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The blood-brain barrier (BBB) protects the central nervous system (CNS) from harmful blood-borne factors. Although BBB dysfunction is a hallmark of several neurological disorders, therapies to restore BBB function are lacking. An attractive strategy is to repurpose developmental BBB regulators, such as Wnt7a, into BBB-protective agents. However, safe therapeutic use of Wnt ligands is complicated by their pleiotropic Frizzled signaling activities. Taking advantage of the Wnt7a/b-specific Gpr124/Reck co-receptor complex, we genetically engineered Wnt7a ligands into BBB-specific Wnt activators. In a “hit-and-run” adeno-associated virus–assisted CNS gene delivery setting, these new Gpr124/Reck-specific agonists protected BBB function, thereby mitigating glioblastoma expansion and ischemic stroke infarction. This work reveals that the signaling specificity of Wnt ligands is adjustable and defines a modality to treat CNS disorders by normalizing the BBB.

Summary Human innate immunity to Trypanosoma brucei involves the trypanosome C-terminal kinesin TbKIFC1, which transports internalized trypanolytic factor apolipoprotein L1 (APOL1) within the parasite. We show that TbKIFC1 preferentially associates with cholesterol-containing membranes and is indispensable for mammalian infectivity. Knockdown of TbKIFC1 did not affect trypanosome growth in vitro but rendered the parasites unable to infect mice unless antibody synthesis was compromised. Surface clearance of Variant Surface Glycoprotein (VSG)-antibody complexes was far slower in these cells, which were more susceptible to capture by macrophages. This phenotype was not due to defects in VSG expression or trafficking but to decreased VSG mobility in a less fluid, stiffer surface membrane. This change can be attributed to increased cholesterol level in the surface membrane in TbKIFC1 knockdown cells. Clearance of surface-bound antibodies by T. brucei is therefore essential for infectivity and depends on high membrane fluidity maintained by the cholesterol-trafficking activity of TbKIFC1., Graphical Abstract, Highlights • The TbKIFC1 kinesin preferentially transports cholesterol-containing membranes • TbKIFC1 is absolutely required for parasite growth in mice • Cholesterol trafficking by TbKIFC1 confers high fluidity to the plasma membrane • High membrane fluidity allows antibody clearance necessary for parasite infectivity, Immunology; Microbiology Parasite; Cell Biology

Human apolipoprotein L1 (APOL1) kills African trypanosomes except Trypanosoma rhodesiense and Trypanosoma gambiense, the parasites causing sleeping sickness. APOL1 uptake into trypanosomes is favoured by its association with the haptoglobin-related protein-haemoglobin complex, which binds to the parasite surface receptor for haptoglobin-haemoglobin. As haptoglobin-haemoglobin can saturate the receptor, APOL1 uptake is increased in haptoglobin-poor (hypohaptoglobinaemic) serum (HyHS). While T. rhodesiense resists APOL1 by RNA polymerase I (pol-I)-mediated expression of the serum resistance-associated (SRA) protein, T. gambiense resists by pol-II-mediated expression of the T. gambiense-specific glycoprotein (TgsGP). Moreover, in T. gambiense resistance to HyHS is linked to haptoglobin-haemoglobin receptor inactivation by mutation. We report that unlike T. gambiense, T. rhodesiense possesses a functional haptoglobin-haemoglobin receptor, and that like T. gambiense experimentally provided with active receptor, this parasite is killed in HyHS because of receptor-mediated APOL1 uptake. However, T. rhodesiense could adapt to low haptoglobin by increasing transcription of SRA. When assayed in Trypanosoma brucei, resistance to HyHS occurred with pol-I-, but not with pol-II-mediated SRA expression. Similarly, T. gambiense provided with active receptor acquired resistance to HyHS only when TgsGP was moved to a pol-I locus. Thus, transcription by pol-I favours adaptive gene regulation, explaining the presence of SRA in a pol-I locus.

