RESUMEN
Porphyromonas gingivalis es un microorganismo fuertemente asociado con la etiología de la periodontitis. Esta bacteria posee varios factores de virulencia, dentro de los que destacan las gingipaínas, debido a sus múltiples acciones relacionadas con la destrucción de la matriz extracelular del tejido conectivo periodontal, la modulación del sistema inmune del hospedero y la estimulación de la expresión de citoquinas pro-inflamatorias. Estas proteinasas tienen afinidades específicas siendo Arg-gingipaínas (RgpA y RgpB, codificadas por los genes rgpA y rgpB, respectivamente) y Lys-gingipaínas (Kgp, codificada por el gen kgp). Se ha descrito que existen polimorfismos en los genes que codifican para esta proteinasas. El objetivo del presente estudio fue describir la frecuencia de los genotipos identificados para los genes rgpA y kgp en aislados clínicos de P. gingivalis, obtenidos desde pacientes con periodontitis. Para ello se utilizó amplificación por PCR de los genes rgpA y kgp, seguido de análisis de restricción. De un total de 47 aislados provenientes de 4 individuos con periodontitis crónica y 2 con periodontitis agresiva, se genotipificaron 38 aislados para el gen rgpA, exhibiendo la totalidad de éstos el patrón electroforético A (100 por ciento). Para el gen kgp se genotipificaron 43 aislados, presentando 28 de ellos (65.2 por ciento) el perfil electroforético kgp-I y 15 aislados (34.8 por ciento) el perfil kgp-II. En los aislados provenientes de un individuo fue posible apreciar ambos genotipos descritos para el gen kgp. Los resultados indican un predominio del patrón electroforético A (rgpA) y que el genotipo kgp-I fue el más frecuentemente encontrado de los genotipos kgp.
Porphyromonas gingivalis is a microorganism strongly associated with the etiology of periodontitis. This periodontal bacterium possesses an array of virulence factors, among which gingipains have a key importance, being involved with extracellular matrix destruction of periodontal tissues, modulation of host immune response and stimulation in the production of pro-inflammatory cytokines by different types of cells. These proteinases have specific affinities, being Arg-gingipains (RgpA and RgpB, encoded by rgpA and rgpB genes, respectively) and Lys-gingipains (Kgp, encoded by the kgp gene). It has been described that there are polymorphisms in the genes encoding for gingipains. Therefore, the aim of the present study was to describe the frequency of rgpA and kgp genotypes in clinical isolates of P. gingivalis obtained from periodontitis patients. For determining the rgpA and kgp genotypes, we used PCR amplification and restriction analysis. From 47 isolates obtained from 4 individuals with chronic periodontitis and 2 subjects with aggressive periodontitis, 38 were typified for rgpA gene and all exhibited the electrophoretic pattern A (100 percent). For kgp gene, we characterized 43 isolates, 28 of them (65.2 percent) with the kgp-I electrophoretic profile and 15 isolates (34.8 percent) with the kgp-II profile. In the isolates belonging to one individual, we found both genotypes of kgp gene. The results indicate a clear predominance of the electrophoretic pattern A (for rgpA gene) and kgp-I genotype was the most frequently found of the kgp genotypes.
Asunto(s)
Humanos , Adhesinas Bacterianas/genética , Cisteína Endopeptidasas/genética , Porphyromonas gingivalis/aislamiento & purificación , Porphyromonas gingivalis/genética , Amplificación de Genes , Genotipo , Reacción en Cadena de la Polimerasa , Periodontitis/genética , Periodontitis/microbiologíaRESUMEN
Objective:To detect and compare the intensity of gingipain K(Kgp)in culture medium and cell extract of Porphyromonas gingivalis(P.gingivalis)isolates in puberty gingivitis,and then to reveal the possible relationship between Kgp and puberty gingivitis.Methods:36 patients with puberty gingivitis aged from 14 to 17 years were enrolled.Clinical parameters including GI,SBI and PD were evaluated before subgingival plaque samples collection.Subgingival plaque samples were collected and then P.gingivalis isolates were obtained.16S rRNA PCR was used to confirm the presence of P.gingivalis in clinical isolates.Bacteria were cultured in BHI agar base and harvested at the end of log-phase growth.Culture fractions of P.gingivalis(culture medium and cell extracts)were performed with SDS-PAGE and Western blot technique using primary antibody against specific anti-Kgp N-terminal IgG subdomain.The data were statistically analyzed using SPSS 11.5 software.The relationship between the Kgp intensity and the clinical parameters was statistically analyzed using sum rank test.Results:There was positive correlation between the intensity of Kgp N-terminal IgG subdomain and the clinical parameters(P