RESUMEN
Vesicular trafficking, which is implicated in secretion of cytolytic molecules as well as in phagocytosis, plays an important role in the pathogenic mechanism of Entamoeba histolytica, the protozoan parasite causative of human amoebiasis. Thus, Rab GTPases, that are key regulators of vesicle trafficking, should be considered as molecules involved in the parasite virulence. EhRabB is a Rab protein located in cytoplasmic vesicles that are translocated to phagocytic mouths during ingestion of target cells, suggesting that this Rab protein is involved in phagocytosis. To prove this hypothesis, we over expressed the wild type EhrabB gene and a mutant gene encoding for a protein (RabBN118I) unable to bind guanine nucleotides and therefore constitutively inactive. The over expression of the mutated protein in E. histolytica trophozoites provoked a dominant negative effect, reflected in a significant decrease of both phagocytosis and cytopathic effect as well as in a failure to produce hepatic abscesses in hamsters. These results confirm that EhRabB is involved in phagocytosis and virulence of E. histolytica.
Asunto(s)
Entamoeba histolytica/patogenicidad , Proteínas Protozoarias/metabolismo , Animales , Western Blotting , Cricetinae , Perros , Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Eritrocitos/parasitología , Técnica del Anticuerpo Fluorescente/métodos , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Regulación de la Expresión Génica , Humanos , Absceso Hepático Amebiano/parasitología , Mutagénesis , Fagocitosis/fisiología , Proteínas Protozoarias/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , VirulenciaRESUMEN
EhADH112 is part of the EhCPADH complex, a protein involved in key events of the Entamoeba histolytica host invasion. EhADH112 participates in trophozoite adherence to target cells and in phagocytosis. We report here the finding of two EhADH112 homologues in the E. histolytica genome (EhADH112-like proteins). EhADH112 and its relatives have a Bro1 domain at their amino-terminus and a consensus context for phosphorylation by Src-tyrosine kinases, both involved in signal transduction processes in other organisms. Our findings associate EhADH112 to supplementary functions related to those reported for the Alix/AIP1 family. To elucidate the precise function of EhADH112, we studied the phenotypes displayed by trophozoites transfected with the Ehadh112 full gene. Transfected trophozoites overexpressed a 78 kDa protein, which was mainly targeted to the EhCPADH complex. Moreover, these trophozoites exhibited enhanced phagocytic rates, providing further evidence of EhADH112 contribution to adhesion and phagocytosis activities.
Asunto(s)
Entamoeba histolytica/genética , Lectinas/fisiología , Glicoproteínas de Membrana/fisiología , Fagocitosis/genética , Proteínas Protozoarias/fisiología , Secuencia de Aminoácidos , Animales , Western Blotting , Adhesión Celular/genética , ADN Protozoario/química , Entamoeba histolytica/fisiología , Regulación de la Expresión Génica , Lectinas/química , Lectinas/genética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Plásmidos/química , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
This paper reports the identification of Ehp53, a p53-like Entamoeba histolytica protein, which binds to the human p53 DNA consensus sequence (oli-p53). Monoclonal antibodies against p53 (Ab-1 and Ab-2) recognized a single 53 kDa spot in two-dimensional gels and inhibited the formation of complexes produced by E. histolytica nuclear extracts and oli-p53. Additionally, E. histolytica gene promoter sequences with high homology to oli-p53 formed complexes with nuclear proteins that were abolished by oli-p53. Ehp53 protein levels increased in UV-irradiated trophozoites. This protein was also detected in Entamoeba moshkovskii and Entamoeba invadens. By confocal microscopy, Ehp53 was located in the nuclei, EhkO organelles and cytoplasm. The Ehp53-encoding gene was cloned and its predicted amino acid sequence showed 30-54 % and 50-57 % homology with important domains of the human and the Drosophila melanogaster p53 proteins, respectively. This homology included the tetramerization domain, the nuclear export signal and a nuclear localization signal. Ehp53 also contains seven of the eight DNA-binding residues and two of the four Zn(2+)-binding sites described for p53. A recombinant Ehp53 was recognized by Ab-2. Ehp53 is believed to be the first p53-like protein found in protozoa and may be the evolutionary ancestor of the mammalian p53.
