Optimization of a protein synthesizing lysate system from Trypanosoma cruzi

A protein-synthesizing lysate system from Trypanosoma cruzi epimastigotes analogous to the rabbit reticulocyte lysate system was established. The system was optimized by the ‘classical’ method where one of the factors is varied while the others are kept constant. With this the following optima were found: [Mg²⁺]: 1.0 mM, [K⁺]: 60 mM, T: 25°C, pH: 7.5. This method was compared with the ‘sequential simplex’ method [Long, D.E. (1969) Anal. Chim. Acta 46, 93–100], a method designed to optimize rationally interdependent factors in biological systems. The optima as determined with this method were: [Mg²⁺]: 1.02 mM, [K⁺]: 63 mM, T: 25.5°C, pH: 7.25. At these values the system incorporated 43% more amino acids into proteins than a system optimized with the ‘classical’ method. Fluorographic analysis of the proteins synthesized by the system shows that while proteins in the molecular weight range between 14 000 and 45 000 are synthesized in amounts comparable to the in vivo situation, the higher molecular weight proteins (>45 000) are synthesized in lesser quantities.     

Galactofuranose-containing glycoconjugates of epimastigote and trypomastigote forms of Trypanosoma cruzi

Antiserum to LPPG, a lipopeptidophosphoglycan originally described on the surface of Trypanosoma cruzi epimastigotes of the Y strain, and antibodies to furanoic galactose (gal_f) were obtained in rabbits. A micromethod for the extraction and purification of LPPG from a limited amount of parasites is described. Analysis by Western blots of the purified glycoconjugate probed with both antisera confirmed the presence of gal_f-containing LPPG-like molecules in 10 different strains and clones of T. cruzi. An analogous approach indicated that trypomastigotes also contain LPPG-like components. Quantitation experiments allowed to calculate an average value of 1.0 × 10⁷ LPPG molecules per epimastigote cell and 0.16 × 10⁷ LPPG-like molecules per trypomastigote cell.

Immunoelectron microscopy has shown a homogeneous distribution of LPPG on the surface of epimastigotes. The trypomastigote population, however, is highly heterogeneous with no more than 15% of the parasites being labeled by the anti-LPPG serum. Intense labeling has also been found in vesicles inside the epimastigote and trypomastigote forms. The distribution of gal_f epitopes among glycoconjugates of epimastigotes and trypomastigotes was further investigated. It was shown that gal_f units in epimastigotes are bound to low molecular mass compounds which co-migrate with LPPG whereas in trypomastigotes they have been found in both low molecular mass LPPG-like molecules and glycoproteins of 80–90 kDa. Direct chemical evidence for the presence of gal_f residues in the N-linked oligosaccharide chains of these surface glycoproteins has been obtained. Finally, the natural antigenicity of LPPG and gal_f in chronic Chagas' disease was investigated. It was found that all chronic chagasic sera investigated recognize this glycoconjugate and that an important part of such recognition can be attributed to gal_f residues. Furthermore, no correlation among reactivity to LPPG, strain zymodeme and clinical forms of the disease was found.     

Purification and characterization of a soluble nucleoside diphosphate kinase in Trypanosoma cruzi

A soluble nucleoside diphosphate kinase (NDP kinase) was purified and characterized in epimastigote forms of Trypanosoma cruzi. The enzyme was purified by affinity chromatography on Blue-agarose and Q-Sepharose columns and by FPLC on a Superose 12 column. A membrane-associated NDP kinase was identified which accounts for 30% of total enzymatic activity. Western blot analysis of the soluble NDP kinase revealed a 16.5-kDa monomer recognized by polyclonal antibodies to NDP kinase from Dictyostelium discoideum, Candida albicans or human. Most of the T. cruzi NDP kinase is found in the cell as a hexamer composed of 16.5-kDa monomers. The K_m values of the enzyme for ATP, GDP and dTDP were 0.2 ± 0.008 mM, 0.125 ± 0.012 mM and 0.4 ± 0.009 mM, respectively. The parasite enzyme was stable, remained active at 65°C and was found to tolerate up to 2.5 M urea. The 16.5-kDa subunit was phosphorylated with [γ-³²P]ATP or thiophosphorylated with [³⁵S]GTPγS. The incubation of the ³²P-labelled phosphoenzyme with unlabelled nucleoside 5′-diphosphates resulted in the formation of ³²P-labelled nucleoside 5′-triphosphates without strict base specificity, indicating that the reaction mechanism of the T. cruzi enzyme is the same as reported for other NDP kinases. When the phosphoenzyme was incubated with a mixture of nucleoside 5′-diphosphates, GTP was preferentially formed.     

