RESUMO
Particulate preparations from epimastigote forms of Trypanosoma cruzi contain an adenylyl cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) which could be stored at --20 degree C and resisted 5 cycles of freezing and thawing over 10 days without significant loss of activity. The enzyme reaction strictly required Mn2+, had a pH optimum of 7.7 and was not inhibited or stimulated by NaF. Particles prepared in the presence of 10 mM Mn2+ or Mg2+ were 3--4 times more active than particles prepared in the absence of these cations. However, Mg2+ could not substitute for Mn2+ during enzyme assay nor did it enhance activity in the presence of saturating concentrations of Mn2+. The binary complex Mn - ATP2- was shown to be the true substrate for the adenylyl cyclase and free ATP was highly inhibitory. Plots of enzyme activity against equimolar concentrations of ATP - Mn gave sigmoid curves with n values in Hill plots ranging between 1.5 and 2.0. Excess Mn2+ activated the cyclase catalyzed reaction at low but not at high concentrations of ATP - Mn. In the presence of an excess of 1 mM Mn2+, which transforms 97% of the added ATP to productive Mn - ATP2- complex, the substrate saturation curve assumed a Michaelian pattern with an apparent Km =0.2 mM.
Assuntos
Adenilil Ciclases/metabolismo , Trypanosoma cruzi/enzimologia , Trifosfato de Adenosina/farmacologia , Animais , Cátions Bivalentes , Ativação Enzimática/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Cinética , Magnésio/farmacologia , Manganês/farmacologiaRESUMO
Membrane vesicles can be obtained from epimastigote forms of Trypansoma cruzi by incubating cells with either cross-linking reagents or acid pH. Acetate, phtalate or citrate, at pH 4.0, but not at higher pH values, were able to induce plasma membrane vesiculation. Vesicles have been purified by sucrose density centrifugation and their membrane origin was demonstrated by the following criteria: (a) Vesicles are 5--10 times richer in protein-bound iodine when they are prepared from cells previously labeled with 131I by the lactoperoxidase catalyzed reaction. (b) Electron microscopy of vesiculating cells shows physical continuity between cell plasma membrane and vesicle membrane. (c) Antibodies prepared against purified vesicles are able to agglutinate epimastigote forms of T. cruzi with sera dilutions up to 1 : 256 to 1 : 512. (d) Freeze-fracture studies of the purified vesicles have shown images of faces P and E compatible with known images of the intact cell plasma membrane. Typical preparations of acetate vesicles present the following characteristics: total carbohydrate : protein=1.5--2.0; orcinol : protein-0.07 and absence of diphenylamine reaction. Vesicles contain 0.2--0.5% and 0.3--1.0% of the total homogenate protein and carbohydrate, respectively. The presence of 10 major protein bands and 30--50-fold enrichment of the four sugar-containing macromolecules present in epimastigote forms of T. cruzi have been demonstrated in these preparations.
Assuntos
Membrana Celular/ultraestrutura , Trypanosoma cruzi/ultraestrutura , Animais , Fracionamento Celular/métodos , Membrana Celular/metabolismo , Glicoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismoRESUMO
Diagnosis of individuals infected by Trypanosoma cruzi is performed mainly by serological tests using crude antigens, which might crossreact with other infections. In the past ten years, many recombinant T. cruzi proteins and synthetic peptides have been described, and some are already on the market. Managers of laboratories and blood banks need to make decisions on a cost-benefit basis whether to include these new-generation tests. Here, we indicate antigens that are likely to prove most useful.
