RESUMEN
Giardia lamblia (also called Giardia intestinalis) is one of the most common intestinal parasites of humans. To evade the host's immune response, Giardia undergoes antigenic variation-a process that allows the parasite to develop chronic and recurrent infections. From a repertoire of approximately 190 variant-specific surface protein (VSP)-coding genes, Giardia expresses only one VSP on the surface of each parasite at a particular time, but spontaneously switches to a different VSP by unknown mechanisms. Here we show that regulation of VSP expression involves a system comprising RNA-dependent RNA polymerase, Dicer and Argonaute, known components of the RNA interference machinery. Clones expressing a single surface antigen efficiently transcribe several VSP genes but only accumulate transcripts encoding the VSP to be expressed. Detection of antisense RNAs corresponding to the silenced VSP genes and small RNAs from the silenced but not for the expressed vsp implicate the RNA interference pathway in antigenic variation. Remarkably, silencing of Dicer and RNA-dependent RNA polymerase leads to a change from single to multiple VSP expression in individual parasites.
Asunto(s)
Variación Antigénica/genética , Antígenos de Protozoos/genética , Antígenos de Superficie/genética , Regulación de la Expresión Génica , Giardia lamblia/genética , Interferencia de ARN , Animales , Animales Modificados Genéticamente , Variación Antigénica/inmunología , Antígenos de Protozoos/inmunología , Antígenos de Superficie/inmunología , Técnicas de Silenciamiento del Gen , Giardia lamblia/inmunología , Datos de Secuencia Molecular , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , ARN Protozoario/metabolismo , Ribonucleasa III/metabolismoRESUMEN
Tritrichomonas foetus is a flagellated and anaerobic parasite able to infect cattle and felines. Despite its prevalence, there is no effective standardized or legal treatment for T. foetus-infected cattle; the vaccination still has limited success in mitigating infections and reducing abortion risk; and nowadays, the diagnosis of T. foetus presents important limitations in terms of sensitivity and specificity in bovines. Here, we characterize the plasma membrane proteome of T. foetus and identify proteins that are represented in different isolates of this protozoan. Additionally, we performed a bioinformatic analysis that revealed the antigenicity potential of some of those proteins. This analysis is the first study to identify common proteins at the plasma membrane of different T. foetus isolates that could be targets for alternative diagnostic or vaccine techniques in the future.
Asunto(s)
Proteómica , Proteínas Protozoarias , Tritrichomonas foetus , Tritrichomonas foetus/aislamiento & purificación , Proteómica/métodos , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/análisis , Animales , Proteoma/análisis , Membrana Celular/metabolismo , Bovinos , Proteínas de la Membrana/metabolismo , Enfermedades de los Bovinos/parasitología , Infecciones Protozoarias en Animales/parasitología , Infecciones Protozoarias en Animales/diagnóstico , Biología Computacional/métodosRESUMEN
Giardia lamblia is a medically important protozoan parasite with a basal position in the eukaryotic lineage and is an interesting model to explain the evolution of biochemical events in eukaryotic cells. G. lamblia trophozoites undergo significant changes in order to survive outside the intestine of their host by differentiating into infective cysts. In the present study, we characterize the previously identified Orf-C4 (G. lamblia open reading frame C4) gene, which is considered to be specific to G. lamblia. It encodes a 22 kDa protein that assembles into high-molecular-mass complexes during the entire life cycle of the parasite. ORF-C4 localizes to the cytoplasm of trophozoites and cysts, and forms large spherical aggregates when overexpressed. ORF-C4 overexpression and down-regulation do not affect trophozoite viability; however, differentiation into cysts is slightly delayed when the expression of ORF-C4 is down-regulated. In addition, ORF-C4 protein expression is modified under specific stress-inducing conditions. Neither orthologous proteins nor conserved domains are found in databases by conventional sequence analysis of the predicted protein. However, ORF-C4 contains a region which is similar structurally to the alpha-crystallin domain of sHsps (small heat-shock proteins). In the present study, we show the potential role of ORF-C4 as a small chaperone which is involved in the response to stress (including encystation) in G. lamblia.
