Summary: Porphyromonas gingivalis 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 cell-associated trypsin-like proteolytic activity of P. gingivalis W50 using mild sonication. Anion-exchange and gel-filtration 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 44 kDa band containing two different proteins as shown by N-terminal sequence analysis. On further chromatography of the 300 kDa complex on Arg-Sepharose 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 octyl–D-glucopyranoside and was shown to be an Arg-specific, 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 haemagglutinins/adhesins. The 45 kDa Arg-specific 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 prtK, 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, PrtRIS, PrtR17, PrtR27 and PrtK39, PrtK15 and PrtK44. We propose that this proteinase-adhesin complex is a major virulence factor for P. gingivalis
Published Online:
Funding
This study was supported by the:
Australian National Health and Medical Research Council Dental Postgraduate Scholarship for P.S.B.
(Award 930687)
Australian National Health and Medical Research Council project
(Award 960231)
CarlssonJ.,
HerrmannB. F.,
HoflingJ. F.,
SundqvistG. K.1984; Degradation of the human proteinase inhibitors alpha-1-anti trypsin and alpha-2-macroglobulin by Bacteroides gingivalis
. Infect Immun 43:644–648
ChristerssonL. A.,
ZambonJ. J.,
DunfordR. G.,
GrossiS. G.,
GencoR. J.1989; Specific subgingival bacteria and diagnosis of gingivitis and periodontitis. J Dent Res 68:1633–1639
CiborowskiP.,
NishikataM.,
AllenR. D.,
LantzM. S.1994; Purification and characterization of two forms of a high-molecular-weight cysteine proteinase (Porphypain) from Porphyromonas gingivalis
. J Bacteriol 176:4549–4557
CurtisM. A.,
Aduse-OpokuJ.,
SlaneyJ.,
M„ RangarajanM.,
BoothV.,
CridlandJ.,
ShepherdP.1996; Characterization of an adherence and antigenic determinant of the ArgI protease of Porphyromonas gingivalis which is present on multiple gene products. Infect Immun 64:2532–2539
EngelmanD. M.,
SteitzT. A.,
GoldmanA.1986; Identifying non-polar transbilayer helices in amino acid sequences of membrane proteins. Annu Rev Biophys Biophys Chem 15:321–353
FujimuraS.,
ShibataY.,
NakamuraT.1993; Purification and partial characterization of a lysine-specific protease of Porphyromonas gingivalis
. FEMS Microbiol Lett 113:133–138
GrenierD.1996; Degradation of host protease inhibitors and activation of plasminogen by proteolytic enzymes from Porphyromonas gingivalis and Treponema denticola
. Microbiology 142:955–961
GrenierD.,
MayrandD.1993 Proteinases. . In Biology of the Species Porphyromonas gingivalis , pp. 227–244 . Edited by
ShahH. N.
Boca Raton, FL: CRC Press;
GrenierD.,
MayrandD.,
McBrideB. C.1989; Further studies on the degradation of immunoglobulins by black-pigmented Bacteroides
. Oral Microbiol Immun 4:12–18
ImamuraT.,
PikeR. N.,
PotempaJ.,
TravisJ.1994; Pathogenesis of periodontitis: a major arginine-specific cysteine proteinase from Porphyromonas gingivalis induces vascular permeability enhancement through activation of the kallikinin pathway. J Clin Invest 94:361–367
JagelsM. A.,
TravisJ.,
PotempaJ.,
PikeR.,
HugliT. E.1996; Proteolytic inactivation of the leukocyte C5a receptor by proteinases derived from Porphyromonas gingivalis
. Infect Immun 64:1984–1991
KadowakiT.,
YonedaM.,
OkamotoK.,
MaedaK.,
YamamotoK.1994; Purification and characterization of a novel arginine-specific cysteine proteinase (argingipain) involved in the pathogenesis of periodontal disease from the culture supernatant of Porphyromonas gingivalis.
