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.     

Evolutionary analysis of synteny and gene fusion for pyrimidine biosynthetic enzymes in Euglenozoa: an extraordinary gap between kinetoplastids and diplonemids

A unique feature of the genome architecture in the parasitic trypanosomatid protists is large-scale synteny. We addressed the evolutionary trait of synteny in the eukaryotic group, Euglenozoa, which consists of euglenoids (earliest branching), diplonemids, and kinetoplastids (trypanosomatids and bodonids). Synteny of the pyrimidine biosynthetic (pyr) gene cluster, which constitutes part of a large syntenic cluster in trypanosomatids and includes four separate genes (pyr1-pyr4) and one fused gene (pyr6/pyr5 fusion), was conserved in the bodonid, Parabodo caudatus. In the diplonemid, Diplonema papillatum, we identified pyr4 and pyr6 genes. Phylogenetic analyses of pyr4 and pyr6 showed the separate origin of each in kinetoplastids and euglenoids/diplonemids and suggested that kinetoplastids have acquired these genes via lateral gene transfer (LGT). Because replacement of genes by non-orthologs within the syntenic cluster is highly unlikely, we concluded that, after separation of the line leading to diplonemids, the syntenic pyr gene cluster was established in the common ancestor of kinetoplastids, preceded by their acquisition via LGT. Notably, we found that diplonemid pyr6 is a stand-alone gene, inconsistent with both euglenoid pyr5/pyr6 and kinetoplastid pyr6/pyr5 fusions. Our findings provide insights into the evolutionary gaps within Euglenozoa and the evolutionary trait of rearrangement of gene fusion in this lineage.     

The lineage and diversity of putative amino acid sensor ACR proteins in plants

Amino acid metabolic enzymes often contain a regulatory ACT domain, named for aspartate kinase, chorismate mutase, and TyrA (prephenate dehydrogenase). Arabidopsis encodes 12 putative amino acid sensor ACT repeat (ACR) proteins, all containing ACT repeats but no identifiable catalytic domain. Arabidopsis ACRs comprise three groups based on domain composition and sequence: group I and II ACRs contain four ACTs each, and group III ACRs contain two ACTs. Previously, all three groups had been documented only in Arabidopsis. Here, we extended this to algae and land plants, showing that all three groups of ACRs are present in most, if not all, land plants, whereas among algal ACRs, although quite diverse, only group III is conserved. The appearance of canonical group I and II ACRs thus accompanied the evolution of plants from living in water to living on land. Alignment of ACTs from plant ACRs revealed a conserved motif, DRPGLL, at the putative ligand-binding site. Notably, the unique features of the DRPGLL motifs in each ACT domain are conserved in ACRs from algae to land plants. The conservation of plant ACRs is reminiscent of that of human cellular arginine sensor for mTORC1 (CASTOR1), a member of a small protein family highly conserved in animals. CASTOR proteins also have four ACT domains, although the sequence identities between ACRs and CASTORs are very low. Thus, plant ACRs and animal CASTORs may have adapted the regulatory ACT domains from a more ancient metabolic enzyme, and then evolved independently.     

Nucleotide sequence of Escherichia coli pyrG encoding CTP synthetase

The amino acid sequence of Escherichia coli CTP synthetase was derived from the nucleotide sequence of pyrG. The derived amino acid sequence, confirmed at the N terminus by protein sequencing, predicts a subunit of 544 amino acids having a calculated Mr of 60,300 after removal of the initiator methionine. A glutamine amide transfer domain was identified which extends from approximately amino acid residue 300 to the C terminus of the molecule. The CTP synthetase glutamine amide transfer domain contains three conserved regions similar to those in GMP synthetase, anthranilate synthase, p-aminobenzoate synthase, and carbamoyl-P synthetase. The CTP synthetase structure supports a model for gene fusion of a trpG-related glutamine amide transfer domain to a primitive NH3-dependent CTP synthetase. The major 5' end of pyrG mRNA was localized to a position approximately 48 base pairs upstream of the translation initiation codon. Translation of the gene eno, encoding enolase, is initiated 89 base pairs downstream of pyrG. The pyrG-eno junction is characterized by multiple mRNA species which are ascribed to monocistronic pyrG and/or eno mRNAs and a pyrG eno polycistronic mRNA.     

