RESUMEN
The protein density in biological membranes can be extraordinarily high, but the impact of molecular crowding on the diffusion of membrane proteins has not been studied systematically in a natural system. The diversity of the membrane proteome of most cells may preclude systematic studies. African trypanosomes, however, feature a uniform surface coat that is dominated by a single type of variant surface glycoprotein (VSG). Here we study the density-dependence of the diffusion of different glycosylphosphatidylinositol-anchored VSG-types on living cells and in artificial membranes. Our results suggest that a specific molecular crowding threshold (MCT) limits diffusion and hence affects protein function. Obstacles in the form of heterologous proteins compromise the diffusion coefficient and the MCT. The trypanosome VSG-coat operates very close to its MCT. Importantly, our experiments show that N-linked glycans act as molecular insulators that reduce retarding intermolecular interactions allowing membrane proteins to function correctly even when densely packed.
Asunto(s)
Glicoproteínas Variantes de Superficie de Trypanosoma/fisiología , Glicosilación , Glicosilfosfatidilinositoles/metabolismo , TrypanosomaRESUMEN
Human African trypanosomiasis (sleeping sickness) is a neglected tropical disease caused by Trypanosoma brucei spp. The parasites are transmitted by tsetse flies and adapt to their different hosts and environments by undergoing a series of developmental changes. During differentiation, the trypanosome alters its protein coat. Bloodstream form trypanosomes in humans have a coat of variant surface glycoprotein (VSG) that shields them from the immune system. The procyclic form, the first life-cycle stage to develop in the tsetse fly, replaces the VSG coat by procyclins; these proteins do not protect the parasite from lysis by serum components. Our study exploits the parasite-specific process of differentiation from bloodstream to procyclic forms to screen for potential drug candidates. Using transgenic trypanosomes with a reporter gene in a procyclin locus, we established a whole-cell assay for differentiation in a medium-throughput format. We screened 7,495 drug-like compounds and identified 28 hits that induced expression of the reporter and loss of VSG at concentrations in the low micromolar range. Small molecules that induce differentiation to procyclic forms could facilitate studies on the regulation of differentiation as well as serving as scaffolds for medicinal chemistry for new treatments for sleeping sickness.
Asunto(s)
Antiprotozoarios/farmacología , Diferenciación Celular/efectos de los fármacos , Proteínas Protozoarias/fisiología , Trypanosoma brucei brucei/efectos de los fármacos , Animales , Animales Modificados Genéticamente , Glucuronidasa/genética , Humanos , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/fisiologíaRESUMEN
Many cases of infection caused by the oral transmission of Trypanosoma cruzi have been reported during the last decade. These have been due to the contamination of food by faeces from sylvatic triatomines or by leakage from reservoirs in areas where domiciliated vectors have been controlled or where there has been no prior background of domiciliation. The United Nations Food and Agriculture Organization (FAO) and the World Health Organization (WHO) have used epidemiological, clinical and socioeconomic criteria for ranking parasites transmitted by the contamination of food in different areas of the world; T. cruzi was placed tenth in importance amongst a group of 24 parasites in such ranking. Environmental changes such as deforestation and global warming have affected ecotopes and the behaviour of T. cruzi vectors and reservoirs so that these have become displaced to new areas, thereby leading to such new transmission scenario caused by the contamination of food, which requires evaluation in Colombia. The current review deals with the oral transmission of Chagas' disease, emphasising studies aimed at identifying the pertinent risk factors, the triatomine species involved, the physiopathology of oral infection, the parasite's genotypes implicated in this type of transmission in Colombia and other Latin American regions, as well as the need for ongoing epidemiological surveillance and control policies.
