Maintenance of B cells during chronic murine Trypanosoma brucei gambiense infection.

African trypanosomosis is a debilitating parasitic disease occurring in large parts of sub-Saharan Africa. Trypanosoma brucei gambiense accounts for 98% of the reported HAT infections and causes a chronic, gradually progressing disease. Multiple experimental murine models for trypanosomosis have demonstrated inflammation-dependent apoptosis of splenic follicular B (FoB) cells and the destruction of B-cell memory against previously encountered pathogens. Here, we report that during murine infection with a chronic T. b. gambiense field isolate, FoB cells are retained. This coincided with reduced levels of IFN-γ and TNF-α during the acute phase of the infection. This result suggests that in chronic infections with low virulent parasites, less inflammation is elicited and consequently no FoB cell destruction occurs.

Erratum to: Molecular characterization and classification of Trypanosoma spp. Venezuelan isolates based on microsatellite markers and kinetoplast maxicircle genes.

Unfortunately, the original version of this article [1] contained an error. Figure 1 in the original article, corresponded to the first coinertia analysis that was carried out with no data on the procyclin PE repeats for the T. brucei brucei strains. After including these data, the coinertia analysis was modified both in the directionality of the arrows in the Y Hyperspace and in the biplot generated by the interaction of the two coinertia axes. The modified coinertia analysis is included in Fig. 1.

Molecular characterization and classification of Trypanosoma spp. Venezuelan isolates based on microsatellite markers and kinetoplast maxicircle genes.

BACKGROUND: Livestock trypanosomoses, caused by three species of the Trypanozoon subgenus, Trypanosoma brucei brucei, T. evansi and T. equiperdum is widely distributed throughout the world and constitutes an important limitation for the production of animal protein. T. evansi and T. equiperdum are morphologically indistinguishable parasites that evolved from a common ancestor but acquired important biological differences, including host range, mode of transmission, distribution, clinical symptoms and pathogenicity. At a molecular level, T. evansi is characterized by the complete loss of the maxicircles of the kinetoplastic DNA, while T. equiperdum has retained maxicircle fragments similar to those present in T. brucei. T. evansi causes the disease known as Surra, Derrengadera or "mal de cadeiras", while T. equiperdum is the etiological agent of dourine or "mal du coit", characterized by venereal transmission and white patches in the genitalia. METHODS: Nine Venezuelan Trypanosoma spp. isolates, from horse, donkey or capybara were genotyped and classified using microsatellite analyses and maxicircle genes. The variables from the microsatellite data and the Procyclin PE repeats matrices were combined using the Hill-Smith method and compared to a group of T. evansi, T. equiperdum and T. brucei reference strains from South America, Asia and Africa using Coinertia analysis. Four maxicircle genes (cytb, cox1, a6 and nd8) were amplified by PCRfrom TeAp-N/D1 and TeGu-N/D1, the two Venezuelan isolates that grouped with the T. equiperdum STIB841/OVI strain. These maxicircle sequences were analyzed by nucleotide BLAST and aligned toorthologous genes from the Trypanozoon subgenus by MUSCLE tools. Phylogenetic trees were constructed using Maximum Parsimony (MP) and Maximum Likelihood (ML) with the MEGA5.1® software. RESULTS: We characterized microsatellite markers and Procyclin PE repeats of nine Venezuelan Trypanosoma spp. isolates with various degrees of virulence in a mouse model, and compared them to a panel of T. evansi and T. equiperdum reference strains. Coinertia analysis of the combined repeats and previously reported T. brucei brucei microsatellite genotypes revealed three distinct groups. Seven of the Venezuelan isolates grouped with globally distributed T. evansi strains, while TeAp-N/D1 and TeGu-N/D1 strains clustered in a separate group with the T. equiperdum STIB841/OVI strain isolated in South Africa. A third group included T. brucei brucei, two strains previously classified as T. evansi (GX and TC) and one as T. equiperdum (BoTat-1.1). Four maxicircle genes, Cytochrome b, Cythocrome Oxidase subunit 1, ATP synthase subunit 6 and NADH dehydrogenase subunit 8, were identified in the two Venezuelan strains clustering with the T. equiperdum STIB841/OVI strain. Phylogenetic analysis of the cox1 gene sequences further separated these two Venezuelan T. equiperdum strains: TeAp-N/D1 grouped with T. equiperdum strain STIB818 and T. brucei brucei, and TeGu-N/D1 with the T. equiperdum STIB841/OVI strain. CONCLUSION: Based on the Coinertia analysis and maxicircle gene sequence phylogeny, TeAp-N/D1 and TeGu-N/D1 constitute the first confirmed T. equiperdum strains described from Latin America.