The trans-sialidase from Trypanosoma cruzi efficiently transfers alpha-(2-->3)-linked N-glycolylneuraminic acid to terminal beta-galactosyl units

The trans-sialidase from Trypanosoma cruzi (TcTS), the agent of Chagas' disease, is a unique enzyme involved in mammalian host-cell invasion. Since T. cruzi is unable to synthesize sialic acids de novo, TcTS catalyzes the transfer of alpha-(2-->3)-sialyl residues from the glycoconjugates of the host to terminal beta-galactopyranosyl units present on the surface of the parasite. TcTS also plays a key role in the immunomodulation of the infected host. Chronic Chagas' disease patients elicit TcTS-neutralizing antibodies that are able to inhibit the enzyme. N-Glycolylneuraminic acid has been detected in T. cruzi, and the trans-sialidase was pointed out as the enzyme involved in its incorporation from host glycoconjugates. However, N-glycolylneuraminic acid alpha-(2-->3)-linked-containing oligosaccharides have not been analyzed as donors in the T. cruzi trans-sialidase reaction. In this paper we studied the ability of TcTS to transfer N-glycolylneuraminic acid from Neu5Gc(alpha2-->3)Gal(beta1-->4)GlcbetaOCH(2)CH(2)N(3) (1) and Neu5Gc(alpha2-->3)Gal(beta1-->3)GlcNAcbetaOCH(2)CH(2)N(3) (2) to lactitol, N-acetyllactosamine and lactose as acceptor substrates. Transfer from 1 was more efficient (50-65%) than from 2 (20-30%) for the three acceptors. The reactions were inhibited when the enzyme was preincubated with a neutralizing antibody. K(m) values were calculated for 1 and 2 and compared with 3'-sialyllactose using lactitol as acceptor substrate. Analysis was performed by high-performance anion-exchange (HPAEC) chromatography. A competitive transfer reaction of compound 1 in the presence of 3'-sialyllactose and N-acetyllactosamine showed a better transfer of Neu5Gc than of Neu5Ac.     

Trypanosoma cruzi: Mutations in the 3' untranslated region of calmodulin gene are specific for lineages T. cruzi I, T. cruzi II, and the Zymodeme III isolates

Populations of Trypanosoma cruzi can be clustered in two main phylogenetic lineages, T. cruzi I and T. cruzi II and a third group denominated Zymodeme III (ZIII) has been described. Using 23 isolates representing the two major T. cruzi groups and the Zymodeme III, the 3' untranslated region (3'UTR) of the calmodulin gene was analyzed. Several mutations located on a 330 bp segment of this 3'UTR were observed, among which three important insertion/deletion events, namely (1) a dinucleotide AG present only in ZIII isolates; (2) a 13 bases purine block missing only in ZIII; and (3) a five base GT block in T. cruzi II. Minimum free energy dot plots show that T. cruzi I and T. cruzi ZIII exhibit similar patterns of optimal and sub-optimal folding of this segment. These mutations in 3'UTR of calmodulin raise the possibility that T. cruzi I and ZIII group are sharing common functional routes.     

Identification of PDZ5, a candidate universal minicircle sequence binding protein of Trypanosoma cruzi

The dodecamer universal minicircle sequence is a conserved sequence present in minicircles of trypanosomatid kinetoplast DNA studied so far. This sequence is recognised by a protein named universal minicircle sequence binding protein, described for Crithidia fasciculata, involved in minicircle DNA replication. We have identified a Trypanosoma cruzi gene homologue of the Crithidia fasciculata universal minicircle sequence binding protein. Similar to the Crithidia fasciculata universal minicircle sequence binding protein, the Trypanosoma cruzi protein, named PDZ5, contains five zinc finger motifs. Pulsed field gel electrophoresis indicated that the pdz5 gene is located in the chromosomal band XX of the Trypanosoma cruzi genome. The predicted amino acid sequence of PDZ5 shows a high degree of similarity with several trypanosomatid zinc finger proteins. Specific antibody raised against Crithidia fasciculata universal minicircle sequence binding protein recognises both the recombinant and endogenous PDZ5. The complete pdz5 coding sequence cloned in bacteria expresses a recombinant PDZ5 protein that binds specifically to the universal minicircle sequence dodecamer. These data strongly suggest that PDZ5 represents a Trypanosoma cruzi universal minicircle sequence binding protein.     

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.     

Chromosomal size conservation through the cell cycle supports karyotype stability in Trypanosoma cruzi

The Trypanosoma cruzi karyotype shows an extensive chromosomal size polymorphism. Absence of condensed mitotic chromosomes and chromatin fragility are characteristic features of T. cruzi which would allow DNA breaks and chromosomal rearrangements during cell proliferation. We have investigated by pulsed field gel electrophoresis (PFGE) eventual changes in chromosomal size during exponential and stationary phases of T. cruzi epimastigotes in culture, in G0 trypomastigotes and throughout the cell cycle in synchronized epimastigotes. T. cruzi molecular karyotype was stable throughout the cell cycle and during differentiation. Thus, the chromosomal size polymorphism previously reported in T. cruzi contrasts with the stability of the molecular karyotype observed here and suggests that chromosomal rearrangements leading to changes in chromosomal size are scarce events during the clonal propagation of this parasite.     

