Highly Specific Protease-Based Approach for Detection of Porphyromonas gingivalis in Diagnosis of Periodontitis
- 1 January 2012
- journal article
- research article
- Published by American Society for Microbiology in Journal of Clinical Microbiology
- Vol. 50 (1), 104-112
- https://doi.org/10.1128/jcm.05313-11
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 .Keywords
This publication has 42 references indexed in Scilit:
- Cysteine Cathepsins in Human Carious DentinJournal of Dental Research, 2011
- The core genome of the anaerobic oral pathogenic bacterium Porphyromonas gingivalisBMC Microbiology, 2010
- Prevalence and simultaneous occurrence of periodontitis and dental cariesJournal of Clinical Periodontology, 2010
- Virulence mechanisms of Tannerella forsythiaPeriodontology 2000, 2010
- A novel matrix metalloprotease-like enzyme (karilysin) of the periodontal pathogen Tannerella forsythia ATCC 43037Biological Chemistry, 2009
- Quantification of periodontal pathogens by paper point sampling from the coronal and apical aspect of periodontal lesions by real-time PCRClinical Oral Investigations, 2009
- Cathepsin L Proteolytically Processes Histone H3 During Mouse Embryonic Stem Cell DifferentiationCell, 2008
- Survival in transport media of Actinobacillus actinomycetemcomitans, Porphymmonas gingivalis and Prevotella intermedia in human subgingival samplesOral Microbiology and Immunology, 1993
- Periodontal DiseaseNew England Journal of Medicine, 1990
- On the substrate specificity of cathepsins B1 and B2 including a new fluorogenic substrate for cathepsin B1Life Sciences, 1975