Objectives: The RgpA-Kgp-HagA proteinase-adhesin complex of Porphyromonas gingivalis is a major virulence factor in periodontitis. Functional whole-cell-associated Kgp is required for colony pigmentation on blood agar. Multiple conserved, repetitive sequences, designated adhesin-binding motifs (ABMs), have been identified throughout the complex domains. ABM1 is found in isolation in the RpgA and Kgp catalytic domains, and was suggested to be responsible for the non-covalent association of these domains with the adhesin domains of the complex. This study aimed to investigate the role of ABM1 within the catalytic domain of Kgp (Kgpcat) in the association of Kgpcat with the RgpA-Kgp-HagA complex.
Methods: Three P. gingivalis recombinant strains were generated that produce recombinant Kgp (rKgpcat) with KgpcatABM1 residues scrambled in three regions. Mutant strains were analysed via colony morphology, Kgp activity assays, sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and peptide mass fingerprinting (PMF) mass spectrometry.
Results:
Mutant colonies pigmented slower than the wild-type, indicating the eventual production of functional Kgp. Mutant whole-cells exhibited no Kgp activity in exponential and stationary phase cultures. However, supernatants of mutant stationary phase (3 day) cultures displayed significantly greater Kgp activity than the wild-type (p < 0.5). SDS-PAGE of exponential phase supernatant proteins revealed three abundant proteins that were produced by each mutant but absent from the wild-type supernatants. PMF revealed that these proteins were rKgpcat with partial prodomain sequences, indicating that these strains are defective in incorporation of rKgpcat into the Kgp-RgpA-HagA complex. PMF also revealed that in stationary phase mutant supernatants the non-precursor form of the rKgpcat was present, which is concordant with the increased activity observed and the delayed colony pigmentation.
Conclusions: Rearrangement of the residues in KgpcatABM1 regions prevented incorporation of active rKgpcat into the cell-associated Kgp-RgpA-HagA complex supporting the hypothesis that ABM1 is involved in the non-covalent association of the Kgpcat domain within the complex.