Acidic ribosomal proteins and histone H3 from Leishmania present a high rate of divergence

Another additional peculiarity in Leishmania will be discussed about of the amino acid divergence rate of three structural proteins: acidic ribosomal P1 and P2b proteins, and histone H3 by using multiple sequence alignment and dendrograms. These structural proteins present a high rate of divergence regarding to their homologous protein in Trypanosoma cruzi. At this regard, L. (V.) peruviana P1 and T. cruzi P1 showed 57.4% of divergence rate. Likewise, L. (V.) braziliensis histone H3 and acidic ribosomal P2 protein exhibited 31.8% and 41.7% respectively of rate of divergence in comparison with their homologous in T. cruzi.     

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.     

Evidence for multiple hybrid groups in Trypanosoma cruzi

A role for parasite genetic variability in the spectrum of Chagas disease is emerging but not yet evident, in part due to an incomplete understanding of the population structure of Trypanosoma cruzi. To investigate further the observed genotypic variation at the sequence and chromosomal levels in strains of standard and field-isolated T. cruzi we have undertaken a comparative analysis of 10 regions of the genome from two isolates representing T. cruzi I (Dm28c and Silvio X10) and two from T. cruzi II (CL Brener and Esmeraldo). Amplified regions contained intergenic (non-coding) sequences from tandemly repeated genes. Multiple nucleotide polymorphisms correlated with the T. cruzi I/T. cruzi II classification. Two intergenic regions had useful polymorphisms for the design of classification probes to test on genomic DNA from other known isolates. Two adjacent nucleotide polymorphisms in HSP 60 correlated with the T. cruzi I and T. cruzi II distinction. 1F8 nucleotide polymorphisms revealed multiple subdivisions of T. cruzi II: subgroups IIa and IIc displayed the T. cruzi I pattern; subgroups IId and IIe possessed both the I and II patterns. Furthermore, isolates from subgroups IId and IIe contained the 1F8 polymorphic markers on different chromosome bands supporting a genetic exchange event that resulted in chromosomes V and IX of T. cruzi strain CL Brener. Based on these analyses, T. cruzi I and subgroup IIb appear to be pure lines, while subgroups IIa/IIc and IId/IIe are hybrid lines. These data demonstrate for the first time that IIa/IIc are hybrid, consistent with the hypothesis that genetic recombination has occurred more than once within the T. cruzi lines.     

Post genomic analysis of permeases from the amino acid/auxin family in protozoan parasites

The "amino acid/auxin permeases" is probably the most represented family of transporters in the Trypanosoma cruzi genome. Using a high-throughput searching routine and preliminary data from the T. cruzi genome project, more than 15,000 sequences were iteratively assembled into contigs, and 60 open reading frames corresponding to different putative amino acid transporters, clustered in 12 groups, were detected and characterized in silico. T. cruzi genomic organization of such sequences showed that these putative amino acid transporter genes are in an unusually large number and arranged in repeat clusters comprising about 0.2% of the genome. These data suggest that the family has evolved following tandem duplication events and constitutes a novel family of variable proteins in protozoan organisms. The mRNA expression of the predicted genes was demonstrated in infective and non-infective parasite forms. Orthologous sequences were also identified in other unicellular parasites such as Leishmania spp., Plasmodium spp., and Trypanosoma brucei.     

NMR structure of the conserved hypothetical protein TM0487 from Thermotoga maritima: implications for 216 homologous DUF59 proteins

The NMR structure of the conserved hypothetical protein TM0487 from Thermotoga maritima represents an alpha/beta-topology formed by the regular secondary structures alpha1-beta1-beta2-alpha2-beta3-beta4-alpha3- beta5-3(10)-alpha4, with a small anti-parallel beta-sheet of beta-strands 1 and 2, and a mixed parallel/anti-parallel beta-sheet of beta-strands 3-5. Similar folds have previously been observed in other proteins, with amino acid sequence identity as low as 3% and a variety of different functions. There are also 216 sequence homologs of TM0487, which all have the signature sequence of domains of unknown function 59 (DUF59), for which no three-dimensional structures have as yet been reported. The TM0487 structure thus presents a platform for homology modeling of this large group of DUF59 proteins. Conserved among most of the DUF59s are 13 hydrophobic residues, which are clustered in the core of TM0487. A putative active site of TM0487 consisting of residues D20, E22, L23, T51, T52, and C55 is conserved in 98 of the 216 DUF59 sequences. Asp20 is buried within the proposed active site without any compensating positive charge, which suggests that its pK(a) value may be perturbed. Furthermore, the DUF59 family includes ORFs that are part of a conserved chromosomal group of proteins predicted to be involved in Fe-S cluster metabolism.