Developmental variation of glycosylphosphatidylinositol membrane anchors in Trypanosoma brucei. In vitro biosynthesis of intermediates in the construction of the GPI anchor of the major procyclic surface glycoprotein.

The African trypanosome, Trypanosoma brucei, expresses two abundant stage-specific glycosylphosphatidylinositol (GPI)-anchored glycoproteins, the procyclic acidic repetitive protein (PARP or procyclin) in the procyclic form, and the variant surface glycoprotein (VSG) in the mammalian bloodstream form. The GPI anchor of VSG can be readily cleaved by phosphatidylinositol (PI)-specific phospholipase C (PI-PLC), whereas that of PARP cannot, due to the presence of a fatty acid esterified to the inositol. In the bloodstream form trypanosome, a number of GPIs which are structurally related to the VSG GPI anchor have been identified. In addition, several structurally homologous GPIs have been described, both in vivo and in vitro, that contain acyl-inositol. In vivo the procyclic stage trypanosome synthesizes a GPI that is structurally homologous to the PARP GPI anchor, i.e. contains acyl-inositol. No PI-PLC-sensitive GPIs have been detected in the procyclic form. Using a membrane preparation from procyclic trypanosomes which is capable of synthesizing GPI lipids upon the addition of nucleotide sugars we find that intermediate glycolipids are predominantly of the acyl-inositol type, and the mature ethanolamine-phosphate-containing precursors are exclusively acylated. We suggest that the differences between the bloodstream and procyclic form GPI biosynthetic intermediates can be accounted for by the developmental regulation of an inositol acylhydrolase, which is active only in the bloodstream form, and a glyceride fatty acid remodeling system, which is only partially functional in the procyclic form.     

Developmental regulation of a novel repetitive protein of Trypanosoma brucei.

Trypanosoma brucei undergoes many morphological and biochemical changes during transformation from the bloodstream trypomastigote to the insect procyclic trypomastigote form. We cloned and determined the complete nucleotide sequence of a developmentally regulated cDNA. The corresponding mRNA was abundant in in vitro-cultivated procyclics but absent in bloodstream forms. The trypanosome genome contains eight genes homologous to this cDNA, arranged as four unlinked pairs of tandem repeats. The longest open reading frame of the cDNA predicts a protein of 15 kilodaltons, the central portion of which consists of 29 tandem glutamate-proline dipeptides. The repetitive region is preceded by an amino-terminal signal sequence and followed by a hydrophobic domain that could serve as a membrane anchor; the mRNA was found on membrane-bound polyribosomes. These results suggest that the protein is membrane associated.     

Stage-specific differences in cell cycle control in Trypanosoma brucei revealed by RNA interference of a mitotic cyclin

African trypanosomes have a tightly coordinated cell cycle to effect efficient segregation of their single organelles, the nucleus, flagellum, and kinetoplast. To investigate cell cycle control in trypanosomes, a mitotic cyclin gene (CYC6) has been identified in Trypanosoma brucei. We show that CYC6 forms an active kinase complex with CRK3, the trypanosome CDK1 homologue, in vivo. Using RNA interference, we demonstrate that absence of CYC6 mRNA results in a mitotic block and growth arrest in both the insect procyclic and mammalian bloodstream forms. In the procyclic form, CYC6 RNA interference generates anucleate cells with a single kinetoplast, whereas in bloodstream form trypanosomes, cells with one nucleus and multiple kinetoplasts are observed. Fluorescence-activated cell sorting analysis shows that bloodstream but not procyclic trypanosomes are able to reinitiate nuclear S phase in the absence of mitosis. Taken together, these data show that procyclic trypanosomes can undergo cytokinesis without completion of mitosis, whereas a mitotic block in bloodstream form trypanosomes inhibits cytokinesis but not kinetoplast replication and segregation nor an additional round of nuclear DNA synthesis. This indicates that there are fundamental differences in cell cycle controls between life cycle forms of T. brucei and that key cell cycle checkpoints present in higher eukaryotes are absent from trypanosomes.     

