Inhibitory action of marine algae extracts on the Trypanosoma cruzi dihydroorotate dehydrogenase activity and on the protozoan growth in mammalian cells

Trypanosoma cruzi, the causative agent of Chagas' disease, replicates in mammalian cells and relies on the de novo pyrimidine biosynthetic pathway that supplies essential precursors for nucleic acid synthesis. The protozoan dihydroorotate dehydrogenase (DHOD), the fourth enzyme of the pathway catalyzing production of orotate from dihydroorotate, markedly differs from the human enzyme. This study was thus aimed to search for potent inhibitors against T. cruzi DHOD activity, and a number of methanol extracts prepared from green, brown, and red algae were assayed. The extracts from two brown algae, Fucus evanescens and Pelvetia babingtonii, yielded 59 and 58% decrease in the recombinant DHOD activity, respectively, at the concentration of 50 microg/ml. Inhibition by these extracts was noncompetitive with respect to dihydroorotate, with apparent Ki values of 35.3+/-5.9 and 10.3+/-4.4 microg/ml, respectively. Further, in an in vitro T. cruzi-HeLa cell infection system, ethanol-reconstituted F. evanescens and P. babingtonii extracts at the concentration of 1 microg/ml, respectively, decreased significantly the infection rate of host cells and the average parasite number per infected cell. These results imply that F. evanescens and P. babingtonii contain inhibitor(s) against the T. cruzi DHOD activity and against the protozoan infection and proliferation in mammalian cells. Identification of inhibitor(s) in these two brown algae and further screening of other marine algae may facilitate the discovery of new, anti-trypanosomal lead compounds.     

Biochemical characterization of proline racemases from the human protozoan parasite Trypanosoma cruzi and definition of putative protein signatures

Proline racemase catalyzes the interconversion of L- and D-proline enantiomers and has to date been described in only two species. Originally found in the bacterium Clostridium sticklandii, it contains cysteine residues in the active site and does not require co-factors or other known coenzymes. We recently described the first eukaryotic amino acid (proline) racemase, after isolation and cloning of a gene from the pathogenic human parasite Trypanosoma cruzi. Although this enzyme is intracellularly located in replicative non-infective forms of T. cruzi, membrane-bound and secreted forms of the enzyme are present upon differentiation of the parasite into non-dividing infective forms. The secreted form of proline racemase is a potent host B-cell mitogen supporting parasite evasion of specific immune responses. Here we describe that the TcPRAC genes in T. cruzi encode functional intracellular or secreted versions of the enzyme exhibiting distinct kinetic properties that may be relevant for their relative catalytic efficiency. Although the Km of the enzyme isoforms were of a similar order of magnitude (29-75 mM), Vmax varied between 2 x 10(-4 )and 5.3 x 10(-5) mol of L-proline/s/0.125 microM of homodimeric recombinant protein. Studies with the enzyme-specific inhibitor and abrogation of enzymatic activity by site-directed mutagenesis of the active site Cys330 residue reinforced the potential of proline racemase as a critical target for drug development against Chagas' disease. Finally, we propose a protein signature for proline racemases and suggest that the enzyme is present in several other pathogenic and non-pathogenic bacterial genomes of medical and agricultural interest, yet absent in mammalian host, suggesting that inhibition of proline racemases may have therapeutic potential.     

The 6-phosphogluconate dehydrogenase of Leishmania (Leishmania) mexicana: gene characterization and protein structure prediction

6-Phosphogluconate dehydrogenase (6PGDH) is a key enzyme of the oxidative branch involved in the generation of NADPH and ribulose 5-phosphate. In the present work, we describe the cloning, sequencing and characterization of a 6PGDH gene from Leishmania (Leishmania) mexicana. The gene encodes a polypeptide chain of 479 amino acid residues with a predicted molecular mass of 52 kDa and a pI of 5.77. The recombinant protein possesses a dimeric quaternary structure and displays kinetic parameter values intermediate between those reported for Trypanosoma brucei and T. cruzi with apparent K(m) values of 6.93 and 5.2 μM for 6PG and NADP(+), respectively. The three-dimensional structure of the enzymes of Leishmania and T. cruzi were modelled from their amino acid sequence using the crystal structure of the enzyme of T. brucei as template. The amino acid residues located in the 6PGDH C-terminal region, which are known to participate in the salt bridges maintaining the protein dimeric structure, differed significantly among the enzymes of Leishmania, T. cruzi, and T. brucei. Our results strongly suggest that 6PGDH can be selected as a potential target for the development of new therapeutic drugs in order to improve existing chemotherapeutic treatments against these parasites.     

