A potential hexose transporter gene expressed predominantly in the bloodstream form of Trypanosoma brucei.

A cDNA cloned from Trypanosoma brucei brucei codes for a putative membrane protein which is homologous to the erythrocyte glucose transporter and several other sugar transporters from Escherichia coli, yeast, algae and Leishmania. This cDNA hybridizes to a 2.3-kb mRNA that accumulates to a much higher degree in the bloodstream mammalian form than in the procyclic insect form of the parasite. The correlation between the expression of this gene and the hexose metabolism of Leishmania enriettii and T. brucei suggest that these 2 related genes probably encode hexose transporters. The gene encoding this mRNA is a member of a multigene family. The putative hexose transporter gene is highly conserved among Kinetoplastidae, indicating an important role for this protein in the parasite life cycle.     

Distinct patterns of tyrosine phosphorylation during the life cycle of Trypanosoma brucei.

Regulation of tyrosine phosphorylation is a critical element in controlling growth and differentiation in higher eukaryotes. We have determined that the protozoan Trypanosoma brucei, which diverged early in the eukaryotic lineage, possesses multiple proteins which react with a specific anti-phosphotyrosine antiserum. Anti-phosphotyrosine immunoprecipitates of [32P]orthophosphate-labeled cells were shown to contain phosphotyrosine by two-dimensional electrophoresis. Western analysis of cells from different stages of the life cycle demonstrates the appearance of tyrosine-phosphorylated proteins at 40-42 kDa during the transition from slender to stumpy blood-forms. Growth of procyclic form cells in orthovanadate resulted in increased levels of specific tyrosine-phosphorylated proteins. The demonstration of phosphotyrosine-containing proteins in T. brucei and their differential regulation during the life cycle suggests that tyrosine kinases and phosphatases may play an important role in the biology of primitive protozoa.     

A novel telomeric gene conversion in Trypanosoma brucei

Gene conversion is one mechanism of antigenic variation in Trypanosoma brucei. Variant surface glycoprotein (VSG) genes are duplicated by this process to telomeric locations from which they may be expressed. We examined four independent antigenic switches in which the IsTaR 1.1 minichromosomal VSG gene is duplicated to a large chromosome where it is expressed. An unusual feature of three of these telomeric gene conversions is that the distance between the VSG gene and the end of the chromosome is identical for both the basic and duplicated copies following the antigenic switch. This suggests that the gene conversion is initiated 5' to the VSG gene and extends to the end of the telomere. The data also suggest that events other than simple nucleotide addition account for telomeric growth.     

Differential regulation of nucleoside and nucleobase transporters in Crithidia fasciculata and Trypanosoma brucei brucei

The regulation of the activity of purine transporters in two protozoan species, Crithidia fasciculata and Trypanosoma brucei brucei, was investigated in relation to purine availability and growth cycle. In C. fasciculata, two high-affinity purine nucleoside transporters were identified. The first, designated CfNT1, displayed a K(m) of 9.4 +/- 2.8 microM for adenosine and was inhibited by pyrimidine nucleosides as well as adenosine analogues; a second C. fasciculata nucleoside transporter (CfNT2) recognized inosine (K(m) = 0.38 +/- 0.06 microM) and guanosine but not adenosine. The activity of both transporters increased in cells at mid-logarithmic growth, as compared to cells in the stationary phase, and was also stimulated 5-15-fold following growth in purine-depleted medium. These increased rates were due to increased Vmax values (K(m) remained unchanged) and inhibited by cycloheximide (10 microM). In the procyclic forms of T. b. brucei, adenosine transport by the P1 transporter was upregulated by purine starvation but only after 48 h, whereas hypoxanthine transport was maximally increased after 24 h. The latter effect was due to the expression of an additional hypoxanthine transporter, H2, that is normally absent from procyclic forms of T. b. brucei and was characterised by its high affinity for hypoxanthine (K(m) approximately 0.2 microM) and its sensitivity to inhibition by guanosine. The activity of the H1 hypoxanthine transporter (K(m) approximately 10 microM) was unchanged. These results show that regulation of the capacity of the purine transporters is common in different protozoa, and that, in T. b. brucei, various purine transporters are under differential control.     

The electrochemical proton gradient in the bloodstream form of Trypanosoma brucei is dependent on the temperature.

The membrane potential and pH gradient over the plasma membrane of the protozoan parasite Trypanosoma brucei were measured with radioactive indicators in combination with the silicone oil centrifugation technique over a range of temperatures. At 37 degrees C a small membrane potential and pH gradient of similar magnitude, but of opposite polarity, were measured. The resulting electrochemical proton gradient was almost zero. However, when the temperature was lowered from 37 degrees C to 22 degrees C, the internal pH was kept constant independent of the external pH and a membrane potential of between -100 and -150 mV was measured, depending on the external pH. Measurements at various temperatures between 15 degrees C and 37 degrees C revealed that above 26 degrees C the membrane potential collapsed and that this collapse correlated with a sudden increase in membrane fluidity. The uptake of 2-deoxy-D-glucose and of pyruvate, which are both mediated by facilitated diffusion carriers in the plasma membrane of the trypanosome, were also affected by this sudden increase in fluidity of the membrane. The overall rate of the conversion of glucose into its metabolites, which is independent of the plasma membrane, varied only gradually. We conclude (i) that major changes occur in the plasma membrane of T. brucei around 26 degrees C, that affect all membrane related processes; (ii) that the electrochemical proton gradient plays a minor role in the energy metabolism of T. brucei when it resides in the bloodstream of the mammalian host at 37 degrees C; and (iii) that below 26 degrees C an electrochemical proton gradient is maintained over the plasma membrane.     