Expression and function of the Trypanosoma brucei major surface protease (GP63) genes

The genome of the African trypanosome Trypanosoma brucei (Tb) contains at least three gene families (TbMSP-A, -B, and -C) encoding homologues of the abundant major surface protease (MSP, previously called GP63), which is found in all Leishmania species. TbMSP-B mRNA occurs in both procyclic and bloodstream trypanosomes, whereas TbMSP-A and -C mRNAs are detected only in bloodstream organisms. RNA interference (RNAi)-mediated gene silencing was used to investigate the function of TbMSP-B protein. RNAi directed against TbMSP-B but not TbMSP-A ablated the steady state TbMSP-B mRNA levels in both procyclic and bloodstream cells but had no effect on the kinetics of cultured trypanosome growth in either stage. Procyclic trypanosomes have been shown previously to have an uncharacterized cell surface metalloprotease activity that can release ectopically expressed surface proteins. To determine whether TbMSP-B is responsible for this release, transgenic variant surface glycoprotein 117 (VSG117) was expressed constitutively in T. brucei procyclic TbMSP-RNAi cell lines, and the amount of surface VSG117 was determined using a surface biotinylation assay. Ablation of TbMSP-B but not TbMSP-A mRNA resulted in a marked decrease in VSG release with a concomitant increase in steady state cell-associated VSG117, indicating that TbMSP-B mediates the surface protease activity of procyclic trypanosomes. This finding is consistent with previous pharmacological studies showing that peptidomimetic collagenase inhibitors block release of transgenic VSG from procyclic trypanosomes and are toxic for bloodstream but not procyclic organisms.     

A function for a specific zinc metalloprotease of African trypanosomes

The Trypanosoma brucei genome encodes three groups of zinc metalloproteases, each of which contains approximately 30% amino acid identity with the major surface protease (MSP, also called GP63) of Leishmania. One of these proteases, TbMSP-B, is encoded by four nearly identical, tandem genes transcribed in both bloodstream and procyclic trypanosomes. Earlier work showed that RNA interference against TbMSP-B prevents release of a recombinant variant surface glycoprotein (VSG) from procyclic trypanosomes. Here, we used gene deletions to show that TbMSP-B and a phospholipase C (GPI-PLC) act in concert to remove native VSG during differentiation of bloodstream trypanosomes to procyclic form. When the four tandem TbMSP-B genes were deleted from both chromosomal alleles, bloodstream B (-/-) trypanosomes could still differentiate to procyclic form, but VSG was removed more slowly and in a non-truncated form compared to differentiation of wild-type organisms. Similarly, when both alleles of the single-copy GPI-PLC gene were deleted, bloodstream PLC (-/-) cells could still differentiate. However, when all the genes for both TbMSP-B and GPI-PLC were deleted from the diploid genome, the bloodstream B (-/-) PLC (-/-) trypanosomes did not proliferate in the differentiation medium, and 60% of the VSG remained on the cell surface. Inhibitors of cysteine proteases did not affect this result. These findings demonstrate that removal of 60% of the VSG during differentiation from bloodstream to procyclic form is due to the synergistic activities of GPI-PLC and TbMSP-B.     

Cyclical differentiation of Trypanosoma brucei involves changes in the cellular complement of calmodulin-binding proteins

The present study was undertaken to evaluate changes in the complement of calmodulin-binding proteins which accompany cyclical differentiation in Trypanosoma brucei. An [125I]trypanosome calmodulin overlay procedure was used to detect calmodulin-binding proteins with Mr of 126,000 and 106,000 that were present in homogenates of slender bloodstream froms but were absent in procyclic culture forms. Competition assays with unlabeled bovine brain or trypanosome calmodulins indicated that the developmentally regulated proteins associated with calmodulins from either source. Moreover, [125I]bovine brain calmodulin associated with the same proteins as trypanosome calmodulin. Homogenates of T. evansi exhibited the same pattern of calmodulin-binding activity as T. brucei slender bloodstream forms; however, T. cruzi and Leishmania tarentolae contained distinct patterns of calmodulin-binding activity. Mouse serum contained no detectable binding proteins while mouse brain contained predominantly proteins of Mr 210,000, 60,000, and 49,000 which were associated with the trypanosome calmodulin probe. The developmentally regulated calmodulin-binding proteins from T. brucei were in the 10,000g pellet. We conclude that the cellular complement of calmodulin-binding proteins varies during the trypanosome life cycle.     