Asunto(s)
Enfermedad de Chagas/transmisión , Heces/parasitología , Parasitología de Alimentos , Frutas/parasitología , Insectos Vectores/parasitología , Carne/parasitología , Rhodnius/parasitología , Trypanosoma cruzi/aislamiento & purificación , Verduras/parasitología , Animales , Animales Salvajes/parasitología , Armadillos/parasitología , Bebidas/parasitología , Donantes de Sangre , Enfermedad de Chagas/congénito , Enfermedad de Chagas/epidemiología , Enfermedad de Chagas/parasitología , Colombia , Reservorios de Enfermedades/parasitología , Femenino , Mucosa Gástrica/parasitología , Genotipo , Vivienda , Humanos , Mucosa Bucal/parasitología , Parasitemia/parasitología , Parasitemia/transmisión , Péptido Hidrolasas/fisiología , Embarazo , Complicaciones Infecciosas del Embarazo/parasitología , Proteínas Protozoarias/química , Proteínas Protozoarias/fisiología , Factores de Riesgo , Reacción a la Transfusión , Trypanosoma cruzi/genética , Trypanosoma cruzi/patogenicidad , Trypanosoma cruzi/fisiología , Glicoproteínas Variantes de Superficie de Trypanosoma/química , Glicoproteínas Variantes de Superficie de Trypanosoma/fisiologíaRESUMEN
Durante la última década se han reportado numerosos casos de infección por Trypanosoma cruzi por vía oral, debidos a la contaminación de alimentos con heces de triatominos silvestres o con secreciones de reservorios en áreas donde los vectores domiciliados han sido controlados o no hay antecedentes de domiciliación. Con base en criterios epidemiológicos, clínicos y socioeconómicos, la Organización de las Naciones Unidas para la Agricultura y la Alimentación (FAO) y la Organización Mundial de la Salud (OMS) establecieron una clasificación de los parásitos transmitidos por contaminación de alimentos en diferentes regiones del mundo, en la cual T. cruzi ocupó el décimo lugar de importancia en un grupo de 24 parásitos. Los cambios ambientales, como la deforestación y el calentamiento global, han afectado los ecotopos y el comportamiento de los vectores y de los reservorios de T. cruzi , de manera que estos se han desplazado a nuevas zonas, generando una nueva forma de transmisión por contaminación de alimentos que requiere su evaluación en el país. La presente revisión aborda la transmisión oral de la enfermedad de Chagas con énfasis en los estudios orientados a identificar los factores de riesgo, las especies de triatominos involucrados, la fisiopatología de la infección oral y los genotipos del parásito que están implicados en esta forma de transmisión en Colombia y en otras regiones de América Latina, así como la necesidad de adoptar políticas para su control y vigilancia epidemiológica.
Many cases of infection caused by the oral transmission of Trypanosoma cruzi have been reported during the last decade. These have been due to the contamination of food by faeces from sylvatic triatomines or by leakage from reservoirs in areas where domiciliated vectors have been controlled or where there has been no prior background of domiciliation. The United Nations Food and Agriculture Organization (FAO) and the World Health Organization (WHO) have used epidemiological, clinical and socioeconomic criteria for ranking parasites transmitted by the contamination of food in different areas of the world; T. cruzi was placed tenth in importance amongst a group of 24 parasites in such ranking. Environmental changes such as deforestation and global warming have affected ecotopes and the behaviour of T. cruzi vectors and reservoirs so that these have become displaced to new areas, thereby leading to such new transmission scenario caused by the contamination of food, which requires evaluation in Colombia. The current review deals with the oral transmission of Chagas´ disease, emphasising studies aimed at identifying the pertinent risk factors, the triatomine species involved, the physiopathology of oral infection, the parasite´s genotypes implicated in this type of transmission in Colombia and other Latin American regions, as well as the need for ongoing epidemiological surveillance and control policies.
Asunto(s)
Animales , Femenino , Humanos , Embarazo , Enfermedad de Chagas/transmisión , Parasitología de Alimentos , Heces/parasitología , Frutas/parasitología , Insectos Vectores/parasitología , Carne/parasitología , Rhodnius/parasitología , Trypanosoma cruzi/aislamiento & purificación , Verduras/parasitología , Animales Salvajes/parasitología , Armadillos/parasitología , Donantes de Sangre , Bebidas/parasitología , Transfusión Sanguínea/efectos adversos , Colombia , Enfermedad de Chagas/congénito , Enfermedad de Chagas/epidemiología , Enfermedad de Chagas/parasitología , Reservorios de Enfermedades/parasitología , Genotipo , Mucosa Gástrica/parasitología , Vivienda , Mucosa Bucal/parasitología , Parasitemia/parasitología , Parasitemia/transmisión , Péptido Hidrolasas/fisiología , Complicaciones Infecciosas del Embarazo/parasitología , Proteínas Protozoarias/química , Proteínas Protozoarias/fisiología , Factores de Riesgo , Trypanosoma cruzi/genética , Trypanosoma cruzi/patogenicidad , Trypanosoma cruzi/fisiología , Glicoproteínas Variantes de Superficie de Trypanosoma/química , Glicoproteínas Variantes de Superficie de Trypanosoma/fisiologíaAsunto(s)
Trypanosoma brucei gambiense/fisiología , Tripanosomiasis Africana/inmunología , Tripanosomiasis Africana/parasitología , Adenilil Ciclasas/inmunología , Adenilil Ciclasas/metabolismo , Adenilil Ciclasas/fisiología , Animales , Humanos , Sistema Inmunológico/fisiología , Modelos Biológicos , Trypanosoma brucei gambiense/inmunología , Trypanosoma brucei gambiense/patogenicidad , Glicoproteínas Variantes de Superficie de Trypanosoma/inmunología , Glicoproteínas Variantes de Superficie de Trypanosoma/fisiologíaRESUMEN
TSSA (trypomastigote small surface antigen) is a polymorphic mucin-like molecule displayed on the surface of Trypanosoma cruzi trypomastigote forms. To evaluate its functional properties, we undertook comparative biochemical and genetic approaches on isoforms present in parasite stocks from extant evolutionary lineages (CL Brener and Sylvio X-10). We show that CL Brener TSSA, but not the Sylvio X-10 counterpart, exhibits dose-dependent and saturable binding towards non-macrophagic cell lines. This binding triggers Ca(2+)-based signalling responses in the target cell while providing an anchor for the invading parasite. Accordingly, exogenous addition of either TSSA-derived peptides or specific antibodies significantly inhibits invasion of CL Brener, but not Sylvio X-10, trypomastigotes. Non-infective epimastigote forms, which do not express detectable levels of TSSA, were stably transfected with TSSA cDNA from either parasite stock. Although both transfectants produced a surface-associated mucin-like TSSA product, epimastigotes expressing CL Brener TSSA showed a ~2-fold increase in their attachment to mammalian cells. Overall, these findings indicate that CL Brener TSSA functions as a parasite adhesin, engaging surface receptor(s) and inducing signalling pathways on the host cell as a prerequisite for parasite internalization. More importantly, the contrasting functional features of TSSA isoforms provide one appealing mechanism underlying the differential infectivity of T. cruzi stocks.
