A CELL-ASSOCIATED PROTEIN COMPLEX OF PORPHYROMONAS-GINGIVALIS W50 COMPOSED OF ARG-SPECIFIC AND LYS-SPECIFIC CYSTEINE PROTEINASES AND ADHESINS

Porphyromonas gingivalis has been associated with the development of a dult periodontitis and cysteine proteinases with trypsin-like specific ity have been implicated as major virulence factors. We have extracted the major cell-associated trypsin-like proteolytic activity of P. gin givalis W50 using mild sonication. Anion-exchange and gel-filtration F PLC of the sonicate revealed that Arg- and Lys-specific proteinase act ivity was associated with a 300 kDa complex which could be dissociated into seven bands (48, 45, 44, 39, 27, 17 and 15 kDa) by SDS-PACE with the 44 kDa band containing two different proteins as shown by N-termi nal sequence analysis. On further chromatography of the 300 kDa comple x on Arg-Sepharose the majority of the complex eluted from the affinit y column as an undissociated complex. However, a small amount dissocia ted such that the Lys- and Arg-specific activities could be separated by eluting first with lysine then arginine, respectively. The 45 kDa p rotein of the complex was purified by further anion-exchange FPLC in t he presence of octyl-beta-D-glucopyranoside and was shown to be an Arg -specific, thiol-activated, calcium-stabilized cysteine proteinase. Th e 48 kDa protein was also further purified in a similar fashion and sh own to be a Lys-specific cysteine proteinase that was not inhibited by EDTA. The two 44 kDa and the 39, 27, 17 and 15 kDa proteins of the co mplex exhibit amino acid sequence homology and are proposed to be haem agglutinins/adhesins. The 45 kDa Arg-specific proteinase and one of th e 44 kDa adhesins as well as the 15, 17 and 27 kDa adhesins are proces sed from the single polyprotein encoded by the gene designated prtR, w ith all proteins preceded by an Arg or Lys residue within the polyprot ein. Similarly, the 48 kDa Lys-specific proteinase, the 39 and 15 kDa adhesins as well as the other 44 kDa adhesin of the 300 kDa complex ar e encoded by a single gene designated prtK, with all proteins preceded by an Arg or Lys residue within the polyprotein. The 39, 15 and 44 kD a adhesins of PrtK all exhibit high homology with the 44, 15, 17 and 2 7 kDa adhesins encoded by prtR, particularly the 15 kDa proteins which are identical. The cell-associated proteinase-adhesin complex, design ated PrtR-PrtK, is therefore composed of the two gene products, the ma ture PrtR (160 kDa) and mature PrtK (163 kDa) that are further proteol ytically processed (most likely autolytically) to release proteinase a nd adhesin domains that remain non-covalently associated. The fully pr ocessed PrtR-PrtK complex comprises the cysteine proteinases PrtR45 an d PrtK48 and seven sequence-related adhesin molecules, PrtR44, PrtR15, PrtR17, PrtR27 and PrtK39, PrtK15 and PrtK44. We propose that this pr oteinase-adhesin complex is a major virulence factor for P. gingivalis involved in the evasion of host defence and in the assimilation of ha em and peptides.