Decoding caspase signaling in host immunity to the protozoan Trypanosoma cruzi

Caspases, a family of cysteinyl-aspartate-specific proteases, induce apoptosis but are also involved in signal transduction in live cells. Caspase activation and apoptosis in T lymphocytes occur following infection with parasites and might affect immune responses. Rapid progress has occurred in the development and testing of caspase inhibitors and other apoptosis blockers, which are potentially useful for treating diseases associated with the pathogenic effects of apoptosis. Pharmacological approaches and the use of genetically modified hosts can be combined in research strategies to understand how apoptosis and caspase signaling affect the immune system.     

Identification of a functional prostanoid-like receptor in the protozoan parasite, Trypanosoma cruzi

Trypanosoma cruzi infection in humans and experimental animals causes Chagas disease which is often accompanied by myocarditis, cardiomyopathy, and vasculopathy. T. cruzi-derived thromboxane A₂ (TXA₂) modulates vasculopathy and other pathophysiological features of Chagasic cardiomyopathy. Here, we provide evidence that epimastigotes, trypomastigotes, and amastigotes of T. cruzi (Brazil and Tulahuen strains) express a biologically active prostanoid receptor (PR) that is responsive to TXA₂ mimetics, e.g. IBOP. This putative receptor, TcPR, is mainly localized in the flagellar membrane of the parasites and shows a similar glycosylation pattern to that of bona fide thromboxane prostanoid (TP) receptors obtained from human platelets. Furthermore, TXA₂-PR signal transduction activates T. cruzi-specific MAPK pathways. While mammalian TP is a G-protein coupled receptor (GPCR); T. cruzi genome sequencing has not demonstrated any confirmed GPCRs in these parasites. Based on this genome sequencing it is likely that TcPR is unique in these protists with no counterpart in mammals. TXA₂ is a potent vasoconstrictor which contributes to the pathogenesis of Chagasic cardiovascular disease. It may, however, also control parasite differentiation and proliferation in the infected host allowing the infection to progress to a chronic state.     

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.     

Gene gun-mediated delivery of an interleukin-12 expression plasmid protects against infections with the intracellular protozoan parasites Leishmania major and Trypanosoma cruzi in mice

An interleukin-12 (IL-12) expression plasmid was transferred, using a gene gun, to mice infected with Leishmania major or Trypanosoma cruzi. Transfer of the IL-12 gene to susceptible BALB/c mice resulted in regression of lesion size and reduced the number of parasites in draining lymph nodes (LN) at the site of L. major infection. Coincident with these protective effects, the T-helper type (Th) response shifted towards Th1, as evaluated by cytokine production in vitro and L. major-specific antibody responses. Protective effects of the IL-12 gene were also observed in T. cruzi infection. Treatment of BALB/c mice infected with T. cruzi enhanced the production of interferon-gamma (IFN-gamma) by spleen cells, while suppressed production of interleukin-10 (IL-10) compared with control mice. Administration of anti-CD4 or anti-CD8 monoclonal antibody (mAb) abolished the protective immunity against T. cruzi infection, and treatment with the IL-12 gene could not restore the resistance in these mice. Mice depleted of natural killer (NK) cells with anti-asialo GM1 also became susceptible to infection, while the resistance was restored when these mice were treated with the IL-12 gene. Thus, target cells for the treatment appear to be CD4+ and CD8+ T cells, which are ordinarily activated by NK cells. These results suggest that the transfer of cytokine genes using a gene gun is an effective method for investigating the roles of cytokines and gene therapy in infectious diseases.     

Gene Discovery through Expressed Sequence Tag Sequencing in Trypanosoma cruzi

Analysis of expressed sequence tags (ESTs) constitutes a useful approach for gene identification that, in the case of human pathogens, might result in the identification of new targets for chemotherapy and vaccine development. As part of the Trypanosoma cruzi genome project, we have partially sequenced the 5′ ends of 1,949 clones to generate ESTs. The clones were randomly selected from a normalized CL Brener epimastigote cDNA library. A total of 14.6% of the clones were homologous to previously identified T. cruzi genes, while 18.4% had significant matches to genes from other organisms in the database. A total of 67% of the ESTs had no matches in the database, and thus, some of them might be T. cruzi-specific genes. Functional groups of those sequences with matches in the database were constructed according to their putative biological functions. The two largest categories were protein synthesis (23.3%) and cell surface molecules (10.8%). The information reported in this paper should be useful for researchers in the field to analyze genes and proteins of their own interest.     

