Generation of Lys-gingipain protease activity in Porphyromonas gingivalis W50 is independent of Arg-gingipain protease activities

Abstract

Porphyromonas gingivalis, a black-pigmenting anaerobe implicated in the aetiology of periodontal disease, contains two loci, rgpA and rgpB, encoding the extracellular Arg-X specific proteases (RGPs, Arg-gingipains), and kgp, which encodes a Lys-X specific protease (KGP, Lys-gingipain). The rgpA and kgp genes encode polyproteins comprising pro-peptide and catalytic domain with large N-and C-terminal extensions which require proteolytic processing at several Arg and Lys residues to generate mature enzymes. The product of rgpB contains only a pro-peptide and the catalytic domain which requires processing at an Arg residue to generate active enzyme. An rgpA rgpB double mutant (E8) of P. gingivalis was constructed to study the role of RGPs in the processing of KGP. A kgp mutant (K1A) was also studied to investigate the role of KGP in the generation of RGPs. E8 was stable in the absence of the antibiotics tetracycline and clindamycin (selection markers for rgpA and rgpB, respectively) and exhibited the same pigmentation, colony morphology and identical growth rates to the parent W50 strain in the absence of antibiotics, in both complex and chemically defined media. The KGP activity of E8, grown in the absence of tetracycline, in whole cultures and in culture supernatants (up to 6 d) was identical to levels in W50. However, in the presence of tetracycline in the growth medium, the level of KGP was reduced to 50 % of levels present in whole cultures of W50. Since tetracycline had no effect on RGP or KGP activity when incorporated into assay buffer, this effect is most likely to be on the synthesis of Kgp polypeptide. K1A was also stable in the absence of antibiotics but was unable to pigment, and remained straw-coloured throughout growth. RGP activity in whole cultures of K1A was identical to levels in W50, but RGP activity in 6 d culture supernatants was reduced to 50 % of levels present in W50. Thus, although KGP is not required for generation of RGP activity from RgpA and RgpB polypeptides, its absence affects the release/transport of RGP into culture supernatant.

Highly Specific Protease-Based Approach for Detection of Porphyromonas gingivalis in Diagnosis of Periodontitis

“…It was found that leupeptin inhibited the cleavage of all substrates, except for BikKam14, the only substrate in which no arginine residue is present (Table 4). This finding is in agreement with findings by Aduse-Opoku and coworkers, who observed inhibition of Arg-gingipain activity in the presence of leupeptin, whereas no effect on Lys-gingipain activity was observed (1). A literature search revealed that the amidolytic activity of both Argand Lys-gingipain on L-BApNA is stimulated by the addition of glycyl-glycine (5, 7).…”

“…1). In addition to the absence of Arg-gingipain in ⌬Rgp, it is known that ⌬Rgp strains secrete Lys-gingipain less efficiently (1,41). This was confirmed by the decreased proteolytic activity of ⌬Rgp culture supernatant on the BikKam14 substrate (Fig.…”

LuxS-dependent quorum sensing in Porphyromonas gingivalis modulates protease and haemagglutinin activities but is not essential for virulence

“…or 1i10"" c.f.u. of P. gingivalis W50, the isogenic luxS mutant or the kgp mutant K1A (Aduse-Opoku et al, 2000). K1A is known to be attenuated in this animal model (M. A. Curtis, unpublished data).…”

Major outer membrane proteins and proteolytic processing of RgpA and Kgp of Porphyromonas gingivalis W50

“…53978) and the mutant W50 strain K1A (kgp : :erm), lacking the Lys-Xaaspecific proteinase Kgp [19], were obtained from the culture collection of the School of Dental Science, The University of Melbourne. Strains W50, K1A and W50AB (see below) were grown anaerobically and maintained ( 10 passages) on horse blood agar (HBA ; Oxoid Blood Agar Base 2, Oxoid, Basingstoke, U.K.) supplemented with 10 % (v\v) defibrinated horse blood, with weekly subculturing.…”

“…The residue preceding the N-terminus of most of the domains is arginine, suggesting the involvement of the arginine-specific proteinases, RgpA and RgpB, in N-terminal processing. To investigate the involvement of these enzymes in N-and Cterminal processing, OMs were prepared from mutant strains lacking either a functional Kgp (K1A) [19] or both RgpA and RgpB in a double mutant (W50AB) that was constructed by insertional inactivation for this study. The correct insertion of the antibiotic-resistance cassettes into the rgpA and rgpB genes in W50AB was confirmed by Southern blot analysis (results not shown).…”