Glycerol kinase of African trypanosomes possesses an intrinsic phosphatase activity.

BACKGROUND: In general, glycerol kinases (GKs) are transferases that catalyze phospho group transfer from ATP to glycerol, and the mechanism was suggested to be random bi-bi. The reverse reaction i.e. phospho transfer from glycerol 3-phosphate (G3P) to ADP is only physiologically feasible by the African trypanosome GK. In contrast to other GKs the mechanism of Trypanosoma brucei gambiense glycerol kinase (TbgGK) was shown to be in an ordered fashion, and proceeding via autophosphorylation. From the unique reaction mechanism of TbgGK, we envisaged its potential to possess phosphatase activity in addition to being a kinase. METHODS: Our hypothesis was tested by spectrophotometric and LC-MS/MS analyses using paranitrophenyl phosphate (pNPP) and TbgGK's natural substrate, G3P respectively. Furthermore, protein X-ray crystallography and site-directed mutagenesis were performed to examine pNPP binding, catalytic residues, and the possible reaction mechanism. RESULTS: In addition to its widely known and expected phosphotransferase (class II) activity, TbgGK can efficiently facilitate the hydrolytic cleavage of phosphoric anhydride bonds (a class III property). This phosphatase activity followed the classical Michaelis-Menten pattern and was competitively inhibited by ADP and G3P, suggesting a common catalytic site for both activities (phosphatase and kinase). The structure of the TGK-pNPP complex, and structure-guided mutagenesis implicated T276 to be important for the catalysis. Remarkably, we captured a crystallographic molecular snapshot of the phosphorylated T276 reaction intermediate. CONCLUSION: We conclude that TbgGK has both kinase and phosphatase activities. GENERAL SIGNIFICANCE: This is the first report on a bifunctional kinase/phosphatase enzyme among members of the sugar kinase family.

Molecular basis for the reverse reaction of African human trypanosomes glycerol kinase.

The glycerol kinase (GK) of African human trypanosomes is compartmentalized in their glycosomes. Unlike the host GK, which under physiological conditions catalyzes only the forward reaction (ATP-dependent glycerol phosphorylation), trypanosome GK can additionally catalyze the reverse reaction. In fact, owing to this unique reverse catalysis, GK is potentially essential for the parasites survival in the human host, hence a promising drug target. The mechanism of its reverse catalysis was unknown; therefore, it was not clear if this ability was purely due to its localization in the organelles or whether structure-based catalytic differences also contribute. To investigate this lack of information, the X-ray crystal structure of this protein was determined up to 1.90 Å resolution, in its unligated form and in complex with three natural ligands. These data, in conjunction with results from structure-guided mutagenesis suggests that the trypanosome GK is possibly a transiently autophosphorylating threonine kinase, with the catalytic site formed by non-conserved residues. Our results provide a series of structural peculiarities of this enzyme, and gives unexpected insight into the reverse catalysis mechanism. Together, they provide an encouraging molecular framework for the development of trypanosome GK-specific inhibitors, which may lead to the design of new and safer trypanocidal drug(s).

Secreted proteases of Trypanosoma brucei gambiense: possible targets for sleeping sickness control?

Human African trypanosomiasis (HAT) is caused by trypanosomes of the species Trypanosoma brucei and belongs to the neglected tropical diseases. Presently, WHO has listed 36 countries as being endemic for sleeping sickness. No vaccine is available, and disease treatment is difficult and has life-threatening side effects. Therefore, there is a crucial need to search for new therapeutic targets against the parasite. Trypanosome excreted-secreted proteins could be promising targets, as the total secretome was shown to inhibit, in vitro, host dendritic cell maturation and their ability to induce lymphocytic allogenic responses. The secretome was found surprisingly rich in various proteins and unexpectedly rich in diverse peptidases, covering more than ten peptidase families or subfamilies. Given their abundance, one may speculate that they would play a genuine role not only in classical "housekeeping" tasks but also in pathogenesis. The paper reviews the deleterious role of proteases from trypanosomes, owing to their capacity to degrade host circulating or structural proteins, as well as proteic hormones, causing severe damage and preventing host immune response. In addition, proteases account for a number of drug targets, such drugs being used to treat severe diseases such AIDS. This review underlines the importance of secreted proteins and especially of secreted proteases as potential targets in HAT-fighting strategies. It points out the need to conduct further investigations on the specific role of each of these various proteases in order to identify those playing a central role in sleeping sickness and would be suitable for drug targeting.

