Natural variation within the principal arginine‐specific protease gene, prpR1, of Porphyromonas gingivalis

Abstract

RI, one of the major extracellular arginine-specific proteases of Porphyromonas gingivalis is a heterodimer composed of catalytic (alpha) and adhesin (beta) chains, encoded by the gene prpR1. The distribution of prpR1 and its variation within 43 isolates of P. gingivalis was determined. Chromosomal DNA was digested with Sma I and probed with a 32P-labeled DNA fragment from within the coding region for the alpha component of P. gingivalis W50. All isolates gave the expected 3.2 kb band, corresponding to the coding region for the alpha and beta components. The presence of a second locus (prR2) homologous to the alpha region of prpR1 was also detected. The 1.7-kb alpha coding region of prpR1 was amplified for subsequent restriction analysis. Following Taq I restriction all isolates gave identical patterns. With Rsa I, the majority of isolates (77%) could be placed into a single group. In conclusion, the prpR1 and prR2 loci are maintained in natural populations of P. gingivalis, and only minor polymorphism is detectable within the catalytic domain.

Identification and Characterization of the Capsular Polysaccharide (K-Antigen) Locus of Porphyromonas gingivalis

“…As a control for these PCR experiments, we also amplified the 1.7-kb region encoding the ␣ catalytic domain of Arg-gingpain A protease, rgpA. This gene could be amplified from 18 of the 20 strains (data not shown); the other two were negative probably because of minor variations in nucleotide sequences at the primer target sites within the genomes; rgpA is known to be highly conserved in clinical and laboratory strains of P. gingivalis (4). The other candidate capsule locus identified by bioinformatics analysis of the P. gingivalis genome, at PG1135-PG1142, appears highly conserved.…”

Genotipificación de los genes rgpA y kgp que codifican para las gingipaínas de Porphyromonas gingivalis

“…(19) , cuyas secuencias fueron F 5' AGT GAG CGA AAC TTC GGA GC 3' y R 5'-GGT ATC ACT GGG TAT AAC CTG TCC-3'. El volumen final de cada reacción fue 50 μl, los cuales contenían 1X de buffer de PCR, 2.5 mM de MgCl2, 0.2 mM de una mezcla equimolar de dNTP, 1μM de cada uno de los partidores, 1.5 U de Taq DNA polimerasa (Roche Diagnostics GmbH, Mannheim, Germany) y 200 ng de DNA genómico de P. gingivalis.…”

“…Todos los aislados genotipificados (100%) para el gen rgpA presentaron el mismo patrón electroforético, posterior a la restricción con Rsa I. Este patrón era similar al patrón electroforético A descrito por Allaker y cols. (19) . Como se observa en la Figura 1, de la digestión con la enzima Rsa I del amplicón de 1.7 kb, se obtuvieron 7 fragmentos de 552, 543, 171, 159, 117, 111 y 49 bp lo que corresponde al patrón A. Cabe destacar que si bien no se alcanzó a visualizar el fragmento de 49 bp, su presencia se comprobó analizando la secuenciación del amplicón del gen rgpA (datos no mostrados).…”

“…Allaker y cols. (19) , utilizando análisis de restricción, describieron 3 patrones electroforéticos (patrones A, B y C) para el gen rgpA y en otro estudio posterior Beikler y cols. (20) , a través de la misma metodología, identificaron 2 genotipos para el gen kgp.…”

Porphyromonas gingivalis infection in gestational diabetes mellitus and survival in tobacco smokers.

“…Gingipains are cysteine proteases that can cleave the proteins at arginine and lysine specific sites [95]. A variation on the genotype level of the proteolytic enzymes (gingipains) has been reported [96,97]. P. gingivalis produces Lys-X and Arg-X peptide bonds specific proteases (Kgp, RgpA, and RgpB gingipains) which are considered as a major virulence determinant.…”