Construction and characterization of Arginine-specific cysteine proteinase (Arg-gingipain)-deficient mutants of Porphyromonas gingivalis: Evidence for significant contribution of Arg-gingipain to virulence

Koji Nakayama, Tomoko Kadowaki, Kuniaki Okamoto, Kenji Yamamoto

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206 Citations (Scopus)

Abstract

Arginine-specific cysteine proteinase (Arg-gingipain; formerly, argingipain) is one of the major extracellular proteinases produced by the oral anaerobic bacterium Porphyromonas gingivalis. To determine whether Arg-gingipain is important for periodontopathogenicity of the organism, Arg-gingipain-deficient mutants were constructed via gene disruption by use of suicide plasmid systems. First, Southern hybridization analyses suggested that two separate Arg-gingipain-encoding genes designated rgpA and rgpB existed on 12.5- and 7.8-kilobase pair HindIII chromosomal fragments of P. gingivalis ATCC33277, respectively. rgpA and rgpB single mutants were constructed by mobilization of a suicide plasmid. Then, an rgpA rgpB double mutant was isolated by electroporation with a second suicide plasmid. No proteolytic activity for Arg-gingipain was observed in either the cell extract or the culture supernatant of the rgpA rgpB mutant. The chemiluminescence response of polymorphonuclear leukocytes, which is closely related to their bactericidal function, was not inhibited by the culture supernatant of the rgpA rgpB mutant, while the wild type parent showed a significant inhibition of the response. The result suggests that Arg-gingipain is responsible for disruption of the function of polymorphonuclear leukocytes. In addition, the rgpA rgpB double mutations caused a marked decrease in the hemagglutination of P. gingivalis, indicating that a major part of the hemagglutinin activity of the organism is associated with the two genes. These findings demonstrate that Arg-gingipain makes a significant contribution to the virulence of P. gingivalis.

Original languageEnglish
Pages (from-to)23619-23626
Number of pages8
JournalJournal of Biological Chemistry
Volume270
Issue number40
Publication statusPublished - Oct 6 1995
Externally publishedYes

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Porphyromonas gingivalis
Cysteine Proteases
Virulence
Arginine
Suicide
Plasmids
Neutrophils
Genes
Gene encoding
Electroporation
Chemiluminescence
Anaerobic Bacteria
Hemagglutination
Hemagglutinins
Luminescence
Porphyromonas gingivalis argingipain
Cell Extracts
Bacteria
Peptide Hydrolases
Mutation

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Construction and characterization of Arginine-specific cysteine proteinase (Arg-gingipain)-deficient mutants of Porphyromonas gingivalis: Evidence for significant contribution of Arg-gingipain to virulence",
abstract = "Arginine-specific cysteine proteinase (Arg-gingipain; formerly, argingipain) is one of the major extracellular proteinases produced by the oral anaerobic bacterium Porphyromonas gingivalis. To determine whether Arg-gingipain is important for periodontopathogenicity of the organism, Arg-gingipain-deficient mutants were constructed via gene disruption by use of suicide plasmid systems. First, Southern hybridization analyses suggested that two separate Arg-gingipain-encoding genes designated rgpA and rgpB existed on 12.5- and 7.8-kilobase pair HindIII chromosomal fragments of P. gingivalis ATCC33277, respectively. rgpA and rgpB single mutants were constructed by mobilization of a suicide plasmid. Then, an rgpA rgpB double mutant was isolated by electroporation with a second suicide plasmid. No proteolytic activity for Arg-gingipain was observed in either the cell extract or the culture supernatant of the rgpA rgpB mutant. The chemiluminescence response of polymorphonuclear leukocytes, which is closely related to their bactericidal function, was not inhibited by the culture supernatant of the rgpA rgpB mutant, while the wild type parent showed a significant inhibition of the response. The result suggests that Arg-gingipain is responsible for disruption of the function of polymorphonuclear leukocytes. In addition, the rgpA rgpB double mutations caused a marked decrease in the hemagglutination of P. gingivalis, indicating that a major part of the hemagglutinin activity of the organism is associated with the two genes. These findings demonstrate that Arg-gingipain makes a significant contribution to the virulence of P. gingivalis.",
author = "Koji Nakayama and Tomoko Kadowaki and Kuniaki Okamoto and Kenji Yamamoto",
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T1 - Construction and characterization of Arginine-specific cysteine proteinase (Arg-gingipain)-deficient mutants of Porphyromonas gingivalis

T2 - Evidence for significant contribution of Arg-gingipain to virulence

AU - Nakayama, Koji

AU - Kadowaki, Tomoko

AU - Okamoto, Kuniaki

AU - Yamamoto, Kenji

PY - 1995/10/6

Y1 - 1995/10/6

N2 - Arginine-specific cysteine proteinase (Arg-gingipain; formerly, argingipain) is one of the major extracellular proteinases produced by the oral anaerobic bacterium Porphyromonas gingivalis. To determine whether Arg-gingipain is important for periodontopathogenicity of the organism, Arg-gingipain-deficient mutants were constructed via gene disruption by use of suicide plasmid systems. First, Southern hybridization analyses suggested that two separate Arg-gingipain-encoding genes designated rgpA and rgpB existed on 12.5- and 7.8-kilobase pair HindIII chromosomal fragments of P. gingivalis ATCC33277, respectively. rgpA and rgpB single mutants were constructed by mobilization of a suicide plasmid. Then, an rgpA rgpB double mutant was isolated by electroporation with a second suicide plasmid. No proteolytic activity for Arg-gingipain was observed in either the cell extract or the culture supernatant of the rgpA rgpB mutant. The chemiluminescence response of polymorphonuclear leukocytes, which is closely related to their bactericidal function, was not inhibited by the culture supernatant of the rgpA rgpB mutant, while the wild type parent showed a significant inhibition of the response. The result suggests that Arg-gingipain is responsible for disruption of the function of polymorphonuclear leukocytes. In addition, the rgpA rgpB double mutations caused a marked decrease in the hemagglutination of P. gingivalis, indicating that a major part of the hemagglutinin activity of the organism is associated with the two genes. These findings demonstrate that Arg-gingipain makes a significant contribution to the virulence of P. gingivalis.

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