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.     

In vitro growth inhibition of bloodstream forms of Trypanosoma brucei and Trypanosoma congolense by iron chelators

African trypanosomes exert significant morbidity and mortality in man and livestock. Only a few drugs are available for the treatment of trypanosome infections and therefore, the development of new anti-trypanosomal agents is required. Previously it has been shown that bloodstream-form trypanosomes are sensitive to the iron chelator deferoxamine. In this study the effect of 13 iron chelators on the growth of Trypanosoma brucei, T. congolense and human HL-60 cells was tested in vitro. With the exception of 2 compounds, all chelators exhibited anti-trypanosomal activities, with 50% inhibitory concentration (IC₅₀) values ranging between 2.1 – 220 μM. However, the iron chelators also displayed cytotoxicity towards human HL-60 cells and therefore, only less favourable selectivity indices compared to commercially available drugs. Interfering with iron metabolism may be a new strategy in the treatment of trypanosome infections. More specifically, lipophilic iron-chelating agents may serve as lead compounds for novel anti-trypanosomal drug development.     

In vitro culture of freshly isolated Trypanosoma brucei brucei bloodstream forms results in gene copy-number changes

Most researchers who study unicellular eukaryotes work with an extremely limited number of laboratory-adapted isolates that were obtained from the field decades ago, but the effects of passage in laboratory rodents, and adaptation to in vitro culture, have been little studied. For example, the vast majority of studies of Trypanosoma brucei biology have concentrated on just two strains, Lister 427 and EATRO1125, which were taken from the field over half a century ago and have since have undergone innumerable passages in rodents and culture. We here describe two new Trypanosoma brucei brucei strains. MAK65 and MAK98, which have undergone only 3 rodent passages since isolation from Ugandan cattle. High-coverage sequencing revealed that adaptation of the parasites to culture was accompanied by changes in gene copy numbers. T. brucei has so far been considered to be uniformly diploid, but we also found trisomy of chromosome 5 not only in one Lister 427 culture, but also in the MAK98 field isolate. Trisomy of chromosome 6, and increased copies of other chromosome segments, were also seen in established cultured lines. The two new T. brucei strains should be useful to researchers interested in trypanosome differentiation and pathogenicity. Initial results suggested that the two strains have differing infection patterns in rodents. MAK65 is uniformly diploid and grew more reproducibly in bloodstream-form culture than MAK98.     

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.     

Synchronous differentiation of Trypanosoma brucei from bloodstream to procyclic forms in vitro.

The differentiation of mammalian stage Trypanosoma brucei bloodstream forms comprising predominantly parasites of intermediate and stumpy morphology to the procyclic forms characteristic for the insect midgut stage was studied in vitro. Differentiation of the cell population occurred synchronously as judged by the synthesis of the surface glycoprotein, procyclin, characteristic of the arising procyclic forms and the loss of the membrane-form variant surface glycoprotein, the coat protein of bloodstream forms. The change in surface antigens took place within 12 h in the absence of cell growth; subsequently, the procyclic cells divided exponentially. As defined in this study, T. brucei may be a useful model to follow other changes in gene expression, metabolism or ultrastructure during differentiation of a unicellular eucaryote.     

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.     

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)     

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.     

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.     

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).     

Trypanosoma brucei: five commonly used trypanocides assayed in vitro with a mammalian feeder layer system for cultivation of bloodstream forms

The in vitro activity of five commonly used trypanocides on bloodstream forms of Trypanosoma brucei brucei TC 221 was examined in 24-well culture plates in the presence of bovine fibroblast feeder layer cells. The minimum effective concentrations determined were as follows: Berenil 1.0 microgram/ml; Samorin 10.0 micrograms/ml; Antrycide dimethylsulfate 0.1 microgram/ml; Arsobal 0.01 microgram/ml; Naganol 1.0 microgram/ml. Contrary to values obtained with other in vitro assays, minimum effective concentrations obtained here were within the range of drug levels reached in blood, plasma, or serum of humans and animals after treatment with curative doses. The trypanocidal activity of Naganol in this assay was of particular interest, since Naganol has been hitherto considered to be inactive in vitro.     

Trypanosoma brucei gambiense: growth in vitro of bloodstream forms inhibited by dichlorodiammineplatinum (II) compounds and hypolipidemic drugs

Infectious bloodstream forms of Trypanosoma brucei gambiense were grown in microcultures of murine bone marrow cells in 96-well tissue culture plates. Limiting dilution studies showed that fewer than 10 cultured trypanosomes developed into populations of about 5 X 10(4) parasites per well in a week. Bloodstream parasites were reisolated with high efficiency from mice infected with cultured parasites; fewer than 10 bloodstream parasites successfully established a trypanosome population in a microculture. Both the cis and trans isomers of dichlorodiammineplatinum (II) (cisplatin and transplatin) and a hypolipidemic agent, Wy 14643, were found to have activity against T. b. gambiense growing in microcultures. The minimum concentration of drug necessary to completely eliminate parasites from microcultures was 4 microM for cisplatin, 40 microM for transplatin, and 500 microM for Wy 14643. A preformed complex of cisplatin and bovine serum albumin and another hypolipidemic agent, chlofibric acid, were inactive. This culture system should be useful for rapid screening of large numbers of compounds for trypanocidal activity.     

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: 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.     

Characterization of two protein disulfide isomerases from the endocytic pathway of bloodstream forms of Trypanosoma brucei

Proteins from the endocytic pathway in bloodstream forms of Trypanosome brucei are modified by the addition of linear poly-N-acetyllactosamine side chains, which permits their isolation by tomato lectin affinity chromatography. Antibodies against this tomato lectin binding fraction were employed to screen a cDNA expression library from bloodstream forms of T. brucei. Two cDNAs were prominent among those selected. These cDNAs coded for two putative protein disulfide isomerases (PDIs) that respectively contained one and two double-cysteine redox-active sites and corresponded to a single domain PDI and a class 1 PDI. Assays of the purified recombinant proteins demonstrated that both proteins possess isomerase activity, but only the single domain PDI had a reducing activity. These PDIs possess a number of unusual features that distinguish them from previously characterized PDIs. The expression of both is developmentally regulated, they both co-localize with markers of the endocytic pathway, and both are modified by N-glycosylation. The larger PDI possesses N-glycans containing poly-N-acetyllactosamine, a modification that is indicative of processing in the Golgi and suggests the presence of a novel trafficking pathway for PDIs in trypanosomes. Although generally PDIs are considered essential, neither activity appeared to be essential for the growth of trypanosomes, at least in vitro.