RESUMEN
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.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Apolipoproteínas/genética , Apolipoproteínas/metabolismo , Haptoglobinas/metabolismo , Lipoproteínas HDL/genética , Lipoproteínas HDL/metabolismo , Mutación , Trypanosoma brucei gambiense/genética , Trypanosoma brucei rhodesiense/genética , Tripanosomiasis Africana/parasitología , Animales , Apolipoproteína L1 , Apolipoproteínas/inmunología , Humanos , Inmunidad Innata , Lipoproteínas HDL/inmunología , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/inmunología , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei gambiense/inmunología , Trypanosoma brucei rhodesiense/inmunología , Tripanosomiasis Africana/inmunologíaRESUMEN
The function of the proteins of the apolipoprotein L (apoL) family is largely unknown. These proteins are classically thought to be involved in lipid transport and metabolism, mainly due to the initial discovery that a secreted member of the family, apoL-I, is associated with high-density lipoprotein particles. However, the other members of the family are believed to be intracellular. The recent unravelling of the mechanism by which apoL-I kills African trypanosomes, as well as the increasing evidence for modulation of apoL expression in various pathological processes, provides new insights about the functions of these proteins. ApoLs share structural and functional similarities with proteins of the Bcl-2 family. Based on the activity of apoL-I in trypanosomes and the comparison with Bcl-2 proteins, we propose that apoLs could function as ion channels of intracellular membranes and be involved in mechanisms triggering programmed cell death.
Asunto(s)
Apolipoproteínas/fisiología , Lipoproteínas HDL/fisiología , Familia de Multigenes , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Apolipoproteína L1 , Apolipoproteínas/genética , Apolipoproteínas/farmacología , Apoptosis , Humanos , Inmunidad Innata , Canales Iónicos/fisiología , Lipoproteínas HDL/genética , Lipoproteínas HDL/farmacología , Lisosomas/fisiología , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas c-bcl-2/genética , Homología de Secuencia de Aminoácido , Trypanosoma brucei brucei/efectos de los fármacosRESUMEN
Procyclins are the major surface glycoproteins of insect-form Trypanosoma brucei. The procyclin expression sites are polycistronic and are transcribed by an alpha-amanitin-resistant polymerase, probably RNA polymerase I (Pol I). The expression sites are flanked by transcription units that are sensitive to alpha-amanitin, which is a hallmark of Pol II-driven transcription. We have analysed a region of 9.5 kb connecting the EP/PAG2 expression site with the downstream transcription unit. The procyclin expression site is longer than was previously realized and contains an additional gene, procyclin-associated gene 4 (PAG4), and a region of unknown function, the T region, that gives rise to trans-spliced, polyadenylated RNAs containing small open reading frames (ORFs). Two new genes, GU1 and GU2, were identified in the downstream transcription unit on the opposite strand. Unexpectedly, the 3' untranslated region of GU2 and the complementary T transcripts overlap by several hundred base pairs. Replacement of GU2 by a unique tag confirmed that sense and antisense transcription occurred from a single chromosomal locus. Overlapping transcription is stage specific and may extend > or = 10 kb in insect-form trypanosomes. The nucleotide composition of the T. brucei genome is such that antisense ORFs occur frequently. If stable mRNAs can be derived from both strands, the coding potential of the genome may be substantially larger than has previously been suspected.
