The molecular analysis of Trypanosoma cruzi metallocarboxypeptidase 1 provides insight into fold and substrate specificity

Trypanosoma cruzi is the aetiological agent of Chagas' disease, a chronic infection that affects millions in Central and South America. Proteolytic enzymes are involved in the development and progression of this disease and two metallocarboxypeptidases, isolated from T. cruzi CL Brener clone, have recently been characterized: TcMCP-1 and TcMCP-2. Although both are cytosolic and closely related in sequence, they display different temporary expression patterns and substrate preferences. TcMCP-1 removes basic C-terminal residues, whereas TcMCP-2 prefers hydrophobic/aromatic residues. Here we report the three-dimensional structure of TcMCP-1. It resembles an elongated cowry, with a long, deep, narrow active-site cleft mimicking the aperture. It has an N-terminal dimerization subdomain, involved in a homodimeric catalytically active quaternary structure arrangement, and a proteolytic subdomain partitioned by the cleft into an upper and a lower moiety. The cleft accommodates a catalytic metal ion, most likely a cobalt, which is co-ordinated by residues included in a characteristic zinc-binding sequence, HEXXH and a downstream glutamate. The structure of TcMCP-1 shows strong topological similarity with archaeal, bacterial and mammalian metallopeptidases including angiotensin-converting enzyme, neurolysin and thimet oligopeptidase. A crucial residue for shaping the S(1') pocket in TcMCP-1, Met-304, was mutated to the respective residue in TcMCP-2, an arginine, leading to a TcMCP-1 variant with TcMCP-2 specificity. The present studies pave the way for a better understanding of a potential target in Chagas' disease at the molecular level and provide a template for the design of novel therapeutic approaches.     

Trypanosoma cruzi: a stage-specific calpain-like protein is induced after various kinds of stress

Calpains are calcium-dependent cysteine proteinases found in all living organisms and are involved in diverse cellular processes. Calpain-like proteins have been reported after in silico analysis of the Tritryps genome and are believed to play important roles in cell functions of trypanosomatids. We describe the characterization of a member of this family, which is differentially expressed during the life-cycle of Trypanosoma cruzi.     

Functional characterization of methionine sulfoxide reductase A from Trypanosoma spp

Methionine is an amino acid susceptible to being oxidized to methionine sulfoxide (MetSO). The reduction of MetSO to methionine is catalyzed by methionine sulfoxide reductase (MSR), an enzyme present in almost all organisms. In trypanosomatids, the study of antioxidant systems has been mainly focused on the involvement of trypanothione, a specific redox component in these organisms. However, no information is available concerning their mechanisms for repairing oxidized proteins, which would be relevant for the survival of these pathogens in the various stages of their life cycle. We report the molecular cloning of three genes encoding a putative A-type MSR in trypanosomatids. The genes were expressed in Escherichia coli, and the corresponding recombinant proteins were purified and functionally characterized. The enzymes were specific for L-Met(S)SO reduction, using Trypanosoma cruzi tryparedoxin I as the reducing substrate. Each enzyme migrated in electrophoresis with a particular profile reflecting the differences they exhibit in superficial charge. The in vivo presence of the enzymes was evidenced by immunological detection in replicative stages of T. cruzi and Trypanosoma brucei. The results support the occurrence of a metabolic pathway in Trypanosoma spp. involved in the critical function of repairing oxidized macromolecules.     

American Leishmania spp. and Trypanosoma cruzi: galactosyl alpha(1-3) galactose epitope localization by colloidal gold immunocytochemistry and lectin cytochemistry.

