Giovanni Salvatore de Simone
Molecular Biology Laboratory, Oswaldo Cruz Institute/Fiocruz
The biochemical foundations of the life of each cell involve their metabolism, which includes both catabolic (degradation) and anabolic (synthesis) processes. Most, if not all, trypanosomatides depend on carbon sources that are present in specific compartments of their hosts to produce their energetic metabolism.
While trypomastigote forms of T. cruzi use glucose, which is abundant in the fluids of their vertebrate hosts, the forms found in the vector insect obtain their energy preferably from amino acids (L-proline, L-aspartic and L-glutamic/glutamine, which make up the hemolymph and the fluids of the vector’s tissues. These amino acids also participate in the epimastigote differentiation process (non-infective and replicating form) into trypomastigotes (infective, non-replicating form), and in particular L-proline in the intracellular differentiation cycle (which takes place in the mammal host). This important catabolic mechanism produces five times more reducing equivalents than the catabolic process of glucose. Amastigotes and epimastigotes are the parasite’s replicating forms; the first inhabit the cytoplasm (adapted to a glucose-based metabolism) of the host cell, and consequently have access to substracts of phosphorylated sugars, while the latter normally grow in environments rich in glucose-amino acids (aerobic fermentation). An interesting fact is that T. cruzi (but not T. brucei or L. major) has the ability to use D-proline, in addition to L-proline (due to the expression of a proline racemase), and L-histidine.
Our current knowledge of the metabolic pathways comes from studies carried out on different parasites, and is accepted, after finalizing the genome of important kinetoplastids, that the simple glucose energetic metabolism is common to all trypanosomatides. However, there are small but significant differences between the various organisms.
The glycolytic pahtway is organized in such a manner that the first seven glucose-conversion enzymes into 3-phosphoglycerate are organized inside the glycosomes (peroxisome-like organelles), while the latter three enzymes are found in the cytosol. In aerobic conditions, pyruvate is the only product excreted, and the liquid synthesis of ATP takes place in the cytosol, due to a reaction catalyzed by pyruvate-kinase (PYK, phase 13), while consumption (phases 1 and 3) and production (phase 9) of ATP in the glycosomes are balanced. Similarly, the glycosomal redox balance is maintained, as the NADH produced by glyceraldehyde 3-phosphate dehydrogenase (GAPDH, phase 8) is reoxidated by the shift of gyicerol 3-phosphate (GP, phases 6 and 45) and the alternative oxidase present in the mitochondrion (phase 47). In contrast, the glucose catabolism in all other trypanosomatides (or adaptive forms) analyzed up to this date involves a complex network of metabolic exchanges between glycosomes and the mitochondrion. This is exemplified by the model proposed for the procyclic stage of T. brucei (Figure 1), which shows the three main differences compared with the slow blood form of T. brucei. First, phosphoglycerate kinase (PGK, phase 10) is located in the cytosol; therefore, 3-phosphoglycerate is produced in the cytosol. Second, glycosomes contain two additional kinases that convert phosphoenolpyruvate (PEP) into malate (PEPCK: PEP carboxykinase, phase 14) or pyruvate (pyruvate phosphate dikinase, phase 15). Second, the CO2 fixation phase catalyzed by PEPCK is the initial stage of the bifurcation pathway that leads to the production of succinate, the main final product excreted by most trypanosomatides. Third, pyruvate is located in a key crossing and can be directed to different final products, such as, l-alanine, ethanol and l-lactate.
Acetate is the main final product formed in the mitochondrion of all trypanosomatides, and it is excreted through a simple diffusion process, through the mitochondrial and plasma membranes. In addition, some adaptative forms, especially the stages present in the insect, use amino acids present in their hosts for energy production. On the other hand, the metacyclic trypomastigote infective stage of the T. cruzi insect lives in an environment rich with l–proline, originally from the insect’s fluids, justifying its preference for this source of carbon. However, it consumes preferably glucose, when both glucose and amino acids are available. On the other hand, the essencial role of l-proline metabolism in energy production during the metacyclic trypomastigote stage was demonstrated in an experiment in which depletion of glucose in the medium resulted in increased consumption of l-proline by T. cruzi. The adaptative capacity of these parasites in culture is intriguing; they can shift and adapt their metabolic pathways rapidly according to the presence or absence of glucose in the medium.
In most trypanosomatides, succinate is the main final excreted product of the glucose metabolism, but the main pathway of its production has been the subject of intense debates. The controversy is due to the importance of FRD (fumarate reductase-NADH-dependent) in the production of succinate. However, it is accepted that succinate is produced in the cytoplasm, based on PEP, through a collateral pathway. PEPCK, a glycosome enzyme (stage 14), and malate dehydrogenase (stage 16) convert PEP into malate, which is converted into fumarate by the two fumarase isoforms. Fumarate is then finally reduced into succinate, which is excreted by the glycosome and mitochondrial FRD isoforms (stages 18 and 20).
The main role of the succinate production pathway (succinate fermentation) is probably maintaining the glycosomal redox balance, supplied by the two glycosomal oxidoredutase enzymes (phases 16 and 18) that allow for the reoxidation of the NADH produced by GAPDH in the glycolytic pathway (phase 8). When compared with lactic fermentation, which involves lactate dehydrogenase (LDH), succinic fermentation has the advantage of requiring only half the PEP produced in order to maintain the NAD+/NADH balance.
Lactate has been detected in several trypanosomatides (ex. T. brucei and Leishmania spp) but frequently appears as a minor final product. This fact is related to the non-existence of its gene in the genome of T. cruzi and to the fact that LDH activity is only detected in parasite extracts, suggesting that this final product must be obtained through other metabolic pathways. In Leishmania, this system consists of two enzymes, glyoxalase I and glyoxalase II, which convert methylglyoxal intod-lactate using trypanothione as co-factor.
The presence of glyconeogenic enzymes in glycosomes is well understood, as they catalyze reversible reactions of the glycolytic pathway, in physiological conditions. However, the existence of a control mechanism that prevents the uncontrolled activity of both processes appears to be crucial, but this mechanism has not yet been identified.
The PPP is an alternative glucose oxidation pathway in most eukaryotic cells, and is a possibility for the cells to adjust their ATP needs, reducing power and nucleotide precursors. There are many reasons that indicate that this pathway is distributed between glycosomes and the cytosol. Glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase activity in T. brucei was detected among glycosomes (15%) and cytosol (50%). It is believed that this also occurs with other enzymes and in T. cruzi and Leishmania spp, as many other sequences containing PTS have been identified in the genomes (e.g. ribulose-5-phosphate-4-epimerase, ribose-5-phosphate epimerase, transketolase).
The final product, ribose 5-phosphate, is converted into phosphoribosyl pyrophosphate, which will move on to the synthesis of purine and pyrimidine nitrogenated bases (in processes that occur in the glycosomes). NADPH is a co-enzyme produced exclusively through this pathway, and has reducing power for the biosynthesis of fatty acids. In addition, this pathway also produces phosphorylated sugars of different sizes, allowing cells to adjust their needs through exchanges with glycolysis.
The PPP includes an initial oxidative phase, in which glucose-6-phosphate is transformed into ribose-5-phosphate and CO2through two oxidations, and an oxidative phase that takes place in the direction NADP– NADPH. This is followed by the transformation of ribulose-5-phosphate into ribose-5-phosphate by the action of an isomerase and the pentose, after various transformations, generates phosphorylated sugars with variable numbers of carbon atoms. All non-oxidative phases are reversible, which makes it possible to interconvert the different sugars. The energy from glucose oxidation is stored in the NADPH molecule, and not as ATP, which is what happens in glycolysis. The PPP regulation mechanism is not yet known, but we know that flow is more intense in proliferative forms.