Asunto(s)
Giardia lamblia/fisiología , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Animales , Regulación de la Expresión Génica , Giardia lamblia/genética , Proteínas de Choque Térmico Pequeñas/genética , Proteínas de Choque Térmico Pequeñas/metabolismo , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Estrés Fisiológico , alfa-Cristalinas/genética , alfa-Cristalinas/metabolismoRESUMEN
Intestinal and free-living protozoa, such as Giardia lamblia, express a dense coat of variant-specific surface proteins (VSPs) on trophozoites that protects the parasite inside the host's intestine. Here we show that VSPs not only are resistant to proteolytic digestion and extreme pH and temperatures but also stimulate host innate immune responses in a TLR-4 dependent manner. We show that these properties can be exploited to both protect and adjuvant vaccine antigens for oral administration. Chimeric Virus-like Particles (VLPs) decorated with VSPs and expressing model surface antigens, such as influenza virus hemagglutinin (HA) and neuraminidase (NA), are protected from degradation and activate antigen presenting cells in vitro. Orally administered VSP-pseudotyped VLPs, but not plain VLPs, generate robust immune responses that protect mice from influenza infection and HA-expressing tumors. This versatile vaccine platform has the attributes to meet the ultimate challenge of generating safe, stable and efficient oral vaccines.
Asunto(s)
Giardia lamblia/química , Vacunas contra la Influenza/inmunología , Proteínas de la Membrana/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Proteínas Protozoarias/inmunología , Vacunas de Partículas Similares a Virus/inmunología , Adyuvantes Inmunológicos , Administración Oral , Animales , Presentación de Antígeno/efectos de los fármacos , Bioingeniería/métodos , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/virología , Femenino , Expresión Génica , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Inmunidad Innata/efectos de los fármacos , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/genética , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos , Neuraminidasa/genética , Neuraminidasa/inmunología , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/virología , Estabilidad Proteica , Proteínas Protozoarias/genética , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología , Trofozoítos/química , Vacunación , Vacunas de Partículas Similares a Virus/administración & dosificación , Vacunas de Partículas Similares a Virus/genéticaRESUMEN
The components of the redox metabolism in Entamoeba histolytica have been recently revisited by Arias et al. (Free Radic. Biol. Med. 42:1496-1505; 2007), after the identification and characterization of a thioredoxin-linked system. The present work deals with studies performed for a better understanding of the localization and identification of different components of the redox machinery present in the parasite. The gene encoding for amoebic thioredoxin 8 was cloned and the recombinant protein typified as having properties similar to those of thioredoxin 41. The ability of these thioredoxins and the specific reductase to assemble a system utilizing NADPH to metabolize hydroperoxides in association with a peroxiredoxin has been kinetically characterized. The peroxiredoxin behaved as a typical 2 cysteine enzyme, exhibiting a ping-pong mechanism with hyperbolic saturation kinetics for thioredoxin 8 (K(m)=3.8 microM), thioredoxin 41 (K(m)=3.1 microM), and tert-butyl hydroperoxide (K(m) about 35 microM). Moreover, the tandem system involving thioredoxin reductase and either thioredoxin proved to be operative for reducing low molecular weight disulfides, including putative physiological substrates as cystine and oxidized trypanothione. Thioredoxin reductase and thioredoxin 41 (by association also the functional redox system) have been immunolocalized underlying the plasma membrane in Entamoeba histolytica cells. These findings suggest an important role for the metabolic pathway involving thioredoxin as a redox interchanger, which could be critical for the maintenance and virulence of the parasite when exposed to highly toxic reactive oxygen species.
