A cell-associated protein complex of Porphyromonas gingivalis W50 composed of Arg- and Lys-specific cysteine proteinases and adhesins

Abstract

Porphyromonas gingiwalis has been associated with the development of adult periodontitis and cysteine proteinases with trypsin-like specificity have been implicated as major virulence factors. We have extracted the major cellassociated trypsin-like proteolytic activity of P. gingiwalis W50 using mild sonication. Anion-exchange and gel-f iltration FPLC of the sonicate revealed that Arg-and Lys-specific proteinase activity 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-PAGE with the 4 4 kDa band containing two different proteins as shown by N-terminal sequence analysis. On further chromatography of the 300 kDa complex on ArgSepharose the majority of the complex eluted from the affinity column as an undissociated complex. However, a small amount dissociated such that the Lys-and Arg-specific activities could be separated by eluting first with lysine then arginine, respectively. The 45 kDa protein of the complex was purified by further anion-exchange FPLC in the presence of octylpD-glucopyranoside and was shown to be an Arg-specif ic, thiol-activated, calcium-stabilized cysteine proteinase. The 48 kDa protein was also further purified in a similar fashion and shown 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 complex exhibit amino acid sequence homology and are proposed to be haemagglutinindadhesins. The 45 kDa Arg-specif ic proteinase and one of the 44 kDa adhesins as well as the 15,17 and 27 kDa adhesins are processed from the single polyprotein encoded by the gene designated prtR, with all proteins preceded by an Arg or Lys residue within the polyprotein. 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 are 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 kDa adhesins of PrtK all exhibit high homology with the 44,15,17 and 27 kDa adhesins encoded by prtR, particularly the 15 kDa proteins which are identical. The cell-associated proteinase-adhesin complex, designated PrtR-PrtK, is therefore composed of the two gene products, the mature PrtR (160 kDa) and mature PrtK (163 kDa) that are further proteolytically processed (most likely autolytically) to release proteinase and adhesin domains that remain non-covalently associated. The fully processed PrtR-PrtK complex comprises the cysteine proteinases PrtR45 and PrtK48 and seven sequence-related adhesin molecules, PrtR44, PrtRl5, PrtRl7, PrtR27 and PrtK39, PrtK15 and PrtK44. We propose that this proteinase-adhesin complex is a major virulence factor for P. gingiwalis On: Sat, 12 May 2018 21:46:32 P. S. BHOGAL, N. SLAKESKI a n d E. C. REYNOLDS involved in the evasion of host defence and in the assimilation of haem and peptides.

Adhesins encoded by the gingipain genes of Porphyromonas gingivalis are responsible for co-aggregation with Prevotella intermedia

“…Since these complexes are on the cell surface (Bhogal et al, 1997;DeCarlo & Harber, 1997), they may play important roles in the attachment of Por. gingivalis to host-cell surfaces.…”

Characterization of proteinase–adhesin complexes of Porphyromonas gingivalis

“…Using sonication of exponentially growing P. gingivalis W50 cells and three chromatographic procedures, Bhogal et al (1997) purified and characterized the major cell-associated Arg-and Lys-specific proteinases as a 300 kDa protein complex containing the Arg and Lys proteinases and their associated adhesins. It was proposed that, after the proteolytic processing of the RgpA and Kgp polyproteins into individual domains, they aggregate via adhesin-binding motifs (ABMs) to form the non-covalently associated cell-surface complex designated the RgpA-Kgp proteinase-adhesin complex (Bhogal et al, 1997;Slakeski et al, 1998). Recently, Takii et al (2005), using sucrose monolaurate to extract P. gingivalis cells, confirmed the presence of this proteinase-adhesin complex and its component proteins from RgpA and Kgp, identified by Bhogal et al (1997), but suggested that it existed as larger aggregates (660 kDa) in the form of 10 nm diameter particles on the cell surface.…”

“…Furthermore, in animal models, subgingival implantation of P. gingivalis in mice (Baker Depending on the P. gingivalis strain, age of culture and purification method, several forms of RgpA and Kgp, both cell-associated and soluble, have been purified and characterized (reviewed by Potempa et al, 2003). The Arg-and Lys-specific proteinases have been purified as monomeric proteins (Bedi & Williams, 1994;Chen et al, 1992) and as multimeric complexes where the proteinase domains are non-covalently associated with a number of adhesin/ haemagglutinin domains (Bhogal et al, 1997;Pike et al, 1994). Using sonication of exponentially growing P. gingivalis W50 cells and three chromatographic procedures, Bhogal et al (1997) purified and characterized the major cell-associated Arg-and Lys-specific proteinases as a 300 kDa protein complex containing the Arg and Lys proteinases and their associated adhesins.…”

“…The Arg-and Lys-specific proteinases have been purified as monomeric proteins (Bedi & Williams, 1994;Chen et al, 1992) and as multimeric complexes where the proteinase domains are non-covalently associated with a number of adhesin/ haemagglutinin domains (Bhogal et al, 1997;Pike et al, 1994). Using sonication of exponentially growing P. gingivalis W50 cells and three chromatographic procedures, Bhogal et al (1997) purified and characterized the major cell-associated Arg-and Lys-specific proteinases as a 300 kDa protein complex containing the Arg and Lys proteinases and their associated adhesins. It was proposed that, after the proteolytic processing of the RgpA and Kgp polyproteins into individual domains, they aggregate via adhesin-binding motifs (ABMs) to form the non-covalently associated cell-surface complex designated the RgpA-Kgp proteinase-adhesin complex (Bhogal et al, 1997;Slakeski et al, 1998).…”

Gingipain-dependent interactions with the host are important for survival of Porphyromonas gingivalis

“…Collectively, these observations indicate that the mature gingipains are derived from extensive processing from the nascent polypeptide precursors. Studies comparing the gene structure with the N-terminal sequences of different isoforms of the purified gingipain have demonstrated specific post-translational cleavage at Arg-Xaa and Lys-Xaa bonds (20). There is emerging evidence that this post-translational process is achieved by an autoproteolytic mechanism (131).…”