How Wnt ligands achieve specificity Wnt signaling is essential for development, tissue homeostasis, and disease. The 19 members of the Wnt family interact promiscuously with the 10 Frizzled receptors, raising the question of how ligand-specific discrimination is achieved in a biological context. Eubelen et al. used experiments in zebrafish to show that cells are equipped with decoding modules that bind Wnt with high specificity and trigger signal amplification via their recruitment into higher-order Frizzled signalosomes (see the Perspective by Kim and Goentoro). Thus, distinct Wnt ligand-receptor pairs can be targeted specifically for therapeutic purposes. Science , this issue p. eaat1178 ; see also p. 643

Normal human serum (NHS) confers human resistance to infection by the parasite Trypanosoma brucei owing to the trypanolytic activity of apolipoprotein L1 (APOL1), present in two serum complexes termed Trypanolytic Factors (TLF-1 and -2). In order to identify parasite components involved in the intracellular trafficking and activity of TLFs, an inducible RNA interference (RNAi) genomic DNA library constructed in bloodstream form T. brucei was subjected to RNAi induction and selection for resistant parasites under NHS conditions favouring either TLF-1 or TLF-2 uptake. While TLF-1 conditions readily selected the haptoglobin-haemoglobin (HP-HB) surface receptor TbHpHbR as expected, given its known ability to bind TLF-1, under TLF-2 conditions no specific receptor for TLF-2 was identified. Instead, the screen allowed the identification of five distinct factors expected to be involved in the assembly of the vacuolar proton pump V-ATPase and consecutive endosomal acidification. These data confirm that lowering the pH during endocytosis is required for APOL1 toxic activity.

The protozoan parasite Trypanosoma brucei is lysed by apolipoprotein L-I, a component of human high-density lipoprotein (HDL) particles that are also characterized by the presence of haptoglobin-related protein. We report that this process is mediated by a parasite glycoprotein receptor, which binds the haptoglobin-hemoglobin complex with high affinity for the uptake and incorporation of heme into intracellular hemoproteins. In mice, this receptor was required for optimal parasite growth and the resistance of parasites to the oxidative burst by host macrophages. In humans, the trypanosome receptor also recognized the complex between hemoglobin and haptoglobin-related protein, which explains its ability to capture trypanolytic HDLs. Thus, in humans the presence of haptoglobin-related protein has diverted the function of the trypanosome haptoglobin-hemoglobin receptor to elicit innate host immunity against the parasite.

Humans resist infection by the African parasite Trypanosoma brucei owing to the trypanolytic activity of the serum apolipoprotein L1 (APOL1). Following uptake by endocytosis in the parasite, APOL1 forms pores in endolysosomal membranes and triggers lysosome swelling. Here we show that APOL1 induces both lysosomal and mitochondrial membrane permeabilization (LMP and MMP). Trypanolysis coincides with MMP and consecutive release of the mitochondrial TbEndoG endonuclease to the nucleus. APOL1 is associated with the kinesin TbKIFC1, of which both the motor and vesicular trafficking VHS domains are required for MMP, but not for LMP. The presence of APOL1 in the mitochondrion is accompanied by mitochondrial membrane fenestration, which can be mimicked by knockdown of a mitochondrial mitofusin-like protein (TbMFNL). The BH3-like peptide of APOL1 is required for LMP, MMP and trypanolysis. Thus, trypanolysis by APOL1 is linked to apoptosis-like MMP occurring together with TbKIFC1-mediated transport of APOL1 from endolysosomal membranes to the mitochondrion., SCOPUS: ar.j, info:eu-repo/semantics/published