Asunto(s)
Entamoeba histolytica/metabolismo , Evolución Molecular , Proteínas Protozoarias , Proteína p53 Supresora de Tumor , Secuencia de Aminoácidos , Animales , Clonación Molecular , Secuencia de Consenso , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Entamoeba histolytica/genética , Entamoeba histolytica/crecimiento & desarrollo , Entamoeba histolytica/efectos de la radiación , Humanos , Microscopía Confocal , Datos de Secuencia Molecular , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transactivadores/química , Transactivadores/genética , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Rayos UltravioletaRESUMEN
We identified here a 576 bp rab-like gene (EhrabB) in Entamoeba histolytica. EhrabB is located 332 bp upstream from the start codon of the Ehcp112 encoding gene, but is transcribed from the complementary strand. The EhrabB open reading frame predicts a 192 amino acid polypeptide (EhRabB) with 40-42% identity to Rab proteins, involved in vesicle docking regulation in endo and exocytic pathways of eukaryotic cells. Transcripts of 0.6 and 0.97 kb were detected by the EhrabB probe in northern blot assays. Using specific antibodies, EhRabB was located in small cytoplasmic vesicles by confocal microscopy. During phagocytosis, EhRabB was initially translocated to the plasma membrane and to the phagocytic mouths. The protein diminished after 10 min phagocytosis, suggesting that EhRabB could be participating in the regulation of the endocytosis process.
Asunto(s)
Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios , Transporte Biológico , Northern Blotting , Western Blotting , Membrana Celular/metabolismo , Citoplasma/metabolismo , Genes Protozoarios , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Microscopía Confocal , Datos de Secuencia Molecular , Fagocitosis , Proteínas Recombinantes/inmunología , Transcripción Genética , Proteínas de Unión al GTP rab/química , Proteínas de Unión al GTP rab/inmunologíaRESUMEN
Here, we present evidence that a cysteine protease (EhCP112) and a protein with an adherence domain (EhADH112) form the Entamoeba histolytica 112 kDa adhesin. Immunoelectron microscopy and immunofluorescence assays using monoclonal antibodies (mAbAdh) revealed that, during phagocytosis, the adhesin is translocated from the plasma membrane to phagocytic vacuoles. mAbAdh inhibited 54% adherence, 41% phagocytosis, and 35% and 62% destruction of MDCK cell monolayers by live trophozoites and their extracts respectively. We cloned a 3587 bp DNA fragment (Eh112 ) with two open reading frames (ORFs) separated by a 188 bp non-coding region. The ORF at the 5' end (Ehcp112 ) encodes a protein with a cysteine protease active site, a transmembranal segment and an RGD motif. The second ORF (Ehadh112 ) encodes a protein recognized by mAbAdh with three putative transmembranal segments and four glycosylation sites. Northern blot, primer extension and Southern blot experiments revealed that Ehcp112 and Ehadh112 are two adjacent genes in DNA. Ehcp112 and Ehadh112 genes were expressed in bacteria. The recombinant peptides presented protease activity and inhibited adherence and phagocytosis, respectively, and both were recognized by mAbAdh. The EhCP112 and EhADH112 peptides could be joined by covalent or strong electrostatic forces, which are not broken during phagocytosis.