Alternative oxidase present in procyclic Trypanosoma brucei may act to lower the mitochondrial production of superoxide

The mitochondrial electron transfer chain present in the procyclic form of the African trypanosome Trypanosoma brucei contains both cytochrome c oxidase and an alternative oxidase (TAO) as terminal oxidases that reduce oxygen to water. By contrast, the electron transfer chain of the primitive mitochondrion present in the bloodstream form of T. brucei contains only TAO as the terminal oxidase. TAO functions in the bloodstream forms to oxidize the ubiquinol produced by the glycerol-3-phosphate shuttle that results in the oxidation of the reduced nicotinamide adenine dinucleotide phosphate produced by glycolysis. The function, however, of TAO in the procyclic forms is unknown. In this study, we found that inhibition of TAO by the specific inhibitor salicylhydroxamic acid stimulates the formation of reactive oxygen species (ROS) in trypanosome mitochondria, resulting in mitochondrial alteration and increased oxidation of cellular proteins. Moreover, the activity and protein content of TAO in procyclic trypanosomes were increased when cells were incubated in the presence of hydrogen peroxide or antimycin A, the cytochrome bc1 complex inhibitor, which also results in increased ROS production. We suggest that one function of TAO in procyclic cells may be to prevent ROS production by removing excess reducing equivalents and transferring them to oxygen.     

Removal or maintenance of inositol-linked acyl chain in glycosylphosphatidylinositol is critical in trypanosome life cycle

The protozoan parasite Trypanosoma brucei is coated by glycosylphosphatidylinositol (GPI)-anchored proteins. During GPI biosynthesis, inositol in phosphatidylinositol becomes acylated. Inositol is deacylated prior to attachment to variant surface glycoproteins in the bloodstream form, whereas it remains acylated in procyclins in the procyclic form. We have cloned a T. brucei GPI inositol deacylase (GPIdeAc2). In accordance with the acylation/deacylation profile, the level of GPIdeAc2 mRNA was 6-fold higher in the bloodstream form than in the procyclic form. Knockdown of GPIdeAc2 in the bloodstream form caused accumulation of an inositol-acylated GPI, a decreased VSG expression on the cell surface and slower growth, indicating that inositol-deacylation is essential for the growth of the bloodstream form. Overexpression of GPIdeAc2 in the procyclic form caused an accumulation of GPI biosynthetic intermediates lacking inositol-linked acyl chain and decreased cell surface procyclins because of release into the culture medium, indicating that overexpression of GPIdeAc2 is deleterious to the surface coat of the procyclic form. Therefore, the GPI inositol deacylase activity must be tightly regulated in trypanosome life cycle.     

Subcellular localization of a variable surface glycoprotein phosphatidylinositol-specific phospholipase-C in African trypanosomes

African trypanosomes contain a membrane-bound enzyme capable of removing dimyristylglycerol from the membrane-attached form of the variable surface glycoprotein (mfVSG; Ferguson, M. A. J., K. Halder, and G. A. M. Cross, 1985, J. Biol Chem., 260:4963-4968). Although mfVSG phospholipase-C has been implicated in the removal of the VSG from the trypanosome surface (Cardoso de Almeida, M. L., and M. J. Turner, 1983, Nature (Lond.)., 302:349-352; Ferguson, M. A. J., K. Halder, and G. A. M. Cross, 1985, J. Biol Chem., 260:4963-4968), its precise function and subcellular location have not been determined. We have developed a procedure for the separation of the cell fractions and organelles of Trypanosoma brucei brucei (and other trypanosome species) by differential sucrose and isopycnic PercollR centrifugation. These fractions were tested for mfVSG phospholipase activity using Trypanosoma brucei mfVSG labeled with 3H-myristic acid as substrate. The highest enzyme-specific activity was associated with the flagella and evidence is presented to suggest that it is localized in the flagellar pocket. Some activity was also associated with the Golgi complex. These results suggest that the mfVSG phospholipase is localized primarily in the membrane of the flagella pocket and possibly other membrane organelles derived from and associated with this structure, and may be part of the VSG-membrane recycling system in African trypanosomes. The activity of mfVSG phospholipase amongst various trypanosome species was determined. We show that, in contrast to the bloodstream forms of Trypanosoma brucei, cultured procyclic Trypanosoma brucei and bloodstream Trypanosoma vivax had little or no mfVSG phospholipase activity. The activity found in bloodstream forms of Trypanosoma congolense was intermediate between Trypanosoma vivax and Trypanosoma brucei.     

A novel CCCH protein which modulates differentiation of Trypanosoma brucei to its procyclic form.

