RESUMEN
Previously, metalloproteinase was isolated and identified from Trichomonas vaginalis, belonging to the aminopeptidase P-like metalloproteinase subfamily A/B, family M24 of clan MG, named TvMP50. The native and recombinant TvMP50 showed proteolytic activity, determined by gelatin zymogram, and a 50 kDa band, suggesting that TvMP50 is a monomeric active enzyme. This was an unexpected finding since other Xaa-Pro aminopeptidases/prolidases are active as a biological unit formed by dimers/tetramers. In this study, the evolutionary history of TvMP50 and the preliminary crystal structure of the recombinant enzyme determined at 3.4 Å resolution is reported. TvMP50 was shown to be a type of putative, eukaryotic, monomeric aminopeptidase P, and the crystallographic coordinates showed a monomer on a "pseudo-homodimer" array on the asymmetric unit that resembles the quaternary structure of the M24B dimeric family and suggests a homodimeric aminopeptidase P-like enzyme as a likely ancestor. Interestingly, TvMP50 had a modified N-terminal region compared with other Xaa-Pro aminopeptidases/prolidases with three-dimensional structures; however, the formation of the standard dimer is structurally unstable in aqueous solution, and a comparably reduced number of hydrogen bridges and lack of saline bridges were found between subunits A/B, which could explain why TvMP50 portrays monomeric functionality. Additionally, we found that the Parabasalia group contains two protein lineages with a "pita bread" fold; the ancestral monomeric group 1 was probably derived from an ancestral dimeric aminopeptidase P-type enzyme, and group 2 has a probable dimeric kind of ancestral eukaryotic prolidase lineage. The implications of such hypotheses are also presented.
Asunto(s)
Aminopeptidasas/metabolismo , Metaloproteasas/metabolismo , Proteínas Protozoarias/metabolismo , Trichomonas vaginalis/enzimología , Secuencia de Aminoácidos , Aminopeptidasas/química , Aminopeptidasas/genética , Cristalografía por Rayos X , Dipeptidasas/química , Dipeptidasas/genética , Dipeptidasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Metaloproteasas/química , Metaloproteasas/genética , Peso Molecular , Filogenia , Conformación Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Trichomonas vaginalis/clasificación , Trichomonas vaginalis/genéticaRESUMEN
Trichomonas vaginalis is a protozoan that parasitizes the human urogenital tract causing trichomoniasis, the most common non-viral sexually transmitted disease. The parasite has unique genomic characteristics such as a large genome size and expanded gene families. Ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) is an enzyme responsible for hydrolyzing nucleoside tri- and diphosphates and has already been biochemically characterized in T. vaginalis. Considering the important role of this enzyme in the production of extracellular adenosine for parasite uptake, we evaluated the gene expression of five putative NTPDases in T. vaginalis. We showed that all five putative TvNTPDase genes (TvNTPDase1-5) were expressed by both fresh clinical and long-term grown isolates. The amino acid alignment predicted the presence of the five crucial apyrase conserved regions, transmembrane domains, signal peptides, phosphorylation and catalytic sites. Moreover, a phylogenetic analysis showed that TvNTPDase sequences make up a clade with NTPDases intracellularly located. Biochemical NTPDase activity (ATP and ADP hydrolysis) is responsive to the serum-restrictive conditions and the gene expression of TvNTPDases was mostly increased, mainly TvNTPDase2 and TvNTPDase4, although there was not a clear pattern of expression among them. In summary, the present report demonstrates the gene expression patterns of predicted NTPDases in T. vaginalis.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , Trichomonas vaginalis/enzimología , Trichomonas vaginalis/genética , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Femenino , Humanos , Datos de Secuencia Molecular , Filogenia , Estructura Terciaria de Proteína , Pirofosfatasas/química , Alineación de Secuencia , Vaginitis por Trichomonas/microbiología , Trichomonas vaginalis/química , Trichomonas vaginalis/clasificaciónAsunto(s)
Antagonistas de Receptores Adrenérgicos alfa 1/uso terapéutico , Técnicas de Laboratorio Clínico/métodos , Doxazosina/uso terapéutico , Tricomoniasis/tratamiento farmacológico , Trichomonas vaginalis/aislamiento & purificación , Orina/parasitología , Anciano , Eritrocitos/ultraestructura , Humanos , Masculino , Resultado del Tratamiento , Tricomoniasis/diagnóstico , Tricomoniasis/orina , Trichomonas vaginalis/clasificaciónRESUMEN
Trichomonas vaginalis can be infected with double-stranded RNA (dsRNA) viruses known as T. vaginalis virus (TVV). This viral infection may have important implications for trichomonal virulence and disease pathogenesis. The objective of this study was to determine the possible correlation between the T. vaginalis genetic polymorphism and the isolate infection with TVV. The Random Amplified Polymorphic DNA (RAPD) technique was used to determine genetic differences among 37 isolates of T. vaginalis using a panel of 30 random primers and these genetic data were correlated with the infection of isolates with TVV. The trees drawn based on RAPD data showed significantly association with the presence of TVV (P = 0.028) demonstrating the existence of concordance between the genetic relatedness and the presence of TVV in T. vaginalis isolates. This result could point to a predisposition of T. vaginalis for the viral enters and/or survival.
