RESUMEN
Glycosylation is one of the most structurally and functionally diverse co- and post-translational modifications in a cell. Addition and removal of glycans, especially to proteins and lipids, characterize this process which has important implications in several biological processes. In mammals, the repeated enzymatic addition of a sialic acid unit to underlying sialic acids (Sia) by polysialyltransferases, including ST8Sia2, leads to the formation of a sugar polymer called polysialic acid (polySia). The functional relevance of polySia has been extensively demonstrated in the nervous system. However, the role of polysialylation in infection is still poorly explored. Previous reports have shown that Trypanosoma cruzi (T. cruzi), a flagellated parasite that causes Chagas disease (CD), changes host sialylation of glycoproteins. To understand the role of host polySia during T. cruzi infection, we used a combination of in silico and experimental tools. We observed that T. cruzi reduces both the expression of the ST8Sia2 and the polysialylation of target substrates. We also found that chemical and genetic inhibition of host ST8Sia2 increased the parasite load in mammalian cells. We found that modulating host polysialylation may induce oxidative stress, creating a microenvironment that favors T. cruzi survival and infection. These findings suggest a novel approach to interfere with parasite infections through modulation of host polysialylation.
Asunto(s)
Enfermedad de Chagas , Ácidos Siálicos , Sialiltransferasas , Trypanosoma cruzi , Trypanosoma cruzi/genética , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/fisiología , Sialiltransferasas/metabolismo , Sialiltransferasas/genética , Enfermedad de Chagas/parasitología , Ácidos Siálicos/metabolismo , Humanos , Animales , GlicosilaciónRESUMEN
This protocol describes the genomic phage (gPhage) display platform, a large-scale antigen and epitope mapping technique. We constructed a gPhage display peptide library of a eukaryotic organism, Trypanosoma cruzi (causative agent of Chagas disease), to map the antibody response landscape against the parasite. Here, we used an organism with a relatively large but intronless genome, although future applications could include other prevalent or (re)emerging infectious organisms carrying genomes with a limited number of introns. For complete details on the use and execution of this protocol, please refer to Teixeira et al. (2021).
Asunto(s)
Técnicas de Visualización de Superficie Celular/métodos , Biblioteca Genómica , Anticuerpos Antiprotozoarios/química , Anticuerpos Antiprotozoarios/metabolismo , Genoma de Protozoos/genética , Trypanosoma cruzi/genéticaRESUMEN
Trypanosoma cruzi, the etiological agent of Chagas disease in humans, infects a wide variety of vertebrates. Trypomastigotes, the parasite infective forms, invade mammalian cells by a still poorly understood mechanism. Adhesion of tissue culture- derived trypomastigotes to the extracellular matrix (ECM) prior to cell invasion has been shown to be a relevant part of the process. Changes in phosphorylation, S-nitrosylation, and nitration levels of proteins, in the late phase of the interaction (2 h), leading to the reprogramming of both trypomastigotes metabolism and the DNA binding profile of modified histones, were described by our group. Here, the involvement of calcium signaling at a very early phase of parasite interaction with ECM is described. Increments in the intracellular calcium concentrations during trypomastigotes-ECM interaction depends on the Ca2+ uptake from the extracellular medium, since it is inhibited by EGTA or Nifedipine, an inhibitor of the L-type voltage gated Ca2+ channels and sphingosine-dependent plasma membrane Ca2+ channel, but not by Vanadate, an inhibitor of the plasma membrane Ca2+-ATPase. Furthermore, Nifedipine inhibits the invasion of host cells by tissue culture- derived trypomastigotes in a dose-dependent manner, reaching 95% inhibition at 100 µM Nifedipine. These data indicate the importance of both Ca2+ uptake from the medium and parasite-ECM interaction for host-cell invasion. Previous treatment of ECM with protease abolishes the Ca2+ uptake, further reinforcing the possibility that these events may be connected. The mitochondrion plays a relevant role in Ca2+ homeostasis in trypomastigotes during their interaction with ECM, as shown by the increment of the intracellular Ca2+ concentration in the presence of Antimycin A, in contrast to other calcium homeostasis disruptors, such as Cyclopiazonic acid for endoplasmic reticulum and Bafilomycin A for acidocalcisome. Total phosphatase activity in the parasite decreases in the presence of Nifedipine, EGTA, and Okadaic acid, implying a role of calcium in the phosphorylation level of proteins that are interacting with the ECM in tissue culture- derived trypomastigotes. In summary, we describe here the increment of Ca2+ at an early phase of the trypomastigotes interaction with ECM, implicating both nifedipine-sensitive Ca2+ channels in the influx of Ca2+ and the mitochondrion as the relevant organelle in Ca2+ homeostasis. The data unravel a complex sequence of events prior to host cell invasion itself.
