Trypsin-stimulated transformation of Trypanosoma brucei gambiense bloodstream forms to procyclic forms in vitro

The effect of trypsin treatment on the transformation of monomorphic Trypanosoma b. gambiense (Wellcome strain) bloodstream forms to procyclic forms was studied in HEPES-buffered RPMI 1640 medium supplemented with 20% inactivated fetal calf serum in the presence of GA-1 cells as feeder layers at 27 C. In this system, 35%–40% of the bloodstream forms transformed to procyclic forms within 24 h, and over 95% of the trypanosomes changed into procyclic forms by day 3 after initiation of the culture. Established cultures of procyclic forms yielded up to 1.5–2×10⁷ trypanosomes/ml. However, transformation of nontreated and inhibited trypsin-treated bloodstream forms were prolonged compared to trypsin-treated populations. In this experiment, the first procyclic forms could be detected on day 7 after initiation of the culture and transformation was complete within 15 days. The transformation of T. b. gambiense from bloodstream to procyclic forms required the living GA-1 cells as feeder layer cells, but established cultures of procyclic forms could be maintained in the culture medium without feeder cells for more than 300 days.     

The selenoproteome is dispensable in bloodstream forms of Trypanosoma brucei

Here we show that absence of Sep-tRNA:Sec-tRNA synthase (SepSecS) a key enzyme required for the synthesis of the three trypanosomal selenoproteins does not affect growth of bloodstream forms of Trypanosoma brucei. Both life cycle stages of T. brucei are highly sensitive to auranofin, a compound known to target selenoproteins. However, the same sensitivity is observed in the SepSecS double knockout cell lines indicating that the trypanocidal action of auranofin is not connected to selenoproteins. Finally, we show that absence of selenoproteins does not increase sensitivity to H₂O₂-induced oxidative stress. Thus in cell culture normal growth of procyclic and bloodstream T. brucei does not depend on selenoproteins.     

Stimulating effect of citrate and cis-aconitate on the transformation ofTrypanosoma brucei bloodstream forms to procyclic forms in vitro

Transformation ofTrypanosoma brucei bloodstream forms to procyclic forms can be stimulated in vitro by adding 2–10 mM citrate and/or cis-aconitate to the culture medium for at least 3 h. Optimal transformation was obtained in the presence of 3 mM cis-aconitate for 48 h. Citrate and cis-aconitate may activate isocitrate dehydrogenase and/or other Krebs' cycle enzymes and thus act as a metabolic trigger for the morphological changes involved in transformation.     

Identification of three ABC transporter genes in Trypanosomabrucei spp.

ABC transporters are key players in the multi-drug resistance of cancer cells and yeast, and they appear to be involved in the drug resistance of various pathogenic protozoa. No member of this ubiquitous protein family has yet been described in Trypanosoma brucei spp., the causative agents of African sleeping sickness and animal trypanosomiases. However, different cases of artificially induced drug resistance were shown to be linked to a reduction in net drug uptake. We used polymerase chain reaction with degenerate oligonucleotide primers corresponding to particularly conserved regions within the ATP-binding cassette to probe the genome of T.brucei spp. for the presence of ABC transporter genes. Three different sequence segments encoding ATP-binding cassettes were identified, which, upon Southern blotting, appeared to belong to distinct genes designated Tbabc1, Tbabc2, and Tbabc3. They appear to be single-copy genes in both drug-susceptible and drug-resistant stocks of T.brucei spp., expressed in bloodstream forms as well as in the procyclic life stage. Whereas Tbabc3 shows moderate homology to various known ABC transporters, Tbabc1 and Tbabc2 are highly homologous to P-glycoprotein A of Leishmaniatarentolae and to the multidrug resistance protein 1 of L.donovani, respectively. Received: 18 July 1997 / Accepted: 13 August 1997     

Cysteine is an essential growth factor for Trypanosoma brucei bloodstream forms

A modified cystine-free minimum essential medium has been used to address the question whether cysteine is an essential growth factor for bloodstream form trypanosomes or if its reducing power is sufficient to support parasite growth in axenic culture. Bloodstream-form trypanosomes, taken either from freshly isolated infected mouse blood or form logarithmically growing axenic cultures were transferred to a medium containing 20% dialysed foctal calf serum, 10 μM bathocuproine sulphonate and 250 μM cysteine. Growth curves of these cultures have been compared to those obtained in identical cultures containing no cysteine but cystine and reducing agents (β-mercaptoethanol, monothioglycerol), or reducing agents alone. The results clearly show that cell growth was only obtained if cysteine was either directly added to the medium or was reduced from cysteine by the action of reducing agents. However, neither reducing agents alone, nor d-cysteine, supported cell growth. Since cysteine is not taken up by bloodstream form trypanosomes, and methionine is a regular constituent of the medium, we conclude from our results that cysteine is an essential growth factor for Trypanosoma brucei.     

