In cellulo crystallization of Trypanosoma brucei IMP dehydrogenase enables the identification of genuine co-factors.

Sleeping sickness is a fatal disease caused by the protozoan parasite Trypanosoma brucei (Tb). Inosine-5'-monophosphate dehydrogenase (IMPDH) has been proposed as a potential drug target, since it maintains the balance between guanylate deoxynucleotide and ribonucleotide levels that is pivotal for the parasite. Here we report the structure of TbIMPDH at room temperature utilizing free-electron laser radiation on crystals grown in living insect cells. The 2.80 Å resolution structure reveals the presence of ATP and GMP at the canonical sites of the Bateman domains, the latter in a so far unknown coordination mode. Consistent with previously reported IMPDH complexes harboring guanosine nucleotides at the second canonical site, TbIMPDH forms a compact oligomer structure, supporting a nucleotide-controlled conformational switch that allosterically modulates the catalytic activity. The oligomeric TbIMPDH structure we present here reveals the potential of in cellulo crystallization to identify genuine allosteric co-factors from a natural reservoir of specific compounds.

Electronic damage in S atoms in a native protein crystal induced by an intense X-ray free-electron laser pulse.

Current hard X-ray free-electron laser (XFEL) sources can deliver doses to biological macromolecules well exceeding 1 GGy, in timescales of a few tens of femtoseconds. During the pulse, photoionization can reach the point of saturation in which certain atomic species in the sample lose most of their electrons. This electronic radiation damage causes the atomic scattering factors to change, affecting, in particular, the heavy atoms, due to their higher photoabsorption cross sections. Here, it is shown that experimental serial femtosecond crystallography data collected with an extremely bright XFEL source exhibit a reduction of the effective scattering power of the sulfur atoms in a native protein. Quantitative methods are developed to retrieve information on the effective ionization of the damaged atomic species from experimental data, and the implications of utilizing new phasing methods which can take advantage of this localized radiation damage are discussed.

Real-time investigation of dynamic protein crystallization in living cells.

X-ray crystallography requires sufficiently large crystals to obtain structural insights at atomic resolution, routinely obtained in vitro by time-consuming screening. Recently, successful data collection was reported from protein microcrystals grown within living cells using highly brilliant free-electron laser and third-generation synchrotron radiation. Here, we analyzed in vivo crystal growth of firefly luciferase and Green Fluorescent Protein-tagged reovirus µNS by live-cell imaging, showing that dimensions of living cells did not limit crystal size. The crystallization process is highly dynamic and occurs in different cellular compartments. In vivo protein crystallization offers exciting new possibilities for proteins that do not form crystals in vitro.

Ergosterone-coupled Triazol molecules trigger mitochondrial dysfunction, oxidative stress, and acidocalcisomal Ca2+ release in Leishmania mexicana promastigotes.

The protozoan parasite Leishmania causes a variety of sicknesses with different clinical manifestations known as leishmaniasis. The chemotherapy currently in use is not adequate because of their side effects, resistance occurrence, and recurrences. Investigations looking for new targets or new active molecules focus mainly on the disruption of parasite specific pathways. In this sense, ergosterol biosynthesis is one of the most attractive because it does not occur in mammals. Here, we report the synthesis of ergosterone coupled molecules and the characterization of their biological activity on Leishmania mexicana promastigotes. Molecule synthesis involved three steps: ergosterone formation using Jones oxidation, synthesis of Girard reagents, and coupling reaction. All compounds were obtained in good yield and high purity. Results show that ergosterone-triazol molecules (Erg-GTr and Erg-GTr2) exhibit an antiproliferative effect in low micromolar range with a selectivity index ~10 when compared to human dermic fibroblasts. Addition of Erg-GTr or Erg-GTr2 to parasites led to a rapid [Ca2+]cyt increase and acidocalcisomes alkalinization, indicating that Ca2+ was released from this organelle. Evaluation of cell death markers revealed some apoptosis-like indicators, as phosphatidylserine exposure, DNA damage, and cytosolic vacuolization and autophagy exacerbation. Furthermore, mitochondrion hyperpolarization and superoxide production increase were detected already 6 hours after drug addition, denoting that oxidative stress is implicated in triggering the observed phenotype. Taken together our results indicate that ergosterone-triazol coupled molecules induce a regulated cell death process in the parasite and may represent starting point molecules in the search of new chemotherapeutic agents to combat leishmaniasis.

