... 1996; revised 9 December, 1996; accepted 19. December, 1996. *For correspondence. E-mail M.A.Curtis@mds. qmw.ac.uk; Tel. (0171) 3770444; Fax (0171) ...
Molecular Microbiology (1997 ) 23(5), 955–965
The prpR1 and prR2 arginine-specific protease genes of Porphyromonas gingivalis W50 produce five biochemically distinct enzymes Minnie Rangarajan, Joseph Aduse-Opoku, Jennifer M. Slaney, Katherine A. Young and Michael A. Curtis* MRC Molecular Pathogenesis Group, Department of Oral Microbiology, St Bartholomew’s and the Royal London School of Medicine and Dentistry, Queen Mary and Westfield College, 32 Newark Street, London E1 2AA.
expression and a maturation pathway which can give rise to a dimer and an unmodifed- or LPS-modified catalytic monomer. Furthermore, RIIA and RIIB, the products of prR2, are exported into the culture supernatant in the absence of prpR1 expression and these forms may also contribute to the pathogenic potential of this organism in destructive disease.
Summary Introduction
The arginine-specific protease activity of Porphyromonas gingivalis is considered to be an important factor in the pathogenic potential of this organism in destructive periodontal disease. Multiple forms of closely related Arg-x proteases are present in the culture supernatants of P. gingivalis W50. RI is a heterodimer (a/b) in which the catalytic a chain is associated with a second b chain which functions as a haemagglutinin. RIA is a single-chain enzyme (a) and RIB is a highly post-translationally lipid-modified enzyme (LPS-a) with reduced solubility compared to the other two forms. The N-terminal sequence of the a chain of all three forms is identical, suggesting that all these enzymes may arise by differential processing of the prpR1 (protease polyprotein for RI ). In the present study we constructed a prpR1¹ strain of P. gingivalis W50 by insertional gene inactivation and characterized the residual extracellular Arg-x protease activity of the resulting mutant. Loss of prpR1 expression led to the abolition of RI, RIA and RIB but the total Arg-x activity in the supernatant of this strain was reduced by only c. 66%. The remaining activity was composed of two novel forms of Arg-x protease (RIIA and RIIB) which appeared to be structurally and kinetically almost identical to RIA and RIB, respectively, except for two amino acid differences in the N-terminus at position 8 (Q→E) and position 17 (A→P) and with respect to their stability to high pH. Confirmation that RIIA and RIIB are the products of a homologous locus ( prR2 ) was obtained by cloning and sequencing the prR2 which showed the predicted substitutions in the deduced translation. These data indicate that RI, RIA and RIB are produced by prpR1
Porphyromonas gingivalis, an anerobic Gram-negative rod, is recognized as an important agent in the aetiology of chronic adult periodontal disease – a destructive inflammatory condition of the tooth-supporting tissues (Slots et al., 1986; Maiden et al., 1990) which affects approximately 10% of most populations studied to date (Johnson et al., 1988). The mechanisms of tissue destruction which can occur at sites in the periodontium colonized by P. gingivalis remain ill-defined. However, extracellular proteolytic activities are frequently implicated in the pathogenic potential of this bacterium in terms of both the deregulation of the host defences and inflammatory response (Imamura et al., 1994; Nilsson et al., 1985) and in the direct proteolytic degradation of host connective-tissue proteins (Kadowaki et al., 1994). Proteolytic enzymes encompassing a number of peptide-bond specificities have been purified from culture supernatants. However, those enzymes with specificity for peptide bonds with arginyl residues in the P1 position have received most attention primarily because of their relative abundance in supernatants of batch culturegrown cells (Curtis et al., 1993) and because of the wide range of host proteins susceptible to proteolytic degradation and inactivation by these activites. Several genes for arginine-specific proteases of P. gingivalis have been cloned and sequenced and include rgp-1 (P. gingivalis HG66), agp (381), prtR (W50), prpR1 (W50), and prtH (W83) (Pavloff et al., 1995; Okamoto et al., 1995; Kirszbaum et al., 1995; Aduse-Opoku et al., 1995; Fletcher et al., 1994). Whilst there are some significant differences in size, physical organization and nucleotide sequence, a general consensus is now emerging that all these genes probably represent a single genetic locus which is either strain variable or has undergone recombination between repeat regions prior to or during cloning (Potempa et al., 1995a; Barkocy-Gallagher et al., 1996). However, based
Received 8 August, 1996; revised 9 December, 1996; accepted 19 December, 1996. *For correspondence. E-mail M.A.Curtis@mds. qmw.ac.uk; Tel. (0171) 3770444; Fax (0171) 2473428. Q 1997 Blackwell Science Ltd
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956 M. Rangarajan et al. on Southern blot hybridization studies a number of reports have suggested the presence of a second genomic locus which shows significant homology to the coding region of the catalytic domain of these homologous genes (AduseOpoku et al., 1995; Barkocy-Gallagher et al., 1996). Furthermore, it has recently been established using insertional gene inactivation in P. gingivalis ATCC33277 that insertions at two distinct genetic loci are sufficient to abolish all the Arg-x protease activity of this strain (Nakayama et al., 1995). Thus despite the confused nomenclature it now seems likely that the arginine-specific protease activity of P. gingivalis is derived from the products of just two genes. Whilst the position at the genetic level is now becoming clearer, our understanding of the contribution of these two genes to the different forms of Arg-x proteases found in the culture supernatants of P. gingivalis remains ill-defined. Purification and characterization of Arg-x proteases from the culture supernatants of P. gingivalis W50 has demonstrated three distinct species: RI, RIA and RIB (Rangarajan et al., 1997). Whilst the N-terminal protein sequence of the catalytic component of all three enzymes is identical over the first 20 cycles, there are significant structural differences between them. RI is a hetero-dimeric enzyme composed of a catalytic (a) chain of
