Chirality: The Key to Specific Bacterial Protease-Based Diagnosis?

Abstract Bacterial proteases play an important role in a broad spectrum of processes, including colonization, proliferation and virulence. In this respect, bacterial proteases are potential biomarkers for bacterial diagnosis and targets for novel therapeutic protease inhibitors. To investigate these potential functions, the authors designed and used a protease substrate fluorescence resonance energy transfer (FRET)-library comprising 115 short D- and L-amino-acid-containing fluorogenic substrates as a tool to generate proteolytic profiles for a wide range of bacteria. Bacterial specificity of the D-amino acid substrates was confirmed using enzymes isolated from both eukaryotic and prokaryotic organisms. Interestingly, bacterial proteases that are known to be involved in housekeeping and nutrition, but not in virulence, were able to degrade substrates in which a D-amino acid was present. Using our FRET peptide library and culture supernatants from a total of 60 different bacterial species revealed novel, bacteria-specific, proteolytic profiles. Although in-species variation was observed for Pseudomonas aeruginosa, Porphyromonas gingivalis and Staphylococcus aureus. Overall, the specific characteristic of our substrate peptide library makes it a rapid tool to high-throughput screen for novel substrates to detect bacterial proteolytic activity

Rapid Colorimetric Detection of Pseudomonas aeruginosa in Clinical Isolates Using a Magnetic Nanoparticle Biosensor

A rapid, sensitive, and specific colorimetric biosensor based on the use of magnetic nanoparticles (MNPs) was designed for the detection of Pseudomonas aeruginosa in clinical samples. The biosensing platform was based on the measurement of P. aeruginosa proteolytic activity using a specific protease substrate. At the N-terminus, this substrate was covalently bound to MNPs and was linked to a gold sensor surface via cystine at the C-terminus of the substrates. The golden sensor appears black to naked eyes because of the coverage of the MNPs. However, upon proteolysis, the cleaved peptide-MNP moieties will be attracted by an external magnet, revealing the golden color of the sensor surface, which can be observed by the naked eye. In vitro, the biosensor was able to detect specifically and quantitatively the presence of P. aeruginosa with a detection limit of 102 cfu/mL in less than 1 min. The colorimetric biosensor was used to test its ability to detect in situ P. aeruginosa in clinical isolates from patients. This biochip is anticipated to be useful as a rapid point-of-care device for the diagnosis of P. aeruginosa-related infections

Salivary Total Protease Activity Based on a Broad-Spectrum Fluorescence Resonance Energy Transfer Approach to Monitor Induction and Resolution of Gingival Inflammation

OBJECTIVE: Salivary total protease and chitinase activities were measured by a broad-spectrum fluorescence resonance energy transfer approach as predictors of induction and resolution of gingival inflammation in healthy individuals by applying an experimental human gingivitis model. METHODS: Dental biofilm accumulated (21 days, Induction Phase) by omitting oral hygiene practices followed by a 2-week Resolution Phase to restore gingival health in an experimental gingivitis study. Plaque accumulation, as assessed by the Turesky Modification of the Quigley-Hein Plaque Index (TQHPI), and gingival inflammation, assessed using the Modified Gingival Index (MGI), scores were recorded and unstimulated saliva was collected weekly. Saliva was analysed for total protein, albumin, total protease activity and chitinase activity (n = 18). RESULTS: The TQHPI and MGI scores, as well as total protease activity, increased until day 21. After re-establishment of oral hygiene, gingival inflammation levels returned to values similar to baseline (day 0). Levels of protease activity decreased significantly, but not to baseline values. Furthermore, 'fast' responders, who responded immediately to plaque, exhibited significantly higher proteolytic activity throughout the experimental course than 'slow' responders, who showed a lagged inflammatory response. CONCLUSION: The results indicate that differential inflammatory responses encompass inherent variations in total salivary proteolytic activities, which could be further utilised in contemporary diagnostic, prognostic and treatment modalities for periodontal diseases

Highly specific protease-based approach for detection of porphyromonas gingivalis in diagnosis of periodontitis

Porphyromonas gingivalis is associated with the development of periodontitis. Here we describe the development of a highly specific protease-b