Adenylate cyclase in bloodstream forms of Trypanosoma (Trypanozoon) brucei sp.

1. The adenylate cyclase in Trypanosoma brucei is located in the plasma membrane. 2. A partial kinetic analysis of the properties of the enzyme revealed a Km for ATP of 1.75 mM and a Km for Mg2+ of 4mM. 3. At low concentrations, Mg2+ activated the enzyme directly in addition to its effect of lowering the concentration of inhibitory free ATP species. 4. At high concentrations, Mg2+ inhibited the enzyme. Furthermore, the enzyme was inhibited at any Mg2+ concentration if the concentration of ATP exceeded that of Mg2+. 5. The opposing effects of Mg2+ at low and high concentrations would be consistent with more than one binding site for Mg2+ on the enzyme. 6. A study of the patterns of product inhibition revealed little or no effect of 3':5'-cyclic AMP, but a profound inhibition by pyrophosphate, which was competitive with respect to ATP (Ki 0.135 mM). This result suggests that the substrate-binding domain on T. brucei adenylate cyclase interacts mainly with the triphosphate portion of the ATP molecule. 7. The enzyme activity was unaffected by the usual mammalian enzyme effectors glucagon, adrenaline, adenosine, GTP and guanyl-5'-yl imidodiphosphate. 8. The enzyme was not activated by fluoride, instead a powerful inhibition was found. The enzyme was also inhibited by relatively high concentrations of Ca2+ (1 mM).     

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.     

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.     

Cysteine proteinase inhibitors kill cultured bloodstream forms of Trypanosoma brucei brucei

Trypanosoma brucei brucei is a causative agent of bovine trypanosomiasis (nagana), a disease of considerable economic significance in much of Africa. Here we report investigations on the effects of various irreversible cysteine proteinase inhibitors, including vinyl sulfones (VS), peptidyl chloromethylketones (CMK), diazomethylketones, and fluoromethyl ketones, on the major lysosomal cysteine proteinase (trypanopain-Tb) of T. b. brucei and on in vitro-cultured bloodstream forms of the parasite. Many of the tested inhibitors were trypanocidal at low micromolar concentrations. Methylpiperazine urea-Phe-homoPhe-VS was the most effective trypanocidal agent, killing 50% of test populations at a work ing concentration of 0.11 microM, while carbobenzoxy-Phe-Phe-CMK was the most trypanocidal of the methylketones with an IC50 of 3.6 microM. Labelling of live and lysed T. b. brucei with biotinylated inhibitor derivatives suggests that trypanopain-Tb is the likely intracellular target for these inhibitors. Kinetic analysis of the inhibition of purified trypanopain-Tb by the inhibitors showed that most had kass values in the 10(6) M-1 s-1 range. We conclude that cysteine proteinase inhibitors have potential as trypanocidal agents and that a major target of these compounds is the lysosomal enzyme trypanopain-Tb.     

Trypanosoma brucei: unexpected azide sensitivity of bloodstream forms

Bloodstream forms of Trypanosoma brucei lack cytochromes and are, therefore, insensitive to cyanide. Azide is a toxic anion that bears chemical and biological properties in common with cyanide and may act in a similar way by inhibition of cytochrome c oxidase. It was, therefore, surprising to find that bloodstream forms of T. brucei are sensitive to azide; growth is reduced by 50% with 0.1 mM azide. So far, the only enzyme known in bloodstream forms of T. brucei to be sensitive to azide is the iron-containing superoxide dismutase. However, because the activity of the superoxide dismutase was not affected in parasites incubated for 16 hr with 0.5 mM azide (a concentration at which no cell proliferates), the toxic action of azide cannot be due to inhibition of this enzyme. These results indicate that the general toxicity of azide is different from that of cyanide.     

Invariant surface proteins in bloodstream forms of Trypanosoma brucei

Antigenic variation of the glycoprotein forming the coat of African trypanosomes has been a dominant field of investigation for many years. The extravagant potential of these parasites to change their surface coat has destroyed hopes for a vaccine based on the variant surface glycoprotein. Recently, there has been a rising interest in the characterization of surface proteins that are not subject to antigenic variation. In this review, Peter Overath, Maliha Chaudhri, Dietmar Steverding and Karl Ziegelbauer summarize the present evidence for the occurrence, cellular localization and function of invariant surface proteins in Trypanosoma brucei.     