Asunto(s)
Trypanosoma cruzi/patogenicidad , Glicoproteínas Variantes de Superficie de Trypanosoma/fisiología , Secuencia de Aminoácidos , Animales , Chlorocebus aethiops , Células HEK293 , Humanos , Datos de Secuencia Molecular , Unión Proteica/fisiología , Glicoproteínas Variantes de Superficie de Trypanosoma/metabolismo , Células VeroRESUMEN
Frequent reports on outbreaks of acute Chagas' disease by ingestion of food contaminated with parasites from triatomine insects illustrate the importance of this mode of transmission. Studies on oral Trypanosoma cruzi infection in mice have indicated that metacyclic trypomastigotes invade the gastric mucosal epithelium. A key molecule in this process is gp82, a stage-specific surface glycoprotein that binds to both gastric mucin and to target epithelial cells. By triggering Ca2+ signalling, gp82 promotes parasite internalisation. Gp82 is relatively resistant to peptic digestion at acidic pH, thus preserving the properties critical for oral infection. The infection process is also influenced by gp90, a metacyclic stage-specific molecule that negatively regulates the invasion process. T. cruzi strains expressing high gp90 levels invade cells poorly in vitro. However, their infectivity by oral route varies considerably due to varying susceptibilities of different gp90 isoforms to peptic digestion. Parasites expressing pepsin-susceptible gp90 become highly invasive against target cells upon contact with gastric juice. Such is the case of a T. cruzi isolate from an acute case of orally acquired Chagas' disease; the gp90 from this strain is extensively degraded upon short period of parasite permanence in the gastric milieu. If such an exacerbation of infectivity occurs in humans, it may be responsible for the severity of Chagas' disease reported in outbreaks of oral infection.
Asunto(s)
Enfermedad de Chagas/transmisión , Mucosa Gástrica/parasitología , Proteínas Protozoarias/fisiología , Trypanosoma cruzi/fisiología , Glicoproteínas Variantes de Superficie de Trypanosoma/fisiología , Animales , Enfermedad de Chagas/parasitología , Células Epiteliales/parasitología , Parasitología de Alimentos , Humanos , Insectos Vectores/parasitología , Ratones , Trypanosoma cruzi/patogenicidadRESUMEN
Th1 cell responses to the variant surface glycoprotein (VSG) of African trypanosomes play a critical role in controlling infection through the production of IFN-gamma, but the role of APCs in the induction and regulation of T cell-mediated protection is poorly understood. In this study, we have investigated the Ag presentation capabilities of dendritic cells (DCs) and macrophages during early trypanosome infection in relatively resistant responder and susceptible nonresponder mouse strains. Splenic DCs appeared to be the primary cell responsible for activating naive VSG-specific Th cell responses in resistant responder animals through the coordinated up-regulation of costimulatory molecules, secretion of IL-12, and presentation of VSG peptides to T cells in vivo. Splenic DC depletion and the down-regulation of costimulatory markers on splenic macrophages were observed in susceptible animals and may be associated with the inability of these animals to elicit a significant VSG-specific T cell response. In contrast to splenic APCs, peritoneal macrophages secreted NO, failed to activate naive Th cells in vitro, and presented relatively low levels of VSG peptides to T cells in vivo. Thus, VSG-specific Th1 cell responses may be determined by tissue- and cell-specific differences in Ag presentation. Additionally, all APCs from resistant and susceptible strains displayed a reduced ability to process and present newly encountered exogenous Ag, including new VSG molecules, during high parasitemia. Thus, initial uptake of VSG (or other trypanosome factors) may interfere with Ag presentation and have dramatic consequences for subsequent T cell responses to other proteins.