Asunto(s)
Enfermedad de Chagas , Trypanosoma cruzi , Animales , Calcio/metabolismo , Señalización del Calcio , Matriz Extracelular/metabolismo , Humanos , Trypanosoma cruzi/metabolismoRESUMEN
Trypanosoma cruzi is a flagellate protozoa being the etiological agent of Chagas disease, a neglected tropical disease, which still poses a public health problem worldwide. The intricate molecular changes during T. cruzi-host interaction have been explored using different largescale omics techniques. However, protein stability is largely unknown. Thermal proteome profiling (TPP) methodology has the potential to characterize proteome-wide stability highlighting key proteins during T. cruzi infection and life stage transition from the invertebrate to the mammalian host. In the present work, T. cruzi epimastigotes and trypomastigotes cell lysates were subjected to TPP workflow and analyzed by quantitative large-scale mass spectrometry-based proteomics to fit a melting profile for each protein. A total of 2884 proteins were identified and associated to 1741 melting curves being 1370 in trypomastigotes (TmAVG 53.53 °C) and 1279 in epimastigotes (TmAVG 50.89 °C). A total of 453 proteins were identified with statistically different melting profiles between the two life stages. Proteins associated to pathogenesis and intracellular transport had regulated melting temperatures. Membrane and glycosylated proteins had a higher average Tm in trypomastigotes compared to epimastigotes. This study represents the first large-scale comparison of parasite protein stability between life stages. SIGNIFICANCE: Trypanosoma cruzi, a unicellular flagellate parasite, is the etiological agent of Chagas disease, endemic in South America and affecting more that 7 million people worldwide. There is an intense research to identify novel chemotherapeutic and diagnostic targets of Chagas disease. Proteomic approaches have helped in elucidating the quantitative proteome and PTMs changes of T. cruzi during life cycle transition and upon different biotic and abiotic stimuli. However, a comprehensive knowledge of the protein-protein interaction and protein conformation is still missing. In order to fill this gap, this manuscript elucidates the T. cruzi Y strain proteome-wide thermal stability map in the epimastigote and trypomastigote life stages. Comparison between life stages showed a higher average melting temperature stability for trypomastigotes than epimastigotes indicating a host temperature adaptation. Both presented a selective thermal stability shift for cellular compartments, molecular functions and biological processes based on the T. cruzi life stage. Membrane and glycosylated proteins presented a higher thermal stability in trypomastigotes when compared to the epimastigotes.
Asunto(s)
Enfermedad de Chagas , Trypanosoma cruzi , Animales , Humanos , Estadios del Ciclo de Vida , Proteoma , Proteómica , Proteínas ProtozoariasRESUMEN
Large-scale mapping of antigens and epitopes is pivotal for developing immunotherapies but challenging, especially for eukaryotic pathogens, owing to their large genomes. Here, we developed an integrated platform for genome phage display (gPhage) to show that unbiased libraries of the eukaryotic parasite Trypanosoma cruzi enable the identification of thousands of antigens recognized by serum samples from patients with Chagas disease. Because most of these antigens are hypothetical proteins, gPhage provides evidence of their expression during infection. We built and validated a comprehensive map of Chagas disease antibody response to show how linear and putative conformation epitopes, many rich in repetitive elements, allow the parasite to evade a buildup of neutralizing antibodies directed against protein domains that mediate infection pathogenesis. Thus, the gPhage platform is a reproducible and effective tool for rapid simultaneous identification of epitopes and antigens, not only in Chagas disease but perhaps also in globally emerging/reemerging acute pathogens.
RESUMEN
Adhesion of T. cruzi trypomastigotes to components of the extracellular matrix (ECM) is an important step in mammalian host cell invasion. We have recently described a significant increase in the tyrosine nitration levels of histones H2A and H4 when trypomastigotes are incubated with components of the ECM. In this work, we used chromatin immunoprecipitation (ChIP) with an anti-nitrotyrosine antibody followed by mass spectrometry to identify nitrated DNA binding proteins in T. cruzi and to detect alterations in nitration levels induced upon parasite incubation with the ECM. Histone H1, H2B, H2A and H3 were detected among the 9 most abundant nitrated DNA binding proteins using this proteomic approach. One nitrated tyrosine residue (Y29) was identified in Histone H2B in the MS/MS spectrum. In addition, we observed a significant increase in the nitration levels of histones H1, H2B, H2A and H4 upon parasite incubation with ECM. Finally, we used ChIP-Seq to map global changes in the DNA binding profile of nitrated proteins. We observed a significant change in the binding pattern of nitrated proteins to DNA after parasite incubation with ECM. This work provides the first global profile of nitrated DNA binding proteins in T. cruzi and additional evidence for modification in the nitration profile of histones upon parasite incubation with ECM. Our data also indicate that the parasite interaction with the ECM induces alterations in chromatin structure, possibly affecting nuclear functions.