Protein geranylgeranyltransferase-I of Trypanosoma cruzi

Protein geranylgeranyltransferase type I (PGGT-I) and protein farnesyltransferase (PFT) occur in many eukaryotic cells. Both consist of two subunits, the common alpha subunit and a distinct beta subunit. In the gene database of protozoa Trypanosoma cruzi, the causative agent of Chagas' disease, a putative protein that consists of 401 amino acids with approximately 20% amino acid sequence identity to the PGGT-I beta of other species was identified, cloned, and characterized. Multiple sequence alignments show that the T. cruzi ortholog contains all three of the zinc-binding residues and several residues uniquely conserved in the beta subunit of PGGT-I. Co-expression of this protein and the alpha subunit of T. cruzi PFT in Sf9 insect cells yielded a dimeric protein that forms a tight complex selectively with [(3)H]geranylgeranyl pyrophosphate, indicating a key characteristic of a functional PGGT-I. Recombinant T. cruzi PGGT-I ortholog showed geranylgeranyltransferase activity with distinct specificity toward the C-terminal CaaX motif of protein substrates compared to that of the mammalian PGGT-I and T. cruzi PFT. Most of the CaaX-containing proteins with X=Leu are good substrates of T. cruzi PGGT-I, and those with X=Met are substrates for both T. cruzi PFT and PGGT-I, whereas unlike mammalian PGGT-I, those with X=Phe are poor substrates for T. cruzi PGGT-I. Several candidates for T. cruzi PGGT-I or PFT substrates containing the C-terminal CaaX motif are found in the T. cruzi gene database. Among five C-terminal peptides of those tested, a peptide of a Ras-like protein ending with CVLL was selectively geranylgeranylated by T. cruzi PGGT-I. Other peptides with CTQQ (Tcj2 DNAJ protein), CAVM (TcPRL-1 protein tyrosine phosphatase), CHFM (a small GTPase like protein), and CQLF (TcRho1 GTPase) were specific substrates for T. cruzi PFT but not for PGGT-I. The mRNA and protein of the T. cruzi PGGT-I beta ortholog were detected in three life-cycle stages of T. cruzi. Cytosol fractions from trypomastigotes (infectious mammalian stage) and epimastigotes (insect stage) were shown to contain levels of PGGT-I activity that are approximately 100-fold lower than PFT activity. The CaaX mimetics known as PGGT-I inhibitors show very low potency against T. cruzi PGGT-I compared to the mammalian enzyme, suggesting the potential to develop selective inhibitors against the parasite enzyme.     

Characterization of Trypanosoma cruzi hexokinase

Properties of hexokinase (EC 2.7.1.1) from Trypanosoma cruzi epimastigote forms (Tulahuen strain) were studied and compared with enzymes from other sources. The enzyme activity was 37 units g-1 of wet cells (1.2 units mg-1 protein). Hexokinase showed Km values for glucose and ATP of 0.09 and 0.4 mM, respectively. The enzyme reacted with other nucleotides too. N-Acetylglucosamine was a competitive inhibitor with respect to glucose (Ki = 0.3 mM). ADP inhibited the enzyme competitively with respect to ATP (Ki = 1.5 mM) and noncompetitively with respect to glucose (Ki = 7 mM). The enzyme was markedly inhibited by 5-thioglucose, its Ki value was 0.4 mM. Hexokinase activity was not affected by glucose 6-phosphate.     

On the DNA content of Trypanosoma cruzi

The DNA contents of three different Trypanosoma cruzi strains were compared by direct microfluorometry. The maximal difference found was 40% of the lowest value. Two of the cloned strains, reported in an earlier study to differ by 48% in their total DNA content, showed a difference of 33%. The kinetoplast of the Y strain made up about one third of its total genome. The absolute DNA content of T. cruzi was estimated at 125 to 200 fg.     