Assuntos
Antígenos de Protozoários/imunologia , Doença de Chagas/diagnóstico , Trypanosoma cruzi/imunologia , Animais , Bancos de Sangue , Análise Custo-Benefício , Ensaio de Imunoadsorção Enzimática , Humanos , Estudos Multicêntricos como Assunto , Kit de Reagentes para Diagnóstico/economia , Proteínas Recombinantes/imunologia , Testes Sorológicos/métodos , Organização Mundial da SaúdeRESUMO
Intact RNAs were isolated from Plasmodium chabaudi growing synchronously in mice at different stages of the erythrocytic cycle. Translation of the mRNAs using rabbit reticulocyte lysate showed stage-specific patterns comparable to those observed in vivo. Many antigens of P. chabaudi are produced by translation in the rabbit reticulocyte lysate. Some other antigens are predominantly synthesized at a particular stage, indicating that the mRNA populations differ from one stage to another.
Assuntos
Antígenos/biossíntese , Plasmodium/imunologia , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA/isolamento & purificação , Animais , Sistema Livre de Células , Feminino , Código Genético , Camundongos , RNA/genética , RNA Ribossômico/isolamento & purificação , Coelhos , Reticulócitos/análiseRESUMO
We have cloned and sequenced a cDNA clone coding for a metacyclic trypomastigote-specific surface glycoprotein with a molecular mass of 82 kDa (MTS-gp82). By immunoblotting and immunoprecipitation, antibodies against the recombinant protein recognized an 82-kDa protein of metacyclic trypomastigotes, without any detectable reaction towards amastigotes, epimastigotes or tissue culture-derived trypomastigotes. The insert of the MTS-gp82 cDNA clone strongly hybridized with a single 2.2-kb metacyclic trypomastigote mRNA, suggesting that the steady-state levels of mRNAs for MTS-gp82 are developmentally regulated. MTS-gp82 is encoded by a multigene family whose members are distributed in several chromosomes. Sequence analysis revealed 40-56% identity at amino acid level between MTS-gp82 and members of Trypanosoma cruzi gp85/sialidase family (TSA-1, Tt34c1, SA85-1.1). MTS-gp82 showed several amino acid motifs that are characteristic of gp85/sialidase family, such as the Asp box (SxDxGxTW), the subterminal (VTVxNVFLYNR) motif and the putative GPI-anchor sequence. On the basis of its structural features, the MTS-gp82 gene could be included in the T. cruzi gp85/sialidase family, but constituting a distinct group which is preferentially expressed in metacyclic trypomastigotes.
Assuntos
Antígenos de Protozoários/genética , Antígenos de Superfície/genética , Genes de Protozoários , Trypanosoma cruzi/genética , Trypanosoma cruzi/imunologia , Glicoproteínas Variantes de Superfície de Trypanosoma , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/química , Antígenos de Superfície/química , Clonagem Molecular , DNA Complementar/genética , DNA de Protozoário/genética , Glicoproteínas/genética , Dados de Sequência Molecular , Peso Molecular , Família Multigênica , Neuraminidase/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Homologia de Sequência de Aminoácidos , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
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.
Assuntos
Telômero/genética , Trypanosoma cruzi/genética , Animais , Sequência de Bases , Clonagem Molecular , Cosmídeos/genética , DNA de Protozoário/genética , Eletroforese em Gel Bidimensional , Vetores Genéticos , Dados de Sequência Molecular , Neuraminidase/genética , Proteínas Recombinantes , Mapeamento por Restrição , Retroelementos/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/genéticaRESUMO
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.
Assuntos
Sequências Repetitivas de Ácido Nucleico , Trypanosoma cruzi/genética , Animais , Sequência de Bases , DNA de Protozoário/genética , DNA Ribossômico/genética , Genoma , Dados de Sequência Molecular , Mapeamento por Restrição , Especificidade da Espécie , Trypanosoma/genéticaRESUMO
DNA probes from the nontranscribed ribosomal spacer (NTS), of Leishmania garnhami and Leishmania braziliensis were constructed and tested for sensitivity and specificity against different Leishmania isolates. The L. garnhami probes were species-specific under hybridization conditions of high stringency, but displayed specificity for the mexicana complex under conditions of intermediate stringency. The L. braziliensis probes showed 'complex' specificity. RFLP for the nontranscribed spacer within the braziliensis complex revealed very homogeneous patterns even for organisms currently accepted as different species. A PCR assay for the detection of Leishmania from the braziliensis complex is presented.
