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.     

Toxoplasma gondii tachyzoites possess an unusual plasma membrane adenosine transporter

Nucleoside transport may play a critical role in successful intracellular parasitism by Toxoplasma gondii. This protozoan is incapable of de novo purine synthesis, and must salvage purines from the host cell. We characterized purine transport by extracellular T. gondii tachyzoites, focusing on adenosine, the preferred salvage substrate. Although wild-type RH tachyzoites concentrated [³H]adenosine 1.8-fold within 30 s, approx. half of the [³H]adenosine was converted to nucleotide, consistent with the known high parasite adenosine kinase activity. Studies using an adenosine kinase deficient mutant confirmed that adenosine transport was non-concentrative. [¹⁴C]Inosine, [¹⁴C]hypoxanthine and [³H]adenine transport was also rapid and non-concentrative. Adenosine transport was inhibited by dipyridamole (IC₅₀ approx. 0.7 μM), but not nitrobenzylthioinosine (15 μM). Transport of inosine, hypoxanthine and adenine was minimally inhibited by 10 μM dipyridamole, however. Competition experiments using unlabeled nucleosides and bases demonstrated distinct inhibitor profiles for [³H]adenosine and [¹⁴C]inosine transport. These results are most consistent with a single, dipyridamole-sensitive, adenosine transporter located in the T. gondii plasma membrane. Additional permeation pathways for inosine, hypoxanthine, adenine and other purimes may also be present.     

The hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus has unique properties

Tritrichomonas foetus, an anaerobic, flagellated protozoan parasite, is incapable of de novo purine nucleotide synthesis, and depends primarily on the salvage of purine bases from the host. The hypoxanthine-guanine-xanthine phosphoribosyl-transferase (HGXPRTase) from this organism has been purified to homogeneity by ammonium sulfate precipitation and Sephacryl-HR100 gel filtration, followed by anion exchange FPLC. Hypoxanthine, guanine and xanthine phosphoribosyl-transferase activities co-eluted in all the purification steps, suggesting that they are associated with the same enzyme protein. The molecular mass of the native protein, as estimated by gel filtration, is 24 kDa. The molecular mass estimated from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is also 24 kDa. Non-denaturing polyacrylamide gel electrophoresis of the purified protein, followed by activity staining with either [¹⁴C]hypoxanthine, [¹⁴C]guanine or [¹⁴C]xanthine, also demonstrates that the enzyme is a monomer of 24 kDa. This monomeric structure is distinctive from all the other reported PRTases which are either dimers or tetramers. Furthermore, unlike the mammalian HGPRTase, which is heat stable, the T. foetus enzyme is heat labile. Kinetic studies with the purified T. foetus HGXPRTase showed that the apparent Kms for hypoxanthine, guanine and xanthine were 4.1 μM, 3.8 μM and 52.4 μM respectively. This recognition of xanthine as a substrate by the parasite enzyme with only about a 10-fold higher Km value than those for hypoxanthine and guanine distinguishes it from the mammalian HGPRTase, which cannot use xanthine as a substrate, as well as the HGXPRTases of Eimeria tenella and Plasmodiumfalciparum, which are dimers, with xanthine about 100-times less proficient as a substrate. T. foetus HGXPRTase is thus a unique enzyme with opportunity for specific inhibitor design.     

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.     

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.     

A proton pumping pyrophosphatase in the Golgi apparatus and plasma membrane vesicles of Trypanosoma cruzi

The proton pumping pyrophosphatase (H⁺-PPase) is an enzyme that has been identified in membranes of plant vacuoles, in the Golgi complex of plants and Chlamydomonas reinhardtii, and more recently in acidocalcisomes of different trypanosomatids and apicomplexan parasites. Immunofluorescence and immunoelectron microscopy studies using antibodies against the plant enzyme also suggested a plasma membrane localization in different stages of Trypanosoma cruzi. In this report we provide immunogold electron microscopy evidence of the presence of the H⁺-PPase in the Golgi complex and plasma membrane of epimastigotes of T. cruzi. Pyrophosphate promoted acidification of plasma membrane vesicles as determined using acridine orange. This activity was stimulated by K⁺ ions, inhibited by the pyrophosphate analogs imidodiphosphate (IDP) and aminomethylenediphosphonate (AMDP) by KF, NaF and DCCD, and it had different responses to ions and inhibitors as compared with the activity present in acidocalcisomes. Surface localization of the H⁺-PPase was confirmed by experiments using biotinylation of cell surface proteins and immunoprecipitation with antibodies against H⁺-PPase. Taken together, these results are consistent with the presence of a functional H⁺-PPase in the plasma membrane of these parasites.     