Asunto(s)
Matriz Extracelular/parasitología , Histonas/análisis , Procesamiento Proteico-Postraduccional , Proteínas Protozoarias/análisis , Trypanosoma cruzi/química , Trypanosoma cruzi/crecimiento & desarrollo , Inmunoprecipitación de Cromatina , Matriz Extracelular/metabolismo , Histonas/metabolismo , Espectrometría de Masas , Nitrosación , Proteómica , Proteínas Protozoarias/metabolismo , Tirosina/análogos & derivados , Tirosina/inmunologíaRESUMEN
Trypanosoma cruzi, the etiological agent of Chagas' disease, affects 8 million people predominantly living in socioeconomic underdeveloped areas. T. cruzi trypomastigotes (Ty), the classical infective stage, interact with the extracellular matrix (ECM), an obligatory step before invasion of almost all mammalian cells in different tissues. Here we have characterized the proteome and phosphoproteome of T. cruzi trypomastigotes upon interaction with ECM (MTy) and the data are available via ProteomeXchange with identifier PXD010970. Proteins involved with metabolic processes (such as the glycolytic pathway), kinases, flagellum and microtubule related proteins, transport-associated proteins and RNA/DNA binding elements are highly represented in the pool of proteins modified by phosphorylation. Further, important metabolic switches triggered by this interaction with ECM were indicated by decreases in the phosphorylation of hexokinase, phosphofructokinase, fructose-2,6-bisphosphatase, phosphoglucomutase, phosphoglycerate kinase in MTy. Concomitantly, a decrease in the pyruvate and lactate and an increase of glucose and succinate contents were detected by GC-MS. These observations led us to focus on the changes in the glycolytic pathway upon binding of the parasite to the ECM. Inhibition of hexokinase, pyruvate kinase and lactate dehydrogenase activities in MTy were observed and this correlated with the phosphorylation levels of the respective enzymes. Putative kinases involved in protein phosphorylation altered upon parasite incubation with ECM were suggested by in silico analysis. Taken together, our results show that in addition to cytoskeletal changes and protease activation, a reprogramming of the trypomastigote metabolism is triggered by the interaction of the parasite with the ECM prior to cell invasion and differentiation into amastigotes, the multiplicative intracellular stage of T. cruzi in the vertebrate host.
Asunto(s)
Matriz Extracelular/parasitología , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/metabolismo , Animales , Cromatografía de Gases y Espectrometría de Masas , Regulación de la Expresión Génica/fisiología , Interacciones Huésped-Parásitos , Humanos , Proteínas Protozoarias/genéticaRESUMEN
Trypanosoma cruzi, the protozoan that causes Chagas disease, has a complex life cycle involving insect and mammalian hosts and distinct developmental stages. During T. cruzi developmental stages, glycoproteins play important role in the host-parasite interaction, such as cellular recognition, host cell invasion and adhesion, and immune evasion. In this study, comprehensive glycoprofiling analysis was performed in the epimastigote and trypomastigote stages of T. cruzi using two glycopeptide enrichment strategies, lectin-based and hydrophilic interaction liquid chromatography, followed by high resolution LC-MS/MS. Following deglycosylation, a total of 1306 N-glycosylation sites in NxS/T/C motifs were identified from 690 T. cruzi glycoproteins. Among them, 170 and 334 glycoproteins were exclusively identified in epimastigotes and trypomastigotes, respectively. Besides, global site-specific characterization of the N- and O-linked glycan heterogeneity in the two life stages of T. cruzi was achieved by intact glycopeptide analysis, revealing 144/466 unique N-linked and 10/97 unique O-linked intact glycopeptides in epimastigotes/trypomastigotes, respectively. Conclusively, this study documents the significant T. cruzi stage-specific expression of glycoproteins that can help to better understand the T. cruzi phenotype and response caused by the interaction with different hosts during its complex life cycle. BIOLOGICAL SIGNIFICANCE: Chagas disease caused by the protozoan Trypanosoma cruzi is a neglected disease which affects millions of people especially in Latin America. The absence of efficient drugs and vaccines against Chagas disease stimulates the search for novel targets. Glycoproteins are very attractive therapeutic candidate targets since they mediate key processes in the host-parasite interaction, such as cellular recognition, host cell invasion and adhesion, and immune evasion. This study aimed to provide an in depth characterization of the N-linked and O-linked glycoproteome of two T. cruzi life stages: epimastigotes and trypomastigotes. Mass spectrometry-based proteomics showed interesting stage-specific glycoproteome signatures that are valuable to better understand the importance of protein glycosylation in epimastigotes and trypomastigotes and to expand the repertoire of potential therapeutic targets against Chagas disease.