Asunto(s)
ADN sin Sentido/genética , Proteínas Protozoarias/genética , Transcripción Genética , Trypanosoma brucei brucei/genética , Regiones no Traducidas 3' , Animales , Regulación de la Expresión Génica , Glicoproteínas/genética , Proteínas Asociadas a Microtúbulos/genética , Datos de Secuencia Molecular , Mutación , ARN Polimerasa I/genética , ARN Polimerasa I/metabolismo , Análisis de Secuencia de ADNRESUMEN
Trypanosomes use antigenic variation of their variant-specific surface glycoprotein (VSG) coat as defense against the host immune system. However, in order to sustain their growth, they need to expose conserved epitopes, allowing host macromolecule binding and receptor-mediated endocytosis. Here we show that Trypanosoma brucei uses the conserved chitobiose-oligomannose (GlcNAc(2)-Man(5-9)) moieties of its VSG as a binding ligand for tumor necrosis factor (TNF), a host cytokine with lectin-like properties. As endocytosis in trypanosomes is restricted to the flagellar pocket, we show that soluble flagellar pocket extracts, and in particular soluble VSG, inhibit the binding of (125)I-TNF to trypanosomes. The interaction between TNF and VSG is confirmed by affinity chromatography, biosensor, and dot-blot affinity measurements, and soluble VSG inhibition of TNF-mediated trypanolysis. In all approaches, removal of N-linked carbohydrates abrogates the TNF-VSG interaction. In addition, synthetic high mannose oligosaccharides can block TNF-VSG interactions, and a VSG glycopeptide carrying the GlcNAc(2)-Man(5-9) moiety is shown to inhibit TNF-mediated trypanosome killing in mixed parasite/macrophage cell cultures. Together, these results support the observation that TNF plays a role in growth control of trypanosomes and, moreover, suggest that, by the use of conserved VSG carbohydrates as lectin-binding epitopes, trypanosomes can limit the necessity to express large numbers of invariant surface exposed receptors.
Asunto(s)
Citocinas/metabolismo , Flagelos/química , Manosa/química , Glicoproteínas Variantes de Superficie de Trypanosoma/química , Animales , Sitios de Unión , Técnicas Biosensibles , Western Blotting , Secuencia de Carbohidratos , Cromatografía , Técnicas de Cocultivo , Disacáridos/química , Relación Dosis-Respuesta a Droga , Endocitosis , Glicosilación , Immunoblotting , Cinética , Ligandos , Macrófagos , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Unión Proteica , Factores de Tiempo , Trypanosoma brucei brucei , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
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?
Asunto(s)
Variación Antigénica/genética , Trypanosoma/genética , Trypanosoma/inmunología , Glicoproteínas Variantes de Superficie de Trypanosoma/genética , Animales , Conversión Génica , Regulación de la Expresión Génica , Genes Protozoarios , Modelos Genéticos , Recombinación Genética , Transcripción GenéticaRESUMEN
The variant surface glycoprotein (VSG) genes of Trypanosoma brucei are transcribed in telomeric loci termed VSG expression sites (ESs). Despite permanent initiation of transcription in most if not all of these multiple loci, RNA elongation is abortive except in bloodstream forms where full transcription up to the VSG occurs only in a single ES at a time. The ESs active in bloodstream forms are polycistronic and contain several genes in addition to the VSG, named ES-associated genes (ESAGs). So far 12 ESAGs have been identified, some of which are present only in some ESs. Most of these genes encode surface proteins and this list includes different glycosyl phosphatidyl inositol (GPI)-anchored proteins such as the heterodimeric receptor for the host transferrin (ESAG7/6), integral membrane proteins such as the receptor-like transmembrane adenylyl cyclase (ESAG4) and a surface transporter (ESAG10). An interesting exception is ESAG8, which may encode a cell cycle regulator involved in the differentiation of long slender into short stumpy bloodstream forms. Several ESAGs belong to multigene families including pseudogenes and members transcribed out of the ESs, named genes related to ESAGs (GRESAGs). However, some ESAGs (7, 6 and 8) appear to be restricted to the ESs. Most of these genes can be deleted from the active ES without apparently affecting the phenotype of bloodstream form trypanosomes, probably either due to the expression of ESAGs from 'inactive' ESs (ESAG7/6) or due to the expression of GRESAGs (in particular, GRESAGs4 and GRESAGs1). At least three ESAGs (ESAG7, ESAG6 and SRA) share the evolutionary origin of VSGs. The presence of these latter genes in ESs may confer an increased capacity of the parasite for adaptation to various mammalian hosts, as suggested in the case of ESAG7/6 and proven for SRA, which allows T. brucei to infect humans. Similarly, the existence of a collection of slightly different ESAG4s in the multiple ESs might provide the parasite with adenylyl cyclase isoforms that may regulate growth in response to different environmental conditions. The high transcription rate and high recombination level that prevail in VSG ESs may have favored the generation and/or recruitment in these sites of genes whose hyper-evolution allows adaptation to a larger variety of hosts.