The expression of RoTat 1.2 variable surface glycoprotein (VSG) in Trypanosoma evansi and T. equiperdum.

In order to define whether the variable antigenic type RoTat 1.2 is restricted to Trypansoma evansi and could be used as antigen in serological tests to differentiate T. evansi from Trypansoma equiperdum, the appearance of RoTat 1.2-specific antibodies in rabbits, experimentally infected with T. evansi and T. equiperdum, respectively, was analyzed. Ten strains of T. evansi and 11 strains of T. equiperdum originating from Asia, Europe, Africa and Latin America were tested. Rabbit pre-infection sera and sera of days 7, 14, 25, 35 post-infection (p.i.) were analyzed for the presence of antibodies reactive with RoTat 1.2 in immune trypanolysis, ELISA/T. evansi and CATT/T. evansi. Within the duration of the infection (maximum 35 days), all T. evansi as well as 9 out of 11 T. equiperdum infected rabbits became positive in all these tests. The rabbits infected with T. equiperdum OVI (South Africa) and BoTat 1.1 (Morocco) remained negative in the immune trypanolysis test although the latter rabbit became positive in the CATT/T. evansi and ELISA/T. evansi. On the contrary, both rabbits were positive in immune trypanolysis when tested against their respective infecting population. From these data, we conclude that most T. equiperdum strains express isoVATs of RoTat 1.2. This explains, in part, why antibody tests based on T. evansi RoTat 1.2 cannot reliably distinguish between infections caused by T. evansi and those caused by T. equiperdum unless it can be proven that most described T. equiperdum are actually misclassified T. evansi.

How does Trypanosoma equiperdum fit into the Trypanozoon group? A cluster analysis by RAPD and multiplex-endonuclease genotyping approach.

The pathogenic trypanosomes Trypanosoma equiperdum, T. evansi as well as T. brucei are morphologically identical. In horses, these parasites are considered to cause respectively dourine, surra and nagana. Previous molecular attempts to differentiate these species were not successful for T. evansi and T. equiperdum; only T. b. brucei could be differentiated to a certain extent. In this study we analysed 10 T. equiperdum, 8 T. evansi and 4 T. b. brucei using Random Amplified Polymorphic DNA (RAPD) and multiplex-endonuclease fingerprinting, a modified AFLP technique. The results obtained confirm the homogeneity of the T. evansi group tested. The T. b. brucei clustered out in a heterogenous group. For T. equiperdum the situation is more complex: 8 out of 10 T. equiperdum clustered together with the T. evansi group, while 2 T. equiperdum strains were more related to T. b. brucei. Hence, 2 hypotheses can be formulated: (1) only 2 T. equiperdum strains are genuine T. equiperdum causing dourine; all other T. equiperdum strains actually are T. evansi causing surra or (2) T. equiperdum does not exist at all. In that case, the different clinical outcome of horse infections with T. evansi or T. b. brucei is primarily related to the host immune response.

A novel C-terminal kinesin is essential for maintaining functional acidocalcisomes in Trypanosoma brucei.

Kinesins are cytoskeletal motor proteins that play roles in a variety of fundamental cellular processes including cell division and the anterograde transport of vesicles and organelles. We purified, cloned, and functionally characterized in Trypanosoma brucei a new member of the C-terminal kinesin family, TbKIFC1. Kinetic constants of the recombinant motor domain of TbKIFC1 were estimated at 0.56 microm for the microtubule dissociation constant (K(d)) with a k(cat) of 0.2 s(-1). Immunolocalization analysis showed an association of TbKIFC1 with punctate structures. Because they were rapidly transported to the negative pole of the microtubule after NH(4)Cl treatment, these structures were considered to be associated with acidic vesicles. To determine the role of the kinesin in vivo, we produced an inducible kinesin-deficient strain by double-stranded RNA interference methodology. Mutant cells were loaded with the fluorescent reagent fura2/acetoxymethylester to measure intracellular free calcium ([Ca(2+)](i)). The resting [Ca(2+)](i) was unchanged in mutant cells; however, alkalinization of acidic vesicles induced by NH(4)Cl or nigericin was not followed by release of Ca(2+). These data and the relative importance of the ionomycin-releasable and the ionomycin-plus-NH(4)Cl-releasable Ca(2+) pools suggest a lower Ca(2+) content in acidocalcisomes and dysfunctional Ca(2+) release.