Asunto(s)
Glicoproteínas/análisis , Interacciones Huésped-Parásitos , Estadios del Ciclo de Vida , Trypanosoma cruzi/química , Enfermedad de Chagas/parasitología , Cromatografía Liquida , Glicoproteínas/fisiología , Proteómica/métodos , Espectrometría de Masas en Tándem , Trypanosoma cruzi/crecimiento & desarrollo , Trypanosoma cruzi/fisiologíaRESUMEN
The OX513A strain of Aedes aegypti, which was developed by the British company Oxitec, expresses a self-limiting transgene that prevents larvae from developing to adulthood. In April 2014, the Brazilian National Technical Commission on Biosafety completed a risk assessment of OX513A and concluded that the strain did not present new biological risks to humans or the environment and could be released in Brazil. At that point, Brazil became the first country to approve the unconstrained release of a genetically modified mosquito. During the assessment, the commission produced a comprehensive list of - and systematically analysed - the perceived hazards. Such hazards included the potential survival to adulthood of immature stages carrying the transgene - should the transgene fail to be expressed or be turned off by exposure to sufficient environmental tetracycline. Other perceived hazards included the potential allergenicity and/or toxicity of the proteins expressed by the gene, the potential for gene flow or increased transmission of human pathogens and the occupation of vacant breeding sites by other vector species. The Zika epidemic both elevated the perceived importance of Ae. aegypti as a vector - among policy-makers and regulators as well as the general public - and increased concerns over the release of males of the OX513A strain. We have therefore reassessed the potential hazards. We found that release of the transgenic mosquitoes would still be both safe and of great potential value in the control of diseases spread by Ae. aegypti, such as chikungunya, dengue and Zika.
La souche OX513A d'Aedes aegypti, qui a été créée par la société britannique Oxitec, exprime un transgène autolimitant qui empêche les larves de se développer et de devenir adultes. En avril 2014, la Commission technique nationale de biosécurité du Brésil a procédé à une évaluation des risques liés à la souche OX513A et conclu qu'elle ne présentait pas de nouveaux risques biologiques pour les êtres humains ou l'environnement et pouvait être lâchée au Brésil. Le Brésil est donc devenu le premier pays à approuver le lâcher non contraint d'un moustique génétiquement modifié. Au cours de l'évaluation, la commission a établi une liste exhaustive des risques perçus, qu'elle a par ailleurs systématiquement analysés. Ces risques incluaient la survie potentielle à l'âge adulte des larves immatures porteuses du transgène si le transgène ne s'exprime pas ou est désactivé par une exposition à la tétracycline suffisante dans l'environnement. Les autres risques perçus incluaient les potentielles propriétés allergisantes et/ou la toxicité des protéines exprimées par le gène, l'éventualité d'un flux de gènes ou d'une transmission accrue d'agents pathogènes pour l'homme et l'occupation de sites de reproduction vacants par d'autres espèces vectrices. L'épidémie d'infections à virus Zika a accentué l'importance accordée par les responsables politiques, les organismes de réglementation ainsi que le grand public à Ae. aegypti en tant que moustique vecteur, et a accru l'inquiétude relative au lâcher de mâles de la souche OX513A. Nous avons donc réévalué les risques potentiels. Nous estimons que le lâcher de moustiques transgéniques serait à la fois sans danger et extrêmement utile pour lutter contre les maladies transmises par Ae. aegypti, telles que le chikungunya, la dengue et le virus Zika.
La cepa OX513A de Aedes aegypti, que desarrolló la empresa británica Oxitec, expresa un transgén autolimitado que impide que las larvas se desarrollen hasta la edad adulta. En abril de 2014, la Comisión Nacional Técnica de Bioseguridad de Brasil realizó una evaluación de riesgos de OX513A y concluyó que la cepa no presentaba nuevos riesgos biológicos para los humanos o el medioambiente y que podría liberarse en Brasil. En ese momento, Brasil se convirtió en el primer país en aprobar la liberación ilimitada de un mosquito modificado genéticamente. A lo largo de la evaluación, la comisión redactó una lista completa, y analizada sistemáticamente, de las posibles contingencias. Entre dichos peligros se encontraba la posible supervivencia hasta la edad adulta de etapas inmaduras que portan el transgén, en caso de que éste no consiga expresarse o se inutilice debido a la exposición a la suficiente tetraciclina medioambiental. Otras posibles contingencias eran la alergia y/o toxicidad de las proteínas expresadas por el gen, la posibilidad de un flujo genético o el aumento de la transmisión de patógenos humanos y la ocupación de lugares de cría desocupados por parte de otras especies vectores. La epidemia por el virus de Zika aumentó la importancia de Ae. aegypti como vector, entre los responsables y reguladores políticos, así como entre el público general, y aumentó las preocupaciones acerca de la liberación de machos de la cepa OX513A. Por lo tanto, se han vuelto a evaluar los posibles riesgos. Se ha descubierto que la liberación de mosquitos transgénicos sería segura y tendría un gran valor potencial en el control de la propagación de enfermedades por Ae. aegypti, como la fiebre chikungunya, el dengue y la enfermedad por el virus de Zika.
