Manuel José Gil Esteves

Changes in cell-surface carbohydrates of Trypanosoma cruzi during metacyclogenesis under chemically defined conditions

Highly purified lectins with specificities for receptor molecules containing sialic acid, N-acetylglucosamine (D-GlcNAc), N-acetylgalactosamine (D-GalNAc), galactose (D-Gal), mannose-like residues (D-Man) or L-fucose (L-Fuc), were used to determine changes in cell-surface carbohydrates of the protozoal parasite Trypanosoma cruzi during metacyclogenesis under chemically defined conditions. Of the D-GalNAc-binding lectins, BS-I selectively agglutinated metacyclic trypomastigotes, MPL was selective for replicating epimastigotes, whereas SBA strongly agglutinated all developmental stages of T. cruzi. WGA (sialic acid and/or D-GlcNAc specific) was also reactive with differentiating epimastigotes and metacyclic trypomastigotes but displayed a higher reactivity with replicating epimastigote forms. A progressive decrease in agglutinating activity was observed for jacaline (specific for D-Gal) during the metacyclogenesis process; conversely, a progressive increase in affinity was observed for RCA-I (D-Gal-specific), although the reactivity of other D-Gal-specific lectins (PNA and AxP) was strong at all developmental stages. All developmental stages of T. cruzi were agglutinated by Con A and Lens culinaris lectins (specific for D-Man-like residues); however, they were unreactive with the L-fucose-binding lectins from Lotus tetragonolobos and Ulex europaeus. These agglutination assays were further confirmed by binding studies using 125I-labelled lectins. Neuraminidase activity was detected in supernatants of cell-free differentiation medium using the PNA hemagglutination test with human A erythrocytes. The most pronounced differences in lectin agglutination activity were observed between replicating and differentiating epimastigotes, suggesting that changes in the composition of accessible cell-surface carbohydrates precede the morphological transformation of epimastigotes into metacyclic trypomastigotes.

CONCANAVALIN A-INDUCED CELL DIFFERENTIATION IN THE PROTOZOAN HERPETOMONAS SAMUELPESSOAI

The plant lectin Concanavalin A (Con A) induces a process of cell differentiation in the protozoan Herpetomonas samuelpessoai cultivated in a chemically defined medium. At concentrations above 0.1 /xg/ml, Con A induces the appearance of paramastigotes. However, at lower concentrations (0.002 Atg/ml), a large number of opisthomastigotes is observed. a-methyl-D-mannoside inhibits the Con A-induced cell differentiation. Plant lectins that bind to specific carbohy- drate residues are useful probes for assess- ing some of the properties of cell surface components such as the relative mobility of intramembranous macromolecules and the lo- calization and organization of carbohydrate- containing receptor sites (Nicolson, 1974). Among lectins, Concanavalin A (Con A) is fre- quently used, being specific for receptor sites containing a-D-mannopyranose-like residues (Goldstein et al., 1963). It has been shown that Con A has a mitogenic effect on lymphocytes

Changes in cell surface anionogenic groups during differentiation ofHerpetomonas samuelpessoai mediated by dimethylsulfoxide

The surface anionic groups of untreated or dimethyl sulfoxide (DMSO)-treatedHerpetomonas samuelpessoai cells were analyzed by cell electrophoresis, ultrastructural cytochemistry, and identification of sialic acids using thin-layer chromatography. Differentiation ofH. samuelpessoai induced by DMSO treatment caused a significant increase in the net negative surface charge. In flagellates exposed to DMSO, more cationized ferritin, colloidal iron hydroxide, and sendai virus particles bound to the cell surface. Treatment of both untreated and DMSO-treated flagellates with neuraminidase decreased markedly the EPM of cells to the cathodic pole. These findings suggest that sialic acid residues are the major anionogenic groups exposed on the surface ofH. samuelpessoai. Thin-layer chromatography showed thatN-acetyl andN,O-diacylneuraminic acids, in equal proportions, were present inH. samuelpessoai. However,N-acetylneuraminic acid predominates in DMSO-treated cells.