Effects on intestinal nutrient uptake and brush border membrane enzymes in response to atherogenic diet in rhesus monkeys

Transport of nutrients and kinetic parameters (Vmax and Km) of brush border membrane (BBM) enzymes were studied in duodenum, jejunum, and ileum from atherogenic diet-fed monkeys. The Km remained unaltered while feeding of atherogenic diet resulted in higher Vmax of sucrase, maltase, and alkaline phosphatase and lower Vmax of gamma-glutamyltranspeptidase and leucine-aminopeptidase compared to controls. Na+-dependent D-glucose transport was higher in duodenum and jejunum and unaltered in ileum. In contrast to D-glucose transport, the transport of amino acids was decreased in all three intestinal segments from atherogenic diet-fed monkeys.     

Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase does not conform to the ''hotspot'' topogenic signal model.

The genes which encode glycosomal glyceraldehyde-phosphate dehydrogenase (gGAPDH) of Trypanosoma cruzi are arranged as a tandemly repeated pair on a single chromosome and are identical at the level of nucleotide sequence. They are separated by an intergenic region which contains a 317 base pair sequence with the properties of a retroposon. The genes express a 1.5 kb mRNA and a 38 kd protein. The amino acid sequence contains features characteristic of glycosomal enzymes such as peptide insertions and a C-terminal extension. However, T. cruzi gGAPDH lacks one of the positively charged 'hotspot' motifs which have been proposed as topogenic signals for import into the glycosome, a unique microbody-like organelle. Molecular modelling of the T. cruzi and T. brucei enzymes suggests that neither structure would fulfil the requirements of the 'hotspot' glycosomal import model.     

Evolutionary implications of the mosaic pyrimidine-biosynthetic pathway in eukaryotes

The de-novo pyrimidine biosynthetic pathway involves six enzymes, in order from the first to the sixth step, carbamoyl-phosphate synthetase II (CPS II) comprising glutamine amidotransferase (GAT) and carbamoyl-phosphate synthetase (CPS) domains or subunits, aspartate carbamoyltransferase (ACT), dihydroorotase (DHO), dihydroorotate dehydrogenase (DHOD), orotate phosphoribosyltransferase (OPRT), and orotidine-5'-monophosphate decarboxylase (OMPDC). In contrast with reports on molecular evolution of the individual enzymes, we attempted to draw an evolutionary picture of the whole pathway using the protein phylogeny. We demonstrate highly mosaic organizations of the pyrimidine biosynthetic pathway in eukaryotes. During evolution of the eukaryotic pathway, plants and fungi (or their ancestors) in particular may have secondarily acquired the characteristic enzymes. This is consistent with the fact that the organization of plant enzymes is highly chimeric: (1) two subunits of CPS II, GAT and CPS, cluster with a clade including cyanobacteria and red algal chloroplasts, (2) ACT not with a cyanobacterium, Synechocystis spp., irrespective of its putative signal sequence targeting into chloroplasts, and (3) DHO with a clade of proteobacteria. In fungi, DHO and OPRT cluster respectively with the corresponding proteobacterial counterparts. The phylogenetic analyses of DHOD and OMPDC also support the implications of the mosaic pyrimidine biosynthetic pathway in eukaryotes. The potential importance of the horizontal gene transfer(s) and endosymbiosis in establishing the mosaic pathway is discussed.     