Asunto(s)
Entamoeba histolytica/enzimología , Peroxidasas/metabolismo , Tiorredoxinas/inmunología , Tiorredoxinas/metabolismo , Animales , Clonación Molecular , Disulfuros/metabolismo , Entamoeba histolytica/genética , Expresión Génica , Inmunohistoquímica , Cinética , Oxidación-Reducción , Tiorredoxinas/genéticaRESUMEN
During evolution, parasitic microorganisms have faced the challenges of adapting to different environments to colonize a variety of hosts. Giardia lamblia, a common cause of intestinal disease, has developed fascinating strategies to adapt both outside and inside its host's intestine, such as trophozoite differentiation into cyst and the switching of its major surface antigens. How gene expression is regulated during these adaptive processes remains undefined. Giardia lacks some typical eukaryotic features, like canonical transcription factors, linker histone H1, and complex promoter regions; suggesting that post-transcriptional and translational control of gene expression is essential for parasite survival. However, epigenetic factors may also play critical roles at the transcriptional level. Here, we describe the most common post-translational histone modifications; characterize enzymes involved in these reactions, and analyze their association with the Giardia's differentiation processes. We present evidence that NAD+-dependent and NAD+-independent histone deacetylases regulate encystation; however, a unique NAD+-independent histone deacetylase modulate antigenic switching. The rates of acetylation of H4K8 and H4K16 are critical for encystation, whereas a decrease in acetylation of H4K8 and methylation of H3K9 occur preferentially during antigenic variation. These results show the complexity of the mechanisms regulating gene expression in this minimalistic protozoan parasite.
Asunto(s)
Variación Antigénica , Giardia lamblia/inmunología , Giardia lamblia/metabolismo , Histonas/metabolismo , Acetilación/efectos de los fármacos , Variación Antigénica/efectos de los fármacos , Eucromatina/metabolismo , Giardia lamblia/citología , Giardia lamblia/genética , Heterocromatina/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Histonas/química , Lisina/metabolismo , NAD/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacosRESUMEN
ABSTRACT Tenderness is considered the most important meat quality trait regarding its eating quality. Post mortem meat tenderization is primarily the result of calpain mediated degradation of key proteins within muscles fibers. The calpain system originally comprised three molecules: two Ca2+-dependent proteases and a specific inhibitor. Numerous studies have shown that the calpain system plays a central role in postmortem proteolysis and meat tenderization. The objective of this review is to describe the last biochemical and molecular findings in connection with this proteolytic system and their relation with meat tenderness in bovine. Findings of DNA polymorphisms and mRNA and protein expression are described as tools to predict meat tenderness. Understanding the molecular basis of meat tenderization may be useful particularly to the meat industry and may allow amendment of pre-slaughter handling practices and postmortem treatments that improves meat quality.
RESUMEN La terneza de la carne es considerada como el atributo de mayor importancia en el concepto de calidad de carne. El proceso de tenderización de la carne post mortem es principalmente el resultado de la degradación de proteínas clave de las fibras musculares, mediado por las proteasas del sistema calpaína. Este sistema proteico está compuesto por tres moléculas: dos proteasas calcio-dependientes y su inhibidor específico. Numerosos estudios han demostrado que el sistema calpaína desempeña un papel central en la proteólisis postmortem y en la tenderización de la carne. El objetivo de esta revisión es describir los últimos descubrimientos bioquímicos y moleculares de este sistema proteolítico y su relación con la terneza de la carne bovina. Se describen los hallazgos de polimorfismos de ADN y de expresión de ARNm y proteínas, como herramientas para predecir la terneza de la carne. La comprensión de las bases moleculares de la tenderización de la carne puede ser de utilidad para la industria cárnica, permitiendo la modificación de las prácticas de manipulación antes del sacrificio y los tratamientos post mortem, mejorando la calidad de la carne bovina.
RESUMEN
Methionine is an amino acid susceptible to being oxidized to methionine sulfoxide (MetSO). The reduction of MetSO to methionine is catalyzed by methionine sulfoxide reductase (MSR), an enzyme present in almost all organisms. In trypanosomatids, the study of antioxidant systems has been mainly focused on the involvement of trypanothione, a specific redox component in these organisms. However, no information is available concerning their mechanisms for repairing oxidized proteins, which would be relevant for the survival of these pathogens in the various stages of their life cycle. We report the molecular cloning of three genes encoding a putative A-type MSR in trypanosomatids. The genes were expressed in Escherichia coli, and the corresponding recombinant proteins were purified and functionally characterized. The enzymes were specific for L-Met(S)SO reduction, using Trypanosoma cruzi tryparedoxin I as the reducing substrate. Each enzyme migrated in electrophoresis with a particular profile reflecting the differences they exhibit in superficial charge. The in vivo presence of the enzymes was evidenced by immunological detection in replicative stages of T. cruzi and Trypanosoma brucei. The results support the occurrence of a metabolic pathway in Trypanosoma spp. involved in the critical function of repairing oxidized macromolecules.