Human apolipoprotein L1 (APOL1) kills African trypanosomes except Trypanosoma rhodesiense and Trypanosoma gambiense, the parasites causing sleeping sickness. APOL1 uptake into trypanosomes is favoured by its association with the haptoglobin-related protein-haemoglobin complex, which binds to the parasite surface receptor for haptoglobin-haemoglobin. As haptoglobin-haemoglobin can saturate the receptor, APOL1 uptake is increased in haptoglobin-poor (hypohaptoglobinaemic) serum (HyHS). While T. rhodesiense resists APOL1 by RNA polymerase I (pol-I)-mediated expression of the serum resistance-associated (SRA) protein, T. gambiense resists by pol-II-mediated expression of the T. gambiense-specific glycoprotein (TgsGP). Moreover, in T. gambiense resistance to HyHS is linked to haptoglobin-haemoglobin receptor inactivation by mutation. We report that unlike T. gambiense, T. rhodesiense possesses a functional haptoglobin-haemoglobin receptor, and that like T. gambiense experimentally provided with active receptor, this parasite is killed in HyHS because of receptor-mediated APOL1 uptake. However, T. rhodesiense could adapt to low haptoglobin by increasing transcription of SRA. When assayed in Trypanosoma brucei, resistance to HyHS occurred with pol-I-, but not with pol-II-mediated SRA expression. Similarly, T. gambiense provided with active receptor acquired resistance to HyHS only when TgsGP was moved to a pol-I locus. Thus, transcription by pol-I favours adaptive gene regulation, explaining the presence of SRA in a pol-I locus.

Transcription of the variant surface glycoprotein (VSG) gene of Trypanosoma brucei occurs in a single of multiple polycistronic expression sites (ESs). Analysis of RNA from proliferative long slender (LS) bloodstream forms demonstrated that initiation of transcription occurs in different ESs, but inefficient RNA processing and elongation is linked to RNA polymerase arrest in all except one unit at a time. The pattern of ES transcripts was analysed during the transformation of dividing LS forms into quiescent short stumpy (SS) forms. The results demonstrated that the mono-allelic control allowing preferential RNA production from a given ES stops during this process. Accordingly, the steady-state ES transcripts, particularly the VSG mRNA, were strongly reduced. However, transcripts from the beginning of different ESs were still synthesized, and in vitro run-on transcription analysis indicated that RNA polymerase was still fully associated with the promoter-proximal half of the 'active' ES. Analysis of transcripts from two central tandem genes confirmed the existence of a residual decreasing transcriptional gradient in the 'active' ES of SS forms. Thus, in these forms the RNA polymerase of the ES is inactivated in situ. This inactivation is accompanied by a strong overall reduction of nuclear DNA transcription. Although cAMP is involved in the LS to SS transformation, no direct effect of cAMP was observed on the VSG ES control.

Normal human serum (NHS) confers human resistance to infection by the parasite Trypanosoma brucei owing to the trypanolytic activity of apolipoprotein L1 (APOL1), present in two serum complexes termed Trypanolytic Factors (TLF-1 and -2). In order to identify parasite components involved in the intracellular trafficking and activity of TLFs, an inducible RNA interference (RNAi) genomic DNA library constructed in bloodstream form T. brucei was subjected to RNAi induction and selection for resistant parasites under NHS conditions favouring either TLF-1 or TLF-2 uptake. While TLF-1 conditions readily selected the haptoglobin-haemoglobin (HP-HB) surface receptor TbHpHbR as expected, given its known ability to bind TLF-1, under TLF-2 conditions no specific receptor for TLF-2 was identified. Instead, the screen allowed the identification of five distinct factors expected to be involved in the assembly of the vacuolar proton pump V-ATPase and consecutive endosomal acidification. These data confirm that lowering the pH during endocytosis is required for APOL1 toxic activity.

The protozoan parasite Trypanosoma brucei develops antigenic variation to escape the immune response of its host. To this end, the trypanosome genome contains multiple telomeric expression sites competent for transcription of variant surface glycoprotein genes, but as a rule only a single antigen is expressed at any time. We used reverse transcription-PCR (RT-PCR) to analyse transcription of different segments of the expression sites in different variant clones of two independent strains of T. brucei. The results indicated that RNA polymerase is installed and active at the beginning of many, if not all, expression sites simultaneously, but that a progressive arrest of RNA elongation occurs in all but one site. This defect is linked to inefficient RNA processing and RNA release from the nucleus. Therefore, functional transcription in the active site appears to depend on the selective recruitment of a RNA elongation/processing machinery.

