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. gingivalisspecific 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.

Evaluation of a novel immunochromatographic device for rapid and accurate clinical detection of Porphyromonas gingivalis in subgingival plaque

“…The clinical detection threshold of the current device was estimated to be approximately 10 4 copies/two paper points, based on our preliminary experiments and the results from this clinical application. This threshold is comparable with those reported for a DNA probe method (Komiya et al, 2000), an antibody-based detection test (Nakagawa et al, 1995;Boyer et al, 1996) and a DNA-strip technology-based method (Eick et al, 2011), and is also considered to be clinically relevant because such a threshold potentially matches the range in which nonsurgical or surgical therapy is unsuccessful and antimicrobial treatment is needed (Preshaw, 2004;Kaman et al, 2012).…”

LuxS signaling in Porphyromonas gingivalis-host interactions

“…This too may lead to lower fibroblast responses, since these proteases are important P. gingivalis virulence factors, and protease mutants indeed induced less strong inflammatory responses in gingival fibroblasts in a previous study [29,33]. However, we tested the protease activity of the P. gingivalis mutants with a specific FRET substrate analysis [34], and although Pg DluxS had decreased protease activity compared to the wild-type, Pg D0482 protease activity was not affected (data not shown). The impaired ability of Pg D0482 to induce a response in fibroblasts is thus not caused by reduced protease activity.…”

In vitrophenotypic differentiation towards commensal and pathogenic oral biofilms

“…To quantify the protease activity of the biofilms the fluorescence resonance energy transfer (FRET) assay essentially as described previously by Kaman et al (2011Kaman et al ( , 2012 was used. Biofilms were removed from the wells and the spent medium was filter sterilized using 0.2 μm filters and stored at -20°C until needed.…”