Trypanosoma brucei: differentiation of in vitro-grown bloodstream trypomastigotes into procyclic forms

Trypanosoma brucei strain 366D trypomastigotes grown at 37 degrees C in the presence of a human fibroblast cell line formed foci underneath the feeder cells whereas trypanosomes grown in the presence of a human epithelial cell line grew only in the culture supernatant. A culture system was developed to study the differentiation of bloodstream trypomastigotes grown in the epithelial cell system into procyclic trypomastigotes at 27 degrees C. The morphological differentiation into the procyclic form was complete by 48 h. Cell division did not occur until 30-40 h after transfer to 27 degrees C. Various characteristics of this system were examined, including the effect of the feeder layer, the type of medium, the presence of the metabolites cis-aconitate and citrate, the preadaptation period, and the trypanosome cell concentration. The respiration of the recently differentiated procyclic cells was less sensitive to inhibition by CN- than that of established procyclic forms, implying a delayed appearance of complete mitochondrial oxidative pathways. This trypanosome differentiation system has the advantage that the animal host is not needed and the entire process is carried out in in vitro culture.     

Trypanosoma brucei: loss of variable antigens during transformation from bloodstream to procyclic forms in vitro.

The loss of variable antigen from Trypansoma brucei during transformation from the bloodstream to the procyclic form in vitro has been monitored by agglutination and immunofluorescence reactions using antisera against both forms. Maximum agglutination of transforming trypanosomes with anti-culture form sera was obtained in 36–48 hr coinciding with loss of the surface coat as seen by electron microscopy. Agglutination with antisera against homologous bloodstream forms, however, reached a constant minimum but still positive level after 7–9 days: absorption of such antisera with culture or heterologous bloodstream forms reduced this period of persistent agglutinability to 72–84 hr, suggesting that the sera contained antibodies to “common” (surface membrane) antigens which became exposed when the surface coat was lost during transformation. The indirect immunofluorescence reaction provided a direct correlation of loss of antigen with loss of coat. The majority of trypanosomes lost detectable variable antigen by 36–48 hr, but a few flagellates, morphologically resembling bloodstream forms, retained the coat and capacity for labeling up to 84 hr; the numbers of such persistent bloodstream forms were shown to be sufficient to give a positive agglutination reaction for the population as a whole up to this time. Variable antigen appeared to be lost by dilution over the entire trypanosome surface rather than in patches or caps and the relevance of this observation to the process of antigenic variation is discussed.     

Novel sterol metabolic network of Trypanosoma brucei procyclic and bloodstream forms

Trypanosoma brucei is the protozoan parasite that causes African trypanosomiasis, a neglected disease of people and animals. Co-metabolite analysis, labelling studies using [methyl-2H3]-methionine and substrate/product specificities of the cloned 24-SMT (sterol C24-methyltransferase) and 14-SDM (sterol C14demethylase) from T. brucei afforded an uncommon sterol metabolic network that proceeds from lanosterol and 31-norlanosterol to ETO [ergosta-5,7,25(27)-trien-3β-ol], 24-DTO [dimethyl ergosta-5,7,25(27)-trienol] and ergosterol [ergosta-5,7,22(23)-trienol]. To assess the possible carbon sources of ergosterol biosynthesis, specifically 13C-labelled specimens of lanosterol, acetate, leucine and glucose were administered to T. brucei and the 13C distributions found were in accord with the operation of the acetate-mevalonate pathway, with leucine as an alternative precursor, to ergostenols in either the insect or bloodstream form. In searching for metabolic signatures of procyclic cells, we observed that the 13C-labelling treatments induce fluctuations between the acetyl-CoA (mitochondrial) and sterol (cytosolic) synthetic pathways detected by the progressive increase in 13C-ergosterol production (control<[2-(13)C]leucine<[2-(13)C]acetate<[1-(13)C]glucose) and corresponding depletion of cholesta-5,7,24-trienol. We conclude that anabolic fluxes originating in mitochondrial metabolism constitute a flexible part of sterol synthesis that is further fluctuated in the cytosol, yielding distinct sterol profiles in relation to cell demands on growth.     

