[Molecular dialogue between African trypanosomes and humans]. / Dialogue moléculaire entre trypanosomes Africains et l'homme.

The evolutionary origin of Man in the African continent has imposed the requirement to resist endemic parasites, in particular African trypanosomes (prototype: Trypanosoma brucei). Therefore, human serum is provided with an efficient system of innate immunity against these parasites, as discovered by A. Laveran in 1902. However, two T. brucei clones, termed T. b. rhodesiense and T. b. gambiense, managed to escape this immunity system, enabling them to grow in humans where they cause sleeping sickness. We have identified the gene allowing T. b. rhodesiense to resist trypanolysis by human serum, which led us to discover that the trypanolytic factor is apolipoprotein L1 (apoL1). ApoL1 is a human-specific serum protein bound to HDL particles that also contain another human-specific protein termed "haptoglobin-related protein " (Hpr). Following the binding of hemoglobin (Hb) to Hpr, the apoL1-bearing HDL particles are avidly taken up by the trypanosome through their binding to a parasite surface receptor for the Hp-Hb complex. After endocytosis apoL1 kills the parasite by generating anionic pores in the lysosomal membrane. In our laboratory, mutant versions of apoL1 have been constructed, which are no longer neutralized by the resistance protein of T. b. rhodesiense and are therefore able to kill this human pathogen. Unexpectedly, we have recently discovered that similar mutants do actually exist in nature : in Africans and Americans of recent African origin, even a single allele of these mutants allows protection against infection by T. b. rhodesiense, but the price to pay is a high frequency of end-stage renal disease when doubly allelic. The evidence of natural selection of these apoL1 mutations despite their deleterious potential for kidneys highlights the importance of the resistance to trypanosomes in the evolution of Man. The mechanism by which mutant apoL1 triggers end-stage renal disease is currently studied.

Specific uptake of tumor necrosis factor-alpha is involved in growth control of Trypanosoma brucei.

Trypanosoma brucei is lysed by tumor necrosis factor-alpha (TNF-alpha) in a dose-dependent way, involving specific binding of the cytokine to a trypanosomal glycoprotein present in the flagellar pocket of the parasite. TNF-alpha-gold particles are endocytosed via coated pits and vesicles and are directed towards lysosome-like digestive organelles. The specific uptake of the cytokine by the parasite results in a developmentally regulated loss of osmoregulatory capacity. TNF-alpha specific lysis is prevented when lysis assays are performed at a temperature <26 degrees C, despite uptake of the cytokine. Inhibition of lysis is also observed when a lysosomotropic agent is added during the first 2 h of incubation. Both monomorphic and pleomorphic trypanosomes are lysed but only when isolated during the peak of parasitaemia. Lysis is not observed with early infection stage parasites or procyclic (insect-specific) forms. Anti-TNF-alpha treatment of T. brucei-infected mice reveals a dramatic increase in parasitaemia in the blood circulation, the spleen, the lymph nodes, and the peritoneal cavity. These data suggest that in the mammalian host, TNF-alpha is involved in the growth control of T. brucei.

The GPI-phospholipase C of Trypanosoma brucei is nonessential but influences parasitemia in mice.

In the mammalian host, the cell surface of Trypanosoma brucei is protected by a variant surface glycoprotein that is anchored in the plasma membrane through covalent attachment of the COOH terminus to a glycosylphosphatidylinositol. The trypanosome also contains a phospholipase C (GPI-PLC) that cleaves this anchor and could thus potentially enable the trypanosome to shed the surface coat of VSG. Indeed, release of the surface VSG can be observed within a few minutes on lysis of trypanosomes in vitro. To investigate whether the ability to cleave the membrane anchor of the VSG is an essential function of the enzyme in vivo, a GPI-PLC null mutant trypanosome has been generated by targeted gene deletion. The mutant trypanosomes are fully viable; they can go through an entire life cycle and maintain a persistent infection in mice. Thus the GPI-PLC is not an essential activity and is not necessary for antigenic variation. However, mice infected with the mutant trypanosomes have a reduced parasitemia and survive longer than those infected with control trypanosomes. This phenotype is partially alleviated when the null mutant is modified to express low levels of GPI-PLC.

