The malate dehydrogenase isoforms from Trypanosoma brucei: subcellular localization and differential expression in bloodstream and procyclic forms

Trypanosoma brucei procyclic forms possess three different malate dehydrogenase isozymes that could be separated by hydrophobic interaction chromatography and were recognized as the mitochondrial, glycosomal and cytosolic malate dehydrogenase isozymes. The latter is the only malate dehydrogenase expressed in the bloodstream forms, thus confirming that the expression of malate dehydrogenase isozymes is regulated during the T. brucei life cycle. To achieve further biochemical characterization, the genes encoding mitochondrial and glycosomal malate dehydrogenase were cloned on the basis of previously reported nucleotide sequences and the recombinant enzymes were functionally expressed in Escherichia coli cultures. Mitochondrial malate dehydrogenase showed to be more active than glycosomal malate dehydrogenase in the reduction of oxaloacetate; nearly 80% of the total activity in procyclic crude extracts corresponds to the former isozyme which also catalyzes, although less efficiently, the reduction of p-hydroxyphenyl-pyruvate. The rabbit antisera raised against each of the recombinant isozymes showed that the three malate dehydrogenases do not cross-react immunologically. Immunofluorescence experiments using these antisera confirmed the glycosomal and mitochondrial localization of glycosomal and mitochondrial malate dehydrogenase, as well as a cytosolic localization for the third malate dehydrogenase isozyme. These results clearly distinguish Trypanosoma brucei from Trypanosoma cruzi, since in the latter parasite a cytosolic malate dehydrogenase is not present and mitochondrial malate dehydrogenase specifically reduces oxaloacetate.     

The NAD-linked aromatic alpha-hydroxy acid dehydrogenase from Trypanosoma cruzi. A new member of the cytosolic malate dehydrogenases group without malate dehydrogenase activity.

Trypanosoma cruzi, the protozoan parasite causing Chagas disease, contains a novel aromatic alpha-hydroxy acid dehydrogenase. This enzyme is responsible, together with tyrosine aminotransferase, for the catabolism of aromatic amino acids, which leads to the excretion of aromatic lactate derivatives into the culture medium. The gene encoding the aromatic alpha-hydroxy acid dehydrogenase has been cloned through a combined approach using screening of an expression genomic library with antibodies, peptide sequencing and PCR amplification. Its sequence shows high similarity to the cytosolic malate dehydrogenases. However, the enzyme has no malate dehydrogenase activity. The gene seems to be present in a single copy per haploid genome and is differentially expressed throughout the parasite's life cycle, the highest levels being found in the insect forms of T. cruzi. The purified recombinant enzyme, expressed in Escherichia coli, was unable to reduce oxaloacetate and had kinetic constants similar to those of the natural aromatic alpha-hydroxy acid dehydrogenase. Sequence comparisons suggest that the aromatic alpha-hydroxy acid dehydrogenase derives from a cytosolic malate dehydrogenase no longer present in the parasite, made redundant by the presence of a glycosomal malate dehydrogenase as a member of a shuttle device involving the mitochondrial isoenzyme.     

Characterization of glycosomal RING finger proteins of trypanosomatids

The glycosomes of trypanosomatids are essential organelles that are evolutionarily related to peroxisomes of other eukaryotes. The peroxisomal RING proteins-PEX2, PEX10 and PEX12-comprise a network of integral membrane proteins that function in the matrix protein import cycle. Here, we describe PEX10 and PEX12 in Trypanosoma brucei, Leishmania major, and Trypanosoma cruzi. We expressed GFP fusions of each T. brucei coding region in procyclic form T. brucei, where they localized to glycosomes and behaved as integral membrane proteins. Despite the weak transmembrane predictions for TbPEX12, protease protection assays demonstrated that both the N and C termini are cytosolic, similar to mammalian PEX12. GFP fusions of T. cruzi PEX10 and L. major PEX12 also localized to glycosomes in T. brucei indicating that glycosomal membrane protein targeting is conserved across trypanosomatids.     

Production of aromatic α-hydroxyacids by epimastigotes of Trypanosoma cruzi, and its possible role in NADH reoxidation

Abstract Epimastigotes of Trypanosoma cruzi in culture produce and excrete into the medium small amounts of phenyllactic acid and p -hydrocyphenyllactic acids, presumbly arising from the catabolism of the aromatic amino acids phenylalanine and tyrosine, respectively. This production might constitute a minor pathway for the reoxidation of cytosolic NADH, through the concerted action of tyrosine aminotransferase and aromatic α-hydroxyacid dehydrogenase.     