Trypanosoma brucei: morphometric changes and loss of infectivity during transformation of bloodstream forms to procyclic culture forms in vitro.

The transformation of Trypanosoma brucei bloodstream forms to procyclic culture forms in the semidefined medium SDM-77 has been studied by light microscopy and quantitative electron microscopy. Stumpy and intermediate forms are able to transform to culture forms whereas slender forms die after approximately 24 hr. The surface coat and infectivity for the mammalian host are lost after 72 hr. Morphometrical analysis of the cells during transformation process revealed: (1) The cytoplasm and the cell surface increased significantly; (2) the volume density of the mitochondrion increased twofold and the surface density of the inner mitochondrial membrane increased threefold; (3) the volume density of the glycosomes remained about constant; (4) the volume density of the lipid inclusions increased up to 72 hr, probably as a result of the complete oxidation of glucose. Transformation as observed by light microscopy was completed in 72 hr. However, quantitative electron microscopy revealed that establishment of the culture form was incomplete even after 11 days.     

Two distinct succinate thiokinases in both bloodstream and procyclic forms of Trypanosoma brucei

Two succinate thiokinase activities specific for either adenine or guanine nucleotides have been found in Trypanosoma brucei. Key glycolytic and citric acid cycle enzymes were measured to show repression of glycolysis and derepression of the citric acid cycle in the procyclic form, relative to the bloodstream form. A marked rise in adenine-linked succinate thiokinase activity accompanied a rise in activity of citric acid cycle enzymes. However, guanine-linked succinate thiokinase was found to increase only slightly in activity. These results implicate the adenine-linked enzyme as an essential component of the citric acid cycle, whereas the guanine-linked enzyme appears to be under separate control. This communication also reports for the first time the occurrence of citrate synthase activity in the bloodstream (long slender) form of T. brucei.     

Trypanosoma brucei: in vitro slender-to-stumpy differentiation of culture-adapted,monomorphic bloodstream forms

Pleomorphic Trypanosoma brucei strains are characterized by their ability to differentiate from replicating long slender forms into non-dividing short stumpy forms in the mammalian host. The differentiation process can be efficiently induced in vitro by treatment with the membrane-permeable cAMP derivative 8-(4-chlorophenylthio)-cAMP (pCPTcAMP). In contrast, monomorphic T. brucei strains do not differentiate to stumpy forms in the host. Here, we show that exposure of monomorphic, culture-adapted T. brucei bloodstream forms to pCPTcAMP allowed their subsequent differentiation into short stumpy forms. The stumpy nature of pCPTcAMP-treated parasites was confirmed by (1) morphological change, (2) inhibition of growth and DNA synthesis, (3) cell cycle arrest in the G(1)/G(0) phase, (4) expression of NADH diaphorase activity and dihydrolipoamide dehydrogenase, (5) disappearance of the small subunit of ribonucleotide reductase, (6) up-regulation of the major lysosomal membrane protein, and (7) efficient transformation into replicating procyclic insect forms after induction with citrate/cis-aconitate. Our results indicate that the inability of monomorphic T. brucei bloodstream forms to differentiate into short stumpy forms in the host may be due to a failure in the signalling pathway rather than in the differentiation process itself. Treatment of monomorphic bloodstream trypanosomes with pCPTcAMP could be a useful method for identifying the genes involved in the slender-to-stumpy differentiation process.     

Comparative proteomics of glycosomes from bloodstream form and procyclic culture form Trypanosoma brucei brucei

Peroxisomes are present in nearly every eukaryotic cell and compartmentalize a wide range of important metabolic processes. Glycosomes of Kinetoplastid parasites are peroxisome-like organelles, characterized by the presence of the glycolytic pathway. The two replicating stages of Trypanosoma brucei brucei, the mammalian bloodstream form (BSF) and the insect (procyclic) form (PCF), undergo considerable adaptations in metabolism when switching between the two different hosts. These adaptations involve also substantial changes in the proteome of the glycosome. Comparative (non-quantitative) analysis of BSF and PCF glycosomes by nano LC-ESI-Q-TOF-MS resulted in the validation of known functional aspects of glycosomes and the identification of novel glycosomal constituents.     