Asunto(s)
Presentación de Antígeno/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/parasitología , Trypanosoma brucei rhodesiense/inmunología , Tripanosomiasis Africana/inmunología , Glicoproteínas Variantes de Superficie de Trypanosoma/metabolismo , Animales , Presentación de Antígeno/genética , Membrana Celular/inmunología , Membrana Celular/metabolismo , Membrana Celular/parasitología , Citocinas/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/parasitología , Células Dendríticas/patología , Femenino , Predisposición Genética a la Enfermedad , Antígenos de Histocompatibilidad Clase II/biosíntesis , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Inmunofenotipificación , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/parasitología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Subgrupos de Linfocitos T/metabolismo , Células TH1/inmunología , Células TH1/metabolismo , Células TH1/parasitología , Tripanosomiasis Africana/parasitología , Tripanosomiasis Africana/patología , Glicoproteínas Variantes de Superficie de Trypanosoma/biosíntesis , Glicoproteínas Variantes de Superficie de Trypanosoma/fisiologíaRESUMEN
Frequent reports on outbreaks of acute Chagas' disease by ingestion of food contaminated with parasites from triatomine insects illustrate the importance of this mode of transmission. Studies on oral Trypanosoma cruzi infection in mice have indicated that metacyclic trypomastigotes invade the gastric mucosal epithelium. A key molecule in this process is gp82, a stage-specific surface glycoprotein that binds to both gastric mucin and to target epithelial cells. By triggering Ca2+ signalling, gp82 promotes parasite internalisation. Gp82 is relatively resistant to peptic digestion at acidic pH, thus preserving the properties critical for oral infection. The infection process is also influenced by gp90, a metacyclic stage-specific molecule that negatively regulates the invasion process. T. cruzi strains expressing high gp90 levels invade cells poorly in vitro. However, their infectivity by oral route varies considerably due to varying susceptibilities of different gp90 isoforms to peptic digestion. Parasites expressing pepsin-susceptible gp90 become highly invasive against target cells upon contact with gastric juice. Such is the case of a T. cruzi isolate from an acute case of orally acquired Chagas' disease; the gp90 from this strain is extensively degraded upon short period of parasite permanence in the gastric milieu. If such an exacerbation of infectivity occurs in humans, it may be responsible for the severity of Chagas' disease reported in outbreaks of oral infection.
Asunto(s)
Animales , Humanos , Ratones , Enfermedad de Chagas/transmisión , Mucosa Gástrica/parasitología , Proteínas Protozoarias/fisiología , Trypanosoma cruzi/fisiología , Glicoproteínas Variantes de Superficie de Trypanosoma/fisiología , Enfermedad de Chagas/parasitología , Células Epiteliales/parasitología , Parasitología de Alimentos , Insectos Vectores/parasitología , Trypanosoma cruzi/patogenicidadRESUMEN
Macrophages express a spectrum of proinflammatory and regulatory mediators during African trypanosomiasis. Microarray analyses revealed similar profiles of induced genes in macrophages stimulated with the trypanosome soluble variant surface glycoprotein in vitro and in macrophages taken from infected mice. Genes associated with the acute phase response and with type I IFN responses were prominent components of the macrophage activation profiles expressed within 72 h in vitro and in vivo. Thus, induction of proinflammatory gene expression is a characteristic of early trypanosome infection that is driven primarily by soluble variant surface glycoprotein exposure, and it may be that IFN-alpha/beta plays a central role in regulation of early resistance to trypanosomes. To test this hypothesis, we assessed parameters of infection in mouse strains with genetic alterations in the IFN-alpha/beta response pathway. We found that Ifnar1(-/-) mice, which lack the receptor for type I IFNs, exhibited delayed control of parasite burden during the first week of infection and died earlier than did wild-type controls. However, infection of Ubp43(-/-) mice, which are hyperresponsive to type I IFNs, did not exhibit enhanced resistance to trypanosomes. Instead, these animals also failed to control parasite burden and were more susceptible than wild-type animals. Additionally, the Ubp43(-/-) mice exhibited a significant defect in IFN-gamma production, which is definitively linked to host resistance in trypanosomiasis. These results show that type I IFNs play a role in early control of parasites in infected mice but may contribute to down-regulation of IFN-gamma production and subsequent loss of host resistance later in infection.
