Two novel nucleobase/pentamidine transporters from Trypanosoma brucei

African trypanosomes are unable to synthesize purines de novo and must salvage preformed purine nucleosides and nucleobases from their hosts. The Trypanosoma brucei genome project has identified 12 members of the equilibrative nucleoside transporter family, most of which have been characterized previously as nucleoside and/or nucleobase transporters. Here the 11th member of this family, TbNT11.1, has been functionally expressed in null mutants of Leishmania that are deficient in purine nucleoside or nucleobase uptake and identified as a high-affinity purine nucleobase transporter. Expression of TbNT11.1 in Xenopus oocytes revealed that it is also a transporter for the diamidine drug pentamidine that is the principal drug employed to treat early stage human African trypanosomiasis and may thus contribute to the uptake of this therapeutically important compound. In addition, characterization of the 12th member of the family, TbNT12.1, reveals that it is an adenine/pentamidine transporter.     

A phosphoglycerate kinase-related gene conserved between Trypanosoma brucei and Crithidia fasciculata.

Trypanosoma brucei and Crithidia fasciculata both contain three different phosphoglycerate kinase (PGK) genes, A, B and C, in a tandem array. The genes B and C encode the major PGKs: the cytosolic and glycosomal PGKs, respectively. The PGK-A genes of both Trypanosomatid species encode open reading frames related to PGK, which have most active site residues conserved, but contain an insert of 80 amino acids at approximately position 80 of the 420 amino acids average PGK sequence. The deduced amino acid sequence of these inserts is conserved between T. brucei and C. fasciculata (48% positional identity), indicating its functional importance. Although we have not been able to demonstrate PGK activity in the PGK-A gene product, we consider it likely that this gene codes for a minor PGK with special function.     

Glucosylation of glycoproteins in Crithidia fasciculata.

High mannose-type, N-linked oligosaccharides devoid of glucose units may be glucosylated directly from UDP-Glc in mammalian, plant, fungal and protozoan cells. The glucosylated compounds thus formed (protein-linked Glc1Man5-9GlcNAc2, depending on the organisms) are immediately deglucosylated by glucosidase II, an enzyme located, the same as the glucosylating activity, in the endoplasmic reticulum. In order to evaluate the molar proportion of N-linked oligosaccharides that are glucosylated in the trypanosomatid Crithidia fasciculata (a microorganism transferring Man7GlcNAc2 in protein N-glycosylation) cells of the parasite were grown in the presence of [14C]glucose and concentrations of the glucosidase II inhibitors deoxynojirimycin and/or castanospermine that were several hundred-fold higher than those required to inhibit 50% of the activity of the protozoan enzyme. The inhibitors did not affect the cell growth rate and, although glucose analogs, did not interfere with the entry of glucose into the cells. About 40-43% of total N-linked oligosaccharides appeared to be glucosylated. As on the average there are several N-linked oligosaccharides per glycoprotein, more than 40-43% (but probably not all of them) are transiently glucosylated in the endoplasmic reticulum.     

Different allele frequencies in Trypanosoma brucei brucei and Trypanosoma brucei gambiense populations

Restriction fragment length polymorphism (RFLP) has been analysed in Trypanosoma brucei DNA following hybridization with different DNA probes. This polymorphism seems to be due to allelic variation, and not to variation between sequence duplicates, since the genomic environment of the probed polymorphic fragments is conserved over considerable distances. In an analysis of 35 non-gambiense stocks, we found different combinations of homozygotes and heterozygotes for the four RFLP probes used, in keeping with previous observations that genetic reassortment occurs in T. b. brucei. Moreover, the non-gambiense populations from West and East Africa can be differentiated according to their characteristic allele frequencies. In sharp contrast, we found that the 49 T. b. gambiense stocks, analysed with the same probes, share the same single allelic combination and are all homozygous for each one of the four markers. This characteristic gambiense allele combination is very common among Western non-gambiense isolates, but rare or absent among Eastern ones. Two stocks isolated from man in West Africa turned out to be non-gambiense by all molecular criteria examined, including total nuclear DNA content. Taken together, these observations suggest that human serum-resistant variants may appear among the West African T. b. brucei population, and that T. b. gambiense evolved from one of these resistant variants as a man-adapted subspecies that became genetically isolated from the rest of the West African trypanosome population.     