Assuntos
DNA de Protozoário/genética , DNA Ribossômico/genética , Leishmania/genética , Animais , Sequência de Bases , Clonagem Molecular , Sondas de DNA , Leishmania/classificação , Leishmania/patogenicidade , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Mapeamento por Restrição , Especificidade da EspécieRESUMO
A Trypanosoma cruzi genomic expression library was screened with a pool of sera obtained from chronic chagasic patients. The recombinant antigen (Tc40) isolated from this library reacted with a large number of serum samples of chronic chagasic patients, suggesting that the presence of anti-Tc40 antibodies may be specifically associated to Chagas' disease. The full-length sequence of the Tc40 gene was determined after isolation of genomic and cDNA clones. The Tc40 cDNA includes a large open reading frame (2745 bp-long) that encodes a polypeptide of 100 kDa without any homology with previously described T. cruzi sequences. In contrast with other T. cruzi antigens whose immunodominant B-cell epitopes are composed by amino acid repetitive motifs, Tc40 does not show any amino acid repetition. Antibodies against the Tc40 recombinant protein reacted with three native polypeptides of 100, 41 and 38 kDa which are tightly associated with membranes or cytoskeleton and expressed in all developmental stages of the parasite life cycle. A transcript of 3.9-kb was detected in Northern blot analysis which is large enough to encode a 100 kDa polypeptide. Tc40 genes were mapped on a chromosomal band of 1.1 Mbp and in a few copies per haploid genome in the G strain.
Assuntos
Antígenos de Protozoários/genética , Genes de Protozoários , Trypanosoma cruzi/genética , Trypanosoma cruzi/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/sangue , Sequência de Bases , Doença de Chagas/imunologia , Clonagem Molecular , Primers do DNA/genética , DNA Complementar/genética , DNA de Protozoário/genética , Humanos , Epitopos Imunodominantes/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
Recombinant proteins and synthetic peptides representing various sequences of gp82, a surface glycoprotein of Trypanosoma cruzi metacyclic trypomastigotes implicated in mammalian cell invasion, were used in this study aiming at the identification of the domain(s) of this molecule required for interaction with target cells. Invasion of cultured HeLa cells by metacyclic trypomastigotes was inhibited by about 80% in the presence of native gp82 or the corresponding recombinant construct J18. Inhibition by recombinant proteins J18a and J18b, containing respectively the N-terminal and the C-terminal portions of gp82, was on the order of 30% and 65%. As compared to J18b (amino acids 224-516), the truncated gp82 fragments J18b1 (amino acids 303-516) and J18b2 (amino acids 357-516) displayed lower inhibitory effect (approximately 40% and approximately 15%, respectively). Compatible with these observations, we found that the recombinant protein J18b, but not J18a or J18b2, binds to HeLa cells in a dose-dependent and saturable fashion. Experiments with ten overlapping synthetic peptides, representing the gp82 portion spanning amino acids 224-333, showed that peptides 4 (amino acids 254-273) and 8 (amino acids 294-313) have significant inhibitory activity on HeLa cell invasion by metacyclic forms. All these results indicate that the portion of gp82 required for mammalian cell attachment and invasion is located in the central domain of the molecule.