Asunto(s)
Inmunidad Innata , Interferón Tipo I/fisiología , Trypanosoma brucei gambiense/inmunología , Tripanosomiasis Africana/inmunología , Animales , Línea Celular , Susceptibilidad a Enfermedades/inmunología , Femenino , Perfilación de la Expresión Génica , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/metabolismo , Interferón Tipo I/biosíntesis , Interferón Tipo I/genética , Interferón beta/genética , Interferón beta/metabolismo , Interferón gamma/biosíntesis , Interferón gamma/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de Señal/inmunología , Factores de Tiempo , Tripanosomiasis Africana/parasitología , Glicoproteínas Variantes de Superficie de Trypanosoma/fisiologíaRESUMEN
The GPI residues of soluble variant surface glycoprotein (sVSG) molecules released from the membrane of African trypanosomes during infection induce macrophage activation events. In this study, we demonstrate that the trypanosome sVSG molecule binds to the membrane of murine RAW 264.7 macrophages and activates the NF-kappaB cascade independently of a TLR-mediated interaction. The binding of fluorochrome-labeled sVSG molecules to macrophage membranes was saturable, was inhibited by the scavenger receptor-specific ligand maleylated BSA, and was followed by rapid intracellular uptake of the molecules and subsequent internalization to lysosomal compartments. Inhibition of cellular phagocytic and endocytic uptake processes by cytochalasin B and monodansylcadaverine, respectively, revealed that sVSG internalization was necessary for IkappaBalpha degradation and occurred by an actin-dependent, clathrin-independent process. Activation of RAW 264.7 cells by sVSG following treatment of the cells with the TRAF6 inhibitory peptide DIVK resulted in enhanced NF-kappaB signaling, suggesting both that TRAF6-dependent TLR activation of the pathway alone is not required for signaling and that TLR pathway components may negatively regulate expression of sVSG-induced signaling. These results demonstrate that stimulation of macrophages by sVSG involves a complex process of receptor-mediated binding and uptake steps, leading to both positive and negative signaling events that ultimately regulate cellular activation.
Asunto(s)
Proteínas I-kappa B/metabolismo , Macrófagos Peritoneales/metabolismo , Receptores Depuradores/metabolismo , Transducción de Señal/inmunología , Factor 6 Asociado a Receptor de TNF/fisiología , Receptores Toll-Like/fisiología , Trypanosoma brucei rhodesiense , Glicoproteínas Variantes de Superficie de Trypanosoma/metabolismo , Actinas/fisiología , Animales , Línea Celular , Clatrina/fisiología , Femenino , Activación de Macrófagos , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/parasitología , Ratones , Ratones Endogámicos BALB C , Inhibidor NF-kappaB alfa , Fagocitosis/inmunología , Solubilidad , Glicoproteínas Variantes de Superficie de Trypanosoma/fisiologíaRESUMEN
African trypanosomes show monoallelic expression of one of about 20 telomeric variant surface glycoprotein (VSG) gene-expression sites (ESs) while multiplying in the mammalian bloodstream. We screened for genes involved in ES silencing using flow cytometry and RNA interference (RNAi). We show that a novel member of the ISWI family of SWI2/SNF2-related chromatin-remodelling proteins (TbISWI) is involved in ES downregulation in Trypanosoma brucei. TbISWI has an atypical protein architecture for an ISWI, as it lacks characteristic SANT domains. Depletion of TbISWI by RNAi leads to 30-60-fold derepression of ESs in bloodstream-form T. brucei, and 10-17-fold derepression in insect form T. brucei. We show that although blocking synthesis of TbISWI leads to derepression of silent VSG ES promoters, this does not lead to fully processive transcription of silent ESs, or an increase in ES-activation rates. VSG ES activation in African trypanosomes therefore appears to be a multistep process, whereby an increase in transcription from a silent ES promoter is necessary but not sufficient for full ES activation.
Asunto(s)
Adenosina Trifosfatasas/fisiología , Factores de Transcripción/fisiología , Trypanosoma brucei brucei/fisiología , Glicoproteínas Variantes de Superficie de Trypanosoma/fisiología , Alelos , Secuencia de Aminoácidos , Animales , Núcleo Celular/metabolismo , Regulación hacia Abajo , Silenciador del Gen , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Interferencia de ARN , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/biosíntesis , Glicoproteínas Variantes de Superficie de Trypanosoma/genéticaRESUMEN
Establishment of infection by Trypanosoma cruzi, the agent of Chagas' disease, depends on a series of events involving interactions of diverse parasite molecules with host components. Here we focus on the mechanisms of target cell invasion by metacyclic trypomastigotes (MT) and mammalian tissue culture trypomastigotes (TCT). During MT or TCT internalization, signal transduction pathways are activated both in the parasite and the target cell, leading to Ca2+ mobilization. For cell adhesion, MT engage surface glycoproteins, such as gp82 and gp35/50, which are Ca2+ signal-inducing molecules. In T. cruzi isolates that enter host cells in gp82-mediated manner, parasite protein tyrosine kinase as well as phospholipase C are activated, and Ca2+ is released from I P3-sensitive stores, whereas in T. cruzi isolates that attach to target cells mainly through gp35/50, the signaling pathway involving adenylate cyclase appears to be stimulated, with Ca2+ release from acidocalciosomes. In addition, T. cruzi isolate-dependent inhibitory signals, mediated by MT-specific gp90, may be triggered both in the host cell and the parasite. The repertoire of TCT molecules implicated in cell invasion includes surface glycoproteins of gp85 family, with members containing binding sites for laminin and cytokeratin 18, enzymes such as cruzipain, trans-sialidase, and an oligopeptidase B that generates a Ca2+-agonist from a precursor molecule.