Asunto(s)
Metionina Sulfóxido Reductasas/genética , Metionina Sulfóxido Reductasas/metabolismo , Trypanosoma/enzimología , Secuencia de Aminoácidos , Animales , Células Cultivadas , Chlorocebus aethiops , Clonación Molecular , Fase I de la Desintoxicación Metabólica/genética , Redes y Vías Metabólicas/genética , Metionina Sulfóxido Reductasas/química , Metionina Sulfóxido Reductasas/aislamiento & purificación , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Estrés Oxidativo/genética , Homología de Secuencia , Trypanosoma/genética , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , Células VeroRESUMEN
Giardia lamblia is one of the most common causes of intestinal disease in humans. To adapt to environments both inside and outside of the host's small intestine, this protozoan parasite undergoes significant developmental changes during its life cycle. In this review, we analyze and discuss the most recent findings regarding the process of Giardia trophozoites differentiation into infective cysts as well as the mechanism of antigenic variation, which allows the parasite to cause chronic and recurrent infections in susceptible individuals.
Asunto(s)
Variación Antigénica , Giardia lamblia/inmunología , Giardia lamblia/fisiología , Estadios del Ciclo de Vida , Animales , Giardia lamblia/patogenicidad , HumanosRESUMEN
Giardia lamblia is a human intestinal pathogen. Like many protozoan microorganisms, Giardia undergoes antigenic variation, a mechanism assumed to allow parasites to evade the host's immune response, producing chronic and/or recurrent infections. Recently, we found that the mechanism controlling variant-specific surface protein (VSP) switching in Giardia involves components of the RNA interference machinery and that disruption of this pathway generates trophozoites simultaneously expressing many VSPs. Here we use these altered trophozoites to determine the role of antigenic variation in a gerbil model of giardiasis. Our results show that either primary infection with trophozoites simultaneously expressing many VSPs or immunization with purified VSPs from the transgenic cells protects gerbils from subsequent Giardia infections. These results constitute, to our knowledge, the first experimental evidence that antigenic variation is essential for parasite survival within hosts and that artificial disruption of this mechanism might be useful in generating vaccines against major pathogens that show similar behavior.
Asunto(s)
Variación Antigénica , Giardia lamblia , Giardiasis/inmunología , Parásitos , Vacunas Antiprotozoos/inmunología , Animales , Animales Modificados Genéticamente , Gerbillinae , Giardia lamblia/genética , Giardia lamblia/inmunología , Giardiasis/parasitología , Humanos , Mucosa Intestinal/metabolismo , Intestinos/inmunología , Ratones , Parásitos/genética , Parásitos/inmunología , Interferencia de ARNAsunto(s)
Fosfatasa Ácida/metabolismo , Giardia lamblia/enzimología , Giardia lamblia/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Animales , Giardia lamblia/citología , Giardia lamblia/crecimiento & desarrollo , Humanos , Datos de Secuencia Molecular , Fosforilación , Homología de Secuencia de AminoácidoRESUMEN
Giardia lamblia undergoes antigenic variation, a process that might allow the parasite to evade the host's immune response and adapt to different environments. Here we show that Giardia muris, a related species that naturally infects rodents, possesses multiple variant-specific surface proteins (VSPs) and expresses VSPs on its surface, suggesting that it undergoes antigenic variation similar to that of G. lamblia.
Asunto(s)
Giardia/inmunología , Giardiasis/inmunología , Proteínas de la Membrana/inmunología , Secuencia de Aminoácidos , Animales , Northern Blotting , Giardia/genética , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Ratas , Ratas WistarRESUMEN
A novel variant-specific surface protein (VSP) from Giardia was identified using the monoclonal antibody 9B10, raised against purified cyst walls. VSP9B10B is preferentially induced during encystation and expressed simultaneously with other VSPs on the surface of encysting trophozoites. These results support the hypothesis that encystation and antigenic variation are processes that are mechanistically related.