A novel calcium-stimulated adenylyl cyclase from Trypanosoma cruzi,which interacts with the structural flagellar protein paraflagellar rod

Trypanosoma cruzi adenylyl cyclases are encoded by a large polymorphic gene family. Although several genes have been identified in this parasite, little is known about the properties and regulation of these enzymes. Here we report the cloning and characterization of TczAC, a novel member of T. cruzi adenylyl cyclase family. The TczAC gene is expressed in all of the parasite life forms and encodes a 1,313-amino acid protein that can complement a Saccharomyces cerevisiae mutant deficient in adenylyl cyclase activity. The recombinant enzyme expressed in yeasts is constitutively active, has a low affinity for ATP (K(m) = 406 microm), and requires a divalent cation for catalysis. TczAC is inhibited by Zn(2+) and the P-site inhibitor 2'-deoxyadenosine 3'-monophosphate, suggesting some level of conservation in the catalytic mechanism with mammalian adenylyl cyclases. It shows a dose-dependent stimulation by Ca(2+) which can be reversed by high concentrations of phenothiazinic calmodulin inhibitors. However, bovine calmodulin fails to stimulate the enzyme. Using a yeast two-hybrid screen it was found that TczAC interacts through its catalytic domain with the paraflagellar rod protein, a component of the flagellar structure. Furthermore, we demonstrate that TczAC can dimerize through the same domain. These results provide novel evidence of the possible localization and regulation of this protein.     

Isolation and characterization of paraflagellar proteins from Trypanosoma cruzi

Two different Trypanosoma cruzi polypeptides, with masses of 70 and 68 kDa were purified and characterized in this work. These two polypeptides designated PAR 1 and PAR 2, respectively, co-purified during each step of the isolation procedure and were found to be located exclusively in T. cruzi flagella by indirect immunofluorescence. A pre-embedding immunoelectron microscopy procedure, with a gold-tagged secondary antibody, permitted direct identification of PAR 2 as a component of the T. cruzi paraflagellar rod. PAR 1 and PAR 2 were found to be immunologically distinct and showed no cross-reactivity with actin, tubulin, intermediate filament proteins, or other proteins present in mammalian cells. The results presented indicate that PAR 1 and PAR 2 are the major components of T. cruzi paraflagellar filaments, and that these filaments have no counterpart in mammalian cells.     

Evidence for two distinct major protein components, PAR 1 and PAR 2, in the paraflagellar rod of Trypanosoma cruzi. Complete nucleotide sequence of PAR.

The previously identified major protein components of the paraflagellar rod in Trypanosoma cruzi, PAR 1 and PAR 2, were analyzed to determine if they are distinct proteins or different conformations of a single polypeptide as has been suggested for other trypanosomatids. Amino acid sequence analysis showed PAR 1 and PAR 2 to be two distinct polypeptides. Antibodies specific against either PAR 1 or PAR 2 were shown to each react with a distinct band in Western blots of paraflagellar isolates of T. cruzi and other trypanosomatids if rigorous protease inhibition was used. The PAR 2 message was isolated and characterized by Northern blot and nucleic acid sequence analysis. Preliminary analysis of the PAR 2 gene indicates that PAR 2 is a member of a multigene family with all members residing on a single chromosome.     

Trypanosoma cruzi: a stage-specific calpain-like protein is induced after various kinds of stress

Calpains are calcium-dependent cysteine proteinases found in all living organisms and are involved in diverse cellular processes. Calpain-like proteins have been reported after in silico analysis of the Tritryps genome and are believed to play important roles in cell functions of trypanosomatids. We describe the characterization of a member of this family, which is differentially expressed during the life-cycle of Trypanosoma cruzi.     

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 characteristic 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 residues 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.     

TcRho1 of Trypanosoma cruzi: role in metacyclogenesis and cellular localization

Here we have investigated the function of TcRho1, a Rho family orthologue from the parasite Trypanosoma cruzi. We have selected parasites overexpressing wild-type TcRho1 and a truncated form of TcRho1 (TcRho1-DeltaCaaX) which is unable to undergo farnesylation and supposed to interfere with recruitment of Rho effectors to membranes. TcRho1 protein was localized at the anterior region of wild-type and TcRho1 overexpressing epimastigotes, suggesting association with the Golgi apparatus. Accordingly, parasites overexpressing TcRho1-DeltaCaaX presented cytoplasmic fluorescence. To address the function of TcRho1 during differentiation, from epimastigotes to trypomastigotes, we submitted parasites overexpressing the above-cited lineages to metacyclogenesis assays. Parasites overexpressing TcRho1-DeltaCaaX generated a discrete number of metacyclic trypomastigotes when compared with other lineages. Strikingly, TcRho1-DeltaCaaX cells died synchronously during the process of metacyclogenesis.     