In silico reconstruction of the amino acid metabolic pathways of Trypanosoma cruzi

Trypanosoma cruzi is the epidemiological agent of Chagas' disease, affecting most of Central and South America, constituting a significant health and socio-economic problem. The parasite has a metabolism largely based on the consumption of amino acids, which participate in a diversity of metabolic pathways, leading to many crucial compounds for the survival of this parasite. Study of its enzymes has the potential to disclose new therapeutic targets and foster the development of new drugs. In this study, we employed computational approaches to reconstruct in silico the amino acid metabolic pathways of T. cruzi, aiming to link genomic information with functional information. For that, protein sequences from 570 EC classes belonging to 25 different pathways in general amino acid metabolism were downloaded from KEGG. A subset of 471 EC classes had at least one sequence deposited. Clustering of the proteins belonging to each EC class was performed using a similarity-based approach implemented in the tool AnEnPi. Reconstruction of the metabolic pathways comprising the amino acid metabolism of T. cruzi was performed by analyzing the output of BLASTP, using as query the dataset of predicted proteins of T. cruzi against all sequences of each individual cluster. This approach allowed us to identify 764 T. cruzi proteins probably involved in the metabolism of amino acids as well as the identification of several putative cases of analogy. Furthermore, we were able to identify several enzymatic activities of T. cruzi that were not previously included in KEGG.     

The role of Leu-190 in the function and stability of adenylate kinase

To elucidate the role of C-terminal region of chicken adenylate kinase (a single polypeptide consisting of 193 amino acid residues) in the catalysis and stability of the enzyme, a series of mutant proteins truncated in the C-terminal region has been prepared by successive replacements of the sense codons by a termination codon via site-directed mutagenesis. Removal of the three C-terminal residues did not affect the apparent Michaelis constants (Km values) for AMP and ATP, although the Vmax values decreased gradually in parallel with the length of the polypeptide chain. A sudden increase in Km values for substrates, in particular for ATP, was observed on removal of one additional residue (Leu-190), the Vmax value also being less than one-half of that of the mutant enzyme with 3 residues shorter than the wild-type enzyme. These results suggest the importance of the highly conservative Leu-190. Therefore, we further prepared the mutant enzymes through replacement of Leu-190 by a variety of other amino acid residues. They all had substantially lower Vmax values and decreased thermostabilities. Their apparent Km values for ATP also changed, whereas those for AMP were affected to a lesser extent. The hydrophobicity of amino acid residues at position 190 was found to positively correlate with the specificity constants (kcat/Km values) for ATP and also with the thermostability of the enzyme. The fluorescence emission of the Trp-190 mutant enzyme was quenched by the addition of ATP. It is suggested that the C-terminal residues, particularly those around Leu-190, are present in a hydrophobic region which may be involved in binding of ATP.     

T cell epitope characterization in tandemly repetitive Trypanosoma cruzi B13 protein

Proteins containing tandemly repetitive sequences are present in several immunodominant protein antigens in pathogenic protozoan parasites. The tandemly repetitive Trypanosoma cruzi B13 protein is recognized by IgG antibodies from 98% of Chagas' disease patients. Little is known about the molecular mechanisms that lead to the immunodominance of the repeated sequences, and there is limited information on T cell epitopes in such repetitive antigens. We finely characterized the T cell recognition of the tandemly repetitive, degenerate B13 protein by T cell lines, clones and PBMC from Chagas' disease cardiomyopathy (CCC), asymptomatic T. cruzi infected (ASY) and non-infected individuals (N). PBMC proliferative responses to recombinant B13 protein were restricted to individuals bearing HLA-DQA1*0501(DQ7), -DR1, and -DR2; B13 peptides bound to the same HLA molecules in binding assays. The HLA-DQ7-restricted minimal T cell epitope [FGQAAAG(D/E)KP] was identified with an overlapping combinatorial peptide library including all B13 sequence variants in T. cruzi Y strain B13 protein; the underlined small residues GQA were the major HLA contact residues. Among natural B13 15-mer variant peptides, molecular modeling showed that several variant positions were solvent (TCR)-exposed, and substitutions at exposed positions abolished recognition. While natural B13 variant peptide S15.9 seems to be the immunodominant epitope for Chagas' disease patients, S15.4 was preferentially recognized by CCC rather than ASY patients, which may be pathogenically relevant. This is the first thorough characterization of T cell epitopes of a tandemly repetitive protozoan antigen and may suggest a role for T cell help in the immunodominance of protozoan repetitive antigens.     

