Cysteine eliminates the feeder cell requirement for cultivation of Trypanosoma brucei bloodstream forms in vitro.

In all previous studies, bloodstream forms of Trypanosoma brucei could be grown in vitro only when supported by a feeder layer of mammalian fibroblasts. We have axenically cultivated bloodstream T. brucei by adding L-cysteine at regular intervals and appropriate concentrations. The optimum cysteine concentration depends on cell density and is close to physiological serum levels. At concentrations greater than 24 mg/liter (2 X 10(-4) M), cysteine was acutely toxic to trypanosome concentrations of 3 X 10(7)/ml. Toxicity was prevented by addition of pyruvate or catalase, which neutralize H2O2 produced by cysteine autoxidation. In uptake studies using [35S]cysteine and [35S]cystine, T. brucei efficiently incorporated only cysteine. The Km for cysteine uptake was 4 X 10(-4) M. Cystine supported axenic growth if low concentrations of 2-mercaptoethanol were added at regular intervals.

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.

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.

Intracellular transport of a variant surface glycoprotein in Trypanosoma brucei.

Trypanosome variant surface glycoproteins (VSGs) have a novel glycan-phosphatidylinositol membrane anchor, which is cleavable by a phosphatidylinositol-specific phospholipase C. A similar structure serves to anchor some membrane proteins in mammalian cells. Using kinetic and ultrastructural approaches, we have addressed the question of whether this structure directs the protein to the cell surface by a different pathway from the classical one described in other cell types for plasma membrane and secreted glycoproteins. By immunogold labeling on thin cryosections we were able to show that, intracellularly, VSG is associated with the rough endoplasmic reticulum, all Golgi cisternae, and tubulovesicular elements and flattened cisternae, which form a network in the area adjacent to the trans side of the Golgi apparatus. Our data suggest that, although the glycan-phosphatidylinositol anchor is added in the endoplasmic reticulum, VSG is nevertheless subsequently transported along the classical intracellular route for glycoproteins, and is delivered to the flagellar pocket, where it is integrated into the surface coat. Treatment of trypanosomes with 1 microM monensin had no effect on VSG transport, although dilation of the trans-Golgi stacks and lysosomes occurred immediately. Incubation of trypanosomes at 20 degrees C, a treatment that arrests intracellular transport from the trans-Golgi region to the cell surface in mammalian cells, caused the accumulation of VSG molecules in structures of the trans-Golgi network, and retarded the incorporation of newly synthesized VSG into the surface coat.

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.

Specific inactivation of glucose metabolism from eucaryotic cells by pentalenolactone.

Pentalenolactone, an antibiotic related to the class of the sesquiterpene-lactones and produced by the strain Streptomyces arenae Tü-469, inhibits specifically the glucose metabolism by inactivation of the enzyme glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD oxidoreductase (phosphorylating) ED 1.2.1.1.2). The sensitivity of several eucaryotic cell-systems for pentalenolactone was shown under in vivo conditions. The glycolytic as well as the gluconeogenetic pathway of mammalian cells can be completely inhibited with low concentrations of the antibiotic. In all cases, the minimum inhibitory concentration is dependent on cell density. The inhibitory effect in vivo and in vitro does not seem to be species-specific. In erythrocytes from rats, in Ehrlich-ascites tumor cells and in Plasmodium vinckei infected erythrocytes from mice glycolysis can be inhibited with concentrations of 18--90 micrometers pentalenolactone. In hepatocytes, glycolysis as well as gluconeogenesis in prevented by the same concentrations. In contrast to these results, in yeast the inhibition depends on growth conditions. The inhibition in glucose medium is cancelled by precultivation on acetate-containing medium.

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.

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.

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.

Influence of Ca2+ depletion on cytoskeleton and nucleolus morphology in Trypanosoma brucei.

Trypanosoma brucei bloodstream forms were incubated in a calcium-free medium containing 10 microM ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). Under these conditions, addition of 5 microM calcium ionophore A23187 led to striking morphological alterations, as judged by light and electron microscopy. The cytoskeleton of trypanosomes consists of a subpellicular corset of microtubules. Characteristically four of these microtubules are attached invariantly to an extension of the endoplasmic reticulum at the flagellar attachment site. Specifically in this area calcium depletion led to the polymerization of additional microtubules and to a retraction of the endoplasmic reticulum extension from its usual position. Additionally, A23187 led to nucleolus segregation, as revealed by immunocytochemistry using antibodies against DNA and fibrillarin, respectively. Nucleolus segregation, but not microtubule accumulation, was also obtained by using 20 microM camptothecin, a specific inhibitor of topoisomerase I. Our data suggest that intracellular calcium regulation might be important for specific depolymerization/polymerization reactions during the course of cell division and the formation of functional ribosomes.

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.

Inhibition of glyceraldehyde-3-phosphate dehydrogenase by pentalenolactone in Trypanosoma brucei.

Pentalenolactone (PL), an antibiotic produced by several strains of Streptomycetes, is a specific irreversible inhibitor of glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12). The effect of this antibiotic was studied in Trypanosoma brucei. In infected mice, due to the rapid metabolic inactivation of PL in vivo, trypanosomes were not affected by concentrations that were lethal to the host. Bloodstream trypanosomes in vitro were killed by low concentrations of PL (1.5 microgram ml-1), suggesting that there is no alternative to the glycolytic pathway for the generation of ATP in the bloodstream forms. In contrast, even high concentrations of PL (75 micrograms ml-1) were unable to inhibit growth of the procyclic form in vitro, presumably due to their ability to generate ATP independently of the glycolytic pathway.

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 from logarithmically growing axenic cultures were transferred to a medium containing 20% dialysed foetal calf serum, 10 microM bathocuproine sulphonate and 250 microM cysteine. Growth curves of these cultures have been compared to those obtained in identical cultures containing no cysteine but cystine and reducing agents (beta-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 cystine by the action of reducing agents. However, neither reducing agents alone, nor D-cysteine, supported cell growth. Since cystine 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.

Differentiation of Trypanosoma brucei bloodstream trypomastigotes from long slender to short stumpy-like forms in axenic culture.

An axenic cultivation system was used to study the differentiation of Trypanosoma brucei bloodstream forms from long slender to short stumpy-like forms. Trypanosomes in the logarithmic phase are similar to long slender bloodstream forms freshly isolated from infected mice, differing only in the rate of oxygen uptake. In contrast, trypanosomes in the stationary phase show a decreased level of glucose oxidation, express pyrroline-5-carboxylate reductase (proline oxidase), are inhibited in oxygen uptake to about 44% by KCN, undergo considerable morphological changes on the cellular and subcellular level, and have a significantly smaller cell volume. These results are comparable to those observed during the differentiation of long slender to short stumpy forms in infected animals, suggesting that the differentiation process towards insect procyclic forms can be initiated in culture at 37 degrees C. As judged from immunofluorescence and electron microscopy analysis, the surface coat remains intact.

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.

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.

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.

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.