title: Highly specific protease-based approach for detection of porphyromonas gingivalis in diagnosis of periodontitis.
abstract: Porphyromonas gingivalis is associated with the development of periodontitis. Here we describe the development of a highly specific protease-based diagnostic method for the detection of P. gingivalis in gingival crevicular fluid. Screening of a proteolytic peptide substrate library, including fluorogenic dipeptides that contain d-amino acids, led to the discovery of five P. gingivalis-specific substrates. Due to the presence of lysine and arginine residues in these substrates, it was hypothesized that the cleavage was mediated by the gingipains, a group of P. gingivalis-specific proteases. This hypothesis was confirmed by the observation that P. gingivalis gingipain knockout strains demonstrated clearly impaired substrate cleavage efficacy. Further, proteolytic activity on the substrates was increased by the addition of the gingipain stimulators dithiothreitol and l-cysteine and decreased by the inhibitors leupeptin and N-ethylmaleimide. Screening of saliva and gingival crevicular fluid of periodontitis patients and healthy controls showed the potential of the substrates to diagnose the presence of P. gingivalis proteases. By using paper points, a sensitivity of approximately 10(5) CFU/ml was achieved. P. gingivalis-reactive substrates fully composed of l-amino acids and Bz-l-Arg-NHPhNO(2) showed a relatively low specificity (44 to 85%). However, the five P. gingivalis-specific substrates that each contained a single d-amino acid showed high specificity (96 to 100%). This observation underlines the importance of the presence of d-amino acids in substrates used for the detection of bacterial proteases. We envisage that these substrates may improve the specificity of the current enzyme-based diagnosis of periodontitis associated with P. gingivalis.
authors.affiliation: Department of Medical Microbiology and Infectious Diseases, Erasmus MC, Rotterdam, The Netherlands. w.kaman@erasmusmc.nl
authors.forename: Wendy E
authors.initials: WE
authors.lastname: Kaman
authors.forename: Fabiano
authors.initials: F
authors.lastname: Galassi
authors.forename: Johannes J
authors.initials: JJ
authors.lastname: de Soet
authors.forename: Sergio
authors.initials: S
authors.lastname: Bizzarro
authors.forename: Bruno G
authors.initials: BG
authors.lastname: Loos
authors.forename: Enno C I
authors.initials: EC
authors.lastname: Veerman
authors.forename: Alex
authors.initials: A
authors.lastname: van Belkum
authors.forename: John P
authors.initials: JP
authors.lastname: Hays
authors.forename: Floris J
authors.initials: FJ
authors.lastname: Bikker
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods*; D005883:Gingival Crevicular Fluid / Q000382:microbiology; D006801:Humans; D010447:Peptide Hydrolases / Q000032:analysis*; D010518:Periodontitis / Q000175:diagnosis* / Q000382:microbiology*; D016966:Porphyromonas gingivalis / Q000201:enzymology* / Q000302:isolation & purification* / Q000378:metabolism; D012680:Sensitivity and Specificity
chemical_list: D010447:Peptide Hydrolases
references.citation: Cell. 2008 Oct 17;135(2):284-94
references.pmid: 18957203
references.citation: Periodontol 2000. 1994 Jun;5:78-111
references.pmid: 9673164
references.citation: Periodontol 2000. 1999 Jun;20:168-238
references.pmid: 10522227
references.citation: J Periodontal Res. 1982 May;17(3):275-83
references.pmid: 6125580
references.citation: Biol Chem. 2010 Jan;391(1):105-17
references.pmid: 19919176