Biochemical characterization of highly active Trypanosoma brucei gambiense glycerol kinase, a promising drug target.

Human African trypanosomes are blood parasites that cause sleeping sickness, a debilitating disease in sub-Saharan Africa. Glycerol kinase (GK) of these parasites additionally possesses a novel property of reverse catalysis. GK is essential to blood stream form trypanosome, and therefore a promising drug target. Here, utilizing recombinant DNA technology an optimized procedure for obtaining large amount of the purified protein was established. Furthermore, biochemical data on its enzymology are reported. The protein was maximally active at pH 6.8 over a temperature range of 25-70°C, with activation energy of 34.02 ± 0.31 kJ mol(-1). The enzyme catalyses a reversible bisubstrate [ADP and glycerol 3-phosphate (G3P)]-biproduct (ATP and glycerol) reaction. It has Km of 0.90 and 5.54 mM for ADP and G3P, respectively, and Vmax of 25.3 and 20.0 µmol min(-1) mg(-1), respectively. Unexpectedly, the enzyme lost more than 50% of its activity in 48 h at 4°C in 0.1 M sodium phosphate buffer pH 6.8 containing 10 mM MgSO4. However, perfect stabilization of the GK for more than 4 weeks was achieved in the presence of its natural ligands and cofactor. Using this stabilized protein, crystals of trypanosome GK with better resolution were obtained. This will accelerate the success of GK inhibitor development for drug design.

Trypanosome alternative oxidase, a potential therapeutic target for sleeping sickness, is conserved among Trypanosoma brucei subspecies.

Trypanosoma brucei rhodesiense and T. b. gambiense are known causes of human African trypanosomiasis (HAT), or "sleeping sickness," which is deadly if untreated. We previously reported that a specific inhibitor of trypanosome alternative oxidase (TAO), ascofuranone, quickly kills African trypanosomes in vitro and cures mice infected with another subspecies, non-human infective T. b. brucei, in in vivo trials. As an essential factor for trypanosome survival, TAO is a promising drug target due to the absence of alternative oxidases in the mammalian host. This study found TAO expression in HAT-causing trypanosomes; its amino acid sequence was identical to that in non-human infective T. b. brucei. The biochemical understanding of the TAO including its 3 dimensional structure and inhibitory compounds against TAO could therefore be applied to all three T. brucei subspecies in search of a cure for HAT. Our in vitro study using T. b. rhodesiense confirmed the effectiveness of ascofuranone (IC(50) value: 1 nM) to eliminate trypanosomes in human infective strain cultures.

Overproduction, purification, crystallization and preliminary X-ray diffraction analysis of Trypanosoma brucei gambiense glycerol kinase.

In the bloodstream forms of human trypanosomes, glycerol kinase (GK; EC 2.7.1.30) is one of the nine glycosomally compartmentalized enzymes that are essential for energy metabolism. In this study, a recombinant Trypanosoma brucei gambiense GK (rTbgGK) with an N-terminal cleavable His(6) tag was overexpressed, purified to homogeneity and crystallized by the sitting-drop vapour-diffusion method using PEG 400 as a precipitant. A complete X-ray diffraction data set to 2.75 A resolution indicated that the crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 63.84, b = 121.50, c = 154.59 A. The presence of two rTbgGK molecules in the asymmetric unit gives a Matthews coefficient (V(M)) of 2.5 A(3) Da(-1), corresponding to 50% solvent content.

Expression, purification, characterization, and in vivo targeting of trypanosome CTP synthetase for treatment of African sleeping sickness.

African sleeping sickness is a fatal disease caused by two parasite subspecies: Trypanosoma brucei gambiense and T. b. rhodesiense. We previously reported that trypanosomes have extraordinary low CTP pools compared with mammalian cells. Trypanosomes also lack salvage of cytidine/cytosine making the parasite CTP synthetase a potential target for treatment of the disease. In this study, we have expressed and purified recombinant T. brucei CTP synthetase. The enzyme has a higher K(m) value for UTP than the mammalian CTP synthetase, which in combination with a lower UTP pool may account for the low CTP pool in trypanosomes. The activity of the trypanosome CTP synthetase is irreversibly inhibited by the glutamine analogue acivicin, a drug extensively tested as an antitumor agent. There is a rapid uptake of acivicin in mice both given intraperitoneally and orally by gavage. Daily injection of acivicin in trypanosome-infected mice suppressed the infection up to one month without any significant loss of weight. Experiments with cultured bloodstream T. brucei showed that acivicin is trypanocidal if present at 1 mum concentration for at least 4 days. Therefore, acivicin may qualify as a drug with "desirable" properties, i.e. cure within 7 days, according to the current Target Product Profiles of WHO and DNDi.