Assuntos
Glicoproteínas de Membrana/fisiologia , Fosfoproteínas/fisiologia , Proteínas de Protozoários/fisiologia , Trypanosoma cruzi/fisiologia , Trypanosoma cruzi/patogenicidade , Sequência de Aminoácidos , Animais , Sítios de Ligação , Adesão Celular/fisiologia , Células HeLa , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Estrutura Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/fisiologia , Fosfoproteínas/química , Fosfoproteínas/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Trypanosoma cruzi/genéticaRESUMO
We have studied the genomic organization and expression of the gene encoding a high molecular mass (300 kDa) repetitive antigen associated with the cytoskeleton of Trypanosoma cruzi. Protease digestion of the native protein, restriction analysis of genomic DNA and sequencing of genomic and cDNA clones indicated that most of the protein is built up by tandemly arranged, nearly identical repeats of 68 amino acids. The gene size was estimated to be approx. 9.4 kb based on the sizes of the transcript and the native protein. The nucleotide sequence conservation among the repeats indicates that selective sequence homogenization, presumably through gene conversion, maintained the amino-acid sequence conservation. Two duplicated allelic forms of this gene were mapped in fragments of about 20 kb. In some strains an additional allele was located in a fragment of 9.4 kb. Our results suggest that this repetitive antigen is a structural protein which could be involved in the attachment of the flagellum to the cell body.
Assuntos
Antígenos de Protozoários/genética , Citoesqueleto/química , Genes de Protozoários/genética , Proteínas de Protozoários/genética , Trypanosoma cruzi/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Sequência Conservada/genética , Proteínas do Citoesqueleto/genética , Regulação da Expressão Gênica , Dados de Sequência Molecular , Polimorfismo de Fragmento de Restrição , Proteínas de Protozoários/análise , RNA Mensageiro/análise , RNA de Protozoário/análise , Sequências Repetitivas de Ácido Nucleico/genética , Mapeamento por Restrição , Análise de Sequência de DNA , Transcrição Gênica , Trypanosoma cruzi/imunologiaRESUMO
The JL8 protein antigen from Trypanosoma cruzi, a dominant immunogen in man, has been characterized as containing tandem amino acid repeats. Here, we describe the use of the LamB protein of Escherichia coli as a carrier of JL8 derived sequences in order to map the immunodominant B cell epitopes in this antigen. Five different sequences of JL8 were inserted in the LamB protein and the JL8-LamB fusion proteins were tested by ELISA with human chronic chagasic sera. The fusion carrying the sequence AEKQKAAEATKVAE was recognized by most sera. This protein was also capable of inhibiting the binding of human chagasic antibodies to GST-JL8 in competitive ELISA suggesting that it contains an immunodominant B cell epitope of JL8.
Assuntos
Antígenos de Protozoários/imunologia , Linfócitos B/imunologia , Proteínas de Bactérias/imunologia , Epitopos/análise , Proteínas de Escherichia coli , Sinais Direcionadores de Proteínas , Proteínas Recombinantes de Fusão/imunologia , Trypanosoma cruzi/imunologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Escherichia coli , Humanos , Immunoblotting , Epitopos Imunodominantes/imunologia , Epitopos Imunodominantes/isolamento & purificação , CoelhosRESUMO
Clone CL Brener is the reference organism used in the Trypanosoma cruzi Genome Project. CL Brener was obtained by cloning procedures from bloodstream trypomastigotes isolated from mice infected with the CL strain. The doubling time of CL Brener epimastigotes cultured at 28 degrees C in liver infusion-tryptose (LIT) medium is 58 +/- 13 h. Differentiation to metacyclic forms is induced by incubation of epimastigotes in LIT-20% Grace's medium. Metacyclics give very low parasitemia in mice, contrary to what is observed for blood forms which promote 100% mortality of the animals with inocula of 5 x 10(3) parasites. CL Brener blood forms are highly susceptible to nifurtimox, benznidazole and ketoconazole. Allopurinol is inefficient in the treatment of mice experimental infection. The clone infects mammalian cultured cells and performs the complete intracellular cycle at 33 and 37 degrees C. The molecular typing of CL Brener has been done by isoenzymatic profiles; sequencing of a 24S alpha ribosomal RNA gene domain and by schizodeme, randomly amplified polymorphic DNA and DNA fingerprinting analyses. For each typing approach the patterns obtained do not change after prolonged parasite subcultivation in LIT medium (up to 100 generations). The stability of the molecular karyotype of the clone was also confirmed.