Effects of IL-4 on macrophage functions: increased uptake and killing of a protozoan parasite (Trypanosoma cruzi).

We studied whether interleukin-4 (IL-4) could modulate two macrophage functions relevant to their microbicidal activity (uptake and killing), using non-invasive [amastigote (AMA)] forms of the protozoan parasite Trypanosoma cruzi. Treatment of cultures of mouse resident peritoneal macrophages (MPM) with the supernatant of cultures of cells transfected with IL-4 cDNA increased both the capacity of the MPM to take up the organisms and the rate of intracellular killing with respect to MPM mock-treated with medium alone. The presence in the medium of a monoclonal antibody specific for IL-4 during MPM treatment inhibited both effects, pointing to recombinant IL-4 (rIL-4) as the active principle in the supernatant. Kinetic studies revealed that at least a 24-hr pretreatment of the MPM with the rIL-4-containing supernatant was required for these effects to be produced. The rate of intracellular parasite killing was also significantly increased when the rIL-4 treatment was applied after AMA ingestion by MPM. This result confirmed that MPM could be activated by rIL-4 for greater intracellular killing and showed that this enhancement was not necessarily dependent on the initial rIL-4-mediated increase in parasite load. The use of scavengers of reactive oxygen reduction intermediates indicated that hydrogen peroxide, superoxide anion and singlet oxygen, but apparently not hydroxyl radicals, were involved in parasite killing modulated by rIL-4. These results document for the first time the capacity of IL-4 to enhance the microbicidal activity of macrophages and suggest that this lymphokine might play a role in host defence against T. cruzi infection.     

Immune system recognition of Trypanosoma cruzi

Innate and adaptive cellular immune recognition is crucial for control of the protozoan parasite Trypanosoma cruzi. T. cruzi triggers both MyD88-dependent and TRIF-dependent innate activation pathways in macrophages and dendritic cells. TLR-2 and TLR-9 recognize GPI anchors and parasite DNA, respectively; however other, as yet undefined receptors and ligands, also appear to be involved in innate recognition. CD8(+) T cells distinguish T. cruzi-infected host cells primarily via robust recognition of MHC-associated peptide epitopes from the large and highly diverse trans-sialidase family of surface proteins. To date there has been minimal investigation of linkages between innate immune recognition in vivo and the generation of adaptive cellular immune responses.     

Localization and Developmental Regulation of a Dispersed Gene Family 1 Protein in Trypanosoma cruzi