Asunto(s)
Aedes/genética , Animales Modificados Genéticamente/crecimiento & desarrollo , Conocimientos, Actitudes y Práctica en Salud , Control de Plagas/métodos , Transgenes , Animales , Brasil , Contención de Riesgos Biológicos , Medición de RiesgoRESUMEN
Trypomastigote forms of Trypanosoma cruzi, the causative agent of Chagas Disease, shed extracellular vesicles (EVs) enriched with glycoproteins of the gp85/trans-sialidase (TS) superfamily and other α-galactosyl (α-Gal)-containing glycoconjugates, such as mucins. Here, purified vesicles from T. cruzi strains (Y, Colombiana, CL-14 and YuYu) were quantified according to size, intensity and concentration. Qualitative analysis revealed differences in their protein and α-galactosyl contents. Later, those polymorphisms were evaluated in the modulation of immune responses (innate and in the chronic phase) in C57BL/6 mice. EVs isolated from YuYu and CL-14 strains induced in macrophages higher levels of proinflammatory cytokines (TNF-α and IL-6) and nitric oxide via TLR2. In general, no differences were observed in MAPKs activation (p38, JNK and ERK 1/2) after EVs stimulation. In splenic cells derived from chronically infected mice, a different modulation pattern was observed, where Colombiana (followed by Y strain) EVs were more proinflammatory. This modulation was independent of the T. cruzi strain used in the mice infection. To test the functional importance of this modulation, the expression of intracellular cytokines after in vitro exposure was evaluated using EVs from YuYu and Colombiana strains. Both EVs induced cytokine production with the appearance of IL-10 in the chronically infected mice. A high frequency of IL-10 in CD4+ and CD8+ T lymphocytes was observed. A mixed profile of cytokine induction was observed in B cells with the production of TNF-α and IL-10. Finally, dendritic cells produced TNF-α after stimulation with EVs. Polymorphisms in the vesicles surface may be determinant in the immunopathologic events not only in the early steps of infection but also in the chronic phase.
RESUMEN
BACKGROUND: Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi, is a disease that affects millions of people most of them living in South and Central Americas. There are few treatment options for individuals with Chagas' disease making it important to understand the molecular details of parasite infection, so novel therapeutic alternatives may be developed for these patients. Here, we investigate the interaction between host cell intermediate filament proteins and the T. cruzi gp85 glycoprotein superfamily with hundreds of members that have long been implicated in parasite cell invasion. METHODOLOGY/PRINCIPAL FINDINGS: An in silico analysis was utilized to identify peptide motifs shared by the gp85 T. cruzi proteins and, using phage display, these selected peptide motifs were screened for their ability to bind to cells. One peptide, named TS9, showed significant cell binding capacity and was selected for further studies. Affinity chromatography, phage display and invasion assays revealed that peptide TS9 binds to cytokeratins and vimentin, and prevents T. cruzi cell infection. Interestingly, peptide TS9 and a previously identified binding site for intermediate filament proteins are disposed in an antiparallel ß-sheet fold, present in a conserved laminin-G-like domain shared by all members of the family. Moreover, peptide TS9 overlaps with an immunodominant T cell epitope. CONCLUSIONS/SIGNIFICANCE: Taken together, the present study reinforces previous results from our group implicating the gp85 superfamily of glycoproteins and the intermediate filament proteins cytokeratin and vimentin in the parasite infection process. It also suggests an important role in parasite biology for the conserved laminin-G-like domain, present in all members of this large family of cell surface proteins.
Asunto(s)
Glicoproteínas/metabolismo , Interacciones Huésped-Parásitos/fisiología , Proteínas de Filamentos Intermediarios/metabolismo , Laminina/metabolismo , Neuraminidasa/metabolismo , Estructura Terciaria de Proteína/fisiología , Trypanosoma cruzi/metabolismo , Secuencias de Aminoácidos/fisiología , Enfermedad de Chagas/metabolismo , Enfermedad de Chagas/parasitología , Cromatografía de Afinidad , Secuencia Conservada/fisiología , Glicoproteínas/química , Humanos , Queratinas/metabolismo , Laminina/química , Neuraminidasa/química , Unión Proteica , Vimentina/metabolismoRESUMEN
BACKGROUND: Adhesion of the Trypanosoma cruzi trypomastigotes, the causative agent of Chagas' disease in humans, to components of the extracellular matrix (ECM) is an important step in host cell invasion. The signaling events triggered in the parasite upon binding to ECM are less explored and, to our knowledge, there is no data available regarding â¢NO signaling. METHODOLOGY/PRINCIPAL FINDINGS: Trypomastigotes were incubated with ECM for different periods of time. Nitrated and S-nitrosylated proteins were analyzed by Western blotting using anti-nitrotyrosine and S-nitrosyl cysteine antibodies. At 2 h incubation time, a decrease in NO synthase activity, â¢NO, citrulline, arginine and cGMP concentrations, as well as the protein modifications levels have been observed in the parasite. The modified proteins were enriched by immunoprecipitation with anti-nitrotyrosine antibodies (nitrated proteins) or by the biotin switch method (S-nitrosylated proteins) and identified by MS/MS. The presence of both modifications was confirmed in proteins of interest by immunoblotting or immunoprecipitation. CONCLUSIONS/SIGNIFICANCE: For the first time it was shown that T. cruzi proteins are amenable to modifications by S-nitrosylation and nitration. When T. cruzi trypomastigotes are incubated with the extracellular matrix there is a general down regulation of these reactions, including a decrease in both NOS activity and cGMP concentration. Notwithstanding, some specific proteins, such as enolase or histones had, at least, their nitration levels increased. This suggests that post-translational modifications of T. cruzi proteins are not only a reflex of NOS activity, implying other mechanisms that circumvent a relatively low synthesis of â¢NO. In conclusion, the extracellular matrix, a cell surrounding layer of macromolecules that have to be trespassed by the parasite in order to be internalized into host cells, contributes to the modification of â¢NO signaling in the parasite, probably an essential move for the ensuing invasion step.