Asunto(s)
Interacciones Huésped-Parásitos/fisiología , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/genética , Animales , Genes Protozoarios , Interacciones Huésped-Parásitos/genética , Humanos , Mamíferos , Transcripción Genética , Trypanosoma brucei brucei/fisiología , Tripanosomiasis Africana/parasitologíaRESUMEN
African trypanosomes escape the host immune response through a periodical change of their surface coat made of one major type of protein, the variant surface glycoprotein. From a repertoire of a thousand variant surface glycoprotein genes available, only one is expressed at a time, and this takes place in a specialised expression site itself selected from a collection of an estimated 20-30 sites. As the specialised expression sites are long polycistronic transcription units, the variant surface glycoprotein is co-transcribed with several other genes termed expression site-associated genes. How do the trypanosomes only use a single specialised expression site at a time? Why are there two dozen specialised expression sites? What are the functions of the other genes of these transcription units? We review the currently available answers to these questions.
Asunto(s)
Variación Antigénica/genética , Regulación de la Expresión Génica/fisiología , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/fisiología , Animales , Variación Antigénica/inmunología , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Transcripción Genética/genética , Trypanosoma brucei brucei/inmunología , Trypanosoma brucei brucei/metabolismo , Glicoproteínas Variantes de Superficie de Trypanosoma/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/inmunologíaRESUMEN
African trypanosomes are extracellular parasites causing sleeping sickness to human or nagana to livestock in sub-Saharan Africa. To gain insight into factors governing resistance/susceptibility to these parasites, the immune responses in mice infected with a Trypanosoma brucei phospholipase C null mutant (PLC(-/-)) or its wild type counterpart (WT) were compared. We found that the T. b. brucei mutant inducing a chronic infection triggers the production of type I cytokines during the early stage of infection, followed by the secretion of type II cytokines in the late/chronic phase of the disease. In contrast, WT-infected mice are killed within 5 weeks and remain locked in a type I cytokine response. The type I/type II cytokine balance may influence the development of different subsets of suppressive macrophages, i.e. classically activated macrophages (type I) versus alternatively activated macrophages (type II) that are antagonistically regulated. Therefore, the phenotype and accessory cell function of macrophages elicited during WT and PLC(-/-) T. b. brucei infections were addressed. Results indicate that classically activated macrophages develop in a type I cytokine environment in the early phase of both WT and PLC(-/-) trypanosome infections. In the late stage of infection, only PLC(-/-)-infected mice resisting the infection develop type II cytokine-associated alternative macrophages. In parallel, we found that mice susceptible to Trypanosoma congolense infection, showing an exponential parasite growth until they die, have a higher level of type II cytokines in the early stage of infection than resistant animals controlling the first peak of parasitaemia. The levels of type I cytokines were comparable in both T. congolense-resistant and -susceptible mice. On the basis of these results, we propose that survival to African trypanosome infection requires a type I cytokine environment and classical macrophage activation in the early stage of infection, enabling mice to control the first peak of parasitaemia. Thereafter, a switch to type II cytokine environment triggering alternative macrophage activation is required to enable progression of the disease into the chronic phase. The possible role of the sequential activation of alternative macrophages in the late/chronic stage of infection in the increased resistance of mice to PLC(-/-) T. b. brucei will be discussed.