Import and assembly of<Emphasis Type="Italic"> Neurospora crassa</Emphasis> Tom40 into mitochondria of<Emphasis Type="Italic"> Trypanosoma brucei</Emphasis> in vivo

The TOM complex (translocase of the mitochondrial outer membrane) is a dynamic, multisubunit protein complex. Tom40 is the major component of the complex and forms the preprotein conducting pore. To determine if a heterologous Tom40 could be properly targeted and assembled into the Trypanosoma brucei mitochondrial outer membrane, an ectopic copy of a gene encoding Neurospora crassa Tom40 (NcTom40) was expressed in procyclic trypanosomes from a tetracycline regulated procyclic acidic repetitive protein promoter. The level of NcTom40 expression was found to be maximal within 20–26 h of induction with tetracycline. Immunoblot analysis of subcellular fractions showed that NcTom40 was enriched in the mitochondrial fraction. Alkali extraction of isolated mitochondria revealed that NcTom40 was assembled as an integral membrane protein and limited proteolysis demonstrated that it was present in the outer membrane of the mitochondria. These data demonstrate that a heterologous mitochondrial protein containing internal targeting information can be correctly targeted to T. brucei mitochondria. Following blue native gel electrophoresis, the NcTom40 protein was found in a 370 kDa complex which may contain T. brucei Tom components. A 16 kDa protein was coimmunoprecipitated from T. brucei mitochondria containing NcTom40 using antisera developed against the N. crassa protein. The 16 kDa protein may represent a component of the T. brucei TOM complex that associates with NcTom40.Communicated by M. BrunnerThis revised version was published in October 2003. Throughout the text, owing to a technical problem, degree signs were erroneously inserted before µg/ml and µl.     

Cyclical transmission of in vitro cultivated bloodstream forms and procyclic trypomastigotes ofTrypanosoma brucei brucei byGlossina morsitans morsitans

In vitro cultivated bloodstream and procyclic forms ofTrypanosoma b. brucei STIB 247 were cyclically transmitted byGlossina m. morsitans. The tsetse flies were infected artificially on a silicon membrane. Metacyclic trypanosomes from mature salivary gland infections were used to initiate bloodstream form cultures. They transformed into slender bloodstream forms and gave rise to established cultures that proved to be infective for the vector. The metacyclic forms retained the strain-specific basic set of variable antigen types.     

Protein phosphatase 5 is required for Hsp90 function during proteotoxic stresses in <Emphasis Type="Italic">Trypanosoma brucei</Emphasis>

Trypanosoma brucei, a parasitic protozoan that causes African trypanosomiasis in human and domestic animals, adapt in various environments during their digenetic life cycle. In this study, we found that Hsp90 is crucial for the survival of this parasite. Inhibition of Hsp90 activity by geldanamycin (GA) reduced cell growth and increased the level of Hsp90. Both the bloodstream and procyclic forms of T. brucei showed a several-fold greater sensitivity than the mammalian cells to GA and also to 17-AAG, a less toxic derivative of GA, suggesting that Hsp90 could be a potential chemotherapeuric target for African trypanosomiasis. T. brucei Hsp90 interacts with the protein phosphatase 5 (PP5) in vivo. Under normal growth conditions, T. brucei PP5 (TbPP5) and Hsp90 are primarily localized in the cytosol. However, with increase in growth temperature and GA treatment, these proteins translocate to the nucleus. Overproduction of TbPP5 by genetic manipulation reduced the growth inhibitory effect of GA, while knockdown of TbPP5 reduced cell growth more in the presence of GA, as compared to parental control. Depletion of TbPP5, however, did not prevent the induction of Hsp90 protein level during GA treatment. Together, these results suggest that TbPP5 positively regulates the function of Hsp90 to maintain cellular homeostasis during proteotoxic stresses in T. brucei.     