The protozoan parasite Trypanosoma brucei relies on trans-splicing of a common spliced leader (SL) RNA to maturate mRNAs. Using the yeast two-hybrid system a protein (TSR1IP) was identified that interacts with the T. brucei serine-arginine (SR) protein termed TSR1. TSR1IP shows homology to U1 70 kDa proteins, and contains an SR rich domain as well as an acidic/arginine domain homologous to the U1 70 kDa poly(A) polymerase inhibiting domain. This protein is localized in the nucleoplasm and excluded from the nucleolus in trypanosomal bloodstream and procyclic forms. Based on structural modelling predictions and on the identification of a RNA recognition motif (RRM), it was possible to demonstrate by the yeast three-hybrid system that TSR1IP interacts with the 5' splice region of the SL RNA. All the above characteristics suggest that TSR1IP could be involved in trans-splicing.

The Trypanosoma brucei transferrin (Tf) receptor is a heterodimer encoded by ESAG7 and ESAG6, two genes contained in the different polycistronic transcription units of the variant surface glycoprotein (VSG) gene. The sequence of ESAG7/6 differs slightly between different units, so that receptors with different affinities for Tf are expressed alternatively following transcriptional switching of VSG expression sites during antigenic variation of the parasite. Based on the sequence homology between pESAG7/6 and the N-terminal domain of VSGs, it can be predicted that the four blocks containing the major sequence differences between pESAG7 and pESAG6 form surface-exposed loops and generate the ligand-binding site. The exchange of a few amino acids in this region between pESAG6s encoded by different VSG units greatly increased the affinity for bovine Tf. Similar changes in other regions were ineffective, while mutations predicted to alter the VSG-like structure abolished the binding. Chimeric proteins containing the N-terminal dimerization domain of VSG and the C-terminal half of either pESAG7 or pESAG6, which contains the ligand-binding domain, can form heterodimers that bind Tf. Taken together, these data provided evidence that the T.brucei Tf receptor is structurally related to the N-terminal domain of the VSG and that the ligand-binding site corresponds to the exposed surface loops of the protein.

The African parasite Trypanosoma brucei gambiense accounts for 97% of human sleeping sickness cases. T. b. gambiense resists the specific human innate immunity acting against several other tsetse-fly-transmitted trypanosome species such as T. b. brucei, the causative agent of nagana disease in cattle. Human immunity to some African trypanosomes is due to two serum complexes designated trypanolytic factors (TLF-1 and -2), which both contain haptoglobin-related protein (HPR) and apolipoprotein LI (APOL1). Whereas HPR association with haemoglobin (Hb) allows TLF-1 binding and uptake via the trypanosome receptor TbHpHbR (ref. 5), TLF-2 enters trypanosomes independently of TbHpHbR (refs 4, 5). APOL1 kills trypanosomes after insertion into endosomal/lysosomal membranes. Here we report that T. b. gambiense resists TLFs via a hydrophobic β-sheet of the T. b. gambiense-specific glycoprotein (TgsGP), which prevents APOL1 toxicity and induces stiffening of membranes upon interaction with lipids. Two additional features contribute to resistance to TLFs: reduction of sensitivity to APOL1 requiring cysteine protease activity, and TbHpHbR inactivation due to a L210S substitution. According to such a multifactorial defence mechanism, transgenic expression of T. b. brucei TbHpHbR in T. b. gambiense did not cause parasite lysis in normal human serum. However, these transgenic parasites were killed in hypohaptoglobinaemic serum, after high TLF-1 uptake in the absence of haptoglobin (Hp) that competes for Hb and receptor binding. TbHpHbR inactivation preventing high APOL1 loading in hypohaptoglobinaemic serum may have evolved because of the overlapping endemic area of T. b. gambiense infection and malaria, the main cause of haemolysis-induced hypohaptoglobinaemia in western and central Africa.