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.     

Comparative analysis of genomic sequences suggests that Trypanosoma cruzi CL Brener contains two sets of non-intercalated repeats of satellite DNA that correspond to T. cruzi I and T. cruzi II types

Approximately 10% of the Trypanosoma cruzi genome is formed by a satellite DNA, composed by 195-bp repeats organized in 30 ± 10 kb clusters in some, but not all chromosomes. Here, the satellite DNA of six representative T. cruzi strains was sequenced and used for phylogenetic inference. The results show that CL Brener contains satellite repeats from T. cruzi I and T. cruzi II strains, although type II sequences are more abundant. The presence of types I and II sequences extends previous propositions that genetic exchange between the two major T. cruzi lineages have occurred in CL Brener, although our data accommodate alternative scenarios of hybridization within T. cruzi II, as proposed by others. Altogether, present data suggest a complex origin for CL Brener. Sequence analysis of satellites isolated from chromosomal bands indicates that satellite DNA sequences are not chromosome specific. Neighbor analysis of in tandem satellite DNAs containing up to five repeats shows that each cluster contains only one type of sequence. Consequently, clusters with intercalated types I and II repeats were not found. We propose that the CL Brener genome contains large pieces of satellite DNA originated mainly from chromosomes of T. cruzi II with introgression of T. cruzi I lineage.     

Inhibitory action of the antitumor agent lonidamine on mitochondrial respiration of Trypanosoma cruzi and T. brucei

The antitumor and antispermatogenic agent lonidamine inhibits Trypanosoma cruzi epimastigotes growth in culture with an ID₅₀ around 80 μM. The main site of action appears to be the mitochondria, where the rate of uncoupled respiration was inhibited in 50% at a similar lonidamine concentration (50 μM). Hexokinase (the other point where lonidamine inhibits tumor energy metabolism) was not sensitive to this drug. Lonidamine also inhibited uncoupled respiration in T. brucei procyclic trypomastigotes, suggesting a common mechanism of action with T. cruzi. When lonidamine was added to T. brucei trypomastigotes, there was little effect on the CN-insensitive respiration, demonstrating that at least in T. brucei glycolysis is not affected by the drug.     

Interferon-gamma suppresses the growth of Toxoplasma gondii in human fibroblasts through starvation for tryptophan

The effect of human recombinant interferon-γ (IFN-γ) on Toxoplasma gondii in cultured human fibroblasts is predominantly parasitostatic. This effect is dependent upon the induction in the host cell of a potent indoleamine 2,3-dioxygenase that converts tryptophan to N-formylkynurenine. This product is, in turn, degraded to kynurenine by a formamidase. Within 24 h of treatment with IFN-γ most of the tryptophan originally present in the medium is converted to these products together with some minor metabolites. When added to the medium of infected cultures at concentrations equimolar to the tryptophan content, neither N-formylkynurenine nor kynurenine suppresses the growth of T. gondii, although at higher concentrations they are effective. The medium of uninfected cultures treated with IFN-γ for 24 h has no effect on the growth of T. gondii, when transferred to fresh cultures provided that the residual IFN-γ is first removed by ultrafiltration or neutralized with a specific monoclonal antibody. Thus minor metabolites produced from tryptophan in response to IFN-γ and excreted into the medium are not parasitostatic. When cultures treated with IFN-γ for 24 h are incubated with medium that contains [³H]tryptophan, the radioactive amino acid is converted to N-formylkynurenine and kynurenine as rapidly as it enters the cell. This degradation not only results in a very low intracellular concentration of tryptophan but also produces intracellular concentrations of tryptophan metabolites that are significantly higher than the tryptophan concentration in control cells. However, it is unlikely that either metabolite reaches intracellular concentrations that are sufficient to suppress the growth of the parasite. The parasitostatic effect of IFN-γ is most likely to result from the starvation of T. gondii for tryptophan.     

