A novel lipase with dual localisation in Trypanosoma brucei.

Phospholipases are esterases involved in lipid catabolism. In pathogenic micro-organisms (bacteria, fungi, parasites) they often play a critical role in virulence and pathogenicity. A few phospholipases (PL) have been characterised so far at the gene and protein level in unicellular parasites including African trypanosomes (AT). They could play a role in different processes such as host-pathogen interaction, antigenic variation, intermediary metabolism. By mining the genome database of AT we found putative new phospholipase candidate genes and here we provided biochemical evidence that one of these has lipolytic activity. This protein has a unique non-canonical glycosome targeting signal responsible for its dual localisation in the cytosol and the peroxisomes-related organelles named glycosomes. We also show that this new phospholipase is excreted by these pathogens and that antibodies directed against this protein are generated during an experimental infection with T. brucei gambiense, a subspecies responsible for infection in humans. This feature makes this protein a possible tool for diagnosis.

Acetate and succinate production in amoebae, helminths, diplomonads, trichomonads and trypanosomatids: common and diverse metabolic strategies used by parasitic lower eukaryotes.

Parasites that often grow anaerobically in their hosts have adopted a fermentative strategy relying on the production of partially oxidized end products, including lactate, glycerol, ethanol, succinate and acetate. This review focuses on recent progress in understanding acetate production in protist parasites, such as amoebae, diplomonads, trichomonads, trypanosomatids and in the metazoan parasites helminths, as well as the succinate production pathway(s) present in some of them. We also describe the unconventional organisation of the tricarboxylic acid cycle associated with the fermentative strategy adopted by the procyclic trypanosomes, which may resemble the probable structure of the primordial TCA cycle in prokaryotes.

N-acetyl D-glucosamine stimulates growth in procyclic forms of Trypanosoma brucei by inducing a metabolic shift.

SUMMARYThe lectin-inhibitory sugars D-glucosamine (GlcN) and N-acetyl D-glucosamine (GlcNAc) are known to enhance susceptibility of the tsetse fly vector to infection with Trypanosoma brucei. GlcNAc also stimulates trypanosome growth in vitro in the absence of any factor derived from the fly. Here, we show that GlcNAc cannot be used as a direct energy source, nor is it internalized by trypanosomes. It does, however, inhibit glucose uptake by binding to the hexose transporter. Deprivation of D-glucose leads to a switch from a metabolism based predominantly on substrate level phosphorylation of D-glucose to a more efficient one based mainly on oxidative phosphorylation using L-proline. Procyclic form trypanosomes grow faster and to higher density in D-glucose-depleted medium than in D-glucose-rich medium. The ability of trypanosomes to use L-proline as an energy source can be regulated depending upon the availability of D-glucose and here we show that this regulation is a graded response to D-glucose availability and determined by the overall metabolic state of the cell. It appears, therefore, that the growth stimulatory effect of GlcNAc in vitro relates to the switch from D-glucose to L-proline metabolism. In tsetse flies, however, it seems probable that the effect of GlcNAc is independent of this switch as pre-adaptation to growth in proline had no effect on tsetse infection rate.

Conservation of metacyclic variant surface glycoprotein expression sites among different trypanosome isolates.

