Protein Uptake and Digestion in Bloodstream and Culture Forms of Trypanosoma brucei*

SYNOPSIS. The mechanisms of ferritin uptake and digestion differ in bloodstream and culture forms of Trypanosoma brucei. Ferritin enters bloodstream forms from the flagellar pocket by pinocytosis in large spiny-coated vesicles. These vesicles become continuous with straight tubular extensions of a complex, mostly tubular, collecting membrane system where ferritin is concentrated. From the collecting membrane system the tracer enters large digestive vacuoles. Small spiny-coated vesicles, which never contain ferritin, are found in the Golgi region, fusing with the collecting membrane system, and around the flagellar pocket. Acid phosphatase activity is present in some small spiny-coated vesicles which may represent primary lysosomes. This enzymic activity is also found in the flagellar pocket, pinocytotic vesicles, the collecting membrane system, the Golgi (mature face), and digestive vacuoles of bloodstream forms. About 50% of the acid phosphatase activity of blood forms is latent. The remaining nonlatent activity is firmly cell-associated and probably represents activity in the flagellar pocket. The structures involved in ferritin uptake and digestion are larger and more active in the short stumpy than in the long slender bloodstream forms. The short stumpy forms also have more autophagic vacuoles. No pinocytotic large, spiny-coated vesicles or Golgi-derived, small spiny-coated vesicles are seen in culture forms. Ferritin leaves the flagellar pocket of these forms and enters small smooth cisternae located just beneath bulges in the pocket membrane. The tracer then passes through a cisternal collecting membrane network, where it is concentrated, and then into multivesicular bodies. In the culture forms, acid phosphatase activity is localized in the cisternal system, multivesicular bodies, the Golgi (mature face), and small vesicles in the Golgi and cisternal regions. The flagellar pocket has no acid phosphatase activity, and almost all the activity is latent in these forms. The culture forms do not release acid phosphatase into culture medium during 4 days growth. Uptake of ferritin by all forms is almost completely inhibited by low temperature. These differences among the long slender and short stumpy bloodstream forms and culture forms are undoubtedly adaptive and reflect different needs of the parasite in different life cycle stages.     

Analysis of the Antigenic Composition of Trypanosoma brucei brucei Bloodstream and Culture Forms by the Quantitative Direct Fluorescent Antibody Methods*

SYNOPSIS The quantitative direct fluorescent antibody (QDFA) methods were employed for the antigenic analysis of bloodstream forms and culture procyclics of 2 variants, TRUM (Trypanosome Research University of Massachusetts) 106 and TRUM 107, of Trypanosoma brucei brucei. Intact and trypsinized trypanosomes were studied. It was demonstrated that: (A) The specific variant antigens are localized in the surface coat of bloodstream trypomastigotes. (B) In addition to the common antigens shared by bloodstream forms and culture procyclics, there are also certain antigens unique to these latter stages. (C) Still another group of antigens. not found in the culture procyclics, appears to be shared by the bloodstream forms, irrespective of their variant-specific antigens. These antigens may be present in part in the coat or on the cell membrane and in part within the cytoplasm. (D) Irrespective of the bloodstream-form variant from which they are derived, the procyclics are antigenically the same. The QDFA results are analyzed statistically and discussed in the light of the available literature.     

Synthesis and Content of Polyamines in Bloodstream Trypanosoma brucei*

SYNOPSIS. The sensitive dansyl procedure was used to detect putrescine and spermidine, but not spermine and cadaverine, in pleomorphic Trypanosoma brucei. The polyamines were synthesized in vitro from [³H]ornithine, [¹⁴C]arginine and [¹⁴C]methionine. Proline, agmatine, and citrulline, but not glutamine, glutamic or pyroglutamic acids, stimulated spermidine formation from [¹⁴C]methionine. Putrescine and spermidine synthesis occurred rapidly from ornithine: putrescine synthesis peaked in 0.5 h, spermidine in 1 h. Trypanosoma brucei assimilated exogenous ¹⁴C-labeled putrescine, spermidine, and spermine; spermidine and spermine were taken up 5 times as rapidly as putrescine. Polyamine syntheses may therefore be a practical target for novel trypanocies.     

m-Chlorobenzhydroxamic Acid–an Inhibitor of Cyanide-Insensitive Respiration in Trypanosoma brucei*

Cyanide-insensitive respiration of bloodstream and culture forms of T. brucei was inhibited by m-chlorobenz-hydroxamic acid (m-CLAM). The cyanide-sensitive respiration of culture forms of this organism was not affected by m-CLAM. This compound is the 1st really effective inhibitor to be described that acts specifically on the cyanide-insensitive respiration of the T. brucei group of trypanosomes; as such it may be of some importance as a trypanocidal drug. Evidence is adduced which suggests that a branched electron transport chain is present in culture forms of T. brucei and that the cyanide-insensitive terminal oxidase found in these stages is the same as that found in bloodstream forms.     