Patients with Chagas' disease or different clinical forms of leishmaniasis (cutaneous or visceral) have elevated galactosyl alpha (1-3)galactose antibodies. Using colloidal gold immunocytochemistry--monoclonal antibody gal-13 (specific for lipid-linked galactosyl alpha (1-3)galactose residues) and anti-nidogen antibodies and lectin cytochemistry (Bandeiraea simplicifolia IB4), both techniques specific for demonstrating galactosyl alpha (1-3)galactose residues--we have found terminal disaccharide residues on the Trypanosoma cruzi external surface of Vero cell-derived trypomastigotes but not in intact epimastigotes (although disrupted epimastigotes strongly stained), in the lips of the flagellar pocket, and on the parasitic side exactly opposite to the flagellar pocket in amastigote and promastigote forms of American Leishmania. These results resemble those obtained using anti-laminin antibodies in both trypanosomatids. In addition, results obtained with anti-nidogen antibodies seem to recognize in Trypanosoma cruzi and American Leishmania culture forms another different unknown terminal disaccharide. These results confirm the presence of terminal galactosyl alpha (1-3)galactose residues in both trypanosomatids, and that rabbit anti-laminin antibodies are indeed also recognizing galactosyl alpha (1-3)galactose residues as demonstrated for human circulating antibody. The presence of abundant galactosyl alpha (1-3)galactose residues on Trypanosomatid family members suggests a specific unknown role in parasite physiology for this terminal disaccharide.     

Characterization of dolichol diphosphate oligosaccharide: protein oligosaccharyltransferase and glycoprotein-processing glucosidases occurring in trypanosomatid protozoa

We have previously reported that the oligosaccharides transferred in vivo from dolichol-P-P derivatives in protein N-glycosylation in trypanosomatids are devoid of glucose residues and contain 2 N-acetylglucosamine and 6, 7, or 9 mannose units depending on the species. In this respect trypanosomatids differ from wild type mammalian, plant, insect, and fungal cells in which Glc3Man9GlcNAc2 is transferred. We are now reporting that incubation of Glc1-3Man9GlcNAc2-P-P-dolichol and Man7-9GlcNAc2-P-P-dolichol with membranes of Trypanosoma cruzi, Leptomonas samueli, Crithidia fasciculata, and Blastocrithidia culicis and an acceptor hexapeptide leads to the transfer of the six above mentioned lipid-linked oligosaccharides at the same rate. Control experiments performed under similar conditions but with rat liver and Saccharomyces cerevisiae membranes showed that, as already known, Glc3Man9GlcNAc2 is preferentially transferred in the latter systems. We have also previously reported that, once transferred to protein, the oligosaccharides become transiently glucosylated in trypanosomatids. Depending on the species, protein-linked Glc1Man5-9GlcNAc2 have been transiently detected in cells incubated with [14C] glucose. We are now reporting that glucosidase activities degrading both Glc1Man9GlcNAc2 and Glc2Man9GlcNAc2 were detected in T. cruzi, L. samueli, and C. fasciculata. The enzymatic activities were associated with a membrane fraction; they had a neutral optimum pH value, and similarly to mammalian glucosidase II, the enzyme acting on the monoglucosylated substrate showed a decreased affinity when the latter contained fewer mannose residues. No glucosidase I-like enzyme acting on Glc3Man9GlcNAc2 was detected in any of the three above-mentioned protozoan species. This result is consistent with the fact that no oligosaccharides containing 3 glucose units occur in trypanosomatids.     

Exploring the role of insect host factors in the dynamics of Trypanosoma cruzi-Rhodnius prolixus interactions

Members of the subfamily Triatominae, family Reduviidae, comprise a large number of insect species of which some are vectors of Trypanosoma cruzi, the causative agent of Chagas' disease. This article outlines research on the process of transformation and the dynamics of developmental stages of Trypanosoma cruzi in the triatomine insect hosts. Special attention is given to the interactions of parasites with gut molecules, and the gut environment, and with host developmental physiology and intestinal organization. The vector insect's permissiveness to Trypanosoma cruzi, which develops in the vector gut, largely depends on the host nutritional state, the parasite strain, trypanolytic compounds, digestive enzymes, lectins, resident bacteria in the gut and the endocrine system of the insect vector. Finally, the mechanisms of these interactions and their significance for Trypanosoma cruzi transmission are discussed.     