The polycistronic procylcin PARP (for procyclic acidic repetitive protein) A transcription unit of Trypanosoma brucei was completely characterized by the mapping of the termination region. In addition to the tandem of procyclin genes and GRESAG 2.1, this 7.5- to 9.5-kb unit contained another gene for a putative surface protein, termed PAG (for procyclin-associated gene) 3. The terminal 3-kb sequence did not contain significant open reading frames and cross-hybridized with the beginning of one or several transcription units specific to the bloodstream form. At least three separate fragments from the terminal region were able to inhibit chloramphenicol acetyltransferase expression when inserted between either the PARP, the ribosomal, or the variable surface glycoprotein promoter and a chloramphenicol acetyltransferase reporter gene. This inhibition was due to an orientation-dependent transcription termination caused by the combination of several attenuator elements with no obvious sequence conservation. The procyclin transcription terminator appeared unable to inhibit transcription by polymerase II.

We describe here the stable transformation of Trypanosoma brucei using a new selectable marker for kinetoplastid protozoa, the Sh ble, or phleomycin, resistance gene. A plasmid containing this gene targeted to the tubulin gene locus by homologous sequences was introduced into procyclic trypanosomes by electroporation and cells selected for antibiotic resistance. Southern analysis of stable transformants showed that the plasmid had been integrated into the tubulin locus by homologous recombination. Analysis of bloodstream stage transformants, produced by transmission through the vector Glossina, showed that the resistance gene was conserved and expressed in these forms in the absence of selective drug pressure. In both procyclic and bloodstream forms, transcription of the ble gene appears to originate from the upstream tubulin promoter, despite the presence of a VSG promoter in the integrated construct. The generation of stable bloodstream transformants for the first time will facilitate the study of gene function and expression during the trypanosome life cycle, and aid in the investigation of genetic exchange in these organisms.

African trypanosomes face a peculiar dilemma in mammalian hosts. They are simultaneously constrained to avoid interactions that might be detrimental to their survival but also must acquire macromolecules. At the moment precisely how they have successfully resolved this conundrum remains elusive but what is clear is that they have developed a very extraordinary and efficacious machinery of endocytosis. Ligand binding and uptake occur only in a specialized region of the cellular surface called the flagellar pocket. Emerging evidence suggests that trypanosomal receptors for host macromolecules are unusual and that mechanisms of their internalization are different to that of higher eukaryotes. This review will focus on areas of endocytosis where there is general agreement, highlight conflicting views and consider general paradigms of the process.

PUBLISHED, Apolipoprotein L-I is the trypanolytic factor of human serum. Here we show that this protein contains a membrane pore-forming domain functionally similar to that of bacterial colicins, flanked by a membrane-addressing domain. In lipid bilayer membranes, apolipoprotein L-I formed anion channels. In Trypanosoma brucei, apolipoprotein L-I was targeted to the lysosomal membrane and triggered depolarization of this membrane, continuous influx of chloride, and subsequent osmotic swelling of the lysosome until the trypanosome lysed.

Transcription of the variant surface glycoprotein (VSG) gene of Trypanosoma brucei occurs in a single of multiple polycistronic expression sites (ESs). Analysis of RNA from proliferative long slender (LS) bloodstream forms demonstrated that initiation of transcription occurs in different ESs, but inefficient RNA processing and elongation is linked to RNA polymerase arrest in all except one unit at a time. The pattern of ES transcripts was analysed during the transformation of dividing LS forms into quiescent short stumpy (SS) forms. The results demonstrated that the mono-allelic control allowing preferential RNA production from a given ES stops during this process. Accordingly, the steady-state ES transcripts, particularly the VSG mRNA, were strongly reduced. However, transcripts from the beginning of different ESs were still synthesized, and in vitro run-on transcription analysis indicated that RNA polymerase was still fully associated with the promoter-proximal half of the 'active' ES. Analysis of transcripts from two central tandem genes confirmed the existence of a residual decreasing transcriptional gradient in the 'active' ES of SS forms. Thus, in these forms the RNA polymerase of the ES is inactivated in situ. This inactivation is accompanied by a strong overall reduction of nuclear DNA transcription. Although cAMP is involved in the LS to SS transformation, no direct effect of cAMP was observed on the VSG ES control.