A comparative study of d-lactate, l-lactate and glycerol formation by four species of Leishmania and by Trypanosoma lewisi and Trypanosoma brucei gambiense

Leishmania braziliensis panamensis, L. donovani, L. major, and L. mexicana amazonensis promastigotes, Trypanosoma lewisi bloodstream forms, and T. brucei gambiense procyclic forms were incubated with glucose as sole carbon source. All species consumed glucose more rapidly under aerobic than anaerobic conditions. All produced glycerol under anaerobic conditions, though the rate of glycerol production by T. lewisi was markedly lower than that by the other species. The four Leishmania species produced d-lactate, but not l-lactate, whereas T. b. gambiense procyclic forms produced l-lactate, but not d-lactate, and T. lewisi produced both isomers.     

Studies on compounds promoting the in vitro transformation ofTrypanosoma brucei from bloodstream to procyclic forms

In vitro differentiation of pleomorphic blood-stream forms ofTrypanosoma brucei to procyclic culture forms occurred rapidly and at high rates at 27°C in a culture medium containing 1 mM cis-aconitate as the transformation-inducing agent. Citrate was required at a much higher concentration (10 mM) to produce a similar transformation rate. The highest percentage of transformed cells was obtained when bloodstream-form trypanosomes were treated with pronase in the absence of a feeder-cell layer. However, under these conditions, the amount of procyclic forms obtained after 72 h was lower than that obtained in the presence ofcis-aconitate. Trypsin was also capable of inducing transformation in the absence of a feeder-cell layer, but this treatment again resulted in low numbers of transformed cells. Blood-stream-form trypanosomes were incapable of taking up citrate to any significant extent and the citrate content of these stages was negligible. After 72 h of exposure to citrate (3 mM), intracellular levels of this compound remained very low (<1 nmol (10⁹ cells)^–1), increasing in established procyclic stages to approximately 1.7 nmol (10⁹ cells)^–1. These observations suggest that the tricarboxylic acid (TCA)-cycle metabolite-dependent transformation may be initiated externally to the trypanosome cell membrane. The ability of both citrate andcis-aconitate to bind calcium and, thus, to reduce the concentration of this cation in the culture medium was found not to be responsible for the triggering effect on trypanosome transformation. Citrate levels in tsetse fly body fluid were found to be far below those required to induce trypanosome transformation in vitro. Since proteolytic enzymes can effectively induce this process, these enzymatic components may be of greater physiological relevance as environmental stimuli for the differentiation process than are the TCA-cycle metabolites.     

On the DNA content and ploidy of trypanosomes

We have determined the nuclear and kinetoplast DNA content of two trypanosomatids by quantitative absorption and fluorescence cytophotometry of individual Feulgen-pararosaniline stained cells. For the insect trypanosomatid Crithidia fasciculata we find nuclear and kinetoplast DNA contents of 0.095 and 0.032 pg per non-replicating cell. For the African trypanosome Trypanosoma brucei these values are 0.097 and 0.004 pg. A sub-population of T. brucei cells with two kinetoplasts and one nucleus was found to contain 0.181 pg/nucleus. The DNA values of bloodstream form T. brucei and the procyclic culture form were not significantly different.In DNA-DNA renaturation experiments the haploid amount of DNA in T. brucei was previously found to be 0.041 pg/nucleus (Borst, P., Fase-Fowler, F., Frasch, A.C.C., Hoeijmakers, J.H.J. and Weijers, P.J. (1980) Mol. Biochem. Parasitol. 1, 221–246). Our data, therefore, indicate that T. brucei is diploid. No sub-population of haploid cells was observed in T. brucei grown in rats or in culture.     

In vitro drug sensitivity ofTrypanosoma congolense isolates

The sensitivity ofTrypanosoma congolense isolates to diminazene and isometamidium was determined using an incorporation assay based on the uptake of [³H]-hypoxanthine in the presence of serial drug dilutions. The bloodstream forms of the different isolates exhibited variation in their sensitivity to the drugs that correlated well with the in vivo drug response. For diminazene, the sensitivity of the most sensitive population was 40 times that of the least sensitive population. For isometamidium, the IC₅₀ values (the drug concentrations that decreased radiolabel incorporation by 50%) lay in a similar range, except for those found for two isolates from lions, which were 10³–10⁴ times more sensitive than the isolates from cattle. The sensitivity of procyclic forms differed markedly from that of the bloodstream stages. Therefore, it must be concluded that the procyclic stage does not reflect the sensitivity of the bloodstream forms ofT. congolense and that the former should not be used for determinations of in vitro drug sensitivity.Dedicated to Prof. Dr. J.Eckert (Zürich) on the occasion of his 60th birthday     