references.citation: J Biol Chem. 1992 Sep 15;267(26):18896-901
references.pmid: 1527017
references.citation: Anal Chem. 2011 Apr 1;83(7):2511-7
references.pmid: 21370823
references.citation: J Periodontol. 2009 Nov;80(11):1809-14
references.pmid: 19905950
references.citation: J Biol Chem. 1996 Dec 13;271(50):31885-93
references.pmid: 8943232
references.citation: Dent Today. 2010 Sep;29(9):60-2, 64-6; quiz 68-9
references.pmid: 20973418
references.citation: J Dent Res. 2011 Apr;90(4):506-11
references.pmid: 21248362
references.citation: Oral Microbiol Immunol. 1993 Dec;8(6):370-4
references.pmid: 8152838
references.citation: Curr Protein Pept Sci. 2003 Dec;4(6):397-407
references.pmid: 14683426
references.citation: N Engl J Med. 1990 Feb 8;322(6):373-82
references.pmid: 2405268
references.citation: Rheumatology (Oxford). 2011 Jul;50(7):1189-93
references.pmid: 21343168
references.citation: J Periodontal Res. 1996 May;31(4):278-84
references.pmid: 8814599
references.citation: Clin Oral Investig. 2010 Oct;14(5):533-41
references.pmid: 19693550
references.citation: J Endod. 2010 Mar;36(3):475-81
references.pmid: 20171366
references.citation: J Periodontol. 2010 Jul;81(7):982-91
references.pmid: 20384462
references.citation: FEMS Microbiol Lett. 1998 Jun 15;163(2):173-9
references.pmid: 9673019
references.citation: J Biol Chem. 1999 Jun 18;274(25):17955-60
references.pmid: 10364243
references.citation: BMC Microbiol. 2010 Sep 29;10:252
references.pmid: 20920246
references.citation: J Periodontol. 2010 Feb;81(2):186-98
references.pmid: 20151796
references.citation: J Clin Periodontol. 2008 May;35(5):405-14
references.pmid: 18433384
references.citation: Periodontol 2000. 2010 Oct;54(1):106-16
references.pmid: 20712636
references.citation: J Clin Microbiol. 1992 Feb;30(2):427-33
references.pmid: 1311335
references.citation: Life Sci. 1975 Oct 15;17(8):1269-76
references.pmid: 577
references.citation: J Bacteriol. 1994 Aug;176(15):4549-57
references.pmid: 8045885
references.citation: Dent Update. 2004 Oct;31(8):448-50, 453-4, 456
references.pmid: 15554049
references.citation: Methods Mol Biol. 2010;666:125-40
references.pmid: 20717782
references.citation: J Clin Periodontol. 2010 Mar;37(3):211-9
references.pmid: 20070861
references.citation: Biochem J. 1991 May 1;275 ( Pt 3):751-7
references.pmid: 2039451
references.citation: J Periodontal Res. 1987 Sep;22(5):335-41
references.pmid: 2961863
references.citation: Microbiol Mol Biol Rev. 1998 Dec;62(4):1244-63
references.pmid: 9841671
references.citation: Microbiology (Reading). 2000 Aug;146 ( Pt 8):1933-1940
references.pmid: 10931897
references.citation: J Dent Res. 1988 Dec;67(12):1505-9
references.pmid: 3198850
references.citation: J Clin Microbiol. 1990 Jul;28(7):1551-9
references.pmid: 2380379
references.citation: Adv Dent Res. 1988 Nov;2(2):275-83
references.pmid: 3271022
references.citation: J Clin Periodontol. 1988 May;15(5):316-23
references.pmid: 3292595
references.citation: J Clin Periodontol. 2010 Nov;37(11):962-7
references.pmid: 20958340
references.citation: J Am Chem Soc. 2005 May 4;127(17):6207-15
references.pmid: 15853325
references.citation: Aust Dent J. 2009 Sep;54 Suppl 1:S62-9
references.pmid: 19737269
references.citation: J Dent Res. 1997 Dec;76(12):1840-4
references.pmid: 9390477
references.citation: Nucleic Acids Res. 2010 Jan;38(Database issue):D227-33
references.pmid: 19892822
references.citation: J Biol Chem. 2006 May 5;281(18):12824-32
references.pmid: 16520377
references.citation: Glycobiology. 2001 Mar;11(3):25R-36R
references.pmid: 11320055