Homeostasis intracelular del calcio: la calmodulina y la Ca2+ -ATPasa de la membrana plasmática de tripanosomatidios / Intracellular calcuim homeostasis calmodulin and the plasma menihane Ca2+ ATPase of trypanosomatids

La concentración citoplasmática de calcio iónico en todas las células eucarióticas estudiadas hasta el presente es de 4 órdenes de magnitud menor que la del exterior celular. En tripanosomatidios se ha determinado que la concentración intracelular de este cation es de alrededor de 50 mm, aun menor que la reportada en eucariotes superiores. Esta diferencia de concentración es mantenida mediante diversos sistemas de transporte ubicados a nivel de la membrana plasmática y en ciertos organelos intracelulares. En el caso de los tripanosomatidios, se ha identificado la presencia de un uniporte electroforético en la membrana interna de la mitocondria única gigante de estos parásitos, la cual presenta propiedades cinéticas esencialmente idénticas a las reportadas en aucariotes superiores. Este sistema presenta una afinidad por el Ca2+ incompatible con la posibilidad de mantener la concentración de este cation a nivel submicromolar. Por otra parte, contrario a lo reportado por otros autores, hemos identificado la presencia de una Ca2+ -ATPasa en la membrana plásmatica de Leishmania braziliensis, Leishmania mexicana, Trypanosoma cruzi y Tripanosona brucei. La enzima presenta alta afinidad por Ca2+ (Kmap Ca2+ = 0.5µM), es dependiente de Mg2+, y es estimulante por la calmodulina purificada de los mismos hemoflagelados. Esta Ca2+ -ATPasa es sensible al vanadato (Ki=1µM), lo cual permite identificarla como bomba iónica del tipo "P". Vesículas provenientes la membrana plasmática de estos parásitos son capaces de acumular Ca2+ en contra de un gradiente de concentración. Las características cinéticas de este transporte son similares a las de la Ca2+ -ATPasa, lo cual apoya que se trata de la misma entidad molecular. Los resultados obtenidos permiten postular que la Ca2+ -ATPasa es la estructura responsable del mantenimiento de la concentración intracelular de Ca2+ a nivel submicromolar a largo en estos parásitos

Trypanosoma rangeli (Tejera, 1920): observations upon pleomorphism

Metatrypomastigotes of Trypanosoma rangeli Tejera, 1920, harvested from LIT medium, were inoculated i.p. or s.c. into 6, 16, and 26g NMRI mice, these representing increasing degrees of immunological maturity. In all cases, similar pleomorphic patterns were observed. Four morphobiometrically differentiable types of trypanosome were encountered in an overlapping temporal sequence. These observations, taken in comparison with those on pleomorphism in this and other species of Trypanosoma by other workers, are consistent with the hypothesis that the pleomorphic types represent the natural development of the parasite, rather than the result of the immune response of the mammal host. Small, slender trypanosomes prevalent at the onset of the parasitemia either reinvade the tissue cells for relatively limited subsequent generations of tissue reproduction, or else differentiate toward the forms that are only capable of colonizing the insect vector

Estudios sobre Trypanosoma rangeli Tejera, 1920 XI-ensayo de protección en ratas "Wistar" y proechymis sp. / Studies on Trypanosoma rangeli Tejera, 1920 XI-protection trials in \"Wistar\" rats and proechymis sp

El seguimiento sistemático del curso de la infección primaria y la respuesta a sucesivas reinfecciones por Trypanosoma rangeli en ratas "Wistar" y Proechymis sp., reveló un comportamiento similar del parásito en ambos modelos vertebrados, observándose durante la primo-infección una parasitemia relativamente baja y de corta duración en todos los animales. Se observa una protección activa contra T.rangeli en ambos modelos a partir de la segunda reinfección con el parásito, evidenciada por ausencia de parásitos sanguícolas y detección de anticuerpos circulantes. Se discuten aspectos epidemiológicos sobre estos hallazgos. Ensayos de protección pasiva por transferencia de suero inmune y células esplénicas como factores inmunológicos en ratas "Wistar" demostró un muy corto período de protección sólo en los animales que recibieron simultáneamente suero inmune y células esplénicas. Se especulan las posibles causas de esta efímera protección

Distribution of carbohydrates recognized by the lectins Euonymus europaeus and concanavalin A in monoxenic and heteroxenic trypanosomatids.