Drug development against sleeping sickness: old wine in new bottles?

Atoxyl, the first medicinal drug against human African trypanosomiasis (HAT), also known as sleeping sickness, was applied more than 100 years ago. Ever since, the search for more effective, more specific and less toxic drugs continued, leading to a set of compounds currently in use against this devastating disease. Unfortunately, none of these medicines fulfill modern pharmaceutical requirements and may be considered as therapeutic ultima ratio due to the many, often severe side effects. Starting with a historic overview on drug development against HAT, we present a selection of trypanosome specific pathways and enzymes considered as highly potent druggable targets. In addition, we describe cellular mechanisms the parasite uses for differentiation and cell density regulation and present our considerations how interference with these steps, elementary for life cycle progression and infection, may lead to new aspects of drug development. Finally we refer to our recent work about CNS infection that offers novel insights in how trypanosomes hide in an immune privileged area to establish a chronic state of the disease, thereby considering new ways for drug application. Depressingly, HAT specific drug development has failed over the last 30 years to produce better suited medicine. However, unraveling of parasite-specific pathways and cellular behavior together with the ability to produce high resolution structures of essential parasite proteins by X-ray crystallography, leads us to the optimistic view that development of an ultimate drug to eradicate sleeping sickness from the globe might just be around the corner.

A compendium of antibiotic-induced transcription profiles reveals broad regulation of Pasteurella multocida virulence genes.

The transcriptional responses of Pasteurella multocida to eight antibiotics with known mode of actions (MoAs) and one novel antibiotic compound with an unknown MoA were collected to create a compendium of transcriptional profiles for MoA studies. At minimal inhibitory concentration the three bactericidal compounds enrofloxacin, cefquinome and the novel compound had a minor impact on gene regulation with approximately 1% of the P. multocida genome affected, whilst the bacteriostatic compounds florfenicol, tilmicosin, rifampin, trimethoprim and brodimoprim regulated 20% of the genome. Novobiocin was special in that it regulated 40% of all P. multocida genes. Regulation of target genes was observed for novobiocin, rifampin, florfenicol and tilmicosin and signature genes were identified for most antibiotics. The transcriptional profile induced by the novel compound was unrelated to the compendium profiles suggesting a new MoA. The transcription of many P. multocida virulence factors, particularly genes involved in capsule synthesis and export, LPS synthesis, competence, adherence and iron transport were altered in the presence of antibiotics. Virulence gene transcription was mainly negatively affected, however the opposite effect was also observed in the case of rifampin where the up-regulation of the tad locus involved in tight adherence was seen. Novobiocin and trimethoprim caused a marked reduction in the transcription of capsule genes, which correlated with a concomitant reduction of the capsular layer on the surface of P. multocida. The broad negative impact on virulence gene transcription supports the notion that the therapeutic effect of some antibiotics could be a combination of growth and virulence inhibition.

Prostaglandin-induced programmed cell death in Trypanosoma brucei involves oxidative stress.

Recently, we reported the induction of a programmed cell death (PCD) in bloodstream forms of Trypanosoma brucei by prostaglandin D(2) (PGD(2)). As this prostanoid is readily metabolized in the presence of albumin, we were prompted to investigate if PGD(2) metabolites rather than PGD(2) itself are responsible for the observed PCD. In fact, J series metabolites, especially PGJ(2) and Delta(12)PGJ(2), were able to induce PCD more efficiently than PGD(2). However, the stable PGD(2) analog 17phenyl-trinor-PGD(2) led to the same phenotype as the natural PGD(2), indicating that the latter induces PCD as well. Interestingly, the intracellular reactive oxygen species (ROS) level increased significantly under J series metabolites treatment and, incubation with N-acetyl-L-cysteine or glutathione reduced ROS production and cell death significantly. We conclude that PGJ(2) and Delta(12)PGJ(2) formation within the serum represents a mechanism to amplify PGD(2)-induced PCD in trypanosomes via ROS production.