Asunto(s)
Señalización del Calcio/fisiología , Células Cultivadas/parasitología , Transducción de Señal/fisiología , Trypanosoma cruzi/patogenicidad , Glicoproteínas Variantes de Superficie de Trypanosoma/fisiología , Animales , Adhesión Celular/fisiología , Interacciones Huésped-Parásitos/genética , Interacciones Huésped-Parásitos/fisiología , Humanos , Factores de Tiempo , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , Glicoproteínas Variantes de Superficie de Trypanosoma/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/metabolismoRESUMEN
O estabelecimento da infecção por Trypanosoma cruzi, o agente da doença de Chagas, depende de uma série de eventos envolvendo interações de diversas moléculas do parasita com componentes do hospedeiro. Focalizamos aqui os mecanismos de invasão celular por tripomastigotas metacíclicos (TM) e por tripomastigotas de cultura de tecido (TCT). Durante a internalização de TM ou TCT, vias de transdução de sinal são ativadas tanto no parasita como na célula alvo, acarretando a mobilização de Ca2+. Para adesão, TM utiliza as glicoproteínas de superfície como a gp82 e gp35/50, que são moléculas indutoras de sinal de Ca2+. Em isolados de T. cruzi que entram na célula hospedeira de maneira dependente de gp82, a proteína tirosina quinase assim como a fosfolipase C do parasita são ativadas, e Ca2+ é liberado de reservatórios sensíveis a IP3, enquanto em isolados de T. cruzi que se ligam às células alvo através de gp35/50, a via de sinalização envolvendo adenilil ciclase parece ser estimulada, com liberação de Ca2+ de acidocalciossomos. Além disso, dependendo do isolado de T. cruzi, sinais inibitórios mediados por gp90 específica de TM podem ser desencadeados tanto na célula hospedeira como no parasita. O repertório de moléculas de TCT implicadas na invasão celular inclui glicoproteínas de superfície da família gp85, com membros contendo sitos de ligação à laminina e citoqueratina 18, enzimas como a cruzipaína, trans-sialidase, e uma oligopeptidase B que gera um agonista de Ca2+ a partir de uma molécula precursora. .
Asunto(s)
Animales , Humanos , Señalización del Calcio/fisiología , Células Cultivadas/parasitología , Transducción de Señal/fisiología , Trypanosoma cruzi/patogenicidad , Glicoproteínas Variantes de Superficie de Trypanosoma/fisiología , Adhesión Celular/fisiología , Interacciones Huésped-Parásitos/genética , Interacciones Huésped-Parásitos/fisiología , Factores de Tiempo , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , Glicoproteínas Variantes de Superficie de Trypanosoma/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/metabolismoRESUMEN
Transmission of a protozoan parasite from a vertebrate to invertebrate host is accompanied by cellular differentiation. The signals from the environment that trigger the process are poorly understood. The model parasite Trypanosoma brucei proliferates in the mammalian bloodstream and in the tsetse fly. On ingestion by the tsetse, the trypanosome undergoes a rapid differentiation that is marked by replacement of the variant surface glycoprotein (VSG) coat with GPI-anchored EP and GPEET procyclins. Here we show that a cold shock of DeltaT > 15 degrees C is sufficient to reversibly induce high-level expression of the insect stage-specific EP gene in the mammalian bloodstream stages of T. brucei. The 3'-UTR of the EP mRNA is necessary and sufficient for the increased expression. During cold shock, EP protein accumulates in the endosomal compartment in the proliferating, slender, bloodstream stage, whereas the EP is present on the plasma membrane in the quiescent, stumpy, bloodstream stage. Thus, there is a novel developmentally regulated cell surface access control mechanism for a GPI-anchored protein. In addition to inducing EP expression, cold shock results in the acquisition of sensitivity to micromolar concentrations of cis-aconitate and citrate by stumpy but not slender bloodstream forms. The cis-aconitate and citrate commit stumpy bloodstream cells to differentiation to the procyclic stage along with rapid initial proliferation. We propose a hierarchical model of three events that regulate differentiation after transmission to the tsetse: sensing the temperature change, surface access of a putative receptor, and sensing of a chemical cue.