Cell differentiation in Trypanosoma brucei involves highly regulated changes in morphology, proliferation and metabolism. However, the controls of these developmental processes are unknown. We have identified two novel proteins from the rare CCCH zinc finger family, each <140 amino acids in length and implicated in life cycle regulation. TbZFP1 is transiently enriched during differentiation from the bloodstream to procyclic form, whereas tbZFP2, when ablated in bloodstream forms by RNA interference, inhibits this developmental step. Moreover, expressing an ectopic copy of tbZFP2 results in a dramatic procyclic stage-specific remodelling of the trypanosome cytoskeleton similar to the morphogenic events of differentiation. This phenotype, we term 'nozzle', involves polar extension of microtubules at the posterior end of the cell and is dependent upon a motif hitherto restricted to E3 ubiquitin ligases. TbZFP1 and tbZFP2 represent the first molecules implicated in the control of trypanosome differentiation to the procyclic form.     

Protein tyrosine phosphatase TbPTP1: A molecular switch controlling life cycle differentiation in trypanosomes

Differentiation in African trypanosomes (Trypanosoma brucei) entails passage between a mammalian host, where parasites exist as a proliferative slender form or a G0-arrested stumpy form, and the tsetse fly. Stumpy forms arise at the peak of each parasitaemia and are committed to differentiation to procyclic forms that inhabit the tsetse midgut. We have identified a protein tyrosine phosphatase (TbPTP1) that inhibits trypanosome differentiation. Consistent with a tyrosine phosphatase, recombinant TbPTP1 exhibits the anticipated substrate and inhibitor profile, and its activity is impaired by reversible oxidation. TbPTP1 inactivation in monomorphic bloodstream trypanosomes by RNA interference or pharmacological inhibition triggers spontaneous differentiation to procyclic forms in a subset of committed cells. Consistent with this observation, homogeneous populations of stumpy forms synchronously differentiate to procyclic forms when tyrosine phosphatase activity is inhibited. Our data invoke a new model for trypanosome development in which differentiation to procyclic forms is prevented in the bloodstream by tyrosine dephosphorylation. It may be possible to use PTP1B inhibitors to block trypanosomatid transmission.     

Polymorphism in the procyclic acidic repetitive protein gene family of Trypanosoma brucei.

The expression of procyclic acidic repetitive protein (PARP) by Trypanosoma brucei is strongly induced during the transition of bloodstream form to cultured procyclic trypomastigotes in vitro. The membrane-associated protein is distinguished by a central domain consisting of tandemly repeated glutamate-proline dipeptides. The trypanosome genome contains eight PARP genes, at least four of which are expressed. A minimum of four distinct PARP mRNA species comprises two classes of PARP mRNA, based upon divergent 3' untranslated region sequences, and these mRNAs encode polypeptides that exhibited an inverse relation between molecular weight and isoelectric point. Comparative analysis of PARP gene structure indicated that these polypeptides differ by variation in size of the dipeptide repeat domain. Comparison of PARP genes and polypeptides of three independent T. brucei isolates suggested that PARP is not a homogeneous species but instead represents a family of polymorphic proteins.     

Trypanosoma rhodesiense: mitochondrial proteins of bloodstream and procyclic trypomastigotes

One- and two-dimensional gel electrophoresis of the solubilized mitochondrial proteins of bloodstream and procyclic trypomastigote Trypanosoma brucei rhodesiense and radiolabeling of proteins in the presence of cycloheximide were used to identify proteins synthesized in the trypanosome mitochondrion. The proteins which comprise the mitochondrion were found to be very similar in both bloodstream and procyclic trypomastigotes, but do differ in their level of synthesis. A protein putatively identified as subunit II of cytochrome oxidase (EC 1.9.3.1) was detected in mitochondria from both the procyclic and bloodstream organisms. The presence of this protein in bloodstream trypomastigotes and the overall similarity of protein content in the trypanosome mitochondria is noteworthy in view of the fact that bloodstream trypomastigotes have a repressed mitochondrion with no detectable tricarboxylic acid cycle or cytochrome electron transport chain.     

Synthesis of a hydrolase for the membrane-form variant surface glycoprotein is repressed during transformation of Trypanosoma brucei

A membrane-bound phospholipase C-like hydrolase present in lysates of bloodstream forms of Trypanosoma brucei rapidly converts the membrane form of the variant surface protein to the soluble form and 1,2-dimyristoylglycerol [(1985) M.A.J. Ferguson et al. J. Biol. Chem., 260, 4963-4968]. The hydrolase is inhibited by p-chloromercuribenzenesulfonate. The synthesis of the enzyme is rapidly repressed upon differentiation of bloodstream forms to procyclic cells and the enzyme activity declines to an undetectable level during subsequent growth of procyclic forms.