Asunto(s)
Adhesinas Bacterianas , Proteínas Bacterianas , Cisteína Endopeptidasas/genética , Entamoeba histolytica/enzimología , Lectinas , Glicoproteínas de Membrana/genética , Proteínas Protozoarias/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/farmacología , Adhesión Celular , Línea Celular , Mapeo Cromosómico , Clonación Molecular , Cisteína Endopeptidasas/química , Perros , Entamoeba histolytica/ultraestructura , Técnica del Anticuerpo Fluorescente , Glicoproteínas de Membrana/química , Proteínas de la Membrana/química , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Fragmentos de Péptidos/farmacología , Fagocitosis , Proteínas Protozoarias/química , ARN Mensajero/metabolismo , Alineación de SecuenciaAsunto(s)
Antígenos de Protozoos/genética , Antígenos de Superficie/genética , Entamoeba histolytica/genética , Genes Protozoarios , Lectinas , Glicoproteínas de Membrana/genética , Estructura Terciaria de Proteína , Proteínas Protozoarias/genética , Animales , Antígenos de Protozoos/metabolismo , Antígenos de Superficie/metabolismo , Adhesión Celular/fisiología , Entamoeba histolytica/metabolismo , Glicoproteínas de Membrana/metabolismo , Peso Molecular , Unión Proteica , Proteínas Protozoarias/metabolismoRESUMEN
We identified some gene linkage groups in Entamoeba histolytica using a 4-M urea improved transversal alternating field electrophoresis (TAFE) method. Complex rosette-structured DNA molecules were found trapped along the gel lanes, explaining the fuzziness of the patterns. Using several episomal probes, including 16 S, 5.8 S, and 25 S ribosomal (r)Dna genes, an autonomous replication sequence (ARS), and EhVR1, we identified a complete ribosomal episome linkage group (CELG) at the 1.2-Mb position. Three other incomplete groups were found: IELG-1, formed by EhVR1, 16 S, 5.8 S, and 25 S genes; IELG-2 formed by EhVR1, 16 S and 25 S; and IELG-3 formed only by 5.8 S. Ehadh3, Ehpfo, and Ehredox genes migrated at the 1.8-Mb position, forming the non-ribosomal linkage group, NRLG-1.8, while the Ehenl-1 gene migrated at 1.6 Mb forming the NRLG-1.6 group. Ehhk was located at 1.2, 0.8, and 0.17 Mb in three different groups: NRLG-1.2, IELG-3-0.8, and NRLG-0.17. Putative lineal chromosomes were also identified using an heterologous telomeric probe. By in situ hybridization experiments, the rDNA and Ehhk genes were located in both nucleus and cytoplasm, while the Ehpfo and Ehredox genes were found mainly in the nucleus. We propose a model hypothezising that the 16 S and 25 S genes are in a linear molecule, duplicated in two inverted repeats, which may be looped out of the linear DNA to form an episome probably lacking or not the 5.8 S sequence, which could be added later by recombination.
Asunto(s)
Entamoeba histolytica/genética , Ligamiento Genético , Animales , Mapeo Cromosómico , Sondas de ADN , ADN Protozoario/genética , ADN Ribosómico/genética , Electroforesis en Gel de Agar , Genes Protozoarios , Hexoquinasa/genética , Cariotipificación , Cetona Oxidorreductasas/genética , Datos de Secuencia Molecular , Oxidorreductasas/genética , Piruvato-Sintasa , Ribonucleasas , UreaRESUMEN
Although several factors are involved in the invasive behavior of E. histolytica, proteinases seem to play a key role. Different proteinases have been found in virulent trophozoites of this parasite. Cytosols of clones A, 32-1 462-1 and L-6 of E. histolytica exhibiting various degrees of virulence were used to study the activity of trypsin-like, plasminogen activator and cathepsin B neutral proteinases with specific synthetic oligopeptides. Cathepsin-B like activity showed the highest values in highly virulent clone A, which is derived from virulent strain HM1:IMSS. On the contrary, non virulent clones had very low activity. Clone L-6, a non virulent subclone of strain HM1:IMSS, retained some cathepsin B-like activity. Trypsin-like and plasminogen activator assays revealed low activity and no differences between virulent and non-virulent clones were found. It is concluded that the Arg-Arg-thiol proteinase (Cathepsin B-like) is a good virulence marker.
Asunto(s)
Endopeptidasas/análisis , Entamoeba histolytica/enzimología , Proteínas Protozoarias/análisis , Secuencia de Aminoácidos , Animales , Catepsina B , Entamoeba histolytica/patogenicidad , Datos de Secuencia Molecular , Activadores Plasminogénicos , Especificidad de la Especie , Especificidad por Sustrato , Tripsina , VirulenciaRESUMEN
The 112 kDa adhesin of E. histolytica is directly involved in the cytopathogenic activity of the parasite. We describe here the purification of the 112 kDa protein by electroelution and immunoaffinity chromatography using a monoclonal antibody against the adhesin. Two proteins of 70 and 50 kDa were eluted from the immunoaffinity column along with the 112 kDa adhesin. The three proteins were recognized by monospecific polyclonal antibodies against the adhesin. The same peptides (72 and 56 kDa) were also observed after incubation of the purified intact adhesin in diethylamine buffer. Proteins of 112 and 72 kDa were found to have protease activity, evidenced by their ability to degrade gelatin. Our results indicate that the 112 kDa adhesin was specifically broken down into two polypeptides of 50-56 and 70-72 kDa. The significance of this in vivo is as yet unclear. The adhesin has proteolytic activity, which is retained in the 70-72 kDa polypeptide but not in the 50-56 kDa one.