Prostaglandin D2 induces programmed cell death in Trypanosoma brucei bloodstream form.

African trypanosomes produce some prostanoids, especially PGD2, PGE2 and PGF2alpha (Kubata et al. 2000, J. Exp. Med. 192: 1327-1338), probably to interfere with the host's physiological response. However, addition of prostaglandin D2 (but not PGE2 or PGF2alpha) to cultured bloodstream form trypanosomes led also to a significant inhibition of cell growth. Based on morphological alterations and specific staining methods using vital dyes, necrosis and autophagy were excluded. Here, we report that in bloodstream form trypanosomes PGD2 induces an apoptosis-like programmed cell death, which includes maintenance of plasma membrane integrity, phosphatidylserine exposure, loss of mitochondrial membrane potential, nuclear chromatin condensation and DNA degradation. The use of caspase inhibitors cannot prevent the cell death, indicating that the process is caspase-independent. Based on these results, we suggest that PGD2-induced programmed cell death is part of the population density regulation as observed in infected animals.

Identification of a novel prostaglandin f(2alpha) synthase in Trypanosoma brucei.

Members of the genus Trypanosoma cause African trypanosomiasis in humans and animals in Africa. Infection of mammals by African trypanosomes is characterized by an upregulation of prostaglandin (PG) production in the plasma and cerebrospinal fluid. These metabolites of arachidonic acid (AA) may, in part, be responsible for symptoms such as fever, headache, immunosuppression, deep muscle hyperaesthesia, miscarriage, ovarian dysfunction, sleepiness, and other symptoms observed in patients with chronic African trypanosomiasis. Here, we show that the protozoan parasite T. brucei is involved in PG production and that it produces PGs enzymatically from AA and its metabolite, PGH(2). Among all PGs synthesized, PGF(2alpha) was the major prostanoid produced by trypanosome lysates. We have purified a novel T. brucei PGF(2alpha) synthase (TbPGFS) and cloned its cDNA. Phylogenetic analysis and molecular properties revealed that TbPGFS is completely distinct from mammalian PGF synthases. We also found that TbPGFS mRNA expression and TbPGFS activity were high in the early logarithmic growth phase and low during the stationary phase. The characterization of TbPGFS and its gene in T. brucei provides a basis for the molecular analysis of the role of parasite-derived PGF(2alpha) in the physiology of the parasite and the pathogenesis of African trypanosomiasis.

Synthesis of alpha-galactosylated fragments related to the core-structure of the GPI anchor of Trypanosoma brucei.

A series of octyl glycosides di- to tetrasaccharides related to the GPI anchor of Trypanosoma brucei was prepared. Treatment of octyl 2-O-benzoyl-4,6-O-(1,1,3,3-tetraisopropyl-1,3-disiloxane-1,3 -diyl)-alpha-D-mannopyranoside with ethyl 2,3,4,6-tetra-O-benzyl-1-thio-beta-D-galactopyranoside under activation with bromine and silver trifluoromethanesulfonate afforded the alpha-linked disaccharide octyl 2-O-benzoyl-3-O-(2,3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl)-4,6-O- (1,1,3,3-tetraisopropyl-1,3-disiloxane-1,3-diyl)-alpha -D-mannospyranoside, the siloxane ring of which was regioselectively opened with a HF-pyridine complex to give the disaccharide acceptor octyl 3-O-(2,3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl)-2-O-benzoyl-4-O-(3 -fluoro-1,1,3,3-tetraisopropyl-1,3-disiloxane-3-yl)-alpha-D- mannopyranoside (4). Mannosylation of 4 with benzobromomannose (7), followed by fluoride catalyzed desilylation gave the trisaccharide octyl 2-O-benzoyl-6-O-(2,3,4,6-tetra-O-benzoyl-alpha-D-mannopyranosyl)-3-O-(2, 3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl)-alpha-D-mannospyranosi de, which was deblocked via the deacylated intermediate octyl 3-O-(2,3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl)-6-O-(alpha-D-manno pyranosyl)-alpha-D-mannospyranoside to afford the octyl glycoside trisaccharide octyl 3-O-(alpha-D-galactopyranosyl)-6-O-(alpha-D-mannopyranosyl)-alpha-D-m annospyranoside. Glycosylation of 4 with 3,4,6-tri-O-acetyl-2-O-(2,3,4,6-tetra-O-benzoyl-alpha-D-mannopyranosyl)- alpha-D-mannopyranosyl trichloroacetimidate resulted in the tetrasaccharide octyl 2-O-benzoyl-4-O-(1-fluoro-1,1,3,3-tetraisopropyl-1,3-disiloxane -3-yl)-3-O-(2,3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl)-6-O-[2-O -(2,3,4,6-tetra-O-benzoyl-alpha-D-mannopyranosyl)-3,4,6-tri-O-acetyl-alp ha-D-mannopyranosyl]-alpha-D-mannospyranoside, sequential desilylation, deacylation and debenzylation, respectively, of which via the intermediate octyl 2-O-benzoyl-3-O-(2,3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl)-6-O-[2 -O-(2,3,4,6-tetra-O-benzoyl-alpha-D-mannopyranosyl)-3,4,6-tri-O-acetyl-a lpha-D-mannopyranosyl]-alpha-D-mannospyranoside afforded the octyl glycoside tetrasaccharide octyl 3-O-(alpha-D-galactopyranosyl)-6-O-[2-O-(alpha-D-mannopyranosyl)-alpha-D -mannopyranosyl]-alpha-D-mannospyranoside.