Lysosomal and non-lysosomal peptidyl hydrolases of the bloodstream forms of Trypanosoma brucei brucei

African trypanosomes have thiol-dependent proteolytic activity that resembles some of the cathepsin-like activity found in mammalian lysosomes [Lonsdale-Eccles, J. D. & Mpimbaza, G. W. N. (1986) Eur. J. Biochem. 155, 469-473]. Here we show that this activity is found in lysosome-like organelles which we have isolated (density = 1.082 g/cm3 in Percoll) from bloodstream forms of Trypanosoma brucei brucei. They are approximately 250 nm in diameter, are bounded by a single limiting membrane, and contain acid phosphatase. The predominant proteolytic and peptidolytic activity of these organelles has a pH optimum about 6.0, exhibits latency, and has the characteristics of mammalian cathepsin L (and possibly cathepsin H) with respect to its hydrolysis of small fluorogenic peptidyl substrates such as benzyloxycarbonyl-phenylalanyl-arginyl-7-amido-4-methylcoumarin. This substrate appears to be a good marker for trypanosomal lysosomes. The cathepsin-L-like activity is inhibited by the thiol-protease inhibitors, E-64, cystatin, leupeptin and mercurial compounds. The proteolytic activity of the lysosome-like fraction is observed as a single band of activity with an approximate molecular mass of 27 kDa when measured after electrophoresis in the fibrinogen-containing sodium dodecyl sulphate/polyacrylamide gels. The addition of mammalian serum to this purified fraction, or to whole trypanosome homogenates, results in the appearance of additional bands of activity, with a concomitant increase in the total observed proteolytic activity. The serum of some species of animal (e.g. goat and guinea pig) appear to lack the ability to generate this new and increased activity, while rat, rabbit, human and bovine sera exhibit varying capacities to generate the new activity, the cow being the most effective. The apparent molecular masses of the new bands of activity are different for each mammalian species, suggesting that the activator is a species-specific molecule or class of molecules. We also show that Trypanosoma brucei contains soluble peptidolytic activity with an alkaline pH optimum. It is inhibited by the serine-protease inhibitor diisopropylfluorophosphate, but not by inhibitors such as phenylmethylsulphonyl fluoride, alpha 1-antitrypsin, or aprotinin. Nor is it inhibited by the thiol-protease-specific inhibitors E-64 or cystatin, although it is susceptible to inhibition by tosyllysylchloromethane, leupeptin, HgCl2 and p-chloromercuribenzoate. This enzymic activity has a preference for arginyl residues in the primary binding site (the P1 position), as also does the activity from the lysosomes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and regulatory properties of phosphofructokinase from Trypanosoma (Trypanozoon) brucei brucei.

Phosphofructokinase (EC 2.7.1.11) from Trypanosoma (Trypanozoon) brucei brucei was purified to homogeneity by using a three-step procedure that may be performed within 1 day. Proteolysis, which removes a fragment of Mr approx. 2000, may occur during the purification, but this can be prevented by including antipain, an inhibitor of cysteine proteinases, in the buffers during the purification. The subunits of the enzyme appear to be identical in size, with an Mr of 49 000. The Mr of the native enzyme was estimated to be approx. 220 000, suggesting a tetrameric structure. Kinetic studies showed the activity to depend hyperbolically on the concentration of ATP but sigmoidally on the concentration of fructose 6-phosphate. Although cyclic AMP, AMP and ADP stimulated the enzyme activity at low concentrations of fructose 6-phosphate, the last two nucleotides were inhibitory at high concentrations of this substrate. Phosphoenolpyruvate behaved as an allosteric inhibitor of the phosphofructokinase. Citrate, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate and Pi did not influence significantly the activity of the enzyme.     

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.     

Mitochondrial translation is essential in bloodstream forms of Trypanosoma brucei

The parasitic protozoa Trypanosoma brucei has a complex life cycle. Oxidative phosphorylation is highly active in the procyclic form but absent from bloodstream cells. The mitochondrial genome encodes several gene products that are required for oxidative phosphorylation, but it completely lacks tRNA genes. For mitochondrial translation to occur, the import of cytosolic tRNAs is therefore essential for procyclic T. brucei. Whether the same is true for the bloodstream form has not been studied so far. Here we show that the steady-state levels of mitochondrial tRNAs are essentially the same in both life stages. Editing of the imported tRNA(Trp) also occurs in both forms as well as in mitochondria of Trypanosoma evansi, which lacks a genome and a translation system. These results show that mitochondrial tRNA import is a constitutive process that must be mediated by proteins that are expressed in both forms of the life cycle and that are not encoded in the mitochondrial genome. Moreover, bloodstream cells lacking either mitochondria-specific translation elongation factor Tu or mitochondrial tryptophanyl-tRNA synthetase are not viable indicating that mitochondrial translation is also essential in this stage. Both of these proteins show trypanosomatid-specific features and may therefore be excellent novel drug targets.     

