Cardiac damage induced by immunization with heat-killed Trypanosoma cruzi is not antibody mediated.

Cardiac inflammation that develops during infection with Trypanosoma cruzi may result in part from autoimmunity, which may occur after bystander activation, after parasite-induced cardiomyocyte damage, or molecular mimicry. A/J mice infected with T. cruzi or immunized with heat-killed T. cruzi (HKTC) develop strong autoimmunity accompanied by cardiac damage. To determine whether this cardiac damage occurs via an antibody-dependent mechanism, we analysed T. cruzi-infected and HKTC-immunized mice for the presence of autoantibodies, cardiac antibody deposition, and serum cardiac troponin I as a measure of cardiac damage. We also performed a serum transfer experiment in which sera from T. cruzi-infected and T. cruzi-immunized mice (and controls) were transferred into naïve recipients, which were then analysed for the presence of antibodies and serum troponin. Unlike T. cruzi-infected mice, T. cruzi-immunized mice did not show significant antibody deposition in the myocardium. These results indicate that antibody deposition does not precede cardiac damage and inflammation in mice immunized with or infected with T. cruzi. Serum adoptive transfer did not induce cardiac damage in any recipients. Based on these findings, we conclude that the cardiac damage induced by immunization with HKTC is not mediated by antibodies.

Cardiac myosin autoimmunity in acute Chagas' heart disease.

Infection with Trypanosoma cruzi, the agent of Chagas' disease, may induce antibodies and T cells reactive with self antigens (autoimmunity). Because autoimmunity is generally thought to develop during the chronic phase of infection, one hypothesis is that autoimmunity develops only after long-term, low-level stimulation of self-reactive cells. However, preliminary reports suggest that autoimmunity may begin during acute T. cruzi infection. The goal of the present study was to investigate whether cardiac autoimmunity could be observed during acute T. cruzi infection. A/J mice infected with the Brazil strain of T. cruzi for 21 days developed severe myocarditis, accompanied by humoral and cellular autoimmunity. Specifically, T. cruzi infection induced immunoglobulin G (IgG) autoantibodies and delayed type hypersensitivity (DTH) to cardiac myosin. This autoimmunity resembles that which develops in A/J mice immunized with myosin in complete Freund's adjuvant in that myosin-specific antibodies and DTH responses both develop by 21 days postinfection or postimmunization. While the levels of myosin IgG in T. cruzi-infected mice were slightly lower than those in myosin-immunized mice, the magnitude of myosin DTH in the two groups was statistically equivalent. In contrast, C57BL/6 mice, which are resistant to myosin-induced myocarditis and its associated autoimmunity, developed undetectable or low levels of myosin IgG and did not exhibit myosin DTH or myocarditis upon T. cruzi infection. Therefore, humoral and cellular cardiac autoimmunity can develop during acute T. cruzi infection in the genetically susceptible host.

The life cycle of Trypanosoma cruzi revisited.

The basic features of the life cycle of Trypanosoma cruzi have been known for nearly a century. Various aspects of the life cycle, however, have been elucidated only recently, whilst others remain either controversial or unstudied. Here, we present a revised life cycle influenced by recent findings and specific questions that remain unresolved.

Autoimmunity in Chagas heart disease.

The possibility that cardiac autoimmunity contributes to the pathogenesis of Chagas heart disease is controversial. In this paper, we address the following questions regarding the genesis of autoimmunity in Chagas heart disease: (i) What mechanism(s) are potentially responsible for the generation of self-directed antibodies and lymphocytes? (ii) What is the evidence that any of these mechanisms actually can occur? (iii) What are the implications of the presence of autoimmunity for other mechanisms of cardiac inflammation?

Prevention of autoimmune myocarditis through the induction of antigen-specific peripheral immune tolerance.

