Detection of hybrid phenotypes in African trypanosomes by high resolution two-dimensional gel electrophoresis.

High resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and autoradiography were used to analyze the protein phenotypes of Trypanosoma brucei (T.b. brucei and T.b. gambiense) clones suspected of being hybrids. Procyclic culture forms of parental and suspected hybrid trypanosomes were biosynthetically labeled with [35S]methionine and labeled proteins were resolved by multiple 2D-PAGE (the ISO-DALT system) to allow accurate inter-gel comparisons. Autoradiography of the gels showed that the parental clones had qualitative differences in at least seven sets of spots. Five of these sets represented charge differences and one represented proteins of altered relative molecular mass (Mr) and charge. Autoradiographs of the gels of the putative hybrid trypanosomes showed both forms of the proteins found separately in the parental clones indicating that new, nonparental phenotypes had been generated by transmission of mixed trypanosome clones through tsetse flies. The 2D-PAGE patterns from parasites cultivated for extended periods were identical, showing that the individual cloned parasites were phenotypically stable. The results indicate that analytical 2-D gels can be used to study the phenotypes of "parental" or "hybrid" African trypanosomes without having any previous knowledge of the molecular characteristics of the parasites. In addition, the technique allows an extension of phenotypic analysis to hundreds of different proteins in populations of cloned parasites.

[The fatty acids of Trypanosoma brucei brucei. A comparative study with those of T. b. rhodesiense and T. b. gambiense]. / Les acides gras de Trypanosoma brucei brucei. Etude comparative avec ceux de T. b. rhodesiense et T. b. gambiense.

Total lipid extracts from Trypanosoma brucei brucei (T.b.b.), Trypanosoma brucei gambiense (T.b.g.) and Trypanosoma brucei rhodesiense (T.b.r.) were hydrolyzed and the liberated fatty acids were methylated before analysis by gas-liquid chromatography on capillary columns. The sums of the percentages for fatty acids from the different series were compared and the following relationships for the different trypanosomes obtained: --saturated fatty acids T.b.b. greater than T.b.g. greater than T.b.r., --oleic series (omega 9) T.b.g. = T.b.r. greater than T.b.b., --linoleic series (omega 6) T.b.r. greater than T.b.g. greater than T.b.b., --linolenic series (omega 3) T.b.g. greater than T.b.b. greater than T.b.r.

Trypanosoma brucei brucei and Trypanosoma brucei gambiense: stage specific differences in wheat germ agglutinin binding and in endoglycosidase H sensitivity of glycoprotein oligosaccharides.

Gel electrophoresis, lectin affinity blotting, and endoglycosidase H digestion have been used to analyze the glycoprotein profiles of bloodstream and procyclic forms of Trypanosoma brucei brucei and T. b. gambiense. Proteins resolved by polyacrylamide gel electrophoresis were stained with silver nitrate or electrophoretically transferred to nitrocellulose and probed with a horseradish peroxidase conjugate of either concanavalin A or wheat germ agglutinin. Silver staining showed, as expected, that the expression of the variant specific glycoprotein was restricted to the bloodstream forms. Twenty-three concanavalin A binding proteins were resolved in blots of bloodstream forms. Concanavalin A binding molecules corresponding in electrophoretic mobility to 21 of these 23 bloodstream form glycoproteins were detected in blots of procyclic forms. The two concanavalin A binding glycoproteins present only in bloodstream form extracts were variant specific glycoprotein and an 81-kDa protein designated glycoprotein 81b. One concanavalin A binding molecule of 84 kDa, glycoprotein 84p, was detected only in procyclic forms. The 19 major wheat germ agglutinin binding glycoproteins expressed by bloodstream forms were not detected in procyclic forms; only small proteins or protein fragments in procyclic form extracts bound wheat germ agglutinin. Incubating transferred proteins in endoglycosidase H eliminated subsequent binding of concanavalin A to most of the 22 common glycoproteins of bloodstream forms. Three major concanavalin A binding glycoproteins of bloodstream forms, variant specific glycoprotein, glycoprotein 81b, and a 110-kDa molecule (glycoprotein 110b), and other minor glycoproteins carried sugar chains that resisted endoglycosidase H digestion. In contrast, concanavalin A did not bind to any procyclic form glycoproteins, including a 110-kDa concanavalin A binding molecule (glycoprotein 110p) after endoglycosidase H treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Trypanosoma brucei brucei, T. brucei gambiense, and T. brucei rhodesiense: common glycoproteins and glycoprotein oligosaccharide heterogeneity identified by lectin affinity blotting and endoglycosidase H treatment.