Assuntos
Genoma de Protozoário , Trypanosoma cruzi/genética , Animais , DNA de Protozoário/análise , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Trypanosoma cruzi/classificação , Trypanosoma cruzi/efeitos dos fármacosRESUMO
The main surface glycoproteins of metacyclic trypomastigotes of Trypanosoma cruzi, gp90, gp82, and gp35/50, were purified and the immune response elicited by these antigens was analyzed. Balb/c mice immunized with antibody-affinity-purified gp82, plus alum as adjuvant, produced antibodies that recognized both the gp82 and the heterologous gp90 and gp35/50. On the other hand, antisera to gp90 reacted only with the homologous antigen, either by immunoprecipitation or by immunoblotting. Neither sera reacted with unrelated proteins in ELISA. Both antisera lysed 90-100% metacyclic forms in a complement-mediated reaction, a property associated with protection. However, in contrast to gp90, previously shown to induce protective immunity against acute T. cruzi infection, gp82 was not immunoprotective. Lymph node (LN) cells of mice primed with gp82 or gp90, which display 40% amino acid sequence identity at the carboxy terminal domain, were strongly stimulated in vitro by either one of these antigens. Proliferation, inhibitable by anti-CD4 but not by anti-CD8 antibodies, was T. cruzi-specific, no activation being observed with irrelevant antigens. LN cells of mice immunized with unrelated proteins did not proliferate in vitro in the presence of gp82 or gp90. The 35/50-kDa glycoconjugate, which was phenol-extracted, did not elicit any detectable antibody or T cell response.
Assuntos
Anticorpos Antiprotozoários/biossíntese , Doença de Chagas/imunologia , Linfócitos T/imunologia , Trypanosoma cruzi/imunologia , Glicoproteínas Variantes de Superfície de Trypanosoma/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/imunologia , Soros Imunes , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Trypanosoma cruzi/crescimento & desenvolvimento , Glicoproteínas Variantes de Superfície de Trypanosoma/químicaRESUMO
We have isolated a clone of Trypanosoma cruzi genomic DNA, lambda 3b2-5, which contains sequences that are reiterated in the genome. Northern blot analysis showed that clone 3b2-5 hybridizes to 1,200-5,000 bases different mRNA species. The number of mRNAs species hybridized to clone 3b2-5 exceeds its coding capacity showing that this clone carries sequences that are common to several mRNAs species and conserved in the poly A(+) RNA. These sequences are not homologous to the T. cruzi spliced leader sequence, since clone 3b2-5 does not hybridize to a synthetic 20 nucleotide complementary to the spliced leader sequence. Clone 3b2-5 does not hybridize to DNA and RNA from several genera of Trypanosomatidae and other Trypanosoma species indicating that it carries T. cruzi species-specific sequences.
Assuntos
DNA/genética , RNA Mensageiro/genética , Sequências Repetitivas de Ácido Nucleico , Trypanosoma cruzi/genética , Animais , Northern Blotting , Southern Blotting , Biblioteca Genômica , Família Multigênica , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
Since the start of the human genome project, a great number of genome projects on other "model" organism have been initiated, some of them already completed. Several initiatives have also been started on parasite genomes, mainly through support from WHO/TDR, involving North-South and South-South collaborations, and great hopes are vested in that these initiatives will lead to new tools for disease control and prevention, as well as to the establishment of genomic research technology in developing countries. The Trypanosoma cruzi genome project, using the clone CL-Brener as starting point, has made considerable progress through the concerted action of more than 20 laboratories, most of them in the South. A brief overview of the current state of the project is given.