. J Biol Chem 269:21371–21378
KesavaluL,,
HoltS. C.,
EbersoleJ. L.1996; Trypsin-like protease activity of Porphyromonas gingivalis as a potential virulence factor in a murine lesion model. Microb Pathog 20:1–10
KirszbaumL,,
SotiropoulosC.,
JacksonC.,
deal. S., SlakeskiN.,
ReynoldsE. C.1995; Complete nucleotide sequence of a gene prtR of Porphyromonas gingivalis W50 encoding a 132 kDa protein that contains an arginine-specific, thiol endopeptidase domain and a haemagglutinin domain. Biochem Biophys Res Commun 207:424–431
LantzM. S.,
AllenR.,
D„ VailT. A.,
SwitalskiL. M.,
HookM.1991; Specific cell components of Bacteroides gingivalis mediate binding and degradation of human fibrinogen. J Bacteriol 173:495–504
LoescheW. J.,
SyedS. A.,
MorrisonE. C.,
LaughonB.,
GrossmanN. S.1981; Treatment of periodontal infections due to anaerobic bacteria with short-term treatment with metronidazole. J Clin Periodontol 8:29–44
MarshP. D.,
McDermidA. S.,
McKeeA. S.,
BaskervilleA.1994; The effect of growth rate and haemin on the virulence and proteolytic activity of Porphyromonas gingivalis W50. Microbiology 140:861–865
McKeeA. S.,
McDermidA. S.,
BaskervilleA.,
DowsettB.,
EllwoodD. C.,
MarshP. D.1986; Effect of hemin on the physiology and virulence of Bacteroides gingivalis W50. Infect Immun 52:349–355
MerrilC. R.,
GoldmanD.,
Van KeurenM. L.1982; Simplified silver protein detection and image enhancement methods in polyacrylamide gels. Electrophoresis 3:14–23
MooreW. E. C.,
MooreL. H.,
RanneyR. R.,
SmibertR. M.,
BurmeisterJ. A.,
SchenkeinH. A.1991; The microflora of periodontal sites showing active destructive progression. J Clin Periodontol 18:729–739
NeuhoffV.,
StammR.,
EiblH.1985; Clear background and highly sensitive protein staining with coomassie blue dyes in polyacrylamide gels: a systematic analysis. Electrophoresis 6:427–448
OkamotoK.,
MisumiY.,
KadowakiT.,
YonedaM.,
YamamotoK.,
IkeharaY.1995; Structural characterization of argingipain, a novel arginine-specific cysteine proteinase as a major periodontal pathogenic factor from Porphyromonas gingivalis
. Arch Biochem Biophys 316:917–925
OkudaK.,
YamamotoA.,
NaitoY.,
TakazoeI.,
SlotsJ.,
GencoR. J.1986; Purification and properties of a hemagglutinin from culture supernatant of Bacteroides gingivalis
. Infect Immun 54:659–665
PikeR. N.,
PotempaJ.,
McGrawW.,
CoetzerT. H. T.,
TravisJ.1996; Characterization of the binding activities of proteinase-adhesin complexes from Porphyromonas gingialis
. J Bacteriol 178:2876–2882
PotempaJ.,
PikeR.,
TravisJ.1995; The multiple forms of trypsin-like activity present in various strains of Porphyromonas gingivalis are due to the presence of either arg-gingipain or lys-gingipain. Infect Immun 63:1176–1182
ReynoldsE. C.1987; The prevention of sub-surface demineralization of bovine enamel and change in plaque composition by casein in an intra-oral model. J Dent Res 66:1120–1127
ReynoldsE. C.,
RileyP. F.,
AdamsonN. J.1994; A selective-precipitation purification procedure for multiple-phosphoseryl containing peptides and methods for their identification. Anal Biochem 217:277–284
SlakeskiN.,
ClealS. M.,
ReynoldsE. C.1996; Characterization of a Porphyromonas gingivalis gene prtR that encodes an arginine-specific thiol proteinase and multiple adhesins. Biochem Biophys Res Commun 224:605–610
SlotsJ.1982 Importance of black pigmented Bacteroides in human periodontal disease. . In Host–Parasite Interactions in Periodontal Diseases , pp. 27–45 . Edited by
GencoR. J.,
MerganhaganS. E.
Washington DC: American Society for Microbiology;
SmalleyJ. W.,
ShuttleworthC. A.,
BirssA. J.1989a; Colla-genolytic activity of the extracellular vesicles of Bacteroides gingivalis W50 and an avirulent variant W50/BE1. Arch Oral Biol 34:579–583
SmalleyJ. W.,
BirssA. J.,
KayH. M.,
McKeeA. S.,
MarshP. D.1989b; The distribution of trypsin-like enzyme activity in cultures of a virulent and an avirulent strain of Bacteroides gingivalis W50. Oral Microbiol Immunol 4:178–181
SocranskyS. S.,
HaffajeeA. D.,
SmithC.,
DibartS.1991; Relation of counts of microbial species to clinical status at the sampled site. J Clin Periodontol 18:766–775
SorsaT.,,
UittoV.-J.,
SuomalainenK.,
TurtoH.,
LindyS.1987; A trypsin-like protease from Bacteroides gingivalis: partial purification and characterization. J Periodontal Res 22:375–380
SundqvistG.,
CarlssonJ.,
HerrmannB.,
TarnvikA.1985; Degradation of human immunoglobulins G and M and complement factors C3 and C5 by black-pigmented Bacteroides
. J Med Microbiol 19:85–94
TodaK.,
OtsukaM.,
IshikawaY.,
SatoM.,
YamamotoY.,
NakamuraR.1984; Thiol-dependent collagenolytic activity in culture media of Bacteroides gingivalis
. J Periodontal Res 19:372–381
Van DykeT. E.,
OffenbacherS.,
PlaceD.,
DowellV. R.,
JonesJ.1988; Refractory periodontitis: mixed infection with Bacteroides gingivalis and other unusual Bacteroides species. J Periodontol 59:184–189
YoshimuraF.,
NishikataM.,
SuzukiT.,
HooverC. I.,
NewbrunE.1984; Characterization of a trypsin-like protease from the bacterium Bacteroides gingivalis isolated from human dental plaque. Arch Oral Biol 29:559–564