N-linked glycans containing linear poly-N-acetyllactosamine as sorting signals in endocytosis in Trypanosoma brucei.

African trypanosomes, such as Trypanosoma brucei, are protozoan parasites that are transmitted by the tsetse fly and cause sleeping sickness in humans and Nagana in cattle. Trypanosomes evade the immune responses of their hosts by varying their surface coat protein (VSG) and restricting exocytosis and endocytosis to an invagination of the plasma membrane called the flagellar pocket (FP). The FP represents only 0.5% of the cellular surface but membrane turnover here occurs at high rates [1] [2] [3]. No model has yet been proposed to account for the sequestration of membrane proteins and the rate of membrane turnover that occur in the FP. Recent data have suggested that glycans are involved in the sorting of membrane proteins in polarized cells [4] [5] [6] [7]. Here, we show that N-linked glycans containing linear poly-N-acetyllactosamine (pNAL) are only associated with proteins of the FP/endocytic pathway in T. brucei and are present only in bloodstream forms of the parasite. These glycoproteins bind to tomato lectin (TL), a property that allowed their single-step isolation. Chito-oligosaccharides that compete specifically for pNAL binding to TL also inhibited receptor-mediated uptake of several ligands. These results suggest a model in which N-linked linear pNAL acts as a sorting signal for endocytosis in trypanosomes.

Transient activity assays of the Trypanosoma brucei variant surface glycoprotein gene promoter: control of gene expression at the posttranscriptional level.

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.

A similar gene is shared by both the variant surface glycoprotein and procyclin gene transcription units of Trypanosoma brucei.

The genes for the variant surface glycoprotein (VSG) and procyclin are expressed in a mutually exclusive manner during the life cycle of Trypanosoma brucei and synthesize the most abundant mRNAs specific to the bloodstream and procyclic stages of the parasite, respectively. Genes belonging to the polycistronic transcription unit of the VSG gene (expression site-associated genes [ESAGs]) are uniquely expressed in the bloodstream form, but some members of ESAG families (genes related to ESAGs [GRESAGs]) are independently transcribed outside the VSG gene expression site. We report here that a gene related to ESAG 2, GRESAG 2.1, is present and expressed in a procyclin gene transcription unit (PARP A locus), which is polycistronic. Members of the ESAG 2 family are thus present in the two major differentially stage-regulated transcription units of this parasite.

Specific binding of proteins to the noncoding strand of a crucial element of the variant surface glycoprotein, procyclin, and ribosomal promoters of trypanosoma 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.

Characterization of a transcription terminator of the procyclin PARP A unit of Trypanosoma brucei.

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.

Trypanosoma brucei: posttranscriptional control of the variable surface glycoprotein gene expression site.

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.

Trypanosoma brucei: enrichment by UV of intergenic transcripts from the variable surface glycoprotein gene expression site.

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.

Structure and transcription of the actin gene of Trypanosoma brucei.

In Trypanosoma brucei, the actin gene is present in a cluster of two, three, or four tandemly linked copies, depending on the strain. Each cluster seems to exist in two allelic versions, as suggested by the polymorphism of both gene number and restriction fragment length in the DNA from cloned trypanosomes. The amplification of the gene copy number probably occurs through unequal sister chromatid exchange. The chromosomes harboring the actin genes belong to the large size class. The coding sequence was 1,128 nucleotides long and showed 60 to 70% homology to other eucaryotic actin genes. Surprisingly, this homology seemed weaker with Trypanosoma congolense, Trypanosoma cruzi, Trypanosoma vivax, Trypanosoma mega, or Leishmania actin-specific sequences. The mRNA was around 1.6 kilobases long and was synthesized at the same level in bloodstream and procyclic forms of the parasite. Large RNA precursors, up to 7.7 kilobases, were found in a pattern identical in strains containing either two or three gene copies. Probing of the flanking regions of the gene with either steady-state or in vitro transcripts, as well as S1 nuclease protection and primer extension experiments, allowed mapping of the 3' splice site of the actin mRNA, 38 nucleotides upstream from the translation initiation codon. A variably sized poly(dT) tract was found about 30 base pairs ahead of the splice site. The largest detected actin mRNA precursor seemed to give rise to at least two additional stable mRNAs. The RNA polymerase transcribing the actin gene exhibited the same sensitivity to inhibition by alpha-amanitin as that transcribing both the spliced leader and the bulk of polyadenylated mRNAs.