Cofactor-independent phosphoglycerate mutase is an essential gene in procyclic form <Emphasis Type="Italic">Trypanosoma brucei</Emphasis>

Glycolysis and gluconeogenesis are, in part, driven by the interconversion of 3- and 2-phosphoglycerate (3-PG and 2-PG) which is performed by phosphoglycerate mutases (PGAMs) which can be cofactor dependant (dPGAM) or cofactor independent (iPGAM). The African trypanosome, Trypanosoma brucei, possesses the iPGAM form which is thought to play an important role in glycolysis. Here, we report on the use of RNA interference to down-regulate the T. brucei iPGAM in procyclic form T. brucei and evaluation of the resulting phenotype. We first demonstrated biochemically that depletion of the steady state levels of iPGM mRNA correlates with a marked reduction of enzyme activity. We further show that iPGAM is required for cell growth in procyclic T. brucei.     

The trypanocidal effect of NO-releasing agents is not due to inhibition of the major cysteine proteinase in Trypanosoma brucei

The lysosomal cysteine proteinase activity of bloodstream forms of Trypanosoma brucei is a validated drug target. Previously, it was reported that nitric oxide (NO)-releasing agents inhibit the catalytic activity of cysteine proteinases of the protozoan parasites Leishmania infantum, Trypanosoma cruzi and Plasmodium falciparum. In this study, we investigated the effect of the NO-donors S-nitrosoglutathione, (±)-(E)-4-ethyl-2[(E)-hydroxyimino]-5-nitro-3-hexenamide, 3-morpholinosydnonimine (SIN-1) and S-nitroso-N-acetyl-dl-penicillamine on the activity of the cysteine proteinase of T. brucei. At a concentration of 1 mM, the NO donors inhibited the catalytic activity of purified T. brucei cysteine proteinase by 50–90%. With the exception of SIN-1, all NO donors displayed trypanocidal activities against bloodstream forms of T. brucei in vitro with 50% growth inhibition values of around 30?μM. However, the NO donors were ineffective in significantly inhibiting the cysteine proteinase activity within the parasites. This finding was confirmed by the ineffectiveness of the NO donors to block proteinolysis in the lysosome of the parasites. The results show that the trypanocidal activity of NO donors cannot be attributed to the inhibition of the major lysosomal cysteine proteinase in bloodstream forms of T. brucei.     

Analysis of expressed sequence tags from the four main developmental stages of Trypanosoma congolense

Trypanosoma congolense is one of the most economically important pathogens of livestock in Africa. Culture-derived parasites of each of the three main insect stages of the T. congolense life cycle, i.e., the procyclic, epimastigote and metacyclic stages, and bloodstream stage parasites isolated from infected mice, were used to construct stage-specific cDNA libraries and expressed sequence tags (ESTs or cDNA clones) in each library were sequenced. Thirteen EST clusters encoding different variant surface glycoproteins (VSGs) were detected in the metacyclic library and 26 VSG EST clusters were found in the bloodstream library, 6 of which are shared by the metacyclic library. Rare VSG ESTs are present in the epimastigote library, and none were detected in the procyclic library. ESTs encoding enzymes that catalyze oxidative phosphorylation and amino acid metabolism are about twice as abundant in the procyclic and epimastigote stages as in the metacyclic and bloodstream stages. In contrast, ESTs encoding enzymes involved in glycolysis, the citric acid cycle and nucleotide metabolism are about the same in all four developmental stages. Cysteine proteases, kinases and phosphatases are the most abundant enzyme groups represented by the ESTs. All four libraries contain T. congolense-specific expressed sequences not present in the Trypanosoma brucei and Trypanosoma cruzi genomes. Normalized cDNA libraries were constructed from the metacyclic and bloodstream stages, and found to be further enriched for T. congolense-specific ESTs. Given that cultured T. congolense offers an experimental advantage over other African trypanosome species, these ESTs provide a basis for further investigation of the molecular properties of these four developmental stages, especially the epimastigote and metacyclic stages for which it is difficult to obtain large quantities of organisms. The T. congolense EST databases are available at: http://www.sanger.ac.uk/Projects/T_congolense/EST_index.shtml.     