The occurrence and distribution of alpha-hydroxy-acid dehydrogenase in some members of the order Kinetoplastida

LDH-X is the isoenzyme of lactate dehydrogenase found in mammalian spermatozoa, occurring in cytosolic and mitochondrial locations. Gossypol strongly inhibits it, and the spermicidal action of this compound is attributed to the disruption of a reducing shuttle. The flagellated protozoan, Trypanosoma cruzi, contains an enzyme activity similar to LDH-X, called alpha-hydroxy-acid dehydrogenase, which is here shown to possess cytosolic and glycosomal components. The glycosome is a microbody-like organelle containing the early glycolytic enzymes. We postulate that the inhibition of replication of T. cruzi by gossypol derives from interference with glycosomal reducing shuttles. T. lewisi resembles T. cruzi in this respect.     

Probing the specificity of a trypanosomal aromatic alpha-hydroxy acid dehydrogenase by site-directed mutagenesis

The aromatic l-alpha-hydroxy acid dehydrogenase (AHDAH) from Trypanosoma cruzi has over 50% sequence identity with cytosolic malate dehydrogenases (cMDHs), yet it is unable to reduce oxaloacetate. Molecular modeling of the three-dimensional structure of AHADH using the pig cMDH as template directed the construction of several mutants. AHADH shares with MDHs the essential catalytic residues H195 and R171 (using Eventoff's numbering). The AHADH A102R mutant became able to reduce oxaloacetate, while remaining fully active towards aromatic alpha-oxoacids. The Y237G mutant diminished its affinity for all of the natural substrates, whereas the double mutant A102R/Y237G was more active than Y237G and had similar activity with oxaloacetate and with aromatic substrates. The present results reinforce our proposal that AHADH arose by a moderate number of point mutations from a cMDH no longer present in the parasite.     

Cloning and heterologous expression of a broad specificity aminotransferase of Leishmania mexicana promastigotes1

We have previously reported that Leishmania mexicana promastigotes possess a broad substrate specificity aminotransferase (BSAT), able to transaminate aspartate, aromatic amino acids, methionine and leucine. We have confirmed now this unusual substrate specificity by cloning its gene and expressing in Escherichia coli the recombinant active protein. The amino acid sequence of BSAT shares over 40% identity with other eukaryotic and prokaryotic aspartate aminotransferases, thus showing that the enzyme belongs to the subfamily Ialpha of aminotransferases, and has only 6% identity with the tyrosine aminotransferase from Trypanosoma cruzi, which has a similar substrate specificity. The production of recombinant active enzyme in good yields opens up the possibility of obtaining its 3D-structure, in order to investigate the structural basis of the broad substrate specificity.     

The topogenic signal of the glycosomal (microbody) phosphoglycerate kinase of Crithidia fasciculata resides in a carboxy-terminal extension.

To determine how microbody proteins enter microbodies, we have previously compared the genes for the cytosolic and glycosomal (microbody) phosphoglycerate kinases (PGKs) of Trypanosoma brucei and found the microbody enzyme to differ from other PGKs and the cytosolic form in two respects: a high net positive charge and a C-terminal extension of 20 amino acids (Osinga et al., 1985). Here we present the comparison of the genes for the cytosolic and glycosomal PGKs of Crithidia fasciculata, another kinetoplastid organism. The amino acid sequences of the two Crithidia isoenzymes are virtually identical, except for a C-terminal extension of 38 amino acids. We conclude that this extension must direct the glycosomal PGK to the glycosome. The extensions of the Crithidia and Trypanosoma enzymes are both rich in small hydrophobic and hydroxyl amino acids.     

Topogenesis of microbody enzymes: a sequence comparison of the genes for the glycosomal (microbody) and cytosolic phosphoglycerate kinases of Trypanosoma brucei.

To determine how microbody enzymes enter microbodies, we are studying the genes for cytosolic and glycosomal (microbody) isoenzymes in Trypanosoma brucei. We have found three genes (A, B and C) coding for phosphoglycerate kinase (PGK) in a tandem array in T. brucei. Gene B codes for the cytosolic and gene C for the glycosomal isoenzyme. Genes B and C are 95% homologous, and the predicted protein sequences share approximately 45% amino acid homology with other eukaryote PGKs. The microbody isoenzyme differs from the cytosolic form and other PGKs in two respects: a high positive charge and a carboxy-terminal extension of 20 amino acids. Our results show that few alterations are required to redirect a protein from cytosol to microbody. From a comparison of our results with the unpublished data for three other glycosomal glycolytic enzymes we infer that the high positive charge represents the major topogenic signal for uptake of proteins into glycosomes.     