Sedoheptulose-bisphosphatase (S1,7BPase) is an enzyme that seems to be present exclusively in chloroplasts and involved in the Calvin cycle. Although this genic sequence has been detected in T. cruzi and in other trypanosomatides (T. brucei and L. major), the genes that codify ribulose-1,5-bisphosphate carboxylase-oxygenasee (“RuBisCo”) and other enzymes of the cycle have not been found yet. On the other hand, as the Calvin cycle and the pentose pathway are intrinsically organized processes and have enzymes in common, it has been postulated that in trypanosomatides the S1,7BPase may be involved in an unusual manner, but up to this date this has not been demonstrated or identified for the pentose pathway. The substrate of S1,7BPase in chloroplasts is formed by the action of a bifunctional aldolase that can carry out the regular reaction of the glygolytic pathway, condensation of glyceraldehyde 3-phosphate and dihydroxyacetone phosphate into fructose 1,6 biphosphate, as well as the condensation of glyceraldehyde 3-phosphate and erythrose 4-phosphate into S1,78BPase. The glycolytic aldolase can only carry out the reaction in the reverse direction.
PPP regulation is still not well understood. However, high flow is indisputably necessary in proliferative forms. In the blood forms of T. brucei, the relative flow through PPP is neglectible, as suggested by the measuring of pyruvate produced through glycolysis. In cultivated cells, the quantity of pyruvate produced is almost two molecules for each molecule of glucose consumed. The interconnection between glycolysis and PPP seems therefore to be small. In addition, PPP enzymes have low specific activity when compared to those of the glycolytic pathway. However, specific activities are considerably high in procyclic forms of T. brucei and in promastigotes of Leishmania. In general, in most eukaryotic organisms, when energy exchanges in the cells are high, the supply of glucose-6-phosphate through PPP is also high and subject to the ATP/ADP and NADPH/NADP ratios. The two dehydrogenases convert NADP into NADPH, and the enzyme can be competitively inhibited by NADPH. When the ATP/ADP ratio is low, glucose is consumed by glycolysis, producing ATP; fatty acid synthesis does not occur, and the NADPH/NADP rate is high, consequently inhibiting the PPP. However, if the ATP/ADP ratio is high, the glycolytic pathway can be inhibited and fatty acid synthesis does occur, consuming NADPH and unblocking the dehydrogenases.
Acetyl-CoA is a key intermediary metabolite of the metabolism of carbohydrates, amino acids and fatty acids. The catabolism of the main sources of carbon (glucose, l-proline and l-threonine) generate the formation of acetyl-CoA. Most of the pyruvate produced by the glycolytic pathway is decarboxylated into acetyl-CoA by the mitochondrial pyruvate dehydrogenase complex (PDH, stage 25). The first observations in different trypanosomatides suggested that the acetyl-CoA produced by glucose metabolism should be converted into CO2 and acetate through the cycle of tricarboxylic acids (ATC). However, recent studies involving the use of procyclic trypanosomes containing the gene of aconitase knocked out (stage 30) revealed that most acetyl-CoA is converted into excreted acetate. It is known that all adaptative forms of trypanosomatides analyzed to this moment (except for the blood forms of T. brucei) produce acetate based on glucose, showing the importance of this pathway in the production of ATP. Acetate is produced by two enzymes: acetate:succinate CoA-transferase (ASCT, stage 26), which transfers the CoA from acetyl-CoA to succinate, providing acetate and succinyl-CoA which is subsequently reconverted into succinate by succinyl-CoA synthetase (SCS, stage 28) with concomitant production of ATP.
On the other hand, the acetyl-CoA produced through the various catabolic pathways can be used for the biosynthesis of lipids. Likewise, glucose andl-threonin are substracts for the biosynthesis of fatty acids, through the production of acetyl-CoA. As this process occurs within the mitochondrion, and the synthesis of fatty acids takes place in the cytoplasm, acetyl-CoA and oxaloacetate are condensed into citrate synthase (stage 29), which is then exchanged with cytoplasmatic malate and converted again into acetyl-CoA and oxaloacetate by citrate lyase (stage 42). Although the genome of trypanosomatides has codified genes of citrato lyase, experimental results suggest the existence of other possible exchange systems that feed the anabolic pathways and the biosynthesis of lipids.
Until recently, it was accepted that most trypanosomatides cultivated in a rich primary medium produced ATP through oxidative phosphorylation. In addition, the entire enzymatic machinery for an oxidative metabolism is present in most adaptative forms. This includes a respiratory chain that contains functional cytochromes capable of generating a proton gradient, as well as two terminal oxidases (IV-cytochrome-oxidase complex sensitive to cyanide and one alterative oxidase sensitive to salicylhydroxamic acid-stage 47).
In this model, the proton gradient generated by the respiratory chain (complexes I, III and IV) is used by F0F1-ATPsynthase(stage 48), considered the main site for ATP formation. However, the essential role played by synthase-F0F1-ATP in energy production, in conditions of excess glucose, has recently been questioned. In addition, an excess of oligomycin, a specific inhibitor of F0F1–ATP synthase, does not affect the viability of procyclic forms, and a large amount is necessary to kill the cells in a glucose-rich medium. On the contrary, when cultivated in a glucose-free medium, the same cells become 1,000 times more sensitive to oligomycin, suggesting that, in the presence of glucose, procyclic cells do not depend on oxidative phosphorylation to produce ATP. In the presence of glucose, when cells shift their metabolism to the catabolism of amino acids, oxidative phosphorylation becomes essential. This also implies that, in a glucose-rich medium, most ATP is produced through phosphorylation at a substrate level, having as key enzymes cytosolic PGK (stage 10), PYK (stage 13) and mitochondrial SCS (stage 28). On the other hand, it has been estimated that ATP production through phosphorylation at a substrate level is at least three times higher in a glucose-rich medium than in a glucose-free medium. Therefore, recent experimental evidence show that mitochondrial ATP-synthase F0F1– plays a neglectible role in procyclic trypanosomes cultivated in glucose-rich medium, and that most ATP is synthetised through phosphorylation at substrate level. However, how trypanosomes regulate oxidative phosphorylation is a question that is still waiting for an answer.
Eukaryote cells have developed various alternatives to control oxidative phosphorylation, including regulation through substrate availability, such as oxygen, ADP, and reducing equivalents. These latter factors may play an important role, as long as L-proline catabolism produces approximately five times more reducing equivalents than glucose catabolism. The degree of coupling between respiration and oxidative phosphorylation can also be under control. Two distinct regulatory processes have been described in eukaryotes: the dissipation system of the proton electrochemical potential, represented by the non-coupling of proteins, and the redox potential dissipation systems represented by alternative oxidases. Only the latter has been described in trypanosomatides (stage 47). Electron transfer through the pathway mediated by cytochromes (complexes III and IV) is coupled to the production of ATP through the generation of a proton gradient. In contrast, electron flow through ubiquinol by means of alternative oxidase pathway is not coupled to ATP production. This ambiguous system can provide enough flexibility to modulate the generation of a proton gradient and therefore be involved in the regulation of oxidative phosphorylation.