DNA sequence of Crithidia fasciculata kinetoplast minicircles

Kinetoplast DNA (kDNA) networks of the insect trypanosomatid Crithidia fasciculata strain CF-C1 contain a nearly homogeneous population of kDNA minicircles as judged by restriction enzyme cleavage analysis. We have determined the entire nucleotide sequence of the major class of minicircles by analyzing M13 phage clones carrying half-length segments of the kDNA minicircle molecules. The 12 nucleotide sequence d(G-G-G-G-T-T-G-G-T-G-T-A) is the longest sequence common to kDNA minicircles from several trypanosome species examined to date. Two copies of this universal minicircle sequence were identified 180 degrees apart as direct repeats within the C. fasciculata kDNA minicircles. In addition, these universal minicircle sequences are contained within direct repeats with nearly identical sequences of 173 and 177 base pairs (bp) in length. These sequences are also conserved in the same arrangement in minor sequence classes of minicircles from this strain. Site-specific discontinuities on both strands of the minicircle, identified previously in minicircle replication intermediates, were localized within the 173 and 177 bp conserved sequences. These sequences were also found to have extensive homology with similar conserved sequences in kDNA minicircles from Leishmania tarentolae. We suggest that the two conserved sequences, each containing a single copy of the universal minicircle sequence, represent replication origins in the Crithidia minicircles.     

Differential expression of the oligomycin-sensitive ATPase in bloodstream forms of Trypanosoma brucei brucei.

We report the differential expression of the oligomycin-sensitive mitochondrial ATPase in pleomorphic bloodstream forms of Trypanosoma brucei brucei as observed with enzymatic assays and electron microscope histochemistry. As the cells differentiate from long slender to short stumpy forms, total specific activity of the mitochondrial ATPase in a crude mitochondrial fraction doubles and the oligomycin-sensitive specific activity increases 5-fold. Upon in vitro differentiation to procyclic forms, there is a further doubling of total specific activity and a further tripling of oligomycin-sensitive specific activity. The oligomycin-insensitive ATPase activity remained essentially constant throughout differentiation. We have attempted to characterize this oligomycin-insensitive activity utilizing inhibitors of several other ATPases.     

A comparative study of the proteolytic enzymes of Trypanosoma brucei, T. equiperdum, T. evansi, T. vivax, Leishmania tarentolae and Crithidia fasciculata

Four types of proteolytic activity were detected in the bloodstream form of each of the four Trypanosoma species: (i) HPAase, active on hide powder azure and detected on polyacrylamide gels containing denatured haemoglobin; (ii) AZCase, active on azocasein; (iii) type 1, active on the chromogenic peptide N-benzoyl-L-prolyl-L-phenylalanyl-L-arginine p-nitroanilide in the presence of dithiothreitol, and (iv) type 2, active against several nitroanilide derivatives in the absence of dithiothreitol. Studies of the pH optimum, dithiothreitol requirement and inhibitor sensitivities of the proteolytic activities suggested that: (a) HPAase and type 1 activities could be due to the same enzymes, probably a family of cysteine proteinases; (b) AZCase had some characteristics of a cysteine proteinase, but was not identical to HPAase, and (c) type 2 activity could be due to a serine proteinase. Procyclic T. brucei contained relatively low cysteine proteinase activities (HPAase, AZCase and type 1) but high type 2 activity. Their proteolytic enzymes thus were apparently more similar to those in Crithidia fasciculata and Leishmania tarentolae promastigotes than those in T. brucei bloodstream forms.     

