MRC Molecular Pathogenesis Group, Department of Medical Microbiology, St Bartholomews and The Royal London School of Medicine & Dentistry, Queen Mary and Westfield College, 32 Newark Street, London E1 2AA, UK1
Author for correspondence: Minnie Rangarajan. Tel: +44 208 377 0444. Fax: +44 208 247 3428. e-mail: mrangarajan{at}mds.qmw.ac.uk
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ABSTRACT |
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Keywords: Porphyromonas gingivalis, extracellular proteases, isogenic mutants, polyprotein maturation/processing
Abbreviations: CDM, chemically defined medium; KGP, Lys-gingipain; RGP, Arg-X gingipain
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INTRODUCTION |
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Two major classes of extracellular cysteine proteases specific for Arg-X and Lys-X peptide bonds [Arg-gingipains (RGPs) and Lys-gingipain (KGP)] have been characterized (Curtis et al., 1999 ). The RGPs are encoded by two homologous genes, rgpA and rgpB, and KGP by kgp. The rgpA and kgp genes encode long polyproteins which require proteolytic processing at multiple sites to generate the mature enzymes. The initial translation product of rgpA contains 1704 or 1706 amino acids, depending on the strain (Aduse-Opoku et al., 1995
; Pavloff et al., 1995
) and is composed of a pro-region, an
catalytic domain, a ß-adhesin domain and a C-terminal
domain. Three mature enzyme isoforms are produced from the precursor: HRgpA (
ß heterodimer, ~110 kDa), RgpAcat (
monomer, ~55 kDa) and mt-RgpAcat (a highly post-translationally modified
monomer, ~7080 kDa) (Rangarajan et al., 1997a
). Proteolytic processing of the RgpA translation product is thought to occur at Arg227-Tyr228 and Arg719-Ser720 to release the
catalytic chain and probably at Arg1262-Phe1263 to generate the ß domain of HRgpA. An alternative form of HRgpA has been described by Pike et al. (1994)
which is composed of the
catalytic domain in association with three different sets of lower molecular mass polypeptides derived from the RgpA C terminus (~95 kDa). In this case, the RgpA polyprotein is likely to be processed at Arg227-Tyr228, Arg719-Ser720, Arg1138-Ala1139, Lys1273-Pro1274 and Arg1431-Ala1432.
KGP is generated as a polyprotein of 1732 amino acids (Curtis et al., 1999 ) or 1723 amino acids which is processed at Arg228-Asp229 and Arg737-Ala738 to generate the catalytic domain and at Arg1155-Ala1156, Lys1290-Pro1291 (Pavloff et al., 1997
; Slakeski et al., 1999
) and Arg1448-Ala1449 (Pavloff et al., 1997
) to produce the C-terminally derived polypeptides analogous to the HRgpA described by Pike et al. (1994)
. Unlike RgpA and Kgp, the product of rgpB contains only a pro-peptide and catalytic domain which requires processing only at the junction of these two domains, Arg230-Tyr231 (Nakayama, 1997
), to release the active monomeric enzyme.
Since generation of mature enzymes from the initial translation products of these three genes involves multiple cleavages at Arg-X and Lys-X peptide bonds, it has been suggested that the maturation pathways of these proteases may be interdependent. Thus the RGPs may be involved in processing the products of all three genes and KGP may have an autoprocessing role (Pavloff et al., 1997 ; Slakeski et al., 1999
) and also be required for the generation of the HRgpA multimer described by Pike et al. (1994)
. The requirement for rgpA rgpB- and kgp-derived products in the production of Arg-X and Lys-X protease activity has been addressed through the construction of single, double and triple mutants of these loci using a variety of antibiotic-resistance cassettes as selection markers. However, the results of these studies have generated contradictory conclusions.
Okamoto et al. (1996) reported that Lys-X protease activity in both cell extracts and supernatants of an RGP-null mutant was reduced to approximately 2030% of levels in the wild-type parent strain. This was confirmed by Kadowaki et al. (1998)
, who suggested that abnormal processing produced KGP in the RGP-null mutant with reduced activity or rapid degradation of the processed enzyme. Conversely, in a more recent report, Shi et al. (1999)
observed that an rgpA rgpB mutant of P. gingivalis (KDP133) produced higher levels of KGP activity compared to the parent strain. Similar discrepancies have been reported with respect to Arg-X activity in kgp mutant strains. An insertion mutant of kgp in strain W83 has been shown to be associated with reduced Arg-X activity (Lewis & Macrina, 1999
), whereas loss of function of kgp in strain ATCC 33277 did not affect the Arg-X specific protease activity (Okamoto et al., 1998
). The variation in the results obtained could be due to differences in strains used, although all the P. gingivalis strains examined so far have been shown to possess rgpA rgpB and kgp which are remarkably similar (Curtis et al., 1999
).
In this report we present the construction and systematic analysis of a P. gingivalis W50 rgpA rgpB double mutant in order to establish the role(s) of RGP(s) in the processing of KGP, and a kgp mutant in order to assess the role of KGP in the processing of the RGPs.