As mentioned earlier, when trypanosomatides grow in a rich medium (contining high concentration of glucose and amino acids), they degrade glucose and amino acids into partially oxidated final products through aerobic fermentation (fermentation in the presence of oxygen). Aerobic fermentation appears to be the consequence of the absence of a Pasteur effect (glycolysis inhibition in the presence of oxygen). However, the basic principle of the metabolic strategy developed by the parasites is not clear. Several organisms use fermentation in the absence of oxygen. However, the stages present in the vector are not used to continuous growth in aerobic conditions. Epimastigotes of T. cruzi have low ability to function in anaerobiosis, and have a low consumption of glucose during hypoxia or oxidative stress; they therefore depend on respiration to proliferate. There is no doubt that aerobic fermentation is neither a preadaptation to, nor the need for an aerobic lifestyle in which the parasite may need to develop in the vector insect. Another remarkable feature is the development, in the insects, of a complex of interconnected tubes (the tracheal system) that transport oxygen and other gases all through the body. Thanks to this system, insect forms can proliferate in aerobic conditions and do not require a long-lasting adaptation in the absence of oxygen. In addition, the high concentration of glucose found in the blood (5 mM) favors ATP production through the glycolytic pathway. Under these conditions, oxidative metabolism is not necessary, and this adaptative form down-regulates the expression of ATC cycle and the components of the respiratory chain, in spite of the absence of oxygen, which is important to remove excess reducing equivalents through alternative oxidase. The stage of the T. brucei insect grows in an environment that is rich in proline, but it prefers glucose to proline when available in a rich medium. As proposed, excess glucose (6 mM) combined with the absence of a Pasteur effect allows for a relatively high glygolytic flow, sufficient to generate ATP through phosphorylation at substrate level. Consequently, to down-regulate the oxidative metabolism of procyclic trypanosomes, they probably need ATP-synthase F0F1, respiratory chain and cycle of functional ATC. This hypothesis is consistent with the dramatic reduction of sensitivity to oligomycin observed in glucose-rich media when compared to glucose-free media, which is interpreted as a down-regulation of oxidative phosphorylation. The down-regulation of l-proline consumption in glucose-rich media strengthens this option, as once the catabolism of l-proline can stimulate oxidative phosphorylation through the generation of five times more reducing equivalents than glucose catabolism (reducing equivalents produced through glycolysis are first reoxidated through succynil fermentation). This model was based on the result of procyclic T. brucei, but they can also be applied to the epimastigote of the insect of T. cruzi, which also prefers glucose to amino acids. The mechanism of this glucose repression effect is not yet known.
There is no evidence that endogenous synthesis of L-arginine or other amino acids occurs in T. cruzi. The parasite obtains L-arginine through an independent high-affinity transporter of Na+ and is accumulated in acidocalcisomes, organelles that function as a reservoir of basic amino acids. L-arginine appears to be a precursor of *NO (a molecule with multiple properties), phosphoserine and possibly polyamines.
Marcia Cristina Paes
Laboratory of Trypanosomatides and Vectors Interaction, Department of Biochemistry – University of the State of Rio de Janeiro
Natália Pereira de Almeida Nogueira
Laboratory of Trypanosomatides and Vectors Interaction, Department of Biochemistry – University of the State of Rio de Janeiro
Iron (Fe) is the fourth most abundant element in the Earth’s crust and is crucial for life, as almost all living organisms need iron to carry out their various biological activities. Iron also works as a cofactor of a variety of proteins present in almost all living organisms, prokaryotes as well as eukaryotes. In addition, iron is important for various biological processes, such as cellular respiration, oxygen transportation, the cycle of tricarboxylic acid, for genic regulation and for DNA synthesis. However, very little is known about how iron is captured or which sources of iron are used by Trypanosoma cruzi. Just like another trypanosomatide, Leishmania donovani, which feeds exclusively on labile iron, it is possible that intracellular amastigotes of T. cruzi also use free iron as nutrient, as an increase in available free iron also leads to an increase in macrophage infection by T. cruzi. However, given the wide range of vectors and host cells, it is possible that T. cruzi explores many different sources of iron, such as myoglobin as it infects cardiomyocytes (a source of iron and heme); or transferrin (an iron-binding protein present in the serum of the vertebrate host), as amastigote forms have a specific transferrin receptor in their flagellar pocket. Amastigote forms of T. cruzi actually absorb the transferrin bound to iron when cultivated in vitro, but the physiological meaning of this observation is not yet clear. Tranasferrin is restricted to the lumen of the endocytic pathway and is not present in the cytosol of the host cell, where intracellular amastigote forms replicate. Therefore, the mechanism through which T. cruziacquires iron during its replication phase in the cytosol of the host cell remains unclear. It is possible that the parasites depend on a transient set of active redox iron complexes. Although they are essencial, reactive oxygen species (ROS) are extremely dangerous if there is no physiological equilibrium. Curiously, when ROS levels are increased in microphages, they promote the intracellular growth of T. cruzi amastigotes by means of a mechanism that may involve the release of iron from ferritin, or of proteins with iron-sulphur core. Studies also show that the proliferation of the parasite in macrophages is inhibited by the depletion of labile iron, indicating a redox regulation. Growth stimulus of T. cruzi by ROS is not restricted to macrophages, as a persistent oxidative environment may be generated by the parasites in other cell types, such as cardiomyocytes. The oxidative environment is very important, especially for epimastigote forms, which multiply inside the gut of the vector insect and use the nutrients in the blood, including the heme bound to the hemoglobin of red cells. After the insect ingests the host’s blood, in the hindgut the hemoglobin is digested into amino acids, peptides and free heme. Heme is a ubiquitous molecule usually associated to polypeptide chains by means of interactions between the iron atom and residues of histidine or methionine. Hemeproteins and heme are involved in basic functions such as cellular respiration (cytochromes), oxygen transportation (hemoglobin), energy metabolism, antioxidant defences (peroxidases), cellular growth, drug detox enzymes (CYP450) and cellular differentiation, all essencial for the survival of organisms. Even then, toxic effects of the heme molecule have been demonstrated in many models, based on its pro-oxidant nature or through its detergent action. In spite of its importance, biochemical studies have demonstrated the absence of a biosynthetic pathway complete with heme in T. cruzi and this was later corroborated by the sequencing of the parasite’s genome. This lack of enzymes therefore indicates that T. cruzi must obtain this molecule from its hosts. We know that proliferative forms capture heme and store it in reservosomes for the infectious forms during their life cycle. In fact, the heme uptake process in epimastigotes of T. cruzi is inhibited by heme-analogous molecules and by ABC-type transport inhibitors, and both proliferative forms, amastigotes and epimastigotes, capture the heme molecule through a transport present in the flagellar pocket, called TcHTE.
Surprisingly, in spite of its potentially harmful effects in high concentrations, heme (but not other porphyrins) has a beneficial effect on epimastigote forms, promoting their proliferation. The heme molecule modulates the activity of an uncommon type of translation initiation factor 2 (eIF2α kinase), called TcK2. Heme binds specifically to the catalytic domain of TcK2, inhibiting its activity, which promotes the proliferation of the parasite. In the absence of heme, TcK2 is activated, blocking cellular growth and inducing the differentiation of epimastigotes into metacyclic trypomastigotes.
Heme-induced epimastigote proliferation has also been observed accompanied by an increase in the concentration of ROS over time. On the other hand, antioxidants such as uric acid or reduced glutathione (GSH) revert heme-induced ROS production and also reduces the proliferation of epimastigote forms. In addition, only specific inhibitors of calmodulin kinase II (CaMKII) are capable of abolishing the formation of heme-induced ROS in epimastigotes, thus hampering parasite growth, an indication that the presence of the heme molecule favors a transient oxidative environment that stimulates the proliferation of epimastigotes of T. cruzi through a mechanism mediated by a CaMKII signaling pathway. Consequently, ROS production seems to be the mechanism through which heme promotes epimastigote proliferation. In fact, heme promotes modifications to the mitochondrial physiology of T. cruzi, diminishing its oxygen consumption, increasing the transfer activity of electrons between mitochondrial complexes I and III, and reducing the activity of cytochrome c oxidase (complex IV). Therefore, heme-induced increase in mitochondrial superoxide seems to be a consequence of the hyperpolarization of the internal mitochondrial membrane, with the goal of maintaining the proliferation and survival of epimastigote forms.
