Mechanism of Long Slender (LS) to Short Stumpy (SS) Transformation in the African Trypanosomes

The transformation of the long slender to the short stumpy stages of the African trypanosomes is an essential part of the trypanosome life cycle. Four possible mechanisms which could control this event have been investigated. It has been shown that (a) the dividing long slender to non-dividing short stumpy transition is not a programmed event in the trypanosome life cycle; nor (b) would it appear to be initiated by some form of cell to cell contact inhibition of growth. In addition, evidence is presented which would suggest that (c) the transition is not started by the depletion of a critical growth nutrient from the environment during the growth of the trypanosomes. The last possibility (d) considered is that during trypanosome growth, a growth inhibitor-short stumpy inducer accumulates in the trypanosomes'environment. Evidence is presented which shows that plasma from infected animals can inhibit the incorporation of thymidine by the trypanosomes. These data are consistent with the suggestion of an exogenous growth inhibitor accumulating during the infection.     

Mechanism of long slender (LS) to short stumpy (SS) transformation in the African trypanosomes

The transformation of the long slender to the short stumpy stages of the African trypanosomes is an essential part of the trypanosome life cycle. Four possible mechanisms which could control this event have been investigated. It has been shown that (a) the dividing long slender to non-dividing short stumpy transition is not a programmed event in the trypanosome life cycle; nor (b) would it appear to be initiated by some form of cell to cell contact inhibition of growth. In addition, evidence is presented which would suggest that (c) the transition is not started by the depletion of a critical growth nutrient from the environment during the growth of the trypanosomes. The last possibility (d) considered is that during trypanosome growth, a growth inhibitor-short stumpy inducer accumulates in the trypanosomes' environment. Evidence is presented which shows that plasma from infected animals can inhibit the incorporation of thymidine by the trypanosomes. These data are consistent with the suggestion of an exogenous growth inhibitor accumulating during the infection.     

Polyamine depletion following exposure to DL-alpha-difluoromethylornithine both in vivo and in vitro initiates morphological alterations and mitochondrial activation in a monomorphic strain of Trypanosoma brucei brucei

DL-alpha-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase (ODC), rapidly depletes cells of intracellular putrescine. When administered to animals and humans, DFMO cures acute infections of trypanosomiasis. In order to determine if the mechanism of drug action is related to initiation of transformation and biochemical alterations subsequent to polyamine depletion, trypanosome morphology and mitochondrial activation were studied in a monomorphic strain of Trypanosoma brucei brucei. Exposure of trypanosomes to DFMO in vivo in infected rodents or in vitro in culture resulted in a depletion of intracellular putrescine and a cessation of cell division without specific cytotoxicity. These events were followed by a transformation of the long slender bloodstream form to a short stumpy form via an intermediate morphology. Putrescine, the product of the ODC reaction, abrogates this effect. When introduced into SDM-79 medium, the intermediate form is capable of further transformation to an "insect" procyclic trypomastigote whereas the long slender form and short stumpy form are not. Short stumpy forms are incapable of binary fission and have lost their infectivity for the vertebrate host. In addition, the mitochondrial marker enzyme, NAD diaphorase, was found only in the short stumpy and intermediate forms. We hypothesize that the short stumpy phenotype may not be a viable stage in the natural transformation of the trypanosome from its mammalian host to the insect vector.     

Polyamine Depletion Following Exposure to DL-α-Difluoromethylornithine Both In Vivo and In Vitro Initiates Morphological Alterations and Mitochondrial Activation in a Monomorphic Strain of Trypanosoma brucei brucei