BACKGROUND: Autoimmunity to cardiac antigens, in particular cardiac myosin, has been observed in humans with myocarditis and in animals with experimental inflammatory heart disease. Current treatments for myocarditis are in many cases immunosuppressive and might lead to increased cardiac damage by reducing host defenses against infectious agents. Therefore, we sought to develop an antigen-specific approach to inhibit autoimmunity in mice with myosin-induced experimental autoimmune myocarditis. METHODS AND RESULTS: Syngeneic splenocytes, coupled with cardiac myosin by use of ethylene carbodiimide, were administered intravenously before disease induction, and the effects of this peripheral tolerization on myosin-induced myocarditis were assessed. This antigen-specific immunotherapy significantly reduced both the incidence and severity of myocarditis, with the prevention of myocyte necrosis, mononuclear cell infiltration, and fibrosis. Myosin-specific delayed-type hypersensitivity and antibody production were significantly reduced, demonstrating that peripheral tolerance affected both T- and B-cell responsiveness to the autoantigen. CONCLUSIONS: These results suggest that the induction of antigen-specific peripheral immune tolerance may be an effective approach for the treatment of myocarditides with autoimmune involvement.

Flagellar elongation induced by glucose limitation is preadaptive for Trypanosoma cruzi differentiation.

Trypanosomes must sense and respond to environmental change in order to progress through their life cycles. The American trypanosome, Trypanosoma cruzi, differentiates from the noninfective epimastigote form to the infective metacyclic form spontaneously in axenic culture. Here, we investigate the initial stimulus for that change and demonstrate that T. cruzi epimastigotes sense limitation of glucose in the medium and respond by undergoing significant morphological and biochemical change. As part of this change, the mean flagellar length of the population triples, which is correlated with an increased ability to maintain interactions with hydrophobic substrates, a requirement for differentiation to the next life cycle stage.

Identification of calcium binding sites in the trypanosome flagellar calcium-acyl switch protein.

The 24 kDa flagellar calcium binding protein (FCaBP) of the protozoan Trypanosoma cruzi is a calcium-acyl switch protein. FCaBP is modified by the addition of myristate and palmitate at its amino terminal segment and both modifications are required for calcium-modulated flagellar membrane association. FCaBP has four sequence motifs for potential calcium binding, and comparison to other calcium-acyl switch proteins, such as recoverin, suggested that only two of these sites are functional. Because it is not possible to predict with certainty the calcium binding affinity or selectivity based on motif analysis alone, we determined the quantitative calcium binding activity of FCaBP by direct ligand binding using the flow dialysis method. The results demonstrated the presence of two calcium binding sites in the full length FCaBP and in a mutant (FCaBPdelta12) lacking the amino terminal pair of sites. FCaBPdelta12 retains its ability to localize to the flagellum. A mutant FCaBP lacking the two carboxyl-terminal sites (FCaBPdelta34), did not bind calcium with high affinity and selectivity under the conditions used. The calcium binding properties of FCaBP are therefore distinct from other myristoyl switch proteins such as recoverin. The results add to a growing body of knowledge about the correlation of sequence motifs with calcium binding activity. Moreover, they demonstrate the need to determine the apparently novel mechanism by which FCaBP undergoes calcium modulated flagellar membrane association and its relation to calcium signal transduction.

Flagellar protein localization mediated by a calcium-myristoyl/palmitoyl switch mechanism.

The mechanisms by which proteins are targeted to flagella and cilia are poorly understood. We set out to determine the basis for the specific localization of a 24 kDa flagellar calcium-binding protein (FCaBP) expressed in all life cycle stages of Trypanosoma cruzi. Through the study of trypanosome transfectants expressing various FCaBP deletion mutants, we found that the N-terminal 24 amino acids of the protein are necessary and sufficient for flagellar localization. Subsequent experiments revealed that FCaBP is myristoylated and palmitoylated and, in fact, is one of very few proteins in the cell possessing these acyl modifications. Both fatty acids are required for flagellar localization, suggesting that FCaBP localization may be mediated through association with the flagellar plasma membrane. Indeed, FCaBP associates with the flagellar membrane in a calcium-dependent manner, reminiscent of the recoverin family of calcium-myristoyl switch proteins. Thus, FCaBP is a novel member of the calcium-acyl switch protein family and is the only member described to date that requires two fatty acid modifications for specific membrane association. Its unique localization mechanism is the first described for any flagellar protein. The existence of such a protein in this protozoan suggests that acylation and calcium switch mechanisms for regulated membrane association are conserved among eukaryotes.

TcDJ1, a putative mitochondrial DnaJ protein in Trypanosoma cruzi.