Although heme-induced ROS plays a crucial role in the proliferation of epimastigote forms, when it comes to the differentiation of these forms into metacyclic trypomastigotes, reactive oxygen species have the opposite effect, hampering metacyclogenesis. This effect can be reverted by antioxidants N-acetylcysteine (NAC) or urate, in vitro as well as in vivo, demonstrating the importance of the redox state for the biology of T. cruzi. In fact, the quantification of parasitic load in the anterior and posterior portions of the gut and in the rectum of triatomines infected with T. cruzi shows that antioxidants lead to an increase in the percentage of trypomastigotes. This indicates that inducing a reducing environment by adding antioxidants, mimicking the rectum of the vector, hampers epimastigote proliferation and stimulates the metacyclogenesis of T. cruzi in vivo.
In spite of its importance for the parasite’s biology, the heme molecule must be strictly regulated due to its potential toxic effects. Epimastigotes have a functional heme oxygenasae-like enzyme (HO) that is responsible for heme catabolism and for the production of classic intermediaries of its degradation: alpha-meso-hydroxyheme, verdoheme, and biliverdin, indicating once more that heme is not toxic for T. cruzi: rather, it is a physiological molecule capable of modulating this parasite’s biology.
Thus, the knowledge generated so far shows that the heme molecule and iron are a key interface between the parasite and its vector and in the infection of vertebrates, suggesting that these interactions may determine a great breakthrough in the understanding of Chagas disease transmission.
Cellular Biology and Parasitology Program, Carlos Chagas Filho Biophysics Institute, Center for Health Sciences, Federal University of Rio de Janeiro
The surface glycoconjugates of pathogenic protozoa contain a carbohydrate domain with uncommon structures. These unique structures suggest a specific relation between the carbohydrates and the virulence of the parasite. Trypanosoma cruzi is covered in a glycocalyx whose components are involved in its survival and infectivity. The most abundant glycoconjugate of T. cruzi is a family of molecules similar to mucins, highly O-aGlcNac-glycosilated, called sialoglycoproteins. The incorporation of sialic acid to the b-galactopyranosyl terminal of O-glycans is independent of CMP-sialic acid and dependent on the activity of the trans-sialidase enzyme, also present on the surface of T. cruzi. O-aGlcNAc-bound carbohydrates of sialoglycoproteins contain new types of glycosylation and can be used as prototypes for the synthesis of compounds, with the goal of developing vaccines against Chagas disease, or as specific inhibitors of the activity of specific glycosyltransferases involved in the biosynthesis of O-glycans in T. cruzi.
Glycobiology is the study of the structural and functional aspects of the glycoconjugates present in the cells. The bio-oligomers and biopolymers that make up the group of glycoconjugates are many. Natural products such as glycoproteins, glycolipids, glycosaminoglycans and glycosylphosphatidylinositol are molecules that contain carbohydrates, which usually cover the surface of cells. The carbohydrate domain (or oligosaccharide, or glycan) is considered a modulator or mediator of the mechanisms involved in cell-cell and molecule-cell interactions, as well as in interaction processes between parasites and host cells.
Unlike peptides and oligonucleotides, whose structures are frequently linear, oligosaccharides can be ramified. As there is no direct genetic control, glycoconjugates are almost always heterogeneous. The amplification techniques or expression systems in bacteria do not exist for glycoconjugates. Extracting carbohydrates from biological systems was, until recently, the only way to obtain these molecules. Although carbohydrate analysis has seen huge breakthroughs in the last few decades, their structural diversity requires different methodologies of analysis to characterize, for instance, the composition of the various classes of sugars in a given glycomolecule.
Pathogenic bacteria and protozoa express carbohydrates on their surface that are different from those of their hosts. These carbohydrates are considered cellular surface markers for these micro organisms and could be the foundation for specific diagnosis, vaccines, chemotherapy and immunopathological studies.
T. cruzi is the agent of American trypanosomiasis or Chagas disease, which affects millions of people in Latin America. The main form of transmission of the disease is vectorial, through the feces of infected triatomine insects. T. cruzi is a protozoan of the Trypanosomatidae family. This species forms a genetically diverse group, with strains that vary as for tissue tropism, multiplication rate, mosphology, and surface glycoconjugates. Trypomastigote forms can invade a variety of mammal cells in vivo and in vitro, including fibroblasts, epithelial cells, endothelial cells and neurons, showing a preference for cells of the mononuclear phagocytic system and muscle cells. The genome of T. cruzi contains hundreds of genes that codify a family of molecules with and without enzymatic activity (active and inactive trans-sialidases) and glycoproteins aceptors of sialic acid, the sialoglycoproteins (molecules similar to mucins). Although the molecular mechanisms responsible for invading the host’s cell have not been completely understood, it is believed that these molecules work as binders or receptors, in some phase of the infection process. Studies of mammal cell proteins that bind to salic acid show that these proteins are involved in different biological processes, such as intracellular adhesions, interaction with the surface of micro organisms, and signalling.
Sialic acids are carboxylated sugars with nine carbons (Figure 1) that can be found in the non-reducing terminal of oligosaccharide chains, forming a2,3; a2,6 bonds with b-ligated galactopyranose (bGalp) or N-acetylgalactosamine (GalNAc) or forming polymers of ligated a2,8 ou a2,9 salic acid. The most common salic acid is N-acetylneuraminic acid (Neu5Ac) (Figure 1), considered the precursor of other members of the family, such as N-glycolylneuraminic acid (Neu5Gc) (Figure 1)and 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en) (Figure 1). Sialic acid is usually added to the bGalp unit by the activity of sialiltransferases, present in the Golgi reticulum, which use the activated form of Neu5Ac, cytidine monophosphate-Neu5Ac (CMP-Neu5Ac) as donating substract.
Sialic acids present in glycoproteins and glycolipids are involved in biological functions such as in cellular recognition processes, in the half-life of cells and plasmatic proteins, in the modulation of the immune system, and in apoptosis. Salic acids are binders for adhesion molecules such as selectins or sialoadhesins, mediating cellular adhesion or signal transduction and possibly having an important role in the parasite-host interaction.
Sialidases or neuraminidases are enzymes that catalyze the hydrolysis of sialic acid. In mammals, sialidases modulate the distribution of sialic acid on the cellular surface, with effects in the immune system, in the half-life of circulating cells, and in apoptosis.
Salidases are found in many pathogenic micro organisms, and are often associated to the virulence of these pathogens. The involvement of sialidases in the pathogenic process is well described for the influenza virus. In bacteria, sialidases are involved in different diseases, such as in cholera, in which the sialidase ofVibrio cholerae catalyzes the conversion of complex gangliosides into GM1, increasing receptors for binding with the cholera toxin.
In T. cruzi sialic acid was first described on the surface of epimastigote forms by using lectins and colorimetric methods. The presence of sialic acid in epimasatigote forms has been proven by thin layer and gas-liquid chromatography and in mass spectrometry. In 1983, Pereira and collaborators observed the capacity of trypomastigotes to release Neu5Ac of human erythrocytes and plasmatic glycoproteins, describing a stage-specific neuraminidasic activity.
In 1985, Previato and collaborators characterized, for the first time, the capacity of epimastigote forms to incorporate sialic acid in the parasite’s surface molecules, using exogen sialoglycoconjugates as donating substrate, and suggested that the incorporation of sialic acid to the surface glycoproteins of T. cruzi did not take place through the conventional pathway, in which CMP-Neu5Ac is the substrate that donates sialic acid. These results strongly suggest that in T. cruzi the sialylation of glycoproteins would occur through a new metabolic route, involving reactions of trans-glycosylation for sialic acid. The sialic acid incorporation capacity in trypomastigotes has been demonstrated in vitro and in vivo. In vivo studies demonstrated the incorporation of Neu5Gc to the surface of trypomastigote forms obtained from infected mice. Finally, the trans-glycosylase activity for sialic acid has been characterized as the trans-sialidase enzyme (TS) responsible for transferring sialic acid from exogenous sustrates into glycoproteins present on the surface of T. cruzi, forming the epitope Ssp-3.