Repetitive sequences in the ribosomal intergenic spacer of Trypanosoma cruzi

A fragment of Trypanosoma cruzi ribosomal intergenic spacer (IGS) located at 6.7 kb from the 3′ end of the 24S rRNA gene was analyzed. This IGS fragment is characterized by the presence of three types of repetitive elements (designated Spacer Repetitive Elements, SRE), short direct repeats (5-6 bp) and chi-like recombinational sequences. SRE elements are composed of relatively short repeats (43–145 bp) which show variabilities consisting of nucleotide changes, insertions and deletions. SRE-1 element (145 bp) has a short oligo(dA) tail at the end of the repeat and can be found flanked by other SRE elements. SRE elements are species-specific, suggesting that probes based on them may be diagnostic for Trypanosoma cruzi.     

Characterization of the lipid moiety of the glycosylphosphatidylinositol anchor of Trypanosoma cruzi 1G7-antigen

The 90-kDa stage-specific 1G7-antigen has been implicated in the invasion of host cells by the metacyclic forms of Trypanosoma cruzi. The antigen is attached to the plasma membrane via glycosylphosphatidylinositol, the partial structure of which was the first to be determined for a protein of this parasite. In this study, the complete structure of the lipid component of the anchor was determined by electrospray mass spectrometry, gas chromatography mass spectrometry, phospholipase sensitivity and high-performance thin-layer chromatography of the diradylglycerol components after benzoylation. These analyses showed that the lipid moiety of 1G7-antigen is composed essentially of 1-O-hexadecyl-2-O-hexadecanoyl-phosphatidylinositol and 1-O-hexadecyl-2-O-octadecanoyl-phosphatidylinositol. The high sensitivity of the electrospray mass spectrometric analysis unexpectedly revealed the presence of a small proportion of putative inositol-phosphoceramide structures, and confirmed the absence of inositol-acylated species. An interesting finding was that the biosynthetic incorporation of [³H]palmitate labelled solely the acyl position, and not the 1-O-alkyl chain in the 1G7-antigen anchor.     

Using simultaneous, tandem gene replacements to study expression of the multicopy ubiquitin-fusion (FUS) gene family of Trypanosoma cruzi

Many genes in trypanosomes exist as members of multicopy gene families. Due to this fact it is frequently difficult to determine if specific members of a gene family are expressed. We describe here a strategy for simultaneous tandem gene replacement in T. cruzi which leads to the replacement of the gene of interest by a silent reporter gene, the expression of which can be assayed in stable transformants. To determine if the FUSI gene (one of 5 copies of the ubiquitin-fusion, FUS, gene family) was expressed, stable G418-resistant transformants were isolated in which the tandemly arrayed CUB2.65 and FUSI genes were precisely replaced by the neomycin phosphotransferase (neo^r) and chloramphenicol acetyltransferase (CAT) genes, respectively. All stable clones carrying the tandem gene replacements were shown to express the CAT activity indicating that FUSI is expressed in mid-log epimastigotes. Northern blot analysis of parasites carrying the tandem gene replacements indicated that at least one other member of the FUS gene family is expressed and that there were no apparent polar effects on the expression of genes downstream of the replacement events. These experiments have demonstrated the utility of tandem gene replacements as a means of inserting a nonselected reporter gene into the chromosome, facilitating the molecular genetic analysis of the expression of multicopy gene families.     

Differential tissue distribution of diverse clones of Trypanosoma cruzi in infected mice

Chagas disease, caused by the protozoan Trypanosoma cruzi, presents variable clinical course but the phenomena underlying this variability remain largely unknown. T. cruzi has a clonal population structure and infecting strains are often multiclonal. T. cruzi genetic variability could be a determinant of differential tissue tropism or distribution and consequently of the clinical forms of the disease. We tested this hypothesis by using low-stringency single specific primer polymerase chain reaction (LSSP-PCR) to type genetically the parasites in tissues of experimental infected mice. BALB/c mice were simultaneously inoculated with two different T. cruzi populations (JG strain and Col1.7G2 clone). Doubly infected animals showed clear differential tissue distribution for the two populations (chronic phase). Our results indicate a significant influence of the genetic polymorphism of infecting T. cruzi populations in the pathogenesis of chronic Chagas disease.     