Asunto(s)
Regulación hacia Abajo , Matriz Extracelular/fisiología , Óxido Nítrico/metabolismo , Transducción de Señal/fisiología , Trypanosoma cruzi/fisiología , Animales , Western Blotting , Enfermedad de Chagas/metabolismo , Humanos , Inmunoprecipitación , Procesamiento Proteico-Postraduccional , Espectrometría de Masas en Tándem , Tirosina/análogos & derivadosRESUMEN
Trypanosoma cruzi strains show distinctive characteristics as genetic polymorphism and infectivity. Large repertoires of molecules, such as the Gp85 glycoproteins, members of the Gp85/Trans-sialidase superfamily, as well as multiple signaling pathways, are associated with invasion of mammalian cells by the parasite. Due to the large number of expressed members, encoded by more than 700 genes, the research focused on this superfamily conserved sequences is discussed. Binding sites to laminin have been identified at the N-terminus of the Gp85 molecules. Interestingly, the T. cruzi protein phosphorylation profile is changed upon parasite binding to laminin (or fibronectin), particularly the cytoskeletal proteins such as those from the paraflagellar rod and the tubulins, which are both markedly dephosphorylated. Detailed analysis of the signaling cascades triggered upon T. cruzi binding to extracellular matrix (ECM) proteins revealed the involvement of the MAPK/ERK pathway in this event. At the C-terminus, the conserved FLY sequence is a cytokeratin-binding domain and is involved in augmented host cell invasion in vitro and high levels of parasitemia in vivo. FLY, which is associated to tissue tropism and preferentially binds to the heart vasculature may somehow be correlated with the severe cardiac form, an important clinical manifestation of chronic Chagas' disease.
Asunto(s)
Proteínas Protozoarias/aislamiento & purificación , Trypanosoma cruzi/metabolismo , Animales , Sitios de Unión , Modelos Moleculares , Fosforilación , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismoRESUMEN
Trypanosoma cruzi is the etiologic agent of Chagas disease. Two main distinct forms are present in the mammalian host: trypomastigote, an infective form, and the amastigote, a typical replicative form. Succeeding the host cell invasion, trypomastigotes differentiate to amastigotes, a process known as amastigogenesis. Amastigogenesis is characterized by parasite body remodeling, with drastic reduction of flagellum, and changes in protein profile and energetic metabolism. Our aim is to explore the role of nitric oxide as a signaling molecule during the amastigogenesis process, which must be strictly regulated. We report herein that acid pH (6.0) is essential for T. cruzi amastigogenesis. Also, during amastigogenesis there is a progressive solubilization of the paraflagellar protein, a flagellum marker. Moreover, the process is dependent on (â¢)NO concentration, since it is suppressed by 1mM SNAP, a (â¢)NO donor, and favored by 10mM L-NAME, a NOS inhibitor. Accordingly, S-nitrosylation of selective proteins occurs in amastigogenesis. Additionally, amastigogenesis is affected by IBMX (PDE inhibitor) treatment, suggesting the importance of cyclic nucleotides signaling. Furthermore, tubulin stability is also affected by the (â¢)NO availability. Along amastigogenesis, flagellum disassembling is accompanied by changes in a-tubulin tyrosylation and polyglutamylation levels. Taken together, these results suggest (â¢)NO participation in trypomastigote differentiation to amastigotes in T. cruzi.