We identified in a Trypanosoma brucei brucei strain (AnTat 1) an expression site for a metacyclic variant surface glycoprotein (MVSG) gene (MVSG) that was previously characterized in a T. b. rhodesiense strain (WRATat 1.1). The 3.4 kb sequences of the two expression sites are 99.6% identical, with no differences in the sequence of the 1.5 kb MVSG. Two other MVSGs in the WRATat 1.1 genome are not present in the AnTat 1 genome. In addition, five other T. b. brucei and T. b. rhodesiense strains, isolated in the same geographic region as the two former strains, do not contain any of these three MVSGs. Two of these five strains, however, appear to possess a very similar MVSG expression site, but with different MVSGs in it. Thus, the presence of the same MVSG in the same expression site in two different isolates is unusual and may be the result of genetic exchange in the field between T. b. brucei and T. b. rhodesiense isolates. Analysis of other African trypanosome strains for the presence of the three WRATat 1.1 MVSG expression sites demonstrated that the expression sites' promoter sequences are much more likely to be present than are specific MVSGs, suggesting that loss of MVSGs is the result of replacement by other VSGs. The promoter region of the MVSG expression site active in the WRATat 1.1 MVAT7 variant was found to be highly conserved among T. b. brucei, T. b. rhodesiense and T. b. gambiense group 2 isolates, whereas it does not occur in the T. b. gambiense group 1 isolates tested. A phylogenetic analysis of this promoter region sequence shows that the T. b. gambiense group 2 isolates form a monophyletic clade well separated from the T. b. brucei/T. b. rhodesiense isolates. Thus, whilst the T. b. brucei, T. b. rhodesiense and T. b. gambiense group 2 isolates are closely related but heterogenous, molecular tools may be developed to distinguish T. b. gambiense group 2 isolates from the others.

Crystallization and preliminary crystallographic investigation of glycosomal pyruvate phosphate dikinase from Trypanosoma brucei.

The PP(i)-dependent glycosomal enzyme pyruvate phosphate dikinase (PPDK) from Trypanosoma brucei is expressed in the insect stage of the parasite. Its precise function there is still unclear, but the enzyme may catalyze the 'reverse reaction' of transfer of phosphate from phosphoenolpyruvate (PEP) to generate pyruvate as a means of scavenging large amounts of pyrophosphate. This protein may represent a target for drug design against diseases caused by trypanosomes and related kinetoplastids. The recombinant protein is 918 amino acids long (predicted molecular mass approximately 100 kDa and pI = 8.9). Crystallization conditions for the recombinant PPDK are reported that result in crystals that diffract X-rays to better than 3.0 A resolution. Their space group is P2(1)2(1)2, with unit-cell parameters a = 121.17, b = 153.5, c = 65.46 A, alpha = beta = gamma = 90 degrees. The crystals, like the protein in solution, are sensitive to temperature and fail to diffract or diffract only to low resolution after ageing for two weeks or longer.

Metabolic aspects of glycosomes in trypanosomatidae - new data and views.

The energy metabolism of Trypanosomatidae has been the subject of many reviews during the past decade. In recent years, however, new data have led to a more complete picture of trypanosomatid metabolism and a reappraisal of the role of some characteristic organelles in the energy supply of these parasites. For years, the glycosome was thought to be a peroxisome-like organelle that had evolved to allow the parasites to carry out glycolysis at a high rate using a relatively small amount of enzyme. However, the results of recent studies of trypanosomatid glycolysis and the detection of various other pathways and enzymes in the organelle necessitate a modification of this view. Here, Paul Michels, Véronique Hannaert and Frédéric Bringaud review the new data and discuss the possible implications for our view on the role of the glycosome.

Synthesis and uptake of nitric oxide-releasing drugs by the P2 nucleoside transporter in Trypanosoma equiperdum.

A series of S-nitrosothiols, structurally related to the NO*-donor S-nitroso-N-acetylpenicillamine, and of organic nitrate esters that contain amidine groups which specify a recognition via the trypanosomal purine transporter P2, were synthesized and tested for their ability to inhibit the uptake of [2-(3)H]adenosine on Trypanosoma equiperdum.

The structure-function relationship of functionally distinct but structurally similar hexose transporters from Trypanosoma congolense.