Crystal structure of the tryparedoxin peroxidase from the human parasite Trypanosoma cruzi

Tryparedoxin peroxidase from Trypanosoma cruzi (TcTXNPx) belongs to the family of typical 2-Cys peroxiredoxins. These enzymes function as antioxidants through their peroxidase and peroxynitrite reductase activities. In T. cruzi, as in all trypanosomatids, this enzyme is the final electron acceptor of a unique system for detoxifying hydroperoxides, constituting a relevant target for drug design. We have determined the crystal structure of TcTXPNx in the reduced active state. The structure comprises 10 subunits in the asymmetric unit, associated to form a decamer of toroidal shape obeying 52 (D5) point group symmetry. We have analyzed the structure of TcTXNPx by comparing it with other structures of typical 2-Cys peroxiredoxins in both redox states, and have identified key residues in the structural rearrangement taking place in the enzymatic cycle. This is the first report of the structure of an active peroxiredoxin that has peroxidase and peroxynitrite reductase activity, and it is noteworthy that it is from a human parasite. This knowledge is of interest for further understanding peroxide metabolism in these parasites, and in the design of new trypanosomatidal drugs against Chagas disease.     

Arginine kinase: a common feature for management of energy reserves in African and American flagellated trypanosomatids

This work reports the characterization of an arginine kinase in the unicellular parasitic flagellate Trypanosoma brucei, the etiological agent of human sleeping sickness and Nagana in livestock. The arginine kinase activity, detected in the soluble fraction obtained from procyclic forms, had a specific activity similar to that observed in Trypanosoma cruzi, about 0.2 micromol min(-1) mg(-1). Western blot analysis of T. brucei extracts revealed two bands of 40 and 45 kDa. The putative gene sequence of this enzyme had an open reading frame for a 356-amino acid polypeptide, one less than the equivalent enzyme of T. cruzi. The deduced amino acid sequence has an 82% identity with the arginine kinase of T. cruzi, and highest amino acid identities of both trypanosomatids sequences, about 70%, were with arginine kinases from the phylum Arthropoda. In addition, the amino acid sequence possesses the five arginine residues critical for interaction with ATP as well as two glutamic acids and one cysteine required for arginine binding. The finding in trypanosomatids of a new phosphagen biosynthetic pathway, which is not present in mammalian host tissues, suggests this enzyme as a possible target for chemotherapy.     

The Streamlined Genome of Phytomonas spp. Relative to Human Pathogenic Kinetoplastids Reveals a Parasite Tailored for Plants

Members of the family Trypanosomatidae infect many organisms, including animals, plants and humans. Plant-infecting trypanosomes are grouped under the single genus Phytomonas, failing to reflect the wide biological and pathological diversity of these protists. While some Phytomonas spp. multiply in the latex of plants, or in fruit or seeds without apparent pathogenicity, others colonize the phloem sap and afflict plants of substantial economic value, including the coffee tree, coconut and oil palms. Plant trypanosomes have not been studied extensively at the genome level, a major gap in understanding and controlling pathogenesis. We describe the genome sequences of two plant trypanosomatids, one pathogenic isolate from a Guianan coconut and one non-symptomatic isolate from Euphorbia collected in France. Although these parasites have extremely distinct pathogenic impacts, very few genes are unique to either, with the vast majority of genes shared by both isolates. Significantly, both Phytomonas spp. genomes consist essentially of single copy genes for the bulk of their metabolic enzymes, whereas other trypanosomatids e.g. Leishmania and Trypanosoma possess multiple paralogous genes or families. Indeed, comparison with other trypanosomatid genomes revealed a highly streamlined genome, encoding for a minimized metabolic system while conserving the major pathways, and with retention of a full complement of endomembrane organelles, but with no evidence for functional complexity. Identification of the metabolic genes of Phytomonas provides opportunities for establishing in vitro culturing of these fastidious parasites and new tools for the control of agricultural plant disease.     

Evidence for two distinct major protein components, PAR 1 and PAR 2, in the paraflagellar rod of Trypanosoma cruzi. Complete nucleotide sequence of PAR.