Cryopreservation of Trypanosoma cruzi Bloodstream Forms*

SYNOPSIS. Recovery rates of T. cruzi bloodstream forms subjected to several methods of cryopreservation in liquid nitrogen and at -73 C are reported. Inoculations of animals with cryopreserved and nonpreserved trypomastigotes revealed that prolonged storage at -196 C apparently did not change the biologic characteristics of different T. cruzi strains. The reproducibility and consistency of results suggest that “cryobanks'’or “reference centers'’may be established.     

The Effect of Diphenylamine on Terminal Respiration in Bloodstream and Culture Forms of Trypanosoma brucei*

SYNOPSIS. Diphenylamine was shown to be a potent inhibitor of cyanide insensitive respiration in both bloodstream and newly established culture forms of the same isolate of Trypanosoma brucei, with the L-α-glycerophosphate oxidase system having the greatest sensitivity to the inhibitor. The NADH oxidase activity of bloodstream forms was at least twice as sensitive to diphenylamine as the corresponding activity in culture forms, suggesting different routes of NADH oxidation in the 2 forms. The oxidation of L-α-glycerophosphate was inhibited to a similar degree in both culture and bloodstream forms. L-α-glycerophosphate oxidation in bloodstream forms differed from that found in culture forms in that the bloodstream system, unlike that in the culture form, was unable to donate electrons to cytochrome c. In culture form trypanosomes there was a distinct difference in the degree of diphenylamine inhibition on the oxidation of L-α-glycerophosphate, NADH, and succinate, suggesting the participation of separate flavoproteins in the oxidation of these substrates.     

Quantitative Ultrastructural Differences between Strains of the Trypanosoma brucei Subgroup During Transformation in Blood*

SYNOPSIS. Differences in the relative and absolute cell organization between strains of the Trypanosoma brucei subgroup were studied during the transformation from slender to stumpy bloodforms. Two pleomorphic and 1 monomorphic T. b. brucei, and 1 pleomorphic T. b. rhodesiense strains were investigated. Volume densities, surface densities and surface to volume ratios showed barely significant differences between the 2 pleomorphic T. b. brucei strains; absolute parameters, however, differ markedly between all the strains investigated. Only the relative parameters of the mitochondrion show notable differences between T. b. brucei and T. b. rhodesiense examined here. During the transformation from slender to stumpy forms the enlargement of the mitochondrial volume in T. b. brucei is achieved by an increase in width of the mitochondrial tube and in T. b. rhodesiense by the formation of a more elaborate network. The ratio of the inner mitochondrial membrane surface area to the mitochondrial matrix volume showed no significant change in all 3 pleomorphic strains examined. Because of their morphometric similarity to slender forms of pleomorphic T. b. brucei strains, it can be assumed that the monomorphic trypanosomes correspond morphologically to slender trypanosomes. Neither pleomorphism nor strain specificity have a significant influence on the relative amount of “vesicles” and lipid inclusions.     

Cyclical Transmission of Trypanosoma brucei rhodesiense and Trypanosoma congolense by Tsetse Flies Infected with Culture-Form Procyclic Trypanosomes*

SYNOPSIS. Culture procyclic forms of Trypanosoma brucei rhodesiense and Trypanosoma congolense were fed to Glossina morsitans morsitans through artificial membranes. A very high percentage of the flies so fed produced established midgut infections, a proportion of which went on to develop into mature metacyclic trypanosomes capable of infecting mammalian hosts. The method offers a safe, clean way of infecting tsetse flies with African trypanosomes which reduces the need for trypanosome-infected animals in the laboratory.     

Phosphatase activity characterization on the surface of intact bloodstream forms of Trypanosoma brucei

Procyclic forms of Trypanosoma brucei possess a phosphatase activity on their external cell surface. This activity, while it dephosphorylates [(32)P]phosphocasein, is inhibited weakly by NaF and tartrate but strongly by vanadate. In this work, we describe the presence of an external phosphatase activity in intact bloodstream forms of T. brucei. With p-nitrophenyl phosphate (pNPP) as substrate, these intact cells produced 3-5 nmol pNP min(-1) mg(-1), linearly for up to at least 30 min. The activity was not significantly increased by Mg(2+), Mn(2+), Ca(2+) and Co(2+), but was inhibited by vanadate, NaF, p-chloromercuribenzoate and Zn(2+) and was insensitive to okadaic acid. Membrane-enriched fractions of parasites contained an acid phosphatase activity, with a pH optimum in the range of 4.5-5.5. This activity hydrolyzed phosphotyrosine (40 nmol phosphate min(-1) mg(-1)) better than phosphothreonine or phosphoserine. Partial purification of this phosphatase yielded a single activity band following gel electrophoresis, a K(m) value of 0.29 mM with pNPP and was insensitive to the Fe(2+)/H(2)O(2)/ascorbate system.     