Putative genes of a variant-specific antigen gene transcription unit in Trypanosoma brucei.

In a 7-kilobase (kb) sequence upstream from the 5' barren region, the Trypanosoma brucei AnTat 1.3A expression site carries two putative genes, named ESAG 2 and ESAG 3 for expression site-associated genes, as well as a copy of ESAG 1 (D.F. Cully, H.S. Ip, and G.A.M. Cross, Cell 42:173-182, 1985). At least 3 kb of this expression site exhibits a high degree of homology with the silent telomere carrying the AnTat 1.3A basic copy, whose ESAG 1 is interrupted by stop codons. Like the antigen gene, the region containing the ESAGs is transcribed only in the bloodstream forms, although transcription of 5' barren- and ESAG 2-related sequences also occurs in cultured procyclics. Analysis of steady-state and nascent transcripts suggests a continuous transcription of the whole expression site by an RNA polymerase resistant to alpha-amanitin, possibly initiating at a polymerase I-like promoter located about 17 kb upstream from the antigen gene. This polymerase seems prone to becoming inactivated upon incubation of the trypanosomes at low temperature. The putative protein encoded by ESAG 3 may carry a hydrophobic signal peptide, suggesting interaction with a membrane.

A gene from the variant surface glycoprotein expression site encodes one of several transmembrane adenylate cyclases located on the flagellum of Trypanosoma brucei.

The bloodstream form of Trypanosoma brucei contains transcripts of at least four genes showing partial sequence homology to the genes for eucaryotic adenylate and guanylate cyclases (S. Alexandre, P. Paindavoine, P. Tebabi, A. Pays, S. Halleux, M. Steinert, and E. Pays, Mol. Biochem. Parasitol. 43:279-288, 1990). One of these genes, termed ESAG 4, belongs to the polycistronic transcription unit of the variant surface glycoprotein (VSG) gene. Whereas ESAG 4 is transcribed only in the bloodstream form of the parasite, the three other genes, GRESAG 4.1, 4.2, and 4.3, are also expressed in procyclic (insect) forms. These genes differ primarily in a region presumed to encode a large extracellular domain. We show here that ESAG 4-related glycoproteins of about 150 kDa can be found in the trypanosome membrane, that they are detected, by light and electron gold immunocytochemistry, only at the surface of the flagellum, and that the products of at least two of these genes, ESAG 4 and GRESAG 4.1, can complement a Saccharomyces cerevisiae mutant for adenylate cyclase. The recombinant cyclases are associated with the yeast membrane fraction and differ with respect to their activation by calcium: while the GRESAG 4.1 and yeast cyclases are inhibited by calcium, the ESAG 4 cyclase is stimulated. ESAG 4 thus most probably encodes the calcium-activated cyclase that has been found to be expressed only in the bloodstream form of T. brucei (S. Rolin, S. Halleux, J. Van Sande, J. E. Dumont, E. Pays, and M. Steinert. Exp. Parasitol. 71:350-352, 1990). Our data suggest that the trypanosome cyclases are not properly regulated in yeast cells.

A single-stranded DNA-binding protein shared by telomeric repeats, the variant surface glycoprotein transcription promoter and the procyclin transcription terminator of Trypanosoma brucei.

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.

Specificity of chromatin transcription in vitro. Asymmetric transcription of the globin gene by Escherichia coli RNA polymerase.