The previously identified major protein components of the paraflagellar rod in Trypanosoma cruzi, PAR 1 and PAR 2, were analyzed to determine if they are distinct proteins or different conformations of a single polypeptide as has been suggested for other trypanosomatids. Amino acid sequence analysis showed PAR 1 and PAR 2 to be two distinct polypeptides. Antibodies specific against either PAR 1 or PAR 2 were shown to each react with a distinct band in Western blots of paraflagellar isolates of T. cruzi and other trypanosomatids if rigorous protease inhibition was used. The PAR 2 message was isolated and characterized by Northern blot and nucleic acid sequence analysis. Preliminary analysis of the PAR 2 gene indicates that PAR 2 is a member of a multigene family with all members residing on a single chromosome.     

Signalling the genome: the Ras-like small GTPase family of trypanosomatids

The genomes of the three principle experimental-model species of Kinetoplastida -Trypanosoma brucei brucei, Trypanosoma cruzi and Leishmania major - are now complete, providing both a milestone for trypanosome biology and an opportunity to consider a multitude of questions at the genome level. Of the >40 members of the Ras-like GTPase family in T. brucei, at least 30 are involved in intracellular transport, whereas fewer than eight are likely to have a classical role in signal transduction. There are no true members of the Ras or Rho subfamilies but divergent Ras- or Rho-like GTPases are present, suggesting that signalling mechanisms in trypanosomatids are highly unusual. Comparisons of T. brucei with T. cruzi and L. major indicate a high degree of conservation among the species. These analyses provide a framework for the functional investigation of small-GTPase-mediated signalling processes in trypanosomes.     

Biochemical studies with DNA polymerase beta and DNA polymerase beta-PAK of Trypanosoma cruzi suggest the involvement of these proteins in mitochondrial DNA maintenance

Mammalian DNA polymerase beta is a nuclear enzyme involved in the base excision and single-stranded DNA break repair pathways. In trypanosomatids, this protein does not have a defined cellular localization, and its function is poorly understood. We characterized two Trypanosoma cruzi proteins homologous to mammalian DNA polymerasebeta, TcPolbeta and TcPolbetaPAK, and showed that both enzymes localize to the parasite kinetoplast. In vitro assays with purified proteins showed that they have DNA polymerization and deoxyribose phosphate lyase activities. Optimal conditions for polymerization were different for each protein with respect to dNTP concentration and temperature, and TcPolbetaPAK, in comparison to TcPolbeta, conducted DNA synthesis over a much broader pH range. TcPolbeta was unable to carry out mismatch extension or DNA synthesis across 8-oxodG lesions, and was able to discriminate between dNTP and ddNTP. These specific abilities of TcPolbeta were not observed for TcPolbetaPAK or other X family members, and are not due to a phenylalanine residue at position 395 in the C-terminal region of TcPolbeta, as assessed by a site-directed mutagenesis experiment reversing this residue to a well conserved tyrosine. Our data suggest that both polymerases from T. cruzi could cooperate to maintain mitochondrial DNA integrity through their multiple roles in base excision repair, gap filling and translesion synthesis.     

Functional homology of chemotactic methylesterases from Bacillus subtilis and Escherichia coli.

The methylesterase enzyme from Bacillus subtilis was compared with that from Escherichia coli. Both enzymes were able to demethylate methyl-accepting chemotaxis proteins (MCPs) from the other organism and were similarly affected by variations in glycerol, magnesium ion, or pH. When attractants were added to a mixture of B. subtilis MCPs and E. coli methylesterase, the rate of demethylation was enhanced. Conversely, when attractants were added to a mixture of E. coli MCPs and B. subtilis methylesterase, the rate of demethylation was diminished. These effects are what would be expected if, in these in vitro systems, the MCPs determined the rate of demethylation. These data suggest that, although the enzymes are from evolutionarily divergent organisms and are different in size, they have considerable functional homology.     

