Leaky transcription of variant surface glycoprotein gene expression sites in bloodstream african trypanosomes.

Trypanosoma brucei undergoes antigenic variation by periodically switching the expression of its variant surface glycoprotein (VSG) genes (vsg) among an estimated 20-40 telomere-linked expression sites (ES), only one of which is fully active at a given time. We found that in bloodstream trypanosomes one ES is transcribed at a high level and other ESs are expressed at low levels, resulting in organisms containing one abundant VSG mRNA and several rare VSG RNAs. Some of the rare VSG mRNAs come from monocistronic ESs in which the promoters are situated about 2 kilobases upstream of the vsg, in contrast to the polycistronic ESs in which the promoters are located 45-60 kilobases upstream of the vsg. The monocistronic ES containing the MVAT4 vsg does not include the ES-associated genes (esag) that occur between the promoter and the vsg in polycistronic ESs. However, bloodstream MVAT4 trypanosomes contain the mRNAs for many different ESAGs 6 and 7 (transferrin receptors), suggesting that polycistronic ESs are partially active in this clone. To explain these findings, we propose a model in which both mono- and polycistronic ESs are controlled by a similar mechanism throughout the parasite's life cycle. Certain VSGs are preferentially expressed in metacyclic versus bloodstream stages as a result of differences in ESAG expression and the proximity of the promoters to the vsg and telomere.     

Identification and characterization of three peroxins--PEX6, PEX10 and PEX12--involved in glycosome biogenesis in Trypanosoma brucei

Protozoan Kinetoplastida such as the pathogenic trypanosomes compartmentalize several important metabolic systems, including the glycolytic pathway, in peroxisome-like organelles designated glycosomes. Genes for three proteins involved in glycosome biogenesis of Trypanosoma brucei were identified. A preliminary analysis of these proteins, the peroxins PEX6, PEX10 and PEX12, was performed. Cellular depletion of these peroxins by RNA interference affected growth of both mammalian bloodstream-form and insect-form (procyclic) trypanosomes. The bloodstream forms, which rely entirely on glycolysis for their ATP supply, were more rapidly killed. Both by immunofluorescence studies of intact procyclic T. brucei cells and subcellular fractionation experiments involving differential permeabilization of plasma and organellar membranes it was shown that RNAi-dependent knockdown of the expression of each of these peroxins resulted in the partial mis-localization of different types of glycosomal matrix enzymes to the cytoplasm: proteins with consensus motifs such as the C-terminal type 1 peroxisomal targeting signal PTS1 or the N-terminal signal PTS2 and a protein for which the sorting information is present in a polypeptide-internal fragment not containing an identifiable consensus sequence.     

The regulation of procyclin expression in Trypanosoma bruceli: making or breaking the rules?

The identification of procyclins as stage-specific coat proteins of procyclic forms of Trypanosoma brucei has not only provided a convenient molecular marker for the differentiation of bloodstream-form trypanosomes into procyclic forms, but has also allowed some important insights into gene regulation in trypanosomes. Here, Adrian Hehl and Isabel Roditi summarize what has been learnt in the past few years about the control mechanisms that may contribute to the stage-specific expression of procyclins.     

The FACT subunit TbSpt16 is involved in cell cycle specific control of VSG expression sites in Trypanosoma brucei

The African trypanosome Trypanosoma brucei monoallelically expresses one of more than 1000 Variant Surface Glycoprotein (VSG) genes. The active VSG is transcribed from one of about 15 telomeric VSG expression sites (ESs). It is unclear how monoallelic expression of VSG is controlled, and how inactive VSG ESs are silenced. Here, we show that blocking synthesis of the T. brucei FACT subunit TbSpt16 triggers a G2/early M phase cell cycle arrest in both bloodstream and insect form T. brucei. Segregation of T. brucei minichromosomes in these stalled cells is impaired, implicating FACT in maintenance of centromeres. Strikingly, knock-down of TbSpt16 results in 20- to 23-fold derepression of silent VSG ES promoters in bloodstream form T. brucei, with derepression specific to the G2/M cell cycle stage. In insect form T. brucei TbSpt16 knock-down results in 16- to 25-fold VSG ES derepression. Using chromatin immunoprecipitation (ChIP), TbSpt16 was found to be particularly enriched at the promoter region of silent but not active VSG ESs in bloodstream form T. brucei. The chromatin remodeler FACT is therefore implicated in maintenance of repressed chromatin present at silent VSG ES promoters, but is also essential for chromosome segregation presumably through maintenance of functional centromeres.     