Asunto(s)
Activación de Macrófagos/fisiología , Macrófagos/inmunología , Trypanosoma brucei brucei/inmunología , Trypanosoma congolense/inmunología , Tripanosomiasis Africana/inmunología , Animales , Anticuerpos Antiprotozoarios/biosíntesis , Anticuerpos Antiprotozoarios/sangre , Secuencia de Carbohidratos , Citocinas/biosíntesis , Citocinas/sangre , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Parasitemia/inmunología , Tripanosomiasis Africana/parasitologíaRESUMEN
Trypanosoma brucei gambiense and T. b. rhodesiense are protozoan parasites causing sleeping sickness in humans due to their resistance to lysis by normal human serum (NHS). Based on the observation that the resistance gene of T. b. rhodesiense encodes a truncated form of the variant specific glycoprotein (VSG), we cloned a similar gene in T. b. gambiense using reverse transcription-linked polymerase chain reaction with VSG-specific primers. This gene, termed TgsGP for T. gambiense-specific glycoprotein, was found to be specific to T. b. gambiense. It is located close to a telomere and is transcribed by a pol II RNA polymerase, only at the bloodstream stage of the parasite development. TgsGP encodes a 47-kDa protein consisting of a N-terminal VSG domain presumably provided with a glycosylphosphatidylinositol (GPI) anchor sequence, similar to the pESAG6 subunit of the trypanosomal transferrin receptor. TgsGP is located in the flagellar pocket, and contains the linear N-linked polyacetyllactosamine characteristic of the endocytotic machinery of T. brucei. These observations strongly suggest that TgsGP is a T. b. gambiense specific receptor. Since stable expression of this protein in T. b. brucei did not confer resistance to NHS, TgsGP may either need another factor to achieve this purpose or fulfils another function linked to adaptation of the parasite to man.
Asunto(s)
Antígenos de Protozoos/genética , Proteínas , Receptores de Transferrina/genética , Trypanosoma brucei gambiense/inmunología , Glicoproteínas Variantes de Superficie de Trypanosoma/genética , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/aislamiento & purificación , Antígenos de Protozoos/metabolismo , Northern Blotting , Resistencia a Medicamentos/genética , Genes Protozoarios , Glicosilfosfatidilinositoles/metabolismo , Humanos , Datos de Secuencia Molecular , Peso Molecular , Proteínas Protozoarias , ARN Polimerasa II/metabolismo , ARN Mensajero/análisis , ARN Protozoario/genética , Receptores de Transferrina/aislamiento & purificación , Receptores de Transferrina/metabolismo , Sensibilidad y Especificidad , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Telómero/genética , Trypanosoma brucei gambiense/citología , Trypanosoma brucei gambiense/efectos de los fármacos , Trypanosoma brucei gambiense/crecimiento & desarrollo , Glicoproteínas Variantes de Superficie de Trypanosoma/aislamiento & purificación , Glicoproteínas Variantes de Superficie de Trypanosoma/metabolismoRESUMEN
The genome of Trypanosoma brucei contains about 120 chromosomes, which do not visibly condense during mitosis. We have analyzed the organization and segregation of these chromosomes by in situ hybridization using fluorescent telomere probes. At the onset of mitosis, telomeres migrate from their nuclear peripheral location and congregate into a central zone. This dense group of telomeres then splits into two entities that migrate to opposite nuclear poles. Segregation continues until the double-sized nucleus divides and, before cytokinesis occurs, the telomeres reorganize into the discrete foci observed at interphase. During migration, the telomeres are located at the free end of the mitotic spindle. Treatment with the microtubule polymerization inhibitor rhizoxin prevents telomere clustering and chromosomal segregation. In the insect-specific procyclic form as well as in the non-dividing bloodstream stumpy form, telomeres tend to cluster close to the nuclear periphery at interphase. In contrast, in the proliferative bloodstream slender form the telomeres preferentially locate in the central zone of the nucleus. Thus, telomeres are closer to the nuclear periphery during those life cycle stages where the telomeric expression sites for the variant surface glycoprotein are all inactive, suggesting that transcriptional inactivation of these sites is related to their subnuclear localization.