We observed a wide distribution of the carbohydrate epitopes galactosyl alpha(1-3)galactose (gal alpha1-3 gal), alpha-glucoside and alpha-mannoside in mono- and heteroxenic trypanosomatids by using fluorescein-labelled lectins of Euonymus europaeus (EE) and Concanavalin A (Con A) as well as sera from acute chagasic patients who have very high levels of anti-gal alpha(1-3)gal antibodies. The direct fluorescence test for gal alpha1-3 gal with EE was positive at minimum concentrations of 6 micrograms/ml for heteroxenic trypanosomatids and 0.7 micrograms/ml for monoxenic ones and for the plant parasite, Phytomonas. On the other hand, heteroxenic trypanosomatids that infect vertebrates bound ten-fold more Con A than monoxenic flagellates and Phytomonas. These data were confirmed in ELISA and Western Blot assays carried out with peroxidase-labelled EE and Con A. Euonymus europaeus recognized several glycoproteins in all trypanosomatids that we tested. Con A, however, recognized a glycoprotein cluster in heteroxenic protozoa, which ranging from 60-120 kDa, seemed to lack monoxenic parasites and Phytomonas. These findings suggest that alpha-D-mannose and alpha-D-glucose might play an important role in the interaction between trypanosomatids and vertebrate hosts.

Purification of cloned trypanosomal calmodulin and preliminary NMR studies.

Cloned trypanosomal calmodulin was expressed in Escherichia coli and purified to homogeneity using hydrophobic interaction chromatography on phenyl-Sepharose. The purified protein was subjected to NMR analysis which allows detailed changes to be observed when, firstly, calcium, and secondly, the drug calmidazolium bind. These spectral changes are the result of conformational changes in the protein and proximity effects due to the drug.

The surface coat of bloodstream forms of Trypanosoma musculi from mice.

Iodination and immunoprecipitation techniques together with indirect fluorescent antibody tests identified two polypeptides (SP) of molecular weights 88,000-92,000 and 66,000-70,000 in the surface coat of bloodstream forms of the mouse trypanosome, Trypanosoma musculi. As parasites multiply and enter the early plateau phase of infection the 88,000-92,000 SP is present while the 66,000-70,000 SP is only detectable after the mid-plateau phase. Western blotting of parasite extracts showed that the 88,000-92,000 SP was present throughout the course of infection, but it appears to become masked by the 66,000-70,000 SP or possibly immunoglobulin from about 16 days after infection. Based on results when Western blots of parasite extracts were probed with antibodies affinity purified against the 88,000-92,000 SP, the two SP appear to be immunologically related and the smaller may be a cleavage product of the larger. This would explain why affinity purified antibodies to each SP bound to trypanosomes collected 8 days after infection, when only the 88,000-92,000 is detectable in parasite extracts. However, the failure of antibodies affinity purified against the 66,000-70,000 SP to bind to the 88,000-92,000 SP in Western blots suggests that the smaller SP has some epitopes that are immunologically distinct from those of the larger SP.

Analyses of surface membrane carbohydrates in parasitic flagellates of the order kinetoplastida using lectins.

Crithidia fasciculata, Leishmania donovani, Leishmania major, Leishmania mexicana amazonensis, Leishmania tropica, Leishmania tarentolae, Trypanosoma sp. from Formosan bats (Tb), Trypanosoma lewisi, Trypanosoma musculi, and different strains of Trypanosoma cruzi (Tc) were cultivated at 27 degrees C in a liquid culture medium. Flagellates harvested from log phase culture were analyzed for their lectin agglutinating characteristics with concanavalin A (Con A), Peanut agglutinin, Ricinus communis agglutinin 120, soybean agglutinin (SBA), Ulex europeus agglutinin (UEA) and wheat germ agglutinin (WGA). Results indicated that all these flagellates might have D-galactose and methyl- alpha-D-manopyranoside on their surface. The presence of L-Fucose, which complexes specifically with UEA, could not be demonstrated on the surface of these flagellates. Results from quantitative comparison of surface molecules of Tb and the Tulahuen strain of Tc suggested that Tb may have more WGA-binding molecules while Tc may have more ConA-binding molecules. Pretreatment of the flagellates with 0.05% trypsin at 37 degrees C for 30 minutes caused some reduction of agglutination titers. Cell agglutination with lectins was completely inhibited or reversed in the presence of the specific lectin-binding monosaccharides.

Differentiation of rodent trypanosomes of the subgenus Herpetosoma by lectins.