Assuntos
Mapeamento Cromossômico , Genoma de Protozoário , Trypanosoma cruzi/genética , AnimaisRESUMO
The protective immune response to asexual blood infection by Plasmodium chabaudi was studied in mice immunized either by drug controlled infection or by vaccination with preparations of merozoïtes or free parasites at different stages of development. Animals immunized by the first method developed a sterile immunity. The passive transfer of their serum protected naïve recipients from the lethal development of the infection, but affected only moderately the initial course of the parasitaemia. Animals immunized with either ring, schizont or merozoïte preparations exhibited a limited but significant resistance to infection: when challenged with 10(6) parasites of the homologous strain they exhibited a reduced parasitaemia as compared to control mice, and in addition, 50% of them recovered from the infection. Immunochemical analysis of parasite antigens showed that a family of high molecular weight proteins synthesized essentially at the schizont stage and conserved in the merozoites are important immunogens. Quantitative rather than qualitative differences were observed in the pattern of parasite proteins immunoprecipitated by serum of animals exhibiting sterile immunity or moderate protective immunity. A schizont specific polypeptide of mol. wt 82 Kd which is found in the surface of the merozoite is preferentially immunoprecipited by serum from animals exhibiting sterile immunity.
Assuntos
Antígenos de Superfície/análise , Malária/imunologia , Animais , Eletroforese em Gel de Poliacrilamida , Feminino , Imunidade Ativa , Radioisótopos do Iodo , Proteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos , Plasmodium/isolamento & purificação , Especificidade da Espécie , Radioisótopos de Enxofre , VacinaçãoRESUMO
Peptides derived from the surface glycoprotein gp82 of Trypanosoma cruzi, previously implicated in the parasite's invasion of host cells, were expressed as fusions to the protein LamB of Escherichia coli in a region known to be exposed on the cell surface. Bacteria expressing these proteins adhered to HeLa cells in a manner that mimics the pattern of parasite invasion of mammalian cells. Purified LamB fusion proteins were shown to bind to HeLa cells and to inhibit infection by T. cruzi, supporting the notion that these gp82-derived peptides can mediate interaction of the parasite with its host.
Assuntos
Aderência Bacteriana/fisiologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Escherichia coli/fisiologia , Glicoproteínas de Membrana/metabolismo , Proteínas de Protozoários/metabolismo , Receptores Virais/metabolismo , Trypanosoma cruzi , Sequência de Aminoácidos , Animais , Proteínas da Membrana Bacteriana Externa/genética , Expressão Gênica , Células HeLa , Humanos , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Peptídeos/metabolismo , Porinas , Proteínas de Protozoários/genética , Receptores Virais/genética , Proteínas Recombinantes de Fusão/genética , Trypanosoma cruzi/patogenicidadeRESUMO
This study provides several pieces of evidence indicating that 3F6-Ag, identified by monoclonal antibody (MAb) 3F6 as a stage-specific glycoprotein of approximately 82 kDa on the surface of metacyclic trypomastigotes of different Trypanosoma cruzi strains, promotes the entry of parasites into host cells through a ligand-receptor type interaction. First, invasion of Vero cells by metacyclic trypomastigotes of both CL and Tulahuen strains was significantly inhibited by MAb 3F6 or its Fab fragments. Second, purified 3F6-Ag bound to Vero cells in a dose-dependent and saturable fashion. Third, soluble 3F6-Ag reduced the infection of Vero cells by metacyclic forms of CL and Tulahuen strains by 90 to 97 and 50%, respectively. Unrelated proteins, as well as extracellular matrix components, such as heparan sulfate and collagen, had no effect. Our studies also show that in the Tulahuen strain, 10D8-Ag, a 35/50-kDa glycoprotein identified by MAb 10D8, participates in target cell invasion, confirming previous observations, but the variant form of 10D8-Ag expressed by highly invasive CL strain metacyclic trypomastigotes appears to be irrelevant. Overall, our results indicate that the surface components of T. cruzi metacyclic trypomastigotes involved in the process of host cell penetration are developmentally regulated molecules, such as 3F6-Ag and 10D8-Ag, that have no counterpart in blood- or tissue culture-derived trypomastigotes.