We have previously characterized, in Trypanosoma brucei, a multigene family encoding two developmentally regulated glucose transporters that are 80% identical at the amino-acid level. We report here the characterization of the homologous glucose transporters (TcoHT1 and TcoHT2) in Trypanosoma congolense, an African trypanosome responsible for disease in domestic animals. Both TcoHT isoforms, which are 92.4% identical, are encoded by a single cluster of genes containing two copies of TcoHT1 and three copies of TcoHT2 arranged alternately. Northern blot analysis revealed that TcoHT2 is expressed in all of the adaptive forms, while mRNA encoding TcoHT1 is only present in the metacyclic and bloodstream forms of T. congolense. When transfected with the TcoHT2 gene, Chinese Hamster Ovary cells express a hexose transporter with properties similar to those of the T. congolense procyclic forms (Km D-glucose = 41 microM versus 64 microM). In contrast to TcoHT2, TcoHT1 expressed in the Chinese hamster ovary cells appeared to be a relatively low affinity glucose transporter (Ki D-glucose = 0.8 mM). To determine the region(s) involved in the different apparent affinities for glucose, a chimera analysis was undertaken on the TcoHT isoforms. This study shows that amino-acid residues important for D-glucose recognition are located in the central region (between transmembrane domains 3 and 7) and in the C-terminal intracellular domain of TcoHT2. Site directed mutagenesis identified Ser193 located within transmembrane helix 4 as a key residue in relaxing the apparent affinity of TcoHT1 for glucose.

Chemical and enzymatic synthesis of fructose analogues as probes for import studies by the hexose transporter in parasites.

Various D-fructose analogues modified at C-1 or C-6 positions were synthesized from D-glucose by taking advantage of the Amadori rearrangement or using the aldol condensation between dihydroxyacetone phosphate and appropriate aldehyde catalyzed by fructose 1,6-diphosphate aldolase from rabbit muscle. The affinities of the analogues for the glucose transporter expressed in the mammalian form of Trypanosoma brucei were determined by inhibition of radiolabelled 2-deoxy-D-glucose (2-DOG) transport using zero-trans kinetic analysis. Interestingly, the analogues bearing an aromatic group (i.e. a fluorescence marker) at C-1 or C-6 positions present comparable apparent affinities to D-fructose for the transporter. This result could find applications for hexose transport studies and also provides criteria for the design of glucose import inhibitors.

Characterization of Trypanozoon isolates using a repeated coding sequence and microsatellite markers.

Genetic variation of microsatellite loci is a widely used method for linkage analysis, individual identification or inter-population studies. Here we analyse a repeated DNA coding sequence and eleven new microsatellites identified within the Trypanosoma (Trypanozoon) brucei genome. Ninety-seven isolates belonging to the five species and subspecies Trypanosoma evansi, T. equiperdum, T. brucei brucei, T. b. rhodesiense and T. b. gambiense were compared regarding the genetic patterns of these markers. The results reveal a great heterogeneity of the genotypes related to the repeated coding sequence and five microsatellites, some of which show a high degree of polymorphism. This allows us to define group-specific genotypes or alleles; in particular, we show that one specific pattern clearly segregates the human pathogen T. b. gambiense group 1.

Characterization of Trypanozoon isolates using a repeated coding sequence and microsatellite markers.

Genetic variation of microsatellite loci is a widely used method for linkage analysis, individual identification or inter-population studies. Here we analyse a repeated DNA coding sequence and eleven new microsatellites identified within the Trypanosoma (Trypanozoon) brucei genome. Ninety-seven isolates belonging to the five species and subspecies Trypanosoma evansi, T. equiperdum, T. brucei brucei, T. b. rhodesiense and T. b. gambiense were compared regarding the genetic patterns of these markers. The results reveal a great heterogeneity of the genotypes related to the repeated coding sequence and five microsatellites, some of which show a high degree of polymorphism. This allows us to define group-specific genotypes or alleles; in particular, we show that one specific pattern clearly segregates the human pathogen T. b. gambiense group I.

Characterization and disruption of a new Trypanosoma brucei repetitive flagellum protein, using double-stranded RNA inhibition.