Trypomastigote forms of Trypanosoma microti, T. evotomys, T. grosi, T. musculi, and T. lewisi and trypomastigote and epimastigote forms of T. acomys were differentiated using 34 lectins and the Aminoff test for N-acetylneuraminic acid (NANA). Twelve of the lectins failed to agglutinate any of the above species. The number of lectins which agglutinated each species differed; T. mciroti, T. evotomys, T. grosi, T. musculi, T. lewisi and T. acomys (trypomastigote and epimastigote forms) were agglutinated by 7, 14, 7, 13, 11 and (11 and 10) lectins respectively. Some of the lectins were common in agglutinating all species of parasites, for example Bauhinia purpurea, Caragana aborescens and Viscum album. The minimum concentration of lectins which agglutinated each parasite was quite different. The agglutinations were cell body-cell body, flagellum-flagellum or flagellum-cell body. Most of the agglutinations were inhibited by their specific carbohydrates. The lowest concentrations of NANA was observed in T. lewisi (0.3 micrograms/ml) and the highest in T. musculi (4.5 micrograms/ml). The concentrations of NANA in T. microti, T. grosi and in T. acomys (trypomastigote and epimastigote forms) were 2, 1.8, 2.9, and (1.4 and 1.2) micrograms/ml respectively.

Characterization of plasma menbrane polypeptides of trypanosoma from bats

As proteínas de superfície de Trypanosoma dionisii, Trypanosoma vespertilionis e Trypanosoma sp. (M238) foram radiodinados e sua distribuiçäo na fase rica em detergente (DRP) e fase pobre em detergente (DPP) foram estudadas pela técnica de separaçäo de fases com Triton X-114 e por eletroforese em gel e policrilamida em presença de dodecil sulfato de sódio (SDS-PAGE). Foram observadas diferenças significativas nas proteínas presentes na DRP quando a três espécies de tripanosomas foram comparadas. Duas bandas com 88 e 70 KDa foram observadas em T. sp. (M238) e näo foram detectadas em T. dionisii e t. vespertilionis. Três polípeptídeos com 96, 77 e 60 KDa foram identificados na fase DRP de T. vespertilionis. Três bandas com 84, 72 e 60 KDa foram visualizadas na fase DRP de T. dionisii. Dois polipeptídeos com 34-36 KDA presentes na fase DPP, foram observados nas três espécies de tripanosomas analisadas. Nossas observaçöes mostraram que T. sp. (M238) possui polipeptídeos de superfície característicos, que näo säo encontrados em T. dionisii e T. vespertilionis

Studies on trypanosomatid actin. I. Immunochemical and biochemical identification.

In this study, the presence of actin in cultured trypanosomatids was investigated using polyclonal antibodies to heterologous actin. Polyclonal antisera to rabbit muscle actin and a monospecific anti-actin antibody react with a 43-kDa polypeptide in extracts of Trypanosoma cruzi, Herpetomonas samuelpessoai and Leishmania mexicana amazonensis on protein immunoblots. The 43-kDa polypeptide co-migrates with skeletal muscle actin and is retained within trypanosomatid cytoskeletons. Attempts to isolate H. samuelpessoai actin through DNase I affinity chromatography showed that the 43-kDa polypeptide did not bind to the column. Instead, low yields of a 47-kDa polypeptide were obtained indicating that the trypanosomatid actin displays unusual DNase I binding behavior when compared to actins from higher eukaryotes. Immunofluorescence studies confirmed that cytoskeletons retain the actin-like protein. In H. samuelpessoai, actin is localized in the region close to the flagellum, whereas in T. cruzi it is more homogeneously distributed. The data presented here show that trypanosomatid actin displays biochemical characteristics similar to actins of other protozoa.

Characterization of plasma membrane polypeptides of Trypanosoma from bats.

Cell surface proteins of Trypanosoma dionisii, Trypanosoma vespertilionis and Trypanosoma sp. (M238) were radiodinated and their distribution both in the detergent-poor (DPP) and detergent-enriched phase (DRP) was studied using a phase separation technique in Triton X-114, as well as polyacrylamide gel electrophoresis in sodium dodecyl sulphate (SDS-PAGE). Significant differences were observed in the proteins present in the DRP when the three species of trypanosoma were compared. Two major bands with 88 and 70 KDa were observed in T. sp. (M238) but were not detectable in T. dionisii and T. vespertilionis. Three polypeptides with 96, 77 and 60 KDa were identified in the DRP of T. vespertilionis. Three major bands with 84, 72 and 60 KDa were observed in the DRP of T. dionisii. Two polypeptides with 34-36 KDa present in the DPP, were observed in the three Trypanosome species analyzed. Our observations show that T. sp. (M238) has characteristic surface polypeptides not found in T. dionisii and T. vespertilionis.