Enhanced prevalence of T cells expressing TCRBV8S2 and TCRBV8S3 in hearts of chronically Trypanosoma cruzi-infected mice

We have analysed the relative T cell receptor (TCR) BV gene usage in T cells from hearts and spleens of CBA/HJ mice chronically infected with the Tulahuén strain of Trypanosoma cruzi. During chronic infection, CBA/HJ mice recruit T cells at the major site of inflammation (i.e. the heart), with over-representation of certain TCRBV gene subfamilies (TCRBV8S2 and TCRBV8S3). In contrast, no signal or a very weak message from a limited number of T cells was recorded from one heart of the control group. No alteration of TCRBV distribution was recorded in spleens of chronically infected CBA/HJ. Our findings indicate that there is a preferential TCRBV gene usage in the T cell response in the hearts of chronically infected mice. Furthermore, the pattern of CDR3 lengths in inflammatory T cells was altered.     

Glucose uptake in the mammalian stages of Trypanosoma cruzi

Trypanosoma cruzi, the agent of Chagas’ disease, alternates between different morphogenetic stages that face distinct physiological conditions in their invertebrate and vertebrate hosts, likely in the availability of glucose. While the glucose transport is well characterized in epimastigotes of T. cruzi, nothing is known about how the mammalian stages acquire this molecule. Herein glucose transport activity and expression were analyzed in the three developmental stages present in the vertebrate cycle of T. cruzi. The infective trypomastigotes showed the highest transport activity (V_max = 5.34 ± 0.54 nmol/min per mg of protein; K_m = 0.38 ± 0.01 mM) when compared to intracellular epimastigotes (V_max = 2.18 ± 0.20 nmol/min per mg of protein; K_m = 0.39 ± 0.01 mM). Under the conditions employed no transport activity could be detected in amastigotes. The gene of the glucose transporter is expressed at the mRNA level in trypomastigotes and in intracellular epimastigotes but not in amastigotes, as revealed by real-time PCR. In both trypomastigotes and intracellular epimastigotes protein expression could be detected by Western blot with an antibody raised against the glucose transporter correlating well with the transport activity measured experimentally. Interestingly, anti-glucose transporter antibodies showed a strong reactivity with glycosome and reservosome organelles. A comparison between proline and glucose transport among the intracellular differentiation forms is presented. The data suggest that the regulation of glucose transporter reflects different energy and carbon requirements along the intracellular life cycle of T. cruzi.     

Kinetoplast DNA and molecular karyotypes of Trypanosoma evansi and Trypanosoma equiperdum from China

We compared 12 stocks of Trypanosoma evansi and 1 recently isolated stock of Trypanosoma equiperdum from different regions of China by analysis of kinetoplast DNA (kDNA), nuclear DNA and molecular karyotypes. The T. equiperdum stock was remarkably similar to the T. evansi stocks, except for the possession of kDNA maxi-circles, suggesting a very close evolutionary relationship between T. evansi and T. equiperdum. The maxi-circles of the Chinese T. equiperdum stock were approximately 14.3 kb in size, i.e., about half the size of those of Trypanosoma brucei. This stock is thus similar to an old laboratory stock of T. equiperdum, which also has maxi-circles with a sizeable deletion. Both T. equiperdum and T. evansi kDNA mini-circles hybridised with a T. evansi-specific mini-circle fragment isolated from a Kenyan T. evansi stock. Our results extend the generality that T. evansi and T. equiperdum mini-circles are microheterogeneous rather than homogeneous. Molecular karyotypes obtained by pulsed field gradient gel electrophoresis provided a more sensitive way of distinguishing the T. evansi stocks than isoenzymes or restriction fragment length polymorphisms in kDNA mini-circles, genes for ribosomal RNAs and variant surface glycoproteins. Our results fit the general idea that T. evansi stocks worldwide have a single origin.     