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METHODS |
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Enzyme assays.
Enzyme assays for Arg-X and Lys-X protease activities using N--benzoyl-DL-arginine-p-nitroanilide (BRpNA) and N-
-acetyllysine-p-nitroanilide (AcKpNA), respectively, were performed as described by Rangarajan et al. (1997a
). One unit of enzyme catalyses an increase in A405 of 1·0 min-1 at 30 °C.
Generation of rgpA rgpB double mutant of P. gingivalis W50.
DNA manipulation techniques were according to Sambrook et al. (1989) . Plasmid pNJR12 (Maley et al., 1992
) was kindly supplied by Dr Abigail Salyers, Microbiology Department, University of Illinois at Urbana-Champaign, USA. Both rgpA and rgpB have previously been cloned in pUC18 and single knockout mutants have been generated by insertion of an Erm cassette within the region encompassing the catalytic domain (Rangarajan et al., 1997b
). A 2·8 kb HpaISphI fragment of pKFT2, encoding TetQ resistance, was excised from a preparative agarose gel and cloned into an EcoRVSphI site of pKpL at the 5' end of rgpA (Aduse-Opoku et al., 1995
). The resulting construct (pAG2) was linearized by restriction with KpnI and electroporated into P. gingivalis D7 (rgpB) to achieve allelic exchange in rgpA via homologous recombination. Mutants were selected following anaerobic growth on blood agar plates containing tetracycline (TetQ selection) and clindamycin (Erm selection).
Generation of kgp knockout mutant, P. gingivalis K1A.
Plasmid pNS1 contains a BamHI fragment of P. gingivalis W50 chromosomal DNA in pUC18 (nt 13516; Slakeski et al., 1999 ). This was linearized with BstXI, blunted and a 2·1 kb KpnIBamHI Erm cassette (blunted) was cloned into the site (nt 1928) to generate pNSE1 to inactivate the catalytic domain of kgp in vitro. The 5·6 kb BamHI fragment was then retrieved for electroporation into W50. Twelve random clindamycin-resistant mutants were further screened by PCR and P. gingivalis K1A was chosen for further analysis. PCR was performed using Reddy Load amplification reagent (Advanced Biotechnologies) with primer pairs: PRTKF1, 5'-AGCGGAGAAAAAGGAAAG-3' and PRTKR1, 5'-GTAGCATCATAAGAACCCATAG-3' with P. gingivalis DNA as the template, using 25 cycles of 94 °C (1 min), 55 °C (1 min) and 72 °C (4 min) (Omnigene, Hybaid). A 680 bp fragment was amplified from the parent strain while all mutants yielded a 2·78 kb fragment indicative of insertions of the 2·1 kb cassette at this locus. Chromosomal DNA was prepared with Puregene DNA isolation reagent (Flowgen).
Southern hybridization.
Chromosomal DNA was restricted using enzymes from Amersham Pharmacia Biotech, and transferred to Hybond-N+ (Amersham Pharmacia Biotech) using Vacu-Aid (Hybaid). Hybridization was performed in Rapid Hyb buffer (Amersham Pharmacia Biotech) as previously described (Aduse-Opoku et al., 1995 ).
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RESULTS |
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P. gingivalis (kgp) was generated following in vitro inactivation of the catalytic domain of kgp. Twelve mutants were analysed by PCR and assayed for Arg-X and Lys-X activities. One mutant, K1A, was selected for detailed analysis.
Characterization of P. gingivalis W50 (rgpA rgpB)/E8
The stability of the tetQ marker in E8 was tested by subculturing the strain every 24 h over 4 d in BHI in the absence of tetracycline and using 10% of inoculum from the previous culture. Chromosomal DNA was prepared from the first and fourth cultures and was subjected to restriction digestion and Southern hybridization. The Southern hybridization profiles were the same for the first and fourth samples, suggesting that the mutants were stable in the absence of antibiotic selection. Similarly, there were no differences in Arg-X and Lys-X protease activities between the first and fourth cultures. Hence, tetracycline and clindamycin were usually omitted from the growth medium after the initial culture on blood agar-antibiotic plates.
P. gingivalis E8 exhibited the same colony morphology and rate of pigmentation as wild-type W50 on blood agar plates. The only obvious phenotypic difference was the delayed ability of E8 to show visible zones of haemolysis but this was not accompanied by any perceptible alteration in growth.
Arg-X and Lys-protease activities in mutants
The results of Arg-X and Lys-X protease assays in several P. gingivalis strains, grown in BHI with added haemin, are shown in Table 2. Inactivation of either rgpA or rgpB reduced the Arg-X activity in whole cultures by 50% without affecting the Lys-X enzyme activity (Rangarajan et al., 1997b
; Aduse-Opoku et al., 1998
). Loss of both rgpA and rgpB in P. gingivalis E8 abolished virtually all Arg-X protease activity without any significant effect on the Lys-X protease activity in both day-old whole cultures and 6 d culture supernatants. Thus, the RGPs are not required for generation of KGP activity. However, growth of the E8 mutant (rgpA rgpB) in the presence of tetracycline, with or without clindamycin, led to a dramatic reduction in Lys-X activity in whole cultures to 50% of the wild-type values. Tetracycline had no effect on the Lys-X activities of pure KGP or of whole cultures of W50 or E8 when it was incorporated into the assay solution at concentrations comparable to those present in the growth medium. Therefore, tetracycline appears to exert a non-antibiotic effect on the synthesis/activation of KGP.