Identification and characterization of trypanocides by functional expression of an adenosine transporter from Trypanosoma brucei in yeast

The causative agents of sleeping sickness, Trypanosoma brucei rhodesiense and T. brucei gambiense, do not synthesize purines de novo but salvage purine bases and nucleosides from their hosts. We used yeast as an expression system for functional characterization of the trypanosomal adenosine transporter TbAT1. A selection of purine analogs and flavonoids were tested for their ability to interfere with adenosine transport, with the aims of identifying (a) trypanocidal TbAT1 substrates, and (b) inhibitors of trypanosomal purine transport. Cordycepin (3'-deoxyadenosine) was a TbAT1 substrate of high activity against T. brucei rhodesiense (IC50 0.2 nM). Inhibitors of mammalian nucleoside transport were not active, while the flavonol silibinin was a potent, noncompetitive inhibitor of TbAT1-mediated adenosine transport in yeast. Silibinin also inhibited melarsen-induced lysis of bloodstream form trypanosomes. IC50 values to T. brucei rhodesiense and to human carcinoma cells were 0.6 and 140 microM, respectively, indicating a good selectivity towards the parasites. Further studies are necessary to elucidate the effects of flavonoids on trypanosomal purine transport and their potential as trypanocides.     

Differential expression of the oligomycin-sensitive ATPase in bloodstream forms of Trypanosoma brucei brucei.

We report the differential expression of the oligomycin-sensitive mitochondrial ATPase in pleomorphic bloodstream forms of Trypanosoma brucei brucei as observed with enzymatic assays and electron microscope histochemistry. As the cells differentiate from long slender to short stumpy forms, total specific activity of the mitochondrial ATPase in a crude mitochondrial fraction doubles and the oligomycin-sensitive specific activity increases 5-fold. Upon in vitro differentiation to procyclic forms, there is a further doubling of total specific activity and a further tripling of oligomycin-sensitive specific activity. The oligomycin-insensitive ATPase activity remained essentially constant throughout differentiation. We have attempted to characterize this oligomycin-insensitive activity utilizing inhibitors of several other ATPases.     

Analysis of the Trypanosoma brucei cell cycle by quantitative DAPI imaging

Trypanosoma brucei has two DNA compartments: the nucleus and the kinetoplast. DNA replication of these two compartments only partially coincides. Woodward and Gull [Woodward R, Gull K. Timing of nuclear and kinetoplast DNA replication and early morphological events in the cell cycle of Trypanosoma brucei. J Cell Sci 1990;95:49-57] comprehensively studied the relative timing of the replication and segregation of nuclear DNA (nDNA) and kinetoplast DNA (kDNA). Others have since assumed the consistency of morphological indicators of cell-cycle stage among strains and conditions. We report the use of quantitative DAPI imaging to determine the cell-cycle stage of individual procyclic cells. Using this approach, we found that kinetoplast elongation occurs mainly during nuclear S phase and not during G2, as previously assumed. We confirmed this finding by sorting cells by DNA content, followed by fluorescence microscopy. In addition, simultaneous quantitative imaging at two wavelengths can be used to determine the abundance of cell-cycle-regulated proteins during the cell cycle. We demonstrate this technique by co-staining for the non-acetylated state of lysine 4 of histone H4 (H4K4), which is enriched during nuclear S phase.     

A repertoire of cell cycle regulators whose expression is coordinated with human cytotrophoblast differentiation

Although placental development depends on careful coordination of trophoblast proliferation and differentiation, little is known about the mitotic regulators that are key to synchronizing these events. We immunolocalized a broad range of these regulators in tissue sections of the maternal-fetal interface (first trimester through term) that contained floating villi (which include cytotrophoblasts differentiating into syncytiotrophoblasts) and anchoring villi (which include cytotrophoblasts differentiating into invasive cells). Trophoblast populations at the maternal-fetal interface stained for 16 of the cell cycle regulators whose expression we studied. The staining patterns changed as a function of both differentiation and gestational age. Differentiation along the invasive pathway was associated with entrance into, then permanent withdrawal from, the cell cycle, as evidenced by the orchestrated expression of cyclins, their catalytic subunits, and inhibitors. Surprisingly, we found coexpression of molecules that regulate different portions of the cell cycle in the syncytium. These data, which constitute one of the few examples to date of in situ localization of an extensive repertoire of mitotic regulators, provide the basis for studies aimed at understanding factors that lead to abnormal placentation.     