Biochemical and functional characterization of histone H1-like proteins in procyclicTrypanosoma brucei brucei

Four variants and/or posttranslational modifications of histone H1-like proteins ofTrypanosoma brucei brucei procyclic culture forms were extracted with 0.25N HCl from isolated nuclei and analyzed by two-dimensional gel electrophoresis. The amino acid composition of these proteins, their ability to space nucleosomes regularly and to induce salt-dependent condensation of the chromatin indicated their histone H1 nature. On the other hand, the histone H1-like proteins clearly differed from their higher-eukaryote counterparts by their weak interaction with DNA under low-salt conditions. As a consequence, intact nucleosome filaments were prepared according to a new preparation protocol especially adapted to the unstable chromatin ofT. b. brucei. Our results indicate that the biochemical properties of the histone H1-like proteins contribute to the structural and functional differences between the chromatin of procyclicT. b. brucei and that of higher eukaryotes.     

Characterization of concanavalin A-binding glycoproteins from procyclic culture forms ofTrypanosoma congolense,T. simiae andT. brucei brucei

Concanavalin A-binding glycoproteins were obtained from procyclic culture forms (PCFs) ofTrypanosoma congolense, T. simiae, andT. b. brucei strains. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that glycoproteins of 38.5, 30.5, and 27 kDa were conserved between the different species and strains of the procyclic parasites. There were few similarities in the profiles of the high-molecular-weight glycoconjugates between the parasites. Monoclonal antibody analysis revealed that the 38.5- and 27-kDa glycoproteins were intracellular molecules and that they contained cross-reactive antigenic determinants. Surface biotinylation of PCFT. congolense K45/1 identified surface-accessible glycoproteins of 81.5, 59, and 38–42 kDa. By use of lectin blots and enzymatic deglycosylation studies, we demonstrated that the 81.5-, 59-, 38.5-, and 27-kDa glycoproteins contained N-linked oligosaccharide chains with both high-mannose-type and complex-type oligosaccharides, and the 81.5- and 59-kDa surface glycoproteins contained sialic acid residues. The glycoproteins identified in this study provide a starting point for further structure and function studies.     

Histone-DNA interactions in the chromatin of procyclicTrypanosoma brucei brucei

The dissociation of histone proteins a-d from the chromatin ofTrypanosoma brucei brucei procyclic culture forms was investigated by removing the proteins from the DNA by centrifugation of soluble chromatin through isokinetic sucrose gradients in the presence of NaCl. The dissociation of theT. b. brucei histones was compared with that of their higher-eukaryote counter-parts H3, H2A, H2B and H4. All four histones ofT. b. brucei remained bound to the DNA at 500mM NaCl, were partially released at 750mm NaCl and were completely dissociated from the DNA at 1m NaCl. These interactions of histones a-d with the DNA were comparable with those of the H2 histones in the chromatin of higher eukaryotes, and histones a and d interacted with the DNA more weakly than did their higher-eukaryote counterparts H3 and H4. Substoichiometric amounts of an additional protein were recovered in the top fractions of the gradients under all dissociation conditions. This protein migrated in the H1 region of rat-liver chromatin in various gel systems. Its early release from the DNA also indicated a resemblance to histone H1. The presence of only small amounts of this protein and the relatively weak interactions of histones a and d with the DNA suggest that the mechanisms involved in chromatin compaction inT. b. brucei are different from those in higher eukaryotes.     

The phospholipases of Trypanosoma brucei bloodstream forms and cultured procyclics

Phopholipase from Trypanosoma brucei bloodstream forms was characterized and subsequently localized. The enzyme had a specific activity of 100 nmol · min^?1 · mg^?1 protein. The major portion (>90%) was a soluble phopholipase A₁ with a pH optimum around 6; the remainder, also phospholipase A₁, was particle-bound and had an optimal activity around pH 5.2. Both enzymes were maximally activated by 0.2% Triton X-100 but differed in their sensitivity towards the inhibitory action of higher concentrations of this detergent and diisopropyl fluorophosphate, the particle-bound activity being more sensitive than the soluble one. Cell fractionation showed that the particle-bound, more acidic phospholipase A₁ was associated with α-mannosidase- and acid proteinase-containing lysosomes. Cultured procyclic trypomastigotes also contained phospholipase A but its specific activity was only 15% of that of bloodstream forms. This drastic reduction in overall activity upon transformation from bloodstream to culture form was the result of a decrease in soluble phospholipase, whereas the lysosomal activity essentially remained unchanged.