A full length cDNA encoding a novel Trypanosoma cruzi DnaJ protein was cloned and characterized. The 324 amino acid protein encoded by the cDNA (TcDJ1) displays a characteristics J-domain, but lacks the Gly-Phe and zinc finger regions present in some other DnaJ proteins. Relative to four other T. cruzi DnaJ proteins, TcDJ1 has an amino terminal extension containing basic and hydroxylated resides characteristic of mitochondrial import peptides. A T. cruzi transfectant expressing epitope-tagged TcDJ1 was generated and subcellular fractions were produced. Western blot analysis revealed that the protein has a molecular mass of 29 kDa and is found in the mitochondrial fraction. The expression of TcDJ1 is developmentally regulated since the levels of both mRNA and protein are much higher in epimastigotes (replicative form) than in metacyclic trypomastigotes (infective form). Thus it may participate in mitochondrial biosynthetic processes in this organism.

The DnaJ family of protein chaperones in Trypanosoma cruzi.

We have molecularly cloned four members of the DnaJ (heat shock protein 40) family of protein chaperones of the protozoan parasite Trypanosoma cruzi--tcj1, tcj2, tcj3 and tcj4. While all the proteins contain defining J domains at their N-termini, only tcj2, tcj3 and tcj4 contain glycine/phenylalanine-rich and zinc finger domains common to many other DnaJ homologues. Furthermore, tcj2 and tcj4 contain C-terminal CaaX motifs, substrates for prenyl modifications, suggesting that they are associated with cellular membranes. tcj1 is a divergent member of the family, containing neither glycine/phenylalanine-rich nor zinc finger domains. All the T. cruzi DnaJ genes are single copy, in contrast to other T. cruzi heat shock genes, which are arranged in multicopy direct tandem arrays. Among the tcj mRNAs, only tcj2 is heat inducible, which may result from posttranscriptional regulation involving a sequence found in the 3' untranslated regions of all heat-inducible T. cruzi mRNAs described to date. Further study of this important family of protein chaperones will aid our understanding of the protein folding and assembly processes in protozoans.

Human antibody responses to Trypanosoma cruzi 70-kD heat-shock proteins.

Heat-shock proteins of the 70-kD (hsp70) family are targets of humoral and cellular immune responses following bacterial or parasitic infections, including Chagas' disease. In the present study, we measured antibodies in human sera reactive with hsp70s from the cytoplasm (cy-hsp70), mitochondrion (mt-hsp70), and endoplasmic reticulum (grp78) of Trypanosoma cruzi. Of the three hsp70s tested, only grp78 detected T. cruzi infection in more than 90% of nontreated (NT) patients, with cy-hsp70 and mt-hsp70 detecting only 78% and 25% of NT patients, respectively. Reactivity of leishmanial sera was 77% with cy-hsp70, 13% with grp78, and 5% with mt-hsp70. Therefore, considering sensitivity and specificity, the best candidate for T. cruzi serodiagnosis is grp78. Combination of grp78 with a T. cruzi 24-kD flagellar calcium binding protein (FCaBP) increased the diagnostic sensitivity from 90% to 97% but increased leishmanial reactivity from 3% to 8%. To determine whether hsp70s are useful for discriminating between cured and noncured patients treated with trypanocidal drugs, we tested sera from treated noncured (TNC) patients and cured patients who have positive conventional serology, termed treated dissociated (TD). The cy-hsp70 and grp78 reacted with 74% and 68% of TNC patient sera, respectively, but these antigens did not discriminate TNC from TD patients (52% and 45% positive, respectively). The mt-hsp70 was detected by sera from few TNC patients (18%) and no TD patients. Although individual hsp70s were not useful for determining the effect of trypanocidal drugs on T. cruzi infection in individual patients, the majority of TNC patient sera (70-80%) reacted with two or three of the hsp70s. In contrast, no TD sera reacted with all three hsp70s, and 40% did not react with any of the hsp70s, indicating that the number of hsp70s detected decreases following successful treatment. Considered together, these results show that grp78 has potential as a diagnostic antigen and that absence of reactivity to all three hsp70s may be indicative of effective treatment.

Homologues of the 24-kDa flagellar Ca(2+)-binding protein gene of Trypanosoma cruzi are present in other members of the Trypanosomatidae family.

