CONSTRUCTION AND CHARACTERIZATION OF ARGININE-SPECIFIC CYSTEINE PROTEINASE (ARG-GINGIPAIN)-DEFICIENT MUTANTS OF PORPHYROMONAS-GINGIVALIS - EVIDENCE FOR SIGNIFICANT CONTRIBUTION OF ARG-GINGIPAIN TO VIRULENCE

Arginine-specific cysteine proteinase (Arg-gingipain; formerly, arging ipain) is one of the major extracellular proteinases produced by the o ral anaerobic bacterium Porphyromonas gingivalis, To determine whether Arg-gingipain is important for periodontopathogenicity of the organis m, Arg-gingipain-deficient mutants were constructed via gene disruptio n by use of suicide plasmid systems, First, Southern hybridization ana lyses suggested that two separate Arg-gingipain-encoding genes designa ted rgpA and rgpB existed on 12.5- and 7.8-kilobase pair HindIII chrom osomal fragments of P. gingivalis ATCC33277, respectively, rgpA and rg pB single mutants were constructed by mobilization of a suicide plasmi d, Then, an rgpA rgpB double mutant was isolated by electroporation wi th a second suicide plasmid, No proteolytic activity for Arg-gingipain was ob served in either the cell extract or the culture supernatant o f the rgpA rgpB mutant, The chemiluminescence response of polymorphonu clear leukocytes, which is closely related to their bactericidal funct ion, was not inhibited by the culture supernatant of the rgpA rgpB mut ant, while the wild type parent showed a significant inhibition of the response, The result suggests that Arg-gingipain is responsible for d isruption of the function of polymorphonuclear leukocytes, In addition , the rgpA rgpB double mutations caused a marked decrease in the hemag glutination of P. gingivalis, indicating that a major part of the hema gglutinin activity of the organism is associated with the two genes, T hese findings demonstrate that Arg-gingipain makes a significant contr ibution to the virulence of P. gingivalis.