The trans-sialidase, the major Trypanosoma cruzi virulence factor: Three decades of studies.

Chagas' disease is a potentially life-threatening disease caused by the protozoan parasite Trypanosoma cruzi. Since the description of Chagas'disease in 1909 extensive research has identified important events in the disease in order to understand the biochemical mechanism that modulates T. cruzi-host cell interactions and the ability of the parasite to ensure its survival in the infected host. Exactly 30 years ago, we presented evidence for the first time of a trans-sialidase activity in T. cruzi (T. cruzi-TS). This enzyme transfers sialic acid from the host glycoconjugates to the terminal ß-galactopyranosyl residues of mucin-like molecules on the parasite's cell surface. Thenceforth, many articles have provided convincing data showing that T. cruzi-TS is able to govern relevant mechanisms involved in the parasite's survival in the mammalian host, such as invasion, escape from the phagolysosomal vacuole, differentiation, down-modulation of host immune responses, among others. The aim of this review is to cover the history of the discovery of T. cruzi-TS, as well as some well-documented biological effects encompassed by this parasite's virulence factor, an enzyme with potential attributes to become a drug target against Chagas disease.

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.

Incorporation of sialic acid into Trypanosoma cruzi macromolecules. A proposal for a new metabolic route.

Sialo- and asialoglycoconjugates were isolated from Trypanosoma cruzi epimastigotes and their composition determined. Sialoglycoconjugates bound to wheat germ agglutinin (WGA)-Sepharose and were precipitated by concanavalin A, Wistaria floribunda hemagglutinin and WGA. Asialoglycoconjugate bound to concanavalin A-Sepharose and precipitated with concanavalin-A and W. floribunda hemagglutinin but not with WGA. Cells grown in the presence of fetal calf serum were agglutinated by WGA but not by peanut agglutinin. The reverse was true for cells grown without fetal calf serum. Neuraminidase-treated cells incorporated sialic acid or its 7-carbon analog, 5-acetamido-3,5-dideoxy-L-arabino-2-heptulosonic acid (AcNeu7) from sialylated compounds such as fetuin or sialyl-lactose but did not incorporate free sialic acid. Restoration of the WGA sialylreceptors in neuraminidase-treated cells, as determined by cell agglutination with WGA, was also obtained by incubation with fetuin or sialyl-lactose but not with free sialic acid. Moreover, restoration of agglutinability by WGA in neuraminidase-treated cells or cells grown in medium without fetal calf serum occurred equally well in energy-rich or energy-depleted cells. A transglycosilase reaction for sialic acid incorporation in T. cruzi epimastigotes is suggested.

Chemical characterisation of glycosylinositolphospholipids of Herpetomonas samuelpessoai.

The structure of two glycosylinositolphospholipids of the cell surface of the monoxenic protozoan Herpetomonas samuelpessoai have been deduced by methylation analysis, fast-atom bombardment mass spectrometry and two dimensional nuclear magnetic resonance spectroscopy. These glycolipids have features in common with the glycoinositolphospholipids of both Leishmania and Trypanosoma cruzi, resembling the former by the presence of the hybrid type core sequence Man alpha 1-->3(Man alpha 1-->6)Man alpha 1-->4GlcN alpha 1-->6 myo-inositol-1-PO4-lipid, while the 2-aminoethylphosphonate substituent on 0-6 of glucosamine and the presence of ceramide in place of glycerol lipids is more reminiscent of T. cruzi. Possible phylogenetic implications of these observations are discussed.

Partial chemical characterization of the carbohydrate moieties in Leishmania adleri glycoconjugates.

Promastigotes of Leishmania adleri were submitted to an extraction procedure providing different carbohydrate-containing extracts. The purified aqueous extract showed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis a complex peptide pattern but carbohydrate was present only in bands of Mr approximately equal to 45 000-50 000 and 13 500. Methylated derivatives of the hexose components in this extract, analysed by mass spectrometry, suggest the presence of short sugar chains of alpha-D-mannopyranose and a branched alpha-D-mannan. The phenol extract, released in the aqueous layer a chloroform/methanol/water soluble complex contained 25% protein, 17% phosphate, 11% glucosamine, uronic acid and 61% neutral carbohydrate, and a chloroform/methanol/water insoluble fraction consisting of a glycoprotein Mr approximately equal to 22 000 and a proteic doublet Mr approximately equal to 58 000-66 000. A polysaccharide, showing galactose as predominant sugar, was released through alkaline extraction corresponding to a branched, mainly 1----3 linked galactan associated with alpha-D-mannopyranosyl units.