Acetate Oxidation by Bloodstream Forms of Trypanosoma cruzi*

Bloodstream forms of Trypanosoma cruzi had a substantial increase in respiration in the presence of acetate. Oxidation of acetate took place via the tricarboxylic acid cycle and involved an antimycin A-sensitive respiratory pathway. Oxygen uptake in the presence of acetate was as sensitive to antimycin A inhibition as was CO₂ production. There was a 6–7% residual O₂ uptake which was not inhibited by high antimycin concentrations. Human anti-T. cruzi sera had no effect on oxygen uptake.     

Quantitative Ultrastructural Investigations of the Life Cycle of Trypanosoma brucei: A Morphometric Analysis*

SYNOPSIS. The quantitative ultrastructure of the developmental stages of Trypanosoma brucei brucei in its vector Glossina morsitans was studied by morphometric analysis. Values from ectoperitrophic midgut forms, proventricular forms, epimastigote and metacyclic forms in the salivary gland are compared with results from bloodstream forms, published previously. Significant differences in the volume densities of the trypanosome's single mitochondrion, of microbody-like organelles and in the surface densities of inner and outer mitochondrial membranes were found throughout the whole life cycle. A great increase in volume density of the mitochondrion was observed after transfer to the insect host; reduction took place during metacyclic development. Parallel to the biogenesis of the mitochondrion a reduction of microbodies was found in proventricular forms and there was a great increase in metacyclic forms concomitant with the regression of the mitochondrion. Metacyclic forms had a close quantitative morphologic similarity to bloodstream forms. The results are discussed in connection with changes in structure and in oxidative metabolism.     

Control of Trypanosoma brucei brucei infections in tsetse, Glossina morsitans

Abstract Numbers of immature Trypanosoma brucei brucei within a tsetse midgut remain remarkably constant after establishment throughout the course of an infection, irrespective of whether the infection eventually matures. These results suggest a system of self regulation of the parasite population in the insect gut based on a form of programmed cell death which would carry advantages for both the parasite and the vector.     

Infectivity of Trypanosoma brucei Cultivated at 28 C with Tsetse Fly Salivary Glands*

SYNOPSIS. When transformed procyclic noninfective trypanosomes of several unrelated stocks of Trypanosoma brucei were cultivated in T-30 Falcon flasks at 28 C in a liquid medium containing head-salivary gland explants of Glossina morsitans morsitans some of the organisms developed into forms infective for mice. Infective trypanosomes were detected 7 to 14 days after the cultures were prepared and they persisted for varying periods of up to 88 days when the cultures were terminated. A few of the salivary glands became invaded with parasites about the time infective organisms appeared in the cultures. Using T. brucei TREU 929, it was shown that trypanosomes grown with between 27 and 50 explants were capable of producing infections consistently for prolonged periods. On the other hand, trypanosomes cultivated with 25 or fewer explants rarely infected mice. Infectivity titrations on trypanosome suspensions from cultures of stocks TREU 1275 and TREU 929 revealed that the maximum number of infective organisms was present 26 to 50 days after initiation of the cultures. Control cultures of trypanosomes grown in medium alone were generally not infective but 2 of the 6 stocks gave rise to a few sporadic infections. A few epimastigote-like and metacyclic-like trypanosomes were seen in stained preparations of infective inocula.     

Freeze-Fracture Study of the Bloodstream Form of Trypanosoma brucei gambiense

The ultrastructure of Trypanosoma brucei gambiense was investigated by the freeze-fracture method. Three different regions of the continuous plasma membrane; cell body proper, flagellar pocket, and flagellum were compared in density and distribution of the intramembranous particles (IMP's). The IMP-density was highest in the flagellar pocket membrane and lowest in flagellum. Intra membranous particles of the cell body membrane were distributed uniformly on both the protoplasmic (P) and exoplasmic (E) faces. On the P face of the flagellar membrane, a single row of IMP-clusters was seen along the juncture of the flagllum to the cell body. Since the spacing of the IMP-clusters was almost equal to the spacing of the paired rivet structures observed in thin section, these clusters likely are related to the junction of flagellum and cell body. At the neck of the flagellar pocket, several linear arrays of IMP's were found on the P face of the flagellar membrane, while on the E face rows of depressions were seen. At the flagellar base, the clusters of IMP's were only seen on the P face. On the flagellar pocket membrane, particle-rich depressions and linear particle arrays were also found on the P face, while on the E face such special particle arrangements were not recognized. These particle-rich depressions may correspond to the sites of pinocytosis of the bloodstream forms which have been demonstrated in thin sections.     

