Characterization of proteinase–adhesin complexes of Porphyromonas gingivalis

Abstract

Proteinase–adhesin complexes ofPorphyromonas gingivaliswild-type and RgpA and Kgp mutants were extracted using a Triton X-114 procedure and purified using arginine-affinity chromatography. The complexes were then characterized by peptide mass fingerprinting (PMF) and their equilibrium binding constants, immunogenicity and ability to induce protection as vaccines in the murine lesion model determined. The Triton X-114 procedure resulted in consistently higher yield and specific activity of the wild-type (wt) complex compared with that produced by the previously published sonication method. PMF and N-terminal sequencing of the purified wt complex showed that it consisted of the previously identified Arg-specific proteinase RgpAcat, the Lys-specific proteinase Kgpcatand adhesin domains RgpAA1, RgpAA2, RgpAA3, KgpA1and KgpA2. However, analysis of the 30 kDa band in the wt complex, previously suggested to be RgpAA4, indicated that this band contained C-terminally truncated KgpA1(which has an identical N-terminus to RgpAA4) as well as the HagAA1* adhesin. Analysis of the Triton X-114 extracted complexes from theP. gingivalisisogenic mutantskgp(RgpA complex) andrgpA(Kgp complex) suggested that the Kgp complex consisted of Kgpcat, KgpA1and KgpA2/HagAA2and that the RgpA complex consisted of RgpAcat, RgpAA1, HagAA1*, RgpAA2and RgpAA3. Each of the complexes was found to have equilibrium binding constants (KD) in the nanomolar range for fibrinogen, fibronectin, haemoglobin, collagen type V and laminin. However, the Triton-wt complex exhibited significantly lowerKDvalues for binding to each host protein compared with the sonication-wt complex, or the Triton-RgpA complex and Triton-Kgp complex. Furthermore, the Triton-wt complex induced a stronger antibody response to the A1 adhesins and tended to be more effective in providing protection in the mouse lesion model compared with the sonication-wt complex.