Nonvariant antigens limited to bloodstream forms of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense

The presence of nonvariant antigens (NVAs) limited to bloodstream forms of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense was demonstrated for the first time by immunodiffusion and immunoelectrophoresis. Noncloned and cloned populations were employed in preparation of polyclonal antisera in rabbits and of antigens to be used in the immunologic reactions. The NVAs could be shown best in systems in which hyperimmune rabbit sera (adsorbed with procyclic forms to eliminate antibodies against antigens common to bloodstream form and procyclic stages) were reacted with trypanosomes characterized by heterologous variant-specific antigens (VSAs). The NVAs demonstrated in this study are very likely different from the common parts of VSAs. As has been suggested by experiments with living trypanosomes, at least a part of the NVAs appears to be located on the surface of the bloodstream forms. In these experiments involving the quantitative indirect fluorescent antibody test, the amount of fluorescence recorded for the heterologous system, i.e. ETat 5 trypanosomes incubated with anti-AmTat 1.1 serum, equalled approximately 3.0% of the fluorescence emitted by the AmTat 1.1 bloodstream forms treated with their homologous antiserum. Evidently, only small amounts of NVAs are present on the surfaces of T. brucei bloodstream forms. In addition to the NVAs, the electrophoresis results suggested the presence of antigenic differences between procyclic stages belonging to different T. brucei stocks.     

In vitro cloning of animal-infective bloodstream forms of Trypanosoma brucei

Clones of animal-infective bloodstream forms of Trypanosoma brucei (stocks S.427 and LUMP 227) were made by transferring a single organism from bloodstream-form cultures into each well of Microtest II Tissue Culture Plates containing bovine fibroblast-like feeder cells. When the number of trypanosomes increased to 10(2)--10(3)/well on days 4--16, they were transferred into plastic T-25 culture flasks also containing feeder cells and fresh medium. Cultures were thereafter maintained by partially replacing the trypanosome suspension with the same volume of fresh medium (diluting the density to 2--5 x 15(5) trypanosomes/ml) every 24 h. Sub-cultivations could be made by transferring 1--2 ml of the trypanosome suspension to a new culture flask at 4--5 day intervals. A total of 42 clones in the 3 series TC221, TC52 and TC227, carrying variable antigen types (VATs) 221, 052 and ILTat 1.4, respectively, have been established. Average population doubling times for clones of TC221, TC52 and TC227 were 8.7, 14.5 and 15.5 h respectively. Of 35 populations examined, 34 clones retained the original specificity of their VATs for at least 8--32 days from cloning. One cloned population of TC52 consisted of 99.8% VAT 052 and 0.2% VAT 221 at the time when the first VAT test was made on day 18.     

The malate dehydrogenase isoforms from Trypanosoma brucei: subcellular localization and differential expression in bloodstream and procyclic forms

Trypanosoma brucei procyclic forms possess three different malate dehydrogenase isozymes that could be separated by hydrophobic interaction chromatography and were recognized as the mitochondrial, glycosomal and cytosolic malate dehydrogenase isozymes. The latter is the only malate dehydrogenase expressed in the bloodstream forms, thus confirming that the expression of malate dehydrogenase isozymes is regulated during the T. brucei life cycle. To achieve further biochemical characterization, the genes encoding mitochondrial and glycosomal malate dehydrogenase were cloned on the basis of previously reported nucleotide sequences and the recombinant enzymes were functionally expressed in Escherichia coli cultures. Mitochondrial malate dehydrogenase showed to be more active than glycosomal malate dehydrogenase in the reduction of oxaloacetate; nearly 80% of the total activity in procyclic crude extracts corresponds to the former isozyme which also catalyzes, although less efficiently, the reduction of p-hydroxyphenyl-pyruvate. The rabbit antisera raised against each of the recombinant isozymes showed that the three malate dehydrogenases do not cross-react immunologically. Immunofluorescence experiments using these antisera confirmed the glycosomal and mitochondrial localization of glycosomal and mitochondrial malate dehydrogenase, as well as a cytosolic localization for the third malate dehydrogenase isozyme. These results clearly distinguish Trypanosoma brucei from Trypanosoma cruzi, since in the latter parasite a cytosolic malate dehydrogenase is not present and mitochondrial malate dehydrogenase specifically reduces oxaloacetate.     

