induced by chemical methods. These microvesicles, released by trypomastigotes and amastigotes, may contain virulence factors involved in: (i) invasion of host cells and development of intracellular parasites; (ii) immune evasion; and (iii) increase in cardiac parasitism, inflammation and arrhythmia that contribute to the pathogenesis of Chagas disease. In addition, some of the segregated/excreted proteins work as diagnostic markers for Chagas disease (Jazin et al., 1995; Umezawa et al., 1996; Agusti et al., 2000; Bernabó et al., 2013).
Técia Ulisses de Carvalho and Emile Barrias
Biophysics Program, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro
Email: email@example.com, firstname.lastname@example.org
In the previous item we saw that trypomastigote forms of T. cruzi use different receptors/binders to enter host cells. Regardless of the mechanism used (fusion of lysosomes at the entrance site, participation of plasma membrane components or fusion of early endosomes at the invasion site), the parasite will end up in a vacuole. The membrane of this vacuole does not have all the components of the plasma membrane. Studies show that some molecules are excluded when the vacuole is formed. Soon after, the vacuole merges with lysosomes of the host cell, forming the phagolysosome (parasitophorous vacuole) and begins to present an acid pH. The parasitophorous vacuoles of this phase contain acid phosphatase as well as lysosome marker glycoproteins (such as LAMPs). Within this phagolysosome, T. cruzi goes through different stages:
1) The parasite secretes transialidase/neuraminidase which removes sialic acid residues from the phagolysosome, making the membrane of this vacuole sensitive to the action of Tc-Tox (a peptide that is homologous to factor 9 of human complement), and as it enters the phagolysosome membrane, it creates small pores that end up destryoing the membrane of the parasitophorous vacuole. The formation of these pores, together with enzymes secreted by the parasite, leads to the fragmentation of the vacuole membrane. During this period, the trypomastigote form begins its differentiation process into amastigote: the parasite begins to look rounded and its long flagellum becomes associated with the parasite’s body. The protein galectin 3 has recently been described as a lyse marker for the parasitophorous vacuole of T. cruzi (Reignault et al, 2013);
2) Fragmentation of the membrane of the parasitophorous vasuole can be observed through transmission electron microscopy about 2 hours after infection (PHOTOS);
3) Release of forms ongoing differentiation into the cytoplasm of host cells. The kinetoplast begins to change shape (from the rounded shape, typical of trypomastigotes, into the rod or bar shape, the organization found in the amastigote form) and to migrate to the region posterior to the nucleus;
4) The amastigote form then goes through a period of about 20 to 35 hours (depending on the strain) before it begins to multiply in the cytoplasm through binary division. During this multiplication process, there is a direct relation with the organelles of the host cell, such as mitochondria (Burleigh, 2018), and use of the host’s lipids (Gazos-Lopes et al, 2017);
5) After successive multiplications, the amastigote forms, which now occupy the entire cytoplam of the cell (except for the nucleus), begin a differentiation process into the trypomastigote form. As the invasion process is not synchronized, and as each cell can be invaded by more than one trypomastigote form, it is possible to find, in a cell filled with parasites, trypomastigote forms (in general, they make up the majority), amastigotes, and different forms ongoing differentiation;
6) The amastigote form is also infective. Therefore, if the cell is infected by amastigote forms, the beginning of the multiplication process is faster, as the trypo/ama transformation stage will not occur. The amastigote form can also use different receptors and signalling pathways used by trypomastigotes to infect, and also lyses the membrane of the parasitophorous vacuole in the same way that the trypomastigote does;
7) During the cellular division process, initially the amastigote cell grows in size; then there is a duplication of the basal body, beginning of kinetoplasm division, and modification of nuclear chromatin distribution. A new flagellum then begins to form. Nuclear chromatin becomes less electron-dense (in observations through transmission electronic microscopy), and bundles of intranuclear microtubules appear together with electron-dense plaques (10 plaques were observed). During the nuclear division phase, the nuclear membrane does not disintegrate: this is a closed mitosis. Trypanosomatides do not condensate chromatin with the formation of chromosomes;
8) Successive divisions occur, leading to the occupation of the entire cytoplasm of the host cell. Depending on the size of the host cell, hundreds of amastigotes may form. Some authors describe that the division stops when a defined number of divisions is reached (9 divisions);9) The differentiation process for trypomastigote forms begins when the division process for amastigote forms is concluded. Not much is known about this trypo/ama differentiation process, but it seems that the lack of nutriens in highly infected cells is significant. These forms are very mobile, and they secrete enzymes. It is believed that these two factors cause the plasma membrane to break, consequently releasing the trypomastigote forms into the extracellular medium.
Mirian Claudia S Pereira, Tatiana Galvão de Melo de Oliveira, Francisco Odencio Oliveira-Jr, Claudia Magalhães Calvet and Maria de Nazareth SL de Meirelles.
