title: Genetic analyses of proteolysis, hemoglobin binding, and hemagglutination of Porphyromonas gingivalis. Construction of mutants with a combination of rgpA, rgpB, kgp, and hagA.
abstract: Porphyromonas gingivalis produces arginine-specific cysteine proteinase (Arg-gingipain, RGP) and lysine-specific cysteine proteinase (Lys-gingipain, KGP) in the extracellular and cell-associated forms. Two separate genes (rgpA and rgpB) and a single gene (kgp) have been found to encode RGP and KGP, respectively. We constructed rgpA rgpB kgp triple mutants by homologous recombination with cloned rgp and kgp DNA interrupted by drug resistance gene markers. The triple mutants showed no RGP or KGP activity in either cell extracts or culture supernatants. The culture supernatants of the triple mutants grown in a rich medium had no proteolytic activity toward bovine serum albumin or gelatin derived from human type I collagen. Moreover, the mutants did not grow in a defined medium containing bovine serum albumin as the sole carbon/energy source. These results indicate that the proteolytic activity of P. gingivalis toward bovine serum albumin and gelatin derived from human type I collagen appears to be attributable to RGP and KGP. The hemagglutinin gene hagA of P. gingivalis possesses the adhesin domain regions responsible for hemagglutination and hemoglobin binding that are also located in the C-terminal regions of rgpA and kgp. A rgpA kgp hagA triple mutant constructed in this study exhibited no hemagglutination using sheep erythrocytes or hemoglobin binding activity, as determined by a solid-phase binding assay with horseradish peroxidase-conjugated human hemoglobin, indicating that the adhesin domains seem to be particularly important for P. gingivalis cells to agglutinate erythrocytes and bind hemoglobin, leading to heme acquisition.
authors.affiliation: Department of Microbiology, Faculty of Dentistry, Kyushu University, Fukuoka 812-8582, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Shi
authors.forename: D B
authors.initials: DB
authors.lastname: Ratnayake
authors.forename: K
authors.initials: K
authors.lastname: Okamoto
authors.forename: N
authors.initials: N
authors.lastname: Abe
authors.forename: K
authors.initials: K
authors.lastname: Yamamoto
authors.forename: K
authors.initials: K
authors.lastname: Nakayama
mesh_terms: D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D002455:Cell Division; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism; D004351:Drug Resistance / Q000235:genetics; D005780:Gelatin; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D000080867:Gingipain Cysteine Endopeptidases; D006384:Hemagglutination; D006388:Hemagglutinins / Q000235:genetics / Q000378:metabolism; D037102:Lectins; D009154:Mutation; D010858:Pigmentation / Q000235:genetics; D016966:Porphyromonas gingivalis / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding
chemical_list: D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins; D037102:Lectins; C114923:hemagglutinin A, Porphyromonas gingivalis; D005780:Gelatin; D003546:Cysteine Endopeptidases