The dispersed gene family 1 (DGF-1) is the fifth largest gene family in the Trypanosoma cruzi genome, with over 500 members (11). Many of the predicted DGF-1 protein products have several transmembrane domains and N-glycosylation and phosphorylation sites and were thought to localize in the plasma membrane. Here, we report that affinity-purified antibodies against a region of one of these proteins (DGF-1.2) localized it intracellularly in different stages of the parasite. DGF-1.2 is more abundant in the amastigote stage than in trypomastigotes and epimastigotes, as detected by immunofluorescence and Western blot analyses. The protein changed localization during intracellular or extracellular differentiation from the trypomastigote to the amastigote stage, where it finally localized to small bodies in close contact with the inner side of the amastigote plasma membrane. DGF-1.2 did not colocalize with markers of other subcellular organelles, such as acidocalcisomes, glycosomes, reservosomes, lipid droplets, or endocytic vesicles. During extracellular differentiation, the protein was detected in the culture medium from 0 to 22 h, peaking at 14 h. The presence of DGF-1.2 in the differentiation culture medium was confirmed by mass spectrometry analysis. Finally, when epimastigotes were subjected to starvation, there was a decrease in the labeling of the cells and, in Western blots, the appearance of bands of lower molecular mass, suggesting its cleavage. These results represent the first report of direct immunodetection and developmental expression and secretion of a DGF-1 protein.Trypanosoma cruzi is the causative agent of Chagas disease, an endemic illness affecting between 16 and 18 million people in North, Central, and South America for which no vaccine or satisfactory treatment is available (22). During its life cycle, the parasite goes through different stages in the vector (epimastigotes and metacyclic trypomastigotes) and in the mammalian host (amastigotes and bloodstream trypomastigotes). As part of its survival strategy in these varying environments, the parasite has developed a large repertoire of multigene families (9, 11, 12, 16). Among these families, the dispersed gene family 1 (DGF-1) has approximately 565 copies, ranging from 6 to 10 kbp, dispersed throughout the parasite genome (11). The members of the DGF-1 family encode proteins that share 85 to 95% sequence identity (11). Wincker and colleagues first identified clones bearing a common repeated sequence from a T. cruzi genome library (24) and later described the nucleotide sequence of a representative gene (DGF-1.1) (23). They concluded, from in silico studies, that DGF-1 genes encoded putative cell surface proteins (23). In 2005, Kim and colleagues (16) described a new member of this family (DGF-1.2) located in the subtelomeric region of a T. cruzi chromosome surrounded by mainly two kinds of sequences: genes encoding the trans-sialidase (TS) and retrotransposon hot spot (RHS) protein families. The sizes of the open reading frames (ORFs) of DGF-1 genes and their abundance in the T. cruzi genome suggested that they are essential sequences for parasite survival. Furthermore, the existence of some telomeric DGF-1 copies that were always flanked by pseudogenes suggested that these genes have been subjected to strong selective pressure and, as a consequence, that they should be expressed at some life cycle stage of the parasite (16).A glycopeptide shared by several members of the DGF-1 family was recently detected in a glycoproteomic study of T. cruzi trypomastigotes, demonstrating that at least a DGF-1 family member protein is actually expressed and N-glycosylated (3). We also detected a number of peptides corresponding to several DGF-1 family member proteins in a proteomic study of acidocalcisome fractions of epimastigotes of T. cruzi (R. Docampo, J. A. Atwood, R. Tarleton, and R. Orlando, unpublished data). However, this family of proteins has no known orthologs in other species, even in trypanosomatids, and little is known about their localization, expression patterns, and functions in T. cruzi.In the present work, we prepared affinity-purified antibodies against a peptide of the DGF-1.2 protein and investigated its subcellular localization by immunofluorescence microscopy. We also used mass spectrometry (MS) to identify specific peptides recognized by anti-DGF-1.2 antibodies by using fingerprinting analysis.We found that the antibodies preferentially labeled amastigotes. The localization of the protein was in intracellular bodies and not on the cell surface and changed during amastigote development. During the in vitro trypomastigote-to-amastigote transition, we detected continuous secretion of DGF-1.2 into the medium, peaking at 14 h. Anti DGF-1 antibodies that recognized the intracellular protein in both differentiation forms also recognized the secreted protein from trypomastigotes and amastigotes. Finally, when epimastigotes were subjected to starvation, there was a decrease in labeling of the cells and the appearance of defined bands of smaller molecular mass in Western blots, suggesting its cleavage.     

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.     

Trypanosoma cruzi TcSRPK,the first protozoan member of the SRPK family,is biochemically and functionally conserved with metazoan SR protein-specific kinases

A novel SR protein-specific kinase (SRPK) from the SRPK family was identified for the first time in a protozoan organism. The primary structure of the protein, named TcSRPK, presents a significant degree of identity with other metazoan members of the family. In vitro phosphorylation experiments showed that TcSRPK has the same substrate specificity relative to other SRPKs. TcSRPK was able to generate a mAb104-recognized phosphoepitope, a SRPK landmark. Expression of TcSRPK in different Schizosaccharomyces pombe strains lead to conserved phenotypes, indicating that TcSRPK is a functional homologue of metazoan SRPKs. In functional alternative splicing assays in vivo in HeLa cells, TcSRPK enhanced SR protein-dependent inclusion of the EDI exon of the fibronectin minigene. When tested in vitro, it inhibited splicing either on nuclear extracts or on splicing-deficient S100 extracts complemented with ASF/SF2. This inhibition was similar to that observed with human SRPK1. This work constitutes the first report of a member of this family of proteins and the existence of an SR-network in a protozoan organism. The implications in the origins and control of splicing are discussed.     