Asunto(s)
Ciclo Celular , Estadios del Ciclo de Vida , Telómero/fisiología , Trypanosoma brucei brucei/citología , Trypanosoma brucei brucei/crecimiento & desarrollo , Animales , División Celular , Núcleo Celular/ultraestructura , Segregación Cromosómica/efectos de los fármacos , Interfase , Lactonas/farmacología , Macrólidos , Microtúbulos/ultraestructura , Mitosis , Huso Acromático/ultraestructura , Telómero/ultraestructura , Transcripción Genética , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/genéticaRESUMEN
A purified flagellar pocket fraction of the Trypanosoma brucei AnTat 1.1E clone was used for the generation of polyclonal antiserum in rats. Anti-flagellar pocket antibodies present in this serum recognized several proteins distinct from the major variant surface glycoprotein (VSG). In Balb/c mice, flagellar pocket immunization resulted in partial resistance towards the challenge with a low dose of parasites. This was accompanied by the induction of specific IgG2a antibodies. In an attempt to discover protective parasite antigens, antiflagellar pocket serum was used for the screening of a T. brucei bloodstream form cDNA library constructed in the lambdagt11 bacteriophage expression system. Through antibody panning and VSG elimination, 15 specific cDNA inserts were selected. Most intriguing was the observation that in addition to two clones encoding the invariant surface glycoprotein 75 (ISG75), 10 out of 15 independently selected cDNA inserts encoded the trypanosome heat shock protein 60 (tHSP60).
Asunto(s)
Anticuerpos Antiprotozoarios , Chaperonina 60/inmunología , Flagelos/inmunología , Trypanosoma brucei brucei/inmunología , Tripanosomiasis Africana/prevención & control , Animales , Especificidad de Anticuerpos , Reacciones Cruzadas , Femenino , Expresión Génica , Biblioteca de Genes , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Proteínas Protozoarias/inmunología , Vacunación , Glicoproteínas Variantes de Superficie de Trypanosoma/inmunologíaAsunto(s)
Genes Protozoarios , Glicoproteínas de Membrana/genética , Proteínas Protozoarias/genética , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Clonación Molecular , Expresión Génica , Biblioteca de Genes , Genoma de Protozoos , Glicosilación , Glicoproteínas de Membrana/química , Datos de Secuencia Molecular , Proteínas Protozoarias/química , Seudogenes , ARN Mensajero/genética , ARN Mensajero/metabolismo , Trypanosoma brucei brucei/metabolismoRESUMEN
During the screening of a Trypanosoma brucei brucei (T. b. brucei) cDNA library constructed from bloodstream form mRNA, we identified a 2.3kb cDNA encoding a proteasome beta subunit (ORF1) and a putative zinc finger protein (ORF2). Northern blot analysis indicated the presence of a digenic transcript as well as the two individual messengers in both procyclic and bloodstream forms of the parasite. Southern blot analysis showed the relevant locus to be unique. ORF1 encoded a 22.7kDa protein sharing over 50% identity with the eukaryotic PRCE (aka beta5) proteasome beta subunit. This protein contained a beta amino acid signature and residues involved in the catalytic activity. Further phylogenetic analysis indicated that this subunit as well as those from other kinetoplastids could be confidentially assigned to extant eukaryotic subfamilies such as beta1, beta2, and beta5. ORF2 encoded a 14.6kDa putative zinc finger protein containing five repeats of a CCHC motif commonly present in retroviral nucleocapsid proteins as well as proteins involved in vertebrate embryogenesis.