Ornithine decarboxylase and S-adenosylmethionine decarboxylase in trypanosomatids.

The production of polyamines has been shown to be an effective target for a drug against the West African form of sleeping sickness caused by Trypanosoma brucei gambiense. T. brucei belongs to the group of protozoan parasites classed as trypanosomatids. Parasitic species of this group are the causative agents of various tropical diseases besides African sleeping sickness, e.g. Chagas' disease (Trypanosoma cruzi), cutaneous (Lesihmania spp.) and visceral (Leishmania donovani) leishmaniasis. The metabolism of polyamines in the parasites is a potential target for the development of new drugs for treatment of these diseases. The key steps in polyamine synthesis are catalysed by ODC (ornithine decarboxylase) and AdoMetDC (S-adenosylmethionine decarboxylase). In the present paper, some of the available information on ODC and AdoMetDC in trypanosomatids will be described and discussed.

Blood-brain barrier traversal by African trypanosomes requires calcium signaling induced by parasite cysteine protease.

In this study we investigated why bloodstream forms of Trypanosoma brucei gambiense cross human brain microvascular endothelial cells (BMECs), a human blood-brain barrier (BBB) model system, at much greater efficiency than do T. b. brucei. After noting that T. b. gambiense displayed higher levels of cathepsin L-like cysteine proteases, we investigated whether these enzymes contribute to parasite crossing. First, we found that T. b. gambiense crossing of human BMECs was abrogated by N-methylpiperazine-urea-Phe-homopheylalanine-vinylsulfone-benzene (K11777), an irreversible inhibitor of cathepsin L-like cysteine proteases. Affinity labeling and immunochemical studies characterized brucipain as the K11777-sensitive cysteine protease expressed at higher levels by T. b. gambiense. K11777-treated T. b. gambiense failed to elicit calcium fluxes in BMECs, suggesting that generation of activation signals for the BBB is critically dependant on brucipain activity. Strikingly, crossing of T. b. brucei across the BBB was enhanced upon incubation with brucipain-rich supernatants derived from T. b. gambiense. The effects of the conditioned medium, which correlated with ability to evoke calcium fluxes, were canceled by K11777, but not by the cathepsin B inhibitor CA074. Collectively, these in vitro studies implicate brucipain as a critical driver of T. b. gambiense transendothelial migration of the human BBB.

Maintaining the protective variant surface glycoprotein coat of African trypanosomes.

The African trypanosome Trypanosoma brucei has a precarious existence as an extracellular parasite of the mammalian bloodstream, where it is faced with continuous immune attack. Key to survival is a dense VSG (variant surface glycoprotein) coat, which is repeatedly switched during the course of a chronic infection. New data demonstrate a link between VSG synthesis and cell cycle progression, indicating that VSG is monitored during the trypanosome cell cycle.

Sequencing, modeling, and selective inhibition of Trypanosoma brucei hexokinase.

For Trypanosoma brucei, a parasite responsible for African sleeping sickness, carbohydrate metabolism is the only source of ATP, and glycolytic enzymes are localized within membrane-bound organelles called glycosomes. Hexokinase, the first enzyme of the glycolytic pathway, was chosen as a target for selective drug design. We have cloned and sequenced the hexokinase gene of T. brucei. In parallel, we have synthesized several inhibitors. Kinetic analysis revealed differences in the binding mode of these compounds toward yeast and T. brucei hexokinases, while the m-bromophenyl glucosamide was found to be selective for T. brucei. The modeled structure of T. brucei hexokinase-inhibitor complex (using the crystal structure of the Schistosoma mansoni hexokinase as a template) allows us to propose a mode of action of this inhibitor for the trypanosome hexokinase and to account for the observed selectivity.

Genetic characterization of Trypanosoma brucei gambiense and clinical evolution of human African trypanosomiasis in Côte d'Ivoire.