Putrescine analogue cytotoxicity against Trypanosoma cruzi

Trypanosoma cruzi is the etiological agent of American trypanosomiasis. Most of the available data on trypanosomatid parasites were obtained from African trypanosomes. Parasitic protozoa polyamine metabolism and transport pathways comprise valuable targets for chemotherapy. T. cruzi cannot synthesize putrescine, but its uptake from the extracellular milieu can promote parasite survival. Nevertheless, little is known about the cell biology of this diamine in T. cruzi. Here we notice that the putrescine analogue 1,4-diamino-2-butanone (DAB) inhibited T. cruzi epimastigotes' in vitro proliferation and produced remarkable mitochondrial destruction and cell architecture disorganization, as assessed by transmission electron microscopy. Mitochondrial damage was confirmed by MTT reduction. We decided to analyze the oxidative stress undergone by DAB-treated parasites. Thiobarbituric-acid-reactive substances were measured to assess lipid peroxidation. Analogue effects were dose-dependent; 5 mM DAB only slightly enhanced peroxidation, whereas 10 mM DAB significantly (P<0.05) diminished it. These data indicate that putrescine uptake by this diamine auxotrophic parasite may be important for epimastigote axenic growth and cellular organization.     

Trypanosoma cruzi: Surface antigenic determinants

A fraction (FAd) capable of inhibiting specific agglutination reactions of anti-epimastigote sera (anti-LE) was obtained by extracting the sediment of lyophilized epimastigote lysates (LE) with 0.05 M phosphate buffered saline, at 37° C for 1 h. These conditions favored the action of parasite proteinase whose presence was detected by tandemcrossed immunoelectrophoresis experiments. As expected from the proteinase properties, the addition of 2-mercaptoethanol or sodium iodoacetate to the extracting solution resulted, respectively, in either increased or decreased amounts of protein in the resulting FAd. FAd components could be precipitated by the addition of Concanavalin A, methylated albumins or 0.1 N HCl. This fraction presented a single component when subjected to electrophoresis in 1% agarose gel with an electrophoretic mobility 1.2 times higher than that of human albumin. FAd component(s) were unable to penetrate 15% polycrylamide gel matrix unless 1% SDS was used. Under this condition four glycopeptide components, with Rm of 0.5, 0.55, 0.6 and 0.86, were detected. The antigenic determinants present in FAd resisted heating at 100° C for 30 min and the prolonged action of pronase. However, these determinants were completely destroyed by the action of 25 mM sodium periodate, thus suggesting polysaccharide characteristics. Immunization of rabbits with FAd induced the production of antibodies that were unable to precipitate with either FAd or with parasite proteinase. These antibodies exhibited positive agglutination reactions with epimastigote forms and positive immunofluorescence and immunoperoxidase reactions with trypomastigote and amastigote forms of the different strains tested. FAd was able to inhibit these reactions as well as those obtained with anti-LE and anti-FA immune sera, whereas purified proteinase was unable to inhibit any of these reactions.     

Repetitive sequences scattered throughout the genome of Trypanosoma cruzi

A clone bank from Trypanosoma cruzi DNA was constructed in the plasmid vector pBR325 and screened with total labelled DNA from the same parasite. The experimental conditions used enable recombinant clones containing repetitive sequences to be detected. 2% of the clones gave a strong positive signal. Half of them carried mini-circle sequences but the other half contained repetitive sequences from the nuclear genome. 50% of all colonies showed up more weakly suggesting that half of the trypanosome DNA fragments carried few repetitive elements. One family of repeats, present in two clones from different genomic regions, hybridized with a broad range of nuclear DNA fragment sizes. Moreover, one of these clones had at least two kinds of elements with no common sequences. A third clone, detected under the same conditions, hybridized with distinct nuclear DNA bands. The number of copies estimated for the latter was much lower than the number of homologous sequences detected in nuclear DNA with the former two. This third recombinant plasmid proved useful to differentiate among closely related trypanosome stocks. Neither poly(A)+ or poly(A)- RNA, nor the 50 kilobase pair band corresponding to the satellite DNA already described in trypanosomes, contribute to the repeats present within these recombinant DNAs. Sequences with some degree of homology were found in the nuclear genome of T. brucei and Crithidia fasciculata.