Chagas' disease: serodiagnosis with purified Gp25 antigen.

Development of a highly specific test system for the diagnosis of Chagas' disease (CD) was sought using Gp25, a surface glycoprotein recently isolated from Trypanosoma cruzi culture forms. Radioimmunoprecipitation assays were performed to screen 567 sera for IgG antibodies to Gp25. Correct diagnosis was attained in 97.8% of the 321 sera collected from chagasic patients in several endemic areas of South America. Sera from patients with various clinical forms of chronic disease displayed similar levels of antibodies (Abs) to Gp25. Moreover, there was little cross-reactivity when assayed against 246 sera from non-chagasic individuals, including 105 samples from individuals infected with unrelated trypanosomatids. Cross-reactivity was found in two such sera; these were used to identify a minor protein contaminant as the nonspecific antigen. Residual cross-reactive molecules were eliminated from Gp25 by affinity purification on Concanavalin A (Con A) columns. We recommend this antigen-antibody system for use in routine screening of blood donors.

NMR assignments for glucosylated and galactosylated N-acetylhexosaminitols: oligosaccharide alditols related to O-linked glycans from the protozoan parasite Trypanosoma cruzi.

We report full 1H and 13C NMR assignments for 13 gluco- or galacto-pyranosylated derivatives of GlcNAc-ol, GalNAc-ol or ManNAc-ol, many of which have been prepared by enzymatic methods. These spectra are reference data to aid the structural analysis by NMR spectroscopy of glycosylated alditols derived from the mucin of the protozoan parasite Trypanosoma cruzi. A series of structural reporter groups for the derivatives from this unusual series of O-glycans are described.

Structure of the repeating oligosaccharide from the lipopolysaccharide of the nitrogen-fixing bacterium Acetobacter diazotrophicus strain PAL 5.

Acetobacter diazotrophicus is an acid-tolerant nitrogen-fixing bacterium found in roots, rhizosphere, stems, and leaves of sugar cane (Saccharum officinarum) cultivated in Brazil. The O-polysaccharide from the lipopolysaccharide of the root isolate strain PAL 5 has been determined by a combination of methylation analysis and two-dimensional high field NMR spectroscopy. The pentasaccharide repeat has the structure: [formula: see text] Minor resonances in the NMR spectra are consistent with the presence of a proportion of repeating units which lack the beta-D-Glc side-chain.

Characterization of phosphoinositol oligosaccharides from parasitic protozoa by fast atom bombardment and collisional activation mass spectrometry.

Fast atom bombardment mass spectrometry (FAB MS) enables the rapid, accurate and sensitive determination of the molecular masses of glycosylphosphatidylinositol (GPI)-derived oligosaccharides, from which the composition in terms of monosaccharide residues and non-carbohydrate substituents can be determined. Interpretation of fragment ions in collisional activation mass spectra further enables the determination of residue sequence, the positions of branch points, and the location of non-carbohydrate substituents. We have applied these techniques to the characterization of phosphoinositol oligosaccharides from Leptomonas samueli, Endotrypanum schaudinni and Leishmania adleri. The mass spectral data permit the postulation of candidate structures for the oligosaccharides, which provide a set of constraints that can assist the interpretation of results from other techniques such as NMR.

The use of NMR spectroscopy in the structure determination of a Leptomonas samueli glycosylphosphosphingolipid-derived oligosaccharide.

Nuclear magnetic resonance (NMR) spectroscopy provides an extremely powerful technique to determine the structure of oligosaccharides, particularly when used in conjunction with other physical techniques such as methylation analysis and fast atom bombardment mass spectrometry (FAB-MS). This brief review describes the application of NMR to the determination of the structure of an oligosaccharide isolated from the glycophosphosphingolipid (GPS) from the monogenetic trypanosomatid Leptomonas samueli. When ambiguities arise in the NMR interpretation, the use of other data will be discussed.