In Trypanosoma brucei, we have cloned a gene approximately 5 kb downstream of the glucose transporter gene cluster, containing a variable number of 102 bp repeats. This gene encodes a protein with no homologues in the data bases. Antibodies raised against the 34 amino acids repeated motif recognized proteins ranging from 145 to 270 kDa, depending on strains, in both bloodstream and procyclic forms of T. brucei. A correlation was established between the apparent molecular mass of the detected proteins and the number of 34 amino acid repeats which varies from 3 to 40. We have called this protein the flagellum transition zone component (FTZC) due to its localization to the proximal region of the axoneme, within the transition zone. FTZC is the only reported example of a trypanosomal protein present in the transition zone. To determine the role of FTZC we developed a new strategy of gene inactivation based on conditional expression of double-stranded RNA. In the presence of tetracycline, expression of the double-stranded RNA, we observed a complete disappearance of FTZC in the EATRO 1125 and EATRO 427 strains of T. hrucei. Molecular ablation of FTZC does not generate any obvious phenotype such as, lethality, modification of growth rate or cellular shape, in the growth conditions used.

Uptake of NO-releasing drugs by the P2 nucleoside transporter in trypanosomes.

Nitric oxide (NO.) has been identified as a principal regulatory molecule of the immune system and the major cytotoxic mediator of activated immune cells. NO. can also react rapidly with a variety of biological species, particularly with the superoxide radical anion O2.- at almost diffusion-limited rates to form peroxynitrite anion (ONOO-). ONOO- and its proton-catalyzed decomposition products are capable of oxidizing a great diversity of biomolecules and can act as a source of toxic hydroxyl radicals. As a consequence, a strategy for the development of molecules with potential trypanocidal activities could be developed to increase the concentration of nitric oxide in the parasites through NO.-releasing compounds. In this way, the rate of formation of peroxynitrite from NO. and O2.- would be faster than the rate of dismutation of superoxide radicals by superoxide dismutases which constitute the primary antioxidant enzymatic defense system in trypanosomes. The adenosine transport systems of parasitic protozoa, which are also in certain cases implicated in the selective uptake of active drugs such as melarsoprol or pentamidine, could be exploited to specifically target these NO.-releasing compounds inside the parasites. In this work, we present the synthesis, characterization and biological evaluation of a series of molecules that contain both a group which would specifically target these drugs inside the parasites via the purine transporter, and an NO.-donor group that would exert a specific pharmacological effect by increasing NO level, and thus the peroxynitrite concentration inside the parasite.

An easy stereospecific synthesis of 1-amino-2,5-anhydro-1-deoxy-D-mannitol and arylamino derivatives.

1-Amino-2,5-anhydro-1-deoxy-D-mannitol and a series of arylamino derivatives were prepared by nitrous acid deamination of 2-amino-2-deoxy-D-glucose and subsequent reductive amination of the resulting 2,5-anhydro-D-mannose. Some of these compounds showed an enhanced affinity for the hexose transporter of Trypanosoma brucei as compared to D-fructose.

Knockout of the glutamate dehydrogenase gene in bloodstream Trypanosoma brucei in culture has no effect on editing of mitochondrial mRNAs.

Glutamate dehydrogenase (GDH) was shown previously to bind the 3' oligo[U] tail of the mitochondrial guide RNAs (gRNAs) of Leishmania tarentolae, apparently in the dinucleotide pocket (Bringaud F, Stripecke R, Frech GC, Freedland S, Turck C, Byrne EM, Simpson L. Mol. Cell. Biol. 1997; 17:3915-3923). Bloodstream Trypanosoma brucei cells in culture represent a good system to investigate the genetic effects of knocking out kinetoplastid nuclear genes to test a role in RNA editing, since editing of several mitochondrial genes occurs but is dispensable for viability (Corell RA, Myler P, Stuart K. Mol. Biochem. Parasitol. 1994; 64:65-74 and Stuart K. In: Benne R, editor. RNA editing--the alteration of protein coding sequences of RNA. New York: Ellis Horwood, 1993:25-52). Both GDH alleles of bloodstream T. brucei in culture were replaced by drug resistant markers without any effect on viability. The ratios of edited to unedited mRNAs for several cryptogenes were assayed by primer extension analysis. The steady state abundances of these edited RNAs were unaffected by the double knockout. This evidence suggests that GDH may not play a role in the editing reaction in bloodstream trypanosomes in culture, but this conclusion is tentative since there could be redundant genes for any biological function. We employed a double allelic replacement technique to generate a tetracycline inducible conditional expression of an ectopic copy of the deleted gene in bloodstream trypanosomes in culture. We used this strategy for genes encoding mitochondrial proteins which are not required during this stage of the life cycle, but as a general strategy it should be appropriate for generation of conditional null mutants for essential genes as well.