Molecular and functional characterization of a spermidine transporter (TcPAT12) from Trypanosoma cruzi

Trypanosoma cruzi, the etiological agent of Chagas' disease, is the only eukaryotic cell which lacks the ability to synthesize polyamines de novo. In this work, we describe for the first time the molecular and biochemical properties of a high-affinity spermidine transporter from T. cruzi. The transporter gene TcPAT12 was functionally expressed in Xenopus laevis oocytes, showing high levels of spermidine uptake. Similar apparent affinity constants for spermidine uptake were obtained when comparing T. cruzi epimastigotes and heterologous expressed TcPAT12 in X. laevis. In addition, TcPAT12 also transports putrescine and the amino acid l-arginine at lower rates than spermidine.     

Critical importance of the de novo pyrimidine biosynthesis pathway for Trypanosoma cruzi growth in the mammalian host cell cytoplasm

The intracellular parasitic protist Trypanosoma cruzi is the causative agent of Chagas disease in Latin America. In general, pyrimidine nucleotides are supplied by both de novo biosynthesis and salvage pathways. While epimastigotes-an insect form-possess both activities, amastigotes-an intracellular replicating form of T. cruzi-are unable to mediate the uptake of pyrimidine. However, the requirement of de novo pyrimidine biosynthesis for parasite growth and survival has not yet been elucidated. Carbamoyl-phosphate synthetase II (CPSII) is the first and rate-limiting enzyme of the de novo biosynthetic pathway, and increased CPSII activity is associated with the rapid proliferation of tumor cells. In the present study, we showed that disruption of the T. cruzi cpsII gene significantly reduced parasite growth. In particular, the growth of amastigotes lacking the cpsII gene was severely suppressed. Thus, the de novo pyrimidine pathway is important for proliferation of T. cruzi in the host cell cytoplasm and represents a promising target for chemotherapy against Chagas disease.     

Mercaptopyridine-N-oxide, an NADH-fumarate reductase inhibitor, blocks Trypanosoma cruzi growth in culture and in infected myoblasts

The enzyme NADH-fumarate reductase is not found in mammalian cells but it is present in several parasitic protozoa including Trypanosoma cruzi, the parasite that causes Chagas' disease. This study shows that the drug 2-mercaptopyridine-N-oxide (MPNO) inhibits NADH-fumarate reductase purified from T. cruzi (ID50 = 35 microM). When added to intact cells, MPNO inhibited the growth of T. cruzi epimastigotes in culture (ID50 = 0.08 microM) as well as the infection of mammalian myoblasts by T. cruzi trypomastigotes (ID50 = 20 microM). At a concentration of 2.4 microM, MPNO also inhibited the growth of amastigotes (intracellular dividing forms) in cultured mammalian myoblasts. Supplementation of culture media with 5 mM succinate, the product of fumarate reductase, partially protected against the inhibition of the growth of epimastigotes by MPNO. Moreover, MPNO inhibited the accumulation of succinate in cultures of epimastigotes, as measured by high performance liquid chromatography. Although MPNO may have other intracellular targets in addition to fumarate reductase, these results support the hypothesis that compounds which inhibit the enzyme fumarate reductase may be potential chemotherapeutic agents against Chagas' disease.     

Trypanosoma cruzi proteins which are antigenic during human infections are located in defined regions of the parasite

Polyclonal antibodies obtained against antigenic proteins encoded by six recombinant DNA clones of Trypanosoma cruzi were used for the ultrastructural localization of the respective antigens in thin sections of parasites (epimastigote, amastigote and trypomastigote forms of T. cruzi) embedded at low temperature in Lowicryl K4M resin. Antigens of high molecular weight containing tandemly repeated amino acid sequence motifs and recognized by sera from patients with Chagas' disease, were located only in the flagellum, where it contacts the parasite cell body. Other antigens were located on the surface of the parasite while still others were found within the flagellar pocket, as is the case with an antigen released during the acute phase of Chagas' disease. Thus, we conclude that some of the T. cruzi proteins which are antigenic in human infections are located in defined regions of the parasite. Some of the antigens were not expressed to the same extent in the three different developmental stages of the parasite.     