Cell-surface sialoglycoconjugate structures in wild-type and mutant Crithidia fasciculata

The cell-surface expression of sialoglycoconjugate structures in wild-type Crithidia fasciculata and its TFR^R1 drug-resistant mutant was analyzed with the aid of an influenza C virus strain, lectin, enzymatic treatment, and flow cytofluorimetry analysis probed with fluorescein isothiocyanate-labeled (FITC) lectins. 9-O-Acetyl-N-acetyl neuraminic acid (Neu5,9Ac₂) structures mediate influenza C virus cell-binding. The SAα2,3Gal and SAα2,6Gal sequences are specifically recognized by Maackia amurensis (MAA) and Sambucus nigra (SNA) lectins, respectively. On the basis of these param- eters the TFR^R1 mutant strain of C. fasciculata was found to contain exposed sialoglycoconjugates bearing Neu5,9Ac₂ surface structures. After the removal of sialic acid residues by neuraminidase activity the marked increases in PNA (peanut agglutinin)-mediated agglutinating activity showed that those acidic units on C. fasciculata cells were glycosidically linked to d-galactose. The bond involves SAα2,6Gal and SAα2,3Gal linkages as suggested by the use of FITC-SNA and FITC-MAA lectins, respectively. Both SAα2,3Gal and SAα2,6Gal sequences were preferentially expressed by the TFR^R1 mutant. The SAα2,6 linkage markedly predominated. In the TFR^R1 mutant, but not in wild-type cells, two distinct populations of cells were distinguished by reactivity with FITC-SNA, one of which was enriched with surface SAα2,6Gal sequences. These diverse findings suggest that sialoglycoconjugate structures present on the flagellate surface may be associated with mutation and the cell growth cycle in C. fasciculata. Received: 17 September 1998 / Accepted: 22 October 1998     

Transport of methionine in Trypanosoma brucei brucei

African trypanosomes live free in the bloodstream and central nervous system of mammalian hosts and also within the midgut of the tsetse fly vectors which transmit them. The parasite plasma membrane represents the interface between both hosts and parasite, and trypanosomes accumulate many essential metabolites via specific transport processes. L-Methionine uptake by procyclic and bloodstream forms of Trypanosoma brucei has been measured and shown to be mediated by a transporter presenting similar characteristics in both forms of the parasite. The carrier shows, in both forms, a relatively high affinity for methionine (Km ca. 30 microM). The effect of inhibitors of ion gradients across the membrane indicated that the uptake process is likely to be dependent upon a proton motive force. Various methionine analogues were tested against the transporter and these have demonstrated that the recognition depends on the motif common to all amino acids, and an electronegative group at the position of the sulphur atom separated from the alpha-carbon atom by a two carbon spacer.     

A new expression vector for Crithidia fasciculata and Leishmania

Crithidia fasciculata is a monogenetic parasite of insects. It grows in fully defined media without requiring serum, which facilitates biochemical analysis. We have constructed a series of expression systems that allows expression of transfected genes in the kinetoplastid protozoa Crithidia and Leishmania. These cells can be readily transfected with plasmid DNA by electroporation and transformants selected with various antibiotic resistance markers. 5'-Trans-splicing signals and poorly defined regions within the 3'-untranslated regions of genes are required for optimal expression of genes in trypanosomatids. We, therefore, inserted the intergenic region of the C. fasciculata phosphoglycerate kinase (PGK) genes A and B, which allows polyadenylation of the target gene and spliced leader addition to the selectable marker gene. Part of the intergenic region of the PGK locus was added upstream of the target gene to permit its trans-splicing. A 3'-untranslated sequence from the Crithidia glutathionylspermidine synthetase (GSPS) was also added to allow the polyadenylation of the selectable marker gene. Genes can be readily inserted using a multiple cloning site and can be expressed as a fusion protein with a poly-histidine sequence at either the N or C-terminus or fused with green fluorescent protein. Biologically active proteins can be expressed in C. fasciculata or L. amazonensis promastigotes and purified by affinity chromatography using a metal chelating column.     

Purification and characterization of a tyrosine aminotransferase from Crithidia fasciculata

Tyrosine aminotransferase (TAT, EC 2.6.1.5) from the kinetoplastid, Crithidia fasciculata, was purified over 2000 fold to electrophoretic homogeneity. The native form of the enzyme had a molecular weight of approximately 100,000, whereas under denaturing conditions it produced two polypeptides of approximately 50,000 and 48,000, respectively. Absence of a reaction with the periodic acid-Schiff stain suggested that the crithidial enzyme was not a glycoprotein. It was relatively stable and remained active over a wide range of pH and temperature. It exhibited a broad substrate specificity and was able to utilize L-tyrosine, L-tryptophan, and L-phenylalanine as amino donors. Antiserum produced against partially purified crithidial tyrosine aminotransferase failed to inhibit the enzymatic activity. The same antiserum cross-reacted with a soluble extract from Trypanosoma brucei gambiense, but not with that from normal mouse liver, confirming evolutionary conservatism between the two protozoa.     