ABSTRACT. DL-α-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase (ODC), rapidly depletes cells of intracellular putrescine. When administered to animals and humans, DFMO cures acute infections of trypanosomiasis. In order to determine if the mechanism of drug action is related to initiation of transformation and biochemical alterations subsequent to polyamine depletion, trypanosome morphology and mitochondrial activation were studied in a monomorphic strain of Trypanosoma brucei brucei. Exposure of trypanosomes to DFMO in vivo in infected rodents or in vitro in culture resulted in a depletion of intracellular putrescine and a cessation of cell division without specific cytotoxicity. These events were followed by a transformation of the long slender bloodstream form to a short stumpy form via an intermediate morphology. Putrescine, the product of the ODC reaction, abrogates this effect. When introduced into SDM-79 medium, the intermediate form is capable of further transformation to an "insect" procyclic trypomastigote whereas the long slender form and short stumpy form are not. Short stumpy forms are incapable of binary fission and have lost their infectivity for the vertebrate host. In addition, the mitochondrial marker enzyme, NAD diaphorase, was found only in the short stumpy and intermediate forms. We hypothesize that the short stumpy phenotype may not be a viable stage in the natural transformation of the trypanosome from its mammalian host to the insect vector.     

Trypanosoma brucei: fluxes of the morphological variants in intact and X-irradiated mice

The number of dividing, slender, intermediate, and stumpy forms of Trypanosoma brucei in the blood of inbred mice changed daily. In both intact mice and mice which were exposed to whole body X-irradiation before infection, slender and dividing forms predominated during the first 72 hr of infection, and the number of intermediate and stumpy forms increased to a maximum between 72 and 140 hr. The rate at which stumpy forms accumulated in the blood and the number of these forms which eventually circulated were the same in both groups. However, the fluxes in the number of slender and dividing forms differed in intact and X-irradiated mice. In intact mice, the number of dividing forms in the blood decreased between 72 and 140 hr, and the number of slender forms decreased between 96 and 140 hr. In X-irradiated mice, the number of both these forms increased throughout infection. Electrophoresis of serum proteins and agglutination tests showed that X-irradiation severely depressed the ability of the mice to make antibody. Homogenates of spleen and bone marrow of intact mice contained many dividing forms throughout the infection. It is concluded that although host antibody does not directly induce the transformation of slender forms into stumpy forms, it may influence the morphological composition of the peripheral blood population of trypanosomes in several ways.     

Morphological changes in trypanosoma brucei rhodesiense following inhibition of polyamine biosynthesis in vivo

The effect of α-difluoromethylornithine (DFMO) treatment on the morphology of African trypanosomes was investigated. For this purpose inbred mice were immunosuppressed and infected with a clone of the protozoan blood parasite Trypanosoma brucei rhodesiense. The mice were then treated with DFMO, an irreversible inhibitor of ornithine decarboxylase, which inhibits polyamine synthesis. DFMO treatment in the absence of host immunity resulted in arrest of cytokinesis of the trypanosomes and many binucleated cells could be seen in blood smears. If mice were infected with a highly virulent trypanosome clone (ETat 1.10), which does not normally transform from long slender (LS) to short stumpy (SS) forms, DFMO treatment caused SS transformation to occur on days 3–4. This morphological SS transformation was substantiated by the presence of diaphorase activity and nuclear and mitochondrial changes. The results suggest a possible involvement of polyamines in the transformation from LS to SS forms.     

Trypanosoma brucei brucei and T. b. gambiense: Stumpy Bloodstream Forms Express More CB1 Epitope in Endosomes and Lysosomes than Slender Forms

CB1-glycoprotein is a component of flagellar pocket, endosome, and lysosome membranes of long, slender bloodstream forms of the Trypanosoma brucei subgroup of African trypanosomes. We have used immunoblotting, immunofluorescence, and cryoimmunoelectron microscopy to study CB1-glycoprotein expression as long, slender bloodstream forms of pleomorphic T. b. brucei and T. b. gambiense transform through intermediate stages into short, stumpy forms. Intermediate and stumpy forms express more CB1-glycoprotein than long, slender forms. These results, coupled with previous work showing that procyclic forms do not express CB1-glycoprotein, show that the expression of lysosomal membrane glycoproteins is regulated coordinately with other aspects of lysosome and endosome function as these trypanosomes go through their life cycle.     