The crystal structure of a complex of Campylobacter jejuni dUTPase with substrate analogue sheds light on the mechanism and suggests the "basic module" for dimeric d(C/U)TPases

The crystal structure of the dUTPase from the important gastric pathogen Campylobacter jejuni has been solved at 1.65 A spacing. This essential bacterial enzyme is the second representative of the new family of dimeric dUTPases to be structurally characterised. Members of this family have a novel all-alpha fold and are unrelated to the all-beta dUTPases of the majority of organisms including eukaryotes such as humans, bacteria such as Escherichia coli, archaea like Methanococcus jannaschii and animal viruses. Therefore, dimeric dUTPases can be considered as candidate drug targets. The X-ray structure of the C.jejuni dUTPase in complex with the non-hydrolysable substrate analogue dUpNHp allows us to define the positions of three catalytically significant phosphate-binding magnesium ions and provides a starting point for a detailed understanding of the mechanism of dUTP/dUDP hydrolysis by dimeric dUTPases. Indeed, a water molecule present in the structure is ideally situated to act as the attacking nucleophile during hydrolysis. A comparison of the dUTPases from C.jejuni and Trypanosoma cruzi reveals a common fold with certain distinct features, both in the rigid and mobile domains as defined in the T.cruzi structure. Homologues of the C.jejuni dUTPase have been identified in several other bacteria and bacteriophages, including the dCTPase of phage T4. Sequence comparisons of these proteins define a new superfamily of d(C/U)TPases that includes three distinct enzyme families: (1) dUTPases in trypanosomatides, C.jejuni and several other Gram-negative bacteria, (2) predicted dUTPases in various Gram-positive bacteria and their phages, and (3) dCTP/dUTPases in enterobacterial T4-like phages. All these enzymes share a basic module that consists of two alpha-helices from the rigid domain, two helices from the mobile domain and connecting loops. These results in concert with a number of conserved residues responsible for interdomain cross-talk provide valuable insight towards rational drug design.     

Genetic diversity and kinetic properties of Trypanosoma cruzi dihydroorotate dehydrogenase isoforms

Dihydroorotate dehydrogenase (DHOD) is the fourth enzyme in the de novo pyrimidine biosynthetic pathway and is essential in Trypanosoma cruzi, the parasitic protist causing Chagas' disease. T. cruzi and human DHOD have different biochemical properties, including the electron acceptor capacities and cellular localization, suggesting that T. cruzi DHOD may be a potential chemotherapeutic target against Chagas' disease. Here, we report nucleotide sequence polymorphisms of T. cruzi DHOD genes and the kinetic properties of the recombinant enzymes. T. cruzi Tulahuen strain possesses three DHODgenes: DHOD1 and DHOD2, involved in the pyrimidine biosynthetic (pyr) gene cluster on an 800 and a 1000 kb chromosomal DNA, respectively, and DHOD3, located on an 800 kb DNA. The open reading frames of all three DHOD genes are comprised of 942 bp, and encode proteins of 314 amino acids. The three DHOD genes differ by 26 nucleotides, resulting in replacement of 8 amino acid residues. In contrast, all residues critical for constituting the active site are conserved among the three proteins. Recombinant T. cruzi DHOD1 and DHOD2 expressed in E. coli possess similar enzymatic properties, including optimal pH, optimal temperature, Vmax, and Km for dihydroorotate and fumarate. In contrast, DHOD3 had a higher Vmax and Km for both substrates. Orotate competitively inhibited all three DHOD enzymes to a comparable level. These results suggest that, despite their genetic variations, kinetic properties of the three T. cruziDHODs are conserved. Our findings facilitate further exploitation of T. cruzi DHOD inhibitors, as chemotherapeutic agents against Chagas' disease.     