Molecular determinants of ciliary membrane localization of Trypanosoma cruzi flagellar calcium-binding protein

The flagellar calcium-binding protein (FCaBP) of Trypanosoma cruzi is localized to the flagellar membrane in all life cycle stages of the parasite. Myristoylation and palmitoylation of the N terminus of FCaBP are necessary for flagellar membrane targeting. Not all dually acylated proteins in T. cruzi are flagellar, however. Other determinants of FCaBP therefore likely contribute to flagellar specificity. We generated T. cruzi transfectants expressing the N-terminal 24 or 12 amino acids of FCaBP fused to GFP. Analysis of these mutants revealed that although amino acids 1-12 are sufficient for dual acylation and membrane binding, amino acids 13-24 are required for flagellar specificity and lipid raft association. Mutagenesis of several conserved lysine residues in the latter peptide demonstrated that these residues are essential for flagellar targeting and lipid raft association. Finally, FCaBP was expressed in the protozoan Leishmania amazonensis, which lacks FCaBP. The flagellar localization and membrane association of FCaBP in L. amazonensis suggest that the mechanisms for flagellar targeting, including a specific palmitoyl acyltransferase, are conserved in this organism.     

Clathrin in Trypanosoma cruzi: in silico gene identification, isolation, and localization of protein expression sites

Clathrin is a scaffold protein found in different types of coated vesicles in most eukaryotic cells. Major forces that drive clathrin coat formation are the adaptor protein complexes. Trypanosoma cruzi is a flagellate protozoan that ingests macromolecules through receptor-mediated endocytosis, but the molecules involved in this process are still poorly known. Bioinformatics was used to identify proteins in the T. cruzi genome database, permitting discrimination of the genes involved in clathrin coat assembly. Clathrin expression was demonstrated in T. cruzi epimastigotes by using several experimental approaches. Western blot analysis showed a single 180-kDa protein band, which corresponds to the molecular mass of mammalian clathrin heavy chain. A flow cytometry assay demonstrated that the clathrin heavy chain was expressed in 97.74% of the cell population analyzed, with a high-fluorescence signal. Immunofluorescence observation showed labeling clustered at the flagellar pocket and Golgi complex region. Coated vesicles budding off from the flagellar pocket and the trans Golgi network membranes were identified by transmission electron microscopy. Our data demonstrate the expression of clathrin in T. cruzi epimastigotes and show the association of this polypeptide with the parasite endocytic and exocytic pathways.     

Flagellar morphogenesis: protein targeting and assembly in the paraflagellar rod of trypanosomes

The paraflagellar rod (PFR) of the African trypanosome Trypanosoma brucei represents an excellent model to study flagellum assembly. The PFR is an intraflagellar structure present alongside the axoneme and is composed of two major proteins, PFRA and PFRC. By inducible expression of a functional epitope-tagged PFRA protein, we have been able to monitor PFR assembly in vivo. As T. brucei cells progress through their cell cycle, they possess both an old and a new flagellum. The induction of expression of tagged PFRA in trypanosomes growing a new flagellum provided an excellent marker of newly synthesized subunits. This procedure showed two different sites of addition: a major, polar site at the distal tip of the flagellum and a minor, nonpolar site along the length of the partially assembled PFR. Moreover, we have observed turnover of epitope-tagged PFRA in old flagella that takes place throughout the length of the PFR structure. Expression of truncated PFRA mutant proteins identified a sequence necessary for flagellum localization by import or binding. This sequence was not sufficient to confer full flagellum localization to a green fluorescent protein reporter. A second sequence, necessary for the addition of PFRA protein to the distal tip, was also identified. In the absence of this sequence, the mutant PFRA proteins were localized both in the cytosol and in the flagellum where they could still be added along the length of the PFR. This seven-amino-acid sequence is conserved in all PFRA and PFRC proteins and shows homology to a sequence in the flagellar dynein heavy chain of Chlamydomonas reinhardtii.     

Ultrastructural localization of basic proteins in Trypanosoma cruzi.

The postformalin ammoniacal silver (AS) and the ethanolic phosphotungstic acid (EPTA) techniques were applied in epimastigote and trypomastigote forms of the pathogenic protozoa Trypanosoma cruzi to detect basic proteins at the ultrastructural level. With both techniques, reaction was observed in the nucleus and in some cytoplasmic vacuoles. In the kinetoplast of epimastigotes, reaction was observed only at its periphery. In trypomastigotes, however, an intense reaction was observed in the spherical kinetoplast. With the ethanolic phosphotungstic acid technique, reaction was also observed in ribosomes and at the peripheral doublet microtubules of the flagellum. The filaments which form the paraflagellar structure did not react.     