Cellular Ultrastructure Laboratory, IOC/Fiocruz
The tropism of Trypanosoma cruzi for muscular cells, especially cardiac muscle cells, can result in progressive cardiomyopathy in the chronic phase of Chagas disease. Understanding the molecular basis of the invasion process of T. cruziinto host cells has been a challenge, but it may help comprehend the alterations that take place during the infectious process. The implementation of cellular culture models, primary cultures (2D and 3D) and cellular lineages has resulted in breakthroughs in the knowledge of the biological and molecular events of the T. cruzi-host cell interaction.
The biomolecular interactions that take place durign the cellular recognition phase are crucial for the establishment of the infection. Different molecules are involved in this process, mediating adhesion by the binding with receptors and/or binders on the surface of the host cell and of the parasite (Calvet et al., 2012). The negative surface of cardiomyocytes modulates the adhesion and invasion of T. cruzi by means of the participation of members of the gp85/trans-sialidase family present on the surface of the parasite. Trans-sialidases (TS) play a double role in the invasion process, removing sialic acid from the surface of the target cell and transfering it to acceptors on the surface of the parasite, as well as working as a multi-adhesive molecule with binding properties with laminin and cytokeratin 18 (Magdesian et al., 2007), through their FLY and TS9 domains (Tonelli et al., 2010, Teixeira et al., 2015). Studies have demonstrated the binding capacity of the FLY domain of TS to molecules on the surface of cardiomyocytes and endothelium of cardiac vessels (Mattos et al., 2014, Tonelli et al., 2010, 2011). Carbohydrates play an important role in the recognition of professional and non-professional phagotytic cells by T. cruzi, and the participation of residues of mannose, galactose, N-acetyl galactosamin and N-acetyl glycosamin has been demonstrated in the parasite’s invasion of cardiomyocytes (Araújo-Jorge et al., 1992, Barbosa & Meirelles 1993). The role of surface receptors and components of the extracellular matrix has also been highlighted during the invasion process. Studies have revealed the participation of mannose receptors in this process, demonstrating a down-regulation of these receptors on the surface of infected cardiomyocytes (Soeiro et al., 2002). Cellular surface receptors, such as neurotrophic receptors (Aridgides et al., 2013), LDL (Nagajyothi et al., 2011) and bradykinins, (Scharfstein et al., 2000), are important in the invasion process by T. cruzi. Blocking receptor TrkC inhibits the entrance of T. cruzi in cardiomyocytes and cardiac fibroblasts (Aridgides et al., 2013). The invasion of cardiovascular cells by T. cruzi appears to be mediated by the interaction of cruzipain, a cysteine protease of T. cruzi, and kininogen, modulating the interface between endothelin-1 receptors (ETAR and ETBR) and bradykinin (B2R) (Andrade et al., 2012). Components of the extracellular matrix, such as proteoglycans of heparan sulfate (PGHS), also play an important role in the adhesion and invasion of T. cruzi in cardiomyocytes (Calvet et al., 2003, Bambino-Medeiros et al., 2011, Oilveira-Jr et al., 2008). The PGHS-T. cruzi interaction is mediated by heparin-binding proteins (HBPs), present on the surface of T. cruzi, which recognize the N-acetylated/N-sulfated domain of the heparan sulfate chain (HS) and participates in the triggering of the parasite invasion process into cardiomyocytes (Oliveira-Jr et al., 2008). The participation of fibronectin, a glycoprotein with adhesive properties, has also been demonstrated during the invasion process of T. cruzi in cardiomyocytes; this interaction pathway is mediataed by the Arg-Gli-Asp sequence (RGD sequence) of the fibronectin molecule (Calvet et al., 2004).