RESUMEN
The putrescine analogue 1,4-diamino-2-butanone (DAB) is highly toxic to various microorganisms, including Trypanosoma cruzi. Similar to other α-aminocarbonyl metabolites, DAB exhibits pro-oxidant properties. DAB undergoes metal-catalyzed oxidation yielding H(2)O(2), NH(4)(+) ion, and a highly toxic α-oxoaldehyde. In vitro, DAB decreases mammalian cell viability associated with changes in redox balance. Here, we aim to clarify the DAB pro-oxidant effects on trypomastigotes and on intracellular T. cruzi amastigotes. DAB (0.05-5 mM) exposure in trypomastigotes, the infective stage of T. cruzi, leads to a decline in parasite viability (IC(50)c.a. 0.2 mM DAB; 4 h incubation), changes in morphology, thiol redox imbalance, and increased TcSOD activity. Medium supplementation with catalase (2.5 µM) protects trypomastigotes against DAB toxicity, while host cell invasion by trypomastigotes is hampered by DAB. Additionally, intracellular amastigotes are susceptible to DAB toxicity. Furthermore, pre-treatment with 100-500 µM buthionine sulfoximine (BSO) of LLC-MK2 potentiates DAB cytotoxicity, whereas 5 mM N-acetyl-cysteine (NAC) protects cells from oxidative stress. Together, these data support the hypothesis that redox imbalance contributes to DAB cytotoxicity in both T. cruzi and mammalian host cells.
Asunto(s)
Oxidantes/farmacología , Putrescina/análogos & derivados , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/metabolismo , Animales , Línea Celular , Modelos Biológicos , Oxidantes/toxicidad , Oxidación-Reducción , Proteínas Protozoarias/metabolismo , Putrescina/farmacología , Putrescina/toxicidad , Compuestos de Sulfhidrilo/metabolismo , Superóxido Dismutasa/metabolismo , Tripanocidas/farmacología , Trypanosoma cruzi/crecimiento & desarrollo , Trypanosoma cruzi/patogenicidadRESUMEN
BACKGROUND: The unicellular parasite Trypanosoma cruzi is the causative agent of Chagas disease in humans. Adherence of the infective stage to elements of the extracellular matrix (ECM), as laminin and fibronectin, is an essential step in host cell invasion. Although members of the gp85/TS, as Tc85, were identified as laminin and fibronectin ligands, the signaling events triggered on the parasite upon binding to these molecules are largely unexplored. METHODOLOGY/PRINCIPAL FINDINGS: Viable infective parasites were incubated with laminin, fibronectin or bovine serum albumin for different periods of time and the proteins were separated by bidimensional gels. The phosphoproteins were envisaged by specific staining and the spots showing phosphorylation levels significantly different from the control were excised and identified by MS/MS. The results of interest were confirmed by immunoblotting or immunoprecipitation and the localization of proteins in the parasite was determined by immunofluorescence. Using a host cell-free system, our data indicate that the phosphorylation contents of T. cruzi proteins encompassing different cellular functions are modified upon incubation of the parasite with fibronectin or laminin. CONCLUSIONS/SIGNIFICANCE: Herein it is shown, for the first time, that paraflagellar rod proteins and α-tubulin, major structural elements of the parasite cytoskeleton, are predominantly dephosphorylated during the process, probably involving the ERK1/2 pathway. It is well established that T. cruzi binds to ECM elements during the cell infection process. The fact that laminin and fibronectin induce predominantly dephosphorylation of the main cytoskeletal proteins of the parasite suggests a possible correlation between cytoskeletal modifications and the ability of the parasite to internalize into host cells.
Asunto(s)
Fibronectinas/metabolismo , Laminina/metabolismo , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/fisiología , Fosforilación , Proteínas Protozoarias/metabolismo , Transducción de Señal/fisiología , Tubulina (Proteína)/metabolismoRESUMEN
Different types of shed vesicles as, for example, exosomes, plasma-membrane-derived vesicles or microparticles, are the focus of intense research in view of their potential role in cell-cell communication and under the perspective that they might be good tools for immunotherapy, vaccination or diagnostic purposes. This review discusses ways employed by pathogenic trypanosomatids to interact with the host by shedding vesicles that contain molecules important for the establishment of infection, as opposed to previous beliefs considering them as a waste of cellular metabolism. Trypanosomatids are compared with Apicomplexa, which circulate parasite antigens bound to vesicles shed by host cells. The knowledge of the origin and chemical composition of these different vesicles might lead to the understanding of the mechanisms that determine their biological function.