N,N′-Thiophene-substituted polyamine analogs inhibit mammalian host cell invasion and intracellular multiplication of Trypahosoma cruzi

We studied the effects of two, N,N′-thiophene-substituted polyamine analogs (MDL 28302 and MDL 29431) on the capacities of Trypanosoma cruzi, the etiologic agent of Chagas' disease, to invade and multiply within a mammalian host cell. Both compounds inhibited infectivity significantly in a time- and concentration-dependent manner. This inhibition resulted from a selective effect on the parasite, because pretreatment of T. cruzi but not host cell cultures with either MDL 28302 or MDL 29431 reduced infectivity. The parasite gradually recovered its infective capacity after removal of unincorporated polyamine analog, denoting the reversible nature of the inhibitory effect. Some biochemical modification of MDL 28302 and MDL 29431 appeared to be required for their inhibitory activities to be exerted, since the effects of these drugs on T. cruzi infectivity were abrogated by MDL 72527, a drug known to inhibit polyamine oxidase (PAO) activity specifically. Supporting the notion of that products of MDL 28302 and MDL 29431 oxidation by PAO were involved in the activity of these compounds was the finding that PAO competitive substrates (N¹Lacetylspermine and N¹-acetylspermidine) also abolished the inhibition of T. cruzi infectivity mediated by MDL 28302 or MDL 29431. However, we can not rule out that MDL 72527 and the PAO competitive substrates might have altered an alternative mechanism because no significant polyamine oxidase activity could be demonstrated in preparations of lysed or intact T. cruzi in assays monitoring conversion of [¹⁴C]spermine to [¹⁴C]spermidine. When either MDL 28302 or MDL 29431 was added to infected cell cultures, a marked reduction in the rate of intracellular parasite growth ensued. The significance of the finding that N,N′-thiophene-substituted polyamine analogs inhibit cell invasion and cytoplasmic replication by T. cruzi resides in the fact that this pathogenic parasite requires a cytoplasmic localization to replicate in mammalian hosts.     

High-density-lipoprotein-independent killing of Trypanosoma brucei by human serum

The cattle pathogen Trypanosoma brucei brucei is morphologically indistinguishable from the human pathogens T.b. rhodesiense and T.b. gambiense. However, unlike the human pathogens, T.b. brucei is lysed by normal human serum (NHS). The trypanolytic factor in NHS co-purifies with high-density lipoproteins (HDL), but its precise nature is unknown. Using a new fluorescence-based viability assay to assess T.b. brucei killing, we find that the HDL-deficient sera from two patients with Tangier disease are as trypanolytic as NHS. Fractionation of the Tangier sera by density ultracentrifugation revealed that the activity resides only in lipoprotein-depleted fractions. Tangier and NHS were also subjected to molecular sieving chromatography, and the activity profiles were identical. Lytic fractions to T. brucei (but not to T. rhodesiense) appeared under two distinct peaks of 100–600 kDa and > 1000 kDa. Neither peak coincided with the position of the major serum lipoproteins, as determined by cholesterol titrations. The high-molecular-mass peak did not contain the HDL-associated apolipoprotein-A1. Further, we did not find that purified apolipoproteins A1 or J are lytic for the trypanosomes. We conclude that the killing of T. brucei by human serum can be independent of HDL.     

Cloning and functional expression of Rpn1, a regulatory-particle non-ATPase subunit 1, of proteasome from Trypanosoma cruzi

Non-lysosomal protein degradation in eukaryotic cells involves a proteolytic complex referred to as 26S proteasome that consists of a 20S core particle and one or two 19S regulatory particles. We have cloned the gene RPN1 encoding Rpn1 (regulatory-particle non-ATPase subunit 1), one of the largest subunits of proteasome, from Trypanosoma cruzi. It contains 2712 bp and encodes 904 amino acid residues with a calculated molecular mass of 98.2 kDa and an isoelectric point of 5.2. The predicted amino acid sequence of the trypanosomatid Rpn1 shares 39.0 and 32.0% overall identities with human Rpn1 and Saccharomyces cerevisiae Nas1 (non-ATPase subunit 1), an Rpn1 homolog, respectively, while the sequence identities among T. cruzi, Plasmodium falciparum, and Entamoeba histolytica Rpn1 are approximately 30%. T. cruzi Rpn1 contains nine repeats of about 36 amino acid residues conserved in Rpn1s from various organisms. T. cruzi RPN1 is located on the 2300- and 1900-kb chromosomal DNA, displays a putative allelic variation as RPN1-1 and RPN1-2 with 98.8% identity between these two putative gene products, and is transcribed from both alleles at a comparable level throughout the three developmental stages of the parasite, epimastigotes, trypomastigotes, and amastigotes. The expression of the trypanosomatid Rpn1 in the temperature-sensitive nas1 yeast mutant rescued the growth defect at the restrictive temperature, indicating that Rpn1 functions as a Nas1 and probably assembles into the 19S regulatory particle of the yeast 26S proteasome.     