These studies have demonstrated that the catalytic site was located in the N-terminal domain of an antigen called SAPA (shed acute phase antigen) present in the serum of patients infected with T. cruzi. They also showed that the C-terminal domain of the SAPA antigen contained repetitive units of amino acids that generated a strong immune response during the acute phase of Chagas disease.
TS preferably catalyzes the transference of sialic acid of sialomolecules containing Neu5Aca2,3Galpb1-x to terminal units of ligated bGalp, forming exclusively a2,3 bonds (Figure 2). The TS of T. cruzi differs from the sialyltransferases found in the Golgi complex that use CMP-Neu5Ac as donating substrate. In the absence of b-galactosides (acceptor substrate), TS catalyzes the transfer of salic acid to water molecules, a hydrolysis reaction (Figure 2) similar to that of neuraminidases of viruses, bacteria and mammals.
In trypomastigotes, TS is anchored by glycosylphospatidylinositol (GPI) to the parasite’s membrane. The presence of the GPI anchor allows the TS to be cleaved and released from the surface of T. cruzi by the action of a phospholipase C present on the surface of the parasite. These data justify the detection of the SAPA antigen and of sialidasic activity in the serum of patients in the acute phase of Chagas disease. Purified TS of trypomastigotes forms multimeric aggregates that give origin to proteins with an apparent molecular weight bewteen 120 and 220 kDa.
In trypomastigotes, TS is formed by two distinct domains. The N-terminal domain, which contains the catalytic site, with approximately 680 amino acids, resulting in a molecular mass of approximately 60 kDa. The C-terminal domain, formed by the repetition of 12 amino acids D-S-S-A-H-(S/G)-T-P-S-T-P-(A/V) not necessary for enzymatic activity. The C-terminal portion of TS allows for the oligomerisation and the binding of the enzyme to the surface of the parasite and induces the production of antibodies directed to the SAPA antigen. The authors suggest that the C-terminal domain of TS would act in a first phase of the infection, stabilizing TS activity in the blood, increasing its half-life in circulation. During a second phase of the infection, these complexes would induce the generation of antibodies that would inhibit the enzyme.
The TS found in epimastigotes presents the same specificities and kinetic properties as the enzyme expressed in trypomastigotes, with significant structural differences. The enzyme is monomeric, does not have the C-terminal portion and is not anchored through GPI to the surface of the parasite.
TS has activity on a great variety of acceptor molecules, such as saccharides, glycolipids and glycoproteins, containing a bGalp unit at the non-reducing end of the molecule. Galactose units are not acceptor substrates for the enzyme, even though we know that when in solution, the bGal anomer is the most stable one. However, methyl b-galactoside (Me-bGalp) is a good substrate for the TS of T. cruzi. In experiments with TS in which different disaccharides were used as acceptor substrates, lactose (Galpb1,4Glc) and N-acetyllactosamine (Galpb1,4GlcNAc) are the best substrates. Oligosaccharides containing terminal units of a-Gal at the non-reducing extremity are not substrates for TS. Oligosaccharides Galpb1,4(Fuca1,3)GlcNAc and Galpb1,3(Fuca1-4)GlcNAc are not recognized by the enzyme; as a consequence, selectin ligants sialyl Lewisx e sialyl Lewisa are not substrates for TS. The incorporation of a unit of Neu5Ac prevents the incorporation of a second unit in the acceptor oligosaccharide, containing two potencial sialylation sites.
The TS enzyme recognizes terminal units of ligated Neu5Ac and Neu5Gc a2,3 and synthesizes exclusively sialosidic a2,3 bonds. Neither ligated a2,6 sialic acid nor ligated a2,8, a2,9 sialic acid polymers are substrates for the enzyme. Synthetic substrates 4-methylumbelliferyl-N-acetyl neuraminic acid (4-MUNeu5Ac) and p-nitrophenyl-N-acetyl-a-neuraminic acid (pNPNeu5Ac) are excellent substrates for TS.
The presence of C8 and C9 of the glycerol group of the sialic acid of the donating substrate does not influence recognition by TS, as donating substrates submitted to soft oxidation through periodate and reduction mantained the activity of the enzyme. On the other hand, acetylation in C4, C7 and C8 inhibited trans-sialydasic activity, suggesting that these are contact sites between sialic acid and the enzyme.
TS has enzymatic activity in a large range of pH. It has optimal activity in pH and it drops by about 50% when nearing a pH of 5.5 or 8.5. The remaining sialidases have optimal activity around pH 5.5 The trans-glycosylation reaction is maximum at around 13 ºC, with 50% of activity near 4 and 37 ºC. However, the sialidasic activity increases with temperature, and its maximum action takes place at around 37 ºC.
The compound Neu5Ac2en, a powerful inhibitor of sialidases in viruses, bacteria and mammals, does not inhibit the transfer reaction catalyzed by TS, nor the sialidasic action of the supernatant of a trypomastigote culture. Compounds that effectively inhibit the catalytic activity of the TS of T. cruzi have not yet been described. The search for possible inhibitors has been the research target of different groups, with the goal of clarifying the role played by TS in the pathogenesis of Chagas disease.
TS substrates acceptors of sialic acid on the surface of T. cruzi are O-glycosylated glycoproteins. In tryptomastigotes derived from cell cultures, sialic acid is incorporated into glycoproteins with molecular weight ranging from 60 to 200 kDa. These molecules are recognized by a stage-specific monoclonal antibody, Ssp3. In metacyclic trypomastigotes of axenic culture and in epimastigote forms, sialoglycoproteins have a molecular weight ranging from 34 to 50 kDa, and are GPI-anchored to the surface of the parasite. O-ligated oligosaccharides of sialoglycoproteins of epimastigotes have great structural diversity between the strains of T. cruzi studied (Figure 3).The first stage of the biosynthesis of O-glycans of sialoglycoproteins of T. cruzi is catalyzed by an O-GlcNAc-polypeptidyl transferase that adds N-acetylglucosamine (GlcNAc) in configuration a, exclusively, to the amino acid threonin (Thr) of the proteic portion of the molecule. Figure 3 shows that, depending on the strain, the sequence Thr-aGlcNAc can be extended by adding units of Galp, galactofuranose (Galf) and Neu5Ac.
The role of sialic acid and TS in the T-cruzi-host interaction
Several works suggest that the adhesion and penetration of T. cruzi in the host cell are modulated by the sialidase/trans-sialidase activity. The control of sialylation of glycoconjugates on the surface of the parasite or the control of the bond between the parasite and the sialic acid unit of the surface proteins of host cells would be responsible for adhesion and penetration. Alternatively, TS could sialylate or desialylate glycoconjugates of the host’s cell, facilitating the adhesion and penetration of the parasite.
The importance of sialic acid on the surface of host cells has been demonstrated by various independent works, through experiments with ovarian cells of hamsters with sialic acid deficiency. These cells are weakly invaded by T. cruzi when compared with wild cells. On the other hand, whether the sialylated epitotes of the surface of trypomastigotes are involved in the invasion of mammal cells is a controversial issue. Some works have demonstrated that the presence of the parasite sialic acid increased infection; others suggest that the sialic acid on the surface of the parasite is not necessary for the invasion.
The first evidence that sialic acid units on the surface of the parasite could participate in the invasion process was demonstrated by Piras and collaborators. Incubating trypomastigotes in a medium containing sialoglycoproteins increased T. cruzi penetration in a fibroblast culture. In another article, using monoclonal antibodoes that recognize sialylated epitopes on the surface of the trypomastigote (Ssp3), Schenkman and collaborators demonstrated that these antibodies were capable of inhibiting parasite adhesion and penetration in the host’s cell.
On the other hand, the absence of sialic acid on the surface of the parasite, increasing the invasion of host cells by trypomastigotes, has been verified by many authors. According to these results, Yoshida and coollaborators demonstrated that removing sialic acid from the surface of metacyclic trypomastigotes increased parasite interaction with HeLa cells.