Role of the long slender to short stumpy transition in the life cycle of the african trypanosomes

It is shown using mouse models that the African trypanosomes exert a significant drain upon their host's carbohydrate (energy) resources; and that the higher the parasitemia, the greater the energy demand. It is, therefore, hypothesized that the long slender (LS) to short stumpy (SS) transition evolved, in part, to help control the parasitemia and to increase host survival time. It is also suggested that the SS population is heterogeneous. One part of the population is tsetse infective, while a second older SS population is undergoing apoptotic-like events, which leads to their cell death and their stimulation of the host's immune response. This immune stimulation by the old dying SS forms would eliminate the major LS and SS variant antigen population, and produce the chronic relapsing infection. It is concluded that the SS stages during the apoptosis-like process are acting altruistically. They give their lives to insure the long-term survival of the host, and to insure renewed growth of the minor LS variants and new infective SS forms. This process is predicted to increase the probability for the successful transmission of the trypanosomes to a new host.     

The bloodstream differentiation-division of Trypanosoma brucei studied using mitochondrial markers.

In the bloodstream of its mammalian host, the African trypanosome Trypanosoma brucei undergoes a life cycle stage differentiation from a long, slender form to a short, stumpy form. This involves three known major events: exit from a proliferative cell cycle, morphological change and mitochondrial biogenesis. Previously, models have been proposed accounting for these events (Matthews & Gull 1994a). Refinement of, and discrimination between, these models has been hindered by a lack of stage-regulated antigens useful as markers at the single-cell level. We have now evaluated a variety of cytological markers and applied them to investigate the coordination of phenotypic differentiation and cell cycle arrest. Our studies have focused on the differential expression of the mitochondrial enzyme dihydrolipoamide dehydrogenase relative to the differentiation-division of bloodstream trypanosomes. The results implicate a temporal order of events: commitment, division, phenotypic differentiation.     

Modelling trypanosome chronicity: VSG dynasties and parasite density

A new mathematical model developed by Lythgoe et al. shows that the semi-predictable order of trypanosome antigenic variation can be generated by two parasite-intrinsic factors. The first is the different probabilities of antigen-gene activation that result from the different molecular mechanisms by which the genes become expressed. The second is the density-dependent differentiation of slender to stumpy cells. The study has important implications for understanding the dynamics of antigenic variation and for modelling the consequences of therapeutic strategies directed against trypanosomes.     

High-Throughput Chemical Screening for Antivirulence Developmental Phenotypes in Trypanosoma brucei

In the bloodstream of mammalian hosts, the sleeping sickness parasite, Trypanosoma brucei, exists as a proliferative slender form or a nonproliferative, transmissible, stumpy form. The transition between these developmental forms is controlled by a density-dependent mechanism that is important for the parasite''s infection dynamics, immune evasion via ordered antigenic variation, and disease transmissibility. However, stumpy formation has been lost in most laboratory-adapted trypanosome lines, generating monomorphic parasites that proliferate uncontrolled as slender forms in vitro and in vivo. Nonetheless, these forms are readily amenable to cell culture and high-throughput screening for trypanocidal lead compounds. Here, we have developed and exploited a high-throughput screen for developmental phenotypes using a transgenic monomorphic cell line expressing a reporter under the regulation of gene control signals from the stumpy-specific molecule PAD1. Using a whole-cell fluorescence-based assay to screen over 6,000 small molecules from a kinase-focused compound library, small molecules able to activate stumpy-specific gene expression and proliferation arrest were assayed in a rapid assay format. Independent follow-up validation identified one hit able to induce modest, yet specific, changes in mRNA expression indicative of a partial differentiation to stumpy forms in monomorphs. Further, in pleomorphs this compound induced a stumpy-like phenotype, entailing growth arrest, morphological changes, PAD1 expression, and enhanced differentiation to procyclic forms. This not only provides a potential tool compound for the further understanding of stumpy formation but also demonstrates the use of high-throughput screening in the identification of compounds able to induce specific phenotypes, such as differentiation, in African trypanosomes.     