Cloning, heterologous expression and kinetic analysis of glycerol kinase (TbGLK1) from Trypanosoma brucei.

We have cloned and sequenced the gene for the glycerol kinase of Trypanosoma brucei (TbGLK1), obtained by RT-PCR. The corresponding mRNA is 2.3 kb in size and contains an ORF encoding a protein with high homology to known glycerol kinases of other organisms. It is 512 amino acids in length with a PTS1-like targeting sequence (AKL) at its C-terminus, suggesting glycosomal compartmentalization of this enzyme. Although Northern blot analysis revealed higher mRNA levels in slender bloodstream forms than in the procyclic insect forms, specific glycerol kinase activities were found to be virtually identical in both life stages. Southern blot analysis suggested a single copy gene, but we were able to clone two alleles utmost similar to each other. Heterologous expression of the trypanosomal glycerol kinase in E. coli enabled us to perform a kinetic analysis of this enzyme. In particular, we have been able to monitor ATP production from glycerol-3-phosphate and ADP, a reaction which, although thermodynamically very unfavorable, is regarded essential for the survival of Trypanosoma brucei under anoxic conditions. Since the unique spatial separation of glycolysis in the kinetoplastida imposes important consequences for the regulation of the energy metabolism in these organisms, we discuss the observed differences between TbGLK1 and glycerol kinases from other organisms in view of its physiological relevance.

In vitro translation in a cell-free system from Trypanosoma brucei yields glycosylated and glycosylphosphatidylinositol-anchored proteins.

African trypanosomes escape many cellular and unspecific immune reactions by the expression of a protective barrier formed from a repertoire of several hundred genes encoding immunologically distinct variant surface glycoproteins (VSGs). All mature VSGs are glycosylphosphatidylionositol-anchored and N-glycosylated. To study trypanosome-specific post-translational modifications of VSG, a cell-free system capable of in vitro translation, translocation into the rough endoplasmic reticulum, N-glycosylation and glycosylphosphatidylinositol-anchor addition was established using lysates of the bloodstream form of Trypanosoma brucei. Monitoring protein synthesis by [35S]methionine incorporation, labeled protein bands were readily detected by fluorography following SDS/PAGE. Appearance of these bands increased during a time-course of 45 min and was sensitive to cycloheximide but not chloramphenicol treatment. Efficiency of this system, in terms of incorporation of radiolabeled amino acids into newly formed proteins, is similar to reticulocyte lysates. The system does not, however, allow initiation of protein synthesis. Depending on the clone used, immunoprecipitation revealed one or two newly formed VSG bands. Upon digestion with N-glycosidase F these bands resulted in a single band of a lower apparent molecular mass, indicating that newly synthesized VSG underwent translocation and glycosylation in the cell-free system. Biotinylation of VSG and a combination of precipitation with immobilized avidin and detection of VSG using antibodies specific for clones and cross-reacting determinants revealed that newly formed VSG contained the glycosylphosphatidylinositol anchor.

