A photoacoustic-fluorescent imaging probe for proteolytic gingipains expressed by Porphyromonas gingivalis

Abstract

Porphyromonas gingivalis is a keystone pathogen in periodontal disease. We herein report a dual-modal fluorescent and photoacoustic imaging probe for the detection of gingipain proteases secreted by P. gingivalis. This probe harnesses the intramolecular dimerization of peptide-linked cyanine dyes to induce fluorescence and photoacoustic off-states. Upon proteolytic cleavage by Arg-specific gingipain (RgpB), five-fold photoacoustic enhancement and >100-fold fluorescence activation was measured with detection limits of 1.1 nM RgpB and 5.0E4 CFU/mL bacteria in vitro. RgpB activity was imaged in the subgingival pocket of porcine jaws with 25 nM sensitivity. The diagnostic efficacy of the probe was evaluated in gingival crevicular fluid (GCF) samples from subjects with (n = 14) and without (n = 6) periodontal disease, wherein activation was correlated to qPCR-based detection of P. gingivalis (Pearson’s r = 0.71). The highest activity was seen in subjects with the most severe disease. Finally, photoacoustic imaging of RgpB-cleaved probe was achieved in murine brains ex vivo, demonstrating relevance and potential utility for animal models of general infection by P. gingivalis, motivated by the recent biological link between gingipain and Alzheimer’s disease.

Activatable prodrug for controlled release of an antimicrobial peptide via the proteases overexpressed inCandida albicansandPorphyromonas gingivalis

“…[18] This protease cleaves between arginine and isoleucine (Arg/Ile), and AGPR/ID is a substrate with a high affinity for RgpB. [18] Hence, the specific substrates for either SAP9 or RgpB were incorporated into the peptide design. Di-glycine spacers (GG) were placed between the three different functional parts to provide flexibility.…”

“…RgpB secretion was confirmed similarly as described in previous work. [18] Next, a Kirby-Bauer disk diffusion assay was conducted to evaluate initial sensitivity of the microorganisms to the prodrugs. For C. albicans, a clear diameter was formed by addition of S1 to the agar showing inhibited growth of the fungus to approximately 70% of the positive control treatment (Figure 2A-B).…”

“…F. nucleatum is an interesting negative control because it is also present in the gingival sulcus but is commensal and not known to produce either secreted aspartyl proteases or gingipains. [18] S1 and G1 were expected to show low toxicity due to the absence of specific cleavage. Mammalian cells were unlikely to be targeted when exposed to the prodrugs due to differences in their membrane structure and composition relative to fungi and bacteria.…”

“…[15] P. gingivalis is a keystone bacteria in periodontal disease and involves an inflammatory process with various degrees of severity ranging from readily curable gingivitis to irreversible periodontitis. [15,[17][18][19] Data suggest that interactions between C. albicans and bacteria may modulate the clinical course of infection and negatively impact treatment. [20] Likewise, the rate of colonization of C. albicans at subgingival sites is significantly higher in subjects with chronic periodontitis; the rate of candidiasis increases with higher isolation frequencies of P. gingivalis.…”

Porphyromonas gingivalis and non-alcoholic fatty liver disease as combined factors of periodontitis

“…P. gingivalis has direct and indirect mechanisms of periodontal tissue destruction. By secreting special enzymes, i.e., gingipains, the bacterium destroys interepithelial contacts, and reduces the viscosity of the main substance of the connective tissue, and can therefore more easily invade [22,23] . Using various antigenic factors, the periodontopathogen attracts neutrophils with a wide spectrum of enzymatic activity, thus ensuring the mediated destruction of periodontal tissues.…”