Asunto(s)
Cisteína Endopeptidasas/genética , ADN Complementario/genética , Complejos Multienzimáticos/genética , Trypanosoma brucei brucei/genética , Dedos de Zinc/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/química , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Filogenia , Complejo de la Endopetidasa Proteasomal , Subunidades de Proteína , ARN Mensajero/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transcripción GenéticaAsunto(s)
Flagelos/química , Glicosilfosfatidilinositoles/metabolismo , Proteínas de la Membrana/análisis , Proteínas Protozoarias/análisis , Trypanosoma brucei brucei/química , Fosfolipasas de Tipo C/metabolismo , Animales , Anticuerpos Antiprotozoarios/inmunología , Membrana Celular/metabolismo , Reacciones Cruzadas , Epítopos/inmunología , Flagelos/inmunología , Glicosilfosfatidilinositoles/genética , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/metabolismo , Conejos , Trypanosoma brucei brucei/crecimiento & desarrollo , Trypanosoma brucei brucei/inmunología , Fosfolipasas de Tipo C/genéticaRESUMEN
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.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Regulación de la Expresión Génica , ARN Protozoario/metabolismo , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/genética , Animales , Núcleo Celular/metabolismo , Variación Genética/genética , Reacción en Cadena de la Polimerasa , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Telómero/genética , Transcripción Genética , Trypanosoma brucei brucei/crecimiento & desarrollo , Glicoproteínas Variantes de Superficie de Trypanosoma/metabolismoRESUMEN
The glycosylphosphatidylinositol-specific phospholipase C or VSG lipase is the enzyme responsible for the cleavage of the glycosylphosphatidylinositol anchor of the variant surface glycoprotein (VSG) and concomitant release of the surface coat in Trypanosoma brucei during osmotic shock or extracellular acidic stress. In Xenopus laevis oocytes the VSG lipase was expressed as a nonacylated and a thioacylated form. This thioacylation occurred within a cluster of three cysteine residues but was not essential for catalytic activity per se. These two forms were also detected in trypanosomes and appeared to be present at roughly equivalent amounts. A reversible shift to the acylated form occurred when cells were triggered to release the VSG by either nonlytic acid stress or osmotic lysis. A wild type VSG lipase or a gene mutated in the three codons for the acylated cysteines were reinserted in the genome of a trypanosome null mutant for this gene. A comparative analysis of these revertant trypanosomes indicated that thioacylation might be involved in regulating enzyme access to the VSG substrate.
Asunto(s)
Cisteína/metabolismo , Trypanosoma brucei brucei/enzimología , Fosfolipasas de Tipo C/metabolismo , Acilación , Animales , Animales Modificados Genéticamente , Electroforesis en Gel de Poliacrilamida , Glicosilfosfatidilinositol Diacilglicerol-Liasa , Mutagénesis Sitio-Dirigida , Ácido Mirístico/metabolismo , Oocitos/metabolismo , Plásmidos , ARN Mensajero/metabolismo , Relación Estructura-Actividad , Compuestos de Sulfhidrilo/metabolismo , Transfección , Trypanosoma brucei brucei/genética , Fosfolipasas de Tipo C/genética , Xenopus laevisRESUMEN
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.
Asunto(s)
Proteínas Protozoarias/genética , Proteínas de Saccharomyces cerevisiae , Trypanosoma brucei brucei/metabolismo , Secuencia de Aminoácidos , Animales , Northern Blotting , Southern Blotting , Núcleo Celular/metabolismo , ADN Protozoario/genética , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Datos de Secuencia Molecular , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/metabolismo , Empalme del ARN , ARN Protozoario/genética , ARN Protozoario/metabolismo , ARN Lider Empalmado/genética , ARN Lider Empalmado/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/genética , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Alineación de Secuencia , Homología de Secuencia , Empalmosomas/metabolismo , Trypanosoma brucei brucei/genéticaRESUMEN
Mechanisms regulating resistance to African trypanosomes were addressed by comparing the immune responses of mice infected with attenuated Trypanosoma brucei brucei lacking the phospholipase C gene (PLC-/-) and those of mice infected with wild-type (WT) parasites. Inhibition of concanavalin A (ConA)-induced T cell proliferation occurred in spleen and lymph nodes of PLC-/-- and WT-infected mice. Although suppressive cells were elicited in spleen and lymph nodes of WT-infected animals, such cells were not detected in lymph nodes of PLC-/--infected mice. PLC-/--infected mice had more interleukin-4 and -10 in their blood than did WT-infected mice. Correspondingly, PLC-/--infected mice had higher IgG1 antibody levels against variant surface glycoprotein than did WT-infected mice. These data indicate that attenuation of T. b. brucei correlates with the absence of cells suppressing ConA-induced T cell proliferation in the lymph nodes, with increased production of Th2 cytokines and a stronger IgG1 antibody response to trypanosome antigens.