Asunto(s)
Exosomas/metabolismo , Infecciones por Protozoos/parasitología , Factores de Virulencia/metabolismo , Animales , Apicomplexa/metabolismo , Apicomplexa/patogenicidad , Exosomas/parasitología , Interacciones Huésped-Parásitos , Humanos , Trypanosomatina/metabolismo , Trypanosomatina/patogenicidadRESUMEN
BACKGROUND: Transmitted by blood-sucking insects, the unicellular parasite Trypanosoma cruzi is the causative agent of Chagas' disease, a malady manifested in a variety of symptoms from heart disease to digestive and urinary tract dysfunctions. The reasons for such organ preference have been a matter of great interest in the field, particularly because the parasite can invade nearly every cell line and it can be found in most tissues following an infection. Among the molecular factors that contribute to virulence is a large multigene family of proteins known as gp85/trans-sialidase, which participates in cell attachment and invasion. But whether these proteins also contribute to tissue homing had not yet been investigated. Here, a combination of endothelial cell immortalization and phage display techniques has been used to investigate the role of gp85/trans-sialidase in binding to the vasculature. METHODS: Bacteriophage expressing an important peptide motif (denominated FLY) common to all gp85/trans-sialidase proteins was used as a surrogate to investigate the interaction of this motif with the endothelium compartment. For that purpose phage particles were incubated with endothelial cells obtained from different organs or injected into mice intravenously and the number of phage particles bound to cells or tissues was determined. Binding of phages to intermediate filament proteins has also been studied. FINDINGS AND CONCLUSIONS: Our data indicate that FLY interacts with the endothelium in an organ-dependent manner with significantly higher avidity for the heart vasculature. Phage display results also show that FLY interaction with intermediate filament proteins is not limited to cytokeratin 18 (CK18), which may explain the wide variety of cells infected by the parasite. This is the first time that members of the intermediate filaments in general, constituted by a large group of ubiquitously expressed proteins, have been implicated in T. cruzi cell invasion and tissue homing.
Asunto(s)
Enfermedad de Chagas/parasitología , Endotelio Vascular/parasitología , Glicoproteínas/química , Glicoproteínas/metabolismo , Neuraminidasa/química , Neuraminidasa/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Tropismo , Trypanosoma cruzi/enzimología , Secuencias de Aminoácidos , Animales , Células Cultivadas , Enfermedad de Chagas/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/parasitología , Endotelio Vascular/metabolismo , Femenino , Glicoproteínas/genética , Humanos , Proteínas de Filamentos Intermediarios/metabolismo , Ratones , Ratones Endogámicos C57BL , Neuraminidasa/genética , Especificidad de Órganos , Unión Proteica , Proteínas Protozoarias/genética , Trypanosoma cruzi/química , Trypanosoma cruzi/genética , Trypanosoma cruzi/fisiologíaRESUMEN
Trypanosoma cruzi, the agent of Chagas' disease, alternates between different morphogenetic stages that face distinct physiological conditions in their invertebrate and vertebrate hosts, likely in the availability of glucose. While the glucose transport is well characterized in epimastigotes of T. cruzi, nothing is known about how the mammalian stages acquire this molecule. Herein glucose transport activity and expression were analyzed in the three developmental stages present in the vertebrate cycle of T. cruzi. The infective trypomastigotes showed the highest transport activity (V(max)=5.34+/-0.54 nmol/min per mg of protein; K(m)=0.38+/-0.01 mM) when compared to intracellular epimastigotes (V(max)=2.18+/-0.20 nmol/min per mg of protein; K(m)=0.39+/-0.01 mM). Under the conditions employed no transport activity could be detected in amastigotes. The gene of the glucose transporter is expressed at the mRNA level in trypomastigotes and in intracellular epimastigotes but not in amastigotes, as revealed by real-time PCR. In both trypomastigotes and intracellular epimastigotes protein expression could be detected by Western blot with an antibody raised against the glucose transporter correlating well with the transport activity measured experimentally. Interestingly, anti-glucose transporter antibodies showed a strong reactivity with glycosome and reservosome organelles. A comparison between proline and glucose transport among the intracellular differentiation forms is presented. The data suggest that the regulation of glucose transporter reflects different energy and carbon requirements along the intracellular life cycle of T. cruzi.
Asunto(s)
Glucosa/metabolismo , Trypanosoma cruzi/metabolismo , Animales , Western Blotting , Perfilación de la Expresión Génica , Prolina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Vesículas TransportadorasRESUMEN
Trypanosoma cruzi trypomastigotes continuously shed into the medium plasma membrane fragments sealed as vesicles enriched in glycoproteins of the gp85 and trans-sialidase (TS) superfamily and alpha-galactosyl-containing glycoconjugates. Injection of a vesicle fraction into BALB/c mice prior to T. cruzi infection led to 40% of deaths on the 16thday post-infection and 100% on day 20th whereas 20% of untreated animals survived for more than 30days. The vesicle-treated animals developed severe heart pathology, with intense inflammatory reaction and higher number of amastigote nests. Analysis of the inflammatory infiltrates 15days after infection showed predominance of TCD4(+) lymphocytes and macrophages, but not of TCD8(+) cells, as well as a decrease of areas labeled with anti-iNOS antibodies as compared to the control. Higher levels of IL-4 and IL-10 mRNAs were found in the hearts and higher IL-10 and lower NO levels in splenocytes of vesicles pretreated animals. Treatment of mice with neutralizing anti-IL-10 or anti-IL-4 antibodies precluded the effects of pre-inoculation of membrane vesicles on infection. These results indicate that T. cruzi shed membrane components increase tissue parasitism and inflammation by stimulation of IL-4 and IL-10 synthesis and thus may play a central role in the pathogenesis of Chagas' disease acute phase.