The Utilization of Glucose and Proline by Culture Forms of Trypanosoma brucei*

Exponentially dividing culture forms of Trypanosoma brucei did not utilize glucose provided in the culture medium. The inclusion of 2-deoxyglucose in the medium had no effect on the growth of the trypanosomes. Glucose could be replaced by proline in the liquid phase of biphasic medium without affecting the doubling time of the organisms. Proline added to the culture medium in this way disappeared during the log phase of growth. Glucose in the culture medium was used by the trypanosomes only when the stationary growth phase had been reached. Lipid accumulated in stationary phase trypanosomes grown in glucose-containing medium, but there was no lipid accumulation in log phase organisms or in those which had been grown in proline-containing medium. Bloodstream trypanosomes transferred to liquid medium rapidly utilized glucose over the first 12 hr of culture, and this was accompanied by an accumulation of free pyruvate in the medium. The rate of glucose utilization fell off over the next 36 hr; this was accompanied by a lowering of free pyruvate in the medium and a rise in the proline oxidase activity of the trypanosomes. The possible biologic significance of proline to trypanosomes developing in the midgut of the tsetse vector is discussed.     

Curative Effects of the Antipiroplasms Amicarbalide and Imidocarb on Trypanosoma brucei Infection in Mice*

SYNOPSIS The babesicides imidocarb and amicarbalide, which have structural similarities to the antitrypanosomatid diamidines, proved active against Trypanosoma brucei mouse infections: both cured infections when doses were administered daily for 3 days 24 h post-inoculation (curative dose imidocarb, 10 mg/kg; amicarbalide, 25 mg/kg). Mice were considered cured after survival 30 days longer than untreated infected controls, with no trypanosomes present in blood or cerebrospinal fluid smears. Both agents also cured when administered 48 and 72 h after challenge with T. brucei and prolonged the lives of animals 94 h after challenge. The results are discussed in respect to the potential of these carbanilides and their precursors, the antitumor phthalanilides, as lead compounds in chemotherapy of mammalian trypanosomiases.     

Cryopreservation of Pathogenic African Trypanosomes In Situ: Metacyclic and Bloodstream Forms

SYNOPSIS. Of several methods developed for cryopreservation of Trypanosoma vivax, Trypanosoma congolense , and Trypanosoma brucei metacyclic forms in tsetse fly organs, as well as bloodstream forms in host blood, one proved the most satisfactory. In this method, infected fly proboscises and salivary glands were placed in glass capillary tubes containing fetal calf serum with 8% (v/v) glycerol as the cryoprotectant. The method for bloodstream forms involved the addition of glycerol directly to infected blood, which was then dispensed into capillary tubes. Next the tubes were placed in paper containers inside a glass test tube with a 5 mm thick plasticine jacket. The insulated assembly was suspended in the liquid nitrogen vapor phase in an LR-35 (Union Carbide) refrigerator for 45 min. Under these conditions, the cooling rate was 2 C/min. The frozen samples were transferred to permanent storage. The viability and infectivity of the preserved organisms were found to be satisfactory upon testing, and no antigenic changes were observed. Laboratory and field applications of the method are discussed.     

Molecular heterogeneity of the isolated surface glycoprotein from variant AnTat 1.1 of Trypanosoma brucei brucei

Variant surface glycoprotein (VSG) of Trypanosoma brucei brucei AnTat 1.1 was released by means of the procedure described by Baltz et al. ([1976], Ann. Immunol. [Inst. Pasteur] 127C, 761-774). The concanavalin-A chromatography yielded 3 VSG fractions according to the addition, in the elution buffer, of alpha-methyl-D-mannopyranoside, beta-mercaptoethanol, and sodium dodecyl sulfate. These VSG fractions showed heterogeneous behaviour on reverse-phase high performance liquid chromatography. The 3 VSG fractions as well as the myristylated VSG of AnTat 1.1 essentially consist of dimer VSG forms linked through a disulfide bridge, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis, under reducing and nonreducing conditions.