Characterization of protein-linked oligosaccharides in trypanosomatid flagellates

Protein-linked, endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides were isolated from several trypanosomatids incubated with [U-14C]glucose. Structural analysis of the compounds revealed that Man9GlcNAc2 was the oligosaccharide transferred from dolichol-P-P derivatives to proteins in Trypanosoma dionisii, Trypanosoma conorhini, Leptomonas samueli and Herpetomonas samuelpessoai and Man6GlcNAc2 in Blastocrithidia culicis and Leishmania adleri. In all cases, transiently glucosylated compounds were detected: Glc1Man7-9GlcNAc2 in T. dionisii, T. conorhini, L. samueli; Glc1Man9GlcNAc2 in H. samuelpessoai, Glc1Man6GlcNAc2 in B. culicis and Glc1Man6GlcNAc2 and Glc1Man5GlcNAc2 in L. adleri. The mechanism of protein glycosylation in T. dionisii and T. conorhini appeared to be similar to that described before for Trypanosoma cruzi epimastigotes, although some differences were found between the structures of the main isomers of Man7GlcNAc2 and Man8GlcNAc2 present in T. conorhini and T. cruzi. Differences between the mechanisms of glycosylation occurring in Leishmania mexicana and L. adleri were also found: Man6GlcNAc2 in the latter microorganism was demannosylated to Man5GlcNAc2, a step not detected in the former parasite. A novel substituent in N-linked high mannose-type oligosaccharides was found in L. samueli and H. samuelpessoai: galactose in the furanose configuration. In the latter trypanosomatid, Man9GlcNAc2 was demannosylated only to Man8GlcNAc2, whereas in all other parasites in which the same oligosaccharide was transferred to proteins, Man5-7GlcNAc2 were also detected.     

Biochemical characterization of stage-specific isoforms of aspartate aminotransferases from Trypanosoma cruzi and Trypanosoma brucei

Three genes encoding putative aspartate aminotransferases (ASATs) were identified in the Trypanosoma cruzi genome. Two of these ASAT genes, presumably corresponding to a cytosolic and mitochondrial isoform, were cloned and expressed as soluble His-tagged proteins in Escherichia coli. The specific activities determined for both T. cruzi isozymes were notably higher than the values previously reported for Trypanosoma brucei orthologues. To confirm these differences, T. brucei mASAT and cASAT were also expressed as His-tagged enzymes. The kinetic analysis showed that the catalytic parameters of the new recombinant T. brucei ASATs were very similar to those determined for T. cruzi orthologues. The cASATs from both parasites displayed equally broad substrate specificities, while mASATs were highly specific towards aspartate/2-oxoglutarate. The subcellular localization of the mASAT was confirmed by digitonin extraction of intact epimastigotes. At the protein level, cASAT is constitutively expressed in T. brucei, whereas mASAT is down-regulated in the bloodstream forms. By contrast in T. cruzi, mASAT is expressed along the whole life cycle, whereas cASAT is specifically induced in the mammalian stages. Similarly, the expression of malate dehydrogenases (MDHs) is developmentally regulated in T. cruzi: while glycosomal MDH is only expressed in epimastigotes and mitochondrial MDH is present in the insect and mammalian stages. Taken together, these findings provide evidence for a metabolically active mitochondrion in the mammalian stages of T. cruzi, and suggest that the succinate excreted by amastigotes more likely represents a side product of an at least partially operative Krebs cycle, than an end product of glycosomal catabolism.     

Genomic variation of Trypanosoma cruzi: involvement of multicopy genes.

By using improved pulsed field gel conditions, the karyotypes of several strains of the protozoan parasite Trypanosoma cruzi were analyzed and compared with those of Leishmania major and two other members of the genus Trypanosoma. There was no difference in chromosome migration patterns between different life cycle stages of the T. cruzi strains analyzed. However, the sizes and numbers of chromosomal bands varied considerably among T. cruzi strains. This karyotype variation among T. cruzi strains was analyzed further at the chromosomal level by using multicopy genes as probes in Southern hybridizations. The chromosomal location of the genes encoding alpha- and beta-tubulin, ubiquitin, rRNA, spliced leader RNA, and an 85-kilodalton protein remained stable during developmental conversion of the parasite. The sizes and numbers of chromosomes containing these sequences varied among the different strains analyzed, implying multiple rearrangements of these genes during evolution of the parasites. During continuous in vitro cultivation of T. cruzi Y, the chromosomal location of the spliced leader gene shifted spontaneously. The spliced leader gene encodes a 35-nucleotide RNA that is spliced in trans from a 105-nucleotide donor RNA onto all mRNAs in T. cruzi. The spliced leader sequences changed in their physical location in both the cloned and uncloned Y strains. Associated with the complex changes was an increase in the infectivity of the rearranged variant for tissue culture cells. Our results indicate that the spliced leader gene clusters in T. cruzi undergo high-frequency genomic rearrangements.     