Protective immunity induced by a Trypanosoma cruzi soluble extract antigen in Experimental Chagas' Disease. Role of Interferon γ

CBA/J mice can be protected against lethal infection with Trypanosoma cruzi by treatment using T. cruzi soluble extract antigen (TCSE). In vivo administration of TCSE (400 μg/mouse) into naive mice increased the cellular proliferative response to Con A and elevated the levels of IFN-γ. The production of IFN-γ was extremely important in controlling the replication of the parasite since the protective activity of TCSE was completely abrogated by in vivo treatment with an anti IFN-γ neutralizing antibody. These results suggest that depending on the level, cytokine production results in the control of replication of the parasite in experimental Chagas'disease.     

Protective immunity induced by a Trypanosoma cruzi soluble extract antigen in experimental Chagas' disease. Role of interferon gamma

CBA/J mice can be protected against lethal infection with Trypanosoma cruzi by treatment using T. cruzi soluble extract antigen (TCSE). In vivo administration of TCSE (400 μg/mouse) into naive mice increased the cellular proliferative response to Con A and elevated the levels of IFN-γ. The production of IFN-γ was extremely important in controlling the replication of the parasite since the protective activity of TCSE was completely abrogated by in vivo treatment with an anti IFN-γ neutralizing antibody. These results suggest that depending on the level, cytokine production results in the control of replication of the parasite in experimental Chagas'disease.     

Absence of Trypanosoma cruzi myocardial infection reactivation in Chagas'' heart transplant

The risk of Trypanosoma cruzi myocardial infection reactivation after immunosuppressive therapy has led to precluding heart transplantation as a therapeutic procedure for patients with end-stage Chagas' heart disease. We report a case of an orthotopic heart transplantation in a 43-year-old critically ill chagasic patient with an uneventful postoperative period. He was treated with azathioprine and cyclosporine to control graft rejection and showed no reactivation of the chagasic infection. One year following surgery, the patient is doing well. Nonsteroidal therapy appears not to reactivate T. cruzi infection in transplant chagasic patients.     

Density gradient purification of human lymphocytes from contaminating trypomastigotes of Trypanosoma cruzi

Mixtures of normal human lymphocytes and T. cruzi trypomastigotes obtained from infected mice were centrifuged over Ficoll-Hypaque (FH) continuous and discontinuous gradients. Trypomastigotes were confined to the range 1.051–1.057 g/ml while lymphocytes ranged between 1.046 and 1.080 g/ml. Over 80% of the lymphocytes were found at 1.060 g/ml or higher densities. A discontinuous gradient of FH with 2 layers of 1.060 and 1.077 g/ml of density respectively was selected to obtain trypomastigotes-free white blood cells from blood samples. The functional capacity of lymphocytes recovered from the lower interface, where no parasites were found, was assessed. The response to phytohaemagglutinin of these high density lymphocytes was as good as of total lymphocytes, suggesting that low density lymphocytes are not necessary for proliferative responses. It is postulated that high density lymphoid populations, free from T. cruzi forms, may be used to study the presence of T cell-mediated immune response in Chagas' disease patients.     

Effect of haloallylamines on polyamine oxidase activity and spermine levels in Ascaris suum

 In parasitic nematodes the rate-limiting step in the polyamine interconversion pathway is catalysed by polyamine oxidase. MDL 72527, the specific inhibitor of mammalian polyamine oxidase, had no effect on the Ascaris suum enzyme, whereas its activity was inhibited in a time-dependent manner by the haloallylamine MDL 72145, originally designed as a specific inhibitor of monoamine oxidase A and B. The dissociation constant (K _i) was found to be 0.9 μM and the enzyme half-life under saturation conditions (t ₅₀) was determined to be 0.8 min. Incubation of A. suum in vitro in the presence of 50 μM MDL 72145 for 6 h resulted in a decrease in polyamine oxidase activity to about 20% of the control value, and spermine concentrations simultaneously increased about 200%. Both results suggest that MDL 72145 might be a chemical lead compound for the design of new chemotherapeutic agents against nematode infections. Received: 15 November 1995 / Accepted: 15 January 1996     