Purification and characterization of an alpha-galactosyltransferase from Trypanosoma brucei.

A membrane-associated galactosyltransferase from Trypanosoma brucei was purified 34000-fold by affinity chromatography on UDP-hexanolamine-Sepharosetrade mark. Using SDS/PAGE under reducing conditions, the isolated enzyme ran as a relatively broad band with apparent molecular masses of 53 kDa and 52 kDa, indicative of glycosylation and the existence of two isoforms. N-Glycosylation of the enzyme was subsequently confirmed using Western blotting and either specific binding of concanavalin A or peptide-N4-(N-acetylglucosaminyl)asparagine amidase digestion. The de-N-glycosylated enzyme ran with apparent molecular masses of 51 kDa and 50 kDa, indicative of a single N-glycosylation site. The galactosyltransferase exhibited a pH optimum at 7.2 and had a pronounced requirement for Mn2+ ions (KM=2.5 mM) for its action. The transferase activity was independent of the concentration of Triton X-100. The enzyme was capable of transferring galactose from UDP-galactose to a variety of galactose-based acceptors in alpha-glycosidic linkages. The apparent KM values for UDP-galactose and for the preferred acceptor substrate N-acetyl-lactosamine are 46 microM and 4.5 mM respectively. From these results we would like to suggest that the galactosyltransferase functions in the processing of terminal N-acetyl-lactosamine structures of trypanosomal glycoproteins.

Specific inhibition of an alpha-galactosyltransferase from Trypanosoma brucei by synthetic substrate analogues.

Since the alpha-D-galactose-(1-->3)-D-galactose epitope has been identified to be the major target in the process of hyperacute rejection of xenografts transplanted from nonprimate donors to humans, specific inhibitors of alpha-galactosyltransferases are of broad interest. Using Trypanosoma brucei, a protozoan parasite causing sleeping sickness and Nagana, we have a very useful model system for the investigation of alpha-galactosyltransferase inhibitors, since the variant surface glycoprotein (VSG) accounts for about 10% of the total cell protein an this parasite expresses many different galactosyltransferases including the one catalysing the formation of the Galalpha1-->3Gal epitope. In order to study inhibition of galactosylation on the VSG from Trypanosoma brucei, we designed, synthesized and tested substrate analogues of trypanosomal alpha-galactosyltransferases. Effective inhibitors were a pair of diastereoisomeric UDP-galactose analogs, in which the galactose residue is linked to UDP via a methylene bridge rather than an ester linkage. Hence, galactose cannot be transferred to the respective acceptor substrate VSG or the synthetic acceptor substrate Manalpha1-->6Manalpha1S-(CH2)7-CH3, which was previously proven to replace VSG effectively [Smith et al. (1996) J Biol Chem 271:6476-82]. Inhibitors have been prepared starting from 1-formyl galactal. The final condensation was performed using UMP morpholidate leading to a pair of diastereomeric compounds in 39% or 30% yield, respectively. These compounds were tested using alpha-galactosyltransferases prepared from T. brucei membranes and lactose synthetase from bovine milk. While the K(M)-value for UDP-galactose was determined as 59 microM on bovine lactose synthetase, the K(I)-values for both inhibitors were 0.3 mM and 1.1 mM respectively, showing that these inhibitors are unable to inhibit enzyme activity significantly. However, using the N-glycan specific alpha-galactosyltransferase from trypanosomes, the K(M)-value was determined as 20 microM, while the K(I)-values were 34 microM and 21 microM respectively. Interestingly, other trypanosomal alpha-galactosyltransferases, which modify the GPI membrane anchor, are 2 orders of magnitude less effected by the inhibitor.

Characterization of glycerol uptake in bloodstream and procyclic forms of Trypanosoma brucei.