Immunological dominance of Trypanosoma cruzi tandem repeat proteins

Proteins with tandem repeat (TR) domains have been found in various protozoan parasites, often acting as targets of B-cell responses. However, the extent of the repeats within Trypanosoma cruzi, the causative agent of Chagas' disease, has not been examined well. Here, we present a systematic survey of the TR genes found in T. cruzi, in comparison with other organisms. Although the characteristics of TR genes varied from organism to organism, the presence of genes having large TR domains was unique to the trypanosomatids examined, including T. cruzi. Sequence analyses of T. cruzi TR genes revealed their divergency; they do not share such characteristics as sequence similarity or biased cellular location predicted by the presence of a signal sequence or transmembrane domain(s). In contrast, T. cruzi TR proteins seemed to possess significant antigenicity. A number of previously characterized T. cruzi antigens were detected by this computational screening, and several of those antigens contained a large TR domain. Further analyses of the T. cruzi genome demonstrated that previously uncharacterized TR proteins in this organism may also be immunodominant. Taken together, T. cruzi is rich in large TR domain-containing proteins with immunological significance; it is worthwhile further analyzing such characteristics of TR proteins as the copy number and consensus sequence of the repeats to determine whether they might contribute to the biological variability of T. cruzi strains with regard to induced immunological responses, host susceptibility, disease outcomes, and pathogenicity.     

Functional characterization of NADP-dependent isocitrate dehydrogenase isozymes from Trypanosoma cruzi

Trypanosoma cruzi exhibits two putative isocitrate dehydrogenases (IDHs). Both idh genes were cloned and the recombinant enzymes expressed in Escherichia coli. Our results showed that T. cruzi IDHs are strictly dependent on NADP(+) and display apparent affinities towards isocitrate and the coenzyme in the low micromolar range. In T. cruzi, IDHs are cytosolic and mitochondrial enzymes, and there is no evidence for the typical Krebs cycle-related NAD-dependent IDH. Hence, like in Trypanosoma brucei, the Krebs cycle is not a canonical route in T. cruzi. However, the citrate produced in the mitochondrion could be isomerized into isocitrate in the cytosol and the mitochondrion by means of the putative aconitase, which would provide the substrate for both IDHs. The cytosolic IDH is significantly more abundant in amastigotes, cell-derived and metacyclic trypomastigotes than in epimastigotes. This observation fits in well with the expected oxidative burst this pathogen has to face when infecting the mammalian host.     

Chromosomal localization of seven cloned antigen genes provides evidence of diploidy and further demonstration of karyotype variability in Trypanosoma cruzi

The karyotype of Trypanosoma cruzi was studied by pulsed field gel electrophoresis (PFGE) in conditions that allowed 20-25 chromosome bands to be detected. However, several of these bands were present in non-equimolar amounts, suggesting that the total chromosome number is considerably higher. The patterns obtained with the different cloned and uncloned strains were unique, suggesting that the karyotype of T. cruzi is highly variable. The chromosomal localizations of seven cloned genes were determined by Southern blotting of PFGE-separated chromosomes. Three of the clones gave rise to similar patterns and mapped on a chromosome or a family of chromosomes larger than 1.6 Mb. Two clones mapped on either single or pairs of chromosomes, which in some cases differed considerably in size between the different strains tested, suggesting that extensive chromosome rearrangements occur in T. cruzi. Another clone hybridized to several chromosomes in most strains and probably represents a family of genes. Lastly, one clone hybridized to nearly all chromosomes. Many of the clones hybridized to pairs of restriction fragments in the different strains, suggesting that they are allelic. For one of the clones it was possible to provide further evidence for the allelic nature of the fragments by establishing detailed restriction maps around them and by showing that the two fragments in a pair hybridized to chromosomes which differed slightly in size. Taken together, the results infer that the genome of T. cruzi epimastigotes is diploid.     