The major component of the paraflagellar rod of Trypanosoma brucei is a helical protein that is encoded by two identical,tandemly linked genes

The flagellum of the parasitic hemoflagellate Trypanosoma brucei contains two major structures: (a) the microtubule axoneme, and (b) a highly ordered, filamentous array, the paraflagellar rod (PFR). This is a complex, three-dimensional structure, of yet unknown function, that extends along most of the axoneme and is closely linked to it. Its major structural component is a single protein of 600 amino acids. This PFR protein can assume two different conformations, resulting in two distinct bands of apparent molecular masses of 73 and 69 kD in SDS-gel electrophoresis. Secondary structure predictions indicate a very high helix content. Despite its biochemical similarity to the intermediate filament proteins (solubility properties, amino acid composition, and high degree of helicity), the PFR protein does not belong in this class of cytoskeletal proteins. The PFR protein is coded for by two tandemly linked genes of identical nucleotide sequence. Both genes are transcribed into stable mRNAs of very similar length that carry the mini-exon sequence at their 5'' termini.     

Trypanosoma cruzi glycoprotein 72: Immunological analysis and cellular localization

Two monoclonal antibodies were used to biochemically characterize glycoprotein 72 (GP72) from Trypanosoma cruzi and to localize the protein in live and fixed parasites by indirect immunofluorescence and in thin section of parasites by immunogold electron microscopy. GP72 was shown in immunoblots to be specific for the epimastigote stage; the protein could not be detected in trypomastigotes. Each antibody reacted with a different epitope on the glycoprotein and deglycosylation of GP72 ablated reactivity with one of the antibodies. Indirect immunofluorescence and electron microscopic evaluation of parasite associated gold particles showed the presence of GP72 in the cell surface membrane including the flagellar pocket and the cytostome. In addition, cytoplasmic membrane vesicles of the endosomal-lysosomal system stained intensely.     

The cAMP receptor protein of Trypanosoma cruzi

The widest used method to determine cAMP-binding activity in cell-free extracts (Gilman, A. G. (1970) Proc. Natl. Acad. Sci. U.S.A. 67, 305-312) underestimated by about 10-fold the total amount of [3H]cAMP bound by crude extracts of cultured forms of Trypanosoma cruzi (epimastigotes), when compared with the results obtained by the modified Millipore filter technique described by Doskeland and Ueland (Doskeland, S. O., and Ueland, P.M. (1977) Biochem. J. 165, 561-573). After column chromatography on DEAE-Sephacel, the cAMP-binding activity eluted as a single symmetrical peak at about 210 mM NaCl, totally separated from two peaks of cAMP-independent phosphotransferase activities which eluted, respectively, at 90 and 270 mM NaCl. These two protein kinases showed similar specificities for exogenous substrates, preferring in this order: protamine greater than casein greater than histone H2b. Photoaffinity labeling of cell-free extracts with 8-azido[32P]adenosine 3':5'-monophosphate and autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis specifically labeled only one protein of Mr = 62,000. From these results it is concluded that T. cruzi epimastigotes possess one single cAMP-binding protein of monomeric Mr = 62,000, not associated with a phosphotransferase activity and probably very different in nature to known regulatory subunits of protein kinases, given the results obtained with the Millipore filtration technique.     

Trypanosoma cruzi and American Leishmania spp: immunocytochemical localization of a laminin-like protein in the plasma membrane

Patients with Chagas' disease or different clinical forms of American cutaneous leishmaniasis have high antilaminin antibody levels. An immunogold technique employing a specific antilaminin antibody was used in the present study to determine the presence, and define the ultrastructural localization, of laminin-like molecule(s) in American Leishmania spp. and Trypanosoma cruzi. Laminin was found located specifically in T. cruzi trypomastigotes on the external surface of the plasma membrane, close to the sites where the flagellar veil attaches to the plasma membrane. Laminin immunoreactivity was rapidly lost when trypomastigotes were cultured in liquid medium and no reactivity was found in fresh epimastigotes. Promastigotes and amastigotes of American Leishmania spp. also showed a specific localization of laminin immunoreactivity, this being limited to the lips of the flagellar pocket and to the parasitic side exactly opposite to the flagellar exit. These results confirm the presence of a laminin-like molecule(s) in both trypanosomatids, the specific localization suggesting a presently unknown function for this protein.