The T. cruzi inivasion process seems to depend on the cell type, the genotype and the development stage of the parasite. Protein kinases have been described as participating in the invasion process of T. cruzi (Burleigh, 2005). Activation of phosphatidylinositol 3-kinases (IP3-K) with recruitment of AKT has been demonstrated in T. cruzi invasion of different cell types, including cardiomyocytes. Focal adhesion kinase (FAK) also triggers invasion by this parasite. The entrance ofT. cruzi,orchestrated by the activation of the FAK/Src complex, is blocked by specific pharmacological inhibitors and by RNA silencing (siRNA) for FAK, leading to a significant reduction of infection levels in cardiomyocytes (Melo et al., 2014). In addition, T. cruziis capable of activating latent transforming growth factor β (TGF-β) and of blocking the TGF-β pathway, with inhibitor SB-431542, reducing parasite invasion of cardiomyocytes (Waghabi et al., 2007) and damages to the myocardium (Waghabi et al., 2009). Studies show that cruzapain modulates the activation of latent TGF-β (Ferrão et al., 2015) and that the costamere location (vinculin costameres) of type II TGF-β receptors (TGFβRII) is essential for the activation of the Smad pathway (Calvet et al., 2016) during the parasite’s invasion process. In addition to participating in the entrance and in the intracellular development of the parasite (Waghabi et al., 2005 a,b), TGF-β is implicated in cardiac fibrosis (Ferreira et al., 2016; Waghabi et al., 2002). Treatment with inhibitors of the TGF-β signalling pathway can revert the remodelling of the extracellular matrix in cardiac microtissue in vitro (Ferrão et al., 2018) and in the myocardium (de Oliveira et al., 2012; Araújo-Jorge et al., 2012). In addition, a lysosome-dependent invasion process has been demonstrated during T. cruzi interaction, sendo showing a transient increase in levels of Ca2+ in the cytoplasm, depolymerization of actin filaments at the invasion site, and also recruiting of cortical lysosomes at the parasite adhesion site (Rodríguez et al., 1995, Hissa & Andrade 2015). This event appears to be regulated by cholesterol expression in cardiomyocyte membrane, as cholesterol depletion by methyl-β-cyclodextrin (MβCD) deregulates the exocytosis of lysosomes and reduces invasion by T. cruzi (Hissa et al., 2012).
Alterations in levels of Ca2+ (Garzoni et al., 2003), probably resulting from the opening of pannexin-1 channels (Barría et al., 2018), have also been described in the primary culture model of cardiac muscle cells, but without evidence of disorganization of actin filaments at the parasite’s adhesion site (Pereira et al., 1993). The actual participation of the cytoskeleton of cardiac muscle cells in the invasion process of T. cruzi was demonstrated through the visualization of plasma membrane extensions of the target cell surrounding the parasite (Barbosa and Meirelles 1995). In addition, treating cardiomyocyte cultures with cytochalasin B and D, drugs that can block actin polymerization, significantly reduces invasion by T. cruzi, thus characterizing an endocytic process. Although cardiac muscle cells are not classic phagocytic cells, this cell type is capable of internalizing large inert particles, including zymosan A (Soeiro et al., 2002). After the interiorization, the parasite finds itself within the parasitophorous vacuole, that merges with endosomes and lysosomes, resulting in the fragmentaton of the vacuole membrane and placing the parasite in the cytoplasm of the host cell (De Souza et al., 2010). Studies with cellular lineages have demonstrated that the merge with lysosomes plays a fundamental role in the retention of the parasite within the host cell and, as a consequence, in the establishment of the infection (Andrade & Andrews 2004). In addition, oxidating products induced by T. cruzi infection of cardiomyocytes seem to modulate the intracellular development of the parasite (Dias et al., 2017).
Alterations in the structure and physiology of cardiomyocytes have been demonstrated during the intracellular development of the parasite. Infection by T. cruzi induces modifications in the expression of surface carbohydrates and modulates intracellular traffic in cardiomyocytes, revealed by the down-regulation of Rabs and its effectors, including EEA1, Rab7 and Rab11, higlighting the commitment of the endocytic pathway in infected cardiomyocytes (Batista et al., 2006). Alterations in the architecture of the cytoskeleton and cellular junctions have also been highlighted during the infectious process. Breaking of myofibrils (Pereira et al., 1993), disorganization of costameres (Melo et al., 2004), communicating and adherent junctions (Adesse et al., 2008; Melo et al., 2008) can all contribute to the anomalies observed in Chagas disease. The changes observed in cytoskeleton components are not limited to structural disorganization. Alterations in the levels of mRNA of cardiac β and α-actin have been demonstrated during infection by T. cruzi (Pereira et al., 2000). In addition to the alterations in the cytoskeleton of cardiomyocytes, studies have demonstrated that infection by T. cruzi induces alterations in the expression of extracellular matrix components, resulting in an expressive reduction of fibronectin on the surface of the infected cell (Calvet et al., 2009). This event is regulated by the alteration of the spatial distribution of TGFBRII induced by cytoskeleton disorganization, and inhibition of Smad2/3 activation (Calvet et al., 2016). Galectin-3 (Gal-3), a member of the family of lectins with affinity for β-galactose, prevents cardiac fibrosis, controls the immune response and the parasitary load in experimental infection by T. cruzi (Silva et al., 2017). Also, there is evidence showing the induction of the cellular death pathway during the T. cruzi-cardiomyocyte interaction, suggesting that the apoptosis of cardiomyocytes infected by T. cruzi can contribute to the silence and persistent infection in the chronic phase of Chagas disease (De Souza et al., 2010).Therefore, in the last decades the study of the interaction between T. cruzi and cardiomyocytes has resulted in breakthroughs in our knowledge about the biological events involved in the interaction of this parasite and its main target cell in human infection, contributing to a better understanding of the pathogenesis of the disease.