Asunto(s)
Diferenciación Celular/fisiología , Regulación del Desarrollo de la Expresión Génica , Glicoproteínas de Membrana/metabolismo , Trypanosoma brucei brucei/citología , Trypanosoma brucei brucei/crecimiento & desarrollo , Moscas Tse-Tse/parasitología , Glicoproteínas Variantes de Superficie de Trypanosoma/fisiología , Regiones no Traducidas 3' , Ácido Aconítico/farmacología , Animales , Membrana Celular/metabolismo , Quelantes/farmacología , Ácido Cítrico/farmacología , Frío , Endocitosis/efectos de los fármacos , Endocitosis/fisiología , Endosomas/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Glicoproteínas de Membrana/genética , Transporte de Proteínas , Proteínas Protozoarias/metabolismo , Choque , Sensación Térmica , Trypanosoma brucei brucei/genética , Tripanosomiasis Africana/parasitología , Moscas Tse-Tse/anatomía & histologíaRESUMEN
The Trypanosoma brucei reference strain TREU927/4 exhibits some resistance to lysis by normal human serum (NHS), but this resistance is never complete even after selection. The genome of this strain contains a minimum of eight sequences related to the T. brucei rhodesiense-specific serum resistance-associated gene (SRA), which encodes a truncated variant surface glycoprotein (VSG) conferring full resistance to lysis by NHS. We selected two sequences showing the highest similarity to SRA and also preceded by a region ("cotransposed region") present immediately upstream from SRA in the VSG expression site termed R-ES, where SRA is expressed in T. brucei rhodesiense. Whereas one of these sequences appears to be a pseudogene, the other, which is contained within a cluster of VSG basic copies (BCs), encodes a VSG truncated in the C-terminal domain. In the latter gene, an inserted region encoding surface-exposed loops similar to those of the BoTat 1.20 VSG interrupts the full SRA sequence. Therefore, this gene was termed SRA-BC, for the putative VSG basic copy from which SRA was derived. Neither this gene nor other SRA-like sequences appeared to be responsible for the relative resistance of TREU927/4 to NHS, since (i) transfection of SRA-BC in T. brucei brucei did not confer increased resistance; (ii) SRA transcripts could not be detected in this strain, even when focusing the search on the limited SRA sequence necessary to confer resistance and when using strain-specific SRA probes on RNA from cells selected in the presence of NHS.
Asunto(s)
Glicoproteínas de Membrana/genética , Proteínas Protozoarias/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/patogenicidad , Trypanosoma brucei rhodesiense/genética , Trypanosoma brucei rhodesiense/patogenicidad , Secuencia de Aminoácidos , Animales , Bovinos , Secuencia Conservada , Orden Génico , Genes Protozoarios , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/fisiología , Datos de Secuencia Molecular , Familia de Multigenes , Fenotipo , Señales de Clasificación de Proteína , Proteínas Protozoarias/química , Proteínas Protozoarias/fisiología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Suero , Trypanosoma brucei brucei/crecimiento & desarrollo , Trypanosoma brucei rhodesiense/crecimiento & desarrollo , Glicoproteínas Variantes de Superficie de Trypanosoma/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/fisiologíaRESUMEN
Macrophages are centrally involved in the host immune response to infection with Trypanosoma brucei rhodesiense, a protozoan parasite responsible for human sleeping sickness in Africa. During trypanosome infections, the host is exposed to parasite-derived molecules that mediate macrophage activation, specifically GPI anchor substituents associated with the shed variant surface glycoprotein (VSG), plus the host-activating agent IFN-gamma, which is derived from activated T cells and is essential for resistance to trypanosomes. In this study, we demonstrate that the level and timing of exposure of macrophages to IFN-gamma vs GPI ultimately determine the macrophage response at the level of induced gene expression. Treatment of macrophages with IFN-gamma followed by GIP-sVSG (the soluble form of VSG containing the glycosylinositolphosphate substituent that is released by parasites) stimulated the induction of gene expression, including transcription of TNF-alpha, IL-6, GM-CSF, and IL-12p40. In contrast, treatment of macrophages with GIP-sVSG before IFN-gamma stimulation resulted in a marked reduction of IFN-gamma-induced responses, including transcription of inducible NO synthase and secretion of NO. Additional experiments revealed that the inhibitory activity of GIP-sVSG was associated with reduction in the level of STAT1 phosphorylation, an event required for IFN-gamma-induced macrophage activation. These results suggest that modulation of specific aspects of the IFN-gamma response may be one mechanism by which trypanosomes overcome host resistance during African trypanosomiasis.
Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Regulación hacia Abajo/inmunología , Glicosilfosfatidilinositoles/fisiología , Interferón gamma/farmacología , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/biosíntesis , Transactivadores/antagonistas & inhibidores , Tripanosomiasis Africana/metabolismo , Glicoproteínas Variantes de Superficie de Trypanosoma/fisiología , Animales , Línea Celular , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta Inmunológica , Regulación hacia Abajo/genética , Femenino , Glicosilfosfatidilinositoles/farmacología , Interferón gamma/antagonistas & inhibidores , Interferón gamma/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosforilación , Ratas , Ratas Sprague-Dawley , Factor de Transcripción STAT1 , Transducción de Señal/genética , Transducción de Señal/inmunología , Solubilidad , Transactivadores/metabolismo , Trypanosoma brucei gambiense/química , Trypanosoma brucei gambiense/crecimiento & desarrollo , Trypanosoma brucei gambiense/inmunología , Tripanosomiasis Africana/inmunología , Tripanosomiasis Africana/parasitología , Glicoproteínas Variantes de Superficie de Trypanosoma/farmacologíaRESUMEN
Recently, proteins linked to glycosylphosphatidylinositol (GPI) residues have received considerable attention both for their association with lipid microdomains and for their specific transport between cellular membranes. Basic features of trafficking of GPI-anchored proteins or glycolipids may be explored in flagellated protozoan parasites, which offer the advantage that their surface is dominated by these components. In Trypanosoma brucei, the GPI-anchored variant surface glycoprotein (VSG) is efficiently sorted at multiple intracellular levels, leading to a 50-fold higher membrane concentration at the cell surface compared with the endoplasmic reticulum. We have studied the membrane and VSG flow at an invagination of the plasma membrane, the flagellar pocket, the sole region for endo- and exocytosis in this organism. VSG enters trypanosomes in large clathrin-coated vesicles (135 nm in diameter), which deliver their cargo to endosomes. In the lumen of cisternal endosomes, VSG is concentrated by default, because a distinct class of small clathrin-coated vesicles (50-60 nm in diameter) budding from the cisternae is depleted in VSG. TbRAB11-positive cisternal endosomes, containing VSG, fragment by an unknown process giving rise to intensely TbRAB11- as well as VSG-positive, disk-like carriers (154 nm in diameter, 34 nm in thickness), which are shown to fuse with the flagellar pocket membrane, thereby recycling VSG back to the cell surface.
Asunto(s)
Vesículas Cubiertas por Clatrina/fisiología , Endocitosis/fisiología , Endosomas/fisiología , Exocitosis/fisiología , Glicosilfosfatidilinositoles/fisiología , Proteínas de Unión al GTP rab/fisiología , Animales , Endosomas/ultraestructura , Flagelos/fisiología , Aparato de Golgi/ultraestructura , Microscopía Electrónica , Trypanosoma brucei brucei/fisiología , Glicoproteínas Variantes de Superficie de Trypanosoma/fisiologíaRESUMEN
African trypanosomes (Trypanosoma brucei) are digenetic parasites whose lifecycle alternates between the mammalian bloodstream and the midgut of the tsetse fly vector. In mammals, proliferating long slender parasites transform into non-diving short stumpy forms, which differentiate into procyclic forms when ingested by the tsetse fly. A hallmark of differentiation is the replacement of the bloodstream stage surface coat composed of variant surface glycoprotein (VSG) with a new coat composed of procylin. An undefined endoprotease and endogenous glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) have been implicated in releasing the old VSG coat. However, GPI hydrolysis has been considered unimportant because (i) GPI-PLC null mutants are fully viable and (ii) cytosolic GPI-PLC is localized away from cell surface VSG. Utilizing an in vitro differentiation assay with pleomorphic strains we have investigated these modes of VSG release. Shedding is initially by GPI hydrolysis, which ultimately accounts for a substantial portion of total release. Surface biotinylation assays indicate that GPI-PLC does gain access to extracellular VSG, suggesting that this mode is primed in the starting short stumpy population. Proteolytic release is up-regulated during differentiation and is stereoselectively inhibited by peptidomimetic collagenase inhibitors, implicating a zinc metalloprotease. This protease may be related to TbMSP-B, a trypanosomal homologue of Leishmania major surface protease (MSP) described in the accompanying paper (LaCount, D. J., Gruszynski, A. E., Grandgenett, P. M., Bangs, J. D., and Donelson, J. E. (2003) J. Biol. Chem. 278, 24658-24664). Overall, our results demonstrate that surface coat remodeling during differentiation has multiple mechanisms and that GPI-PLC plays a more significant role in VSG release than previously thought.