Oxidative stress and antioxidant defenses: a target for the treatment of diseases caused by parasitic protozoa

Parasitic protozoa cause several diseases, affecting hundreds of millions, particularly in underdeveloped countries. Although these organisms are eukaryotic cells, some of them present major differences with their mammalian host in selected metabolic pathways. These differences may be exploited as targets for developing better pharmacological agents for the treatment of specific parasitic diseases. This review describes some of the differences in terms of antioxidant defenses between these organisms and their mammalian host, which may provide useful targets for the treatment of these diseases. Some of the potential targets are: (i). iron metabolism in Plasmodium, (ii). the presence of a Fe-containing form of superoxide dismutase in trypanosomatids and malaria-causing parasites, (iii). the unique trypanothione-dependent antioxidant metabolism in trypanosomatids, (iv). the ascorbate peroxidase found in Trypanosoma cruzi and perhaps present in other trypanosomatids.     

Mitochondrial localization of the mevalonate pathway enzyme 3-Hydroxy-3-methyl-glutaryl-CoA reductase in the Trypanosomatidae

3-Hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) is a key enzyme in the sterol biosynthesis pathway, but its subcellular distribution in the Trypanosomatidae family is somewhat controversial. Trypanosoma cruzi and Leishmania HMGRs are closely related in their catalytic domains to bacterial and eukaryotic enzymes described but lack an amino-terminal domain responsible for the attachment to the endoplasmic reticulum. In the present study, digitonin-titration experiments together with immunoelectron microscopy were used to establish the intracellular localization of HMGR in these pathogens. Results obtained with wild-type cells and transfectants overexpressing the enzyme established that HMGR in both T. cruzi and Leishmania major is localized primarily in the mitochondrion and that elimination of the mitochondrial targeting sequence in Leishmania leads to protein accumulation in the cytosolic compartment. Furthermore, T. cruzi HMGR is efficiently targeted to the mitochondrion in yeast cells. Thus, when the gene encoding T. cruzi HMGR was expressed in a hmg1 hmg2 mutant of Saccharomyces cerevisiae, the mevalonate auxotrophy of mutant cells was relieved, and immunoelectron analysis showed that the parasite enzyme exhibits a mitochondrial localization, suggesting a conservation between the targeting signals of both organisms.     

DNA Repair Pathways in Trypanosomatids: from DNA Repair to Drug Resistance

SUMMARY

All living organisms are continuously faced with endogenous or exogenous stress conditions affecting genome stability. DNA repair pathways act as a defense mechanism, which is essential to maintain DNA integrity. There is much to learn about the regulation and functions of these mechanisms, not only in human cells but also equally in divergent organisms. In trypanosomatids, DNA repair pathways protect the genome against mutations but also act as an adaptive mechanism to promote drug resistance. In this review, we scrutinize the molecular mechanisms and DNA repair pathways which are conserved in trypanosomatids. The recent advances made by the genome consortiums reveal the complete genomic sequences of several pathogens. Therefore, using bioinformatics and genomic sequences, we analyze the conservation of DNA repair proteins and their key protein motifs in trypanosomatids. We thus present a comprehensive view of DNA repair processes in trypanosomatids at the crossroads of DNA repair and drug resistance.     

The effects of time,temperature, and pH on the stability of PDU bacterial microcompartments

Bacterial microcompartments (MCPs) are subcellular organelles that are composed of a protein shell and encapsulated metabolic enzymes. It has been suggested that MCPs can be engineered to encapsulate protein cargo for use as in vivo nanobioreactors or carriers for drug delivery. Understanding the stability of the MCP shell is critical for such applications. Here, we investigate the integrity of the propanediol utilization (Pdu) MCP shell of Salmonella enterica over time, in buffers with various pH, and at elevated temperatures. The results show that MCPs are remarkably stable. When stored at 4°C or at room temperature, Pdu MCPs retain their structure for several days, both in vivo and in vitro. Furthermore, Pdu MCPs can tolerate temperatures up to 60°C without apparent structural degradation. MCPs are, however, sensitive to pH and require conditions between pH 6 and pH 10. In nonoptimal conditions, MCPs form aggregates. However, within the aggregated protein mass, MCPs often retain their polyhedral outlines. These results show that MCPs are highly robust, making them suitable for a wide range of applications.