The transcription of globin genes in mouse foetal liver chromatin and nuclei by exogenous Escherichia coli RNA polymerase is prone to artifacts due to RNA-dependent transcription of endogenous mRNA sequences. This is particularly evident when Mn2+ is used as divalent cation in the RNA transcription reaction. However, substitution or supplementation with Mg2+ eliminates this artifact and gives essentially asymmetric DNA-dependent transcription by the polymerase. In this paper we discuss a number of general criteria which can be applied to test the validity of specific gene transcription in vitro.

Specificity of chromatin transcription in vitro. Anomalies due to RNA-dependent RNA synthesis.

In the presence of Mn2+, globin mRNA can be transcribed into a partial RNA copy by Escherichia coli RNA polymerase. This process also occurs when the mRNA is transcribed together with chromatin. A fraction, at least, of the newly synthesized RNA copy (anti-globin RNA) can serve as a template for the synthesis of globin sequences of the same polarity as the original mRNA. This process is sufficient to explain the specific synthesis of a subset of the globin RNA on mouse foetal liver chromatin. It also accounts for the synthesis of double-stranded RNA sequence by E. coli RNA polymerase, on chromatin as well as on pure mRNA. Results are presented suggesting that the poly(A) tract of the mRNA could be preferentially transcribed. In the presence of Mg2+, the RNA-dependent transcription is strongly inhibited, as well as the synthesis of double-stranded RNA. Under these conditions, the transcription on chromatin appears to be largely DNA dependent, and the synthesis of globin sequences is completely asymmetric. Spermine (0.3 mM) seems to improve the specificity of transcription. The transcription of chromatin in vitro is thus largely dependent on the nature of the divalent cation present in the in the incubation mixture.

DNA-binding fingers encoded by a trypanosome retroposon.

A Trypanosoma brucei repeated DNA element (TRS-1, for trypanosome repeated sequence), which seems transposable, may encode a 1651 amino acid polypeptide showing homology with reverse transcriptase. This polypeptide would also carry a DNA-binding domain, as suggested by the presence of five DNA-binding "fingers" homologous to those of the transcription factor TFIIIA of Xenopus laevis and retroviral DNA-binding proteins.

Trypanosoma brucei repeated element with unusual structural and transcriptional properties.

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.

An update on antigenic variation in African trypanosomes.

African trypanosomes can spend a long time in the blood of their mammalian host, where they are exposed to the immune system and are thought to take advantage of it to modulate their own numbers. Their major immunogenic protein is the variant surface glycoprotein (VSG), the gene for which must be in one of the 20--40 specialized telomeric expression sites in order to be transcribed. Trypanosomes escape antibody-mediated destruction through periodic changes of the expressed VSG gene from a repertoire of approximately 1000. How do trypanosomes exclusively express only one VSG and how do they switch between them?

Isolation of a mitotic-like cyclin homologue from the protozoan Trypanosoma brucei.

Activation of the p34cdc2 protein kinase (PK) at different stages of the eukaryotic cell cycle is controlled by interaction with regulatory proteins known as cyclins (CYCs). Using a probe obtained by PCR amplification, we have isolated from the protozoan, Trypanosoma brucei, a cDNA clone encoding a CYC homologue. The amino acid sequence deduced for this gene (CYC1) shares structural homology with A- and B-type CYCs of other organisms, including a motif, the destruction box, which has been related to the rapid turnover of these CYC proteins in mitosis. When expressed in fission yeast, CYC1 is able to rescue the defect of a temperature-sensitive cdc13 mutant, demonstrating that it is functional as a cell-cycle regulator. In trypanosome cells, CYC1 associates with a 34-kDa protein that cross-reacts with a monoclonal antibody against the conserved 'PSTAIR' epitope of p34cdc2, and the complex displays histone H1 PK activity. Furthermore, when trypanosome cells are synchronized by hydroxyurea treatment, CYC1 accumulates as cells progress towards mitosis. These observations, taken together, suggest that CYC1 is a component of the active PK complex required for the control of trypanosome mitosis.