SIR2-like proteins have been implicated in a wide range of cellular events including chromosome silencing, chromosome segregation, DNA recombination and the determination of life span. We report here the molecular and functional characterization of a SIR2-related protein from the protozoan parasite Trypanosoma brucei, which we termed TbSIR2RP1. This protein is a chromosome-associated NAD-dependent enzyme which, in contrast to other known proteins of this family, catalyses both ADP-ribosylation and deacetylation of histones, particulary H2A and H2B. Under- or overexpression of TbSIR2RP1 decreased or increased, respectively, cellular resistance to DNA damage. Treatment of trypanosomal nuclei with a DNA alkylating agent resulted in a significant increase in the level of histone ADP-ribosylation and a concomitant increase in chromatin sensitivity to micrococcal nuclease. Both of these responses correlated with the level of TbSIR2RP1 expression. We propose that histone modification by TbSIR2RP1 is involved in DNA repair.

In Trypanosoma brucei the genes are organised into long polycistronic transcription units and only three promoters for protein-encoding genes and a single terminator have been characterised. These promoters recruit a polI-like RNA polymerase for the transcription units encoding the two major stage-specific antigens of the parasite, the variant surface glycoprotein (VSG) of the bloodstream form and procyclin of the insect-specific procyclic form, while the terminator is that of a procyclin transcription unit. By deletional and mutational analysis we defined the two DNA sequences essential for the activity of the VSG promoter from a bloodstream form transcription unit and one of the functional elements of the procyclin terminator. These three short sequences are similar, and their C-rich strand binds the same protein of 40 kDa. In addition, this factor also binds to the C-rich strand of the telomeric repeats, the consensus target sequence being 5′-CCCTNN-3′. The factor-binding sequences are functionally interchangeable in chimeric promoter or terminator constructs, although additional elements are required for full activity.

The protozoan parasite Trypanosoma brucei relies on trans-splicing to process its mRNAs. A novel nuclear serine/arginine (SR)-rich trypanosomal protein (TSR1) was characterized which contains two RNA recognition motifs. The TSR1 protein appears to be homologous to RNA-binding SR proteins of the cis-splicing machinery from higher eukaryotes. Moreover, in the yeast two-hybrid system, TSR1 is able to interact with the human splicing factors involved in the recognition of the 3' splicing site (U2AF35/U2AF65). In both procyclic and bloodstream forms of T. brucei, TSR1 was found to localize in the nucleus. In the bloodstream stage TSR1 showed the speckles pattern characteristic of SR proteins involved in cis-splicing. Moreover, TSR1 was able to specifically bind the spliced leader (SL) RNA involved in trans-splicing in trypanosomes by the yeast three-hybrid system. These and other observations suggest that TSR1 may be involved in trans-splicing in T. brucei.

The variant surface glycoprotein (VSG) and procyclin promoters of Trypanosoma brucei recruit an RNA polymerase sharing characteristic with polymerase I, but there is no sequence homology between them nor between these promoters and ribosomal promoters. We report the detailed characterization of the VSG promoter. The 70-bp region upstream of the transcription start site was sufficient for full promoter activity. Mutational analysis revealed three short critical stretches at positions -61 to -59 (box 1), -38 to -35 (box 2), and -1 to +1 (start site), the spacing of which was essential. These elements were conserved in the promoter for a metacyclic VSG gene. Hybrid sequences containing box 1 of the VSG promoter and box 2 of the ribosomal promoter were active. A specific binding of proteins to the noncoding strand of box 2, but not to double-stranded DNA, occurred. Competition experiments indicated that these proteins also bind to the corresponding region of the metacyclic VSG, procyclin, and ribosomal promoters. Binding of such a protein, of 40 kDa, appeared to be shared by these promoters.