Isolation and partial characterization of DNA polymerases from Crithidia fasciculata

Two types of DNA polymerase activity were partially purified from Crithidia fasciculata. The alpha-type, DNA polymerase A, was of high molecular weight and sensitive to N-ethylmaleimide, whereas the beta-type, DNA polymerase B, was of low molecular weight and resistant to N-ethylmaleimide. Phosphocellulose chromatography revealed multiple peaks of DNA polymerase A activity the properties of which, such as pH optimum, salt sensitivity, utilization of synthetic template-initiator complexes and response to DNA polymerase inhibitors were similar. The response of the C. fasciculata DNA polymerase A enzymes to some of these inhibitors and utilization of poly(rA) X oligo(dT)11 showed these enzymes to be markedly different from mammalian DNA polymerase alpha.     

The biosynthesis of trypanothione and N1-glutathionylspermidine in Crithidia fasciculata

Studies on the biosynthesis of trypanothione [N1,N8-bis(glutathionyl)-spermidine] in the insect trypanosomatid Crithidia fasciculata have led to the discovery of an additional sulfur-containing peptide conjugated to spermidine. Labelling studies with [3H]spermidine show that 50% of the total intracellular spermidine is incorporated into peptide conjugates, the major component being N1-glutathionylspermidine. This compound has previously been identified in Escherichia coli, as the principal low molecular weight thiol in stationary phase, but not the logarithmic phase of growth. In contrast, in C. fasciculata, this compound is present in all phases of growth. In the presence of glutathione and ATP, extracts of C. fasciculata can catalyse conversion of spermidine to N1-glutathionylspermidine and trypanothione. Both N1- and N8-regioisomers of glutathionylspermidine will replace spermidine in the reaction, suggesting they may be intermediates in the biosynthetic pathway to trypanothione. The antiprotozoal drugs berenil, pentamidine, ethidium bromide, imidocarb, methylglyoxal-bis(guanylhydrazone) and 1,3-diacetylbenzene-bis(guanylhydrazone) had no effect on the synthesis of N1-glutathionylspermidine or trypanothione in vitro.     

Serum lipoprotein and Trypanosoma brucei brucei interactions in vitro

Trypanosoma brucei brucei IL3201 and IL3202, which are dependent on serum high or low-density lipoproteins to multiply under axenic culture conditions, acquired lipoprotein-associated 3H-lipids without binding, accumulating or degrading apolipoproteins. Uptake by the T. b. brucei of lipoprotein-associated [1 alpha, 2 alpha(n)-3H]cholesterol, [1 alpha, 2 alpha(n)-3H]cholesteryl linoleate, [1 alpha, 2 alpha(n)-3H]cholesteryl oleoyl ether and L-3-phosphatidyl [N-methyl-3H]choline, 1,2-dipalmitoyl, occurred at 37 degrees C but not at 0 degree C, and tended towards saturation with increasing concentrations of 3H-lipid-labelled lipoproteins in the incubation mixture. The uptake processes did not discriminate between high- or low-density lipoproteins, did not require exogenous divalent ions and were not inhibited by the presence of acidotropic agents (chloroquine, ammonium chloride) in the incubation mixture. Uptake by T. b. brucei of lipoprotein cholesterol was likely to result mainly from desorption and diffusion processes, whereas specific binding sites were probably involved in the uptake by T. b. brucei of lipoprotein cholesteryl linoleate, cholesteryl oleoyl ether and possibly phosphatidylcholine. Exponentially growing T. b. brucei hydrolysed cholesteryl linoleate to cholesterol and had only a small capacity to reesterify cholesterol, whereas committed non-dividing stumpy form T. b. brucei had a large capacity to esterify cholesterol. Conversion products of phosphatidylcholine were generated during or after uptake of this phospholipid by exponentially growing T. b. brucei.