Sialic acid may also be involved in T. cruzi escaping from the phagosomal vacuole formed during the invasion of the host cell. The de-sialylation of sialoglycoproteins of the phagosome membrane by TS could be involved in the escaping of T. cruzi into the cytoplasm. More recently it was demonstrated that the higher expression of TS in trypomastigotes of cell cultures favored the more rapid release of these forms of the parasite from the parasitophorous vacuole into the cytoplasm of the host cell and its subsequent differentiation into amastigotes.
Parasite-host interactions mediated by carbohydrates can be of the lectinic type. Lectins participate as receptors or binders for interactions between parasites and host cells, resulting in the recognition, adhesion and invasion of the host cell and in immunoregulation, allowing the parasite to replicate and to establish infection. It was recently demonstrated that enzymatically inactive TS is a lectin that binds to sialic acid and co-stimulates T cells through its bond with leukosialin (CD43). It was also demonstrated that the inactive form of TS has an additional binding site for units of b-galactopyranosyl. Initially, the presence of this additional site was suggested by the inhibition of the co-mythogenic effect of the inactive enzyme on T cells through the addition of N-acetyllactosamine. Later, the interaction between the inactive TS and b-galactosides was investigated through spectroscopy of nuclear magnetic resonance (RMN). Todeschini and collaborators directly demonstrated, through RMN, that the recognition domain for the unit of b-galactoside only occurred after the enzyme bond with a molecule containing ligated a2,3 sialic acid. The bivalent property of inactive TS allows for a cross-bond between glycans that is considered an essential property for the transduction of cellular signals and can exacerbate immunopathological reactions in Chagas disease.
For many years, the lack of knowledge about methodologies to study glycobiology made it difficult for biologists and medical researchers to solve various problems involving carbohydrates. In the last decade, sequencing and synthesis technologies, which are commonly used to study nucleic acids and proteins, have become available to study glycoconjugates. Today, in spite of the difficulties still in place when it comes to purifying glycoconjugates, due to the micro heterogenicity and the absence of procedures with which it is possible to amplify these molecules, the primary sequence of carbohydrates has been done systematically.
The characterization of specific oligosaccharides and the comparison of their sequence with that of synthetic oligosaccharides has made it possible to study the interactions between glycans and proteins. The involvement of oligosaccharides in signalling pathways has been determined and understood at the molecular level of the carbohydrate-protein bond. Thanks to X-ray chrystallography and nuclear magnetic resonance spectroscopy, protein-carbohydrate interactions have been determined and, as a consequence, better knowledge of the biological functions of carbohydrates has been acquired as well, resulting in breakthroughs in the diagnosis and treatment of certain diseases. There are already concrete examples regarding bacterial and viral infections. The same is taking place with parasitic diseases such as malaria, leishmaniasis and trypanosomiasis. Recently, relevant structural and immunobiological results obtained with purified glycomolecules of T. cruzi have turned glycobiology into an extremely exciting field of study, with applications in the control and prevention of Chagas disease.
José Franco da Silveira
Department of Microbiology, Immunobiology and Parasitology, Federal University of São Paulo
Email: firstname.lastname@example.orgUNDER CONSTRUCTION.
Proteomic analysis of T. cruzi: over a decade of studies
Rubem F. S. Menna-Barreto
CellularBiologyLaboratory, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro – RJ, 21040-360, Brazil
High-throughput proteomics became especially attractive for studies in trypanosomatids due to the presence of open reading frames in long polycistronic regions in these parasites that leads to a peculiar post-transcriptional gene expression regulation. In T. cruzi, proteomic approaches have been employed to increase accuracy of genome annotations as well as the description of physiological role of protozoa proteins from different evolutive stages as we will review below.
Chronologically, the first T. cruzi study using proteomics was performed in 2004 by Paba and co-workers, analyzing all culture-derived stages by two-dimensional electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) mass spectrometry peptide mass fingerprinting. The comparisons among the parasite forms were performed, pointing to the relative abundance of distinct stage-specific proteins. As an example, paraflagellar rod proteins were more abundant in infective trypomastigotes than in the proliferative forms, suggesting a possible biological role for this structure during the infection and/or parasite dissemination. In figure 1, the proteomic profile of epimastigotes could be observed in 2-DE. One year later, in 2005, the first shotgun analysis was performed by Atwood group. In this study, proteomic profiles of epimastigotes, amastigotes, culture-derived trypomastigotes and metacyclic trypomastigotes were assessed. The approach of liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) led to the identification of 2,784 T. cruzi proteins, being only 30% described in all four stages. Surprisingly, chaperones, ribosomal and surface proteins (mainly trans-sialidases and GP63) were the most abundant. Important differences were detected among the trypanosomatid stages and some hypotheses were raised. For example, antioxidant enzymes such as ascorbate peroxidase and tryparedoxin peroxidase were highly expressed in infective forms, suggesting the exposure of these stages to oxidative burst in the vertebrate hosts. However, These hypotheses remain to be confirmed and validated by orthogonal techniques.
T. cruzi genomic diversity encouraged the description proteomic evaluation of different DTUs. 2-DE/MALDI-TOF analysis of CL Brener strain epimastigotes (hybrid organism derived from DTU I and DTU II) demonstrated some proteins previously detected in this proliferative form, and also an enzyme from polyamine pathway, arginine kinase,identified for the first time. Really comparing DTUs, only one study was performed up to now. In 2010, epimastigotes proteins profiles from isolates 3663 (DTU III) and 4167 (DTU IV) were assessed by 2-DE/MALDI-TOF, and the comparisons pointed to the possibility of the correlation between the virulence and heterogeneity of the parasite cell surface composition. However, additional studies must be done with different DTUs in order to comprehend crucial characteristics of this parasite in nature.
Regarding to the diversity of environmental conditions during T. cruzi life cycle, the success of infection depends on the parasite adaptations to distinct hosts, including the temperature variation from 28°C (insect) to 37°C (mammals). In this context, 2-DE/MALDI-TOF analysis was realized in epimastigotes incubated at 28 to 42°C, and 24 proteins presented differential abundance relative to the control (28°C) such as chaperones and enzymes associated with distinct metabolic pathways. Interestingly, the increase in temperature also led to an overexpression of surface proteins related to infectivity and virulence, correlating infection and stress.
Some years later, in 2012, metacyclogenesis was reanalyzed by LC online with mass spectrometer (LC-MS/MS). This accurate method allowed the quantification of 3,000 proteins involved in the differentiation process. The high abundance of trans-sialidases isoforms in metacyclic trypomastigotes, corroborating to the increase in infectivity, as well as the overexpression of structural proteins during the differentiation, resulting in the strong morphological changes in the parasite, are the most expressive findings.After one decade, the first proteomic assessment of bloodstream trypomastigotes profile was published by our group, and 3,716 bloodstream form proteins identified. Many surface proteins, enzymes related to all kind of metabolic pathways and structural proteins were identified. In this study, proteins identified in bloodstream form and other trypomastigotes (metacyclic or culture-derived forms) profiles previously analyzed by proteomics was also compared, showing 2,202 proteins only present in bloodstream form, reinforcing the differences in trypomastigotes proteomic profiles possibly due to their exposure to the host immune system. Bloodstream trypomastigote descriptive evaluation can contribute to comprehension of disease pathogenesis in the vertebrate hosts. In figure 1, it could be observed an chronological summary of all T. cruzi proteomic studies.
The first proteomic evaluation of T. cruzi isolated organelles was done in epimastigotes employing LC-MS/MS in order to provide subcellular proteins localization. After subfractionation, 38 proteins not previously identified in the whole insect form proteome were detected, demonstrating that the use of enriched fractions for mass spectrometry analysis led to the identification of novel proteins. Some years later in 2009, the first T. cruzi organelle specific proteome was performed. Sub-proteome evaluation of epimastigote-exclusive endocytic organelle named reservosomes by 2D-LC-MSMS pointed to the identification of 709 reservosome proteins such as cruzipain, serine carboxypeptidase, acid phosphatases, calpains, among others, reinforcing reservosomes are the main location of lysosomal hydrolases in this parasite form.