The putative promoter of the variant surface glycoprotein (VSG) gene of Trypanosoma brucei was cloned into a plasmid containing the chloramphenicol acetyltransferase (CAT) gene. After electroporation into trypanosomes, this construct directed the expression of the CAT reporter gene. The essential region for promoter activity was found to reside within 88 bp upstream of the putative transcription start site. Transcription of the CAT construct occurred at approximately the same level in both bloodstream and procyclic forms and was resistant to alpha-amanitin. However, CAT expression appeared to be modulated in the two forms of the parasite. Sequences 3' to the gene seemed to be important in this respect, as CAT activity in bloodstream forms was readily detectable only when the 3' region of a VSG cDNA was placed downstream of the CAT gene. Two separate VSG gene promoter sequences, both cloned from T. brucei AnTat 1.3A, were equally able to direct CAT expression, which suggests that there are a number of potential VSG gene promoters in the genome, although usually only one expression site is fully active at any one time.

The expression site for the variant surface glycoprotein (VSG) gene of Trypanosoma brucei contains several genes of unknown function (ESAGs, for expression site-associated genes). Among these, ESAG 4 shows homology to eukaryotic adenylate/guanylate cyclase genes, in the region encoding the presumptive enzyme catalytic domain. This gene belongs to a family of related sequences, and hybridizes to the genomic DNA of other trypanosomatids, such as Trypanosoma congolense, Trypanosoma vivax and Trypanosoma mega. While ESAG 4 is transcribed only in bloodstream forms by a RNA polymerase resistant to alpha-amanitin, at least three other members of this family are transcribed in both bloodstream and procyclic forms, by a RNA polymerase sensitive to the drug. These genes encode different putative transmembrane proteins showing high sequence conservation in the region corresponding to the adenylate/guanylate cyclase catalytic domain.

The variant surface glycoprotein (VSG) and procyclin are the major surface proteins of the bloodstream and procyclic stages, respectively, of Trypanosoma brucei. The promoter regions of the VSG and procyclin gene transcription units could be mapped thanks to the specific enrichment of initial transcripts that occurs following UV irradiation. Whereas the VSG gene is 45 kb distant from its promoter, procyclin genes are located immediately downstream. We show, by run-on assays on isolated nuclei and by cDNA analysis, that transcription occurs from both promoters in bloodstream as well as in procyclic forms. It is inferred that the control of the stage-specific expression of VSG and procyclin genes is not effected at the level of transcription initiation, but most probably by interfering with the elongation and stability of the specific transcripts.

The expression site for the variable surface glycoprotein (VSG) gene AnTat 1.3A of Trypanosoma brucei is 45 kilobases long and encompasses seven expression site-associated genes (ESAGs) (E. Pays, P. Tebabi, A. Pays, H. Coquelet, P. Revelard, D. Salmon, and M. Steinert, Cell 57:835-845, 1989). After UV irradiation, several large transcripts from the putative promoter region were strongly enriched. We report that one such major transcript starts near the poly(A) addition site of the first gene (ESAG 7), spans the intergenic region, and extends to the poly(A) addition site of the second gene (ESAG 6), thus bypassing the normal 3' splice site of the ESAG 6 mRNA. Since this transcript is spliced, we conclude that UV irradiation does not inhibit splicing but stabilizes unstable processing products. This demonstrates that at least some intergenic regions of the VSG gene expression site are continuously transcribed in accordance with a polycistronic transcription model.

The arrest of variable surface glycoprotein (VSG) synthesis is one of the first events accompanying the differentiation of Trypanosoma brucei bloodstream forms into procyclic forms, which are characteristic of the insect vector. This is because of a very fast inhibition of VSG gene transcription which occurs as soon as the temperature is lowered. We report that this effect is probably not controlled at the level of transcription initiation, since the beginning of the VSG gene expression site, about 45 kilobases upstream from the antigen gene, remains transcribed in procyclic forms. The permanent activity of the promoter readily accounts for the systematic reappearance, upon return to the bloodstream form after cyclical transmission, of the antigen type present before passage to the tsetse fly. The abortive transcription of the VSG gene expression site appears linked to RNA processing abnormalities. Such posttranscriptional controls may allow the modulation of gene expression in a genome organized in large multigenic transcription units.