Apoptosis in procyclic Trypanosoma brucei rhodesiense in vitro

Apoptosis is a phenomenon previously associated exclusively with metazoan organisms. We show here that procyclic insect form Trypanosoma brucei rhodesiense, a protozoan parasite, when treated in vitro with concanavalin A displayed several features normally associated with apoptosis in metazoan cells. Lectin treatment induced cleavage of nuclear DNA into oligonucleosomal fragments, suggesting activation of an endogenous nuclease in the parasite. Treated trypanosomes, although agglutinated and non-motile, exhibited fluorescence after treatment with the vital stain fluorescein diacetate and retained (3)H-uridine indicating that their cell membranes remained intact during the period of DNA fragmentation. Electron micrographs showed characteristic morphology of cells undergoing apoptosis, including surface membrane vesiculation and migration of chromatin to the periphery of the nuclear membrane while mitochondria remained intact. These results suggest that treatment with concanavalin A triggers a cell death mechanism in T. b. rhodesiense similar to the process of apoptosis described in metazoa.     

Lipid composition of bloodstream forms of Trypanosoma brucei brucei

The qualitative and quantitative lipid composition of bloodstream forms of Trypanosoma brucei brucei S42 was compared with that of rat plasma and erythrocytes. The concentrations of lipid-bound phosphate and lipid-bound sialic acid in the trypanosomes were markedly higher than those of the rodent tissues. There was no trace in the trypanosomes of dolichol or dolichol phosphate. Trypanosomes contained two unidentified lipids: an acidic phospholipid and a highly polar glycolipid.     

A Mitogen-activated protein kinase controls differentiation of bloodstream forms of Trypanosoma brucei

African trypanosomes undergo differentiation in order to adapt to the mammalian host and the tsetse fly vector. To characterize the role of a mitogen-activated protein (MAP) kinase homologue, TbMAPK5, in the differentiation of Trypanosoma brucei, we constructed a knockout in procyclic (insect) forms from a differentiation-competent (pleomorphic) stock. Two independent knockout clones proliferated normally in culture and were not essential for other life cycle stages in the fly. They were also able to infect immunosuppressed mice, but the peak parasitemia was 16-fold lower than that of the wild type. Differentiation of the proliferating long slender to the nonproliferating short stumpy bloodstream form is triggered by an autocrine factor, stumpy induction factor (SIF). The knockout differentiated prematurely in mice and in culture, suggestive of increased sensitivity to SIF. In contrast, a null mutant of a cell line refractory to SIF was able to proliferate normally. The differentiation phenotype was partially rescued by complementation with wild-type TbMAPK5 but exacerbated by introduction of a nonactivatable mutant form. Our results indicate a regulatory function for TbMAPK5 in the differentiation of bloodstream forms of T. brucei that might be exploitable as a target for chemotherapy against human sleeping sickness.     