Molecular characterisation of mitochondrial and cytosolic trypanothione-dependent tryparedoxin peroxidases in Trypanosoma brucei.

In trypanosomatids, removal of hydrogen peroxide and other aryl and alkyl peroxides is achieved by the NADPH-dependent trypanothione peroxidase system, whose components are trypanothione reductase (TRYR), trypanothione, tryparedoxin (TRYX) and tryparedoxin peroxidase (TRYP). Here, we report the cloning of a multi-copy tryparedoxin peroxidase gene (TRYP1) from Trypanosoma brucei brucei encoding a protein with two catalytic VCP motifs similar to the cytosolic TRYP from Crithidia fasciculata. In addition, we characterise a novel single copy gene encoding a second tryparedoxin peroxidase (TRYP2). TRYP2 shows 51% similarity to TRYP1, possesses a putative mitochondrial import sequence at its N-terminus and has a variant IPC motif replacing the second VCP motif implicated in catalysis in other 2-Cys peroxiredoxins. TRYP1 and TRYP2 were expressed in Escherichia coli, and the purified recombinant proteins shown to utilise hydrogen peroxide in the presence of NADPH, trypanothione, TRYR and TRYX from T. brucei, similar to the C. fasciculata cytoplasmic system. Western blots showed that TRYX, TRYP1 and TRYP2 are expressed in both bloodstream and procyclic forms of the life cycle. To determine the precise localisation of TRYX, TRYP1 and TRYP2 in the parasite, polyclonal antibodies to purified recombinant TRYX and TRYP1 and monoclonal antibody to TRYP2 were generated in mice. In-situ immunofluorescence and immunoelectron microscopy revealed a colocalisation of TRYX and TRYP1 in the cytosol, whereas TRYP2 was principally localised in the mitochondrion.

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.

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.

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.

Purification, cloning, and characterization of an acidic ectoprotein phosphatase differentially expressed in the infectious bloodstream form of Trypanosoma brucei.

We purified an ecto-phosphatase of 115 kDa (TryAcP115) specifically expressed by bloodstream forms of Trypanosoma brucei. The corresponding gene coded for a 45-kDa protein potentially including a signal peptide, a membrane-spanning domain and an N-terminal domain containing 8 N-glycosylation sites. There was no significant sequence homology with other phosphatases. Antiserum to the Escherichia coli recombinant N-terminal domain, Petase7, recognized a protein of 55 kDa in Western blots after deglycosylation of the TryAcP115 protein by N-glycosidase F. Immunofluorescence and trypsin treatment of living parasites showed that TryAcP115 was localized to the surface of the parasite and that its N-terminal domain was oriented extracellularly. The recombinant N-terminal domains, expressed in E. coli and Leishmania amazonensis, harbored phosphatase activity against Tyr(P)-Raytide, Ser(P)-neurogranin, and ATP. The enzymatic properties of native TryAcP115 and the recombinant proteins for the substrate Tyr(P)-Raytide were virtually identical and included: (i) K(m) and V(max) values of 15 nM and 200 pmol/min/mg, (ii) no requirement for divalent cations, and (iii) sensitivity to vanadate, sodium fluoride, and tartrate, but insensitivity to okadaic acid and tetramisole. Although the function of TryAcP115 remains unknown, a differentially expressed, unique ecto-phosphatase could regulate growth or influence parasite-host interactions and might provide a useful target for chemotherapy.

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.

Affinity chromatography using trypanocidal arsenical drugs identifies a specific interaction between glycerol-3-phosphate dehydrogenase from Trypanosoma brucei and Cymelarsan.