Protein tyrosine phosphatase TbPTP1: A molecular switch controlling life cycle differentiation in trypanosomes

Differentiation in African trypanosomes (Trypanosoma brucei) entails passage between a mammalian host, where parasites exist as a proliferative slender form or a G0-arrested stumpy form, and the tsetse fly. Stumpy forms arise at the peak of each parasitaemia and are committed to differentiation to procyclic forms that inhabit the tsetse midgut. We have identified a protein tyrosine phosphatase (TbPTP1) that inhibits trypanosome differentiation. Consistent with a tyrosine phosphatase, recombinant TbPTP1 exhibits the anticipated substrate and inhibitor profile, and its activity is impaired by reversible oxidation. TbPTP1 inactivation in monomorphic bloodstream trypanosomes by RNA interference or pharmacological inhibition triggers spontaneous differentiation to procyclic forms in a subset of committed cells. Consistent with this observation, homogeneous populations of stumpy forms synchronously differentiate to procyclic forms when tyrosine phosphatase activity is inhibited. Our data invoke a new model for trypanosome development in which differentiation to procyclic forms is prevented in the bloodstream by tyrosine dephosphorylation. It may be possible to use PTP1B inhibitors to block trypanosomatid transmission.     

Adaptations in the Glucose Metabolism of Procyclic Trypanosoma brucei Isolates from Tsetse Flies and during Differentiation of Bloodstream Forms