Characterization of a divergent glycosomal microbody phosphoglycerate kinase from Trypanosoma brucei

There are 3 loci in the phosphoglycerate kinase (PGK) gene complex of Trypanosoma brucei. The PGK-A gene product, which we term 56PGK, is targeted to glycosomal microbodies and is highly homologous to the parasite's 2 known PGKs (one cytoplasmic and one glycosomal). However, 56PGK contains an 80 amino acid insertion as well as numerous substitutions compared to the other PGKs. The complementation and kinetic analyses described here demonstrate that 56PGK is an authentic phosphoglycerate kinase - the largest yet described. When expressed in Escherichia coli, 56PGK complements the pgk⁻ phenotype. 56PGK was expressed as a fusion protein and purified to near homogeneity. The Michaelis constants are similar to those of other PGKs, being 0.12 and 2.4 mM for Mg-ATP and 3-phosphoglycerate, respectively. As with other T. brucei PGKs, ATP but not GTP or ITP can serve as a phosphate donor during catalysis. No evidence was obtained for phosphate transfer to atypical substrates. 56PGK shows sulfate inhibition at all concentrations tested, rather than the sulfate activation observed with yeast PGK.     

Organization of telomeric and sub-telomeric regions of chromosomes from the protozoan parasite Trypanosoma cruzi

We present here a characterization of the telomeric and subtelomeric regions of Trypanosoma cruzi chromosomes, using three types of recombinants: cosmids from a genomic library, clones obtained by a vector–adaptor protocol, and a recombinant fragment cloned by a Bal31 trimming protocol. The last nine nucleotides of the T. cruzi overhang are 5′-GGGTTAGGG-3′, and there are from 9 to 50 copies of the hexameric repeat 5′-TTAGGG-3′, followed by a 189-bp junction sequence common to all recombinants. The subtelomeric region is made of sequences associated with the gp85/sialidase gene family, and/or sequences derived from SIRE, a retrotransposon-like sequence, and also the retrotransposon L1Tc. We discuss the possible implications of this genome organization.     

The major cysteine proteinase (cruzipain) from Trypanosoma cruzi is encoded by multiple polymorphic tandemly organized genes located on different chromosomes

We demonstrate that cruzipain, the major cysteine proteinase of Trypanosoma cruzi epimastigotes, is encoded by a large number of tandemly arranged genes. Restriction enzyme analysis of 20 clones containing complete repeat units of the gene, as well as sequencing of 2 of these clones, and comparison with previously published partial sequences, indicated that the sequence is conserved among the repeat units, although polymorphisms clearly exist. The repeat units contain an intergenic region of 528 bp and coding regions for pre- and pro-enzyme, a central domain and a C-terminal extension. The predicted amino acid sequences of these regions indicated a sequence identity of 30, 60, 70 and 36%, respectively, when the T. cruzi sequence was compared with the sequence of a similar cysteine proteinase from Trypanosoma brucei [14]. Studies by pulsed field gel electrophoresis, complemented with restriction analysis, indicated that the clusters are located on 2 4 different chromosomes in several parasite isolates.     

The selenoproteome is dispensable in bloodstream forms of Trypanosoma brucei

Here we show that absence of Sep-tRNA:Sec-tRNA synthase (SepSecS) a key enzyme required for the synthesis of the three trypanosomal selenoproteins does not affect growth of bloodstream forms of Trypanosoma brucei. Both life cycle stages of T. brucei are highly sensitive to auranofin, a compound known to target selenoproteins. However, the same sensitivity is observed in the SepSecS double knockout cell lines indicating that the trypanocidal action of auranofin is not connected to selenoproteins. Finally, we show that absence of selenoproteins does not increase sensitivity to H₂O₂-induced oxidative stress. Thus in cell culture normal growth of procyclic and bloodstream T. brucei does not depend on selenoproteins.