Structures of four oligosaccharides derived from the glycoinositolphospholipid of Leishmania adleri.

Glycoinositolphospholipids (GIPLs) were extracted from the trypanosomatid Leishmania adleri by hot phenol extraction and the carbohydrate moieties isolated after base cleavage. Purification of the crude oligosaccharides by high performance anion exchange (HPAE) chromatography yielded four fractions whose structures were determined by a combination of methylation analysis, fast atom bombardment (FAB) mass spectrometry and two-dimensional nuclear magnetic resonance (NMR) spectroscopy.

Synthesis of monorhamnosyl L-rhamno-D-mannans by conidia of Sporothrix schenckii.

A rhamnomannan containing single-unit alpha-L-rhamnopyranose side chains was identified in isolated conidia from Sporothrix schenckii. Such a rhamnomannan differed from the dirhamnosyl rhamnomannan synthesized by the hyphae but was very similar to the monorhamnosyl rhamnomannan formed in yeastlike cells. Nuclear magnetic resonance spectroscopy and chemical analysis were used to compare these polysaccharides. Based on the distribution of different rhamnomannans in different S. schenckii cell types and in view of the reactivity of some human antisera previously reported (6), the formation of hyphae in vivo is suggested.

Galactose-containing polysaccharides from the human pathogens Sporothrix schenckii and Ceratocystis stenoceras.

Galactose-containing polysaccharides from three strains of Sporothrix schenckii and one strain of Ceratocystis stenoceras were isolated, and their structures were paritally characterized by chemical analysis and 13C-nuclear magnetic resonance spectroscopy (13C-NMR). S. schenckii polysaccharide preparations from all strains were not precipitated by Fehling solution and contained galactomannan (or a mixture of galactan and galactomannan), amylose, and minor amounts of rhamnomannan. C. stenoceras polysaccharide contained galactomannan and a smaller proportion of amylose. Conventional chemical techniques and 13C-NMR spectroscopy showed that the structures of the two preparations were closely related. The core of the galactomannan consisted principally of nonreducing end units and 2-O-, 2,6-di-O-, and perhaps 2,3-di-O-substituted alpha-D-mannopyranosyl units. The core was substituted by beta-D-galactofuranosyl chains; the units are interlinked (1 leads to 6). 13C-NMR evidence shows that the alpha-D-mannopyranosyl units are substituted in the two positions by the beta-D-galactofuranosyl residues. Galactomannans present at the cell surface of S. schenckii represent other potential fungal antigens in addition to the already recognized rhamnomannans and their peptide complexes.

Sexual agglutination factors from the yeast Pichia amethionina.

Pichia amethionina is a heterothallic yeast isolated from necrotic cactus tissue. Haploid cells of opposite mating type, designated alpha and alpha, agglutinate strongly when mixed. The agglutination factors of the two cell types have been solubilized from the cell walls by beta-glucanase digestion and then partially purified by affinity adsorption to the opposite cell type and by gel filtration. From alpha-cells was obtained a large, heat-stable glycoprotein with the ability to agglutinate alpha-cells. This alpha-agglutinin was inactivated by mercaptoethanol, probably because the recognition sites are linked to the glycoprotein core by disulfide bonds. Digestion of alpha-cells with beta-glucanase released a large heat-labile glycoprotein that did not agglutinate alpha-cells but did inhibit agglutination of alpha-cells by alpha-agglutinin. Subtilisin digestion of this alpha-factor released a carbohydrate-free protein of 27,000 daltons that retained the biological activity of the factor. These agglutination factors are sex- and species-specific and are not found on the surface of heterozygous diploid cells.

Cell-cell recognition in yeast: purification of Hansenula wingei 21-cell sexual agglutination factor and comparison of the factors from three genera.