Herpetomonas samuelpessoai: Changes in cell shape and induction of differentiation by local anesthetic

Abstract Lidocaine, a local anesthetic, induces changes in morphology and motility of Herpetomonas samuelpessoai when incubated under nonproliferative conditions. The cells become rounded and immotile. These effects are dependent on time and temperature of incubation, concentration of lidocaine, and pH. Divalent cations (Ca 2+ , Mg 2+ , Ba 2+ , and Sr 2+ ) reversed the effect of lidocaine. Lidocaine also induces the formation of membrane-bound cytoplasmic vacuoles as determined by morphometry applied to electron micrographs. Lidocaine had a dose-dependent effect on the growth of H. samuelpessoai in a chemically defined medium. At concentrations which did not interfere with cell growth, it induces the transformation of promastigote into opisthomastigote via paramastigote. It is suggested that lidocaine may be used as an inductor of differentiation in H. samuelpessoai opening the possibility of obtaining the three developmental stages of this trypanosomatid.

Effect of lipopolysaccharide (LPS) on the metabolism and cell surface of the trypanosomatid Herpetomonas megaseliae

Abstract 1. 1. The effect of lipopolysaccharide (LPS) on the surface and metabolism of Herpetomonas megaseliae was studied. 2. 2. No inhibition of growth was detected even with the use of high concentrations of LPS (100 μg/ml). 3. 3. LPS stimulated the differentiation-transformation of pro- to opisthomastigote forms at low concentrations (2 × 10 −3 μ g/ml). 4. 4. LPS-treated cells showed an increased O 2 uptake when glucose and glycerol were used as substrate. In addition such cells induce O 2 consumption when proline was present. 5. 5. LPS-treated cells showed an increased content of galactose and xylose and a marked decrease of rhamnose and mannose, whereas glucose amount remained unchanged. 6. 6. LPS-treated cells showed an increased agglutination in the presence of Con A and a decrease in the affinity for anti-flagellate serum. 7. 7. The results of items 3, 4, 5 and 6 are discussed in relation to the cell differentiation of Herpetomonas .

Occurrence ofN-acetyl-andN-O-diacetyl-neuraminic acid derivatives in wild and mutantCrithidia fasciculata

The cell-surface expression of sialic acids in wild-typeCrithidia fasciculata and three drug-resistant mutants (FUR11, TR3, and TFRR1) was analyzed using fluorescein-labeledLimulus polyphemus agglutinin (LPA) binding, glycosidase of known sugar specificity, and thin-layer chromatography (TLC). Gas-liquid chromatography-mass spectrometry (GC-MS) analysis using both electron-impact (EI-MS) and chemical ionization (CI-MS) by isobutane with selected ion monitoring (SIM) was also used. The surface location of sialic acid was inferred from LPA binding to whole cells abrogated by previous treatment with neuraminidase. An exception occurred with the TFRR1 strain, which after incubation with neuraminidase showed increased reactivity with the fluorescent lectin. BothN-acetyl- andN-O-diacetyl-neuraminic acids were identified in the flagellates by TLC, with a clear predominance being noted for the former derivative. However, the content ofN-O-diacetyl-neuraminic acid was preferentially found in the TFRR1 strain. The GC-MS analysis of the acidic component of the TERR1 mutant strain confirmed the occurrence ofN-acetyl-neuraminic acid (Neu5Ac) by the presence of the diagnostic ions (m/z values: 684 and 594 for CI-MS and 478, 298, and 317 for EI-MS) and also by comparison with the standard Neu5Ac retention time. GC-MS analysis also showed fragments (m/z values: 654 and 564 for CI-MS and 594, 478, 298 and 317 for EI-MS) expected for the 7-O- and 9-O-acetyl-N-acetyl-neuraminic acids (Neu5,7Ac2 and Neu 5,9Ac2, respectively).