The primate-specific serum protein apolipoprotein L1 (APOL1) is the only secreted member of a family of cell death promoting proteins 1-4 . APOL1 kills the bloodstream parasite Trypanosoma brucei brucei, but not the human sleeping sickness agents T.b. rhodesiense and T.b. gambiense 3 . We considered the possibility that intracellular members of the APOL1 family, against which extracellular trypanosomes could not have evolved resistance, could kill pathogenic T. brucei subspecies. Here we show that recombinant APOL3 (rAPOL3) kills all African trypanosomes, including T.b. rhodesiense, T.b. gambiense and the animal pathogens Trypanosoma evansi, Trypanosoma congolense and Trypanosoma vivax. However, rAPOL3 did not kill more distant trypanosomes such as Trypanosoma theileri or Trypanosoma cruzi. This trypanolytic potential was partially shared by rAPOL1 from Papio papio (rPpAPOL1). The differential killing ability of rAPOL3 and rAPOL1 was associated with a distinct dependence on acidic pH for activity. Due both to its instability and toxicity when injected into mice, rAPOL3 cannot be used for the treatment of infection, but an experimental rPpAPOL1 mutant inspired by APOL3 exhibited enhanced trypanolytic activity in vitro and the ability to completely inhibit T.b. gambiense infection in mice. We conclude that pH dependence influences the trypanolytic potential of rAPOLs.

The genome of Trypanosoma brucei contains up to 400 copies of a conserved sequence (TRS, trypanosome repeated sequence). The majority of TRS copies (TRS1) are 5.2 X 10(3) base-pairs (kb) and are flanked by different separate halves of the previously described transposable element RIME (ribosomal mobile element), although a variant copy (TRS2) contains only the central 1.45 kb portion and lacks RIME. TRS1 elements can probably undergo transposition, since they are dispersed in all chromosome size classes and are bordered by direct repeats of about four base-pairs. Some TRS1 elements may contain an open reading frame over almost their entire length (1651 codons), encoding a protein showing homology with reverse transcriptase. TRS probes detect poly(A)+ transcripts of 5 to 9 kb, generated by a polymerase moderately sensitive to alpha-amanitin. Transcription is developmentally regulated. Both TRS and RIME sense transcripts are preferentially synthesized compared to anti-sense transcripts, and are much more abundant in bloodstream forms than in cultured procyclics.

The AnTat 1.3A antigen gene expression site of T. brucei was cloned from genomic libraries of the 200 kb expressor chromosome. In addition to the antigen gene, it contains seven putative coding regions (ESAGs, for expression site-associated genes), as well as a RIME retroposon. The polypeptide encoded by ESAG 4 shows homology to yeast adenylate cyclase, and possesses structural features of a transmembrane protein. The expression site is transcribed by a pol l-like polymerase in the parasite bloodstream form only, but sequences similar to ESAGs 5, 4, and 2 are also transcribed constitutively elsewhere, by a polymerase sensitive to alpha-amanitin. Ultraviolet irradiation, which seems to block RNA processing, allows the tentative mapping of a transcription promoter about 45 kb upstream of the antigen gene.

The variant surface glycoprotein (VSG) and procyclin are the respective major surface antigens of the bloodstream and the procyclic forms of Trypanosoma brucei. These proteins and their mRNAs are both the most abundant and absolutely characteristic of their respective life cycle stages. We show that the 3'-terminal region of these mRNAs regulates expression of a reporter gene in an inverse manner, depending on the developmental form of the parasite. In the case of VSG mRNA, the 97 nt sequence upstream from the polyadenylation site is responsible for these effects. The regulation occurs through a variation of mRNA abundance which is not due to a change in primary transcription. In the bloodstream form this effect is manifested by an increase in RNA stability, whereas in the procyclic form it seems to be related to a reduction in the efficiency of mRNA maturation. The 3'-end of VSG mRNA can obviate the 5- to 10-fold stimulation of transcription driven by the procyclin promoter during differentiation from the bloodstream to the procyclic form. The predominance of posttranscriptional over transcriptional controls is probably linked to the organization of the trypanosome genome in polycistronic transcription units.