Trypanosomes are monophyletic: evidence from genes for glyceraldehyde phosphate dehydrogenase and small subunit ribosomal RNA

The genomes of Trypanosoma brucei, Trypanosoma cruzi and Leishmania major have been sequenced, but the phylogenetic relationships of these three protozoa remain uncertain. We have constructed trypanosomatid phylogenies based on genes for glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) and small subunit ribosomal RNA (SSU rRNA). Trees based on gGAPDH nucleotide and amino acid sequences (51 taxa) robustly support monophyly of genus Trypanosoma, which is revealed to be a relatively late-evolving lineage of the family Trypanosomatidae. Other trypanosomatids, including genus Leishmania, branch paraphyletically at the base of the trypanosome clade. On the other hand, analysis of the SSU rRNA gene data produced equivocal results, as trees either robustly support or reject monophyly depending on the range of taxa included in the alignment. We conclude that the SSU rRNA gene is not a reliable marker for inferring deep level trypanosome phylogeny. The gGAPDH results support the hypothesis that trypanosomes evolved from an ancestral insect parasite, which adapted to a vertebrate/insect transmission cycle. This implies that the switch from terrestrial insect to aquatic leech vectors for fish and some amphibian trypanosomes was secondary. We conclude that the three sequenced pathogens, T. brucei, T. cruzi and L. major, are only distantly related and have distinct evolutionary histories.     

A second class of peroxidases linked to the trypanothione metabolism

Trypanosoma brucei, the causative agent of African sleeping sickness, has three nearly identical genes encoding cysteine homologues of classical selenocysteine-containing glutathione peroxidases. The proteins are expressed in the mammalian and insect stages of the parasite. One of the genes, which contains a mitochondrial as well as a glycosomal targeting signal has been overexpressed. The recombinant T. brucei peroxidase has a high preference for the trypanothione/tryparedoxin couple as electron donor for the reduction of different hydroperoxides but accepts also T. brucei thioredoxin. The apparent rate constants k(2)' for the regeneration of the reduced enzyme are 2 x 10(5) m(-1) s(-1) with tryparedoxin and 5 x 10(3) m(-1) s(-1) with thioredoxin. No saturation kinetics was observed and the rate-limiting step of the overall reaction is reduction of the hydroperoxide. With glutathione, the peroxidase has marginal activity and reduction of the enzymes becomes limiting with a k(2)' value of 3 m (-1) s(-1). The T. brucei peroxidase, in contrast to the related Trypanosoma cruzi enzyme, also accepts hydrogen peroxide as substrate. The catalytic efficiency of the peroxidase studied here is comparable with that of the peroxiredoxin-like tryparedoxin peroxidases, which shows that trypanosomes possess two distinct peroxidase systems both dependent on the unique dithiol trypanothione.     

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.     

Enzymatic inhibitory activity and trypanocidal effects of extracts and compounds from Siphoneugena densiflora O. Berg and Vitex polygama Cham

Hexanic, methanolic, and hydroalcoholic extracts, and 34 isolated compounds from Vitex polygama Cham. (Lamiaceae, formely Verbenaceae) and Siphoneugena densiflora O. Berg (Myrtaceae) were screened for their trypanocidal effects on bloodstream forms of Trypanosoma cruzi and T brucei, as well as for their enzymatic inhibitory activities on glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) and trypanothione reductase (TR) enzymes from T cruzi and adeninephosphoribosyl transferase (APRT) enzyme from Leishmania tarentolae. In general, polar extracts displayed strong effects and some of the tested compounds have shown good results in comparison to positive controls of the bioassays.     

Comparative codon and amino acid composition analysis of Tritryps-conspicuous features of Leishmania major

Comparative analyses of codon/amino acid usage in Leishmania major, Trypanosoma brucei and Trypanosoma cruzi reveal that gene expressivity and GC-bias play key roles in shaping the gene composition of all three parasites, and protein composition of L. major only. In T. brucei and T. cruzi, the major contributors to the variation in protein composition are hydropathy and/or aromaticity. Principle of Cost Minimization is followed by T. brucei, disregarded by T. cruzi and opposed by L. major. Slowly evolving highly expressed gene-products of L. major bear signatures of relatively AT-rich ancestor, while faster evolution under GC-bias has characterized the lowly expressed genes of the species by higher GC12-content.     

Trypanosome telomeres are protected by a homologue of mammalian TRF2

Putative TTAGGG repeat-binding factor (TRF) homologues in the genomes of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major were identified. They have significant sequence similarity to higher eukaryotic TRFs in their C-terminal DNA-binding myb domains but only weak similarity in their N-terminal domains. T. brucei TRF (tbTRF) is essential and was shown to bind to duplex TTAGGG repeats. The RNA interference-mediated knockdown of tbTRF arrested bloodstream cells at G(2)/M and procyclic cells partly at S phase. Functionally, tbTRF resembles mammalian TRF2 more than TRF1, as knockdown diminished telomere single-stranded G-overhang signals. This suggests that tbTRF, like vertebrate TRF2, is essential for telomere end protection, and this also supports the hypothesis that TRF rather than Rap1 is the more ancient DNA-binding component of the telomere protein complex. Identification of the first T. brucei telomere DNA-binding protein and characterization of its function provide a new route to explore the roles of telomeres in pathogenesis of this organism. This work also establishes T. brucei as an attractive model for telomere biology.     