Trypsin digestion of Hansenula wingei 21-cells releases a protein (21-factor-T) that inhibits the agglutination of 21-cells by purified 5-agglutinin obtained from 5-cells by subtilisin digestion [Crandall, M. A. & Brock, T. D. (1968) Bacteriol. Rev. 32, 139-163]. We have purified this inhibitor 415-fold by ion-exchange chromatography, affinity adsorption to 5-cells, and gel permeation chromatography. The material shows a diffuse band, on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, with an apparent M(r) of 27,000. It has a pI of 3.8, is rich in acidic amino acids, contains 5% mannose and a trace of glucosamine, and is stable to reducing agents but is inactivated by heat. Zymolyase (beta1-->3-glucanase) digestion of 21-cells releases a similar inhibitor that, after purification, has a larger size than 21-factor-T. This 21-factor-Z appears to contain an additional portion that may serve to anchor 21-factor in the cell wall. Haploid cells of the yeasts Pichia amethionina and Saccharomyces kluyveri also show a constitutive sexual agglutination, and little or no crossreactivity is observed in heterologous mixtures. The agglutination factors in all three genera, however, have parallel properties; one cell type of each pair is heat stable and is inactivated by reducing agents (H. wingei 5-cells, P. amethionina alpha-cells, and S. kluyveri 16-cells), and the other is heat labile and is unaffected by reducing agents H. wingei 21-cells, P. amethionina a-cells, and S. kluyveri 17-cells). Because S. kluyveri 16-cells respond to Saccharomyces cerevisiae alpha-factor with the typical morphogenetic change of a mating half-reaction, the heat-stable agglutinin appears related to the S. cerevisiae a mating type and the heat-labile factor to the S. cerevisiae alpha mating type.

Investigations on polysaccharide components of cells of Herpetomonas samuelpessoai grown on various media.

Herpetomonas samuelpessoai, when cultured in various media, forms a linear beta-D-(1 leads to 2)-mannopyranan and a branched-chain glucuronoxylan containing D-glucopyranosyluronic acid nonreducing end units connected with alpha- and beta-linked D-xylopyranose units, as depicted in fragmental structures I, II, and III. Traces of amylose and alpha-linked mannopyranose moieties are also present. Mannose-containing materials predominate over glucuronoxylan in cells grown with proline as the carbon source (medium C). In a medium with sucrose as the carbon source and complex supplements (medium A) the proportion of glucuronoxylan is greater, and when the supplements were all chemically defined (medium B), a galactose-containing component is also formed. Glucuronoxylan is liberated from cells with hot aqueous alkali and it could be freed from lower molecular weight mannan by fractional precipitation. Mannan was obtained as the only polymeric component, with lower molecular weight homologues, by extraction of cells with hot water. In terms of ratios of component xylose, mannose, and galactose, the flagella resemble whole cells. However, flagella contain polysaccharide with alpha-D-linked mannopyranose side chains rather than beta-linked linear forms. These data are considered from standpoints of immunochemistry and electron microscopy.

Heterogeneity of the rhamnomannans from one strain of the human pathogen Sporothrix schenckii determined by 13C nuclear magnetic resonance spectroscopy.

The synthesis of different rhamnomannans in a strain of Sporothrix schenckii (1099.12) was shown by use of 13C nuclear magnetic resonance spectroscopy. Fractionation of a polysaccharide preparation from cells grown at 25 degrees C provided a neutral monorhamnosyl rhamnomannan and an acidic rhamnomannan containing 4-O-substituted glucuronic acid units and also (1 leads to 2)-linked dirhamnosyl side chains.

Presence of a lipophosphoglycan in two variants of Trypanosoma brucei brucei.

For the family of Trypanosomatidae (Trypanosoma and Leishmania) the organization of the glycoproteins on the cell surface is a well documented structural feature, because their plasma membranes are potential target for chemotherapy. By using metabolic labeling ( [32P] phosphate, [3H]-myristic acid, [3H]-galactose) and by appropriate fractionated extraction, we have found a trypanosomal molecule which has electrophoretic and chromatographic properties consistent with the lipophosphoglycan of Leishmania donovani defined by Turco et al (1987) Biochemistry 26, 6233-6238 (1). In addition, the trypanosomal lipophosphoglycan, appears to have chromatographic behaviour similar to the glycolipid C of Trypanosoma brucei brucei described by Krakow et al (1986) J. Biol. Chem. 261, 12147-12153 (2). Our results suggest that the role of the trypanosomal lipophosphoglycan may take place in the orientation of the glycoproteins in the surface coat and/or corresponds to the glycolipid precursor for the anchor of variant surface glycoprotein.