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DISCUSSION |
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Data in the current report confirmed that inactivation of kgp leads to loss of the black-pigmenting, haemolytic phenotype on blood agar, suggesting that KGP is involved in the acquisition/storage of haemin. Support for this hypothesis has emerged from the work of Lewis et al. (1999) , which demonstrates that this enzyme is an efficient haemoglobinase and hence may serve to release protein-bound haemin into the extracellular environment. Furthermore, Nakayama et al. (1998)
have shown that a haemin-binding peptide is intragenically encoded by kgp and this may function as part of a haemin storage mechanism at the cell surface. However, data in this report demonstrate that KGP is not required for growth in complex medium with added haemin. In addition to a role in haemin acquisition, KGP also appears to be important for growth of P. gingivalis on large proteins since kgp mutants grow very poorly in a defined medium containing albumin as the sole source of carbon and nitrogen (Fig. 3
) and this effect can be reversed by pretreatment of the medium with trypsin (Shi et al., 1999
). Hence there is reasonable evidence that KGP has an important nutritional role.
In contrast, characterization of a P. gingivalis rgpA rgpB double mutant has failed to demonstrate a similar role in nutrition for the RGPs either in this study or in an earlier report (Shi et al., 1999 ). Clearly the possibility that the RGPs play an important nutritional role in vivo through degradation of specific host proteins or release of important protein-bound micronutrients cannot be excluded. However, it is equally plausible that an important function of these enzymes in vivo is an immunosubversive role through the proteolytic inactivation of neutrophil opsonins and other agents of the hosts defensive armoury.
Whilst the extracellular functions of the RGPs remain uncertain there are significant data to support a role in cell-associated house-keeping duties. Kadowaki et al. (1998) described the importance of RGPs in the processing of fimbrillin, a major component of fimbriae, which remained in an unassembled precursor form in an rgpA rgpB double mutant. In addition, the proteolytic maturation of a 75 kDa outer-membrane protein was also shown to be dependent on functional rgpA/rgpB. It has also been suggested that the RGPs are required for appropriate processing and activation of KGP (Lewis & Macrina, 1999
). The data in the present report conclusively demonstrate that processing and activation of KGP is not dependent on products of rgpA and rgpB and also that processing and activation of the RGPs does not require kgp function. Western blotting experiments did not demonstrate a discernible difference in the size of fragments derived from RgpA and Kgp polyproteins in the K1A and E8 mutants, respectively, versus the parent strain. It is possible that there are minor differences in the processing sites of the polyproteins in the mutants that do not result in significant alterations to the apparent molecular mass of the resultant polypeptides on SDS-PAGE. However, if this is the case, altered processing does not influence the total activity of the mature enzymes as was suggested by Kadowaki et al. (1998)
, who reported that KGP was abnormally processed in an rgpA rgpB double mutant of P. gingivalis ATCC 33277 at Leu225-Phe226 instead of Arg228-Asp229and that this resulted in a significant reduction in total KGP activity.
Whilst RGP activity in day-old whole cultures of P. gingivalis (kgp) was identical to those of the parent W50, enzyme activity in 6 d culture supernatants was reduced to 50% of wild-type levels. Hence, although KGP activity is not required for the processing and activation of the RGPs, it may be involved in the export of this activity into culture supernatant.
A plausible explanation for the differences between the present study and previous examination of the P. gingivalis (rgpA rgpB) mutant phenotype concerns a non-antibiotic effect of tetracycline on KGP activity in the RGP-null background. Stability testing of the E8 mutant confirmed that it was not necessary to maintain antibiotic selection pressure during growth of this mutant to maintain the rgpA and rgpB insertions. This enabled a comparison of the effects of the antibiotics on enzyme activity, which demonstrated that tetracycline in the growth medium causes a significant decrease in KGP activity. Since tetracycline has no effect on pre-formed KGP (or on RGP) activities when incorporated into the assay solution at levels present in the growth medium, the reduction of KGP activity in the rgpA rgpB mutant probably represents either a direct effect on synthesis or an indirect effect on activity. This non-antibiotic effect of tetracycline may explain some of the discrepancies in the earlier literature on the inter-relationships between RGP and KGP activity. Hence, the absence of an effect on KGP activity in the parent strain at subantibiotic concentrations suggests it is unlikely to be clinically relevant.
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ACKNOWLEDGEMENTS |
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Received 7 February 2000;
revised 25 April 2000;
accepted 3 May 2000.