The phosphoglucose isomerases of the bloodstream forms of Trypanosoma brucei and Trypanosoma vivax.

1. The phosphoglucose isomerases (PGI's) of the bloodstream forms of Trypanosoma brucei and T. vivax have been purified some 150-fold, using cellulose ion-exchange chromatography, gel filtration and isoelectric focussing. 2. The two trypanosome enzymes showed many similarities in kinetic properties, but differed from each other somewhat in thermal stability and in isoelectric point. 3. Both trypanosome enzymes differ from PGI's from other sources in having a higher Ki for the competitive inhibitor 6-phosphogluconate.     

Enigmatic presence of mitochondrial complex I in Trypanosoma brucei bloodstream forms

The presence of mitochondrial respiratory complex I in the pathogenic bloodstream stages of Trypanosoma brucei has been vigorously debated: increased expression of mitochondrially encoded functional complex I mRNAs is countered by low levels of enzymatic activity that show marginal inhibition by the specific inhibitor rotenone. We now show that epitope-tagged versions of multiple complex I subunits assemble into α and β subcomplexes in the bloodstream stage and that these subcomplexes require the mitochondrial genome for their assembly. Despite the presence of these large (740- and 855-kDa) multisubunit complexes, the electron transport activity of complex I is not essential under experimental conditions since null mutants of two core genes (NUBM and NUKM) showed no growth defect in vitro or in mouse infection. Furthermore, the null mutants showed no decrease in NADH:ubiquinone oxidoreductase activity, suggesting that the observed activity is not contributed by complex I. This work conclusively shows that despite the synthesis and assembly of subunit proteins, the enzymatic function of the largest respiratory complex is neither significant nor important in the bloodstream stage. This situation appears to be in striking contrast to that for the other respiratory complexes in this parasite, where physical presence in a life-cycle stage always indicates functional significance.     

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.     

Growth inhibition of bloodstream forms of Trypanosoma brucei by the iron chelator deferoxamine

Treatment of bloodstream forms of Trypanosoma brucei with the iron chelator deferoxamine inhibits the proliferation of the parasites. Compared with mammalian cells, bloodstream forms of Trypanosoma brucei are 10 times more sensitive to iron depletion. The primary target of the chelator is obviously the intracellular iron as the toxicity of deferoxamine is abolished by addition of holotransferrin, the exogenous source of iron for the parasite. To identify probable target sites, the effect of deferoxamine on ribonucleotide reductase, alternative oxidase and superoxide dismutase, three iron-dependent enzymes in bloodstream-form trypanosomes, was studied. Incubation of the parasites with the chelator leads to inhibition of DNA synthesis and lowers oxygen consumption indicating that deferoxamine may affect ribonucleotide reductase and alternative oxidase. The compound does not inhibit the holoenzymes directly but probably acts by chelating cellular iron thus preventing its incorporation into the newly synthesised apoproteins. Treatment of the parasites with deferoxamine for 24 h has no effect on the activity of superoxide dismutase. The results have implications for antitrypanosomal drug development based on specific intervention with the parasite's iron metabolism.     

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.     

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.     

Factors that determine the plasma-membrane potential in bloodstream forms of Trypanosoma brucei.

The plasma-membrane potential (Delta(psi)p) in bloodstream forms of Trypanosoma brucei was studied using several different radiolabelled probes: 86Rb+ and [14C]SCN- were used to report Delta(psi)p directly because they distribute in easily measured quantities across the plasma membrane only, and [3H]methyltriphenylphosphonium (MePh3P+) was used to report Delta(psi)p only when Delta(psi)m had been abolished with FCCP because it reports the algebraic sum of the two potentials when used alone. The unperturbed Delta(psi)p had a value of -82 mV and was found to be essentially identical with, and determined almost completely by, the potassium diffusion potential, as evidenced by: (a) the lack of effect of valinomycin on the value obtained under appropriate conditions when any of these probes were used; (b) the close agreement of this measured value with that predicted from the measured distribution of K+ across the plasma membrane (-76 mV); (c) the large effect of changes in the extracellular K+ concentration by substitution with Na+ on Delta(psi)p together with the complete lack of effect of substitution of extracellular Na+ by the choline cation or substitution of extracellular Cl- by the gluconate anion on Delta(psi)p. The contribution to Delta(psi)p by electrogenic pumping of Na+/K+-ATPase was found to be small (of the order of 6 mV). H+ was not found to be pumped across the plasma membrane or to contribute to Delta(psi)p.