Chitin: a cell-surface component ofphytomonas françai

The occurrence of chitin as a structural component of the surface of the phytopathogenic protozoanPhytomonas françai was demonstrated by paper and gasliquid chromatographic analysis of the products of enzymatic and chemical hydrolysis of alkali-resistant polysaccharides, lectin binding, glycosidase digestion, and infrared spectra. Chitin was characterized by its insolubility in hot alkali and chromatographic immobility as well as by the release of glucosamine on hydrolysis with strong acid and ofN-acetylglucosamine (GlcNAc) on hydrolysis with chitinase. The presence of chitin was also shown directly by binding of wheat-germ agglutinin (WGA), which recognizes GlcNAc units, to the parasite surface. Fluorescein-labeled WGA binding was completely abolished by treatment with chitinase. This effect was specific since it could be prevented by incubating the enzyme with chitin before treatment of the phytomonads. These findings indicate that chitin is an exposed cell-surface polysaccharide inPhytomonas françai. The data were confirmed by the infrared spectrum of an alkali-insoluble residue, which showed a pattern typical of chitin.

Surface charge and hydrophobicity of wild and mutantCrithidia fasciculata

Surface charge of wild-typeCrithidia fasciculata and three drug-resistant mutants (TR3, TFRR1, and FUR11) was studied by direct zeta-potential determination and ultrastructural cytochemistry. Surface tension was also investigated by measurements of the advancing contact angle formed by the protozoa monolayers with drops of liquids of different polarities. The individual zeta potential varies markedly among theC. fasciculata cells. The wild and FUR11 mutant strains displayed lower negative surface charge (−12.5 and −9.5 mV, respectively), as compared with the TR3 (−14.8 mV) and TFRR1 (−14.7 mV) mutant strains. Binding of cationized ferritin (CF) was observed at the cell surface of wild and mutant strains ofC. fasciculata. Neuraminidase treatment reduced the negative surface charge in the TFRR1 and TR3 mutants in about 37 and 29%, respectively, whereas no significant change was observed with the wild and FUR11 mutant strains. These findings suggest that sialic acid residues are the major anionogenic groups on the surface ofC. fasciculata. The density of sialic acid residues per cell in wild and mutant strains ofC. fasciculata falls in a range of 1.4×104 to 3.6×104. Marked differences of hydrophobicity were also observed. For example, the TFRR1, FUR11, and TR3 drug-resistant mutant strains showed higher contact angle values (55.4, 54.2, and 49.3, respectively) than the wild-type (35.6), as assessed by α-bromonaphtalene.

Phospholipase C-mediated release of neuraminidase from Tritrichomonas foetus cell surface

The release of theTritrichomonas foetus plasma-membrane ectoenzyme neuraminidase by exogenous specific phospholipase C (PI-PLC) was investigated. Neuraminidase activity was determined using both the peanut agglutinin (PNA) hemagglutination test and the specific substrateN-acetylneuramin-lactose in a colorimetric assay. The release of the neuraminidase by PI-PLC was dependent on the reaction time and the concentration of PI-PLC. Neuraminidase activity was also detected in supernatant of untreatedT. foetus. Spontaneous or PI-PLC-induced release of neuraminidase from protozoan cells was markedly decreased by 10 mM ZnCl2, suggesting the occurrence of an endogenous PI-PLC in the parasite. AfterT. foetus lysis at 37°C with a solution of Triton X-114, neuraminidase activity was preferentially found in the aqueous phase rather than in the detergent phase, again suggesting that the parasite contains an endogenous PI-PLC that converts the hydrophobic form of neuraminidase anchored to theT. foetus cell membrane into a hydrophilic form. These results show that neuraminidase is linked to theT. foetus plasma membrane via a glycosylphosphatidylinositol anchor.

Fatty acid composition of amastigote and trypomastigote forms of Trypanosoma cruzi

The fatty acid composition of total lipids from trypomastigote and amastigote forms of Trypanosoma cruzi and of Vero cells before and after parasite infection were analyzed by gas-liquid chromatography and mass spectrometry. Even-numbered, saturated, monoenoic and polyenoic acids ranging from C-12 to C-18 were characterized in both T. cruzi development stages. Significant changes in the fatty acid composition occurred during the T. cruzi life cycle. Oleic and linoleic acids were prominent in trypomastigote forms, whereas palmitic acid was the major fatty acid of amastigotes. Other differences include higher stearic acid and lower palmitoleic and linolenic acid levels as well as the absence of lauric acid in amastigotes as compared with trypomastigote forms. The fatty acid pattern of Vero cells before T. cruzi infection as compared with that after infection showed mostly qualitative differences. Linoleic and linolenic acids were observed only in T. cruzi infected cells.