Sugar transporters from bacteria, parasites and mammals: structure-activity relationships.

Sugar transport across the plasma membrane is one of the most important transport processes. The cloning and expression of cDNAs from a superfamily of related sugar transporters that all adopt a 12-membrane-spanning-domain structure has opened new avenues of investigation, including presteady-state kinetic analysis. Structure-function analyses of mammalian and bacterial sugar transporters, and comparisons of these transporters with those of parasitic trypanosomatids, indicate that different environmental pressures have tailored the evolution of the various members of the sugar-transporter superfamily. Subtle distinctions in the function of these proteins can be related to particular amino acid residue substitutions.

Conserved organization of genes in trypanosomatids.

Trypanosomatids are unicellular protozoan parasites which constitute some of the most primitive eukaryotes. Leishmania spp, Trypanosoma cruzi and members of the Trypanosoma brucei group, which cause human diseases, are the most studied representatives of this large family. Here we report a comparative analysis of a large genomic region containing glucose transporter genes in three Salivarian trypanosomes (T. brucei, T. congolense and T. vivax), T. cruzi and Leishmania donovani. In T. brucei, the 8 kb (upstream) and 14 kb (downstream) regions flanking the glucose transporter genes cluster contain two and six new genes, respectively, six of them encoding proteins homologous to known eukaryotic proteins (phosphatidylinositol 3 kinase, ribosomal protein S12, DNAJ and three small G-proteins--Rab1, YPT6 and ARL3). This gene organization is identical in T. brucei, T. congolense and T. vivax suggesting that Salivarian trypanosomes have a high level of conservation in gene organization. In T. cruzi and Leishmania, the overall organization of this cluster is conserved, with insertion of additional genes when compared with T. brucei. Phylogenetic reconstitution based on glucose transporters is in accord with the monophyly of the genus Trypanosoma and the early separation of T. vivax within Salivarian trypanosomes. On the basis of gene organization, biochemical characteristics of isoforms and phylogeny, we discuss the genesis of the glucose transporter multigene family in Salivarian trypanosomes.

Functional and molecular characterization of a glycosomal PPi-dependent enzyme in trypanosomatids: pyruvate, phosphate dikinase.

Trypanosomatids are parasitic protists that have an ATP-dependent glycolysis with no indication of PPi-dependent metabolism. Most of the glycolysis takes place in peroxisome-like organelles, the glycosomes. We characterized in Trypanosoma brucei a single-copy gene encoding a PPi-dependent enzyme, pyruvate, phosphate dikinase (PPDK), which was expressed functionally in Escherichia coli. Specific antibodies detected a 100-kDa protein in procyclic forms but not in mammalian forms of T. brucei, indicating a differential expression. Glycosomal localization of PPDK was determined by immunofluorescence analysis and was confirmed by Western blot analysis on glycosomal fractions by using anti-PPDK antibodies. Expression and localization of recombinant PPDKs in procyclic forms of T. brucei showed that the AKL motif at the C-terminal extremity of PPDK is necessary for glycosomal targeting. PPDK was detected in every trypanosomatid tested-Trypanosoma congolense, Trypanosoma vivax, Trypanosoma cruzi, Phytomonas, Crithidia and Leishmania-with a good correlation between amount of protein and enzymatic activity. The precise role of PPDK in trypanosomatid carbohydrate metabolism remains to be clarified.