In parallel, two-dimensional liquid chromatography (2D-LC) coupled with tandem mass spectrometry (MSMS) was used to analyze the detergent-solubilized membrane fraction of epimastigotes and metacyclic trypomastigotes.98 and 280 proteins were described in epimastigotes and metacyclic forms, respectively, being about 60% showed post-translational modifications such as myristoylation, GPI-anchor, or even prenylation. Once again, metacyclic repertoire of surface glycoproteins was huge, being related to the parasite adhesion/invasion processes. In 2013, protein surface trypsinization of intact living and biotin labeling of surface proteins followed by affinity chromatography purification were used to assess epimastigotes surface subproteome, leading to the identification of 2,095 proteins. The subsequent work of this research group applied the same approach to characterize vertebrate forms of the parasite, detecting antioxidant enzymes in trypomastigotes, suggestive of the virulence factor role of these molecules.On the other hand, numerous enzymes related to lipid and/or carbohydrate metabolisms were detected in amastigotes, indicating the interference in host metabolism for intracellular self-maintenance.
In 2011, the protein composition of contractile vacuole complexes of epimastigotes was assessed by one-dimensional gel electrophoresis and LC-MS/MS, and 220 proteins were identified such as glycoproteins, calpains, amastins, intracellular transport-related proteins and vacuolar-H+-pyrophosphatases, a well-established acidocalcisome marker, confirming once more the participation of this organelle in osmotic regulation in this parasite. It is worthy to mention that 109 novel proteins were detected in the parasite insect form during this subproteome study, reinforcing the subfractionation as a crucial method for the increase in the number of identifications.
During its life cycle, the parasite differentiates into proliferative or non-proliferative forms, depends on the host and/or environmental conditions, and such event directly influence transcriptional control. In this scenario, the chromatin proteomic map of epimastigotes and trypomastigotes was investigated in 2017. Surprisingly, this analysis not only evidenced well-known nuclear proteins but also carbohydrate pathway enzymes and cytoskeletal proteins. Remarkable differences could be observed between the protozoa stages, especially related to histones abundance in trypomastigotes (H2B variant in particular) and to DNA dynamics associated proteins such as topoisomerases in epimastigotes.
The first proteomic analysis focused on post-translational modifications (PTM) was done in 2006 using LC separation-MS/MS in culture-derived trypomastigotes after subcellular fractionation. The isolation of glycoproteins was performed by lectin affinity capture and the quantification by isotopic labeling, leading to the identification of 29 glycoproteins, especially mucin-associated surface proteins (MASPs) showing N-linked glycan PTM. In 2017, the glycopeptide enrichment approach coupled to LC-MS/MS was employed and the parasite glycoproteome was further analyzed in trypomastigotes and also epimastigotes. 690 glycoproteins were identified presenting 1,309 N-glycosylation sites, being 334 detected only in trypomastigotes (mucin and MASP family members).
Phosphoproteome of epimastigotes was assessed using phosphopeptide/phosphoprotein fractionation and LC-MS/MS analysis. In 2009, 119 proteins were identified with 237 phosphopeptides involved of different biological processes such as differentiation and motility. In 2011, another study pointed to 753 phosphoproteins with 2,572 phosphorylation sites. The first assessment of phosphoproteomic profile of a parasite vertebrate form was only performed in 2014, during amastigogenesis process, evidencing 165 and 18 mono-phosphorylated and multi-phosphorylated peptides, respectively.
In 2016, N-myristoylation was investigated by bead-based click-chemistry and LC-MS/MS. Such approach led to the identification of 56 proteins, being 32 confirmed in stable isotopic labeling with amino acids in cell culture (SILAC) experiments. Interestingly, the great majority of the myristoylated proteins in the parasite was hypothetical or uncharacterized; some proteins participate in metabolic processes regulation, cell trafficking or signaling.
In 2017, acetylation was analyzed in epimastigotes by LC-MS/MS approach, and 389 lysine-acetylated sites in 235 proteins were detected. These data also strongly suggested the participation of this PTM in the regulation of innumerous metabolic processes, being postulated that the activity of crucial enzymes involved in oxidative defenses depends on acetylation. These findings could point to the development of specific deacetylases and/or acetyltransferases inhibitors as an alternative for Chagas disease treatment.
Associating subfractionation and PTM proteomic analysis, our group evaluated T. cruzi secretome by 2D-LC-MS/MS, reaching 367 distinct proteins, 102 and 22 only identified in epimastigotes and metacyclic trypomastigotes, respectively. These proteins were involved in biological events such as gene expression, DNA binding, stress responses, and could play an essential role in disease pathogenesis. Using label-free quantification, the authors demonstrated GP82 and calcium-binding protein in vesicle-enriched fraction. Complementarily, immunoprecipitation of trypomastigotes and amastigotes secreted exovesicles with Chagas disease patients sera followed by subsequent LC-MS/MS analysis. Among 766 identified proteins, trans-sialidases are the most recurrent immunoreactive proteins, together with amastigote surface, complement regulatory, polyubiquitin or transporter proteins. In 2017, secretome of trypomastigotes was revisited, and the comparison of culture-derived forms from distinct DTUs was performed, identifying 591 secreted proteins such as trans-sialidases, MASPs and GP63, as previously demonstrated for metacyclic forms.
As it was mentioned in treatment section (http://chagas.fiocruz.br/tratamento/#terapias), many efforts have been made in order to develop alternatives for the current treatment of Chagas disease. In this way, promising molecular drug targets could be identified by high throughput techniques such as proteomics. However, up to now only three proteomic studies were published in this direction. In 2008, the resistance of epimastigotes to benznidazole was evaluated by 2-DE and MALDI-TOF/TOF, showing 36 proteins modulated in resistant parasites, including peptidases, peroxiredoxin and iron superoxide dismutase, among others. Subsequently, the direct binding of the parasite proteins to benznidazole was evaluated by chemical proteomics, demonstrating the interaction of aldo-ketoreductase family proteins with the drug, probably derived from benznidazole reduction by nitroreductase type-I, but further experiments must be done to prove it. In 2010 and 2016, our group investigated the mode of action of beta-lapachone derivatives in epimastigotes (2-DE and MALDI-TOF) and bloodstream trypomastigotes (2D-DIGE and LC-MS/MS, Figure 3), respectively. Both studies pointed to the mitochondrion as the main target of these compounds. The treatment of epimastigotes and bloodstream trypomastigotes also induced a remarkable reduction in tubulin levels, especially in labile microtubules content, impairing intracellular traffic and also epimastigotes mitosis. Treated epimastigotes showed an increase in the abundance of trypanothione synthetase, an enzyme involved in antioxidant defenses, however these derivatives do not present redox potential to justify the directly ROS production. These mechanisms are under investigation right now in our lab. On the other hand, our data indicated differences in the mechanisms of action of these derivatives between two parasite stages, involving a great variety of biological processes and pathways. More proteomic studies of anti-T. cruzi drugs must be done in order to classify the best molecular targets in the clinical relevant forms of the parasite. Table 1 compares the biological processes involved in these three drugs mode of action in T. cruzi identified by proteomic analysis. Figure 2 summarizes all molecular drug targets suggested by proteomic approaches.