A 36-kDa protein was isolated by affinity chromatography using Cymelarsan, an arsenical drug currently used in African trypanosomiasis treatment, as ligand. This protein was identified as glycerol-3-phosphate dehydrogenase. Trypanosomal glycerol-3-phosphate was bound covalently, whereas its counterpart from rabbit muscle bound by ionic interaction. Arsenical drugs inhibit the enzyme in a dose-dependent manner. Oxidation of cysteine residues protects against inactivation without significantly diminishing enzymic activity. Drug concentrations giving 50% inhibition of the dehydrogenase activity were determined for the enzyme from both Trypanosoma brucei and rabbit and indicate a higher sensitivity of the trypanosomal enzyme to arsenical drugs and thiol reagents. MS was used to identify residues of glycerol-3-phosphate dehydrogenase bound by Cymelarsan; they are not conserved in the mammalian enzyme.

Synthesis and evaluation of the in vivo trypanocidal activity of water soluble organotin compounds.

A series of (3-(2-methoxy)ethoxypropyl)tin derivatives were synthesized as potential trypanocidal drugs. The series included an alkyltin trichloride, a dialkyltin dichloride and the corresponding dialkyltin oxide, and six dialkyltin dithio derivatives. Compounds were evaluated for trypanocidal activity using in vitro cultures of Trypanosoma equiperdum and mice infected with the same strain of parasite for in vivo tests. Two of the title derivatives, the bis (3-(2-methoxy)ethoxypropyl)tin dichloride 2 and the corresponding bis (3-(2-methoxy)ethoxypropyl)tin oxide 3, appeared to be water soluble reagents. Furthermore, they are the first examples of organotin compounds presenting interesting in vivo trypanocidal activity.

Mini radial keratotomy reduces ocular integrity. Axial compression in a postmortem porcine eye model.

OBJECTIVE: This study aimed to examine ocular rupture force in pig eyes after "minimally invasive radial keratotomy" (MRK) and standard radial keratotomy (SRK). DESIGN: Experimental study. MATERIALS: A total of 71 pairs of pig eyes (51 control eyes) were examined. INTERVENTION: An axial-torsional Materials Testing System (MTS, Eden Prairie, MN) was used to apply blunt force to the corneal surface. A force transducer measured the rupture forces in control eyes and in eyes with MRK or SRK. Five groups of paired eyes were compared: 2.0-mm MRK versus control (N = 12), 3.5-mm MRK versus control (N = 21), 6.5-mm SRK versus control (N = 18), SRK versus 3.5-mm MRK versus 2.0-mm MRK (N = 10). MAIN OUTCOME MEASURE: Ocular rupture force (newtons) was measured. RESULTS: The mean rupture force in newtons was 746.3 for control eyes, 514.2 for 2.0-mm MRK, 353.1 for 3.5-mm MRK, and 246.2 for SRK. Analysis of variance showed a statistically significant difference (P < or = 0.04) between paired comparisons. CONCLUSION: The MRK and SRK significantly weakened ocular integrity compared with control eyes not operated on. MRK required significantly more force to rupture than SRK. MRK eyes, however, ruptured at 50% to 70% of the force required to rupture eyes not operated on. Any patient considering radial keratotomy should be counseled about the risk of greater ocular damage in trauma.

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.

Assessment of central nervous system involvement in gambiense trypanosomiasis: value of the cerebro-spinal white cell count.

OBJECTIVE: To assess, in a clinical setting, the comparative values of conventional criteria used in the diagnosis of central nervous system (CNS) involvement in Trypanosoma brucei gambiense sleeping sickness: white cell count (WCC) in cerebrospinal fluid (CSF) > 5 x 10(6) cells/l; total protein concentration in CSF > 40 mg/100 ml); evidence of trypanosomes in CSF following double centrifugation (DC). METHOD: In vitro culture of CSF was used as the gold standard. RESULTS: The study showed that WCC is, by itself, as sensitive for the diagnosis of the CNS involvement as the usually recommended combination of three conventional criteria. The specificity of WCC is improved while the sensitivity is reduced when the cut-off point is set at a higher value (WCC > 10 X 10(6)/l). CONCLUSION: In poorly equipped laboratories, the diagnosis of CNS involvement in patients with confirmed systemic infection should be based only on the WCC. However, a pilot study is needed to assess the feasibility and reliability of the WCC handled by 'front line' personnel, for different cut-off values.

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.