Procyclic forms of Trypanosoma brucei isolated from the midguts of infected tsetse flies, or freshly transformed from a strain that is close to field isolates, do not use a complete Krebs cycle. Furthermore, short stumpy bloodstream forms produce acetate and are apparently metabolically preadapted to adequate functioning in the tsetse fly.African trypanosomatids comprise various pleomorphic trypanosome species that proliferate in the bloodstream of their mammalian hosts as long slender bloodstream form (BSF) trypanosomes, and at the peak of parasitemia they differentiate into nondividing short stumpy form trypanosomes (1). After being ingested during a bloodmeal by a tsetse fly (Glossina sp.), short stumpy form trypanosomes differentiate into procyclic form (PCF) trypanosomes, which actively multiply and colonize the midgut of the fly. Subsequently, PCF Trypanosoma brucei migrates to the salivary glands while undergoing a complex differentiation (22). Here, attached epimastigote forms start multiplying, after which nondividing metacyclic trypomastigotes develop. The life cycle of T. brucei is completed when these metacyclic trypomastigotes are injected into a mammal through the bite of an infected fly, after which they transform into long slender BSF trypanosomes. During this life cycle, trypanosomes encounter different environments to which they have adapted, resulting in distinct stages, characterized by morphological as well as metabolic changes. Long slender BSF trypanosomes degrade glucose by glycolysis and excrete pyruvate as the sole metabolic end product (12, 13, 23). On the other hand, PCF trypanosomes do not excrete pyruvate but degrade glucose to acetate and succinate as main end products (25). Krebs cycle activity was thought previously to be present in trypanosomatids, at least in insect stages of some African trypanosomatids (3, 9, 10, 12, 21). However, this presumed flux through the Krebs cycle is supported only poorly by direct experimental evidence and was based mainly on the presence of certain enzyme activities. Although genes for all enzymes of this cycle are indeed present in the genome and expressed in the insect stages, recent studies revealed that at least in T. brucei, the cycle is not used for the complete oxidation of acetyl-coenzyme A (CoA) to carbon dioxide (2, 26). Instead, parts of the cycle are most likely used in anabolic pathways, such as gluconeogenesis and fatty acid formation, and also for the final steps in the degradation of amino acids (26). It is possible that the reported discrepancies on the presence or absence of full-circle Krebs cycle activity are caused by differences in the number of passages through mice after the isolation of the strain from the field. Such passages may have been ongoing for many years, during which the parasites were continuously propagated as BSF trypanosomes. Furthermore, most insect form trypanosomes that were investigated up to now have been propagated for many years as PCF trypanosomes in rich culture media. Hence, the reported discrepancies could be due to differences between freshly differentiated PCF trypanosomes and those well adapted to in vitro culture, and the absence of an active Krebs cycle in PCF trypanosomes could be the result of an adaptation caused by the prolonged in vitro culturing. To investigate these possibilities, we analyzed the glucose metabolism of PCF T. brucei directly after isolation from the midguts of tsetse flies. We also studied freshly differentiated PCF trypanosomes from the AntAR 1 strain, a T. brucei strain that has had a minor history of animal passaging since its field isolation (15, 17).To investigate the cause of the conflicting reports on Krebs cycle activity in PCF trypanosomes, we first analyzed the effect of environmental factors by comparing the carbohydrate metabolism of PCF trypanosomes well adapted to in vitro culturing and PCF trypanosomes isolated from their natural environment, the midguts of tsetse flies. These experiments were performed with PCF TREU 927 T. brucei, a pleomorphic strain that has been thoroughly characterized and is still able to infect Glossina morsitans, performing a complete physiological life cycle (2). For the infection of tsetse flies, male G. morsitans flies originating from the colony maintained at the Institute of Tropical Medicine in Antwerp, Belgium, were infected with procyclic TREU 927 T. brucei by in vitro membrane feeding and subsequently maintained for 10 days by feeding on rabbit blood (15). Then, flies were dissected on a sterile glass slide and the infected midguts were isolated and incubated for at least 30 min at 28°C in SDM-79 medium that was gently rotated. After sedimentation of the midguts by gravity, insect gut debris was removed by centrifugation at 300 × g for 5 min. PCF trypanosomes were then isolated from the collected supernatant by centrifugation at 1,500 × g for 10 min. Since PCF trypanosomes could not be isolated from the midgut without minor amounts of contaminating insect gut material, such as gut cells and debris, we also investigated the glucose metabolism of this fraction. Analysis of metabolic end products produced from [6-¹⁴C]glucose in this control incubation of insect gut debris, which also contained minor amounts of trypanosome cells, showed the formation of ¹⁴C-labeled pyruvate, CO₂, acetate, and lactate (Fig. ​(Fig.1A).1A). Minor amounts of lactate were also produced in the incubations with PCF trypanosomes isolated from the midgut, which also contained minor amounts of insect gut debris. Since lactate is not an excreted end product of T. brucei, this labeled lactate is indicative for the glucose degradation activity of insect gut debris. Therefore, end product formation in the incubations with PCF trypanosomes isolated from the midgut was corrected for end products produced by the contaminating insect gut debris by subtracting all produced lactate and the calculated accompanying amounts of other end products produced in the insect gut debris incubation. The metabolic incubations with PCF trypanosomes directly