Characterization and developmentally regulated localization of the mitochondrial carrier protein homologue MCP6 from Trypanosoma brucei

Proteins of the mitochondrial carrier family (MCF) are located mainly in the inner mitochondrial membrane and mediate the transport of a large range of metabolic intermediates. The genome of Trypanosoma brucei harbors 29 genes encoding different MCF proteins. We describe here the characterization of MCP6, a novel T. brucei MCF protein. Sequence comparison and phylogenetic reconstruction revealed that MCP6 is closely related to different mitochondrial ADP/ATP and calcium-dependent solute carriers, including the ATP-Mg/Pi carrier of Homo sapiens. However, MCP6 lacks essential amino acids and sequence motifs conserved in these metabolite transporters, and functional reconstitution and transport assays with E. coli suggested that this protein indeed does not function as an ADP/ATP or ATP-Mg/Pi carrier. The subcellular localization of MCP6 is developmentally regulated: in bloodstream-form trypanosomes, the protein is predominantly glycosomal, whereas in the procyclic form, it is found mainly in the mitochondria. Depletion of MCP6 in procyclic trypanosomes resulted in growth inhibition, an increased cell size, aberrant numbers of nuclei and kinetoplasts, and abnormal kinetoplast morphology, suggesting that depletion of MCP6 inhibits division of the kinetoplast.     

Deletion of a novel protein kinase with PX and FYVE-related domains increases the rate of differentiation of Trypanosoma brucei

Growth control of African trypanosomes in the mammalian host is coupled to differentiation of a non-dividing life cycle stage, the stumpy bloodstream form. We show that a protein kinase with novel domain architecture is important for growth regulation. Zinc finger kinase (ZFK) has a kinase domain related to RAC and S6 kinases flanked by a FYVE-related zinc finger and a phox (PX) homology domain. To investigate the function of the kinase during cyclical development, a stable transformation procedure for bloodstream forms of differentiation-competent (pleomorphic) Trypanosoma brucei strains was established. Deletion of both allelic copies of ZFK by homologous recombination resulted in reduced growth of bloodstream-form parasites in culture, which was correlated with an increased rate of differentiation to the non-dividing stumpy form. Growth and differentiation rates were returned to wild-type level by ectopic ZFK expression. The phenotype is stage-specific, as growth of procyclic (insect form) trypanosomes was unaffected, and Deltazfk/Deltazfk clones were able to undergo full cyclical development in the tsetse fly vector. Deletion of ZFK in a differentiation-defective (monomorphic) strain of T. brucei did not change its growth rate in the bloodstream stage. This suggests a function of ZFK associated with the trypanosomes' decision between either cell cycle progression, as slender bloodstream form, or differentiation to the non-dividing stumpy form.     

Turnover of glycosomes during life-cycle differentiation of Trypanosoma brucei

Protozoan Kinetoplastida, a group that comprises the pathogenic Trypanosoma brucei, compartmentalize several metabolic systems such as the major part of the glycolytic pathway, in multiple peroxisome-like organelles, designated glycosomes. Trypanosomes have a complicated life cycle, involving two major, distinct stages living in the mammalian bloodstream and several stages inhabiting different body parts of the tsetse fly. Previous studies on non-differentiating trypanosomes have shown that the metabolism and enzymatic contents of glycosomes in bloodstream-form and cultured procyclic cells, representative of the stage living in the insect's midgut, differ considerably. In this study, the morphology of glycosomes and their position relative to the lysosome were followed, as were the levels of some glycosomal enzymes and markers for other subcellular compartments, during the differentiation from bloodstream-form to procyclic trypanosomes. Our studies revealed a small tendency of glycosomes to associate with the lysosome when a population of long-slender bloodstream forms differentiated into short-stumpy forms which are pre-adapted to live in the fly. The same phenomenon was observed during the short-stumpy to procyclic transformation, but then the process was fast and many more glycosomes were associated with the dramatically enlarged degradation organelle. The observations suggested an efficient glycosome turnover involving autophagy. Changes observed in the levels of marker enzymes are consistent with the notion that, during differentiation, glycosomes with enzymatic contents specific for the old life-cycle stage are degraded and new glycosomes with different contents are synthesized, causing that the metabolic repertoire of trypanosomes is, at each stage, optimally adapted to the environmental conditions encountered.     

Characterization of Trypanosoma brucei PEX14 and its role in the import of glycosomal matrix proteins.

It has been shown previously in various organisms that the peroxin PEX14 is a component of a docking complex at the peroxisomal membrane, where it is involved in the import of matrix proteins into the organelle after their synthesis in the cytosol and recognition by a receptor. Here we present a characterization of the Trypanosoma brucei homologue of PEX14. It is shown that the protein is associated with glycosomes, the peroxisome-like organelles of trypanosomatids in which most glycolytic enzymes are compartmentalized. The N-terminal part of the protein binds specifically to TbPEX5, the cytosolic receptor for glycosomal matrix proteins with a peroxisome-targeting signal type 1 (PTS-1). TbPEX14 mRNA depletion by RNA interference results, in both bloodstream-form and procyclic, insect-stage T. brucei, in mislocalization of glycosomal proteins to the cytosol. The mislocalization was observed for different classes of matrix proteins: proteins with a C-terminal PTS-1, a N-terminal PTS-2 and a polypeptide internal I-PTS. The RNA interference experiments also showed that TbPEX14 is essential for the survival of bloodstream-form and procyclic trypanosomes. These data indicate the protein's great potential as a target for selective trypanocidal drugs.