Comparison of several lizard Leishmania species and strains in terms of kinetoplast minicircle and maxicircle DNA sequences, nuclear chromosomes, and membrane lipids.

Eight strains of a lizard Leishmania species, L. tarentolae, were compared with four other saurian species [L. hoogstrali, L. adleri, L. agamae and Leishmania sp. LizS], with L. major from man and with Trypanosoma platydactyli, a putative lizard trypanosome, in terms of kinetoplast DNA minicircle and maxicircle sequences and in terms of nuclear chromosome patterns on orthogonal gel electrophoresis. The L. tarentolae strains fell into two major groups, one (group A) consisting of the L. tarentolae strains, UC, Krassner and Trager, derived from an Algerian gecko isolate and the other (group B) consisting of five L. tarentolae LEM strains isolated from geckos in southern France. T. platydactyli TPCL2, which was postulated by Wallbanks et al. to represent the lizard form of a French L. tarentolae strain, was closely related to the UC strain and not to the LEM strains, in all respects analyzed. Leishmania sp. LizS from a Mongolian gecko and L. hoogstrali from a Sudanese gecko showed some sequence similarities to the L. tarentolae strains, but the leishmanias said to be L. adleri from a Kenyan lacertid and L. agamae from an Israeli agamid showed no minicircle sequence similarities with lizard Leishmania and in fact were probably the same species. The maxicircle divergent region was larger in the group B strains than in the group A strains, but there were sequences in common with both groups, and not with L. hoogstrali and L. major. Four strains of L. tarentolae, the four other supposed saurian Leishmania species, three mammalian leishmanias, T. platydactyli and four other trypanosomes, T. cyclops (Malaysian macaque), T. conorrhini (Hawaiian reduviid bug), T. cruzi (man) and T. lewisi (feral rat) were analyzed for their contents of sterols and phosphoglyceride fatty acyl groups. T. platydactyli TPCL2 contained a sterol (5-dehydroepisterol), a phosphatidylcholine fatty acyl group (alpha-linolenic acid) and a phosphatidylethanolamine fatty acyl group (dihydrosterculic acid) characteristic of members of the genus Leishmania and not the genus Trypanosoma. The proportions of those lipids in the free sterol and phosphoglyceride fractions of T. platydactyli TPCL2 most closely resembled those seen in the Leishmania strains from Algerian, French, Mongolian and Sudanese geckos.

Structural features and antigenic properties of carbohydrate-containing components of Trypanosoma conorhini.

Aqueous and phenolic extracts of Trypanosoma conorhini were fractionated and high molecular weight, carbohydrate-rich fractions obtained. Their antigenic characteristics, reactivity with lectins and partial chemical structure were determined. The major component, the phenolic extract, was electrophoretically diffuse and consisted of 15% protein, 5% phosphorus, hexosamine, and 67% neutral carbohydrate, which contained mannose, galactose, and xylose in a molar ratio of 1.0:1.8:1.8. Chemical analyses and lectin agglutination experiments showed nonreducing end-groups of beta-D-galactopyranose, beta-xylopyranose, and alpha-D-mannopyranose. Phosphate esters occurred, apparently, at O-6 of hexopyranosyl units. Hexosamine was present as nonacetylated units of 2-amino-2-deoxy-alpha-D-glucopyranosyl units that were extremely resistant to acid hydrolysis. On double immunodiffusion tests, the major component gave a precipitation line with rabbit serum against whole cells of Trypanosoma cruzi, suggesting the presence of common antigenic determinant(s) on the cell surface of each trypanosomatid.

Further characterization of carbohydrate-containing fractions from Trypanosoma mega.