Use of full-length recombinant calflagin and its c fragment for improvement of diagnosis of Trypanosoma cruzi infection

Serological diagnosis of Trypanosoma cruzi infection is hampered by issues related to test specificity due to the cross-reactivity of most antigens with proteins of related parasites such as Leishmania spp. The recombinant calflagins are considered relevant antigens for the diagnosis of infection by Trypanosoma cruzi. In the present work, we describe two genes coding for putative calflagins in Leishmania major with the N-terminal moieties presenting high similarity with T. cruzi genes. This fact raised questions about their role in some cross-recognition of this antigen by sera from Leishmania spp.-infected individuals. The complete T. cruzi calflagin and two fragments of the protein, consisting of 146 amino acids of the N-terminal and 65 amino acids of the C-terminal regions, were expressed and evaluated against a panel of sera, which included well-characterized samples from T. cruzi, and Leishmania-infected patients. We were able to show that sera from Leishmania (Viannia) braziliensis-infected individuals recognized the recombinant full-length calflagin. Both the N-terminal and the complete protein presented the same high sensitivity (98.5% of sera from T. cruzi-infected patients was detected) but different specificities (94% and 98%, respectively, when evaluated against sera from people not infected by T. cruzi, including 15 sera from people infected with L. braziliensis). The C-terminal fragment presented low sensitivity (70%) but 100% specificity. We propose the use of these antigens in two sequential assays to optimize the serological diagnosis of T. cruzi infection in humans in geographic areas where Leishmania spp. infection is coendemic.     

Genomic variation in Trypanosoma cruzi clonal cultures

 Spontaneous changes in restriction DNA profiles and pulsed-field gel electrophoresis (PFGE) patterns, along with a concomitant loss of infectivity, were observed in infective clones of Trypanosoma cruzi strain Y either following a number of passages during the exponential growth phase or after subcloning in liver infusion tryptone (LIT) medium using as the probe a genomic fragment of the parasite (pMYP16), indicating naturally occurring rearrangements of DNA sequences. No variation could be detected when the genomic DNA was probed with conserved T. cruzi tubulin and actin genes. There was no correlation between such rearrangements and the life-cycle forms of the parasites, since trypomastigote forms showed the same karyotype and hybridization patterns as did epimastigote forms. The variations observed could be reverted and infectivity, recovered after inoculation of the parasites in newborn mice. Received: 10 March 1995 / Accepted: 1 August 1995     

Structural features and antigenic properties of carbohydrate-containing components of Trypanosoma conorhini

Aqueous and phenolic extracts of Trypanosoma conorhini were fractionated and high molecular weight, carbohydrate-rich fractions obtained. Their antigenic characteristics, reactivity with lectins and partial chemical structure were determined. The major component, the phenolic extract, was electrophoretically diffuse and consisted of 15% protein, 5% phosphorus, hexosamine, and 67% neutral carbohydrate, which contained mannose, galactose, and xylose in a molar ratio of 1.0:1.8:1.8. Chemical analyses and lectin agglutination experiments showed nonreducing end-groups of beta-D-galactopyranose, beta-xylopyranose, and alpha-D-mannopyranose. Phosphate esters occurred, apparently, at O-6 of hexopyranosyl units. Hexosamine was present as nonacetylated units of 2-amino-2-deoxy-alpha-D-glucopyranosyl units that were extremely resistant to acid hydrolysis. On double immunodiffusion tests, the major component gave a precipitation line with rabbit serum against whole cells of Trypanosoma cruzi, suggesting the presence of common antigenic determinant(s) on the cell surface of each trypanosomatid.     

Tests of cytoplasmic RNA interference (RNAi) and construction of a tetracycline-inducible T7 promoter system in Trypanosoma cruzi

The technique of RNA interference (RNAi) is exceedingly useful for knocking down the expression of a specific mRNA in African trypanosomes and other organisms for the purpose of examining the function of its gene. However, when we attempted to apply RNAi in the Latin American trypanosome, Trypanosoma cruzi, to diminish expression of mRNA encoding the surface protein amastin, we found that the amastin double-stranded RNA (dsRNA) was not efficiently degraded in either epimastigotes or amastigotes, and the level of amastin mRNA remained unchanged. We generated a strain of T. cruzi CL-Brener in which the T7 promoter and tetracycline operator could be used to maximize tetracycline-regulated dsRNA synthesis and constructed plasmids that direct dsRNA against four different T. cruzi endogenous genes (encoding beta-tubulin, GP72 (flagellar adhesion protein), ribosomal protein P0 and amastin) and an exogenously added gene (GFP; green fluorescent protein). After either stable or transient transfection of these plasmids into T. cruzi, the expected RNAi phenotype was not observed for any of the five genes, although the T. cruzi beta-tubulin RNAi plasmid did give the expected FAT cell phenotype in the African trypanosome, Trypanosoma brucei. These data indicate that, similar to Leishmania, T. cruzi lacks one or more components necessary for the RNAi pathway and that these components will need to be engineered into T. cruzi, or compensated for, before RNAi can be used to study gene function in this organism.     