The Origin of Dihydroorotate Dehydrogenase Genes of Kinetoplastids, with Special Reference to Their Biological Significance and Adaptation to Anaerobic, Parasitic Conditions

Trypanosoma cruzi dihydroorotate dehydrogenase (DHOD), the fourth enzyme of the de novo pyrimidine biosynthetic pathway, is localized in the cytosol and utilizes fumarate as electron acceptor (fumarate reductase activity), while the enzyme from other various eukaryotes is mitochondrial membrane-linked. Here we report that DHOD-knockout T. cruzi did not express the enzyme protein and could not survive even in the presence of pyrimidine nucleosides, substrates for the potentially active salvage pathway, suggesting a vital role of fumarate reductase activity in the regulation of cellular redox balance. Cloning and phylogenetic analysis of euglenozoan DHOD genes showed that the euglenoid Euglena gracilis had a mitochondrial DHOD and that biflagellated bodonids, a sister group of trypanosomatids within kinetoplastids, harbor the cytosolic DHOD. Further, Bodo saliens, a bodonid, had an ACT/DHOD gene fusion encoding aspartate carbamoyltransferase (ACT), the second enzyme of the de novo pyrimidine pathway, and DHOD. This is the first report of this novel gene structure. These results are consistent with suggestions that an ancient common ancestor of Euglenozoa had a mitochondrial DHOD whose descendant exists in E. gracilis and that a common ancestor of kinetoplastids (bodonids and trypanosomatids) subsequently acquired a cytosolic DHOD by horizontal gene transfer. The cytosolic DHOD gene thus acquired may have contributed to adaptation to anaerobiosis in the kinetoplastid lineage and further contributed to the subsequent establishment of parasitism in a trypanosomatid ancestor. Different molecular strategies for anaerobic adaptation in pyrimidine biosynthesis, used by kinetoplastids and by euglenoids, are discussed. Evolutionary implications of the ACT/DHOD gene fusion are also discussed.Sequence availability: The nucleotide sequence data reported here appear in the GenBank, EMBL, and DDBJ databases with the accession numbers AB120414, AB159227, and AB159228 for Euglena gracilis dihydroorotate dehydrogenase (DHOD), Bodo saliens aspartate carbamoyltransferase/dihydroorotate dehydrogenase (ACT/DHOD), and B. caudatus DHOD, respectively.Reviewing Editor: Dr. Patrick Keeling     

Human humoral immunity to hsp70 during Trypanosoma cruzi infection

Immunologic screening of cDNA expression libraries has been widely used for the identification of DNA sequences encoding the immunologically relevant proteins of many pathogenic microorganisms. For reasons that are not entirely clear, sequences encoding 70-kDa heat shock and related proteins (hsp70), which are among the most highly conserved proteins known, have routinely been identified by this approach. Consequently, hsp70 proteins have been proposed to be involved in the autoimmune processes thought responsible for the pathogenesis of the diseases caused by some of these organisms, e.g., chronic Trypanosoma cruzi infection (Chagas' disease). Therefore, we investigated whether hsp70 might be a specific target of the human humoral immune response to T. cruzi infection, and, if so, whether humoral autoimmunity to hsp70 might play a role in pathogenesis. We found that hsp70 is indeed a major polypeptide Ag in Chagas' disease, but that the antibodies to T. cruzi hsp70 do not react with human hsp70--even though the proteins display 73% amino acid sequence identify. These results indicate that self-tolerance to hsp70 is maintained during chronic T. cruzi infection and strongly argue against a role for humoral autoimmunity to hsp70 in the pathogenesis of Chagas' disease.     

Serine hydroxymethyltransferase activity in Trypanosoma cruzi, Trypanosoma rangeli and American Leishmania spp

Serine hydroxymethyltransferase (SHMT) was studied in several American trypanosomatids, Trypanosoma cruzi epimastigotes displaying, in contrast with T. rangeli, high enzymatic activity. Several Leishmania spp. members, including L. braziliensis, L. mexicana and L. garnhami promastigotes, under identical assay conditions, showed low enzymatic activity. The T. cruzi and leishmanial enzymes presented several different kinetic properties, and thus apparent Km for THF was 0.30 mM for the trypanosomal SHMT vs 0.60 mM for the leishmanial enzyme, while the apparent Km for serine was 0.40 mM for trypanosomal SHMT vs 0.15 mM for leishmanial enzyme. There were significant variations in the specific activity of SHMT between the several different trypanosomatids strains studied, but the meaning of these results is not clear because they showed no correlation either with taxonomy or infectivity.     