SfiI and NotI polymorphisms in Theileria stocks detected by pulsed field gel electrophoresis

DNAs of Theileria parva parva, T. p. lawrencei, T. p. bovis and Theileria mutans stocks, from Kenya, Uganda, Zanzibar and Zimbabwe were digested with either SfiI or NotI and analysed using contour-clamped homogeneous electric field (CHEF) and field-inversion gel electrophoresis (FIGE). The SfiI-digested T. parva genomic DNA resolved into approximately 30 fragments while the NotI digestion produced between 4–7 bands. The summation of the sizes of SfiI fragments gave an estimate of 9–10×10⁶ base pairs for the size of the T. parva genome. Heterogeneity within T. p. parva Muguga, Pemba/Mnarani and Mariakani stocks was detected. All the T. parva stocks analysed showed SfiI and NotI restriction fragment length polymorphisms (RFLP). Hybridisation of 5 SfiI-digested T. parva DNAs with a Plasmodium berghei telomeric repeat probe suggested that most of the polymorphisms and heterogeneity occurred in the telomeric or sub-telomeric regions of the genome. The recognition by the Plasmodium telomeric probe of 7–8 strongly hybridising SfiI bands indicates that the T. parva genome may possess at least 4 chromosomes. The T. mutans genome was cut frequently with the above enzymes resulting in large numbers of fragments predominantly below 50 kb, thus suggesting either a much higher G+C content than T. parva or the presence of highly reiterated G+C-rich regions.     

Post-translational modifications of Trypanosoma cruzi histone H4

Histone tails provide sites for a variety of post-translational modifications implicated in the control of gene expression and chromatin assembly. As both histones and control of gene expression in trypanosomes are highly divergent compared to most eukaryotes, post-translational modifications of Trypanosoma cruzi histones were investigated. After in vivo incubation of live parasites with radiolabeled precursors, histone H4 mainly incorporates [³H]-acetyl, and to a lesser extent [³H]-methyl residues. In contrast, histone H3 preferentially incorporates [³H]-methyl residues. The modifications of histone H4 were further characterized by mass spectrometry. MALDI-TOF–TOF-MS analysis revealed that peptides from histone H4 amino-terminus, obtained by either endoproteinase Glu-C or endoproteinase Arg-C digestion, contain isoforms with 14 and 42 Da additions, suggesting the presence of simultaneous acetylations and/or methylations. Tandem mass spectrometry analysis demonstrated that the N-terminal alanine is methylated, and lysine residues at positions 4, 10, 14 and 57 are acetylated; lysine at position 18 is mono-methylated, while arginine at position 53 is dimethylated. Immunoblotting analyses using specific antibodies raised against synthetic and acetylated peptides of T. cruzi histone H4 indicate that lysine 4 is acetylated in the majority of histone H4, while other acetylations at the N-terminus portion of histone H4 are less abundant.     

The humoral immune response to the kinetoplastid membrane protein-11 in patients with American Leishmaniasis and Chagas disease: prevalence of IgG subclasses and mapping of epitopes

The kinetoplastid membrane protein-11 (KMP-11) is a major target of the humoral immune response during Leishmania-infections. The majority of sera from visceral leishmaniasis, mucocutaneous leishmaniasis and even some cutaneous leishmaniasis patients contain detectable IgG antibodies against KMP-11. We also provide evidence that this protein may act as a potent antigen in T. cruzi infections, since most Chagas sera show immunological cross-reactivity. Therefore, KMP-11 cannot be used as a specific diagnostical tool for the serodiagnosis of leishmaniasis in those regions where both, Leishmania and T. cruzi infections overlap geographically. When analyzing the subclass specificity of the antibody response to KMP-11 we observed the following order of reactivity: IgG1 >>IgG3 >IgG2 >IgG4, which is similiar to that seen in crude parasite extract. The mapping of antigenic determinants by using synthetic 20-mer peptides revealed the existence of predominantly conformational epitopes in leishmaniasis, while 50% of sera from Chagas patients reacted with a particular KMP-11 peptide. These results therefore suggest the presence of disease-specific B-cell epitopes.     

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.