Human African trypanosomiasis is a parasitic infection caused by protozoa belonging to Trypanosoma brucei subspecies. The clinical evolution of this disease is complex and might be because of the parasite itself, as genetic diversity has been observed in T. brucei ssp. We investigated the relationship between the genetic diversity of trypanosomes and the diversity of clinical patterns in Côte d'Ivoire. We studied clinical sleeping sickness cases, and genetically analysed the trypanosomes isolated from these patients. An important genetic monomorphism among stocks isolated in Côte d'Ivoire was observed by using various markers: isoenzymes electrophoresis, random amplified polymorphism DNA and PCR of microsatellite sequences. At the same time, the diversity of clinical patterns and evolutions was confirmed by clinical analysis. The existence of an individual susceptibility to disease (human trypanotolerance) should be taken into account even if our genetic conclusions might be distorted because the isolation success rates were particularly poor. In fact, we observed that the isolation success rate varied significantly depending both on the focus of origin (P=0.0002) and on the ethnic group (P=0.0317) of the patient. Further investigations are required in order to study a possible selective impact of the use of the kit for in vitro isolation of trypanosomes as an isolation technique.

Phospholipase A2 from Trypanosoma brucei gambiense and Trypanosoma brucei brucei: inhibition by organotins.

Activity and kinetics of phospholipase A2 (PLA2) from Trypanosoma brucei gambiense (Wellcome strain) and Trypanosoma brucei brucei (GUTat 3.1) were examined using two different fluorescent substrates. The activity in the supernatants of sonicated parasites was Ca2+-independent, strongly stimulated by Triton X-100 with optimum activity at 37 degrees C and pH 6.5-8.5. To encourage a possible interaction between the parasite enzyme and organotin compounds, fatty acid derivatives of dibutyltin dichloride were synthesized and evaluated as potential inhibitors of PLA2. The enzyme from the two-trypanosome species differ with respect to kinetic parameters and are noncompetitively inhibited by the organotin compounds. The Michaelis constant (KM) for PLA2 from T. b. brucei is 63.87 and 30.90 microM while for T. b. gambiense it is 119.64 and 32.91 microM for the substrates 1,2-bis-(1-pyrenebutanoyl)-sn-glycero-3-phosphocholine (PBGPC) and 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBDC12-HPC), respectively.

Purified reconstituted inositol 1,4,5-trisphosphate receptors. Thiol reagents act directly on receptor protein.

Thimerosal, a sulfhydryl oxidizing reagent, has been shown to induce Ca2+ mobilization in several cell types and to increase the sensitivity of intracellular Ca2+ stores to inositol 1,4,5-trisphosphate (IP3). Using purified IP3 receptor (IP3R) protein reconstituted in vesicles, we demonstrate pronounced stimulation by thimerosal of its Ca2+ channel activity. Effects of thimerosal are dependent on the redox state of the receptor, implying an action of thimerosal on a critical sulfhydryl group(s) of IP3R. Thimerosal enhances the affinity of IP3R for IP3 binding. The manner in which thimerosal modulates IP3R responsiveness to IP3 provides evidence for receptor heterogeneity with implications for mechanisms of quantal Ca2+ release. These results clarify regulation of IP3R activity by redox modulation.

The isolation and genetic heterogeneity of Trypanosoma brucei gambiense from north-west Uganda.

Fifty-two samples of blood were taken from sleeping sickness patients in north-west Uganda. All samples failed to infect immunosuppressed mice. Ten cryopreserved blood samples were fed to laboratory bred Glossina morsitans morsitans; eight flies developed midgut infections from which procyclic cultures were established in vitro. Isoenzyme electrophoretic analysis of 9 enzymes revealed that 7 of the 8 trypanosome isolates had a combination of enzyme patterns already described for Trypanosoma brucei gambiense. The eighth isolate had a different aspartate aminotransferase polymorphism which placed it in a new zymodeme. Analysis of polymorphisms in genes for 3 variant surface glycoproteins (VSGs) confirmed that the 8 Ugandan trypanosome isolates were T.b.gambiense and revealed further heterogeneity. The VSG 117 gene was present in all the isolates in a pattern of fragments (equivalent to AnTat 1.8) characteristic for T.b.gambiense. For two other VSG genes characteristic of T.b.gambiense, the LiTat 1.3 gene was present in all the isolates, while the AnTat 11.17 gene was present in only 2 of the 8 isolates.

Genetic characterization of six parasitic protozoa: parity between random-primer DNA typing and multilocus enzyme electrophoresis.