Asunto(s)
Polimorfismo Genético , Totiviridae/fisiología , Trichomonas vaginalis/genética , Trichomonas vaginalis/virología , Adolescente , Femenino , Humanos , Filogenia , Técnica del ADN Polimorfo Amplificado Aleatorio , Vaginitis por Trichomonas/parasitología , Trichomonas vaginalis/clasificaciónRESUMEN
Trichomoniasis presents a broad spectrum of clinical patterns ranging from asymptomatic to severe vaginitis and cervicitis. Despite its importance, very little is known about the genetic relatedness of its causative agent, Trichomonas vaginalis, and the clinical phenotypes. To address this question, analysis of restriction length polymorphism (RFLP) within the intergenic spacer of the ribosomal DNA (IGS) from 60 clinically defined isolates of T. vaginalis was performed. This is the first description of the IGS polymorphism of T. vaginalis. As expected, a considerable number of patients were asymptomatic (28%) while only 12% presented both leukorrhea and macular colpitis, the most evident symptoms of trichomoniasis. The IGS-RFLP with the use of eight restriction enzymes showed absence of correlation between the genetic relatedness of the isolates and symptomatology. Further studies are necessary to evaluate the importance of the IGS polymorphism to the parasite virulence and clinical phenotype.
Asunto(s)
ADN Espaciador Ribosómico/genética , Polimorfismo de Longitud del Fragmento de Restricción , Vaginitis por Trichomonas/parasitología , Trichomonas vaginalis/genética , Animales , Cuello del Útero/patología , ADN Espaciador Ribosómico/química , Dispareunia/parasitología , Femenino , Humanos , Leucorrea/parasitología , Filogenia , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/genética , ARN Ribosómico 28S/genética , Mapeo Restrictivo , Vaginitis por Trichomonas/patología , Vaginitis por Trichomonas/fisiopatología , Trichomonas vaginalis/clasificación , Trichomonas vaginalis/patogenicidad , Trastornos Urinarios/parasitología , Vagina/patología , Vulva/patologíaRESUMEN
Se presenta el caso de una RN de 20 días con diagnóstico presuntivo de sepsis, en la que se destacó infección vaginal y urinaria por Trichomonas vaginales resultante de transmisión vertical
Asunto(s)
Lactante , Humanos , Recién Nacido , Niño , Femenino , Trichomonas vaginalis/clasificación , Transmisión Vertical de Enfermedad Infecciosa/clasificación , Enfermedades Vaginales , Recién Nacido , Trichomonas/clasificaciónRESUMEN
The in vitro cytopathic effect of four strains of Trichomonas vaginalis on cultured epithelial monolayers was analyzed through electrophysiology and electron microscopy. Interaction of trichomonads of two virulent strains (GT-10 and GT-13) with cultured MDCK cell monolayers mounted in Ussing chambers produced a rapid decrease in transepithelial resistance to less than 30% of control values after only 15 min. By 30 min the electrical resistance was practically abolished by the virulent parasites. In contrast, of two attenuated strains of trichomonads (GT-3 and GT-7) analyzed under similar conditions, GT-3 trophozoites required 180 min to reduce transepithelial resistance to 9% of control values, while monolayers in contact with GT-7 parasites still showed 28% of control values at this time of incubation. Sequential scanning electron microscopy confirmed the much faster and widespread cytopathic effect of virulent parasites. In contrast, the slow lytic process produced by attenuated trophozoites was reduced to focal areas of direct contact with epithelial cells. Another difference was found by measurement of the surface charge of the four strains of T. vaginalis by means of cell microelectrophoresis. While the two virulent strains showed a negative surface charge, the two attenuated strains had no detectable surface charge at neutral pH. When parasites were incubated with cationized ferritin and studied with transmission electron microscopy the surface of virulent trichomonads appeared heavily labeled, whereas the surface of attenuated parasites had only sparse and irregular ferritin binding.
Asunto(s)
Extensiones de la Superficie Celular/ultraestructura , Propiedades de Superficie , Trichomonas vaginalis/patogenicidad , Trichomonas vaginalis/ultraestructura , Factores de Virulencia , Animales , Línea Celular , Impedancia Eléctrica , Electroforesis , Electrofisiología , Femenino , Humanos , Ratones , Microscopía Electrónica de Rastreo , Especificidad de la Especie , Vaginitis por Trichomonas/parasitología , Trichomonas vaginalis/clasificaciónRESUMEN
The random amplified polymorphic DNA (RAPD) technique was used to determine genetic differences among isolates of Trichomonas vaginalis and these genetic data were correlated with patient records. A panel of 10 random primers was used to determine the type and extent of intraspecific polymorphism in 40 isolates of T. vaginalis. All primers detected DNA polymorphism among isolates. Numerical analysis of 124 RAPD amplified bands generated by these 10 primers were carried out with the unweighted pair group methods analysis (UPGMA) using Jaccard's Similarity Coefficient and data were used to construct a dendrogram. Four main groups can be distinguished by RAPD data, these groups coincide with four different patient categories (asymptomatic and symptomatic: light, moderate, and severe infection). These patients did not have any concomitant vaginal infection. Each of the four groups can be characterized by specific genetic markers, but a specific 490bp marker was found to be specific for all symptomatic isolates, not the asymptomatic isolates. This is the first description of a possible virulence marker for T. vaginalis. Further studies will be necessary to ascertain the importance and function of this genetic marker in clinical infection.