Glucosephosphate isomerase from Trypanosoma brucei. Cloning and characterization of the gene and analysis of the enzyme

In Trypanosoma brucei the enzyme glucose-6-phosphate isomerase, like most other enzymes of the glycolytic pathway, resides in a microbody-like organelle, the glycosome. Here we report a detailed study of this enzyme, involving a determination of its kinetic properties and the cloning and sequence analysis of its gene. The gene codes for a polypeptide of 606 amino acids, with a calculated Mr of 67280. The protein predicted from the gene sequence has 54-58% positional identity with its yeast and mammalian counterparts. Compared to those other glucose-6-phosphate isomerases the trypanosomal enzyme contains an additional 38-49 amino acids in its N-terminal domain, as well as a number of small insertions and deletions. The additional amino acids are responsible for the 5-kDa-larger subunit mass of the T. brucei enzyme, as measured by gel electrophoresis. The glucose-6-phosphate isomerase of the trypanosome has no excess of positive residues and, consequently, no high isoelectric point, in contrast to the other glycolytic enzymes that are present in the glycosome. However, similar to other glycosomal proteins analyzed so far, specific clusters of positive residues can be recognized in the primary structure. Comparison of the kinetic properties of the T. brucei glucose-6-phosphate isomerase with those of the yeast and rabbit muscle enzymes did not reveal major differences. The three enzymes have very similar pH profiles. The affinity for the substrate fructose 6-phosphate (Km = 0.122 mM) and the inhibition constant for the competitive inhibitor gluconate 6-phosphate (Ki = 0.14 mM) are in the same range as those of the similar enzymes. The Km shows the same strong dependence on salt as the rabbit muscle enzyme, although somewhat less than the yeast glucose-6-phosphate isomerase. The trypanocidal drug suramin inhibits the T. brucei and yeast enzymes to the same extent (Ki = 0.29 and 0.36 mM, respectively), but it had no effect on the rabbit muscle enzyme. Agaricic acid, a potent inhibitor of various glycosomal enzymes of T. brucei, has also a strong, irreversible effect on glucose-6-phosphate isomerase, while leaving the yeast and mammalian enzymes relatively unaffected.     

Cloning and characterization of the phosphoglucomutase of Trypanosoma cruzi and functional complementation of a Saccharomyces cerevisiae PGM null mutant

Trypanosoma cruzi is the etiological agent of Chagas' disease, a chronic illness characterized by progressive cardiomyopathy and/or denervation of the digestive tract. The parasite surface is covered with glycoconjugates, such as mucin-type glycoproteins and glycoinositolphospholipids (GIPLs), whose glycans are rich in galactopyranose (Galp) and/or galactofuranose (Galf) residues. These molecules have been implicated in attachment of the parasite to and invasion of mammalian cells and in modulation of the host immune responses during infection. In T. cruzi, galactose (Gal) biosynthesis depends on the conversion of uridine diphosphate (UDP)-glucose (UDP-Glc) into UDP-Gal by an NAD-dependent reduction catalyzed by UDP-Gal 4-epimerase. Phosphoglucomutase (PGM) is a key enzyme in this metabolic pathway catalyzing the interconversion of Glc-6-phosphate (Glc-6-P) and Glc-1-P which is then converted into UDP-Glc. We here report the cloning of T. cruzi PGM, encoding T. cruzi PGM, and the heterologous expression of a functional enzyme in Saccharomyces cerevisiae. T. cruzi PGM is a single copy gene encoding a predicted protein sharing 61% amino acid identity with Leishmania major PGM and 43% with the yeast enzyme. The 59-trans-splicing site of PGM RNA was mapped to a region located at 18 base pairs upstream of the start codon. Expression of T. cruzi PGM in a S. cerevisiae null mutant-lacking genes encoding both isoforms of PGM (pgm1Delta/pgm2Delta) rescued the lethal phenotype induced upon cell growth on Gal as sole carbon source.