Asunto(s)
Citocinas/biosíntesis , Tolerancia Inmunológica , Células Th2/inmunología , Tripanosomiasis Africana/inmunología , Animales , Anticuerpos Antiprotozoarios/biosíntesis , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones SCID , Parasitemia/inmunología , Fosfolipasas de Tipo C/fisiologíaRESUMEN
Heterologous expression in COS cells followed by orientation-specific polymerase chain reaction to select and amplify cDNAs encoding surface proteins in Trypanosoma brucei resulted in the isolation of a cDNA ( approximately 1.4 kilobase) which encodes an acidic, alanine-rich polypeptide that is expressed only in bloodstream forms of the parasite and has been termed bloodstream stage alanine-rich protein (BARP). Analysis of the amino acid sequence predicted the presence of a typical NH(2)-terminal leader sequence as well as a COOH-terminal hydrophobic extension with the potential to be replaced by a glycosylphosphatidylinositol anchor. A search of existing protein sequences revealed partial homology between BARP and the major surface antigen of procyclic forms of Trypanosoma congolense. BARP migrated as a complex, heterogeneous series of bands on Western blots with an apparent molecular mass ( approximately 50-70 kDa) significantly higher than predicted from the amino acid sequence ( approximately 26 kDa). Confocal microscopy demonstrated that BARP was present in small discrete spots that were distributed over the entire cellular surface. Detergent extraction experiments revealed that BARP was recovered in the detergent-insoluble, glycolipid-enriched fraction. These data suggested that BARP may be sequestered in lipid rafts.
Asunto(s)
Proteínas Protozoarias/química , Trypanosoma brucei brucei/química , Secuencia de Aminoácidos , Animales , Células COS , Clonación Molecular , Escherichia coli , Técnica del Anticuerpo Fluorescente , Glucolípidos/química , Proteínas de la Membrana/química , Microscopía Confocal , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Mapeo Restrictivo , Homología de Secuencia de AminoácidoRESUMEN
During Trypanosoma brucei infections, the response against the variant surface glycoprotein (VSG) of the parasite represents a major interaction between the mammalian host immune system and the parasite surface. Since immune recognition of other parasite derived factors also occurs, we examined the humoral host response against trypanosome heat shock protein 60 (HSP60), a conserved antigen with an autoimmune character. During experimental T. brucei infection in BALB/c mice, the anti-HSP60 response was induced when parasites differentiated into stumpy forms. This response was characterized by a stage-specific immunoglobulin isotype switching as well as by the induction of an autoimmune response. Specific recognition of trypanosome HSP60 was found to occur during the entire course of infection. Immunoglobulin G2a (IgG2a) and IgG2b antibodies, induced mainly in a T-cell-independent manner, were observed during the first peak of parasitemia, whereas IgG1 and IgG3 antibodies were found at the end of the infection, due to a specific T-cell-mediated response. Comparative analysis of the kinetics of anti-HSP60, anti-invariant surface glycoprotein 70 (ISG70), and anti-VSG antibody responses indicated that the three trypanosome antigens give rise to specific and independent patterns of immunoglobulin isotype switching.