Uptake of glycerol was studied in bloodstream and insect forms of the African parasite Trypanosoma brucei using [14C]glycerol in combination with the oil centrifugation technique. Our kinetic measurements revealed that in bloodstream forms glycerol appeared to be transported by two different mechanisms: firstly by a facilitated-diffusion carrier showing a Km of 0.17 mM and a Vmax of 44 nmol 10(-8) cells min(-1) that predominates at low glycerol concentrations, and secondly by simple diffusion. The effects induced by various inhibitors suggest that uptake is neither sodium dependent nor proton-motive-force driven. The saturable component of transport was phloretin and cytochalasin B sensitive and could also be inhibited by the substrate analogue glyceraldehyde, which led to a 74% decrease in glycerol uptake. In insect forms, however, glycerol is taken up by simple diffusion only. Uptake was insensitive to mercury ions and was not influenced by a variety of different channel inhibitors. Our data show that in T brucei glycerol transport across the plasma membrane occurs by simple diffusion. In addition, bloodstream forms express a carrier protein which promotes a rapid transport at low glycerol concentrations. Expression of this transport protein may account for a selective secretion of intracellular glycerol which otherwise could become toxic for the parasite due to its specific compartmentation of glycolysis.

Synthesis of octyl O- and S-glycosides related to the GPI anchor of Trypanosoma brucei and their in vitro galactosylation by trypanosomal alpha-galactosyltransferases.

Octyl O- and S-glycosides of mono- to tri-saccharides related to the core structure alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->6)-alpha-D-Manp of the GPI anchor of Trypanosoma brucei have been prepared via regioselective protodesilylation and glycodesilylation of octyl O- and S-glycosides of 2-O-benzoyl-4,6-O-(1,1,3,3-tetraisopropyl-1,3-disiloxane-1, 3-diyl)-alpha-D-mannopyranoside. The synthetic saccharides have been used as substrates for enzymatic alpha-galactosylation with membrane fractions of bloodstream forms of T. brucei strain 427 variants MITat 1.4, MITat 1.2, and MITat 1.5, respectively.

Down regulation of S-adenosyl-L-methionine decarboxylase activity of Trypanosoma brucei during transition from long slender to short stumpy-like forms in axenic culture.

Long slender trypanosomes, isolated from infected mouse blood or from cryopreserved stabilates, respectively, were unable to grow in conditioned media (cMEM), prepared from the declining phase of axenic bloodstream form cultures. Additionally, mixtures of fresh medium and cMEM led to decreased growth rates and, in accordance to the amount of cMEM used, to a decreased S-adenosyl-L-methionine decarboxylase (Ado-MetDC; E.C. 4.1.1.50) activity. Since addition of polyamines could not overcome the process of transition from dividing to non-dividing cells and the intracellular S-adenosyl-L-methionine (AdoMet), ornithine and putrescine concentrations seemed unaltered during the course of cultivation, we questioned if polyamine metabolism is involved in this transition process. Activities of two key enzymes of polyamine metabolism, AdoMetDC and ornithine decarboxylase (ODC; E.C. 4.1.1.17) were therefore monitored during different growth stages. Our results revealed a specific activity of 44 pmol min-1 mg protein-1 for AdoMetDC and a KM of 10 mu M for AdoMet. Methylglyoxal bis(guanylhydrazone) showed a Ki of 6 mu M. The constant activity of the enzyme during a 7 h time-course in the presence of cycloheximide indicates a t1/2 of more than 7 h for the trypanosomal enzyme. Enzyme activity in trypanosomes isolated from infected laboratory animals and from logarithmic phase bloodstream or procyclic form cultures was high according to a high dividing rate, whereas enzyme activity in parasites isolated from the stationary phase of bloodstream from culture was negligible. In these cultures, AdoMetDC activity decreased with a t1/2 of 7 h during transition from long slender to short stumpy-like forms as soon as the stationary phase was reached. ODC activity was high (approximately 300 pmol min-1 mg protein-1) in dividing trypanosomes isolated from infected animals as well as from logarithmic phase bloodstream or procyclic form cultures and decreased also during transition with a t1/2 of 10 h.

Glucose uptake occurs by facilitated diffusion in procyclic forms of Trypanosoma brucei.