We have assayed genetic polymorphisms in several species of parasitic protozoa by means of random amplified polymorphic DNA (RAPD). One goal was to ascertain the suitability of RAPD markers for investigating genetic and evolutionary problems, particularly in organisms, such as the parasitic protozoa, unsuitable for traditional methods of genetic analysis. Another goal was to test certain hypotheses concerning Trypanosoma cruzi, and other protozoa, that have been established by multilocus enzyme electrophoresis. The RAPD results corroborate the hypothesis that the population structure of T. cruzi is clonal and yield a phylogeny of the clonal lineages in agreement with the one obtained by enzyme electrophoresis. This parity between the two sets of results confirms that RAPD markers are reliable genetic markers. The RAPD markers are also suitable for reconstructing species phylogenies and as diagnostic characters of species and subspecific lineages. The number of DNA polymorphisms that can be detected by the RAPD method seems virtually unlimited, since the number of primers can be increased effectively at will. The RAPD method is well suited for investigating genetic and evolutionary questions in certain organisms, because it is cost effective and demands no previous genetic knowledge about the organism.

Numerical taxonomy of Trypanozoon based on polymorphisms in a reduced range of enzymes.

Numerical analyses of Trypanozoon taxonomy are presented, based on the isoenzyme data of Stevens et al. (1992). The previous study used a reduced range of enzymes compared with earlier work; the analyses indicate the value of this rationalized system. Both recently isolated trypanosome stocks and previously studied populations were included, allowing detailed comparison with earlier studies. Relationships between zymodemes were calculated with an improved similarity coefficient program, using Jaccard's coefficient (1908), and by Nei's method (1972). Dendrograms were constructed from the matrices produced with the group-average method. The groupings produced by both numerical methods were in close agreement, and the clusters of related principal zymodemes largely matched the species, subspecies and strain groups proposed by previous workers. Trypanozoon biochemical taxonomy is reviewed and the groupings reinforced by this study are: the mainly East African strain groups, busoga, zambezi, kakumbi, kiboko and sindo; T.b. gambiense and the bouaflé strain group from West Africa, and T. evansi; an intermediate bouaflé/busoga group was also recognized.

Characterization by isoenzyme electrophoresis of Trypanozoon stocks from sleeping sickness endemic areas of south-east Uganda.

An epidemic of sleeping sickness, which started in 1976 in a focus within the county of Luuka in Central Busoga, has spread to cover the three districts of Busoga and large parts of the neighbouring districts of Tororo and Mukono. Forty-three isolates of the subgenus Trypanozoon from Busoga and Tororo (27 from man, 9 from cows, 2 from pigs and 5 from tsetse flies) were compared by thin-layer starch-gel electrophoresis for seven enzymes. Thirty zymodemes were identified; 17 of them were found circulating in the human population. The zymodemes seen previously in Busoga were still circulating together with several new ones. Of the 16 isolates from cattle, pigs and tsetse flies, only two had the same profile, indicating a high degree of diversity. Two zymodemes from cows and a pig were identical to those found in man, implicating domestic stock in the transmission of human disease in south-east Uganda. A computer analysis of the results produced six main zymodeme groups. One comprised only isolates from man; two were composed of isolates from man, domestic animals and tsetse; and three consisted of stocks from domestic animals only. These groups quite probably indicate the different cycles of transmission involving man, tsetse fly and domestic stock.

The substitution of procyclic for bloodstream form Trypanosoma brucei gambiense in isoenzyme studies.

Examination of 10 enzymes from 8 stocks of Trypanosoma brucei showed that procyclic forms could be substituted for bloodstream forms in isoenzyme studies. T. b. gambiense procyclic forms cultured in vitro offer a better source of material for genetic investigations because this species is usually of low infectivity and virulence to laboratory rodents. Using 6 stocks of T. b. gambiense and 2 stocks of T. b. brucei, enzyme patterns of bloodstream and procyclic forms were identical for isocitrate dehydrogenase, malic enzyme, two nucleoside hydrolases (utilizing inosine and deoxyinosine respectively), phosphoglucomutase and superoxide dismutase. Procyclic forms appeared to have greater threonine dehydrogenase activity than bloodstream forms. Consistent differences between bloodstream and culture forms were observed for alanine aminotransferase, aspartate aminotransferase and malate dehydrogenase. These agreed with known differences in the metabolism of procyclic and bloodstream forms.