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.     

Elongation of polyunsaturated fatty acids in trypanosomatids

Leishmania major synthesizes polyunsaturated fatty acids by using Delta6, Delta5 and Delta4 front-end desaturases, which have recently been characterized [Tripodi KE, Buttigliero LV, Altabe SG & Uttaro AD (2006) FEBS J273, 271-280], and two predicted elongases specific for C18 Delta6 and C20 Delta5 polyunsaturated fatty acids, respectively. Trypanosoma brucei and Trypanosoma cruzi lack Delta6 and Delta5 desaturases but contain Delta4 desaturases, implying that trypanosomes use exogenous polyunsaturated fatty acids to produce C22 Delta4 fatty acids. In order to identify putative precursors of these C22 fatty acids and to completely describe the pathways for polyunsaturated fatty acid biosynthesis in trypanosomatids, we have performed a search in the three genomes and identified four different elongase genes in T. brucei, five in T. cruzi and 14 in L. major. After a phylogenetic analysis of the encoded proteins together with elongases from a variety of other organisms, we selected four candidate polyunsaturated fatty acid elongases. Leishmania major CAJ02037, T. brucei AAX69821 and T. cruzi XP_808770 share 57-52% identity, and group together with C20 Delta5 polyunsaturated fatty acid elongases from algae. The predicted activity was corroborated by functional characterization after expression in yeast. T. brucei elongase was also able to elongate Delta8 and Delta11 C20 polyunsaturated fatty acids. L. major CAJ08636, which shares 33% identity with Mortierella alpinaDelta6 elongase, showed a high specificity for C18 Delta6 polyunsaturated fatty acids. In all cases, a preference for n6 polyunsaturated fatty acids was observed. This indicates that L. major has, as predicted, Delta6 and Delta5 elongases and a complete pathway for polyunsaturated fatty acid biosynthesis. Trypanosomes contain only Delta5 elongases, which, together with Delta4 desaturases, allow them to use eicosapentaenoic acid and arachidonic acid, a precursor that is relatively abundant in the host, for C22 polyunsaturated fatty acid biosynthesis.     

Localisation of a 3-hydroxy-3-methylglutaryl-coenzyme A reductase in the mitochondrial matrix of Trypanosoma brucei procyclics

Contrary to Leishmania spp. and Trypanosoma cruzi, Trypanosoma brucei bloodstream forms do not synthesise their own sterols but take these compounds in the form of cholesterol directly from the mammalian host. However, procyclic insect stages synthesise ergosterol rather than cholesterol. Here the sub-cellular localisation of the first committed enzyme of this pathway of isoprenoid synthesis 3-hydroxy-3-methylglutaryl-coenzyme A reductase in T. brucei procyclics (0.9 nmol x min(-1) x mg(-1) protein) was carried out using both cell-fractionation by isopycnic centrifugation and digitonin-titration experiments. The majority of the NADP+-linked 3-hydroxy-3-methylglutaryl-coenzyme A reductase is a soluble enzyme present in the mitochondrial matrix with some additional membrane-associated activity in glycosomes and possibly in the endoplasmic reticulum. It is suggested that the active metabolism of threonine and/or leucine as preferred 2-carbon source for the incorporation of acetyl units into lipids and/or sterols in the mitochondrion of T. brucei procyclics is the explanation for a high 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in these protozoan organelles.     

Structures of type 2 peroxisomal targeting signals in two trypanosomatid aldolases

Trypanosomatids, unicellular organisms responsible for several global diseases, contain unique organelles called glycosomes in which the first seven glycolytic enzymes are sequestered. We report the crystal structures of glycosomal fructose-1,6-bisphosphate aldolase from two major tropical pathogens, Trypanosoma brucei and Leishmania mexicana, the causative agents of African sleeping sickness and one form of leishmaniasis, respectively. Unlike mammalian aldolases, the T. brucei and L. mexicana aldolases contain nonameric N-terminal type 2 peroxisomal targeting signals (PTS2s) to direct their import into the glycosome. In both tetrameric trypanosomatid aldolases, the PTS2s from two different subunits form two closely intertwined structures. These "PTS2 dimers", which have very similar conformations in the two aldolase structures, are the first reported conformations of a glycosomal or peroxisomal PTS2, and provide opportunities for the design of trypanocidal compounds.