The glucose transporter of Trypanosoma brucei procyclic forms was characterized and compared with its bloodstream form counterpart. Measuring the glucose consumption enzymatically, we determined a saturable uptake process of relatively high affinity (Km = 80 microM, Vmax = 4 nmol min-1 10(-8) cells), which showed substrate inhibition at glucose concentrations above 1.5 mM (Ki = 21 mM). Control experiments measuring deoxy-D-[3H]Glc uptake under zero-trans conditions indicated that substrate inhibition occurred on the level of glycolysis. Temperature-dependent kinetics revealed a temperature quotient of Q10 = 2.33 and an activation energy of Ea = 64 kJ mol-1. As shown by trans-stimulation experiments, glucose uptake was stereospecific for the D isomer, whereas L-glucose was not recognized. Inhibitor studies using either the uncoupler carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone (5 microM), the H+/ATPase inhibitor N,N'-dicyclohexylcarbodiimide (20 microM), the ionophor monensin (1 microM), or the Na+/K+-ATPase inhibitor ouabain (1 mM) showed insignificant effects on transport efficiency. The procyclic glucose transporter was subsequently enriched in a plasma-membrane fraction and functionally reconstituted into proteoliposomes. Using Na+-free conditions in the absence of a proton gradient, the specific activity of D-[14C]glucose transport was determined as 2.9 nmol min-1 (mg protein)-1 at 0.2 mM glucose. From these cumulative results, we conclude that glucose uptake by the procyclic insect form of the parasite occurs by facilitated diffusion, similar to the hexose-transport system expressed in bloodstream forms. However, the markedly higher substrate affinity indicates a differential expression of different transporter isoforms throughout the lifecycle.

The hydrophobic mannoside Man alpha 1-6Man alpha 1-S-(CH2)7-CH3 acts as an acceptor for the UDP-Gal:glycosylphosphatidylinositol anchor alpha 1,3-galactosyltransferase of Trypanosoma brucei.

The variant surface glycoproteins (VSGs) of Trypanosoma brucei are attached to the plasma membrane via a glycosylphosphatidylinositol (GPI) membrane anchor. This anchor contains the core sequence ethanolamine-PO4-6Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN alpha 1-6myo-inositol, which is conserved in all GPI anchors, and a unique alpha Gal side chain attached to the 3-position of the alpha Man residue adjacent to the alpha GlcN residue. Here we report that trypanosome membranes can catalyse the transfer of Gal from UDP-Gal to the hydrophobic thioglycoside Man alpha 1-6Man alpha 1-S-(CH2)7-CH3. Characterization of the galactosylated products by electrospray mass spectrometry, exoglycosidase digestion and periodate-oxidation studies revealed that the major product was Man alpha 1-6(Gal alpha 1-3)Man alpha 1-S-(CH2)7-CH3. The similarity of this product to part of the mature VSG GPI anchor suggests that the thioglycoside is able to act as an acceptor for the trypanosome-specific UDP-Gal-GPI anchor alpha 1,3-galactosyltransferase.

A novel cultivation technique for long-term maintenance of bloodstream form trypanosomes in vitro.

We used an axenic cultivation system to grow African trypanosomes in vitro. Long-term cultivation for more than 60 days has been achieved by replacing the culture medium at regular intervals between 6 and 48 h. In contrast to a control culture without medium replacement, increasing amounts of maximum cell concentrations have been obtained, ranging from 5 x 10(6) to 2 x 10(7) trypanosomes ml-1, whereas the generation doubling time remained constant (about 6 h). Higher cell concentrations have only been obtained by total medium replacement; neither addition of fresh medium nor serum led to a higher cell yield, suggesting that a trypanosome-derived factor or metabolite accumulated in the medium rather than medium was depleted of an essential nutrient. Most interestingly, however, successive waves have been obtained which eventually led to a damped oscillation curve with a constant high population density after about 40 days of cultivation. Cultures were started with a homogeneous population of the long-slender form. As judged by light microscopy, cells showed a stumpy morphology during the declining phase and became slender again in the following growth phase. At later time points, when cells remained in a stationary phase at high population density, many different morphological stages have been observed, similar to those described by early authors as intermediate forms [Ormerod, W. E. (1979) In: Biology of the Kinetoplastida, Vol. 2, pp. 340-393], although many dividing forms are still present at that time. In contrast, identically treated procyclic cultures were unable to produce cyclic growth waves. Based on these results, a novel concept considering a possible differentiation mechanism is discussed.