A full-length cDNA encoding the 24-kDa flagellar Ca(2+)-binding protein (FCaBP) of the Dm28c clone of Trypanosoma cruzi was cloned and characterized. Comparison of the deduced amino acid sequence with those of the FCaBPs of other T. cruzi strains revealed greater than 97% sequence conservation. FCaBP-like genes are found in Trypanosoma conorhini, Trypanosoma freitasi, Trypanosoma lewisi, Herpetomonas megaseliae, Leptomonas seymouri, and Phytomonas serpens, but not in Crithidia deanei, Leishmania amazonensis, or Endotrypanum schaudinni: Among various T. cruzi strains, FCaBP genes are located on chromosomes of different size, although all strains possess multiple FCaBP genes organized as tandemly arranged gene families. Northern and Western blot analyses revealed that FCaBP mRNAs are produced in all organisms possessing FCaBP-hybridizing sequences, indicating that expression of FCaBP or an FCaBP-like protein is common to a number of trypanosomatid species.

Molecular comparison of the mitochondrial and cytoplasmic hsp70 of Trypanosoma cruzi, Trypanosoma brucei and Leishmania major.

We compared the expression and localization of the mitochondrial and cytoplasmic hsp70 of the protozoans Trypanosoma cruzi, Trypanosoma brucei and Leishmania major. The mitochondrial protein is encoded by multiple mRNA in all species, while the cytoplasmic protein is encoded by a single mRNA. In all three species, the mitochondrial hsp70 is concentrated in the kinetoplast, a submitochondrial structure that houses the unusual DNA (kDNA) that characterizes this group of organisms, while the cytoplasmic protein is distributed throughout the cell. These results suggest that, in all kinetoplastid species, mt-hsp70 has a specific function in kDNA biology, possibly in the processes of kDNA replication, RNA editing or kinetoplast structure.

Utility of recombinant flagellar calcium-binding protein for serodiagnosis of Trypanosoma cruzi infection.

The protozoan Trypanosoma cruzi is the causative agent of Chagas' disease, a major public health problem in Latin America and of growing concern in the United States as the number of infected immigrants increases. There is currently no testing of U.S. blood products for T. cruzi infection, and the best tests available, although highly sensitive, are not of high enough specificity to be useful for widespread screening of the blood supply in this country. Among the parasite antigens detected by sera of infected humans and mice, those in the range of 24 to 26 kDa are particularly reactive. With an aim of developing a sensitive, specific, recombinant antigen-based serologic test for T. cruzi infection, we used two antibody reagents specific for these 24- to 26-kDa antigens to isolate cDNA clones from a T. cruzi expression library. One clone was found to encode a previously characterized T. cruzi antigen, a 24-kDa flagellar calcium-binding protein (FCaBP). Recombinant FCaBP was found to be a sensitive, specific reagent for distinguishing T. cruzi-infected individuals from uninfected persons, and it therefore could potentially be used for screening purposes, especially if combined with other recombinant T. cruzi antigens that have similarly high degrees of diagnostic sensitivity and specificity.

Comparison of the 24 kDa flagellar calcium-binding protein cDNA of two strains of Trypanosoma cruzi.

DNA sequences encoding the 24 kDa flagellar calcium binding protein (FCaBP) of two strains of Trypanosoma cruzi were found to differ at fourteen positions, six of which result in amino acid differences. Four of the amino acid differences are located within the calcium-binding domains of FCaBP; however, none is predicted to affect the calcium-binding ability of the protein. Chromosomes harboring the FCaBP gene clusters differ in size among T. cruzi strains.

Expression and localization of Trypanosoma cruzi hsp60.

A 60-kDa heat shock protein (hsp60) is involved in mitochondrial protein folding and assembly of oligomeric protein complexes in the mitochondrial matrix. Here we report the isolation of Trypanosoma cruzi hsp60 cDNAs, the determination of the organization and chromosomal location of the genes, and the assessment of the heat-regulated expression and subcellular location of the protein. T. cruzi hsp60 is encoded by a multigene family organized in two allelic direct tandem arrays on a chromosome of 1.6 Mb. The regulation of hsp60 expression by heat is complex. While the hsp60 mRNA level is 6-fold higher at 37 degrees C than at either 26 degrees C, the hsp60 protein level remains essentially constant across all temperatures examined. Further analysis of the protein by two-dimensional immunoblotting revealed the existence of multiple isoforms that, with increasing temperature, shift in relative abundance from the more basic to the more acidic. A combination of immunofluorescence microscopy and cell fractionation was used to show that hsp60 is distributed throughout the matrix of the mitochondrion--a location distinct from that of the 70-kDa mitochondrial hsp, mtp70, which is associated with the kinetoplast.