Whole cell extracts of 10 clones of bloodstream forms of African trypanosomes representing two strains of Trypanosoma brucei gambiense, one strain of T. b. rhodesiense and one strain of T. b. brucei were fractionated on sodium dodecyl sulfate-polyacrylamide gels, electrophoretically transferred to nitrocellulose paper, and probed with horseradish peroxidase conjugated lectins to detect glycoproteins. Variant specific glycoproteins of all 10 clones bound peroxidase labeled concanavalin A, but peroxidase labeled wheat germ agglutinin bound to the variant specific glycoproteins of only 3 of the 10 clones examined. In addition, 22 other glycoproteins expressed in common by all clones bound peroxidase labeled concanavalin A; 19 common glycoproteins bound peroxidase labeled wheat germ agglutinin. Lectin binding to transferred glycoproteins was specifically inhibited by appropriate monosaccharides, alpha-methyl mannoside for concanavalin A and N-acetyl glucosamine for wheat germ agglutinin. Prior incubation of blots in endo-beta-N-acetylglucosaminidase H eliminated binding of peroxidase-labeled concanavalin A to most of the 22 common glycoproteins. Two glycoproteins, designated Gp 81 and Gp 110, were the major Endoglycosidase H resistant components. Endoglycosidase H treatment also reduced binding of peroxidase labeled concanavalin A to the variant specific glycoproteins of 7 clones. The variant specific glycoproteins from the 3 clones that bound peroxidase labeled concanavalin A following enzyme treatment were those that bound peroxidase labeled wheat germ agglutinin. These results show that African trypanosomes express a greater number of glycoproteins than has been reported previously and that only a limited number of these glycoproteins bear Endoglycosidase H resistant oligosaccharides.

Rapid preparative scale purification of myristylated variant surface glycoproteins from African trypanosomes.

A rapid method for the preparative-scale purification of variant surface glycoproteins (VSG) from African trypanosomes has been developed to investigate the recently described myristylated form of this molecule. This high-performance liquid chromatography (HPLC) procedure gives high yields and can be achieved in as little as 1 h from the preparation of a cell lysate in 0.1% trifluoroacetic acid (TFA). Solubilization of myristylated VSG in 0.1% TFA was shown to be much more effective than in neutral or zwitterionic detergents. Surface iodination of bloodstream trypanosomes and subsequent lysis in TFA showed that no proteolytic degradation of VSG occurred during solubilization and purification. Biosynthetic labelling of trypanosomes with [3H]myristic acid and purification of VSG by the described method, demonstrated that the VSG possessed covalently linked fatty acid and can therefore be defined as the membrane form of VSG. Evidence was provided that the myristic acid was covalently associated with the C-terminal portion of the VSG via a hydroxyester bond. Solubilization of the myristylated VSG by 0.1% TFA could be interpreted to indicate that the fatty acid was not embedded in the trypanosome membrane.

The compaction pattern of the chromatin of trypanosomes.

Protozoa of the family Trypanosomatidae are pathogenic agents of human and animal diseases. Fine structure, compaction pattern, and histone content of the soluble chromatin were investigated in procyclic forms of Trypanosoma cruzi (Chagas disease, S. America) and T. brucei brucei (Nagana disease, Africa) in comparison with rat liver chromatin. At low ionic strength chromatin was present as nucleosome filaments. Condensation into compact fibers (solenoid) was complete for rat chromatin at 100 mM salt concentration while chromatin of T. cruzi showed less condensation (tangle formation), and that of T.b. brucei did barely condense under identical experimental conditions. In general, the nucleosomes of trypanosomes, especially T.b. brucei, seemed to be less regularly arranged than those of the higher eukaryote. Addition of histone H1-containing fractions of rat liver chromatin increased the compaction of T. cruzi chromatin but had no influence on T.b. brucei chromatin. SDS-polyacrylamide gel electrophoresis revealed histone H1 and the 4 core histones in rat liver chromatin. Neither in T. cruzi nor T.b. brucei were proteins identical to rat histone H1 present. Differences existed also in molecular weight of core histones between rat and trypanosomes, as well as between T. cruzi and T.b. brucei. These differences might explain species-specific differences in the fine structural organization and compaction pattern of the chromatin of the rat, T. cruzi, and T.b. brucei.

In situ microfluorometry of whole kinetoplast and nuclear DNA in a single cell of Trypanosoma gambiense and Trypanosoma cruzi.

In situ microfluorometry of whole kinetoplast DNA (K-DNA) and nuclear DNA (N-DNA) from a single trypomastigote cell of Trypanosoma gambiense (strain Wellcome) and Trypanosoma cruzi (strain Tulahuen) was attempted by using the microfluorometer combined with a photon counter. The results obtained were summarized as follows; 1. The K-DNA value of 2K 1N (2 kinetoplasts and 1 nucleus) T. gambiense was about twofold as much as that of 1K 1N on. 2. The N-DNA value of 2K 1N T. gambiense was a little less than two times as much as that of 1K 1N T. gambiense, showing that the N-DNA had already bee increased nearly twofold before the nuclear division was initiated. 3. The K-DNA value of 1K 1N T. cruzi was about fivefold as much as that of 1K 1N T. gambiense. The N-DNA value was contrarily lower than that of T. gambiense, being approximately 70% of the latter. 4. Percent K-DNA/N-DNA value was, in average, about 7 in either 1K 1N or 2K 1N T. gambiense and about 50 in T. cruzi. 5. Percent K-DNA/N-DNA value in individual trypomastigote cell was variable, showing that the increase of K-DNA and N-DNA was not synchronized at least in the two species of trypanosomes used here. 6. The fact that K-DNA and N-DNA values varied from cell to cell although they do not divide, suggested the possibility of DNA increase in the trypomastigote of T. cruzi.