after isolation from the tsetse midgut showed that these trypanosomes degrade glucose to the same metabolic end products, acetate, succinate, and pyruvate, as the in vitro culture-adapted PCF trypanosomes (Fig. ​(Fig.1A).1A). Furthermore, the ratio of acetate and succinate produced by PCF trypanosomes isolated from the midgut were similar to that of in vitro-cultured PCF trypanosomes (Fig. ​(Fig.1A).1A). On the other hand, a major difference was observed in the amount of glucose consumed since the PCF trypanosomes isolated from the midguts of tsetse flies consumed 16-fold less glucose than PCF trypanosomes that were derived from in vitro cultures. This difference in glucose consumption can probably be explained by our observation that both motility and especially growth of PCF trypanosomes isolated from the midgut were significantly reduced compared to the in vitro culture-derived PCF trypanosomes. Apparently, the environmental conditions in the midgut of the fly did affect the PCF trypanosomes, but they did not significantly alter the metabolic pathways used for energy metabolism. However, PCF trypanosomes isolated from the midgut of the fly excreted more pyruvate (Fig. ​(Fig.1A),1A), which suggests that pyruvate is a more important metabolic end product for PCF trypanosomes under physiological conditions than acknowledged thus far. Most importantly, however, just like continuously in vitro-cultured ones, PCF trypanosomes isolated from the midgut of the fly did not degrade [6-¹⁴C]glucose to labeled CO₂ (Fig. ​(Fig.1A),1A), which demonstrates the absence of a functional Krebs cycle in these tsetse fly-derived PCF trypanosomes.Open in a separate windowFIG. 1.Radioactive end products of [6-¹⁴C]glucose metabolism of procyclic TREU 927 T. brucei cells grown in vitro or isolated from the midguts of tsetse flies (A) and that of AntAR 1 T. brucei during differentiation of BSF to PCF trypanosomes (B). (A) The results of a single experiment for PCF trypanosomes isolated from the midgut and for insect gut debris and the mean + the standard deviation (SD) of three parallel incubations for in vitro-cultured PCF trypanosomes are shown. Total end product formation from [6-¹⁴C]glucose was 2.08 ± 0.19 μmol/h per 10⁸ cells and 0.23 μmol/h per 10⁸ cells for in vitro-cultured and midgut-isolated PCF trypanosomes, respectively, and was calculated using the number of trypanosome cells present at the beginning of the incubation. End product formation in the incubation with PCF trypanosomes isolated from the midgut was corrected for end products produced by contaminating insect gut debris (see text for details). (B) Metabolic incubations using 6-¹⁴C-labeled glucose were performed during differentiation from short stumpy BSF trypanosomes to insect stage PCF trypanosomes. Incubations with PCF trypanosomes were started at 24, 48, and 96 h after induction of differentiation (PCF trypanosomes on day 1, PCF trypanosomes on day 2, and PCF trypanosomes on day 4, respectively); means + SDs of three parallel incubations are shown (for the short stumpy form, six incubations in two independent experiments). Total glucose consumption in incubations with long slender BSF trypanosomes, short stumpy BSF trypanosomes, PCF trypanosomes on day 1, PCF trypanosomes on day 2, and PCF trypanosomes on day 4 was 4.8, 3.4, 1.5, 1.1, and 0.79 μmol/h per 10⁸ cells, respectively. Excreted labeled end products shown in panels A and B were analyzed as described previously (25) and are expressed as the percentage of the total amount of radioactive end products produced (in the incubation of gut debris, one other unidentified end product was produced, which explains why this total in the figure does not add up to 100%). The decrease in pyruvate production between long slender and short stumpy BSF trypanosomes as well as the increase in acetate production is significant as calculated using an unpaired t test (P < 0.01 for pyruvate and P < 0.001 for acetate).Although TREU 927 T. brucei is a pleomorphic trypanosome strain, it cannot be excluded that these trypanosomes have adapted their energy metabolism during the substantial period that this strain has been cultured in vitro. Therefore, we also studied the carbohydrate metabolism of freshly transformed PCF of the T. brucei AntAR 1 strain, a well-characterized pleomorphic strain that is close to the wild isolate (17). To investigate the energy metabolism of these freshly differentiated PCF trypanosomes, AntAR 1 BSF trypanosomes were harvested from the blood of infected immune-suppressed NMRI mice as described previously (16) and either directly incubated with [6-¹⁴C]glucose or differentiated to PCF trypanosomes, by addition of 6 mM cis-aconitate and incubation at 27°C (7). These trypanosomes were then incubated with [6-¹⁴C]glucose at different time points after the initiation of differentiation. Our experiments (Fig. ​(Fig.1B)1B) confirmed that differentiation of trypanosomes from BSF to PCF is accompanied by a metabolic shift in excreted end products from pyruvate to acetate and succinate (3, 14, 25). This metabolic shift during differentiation of BSF to PCF trypanosomes was complete after 1 to 2 days (Fig. ​(Fig.1B),1B), which is in agreement with previous observations (9). A subsequent switch in medium from HMI-9, a medium used to culture BSF T. brucei, to SDM-79, a medium used for the culture of PCF T. brucei, did not result in further changes in excreted end products (data not shown).Our experiments, however, did not show any significant production of labeled CO₂ and certainly not the massive increase in CO₂ formation upon differentiation of BSF into PCF trypanosomes that was reported in a comparable study by Durieux et al. (9). We cannot exclude that this difference in Krebs cycle activity between our study and that of Durieux et al. is caused by a strain difference, but since the AntAR 1 strain we used can be considered to be close to the field isolate, the results presented here are indicative of wild-type T. brucei metabolism and strongly suggest that a functional Krebs cycle is absent