Trypanosoma brucei brucei: regulation of ornithine decarboxylase in procyclic forms and trypomastigotes

Ornithine decarboxylase (ODC) activity was measured in procyclic forms of Trypanosoma brucei brucei grown in semidefined medium. ODC activity rapidly increased in late log-phase cells which were resuspended in fresh medium. A biphasic induction curve similar to that observed in mammalian cells was observed over an 18-hr period. ODC activity increased 4.5- to 25-fold over control levels measured at zero time. Actinomycin D and cycloheximide inhibited induction by greater than 90%. Polyamines at a level not inhibitory to growth (10 microM) inhibited ODC induction, but only by 30-50%, late in the induction period. Putrescine inhibited the first peak of induction and suppressed activity at 14 hr by 75%. Polyamine analogs such as bis(ethyl)spermidine were not effective suppressors of ODC activity. The half-life of ODC in procyclic forms grown in the presence of cycloheximide was greater than 6 hr, while that of bloodstream trypomastigotes in mice treated with cycloheximide was 5 hr. A single dose of the ODC inhibitor DL-alpha-difluoromethylornithine given to infected rats or mice suppressed trypanosome ODC activity greater than 90% for more than 7 hr. These studies indicate that although trypanosome ODC increases rapidly under log growth conditions, it is less susceptible to fluctuation and external control than the enzyme from mammalian sources. The latter may be a factor in the clinical efficacy of ODC inhibitors.     

Lipoamide dehydrogenase is essential for both bloodstream and procyclic Trypanosoma brucei

Lipoamide dehydrogenase (LipDH) is a component of four mitochondrial multienzyme complexes. RNA interference or the deletion of both alleles in bloodstream Trypanosoma brucei resulted in an absolute requirement for exogenous thymidine. In the absence of thymidine, lipdh-/- parasites showed a severely altered morphology and cell cycle distribution. Most probably, in bloodstream cells with their only rudimentary mitochondrion, LipDH is required as component of the glycine cleavage complex which generates methylene-tetrahydrofolate for dTMP and thus DNA synthesis. The essential role of LipDH in bloodstream parasites was confirmed by an in vivo model. Lipdh-/- cells were unable to infect mice. Our data further revealed that degradation of branched-chain amino acids takes place but is dispensable. In cultured bloodstream--but not procyclic--African trypanosomes, the total cellular concentration of LipDH increases with increasing cell densities. In procyclic parasites, LipDH mRNA depletion caused an even stronger proliferation defect that was not reversed by thymidine suggesting that in the fully elaborated mitochondrion of these cells the primary effect is not on the glycine cleavage complex. Since the medium used for the cultivation of procyclic cells was not supplemented with glucose, impairment of the 2-ketoglutarate dehydrogenase complex is probably the main effect of LipDH depletion.     

Calcium homeostasis in procyclic and bloodstream forms of Trypanosoma brucei. Lack of inositol 1,4,5-trisphosphate-sensitive Ca2+ release.

When Trypanosoma brucei procyclic trypomastigotes were permeabilized with digitonin in a reaction medium containing MgATP, succinate, and 3.5 microM free Ca2+, they lowered the medium Ca2+ concentration to the submicromolar level (0.05-0.1 microM), a range that correlates favorably with that detected in the intact cells with fura-2. The carbonyl cyanide p-trifluoromethoxyphenylhydrazone-insensitive Ca2+ uptake, certainly represented by the endoplasmic reticulum, was completely inhibited by 500 microM vanadate. When vanadate instead of carbonyl cyanide p-trifluoromethoxyphenylhydrazone was present, the Ca2+ set point was increased to 0.6-0.7 microM. The succinate dependence and carbonyl cyanide p-trifluoromethoxyphenylhydrazone sensitivity of the later Ca2+ uptake indicate that it may be exerted by the mitochondria. When bloodstream trypomastigotes were used, neither succinate nor alpha-glycerophosphate stimulated the mitochondrial Ca2+ uptake. The mitochondrial Ca2+ transport could be measured only in the presence of ATP and 500 microM vanadate to inhibit the endoplasmic reticulum uptake. Bloodstream trypomastigotes have a lower cytosolic Ca2+ concentration, as detected with fura-2 and a smaller extramitochondrial Ca2+ pool than procyclic trypomastigotes. Despite the presence of inositol phosphates, as determined by [3H]inositol incorporation, and the large extramitochondrial Ca2+ pool of procyclic trypomastigotes (61.7 nmol of Ca2+/mg of protein), no inositol 1,4,5-trisphosphate-sensitive Ca2+ release could be detected in these parasites.