Electrophoretic detection of Trypanosoma cruzi peptidases

Peptidases of Trypanosoma cruzi epimastigotes were examined by polyacrylamide gel electrophoresis in gels containing gelatin as peptidase substrate. Mini-gels were far superior to large gels in their sensitivity of peptidase detection. Patterns of peptidases were similar between different strains of T. cruzi, although some inter-strain heterogeneity was found. In strain Y, at least five peptidases were detected: four of these enzymes were shown to be cysteine-type peptidases with acidic pH optima. The other peptidase was a 60-kDa membrane-associated peptidase that was sensitive to o-phenanthroline; it was tentatively characterised as a metallopeptidase, and was optimally active at alkaline pH. This membrane-associated peptidase was conserved between strains of T. cruzi.     

Identification of six Trypanosoma cruzi lineages by sequence-characterised amplified region markers

Six discrete phylogenetic lineages were recently identified in Trypanosoma cruzi, on the basis of multilocus enzyme electrophoresis and random amplified polymorphic DNA (RAPD) characterisation. The objective of the present study was to develop specific PCR-based markers for the identification of each of the six lineages. Eighty-seven T. cruzi stocks representative of all the lineages were characterised by RAPD with three primers, resulting in the identification of three fragments that were specifically amplified in the given sets of lineages. After cloning and sequencing these fragments, three pairs of sequence-characterised amplified region (SCAR) primers were designed. After PCR amplification using the SCAR primers, the initial polymorphism was retained either as the presence or absence of amplification, or as size variation between the PCR products. Although most PCR products, taken individually, were distributed across several lineages, the combination of the three SCAR markers resulted in characteristic patterns that were distinct in the six lineages. Furthermore, T. cruzi lineages were distinguished from Trypanosoma rangeli, T. cruzi marinkellei and T. cruzi-like organisms. The excellent correspondence of these new PCR markers with the phylogenetic lineages, allied with their sensitivity, makes them reliable tools for lineage identification and strain characterisation in T. cruzi. The approach described here could be generalised to any species of microorganism harbouring clear-cut phylogenetic subdivisions.     

Characterization of cDNA clones encoding ribonucleoprotein antigens expressed in Trypanosoma cruzi amastigotes

Sera from patients with Chagas' disease were used to screen a Trypanosoma cruzi amastigote cDNA library. Characterization of 50 positive clones showed that 21 (42%) encode previously identified T. cruzi ribosomal and flagellar proteins, heat-shock proteins or proteins with repetitive motifs. Twenty-nine clones (58%) correspond to nine genes not previously described in T. cruzi. Three cDNAs, encoding novel repetitive antigens with homology to ribosomal proteins and to other RNA binding proteins, were further characterized. Patient humoral responses against the recombinant proteins encoded by these cDNAs were evaluated in anticipation that they may constitute potential new targets for serodiagnostic assays.     

Upregulation of sterol C14-demethylase expression in Trypanosoma cruzi treated with sterol biosynthesis inhibitors

Infection with the protozoan, Trypanosoma cruzi, is the cause of Chagas disease that occurs widely throughout Latin America. T. cruzi contains sterol biosynthesis enzymes, and produces sterol products similar to those found in fungi. Antifungal drugs that inhibit ergosterol biosynthesis have potent anti-T. cruzi activity in vitro and in animal models. In this report, we describe the effects of sterol biosynthesis inhibitors (simvistatin, zaragosic acid, terbinafine, a lanosterol synthase inhibitor, ketoconazole, and tridemorph) on the regulation of two sterol biosynthesis genes and their protein products. Culturing T. cruzi in the presence of the lanosterol synthase inhibitor, terbinafine, or ketoconazole increased mRNA levels of the sterol C14-demethylase gene approximately 7-12-fold. The sterol C14-demethylase protein levels were also elevated. The effects of the sterol biosynthesis inhibitors on hydroxymethylglutaryl-CoA reductase expression were minimal. Control of the upregulation of sterol C14-demethylase appears to be mediated through the 3'-untranslated region of the gene. The findings demonstrate that T. cruzi can specifically regulate gene expression in response to derangements in its cellular functions.     