in PCF T. brucei cells in vivo.Next to the absence of carbon dioxide formation via Krebs cycle activity during differentiation of BSF to PCF trypanosomes, our metabolic experiments also demonstrated that acetate accounted for 30% of the glucose-derived excreted labeled end products in freshly isolated BSF AntAR 1 T. brucei cells (Fig. ​(Fig.1B).1B). This is a surprising observation since BSF trypanosomes are reported to rely on glycolysis only and to excrete pyruvate and minor amounts of glycerol (12, 13, 23). However, the BSF trypanosomes that we tested in our incubations were predominantly short stumpy BSF cells, whereas nearly all previously performed metabolic studies of BSF trypanosomes were performed with long slender BSF cells. In order to investigate whether differentiation from long slender to short stumpy form trypanosomes indeed shifts the metabolism toward acetate formation, we analyzed the energy metabolism of BSF trypanosomes harvested from mice at two different time points after infection. At day 4 after infection, predominantly long slender BSF trypanosomes were isolated (94% long slender versus 6% short stumpy), whereas at day 7 after infection, predominantly short stumpy BSF trypanosomes were isolated (92% short stumpy versus 8% long slender). Analysis of glucose-derived metabolic end products from incubations with BSF AntAR 1 trypanosomes isolated at day 4 or at day 7 after infection showed that short stumpy BSF trypanosomes indeed produce significant amounts of acetate as an end product of glucose metabolism (Fig. ​(Fig.1B).1B). In the incubations with predominantly long slender BSF AntAR 1 T. brucei cells, some acetate was also produced, but this relatively small amount of acetate formation can be explained by the presence of a certain amount of short stumpy cells. Although the incubations were started with nearly 95% long slender BSF cells, BSF cells from the AntAR 1 strain are highly pleomorphic and rapidly differentiate to short stumpy forms during in vitro culture conditions. Therefore, increasing amounts of short stumpy form T. brucei were formed during our incubations (up to 40 to 50% at the end of incubation), which accounts for the amount of acetate formed during these incubations.Since acetate production in Trypanosomatidae is catalyzed by the mitochondrial enzyme acetate:succinate CoA transferase (ASCT), which was previously shown not to be expressed in in vitro-cultured BSF T. brucei (20), we examined the ASCT enzyme activity in lysates derived from either over 92% short stumpy cells or 94% long slender cells. These experiments showed that the ASCT enzyme is present in short stumpy BSF trypanosomes in an amount equivalent to around 15% of that of PCF trypanosomes (Fig. ​(Fig.2).2). This is in agreement with the observation that acetate is a more prominent excreted end product in PCF trypanosomes than in short stumpy BSF cells. On the other hand, ASCT activity was nearly absent in long slender BSF trypanosomes (Fig. ​(Fig.2),2), which confirms the conclusion that in our incubations acetate is not produced by long slender BSF trypanosomes but by short stumpy BSF trypanosomes.Open in a separate windowFIG. 2.ASCT activity in total lysates of T. brucei AntAR 1. Enzymatic activity of ASCT was determined in total lysates derived from cultures containing predominantly long slender BSF trypanosomes (BSF LS; 94%), predominantly short stumpy BSF trypanosomes (BSF SS; 92%), or exclusively PCF trypanosomes (PCF). Shown are the means + standard deviations of three experiments.Hence, our experiments show that short stumpy BSF trypanosomes do not only degrade glucose by glycolysis but additionally produce acetate. Acetate formation in trypanosomes occurs via the mitochondrial enzyme ASCT and involves transfer of a CoA moiety from acetyl-CoA to succinate, yielding succinyl-CoA (24). This succinyl-CoA can then be converted back into succinate by succinyl-CoA synthetase, a reaction concomitantly converting ADP in ATP (6, 24). Therefore, our observations that short stumpy BSF trypanosomes produce acetate and express ASCT demonstrate that these stages in addition to glycolysis also use a mitochondrial pathway for the degradation of glucose and production of ATP.Multiple mitochondrial adaptations have been reported to occur during the transition from long slender BSF to short stumpy BSF T. brucei. Differential gene expression and the formation of cristea in the inner mitochondrial membrane have been shown to occur during this transition (8, 11, 19). Furthermore, the trypanosomal homologue of complex I of the respiratory chain is expressed in short stumpy BSF trypanosomes (4, 5, 18). Our experiments show that this more elaborate composition of the electron transport chain is also used by this stage, as the production of acetate implies that acetyl-CoA is formed, which is catalyzed by the pyruvate dehydrogenase complex and results in the production of NADH inside the mitochondrion. This means that either complex I or the alternative NADH dehydrogenase is active in this stage (18). Moreover, our experiments show that the previously reported mitochondrial adaptations in short stumpy BSF trypanosomes are not restricted to morphological changes and to changes in the composition of the electron transport chain but also result in a functionally altered energy metabolism.In conclusion, the data described in this paper demonstrate the absence of a functional Krebs cycle in the mitochondria of PCF T. brucei, isolated from the tsetse midgut or freshly differentiated from BSF trypanosomes. Furthermore, we show that short stumpy BSF T. brucei cells produce large amounts of acetate. Therefore, the mitochondria of short stumpy trypanosomes are metabolically divergent from the mitochondria in long slender BSF T. brucei cells. These results are consistent with prior work (4, 5, 8, 11). The functional changes might be a preadaptation that allows short stumpy BSF T. brucei to function in the intestines of infected tsetse flies and enables them to differentiate further into PCF trypanosomes.     