Epimastigotes of Trypanosoma mega were submitted to phenol extraction after lipid extraction, providing an extract whose carbohydrate portion (30%) contained fucose, ribose, xylose, mannose, galactose, and glucose. The purified fraction recovered in the void volume of Bio Gel P-150 gave on SDS-PAGE a band of Mr approximately equal to 55,000 positive for protein and carbohydrate and a diffuse band strongly positive for carbohydrate and lipids (Mr approximately equal to 22,000). The structural analysis of the carbohydrate moiety of this fraction by GLC-MS indicated the presence of nonreducing end groups of fucopyranose, mannopyranose, and galactopyranose, 3-O- and 4-O-substituted and 2,3- and 2,4-di-O-substituted galactopyranosyl units. Extraction of this fraction with chloroform/methanol/water provided a soluble fraction that on SDS-PAGE gave rise to a carbohydrate and lipid-positive band (Mr approximately equal to 22,000). This fraction contained fucose, mannose, and galactose (1:1:1). As main branch points, 2,3-di-O-substituted galactopyranosyl units were present according to methylation data. Similar proportions of fucopyranosyl, mannopyranosyl, galactopyranosyl end units were present. The presence of lipids in this fraction was confirmed by methanolysis following isolation and characterization of the corresponding fatty acid methyl esters. Palmitic acid (16:0) and an 18:1 fatty acid were the predominant fatty acids.

Secondary structure of the variant surface glycoproteins of trypanosomes.

The secondary structure of seven variant surface glycoproteins (VSGs) of trypanosomes has been determined by Raman spectroscopy. They are all predominantly alpha-helical, the alpha-helix content varying between 50 and 60%. The beta-strand content varies between 20 and 25%, and the content of beta-turn and nonregular structures is about 25%. For three VSGs the N-terminal domain obtained by proteolytic cleavage was found to have essentially the same secondary structure as the complete VSGs. For three VSGs a secondary structure prediction has been performed applying the rules of Chou and Fasman. In all cases, two long alpha-helices extending over about 50 residues or 80 A are predicted in agreement with the X-ray diffraction data of Freymann et al. [(1984) Nature 311, 167-169] and Metcalf et al. [(1987) Nature 325, 84-86]. The region between the two alpha-helical segments exhibits a high potential of beta-turns, suggesting that this segment may be exposed on the cell surface and carry major antigenic determinants.

Glycosyl-sn-1,2-dimyristylphosphatidylinositol is the membrane anchor for Trypanosoma equiperdum and T. (Nannomonas) congolense variant surface glycoproteins.

We have analysed the structures of the Trypanosoma (Nannomonas) congolense and T. equiperdum variant surface glycoprotein (VSG) membrane anchors. Myristic acid uptake, phospholipase treatment, and nitrous acid deamination showed that, for each species, the anchor is glycosyl-sn-1,2-dimyristylphosphatidylinositol, as has been previously described for T. brucei. Osmotic lysis of these trypanosomes resulted in the release of soluble VSG, lacking fatty acid. In both species and in T. evansi, an endogenous phospholipase C, which cleaved diacylglycerol from membrane form VSG, was identified.

Ultrastructural visualization of lipids in trypanosomatids.

An imidazole-buffered osmium tetroxide solution was used to visualize lipids at the ultrastructural level in the following members of the family Trypanosomatidae: Trypanosoma cruzi, T. dionisii, T. vespertilionis. T. rangeli, Crithidia deanei, C. fasciculata, C. oncopelti, and Blastocrithidia culicis. Electron-dense material was seen in various lipid droplets found in all parasites and in the multivesicular structure of members of the sub-genus Schizotrypanum. High contrast of some membranes, mainly those which enclose the mitochondrion, the nucleus, and the endoplasmic reticulum, was observed even in unstained sections. X-ray microanalysis confirmed that the electron density of lipid droplets of B. culicis and membrane-bounded dense granules of C. oncopelti was due to the presence of osmium.

Identification and isolation of a variant surface glycoprotein from Trypanosoma vivax.

The protozoan Trypanosoma vivax is one of the most important agents of African trypanosomiasis, a disease that hinders the productive use of livestock in one-third of the African continent. Trypanosoma vivax is also present in the Caribbean and in South America, posing a threat to the livestock industries of the tropical and subtropical world. Much less is known of the biology of this trypanosome than of the better studied T. brucei and T. congolense. One of the variant surface glycoproteins (VSGs) of a West African stock of T. vivax was identified, purified, and partially characterized by the use of a combination of highly resolving techniques to maximize information from the relatively small amount of parasite material available. The molecular weight of the isolated protein (46,000) is smaller than that of VSGs from other species. As with T. brucei VSGs the protein from T. vivax is complexed with sugars and incorporates 3H when living trypanosomes are incubated with [3H]myristic acid, but the T. vivax molecule is more hydrophobic than the T. brucei molecule. The small size of the T. vivax VSG may have a bearing on the functional and evolutionary relationships of variant antigens in trypanosomes.