Evaluation of Trypanosoma cruzi hybrid stocks based on chromosomal size variation

Although all classical lines of evidence point to the fact that Trypanosoma cruzi has a predominantly clonal evolution, accumulating data show that some T. cruzi stocks are the result of hybridisation events. We evaluated whether chromosomal polymorphism would give evolutionary information on hybrid isolates. Twenty-three coding sequences were mapped on the chromosomes of nine parasite stocks, four of which are putative hybrids (CL Brener and rDNA group 1/2). Phenetic analyses of karyotype data were based on the absolute chromosomal size difference index (aCSDI), a method that assumes that the genomic distance between two organisms is the sum of the size differences between their homologous chromosomes. aCSDI-based dendrograms obtained from a variable number of probes (3-18 probes) defined in all the cases three clusters: two corresponding, respectively, to T. cruzi I and T. cruzi II groups; and a third one, to rDNA group 1/2. CL Brener was alternatively positioned in T. cruzi II or rDNA group 1/2 clusters. Three clusters were also observed in the dendrogram constructed with restriction fragment length polymorphism (RFLP) data from 18 probes. The topology of the chromosome and RFLP dendrograms is similar, with a significant correlation coefficient (r=0.86062; P<0.0001), supporting a strong structuring of the clusters. This study also revealed that hybrid stocks have a larger proportion of two different-sized homologous chromosomes, as compared with non-hybrid strains. Overall, our results show that chromosomes are valuable characters for identification of evolutionary groups, in particular, T. cruzi hybrid organisms.     

Characterization of tubulin genes in Trypanosoma rangeli

Tubulin genes in Trypanosoma rangeli, the only trypanosome besides T. cruzi to infect humans in America, are organized in homogeneous, alternate alpha and beta gene tandem repeats of 3.8 kb. The basic repeat was cloned, mapped and partially sequenced. In contrast to most other eukaryotes, where tubulin genes are scattered throughout the genome, trypanosomatids so far studied are characterized by tandem arrangements of these genes with the genus Trypanosoma displaying an alternating alpha- and beta-tubulin tandem repeat.     

Differential accumulation of mutations localized in particular domains of the mucin genes expressed in the vertebrate host stage of Trypanosoma cruzi

The surface of Trypanosoma cruzi is covered by mucin-type glycoproteins involved in parasite protection, attachment and immunoevasion. The gene family coding for the mucins expressed by the parasite in the vertebrate host, named TcMUC, is composed of several hundred members and presents high variability. The genes encoding mucins expressed in the insect-dwelling parasite stages are part of a much more homogeneous family, named TcSMUG. Here, we addressed the organization and evolution of physically linked T. cruzi mucin genes by sequencing large chromosomal fragments containing these genes. Specific accumulation of mutations was restricted to particular domains of TcMUC genes, showing that these regions have, or have had, an accelerated evolution rate. Sequence analysis of several TcMUC genes allowed for the identification of members sharing features of TcMUC I and II, thus evidencing that one group of genes was generated from the other. The highly conserved intergenic regions of both TcMUC and TcSMUG families contained TG-rich microsatellites that were not present in unrelated genes in the cosmids, suggesting a role for homologous recombination in shuffling and/or amplification of T. cruzi mucin genes. The comparison of putative homologous TcMUC II genes from different strains of T. cruzi showed that their central variable domains are conserved. This conservation was always higher at the DNA level suggesting positive selection in these particular regions of TcMUC II genes.     

Primary structure of Trypanosoma cruzi small-subunit ribosomal RNA coding region: comparison with other trypanosomatids

A Trypanosoma cruzi small subunit ribosomal RNA gene was sequenced from genomic recombinant plasmid clones. The assigned coding region was 2319 bp, the longest SSU rRNA gene described to date. On the basis of comparisons with published sequences from Crithidia fasciculata, Trypanosoma brucei, and Leishmania donovani, we conclude that the extra nucleotides in the T. cruzi gene occur in highly variable regions of rRNA genes. A phylogenetic analysis was performed with the SSU rRNA sequences from these four trypanosomatids and from Euglena gracilis as an outgroup. The Leishmania and Crithidia sequences were remarkably similar to each other (not separable statistically). Given the standard errors associated with the branching order of the two Trypanosoma species and the Leishmania-Crithidia branch, the actual topology cannot be unequivocally determined using only the ribosomal sequences analyzed here.