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.     

Differential protein synthesis during the life cycle of the protozoan parasite Trypanosoma brucei

Two-dimensional polyacrylamide gel electrophoresis has been used to analyze changes in protein content and protein synthesis in three stages of the life cycle of the protozoan parasite Trypanosoma brucei. The stages examined were slender and stumpy mammalian bloodstream forms and procyclic forms, which are analogous to the tsetse fly midgut stage. Two-dimensional gels of 35S-methionine-labeled proteins were examined by autoradiography to analyze newly synthesized protein, and gels were stained with ammoniacal silver to analyze proteins present. Several stage-specific molecules were noted. The most obvious was the variant surface glycoprotein, which was only present in bloodstream forms. Some other proteins were also bloodstream form specific; they had molecular weights of 120,000 and 38,000. Proteins of 52,000, 46,000, 25-30,000, and 16,000 daltons were present both in stumpy forms and procyclics but not in slender-form trypanosomes. Several proteins (molecular weights of 50-70,000, 43,000, 40,000, 26-24,000, 20-25,000, and 15,000) were present only in one of the three stages. One protein, a molecule of about 18,000 daltons present in both slender and stumpy parasites, did not appear to be synthesized in the stumpy stage. In vitro translation products of mRNA purified from the three stages were also examined. The abundance of mRNA encoding a protein of about 40,000 daltons appeared to be greater in slender than in stumpy parasites although the stumpy forms contained more of the protein and synthesized it at a higher rate.     

Deletion of a novel protein kinase with PX and FYVE-related domains increases the rate of differentiation of Trypanosoma brucei

Growth control of African trypanosomes in the mammalian host is coupled to differentiation of a non-dividing life cycle stage, the stumpy bloodstream form. We show that a protein kinase with novel domain architecture is important for growth regulation. Zinc finger kinase (ZFK) has a kinase domain related to RAC and S6 kinases flanked by a FYVE-related zinc finger and a phox (PX) homology domain. To investigate the function of the kinase during cyclical development, a stable transformation procedure for bloodstream forms of differentiation-competent (pleomorphic) Trypanosoma brucei strains was established. Deletion of both allelic copies of ZFK by homologous recombination resulted in reduced growth of bloodstream-form parasites in culture, which was correlated with an increased rate of differentiation to the non-dividing stumpy form. Growth and differentiation rates were returned to wild-type level by ectopic ZFK expression. The phenotype is stage-specific, as growth of procyclic (insect form) trypanosomes was unaffected, and Deltazfk/Deltazfk clones were able to undergo full cyclical development in the tsetse fly vector. Deletion of ZFK in a differentiation-defective (monomorphic) strain of T. brucei did not change its growth rate in the bloodstream stage. This suggests a function of ZFK associated with the trypanosomes' decision between either cell cycle progression, as slender bloodstream form, or differentiation to the non-dividing stumpy form.