Role of RgpA, RgpB, and Kgp proteinases in virulence of Porphyromonas gingivalis W50 in a murine lesion model

Extracellular Arg-x- and Lys-x-specific cysteine proteinases are considered important virulence factors and pathogenic markers for Porphyromonas gingi valis, a bacterium implicated as a major etiological agent of chronic perio dontitis. Three genes. rgpA, rgpB, and kgp, encode an Arg-x-specific protei nase and adhesins (RgpA), an Arg-x-specific proteinase (RgpB), and a Lys-x- specific proteinase and adhesins (Kgp), respectively. The contribution to p athogenicity of each of the proteinase genes of P. gingivalis W50 was inves tigated in a murine lesion model using isogenic mutants lacking RgpA, RgpB, and Kgp. Whole-cell Arg-x-specific proteolytic activity of both the RgpA(- ) and RgpB(-) isogenic mutants was significantly reduced (3- to 4-fold) rel ative to that of the wild-type W50. However, for the Kgp(-) isogenic mutant , whole-cell Arg-x activity was similar to that of the wild-type strain. Wh ole-cell Lys-x proteolytic activity of the RgpA- and RgpB- mutants was not significantly different from that of wild-type W50, whereas the Kgp- mutant was devoid of Lys-x whole-cell proteolytic activity. Sodium dodecyl sulfat e-polyacrylamide gel electrophoresis and Western blot analysis using protei nase-specific antibodies of cell sonicates of the wild-type and mutant stra ins showed that the proteinase catalytic domain of each of the mutants was not expressed. This analysis further showed that RgpB appeared as 72- and 8 0-kDa bands, and the catalytic domains of RgpA and Kgp appeared as processe d 45-kDa and 48-kDa bands, respectively. In the murine lesion model, mice w ere challenged with three doses of each mutant and wild-type strain. At the lower dose (3.0 x 10(9) viable-cells), no lesions were recorded for each o f the mutants, whereas wild-type W50 induced large ulcerative lesions. At a dose of 6.0 x 10(9) viable-cells, all the mice challenged with the wild-ty pe strain died, whereas mice challenged with the RgpA- and RgpB- isogenic m utants did not die but developed lesions. Mice challenged with the Kgp- iso genic mutant at this dose did not develop lesions. At a 1.2 x 10(10) viable -cell dose, only 40% of mice challenged with the Kgp- mutant developed lesi ons, and these lesions were significantly smaller than lesions induced by t he wild-type strain at the 3.0 x 109 viable-cell dose. All the mice challen ged with the RgpA- mutant died at the 1.2 x 10(10) viable-cell dose, wherea s only 20% died when challenged with the RgpB- mutant at this dose. Wild-ty pe phenotype was restored to the RgpB- mutant by complementation with plasm id pNJR12::rgpB containing the rgpB gene. There was no difference between t he pNJR12::rgpB-complemented RgpB- mutant and the wild-type W50 strain in w hole-cell Arg-x activity, protein profile, or virulence in the murine lesio n model. These results show that the three proteinases, RgpA, RgpB, and Kgp , all contributed to virulence of P. gingivalis W50 in the murine lesion mo del and that the order in which they contributed was Kgp much greater than RgpB greater than or equal to RgpA.