Cyclic 3',5'-adenosine monophosphate levels during the developmental cycle of Trypanosoma brucei brucei in the rat

Intracellular content of cyclic 3',5'-adenosine monophosphate was determined in several strains of Trypanosoma brucei brucei during their growth in rats. In non-relapsing infections with the cloned monomorphic strain 110M and with the cloned pleomorphic strain YTatl, the amount of cyclic AMP per 10(9) trypanosomes increased from 30 to over 90 pmol as the parasitemia increased from patency to over 10(9) organisms per ml. This increase was not observed during non-relapsing infections with strain 427. During infections with strain YTatl in both immunocompetent and lethally X-irradiated rats, cyclic AMP content of the parasite increased from 20--20 pmol per 10(9) cells early in logarithmic growth to 65--70 pmol per 10(9) cells at peak parasitemia, then decreased as the transition to intermediate and short stumpy forms commenced. At crisis, basal levels were reestablished when log slender forms were the lowest percentage of the total population and intermediate and short stumpy forms predominated, suggesting a correlation between morphologic type and level of cyclic AMP per cell during fluctuations in parasitemia. Increases in intracellular cyclic AMP were measured during in vitro incubation of the parasite in medium containing potential effectors of the trypanosome cyclic AMP system. Sodium fluoride, adenosine and methyl xanthines stimulated increases in cyclic AMP content while isoproterenol, prostaglandin E1, serotonin, histamine and several trypanocidal drugs were ineffective. The results are discussed in terms of the possible regulatory role of cyclic AMP in differentiation of trypanosomes.     

Functional expression of the ascofuranone-sensitive Trypanosoma brucei brucei alternative oxidase in the cytoplasmic membrane of Escherichia coli

Trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain of long slender bloodstream forms (LS forms) of African trypanosoma, which causes sleeping sickness in human and nagana in cattle. TAO is a cytochrome-independent, cyanide-insensitive quinol oxidase and these properties are quite different from those of the bacterial quinol oxidase which belongs to the heme-copper terminal oxidase superfamily. Only little information concerning the molecular structure and enzymatic features of TAO have been available, whereas the bacterial enzyme has been well characterized. In this study, a cDNA encoding TAO from Trypanosoma brucei brucei was cloned into the expression vector pET15b (pTAO) and recombinant TAO was expressed in Escherichia coli. The growth of the transformant carrying pTAO was cyanide-resistant. A peptide with a molecular mass of 37 kDa was found in the cytoplasmic membrane of E. coli, and was recognized by antibodies against plant-type alternative oxidases from Sauromatum guttatum and Hansenula anomala. Both the ubiquinol oxidase and succinate oxidase activities found in the membrane of the transformant were insensitive to cyanide, while those of the control strain, which contained vector alone, were inhibited. This cyanide-insensitive growth of the E. coli carrying pTAO was inhibited by the addition of ascofuranone, a potent and specific inhibitor of TAO ubiquinol oxidase. The ubiquinol oxidase activity of the membrane from the transformant was sensitive to ascofuranone. These results clearly show the functional expression of TAO in E. coli and indicate that ubiquinol-8 in the E. coli membrane is able to serve as an electron donor to the recombinant enzyme and confer cyanide-resistant and ascofuranone-sensitive growth to E. coli. This system will facilitate the biochemical characterization of the novel terminal oxidase, TAO, and the understanding on the mechanism of the trypanocidal effect of ascofuranone.     

Conditional expression of glycosylphosphatidylinositol phospholipase C in Trypanosoma brucei.

Trypanosoma brucei glycosylphosphatidylinositol phospholipase C (GPIPLC) is expressed in the bloodstream stage of the life cycle, but not in the procyclic form. It is capable of hydrolyzing GPI-anchored proteins and phosphatidylinositol (PI) in vitro. Several roles have been proposed for GPIPLC in vivo, in the release of variant surface glycoprotein during differentiation or in the regulation of GPI and PI levels, but none has been substantiated. To explore GPIPLC function in vivo, tetracycline-inducible GPIPLC gene (GPIPLC) conditional knock-out bloodstream form and tetracycline-inducible GPIPLC-expressing procyclic cell lines were constructed. We were unable to generate GPIPLC null mutants. Cleavage of GPI-anchored proteins was abolished in extracts from uninduced conditional knock-outs and was restored upon induction. Despite the barely detectable level of GPIPLC activity in uninduced conditional knock-out bloodstream forms, their growth was not affected. GPI-protein cleavage activity could be induced in procyclic cell extracts, up to wild-type bloodstream levels. Myo-[3H]inositol incorporation into [3H]inositol monophosphate was about 14-fold lower in GPIPLC conditional knock-out bloodstream forms than in the wild type. Procyclic cells expressing GPIPLC showed a 28-fold increase in myo-[3H]inositol incorporation into [3H]inositol monophosphate and a 1.5-fold increase in [3H]inositol trisphosphate levels, suggesting that GPIPLC may regulate levels of inositol phosphates, by cleavage of PI and phosphatidylinositol 4,5-bisphosphate.