Table 1. Biochemical pathways described in proteomic studies of drugs against T. cruzi
|Biochemical pathway||Epimastigotes treated with BZᵃ||Epimastigotes treated with NIᵇ||Trypomastigotes treated with NI|
|Metabolism of nucleic acids and transcription||14.3||0.0||3.6|
|Metabolism of proteins and biosynthesis||14.3||42.4||32.1|
ᶜPercentage of differential proteins
The search for novel antigens for vaccines or diagnosis historically depends on the discovery of promising candidates especially presented on the parasite surface, or most recently on secreted profile (in vesicles or not). As discussed above, glycoproteomic studies pointed to mucin-associated surface proteins and other glycoproteins as interesting antigens. LC-MS/MS analysis of trypomastigotes showed 45 immunogenic epitopes and GPI-anchored surface proteins involved in pathogenesis, being deposited in a vaccine antigens database to be validated in the future. Using next-generation peptide microarrays to identify serologic antibody affinities to screen antigens, 457 proteins of the parasite were recognized by antibodies from patients, revealing 97 novel antigens and 2,031 disease-specific linear epitopes. This study could contribute to new peptidic markers for Chagas disease and the potential description of serologic biomarkers for diagnosis.
Department of Microbiology, Immunobiology and Parasitology, Sao Paulo Medical School, Federal University of São Paulo
Trypanosoma cruzi, the flagellated protozoan that causes Chagas disease in humans, has two hosts. The intermediary host, which is a mammal, or a human being, and definitive hosts, hemiptera insects that feed on blood (hematophagous) and belong to the Reduviidae family, more specifically to the Triatominae subfamily. The protozoan assumes different morphological forms in these hosts.
T. cruzi reproduces in the midgut of insects through binary fission every 20 to 24 hours. To do this, it acquires nutrients from the blood the insect ingested. During this phase, the parasite assumes a form called epimastigote, defined by the projection of the flagellum from the side of its unicellular body. It has adapted to survive the action of the enzymes that digest the blood inside the insect’s gut. For this purpose, it has, on its surface, a casing resistant to proteases and glycosidases. This casing consists of peculiar glycoproteins and glycolipids anchored to the membrane by phosphatidylinositol. Glycoproteins are similar to mucins, as they are formed by a skeleton of amino acids rich in threonins, to which glycosidic chains are linked. This glycosidic structure prevents the approximation of proteolytic enzymes. The glycolipids project from the surface towards the outside of the parasite and they also help retain the parasite in the insect’s intestinal cells.
In the insect’s gut, blood digestion generates free radicals, because blood is rich in hemoglobin, containing iron, a metal that is highly susceptible to oxidation. To protect itself from the damage produced by free radicals, the parasite has an efficient detox system using oxidoreductase enzymes. The proteins and hemoglobin produced are used as source of energy and carbon by the parasite by means of its elaborate system of endocytosis and digestive vacuoles. The excess nutrients absorbed are accumulated in modified vacuoles called reservosomes.
When the food from the bloodmeal begins to become scarce in the insect’s gut, the protein contents in the reservosomes diminishes, and the parasite begins a cellular differentiation program called metacyclogenesis, which involves morphological and metabolical changes that take approximately 48 hours. Cellular division stops, cellular volume diminishes, and the flagellum insertion position gradually shifts from the posterior part, where the reservosomes were, to the anterior part of the parasite. When the process is concluded, the flagellum remains attached to the entire body of the parasite, defining the trypomastigote form. At this stage, protein synthetis is reduced, and only new proteins necessary for interaction with the mammal host’s cells are expressed. The surface mucin ratio increases and glycolipids diminish, making the parasite detach itself from the intestinal wall and migrate to the hindgut and rectum of the insect. These trypomastigote forms are finally released with feces and urine when the insect makes another bloodmeal, and can therefore come into contact with the intermediary host.
The trypomastigotes released on the skin can come into contact with the mucosa of the eye, mouth, lesions, or can simply be ingested by the mammal. In the mucosas, trypomastigotes can move like a snake, thanks to the way the flagellum is connected to the entire cellular body, until it meets the surface of the host’s cells. This contact involves the participation of another type of glycoprotein, measuring between 80 and 90 kDa, produced only by trypomastigotes. These glycoproteins apparently recognize different types of molecules from the surface of the host’s cell and elements of the extracellular matrix, and trigger signals both on the parasite and on the host cell, culminating in the invasion of the cell. Trypomastigotes release proteases, peptides and glycosidases that also cause alterations on the membrane, on the cytoskeleton and on the organelles, facilitating the deformation of the plasma membrane and the formation of a vacuole containing the parasite inside the host cell.
The vacuole with the parasite is acidified by the fusion with cellular lysosomes, thanks to its proton pump. Lysosomes also deposit membrane glycoproteins in the parasitary vacuole. These glycoproteins are rich in sialic acid, a carbohydrate with negative charge. In lysosomes, sialic acid protects the membrane from self-digestion by lysosome enzymes. The parasite then releases an enzyme called trans-sialidaase, which removes sialic acid from the lysosomal proteins. This, together with the acidification cause by another parasite protein (TcTox), breaks the membrane of the vacuole, and the parasite escapes into the cytosol of the host cell.
The invasion process and the acidification induce the dissolution of the flagellum in the trypomastigote, through a mechanism that involves the action of endogenous proteases. The resulting forms, with a tiny flagellum, are called amastigotes. Located in the cytosol of the host cell, they once again can rely on a nutrient-rich environment. Some of these nutrients are intracellular proteins which are hydrolysed by parasite proteases, supplying the amino acids it requires to proliferate. Therefore, inhibitors of these proteases can inhibit the intracellular growth of the parasite. The amastigote form then begins to divide through binary fission, once every 16 hours, until they occupy the entire cellular space, with the exception of the nucleus. These forms interact closely with each other and/or with structures not yet identified, as they occupy a well-defined position in the cytoplasm of the host cell. When the parasite occupies the entire cell, it begins to elongate its flagellum, turning into a form that is similar to the epimastigote form. During this phase, the host’s nutrients begin to become scarce, signalling to the parasite so that new surface proteins are synthesized. These will be necessary for its extracellular phase inside the mammal host. The parasite begins to express trans-salidase and mucins that will cover its surface. The trans-sialidase that is produced by these forms has, in its carboxi-terminal region, a variable number of repetitions of 12 amino acids necessary for the oligomerization of the enzyme and to extend its catalytic part located in the amino-terminal of the membrane of the parasite, away from the parasite. As for mucins, these have longer protein chains, more extense glycidic portions, and a glycophosphatidylinositol anchor that is different from that of the mucins found in the insect forms.
The parasite induces the breakage of the host cell through a mechanism that is still not well understood, and, already differentiated into its trypomasatigote form, it escapes into the extracellular environment. The great amount of trans-sialidase present on the surface of the parasite captures the sialic acid present in proteins of the extracellular medium, which are then transferred to the terminal galactoses of the parasite’s mucins. This sialylation of the parasite helps protect the recently ecloded parasite from the action of natural antibodies, so it can reach the bloodstream and disseminate the infection. Trans-sialidase is also an important factor in the passage of the parasite through endothelial cells, and as it is released into the bloodstream it modulates the host’s immune system by inducing apoptosis in different tissues. At the same time, the lipidic portion of the mucins produced by these trypomastigotes and the great amount of proteases released by the circulating parasites work on different receptors, modulating the host’s immune response.
The circulating trypomastigotes now begin expressing different proteins of the glycoprotein family, measuring from 80 to 90 kDa, like the insect’s trypomastigotes. These also interact with proteins of the extracellular matrix, such as collagen, fibronectin and laminin, and with cellular surface receptors such as cyokeratins, promoting the retention and re-invasion of cells just like described above. Unlike other insect forms, these trypomastigotes released from the cells express large amounts of trans-sialidase, and rapidly escape the parasitophorous vacuole, which may ensure the survival of the parasite in a host that has already been immuno-stimulated.When, after a few infection cycles (around 7 to 30 days), the number of circulating parasites increases, the host begins to develop a robust immune response, and the infection begins to be controlled. However, some parasites seem to remain, contributing to the pathology of Chagas disease. The life cycle is completed when the blood of an infected mammal host is once again sucked by the vector insect.