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

The Oxidation Stability, Light Absorbing Power, and Component of Humic Acids from Different Origins, and Their Mutal Relations

Toxin-antitoxin modules are necessary for the mode of action of several antibiotics. One of the most studied toxin-antitoxin modules is the quorum sensing - dependent MazEF system in Escherichia coli. The quorum sensing factor in this system is called the extracellular death factor (EDF), a linear pentapeptide with the sequence NNWNN. In spite of the extensive research on the mazEF system and the involvement of the quorum sensing factor EDF, the effect of EDF itself on bacteria has not yet been studied. In this research, we determined the effect of EDF and variants on cell growth in the Gram-negative bacterium E. coli and the Gram-positive Bacillus globigii. By aligning the zwf gene (from where EDF originates) of different bacterial species, we found 27 new theoretical variants of the peptide. By evaluating growth curves and light microscopy we found that three EDF variants reduced bacterial cell size in B. globigii, but not in E. coli. The D-peptides did not affect cell size, indicating that the effect is stereospecific. Peptides wherein tryptophan was substituted by alanine also did not affect cell size, which indicates that the effect seen is mediated by an intracellular target. © 2013 Springer Science+Business Media Dordrecht

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

Natural and synthetic Sortase A substrates are processed by Staphylococcus aureus via different pathways

AbstractEndogenous Staphylococcus aureus sortase A (SrtA) covalently incorporates cell wall anchored proteins equipped with a SrtA recognition motif (LPXTG) via a lipid II-dependent pathway into the staphylococcal peptidoglycan layer. Previously, we found that the endogenous S. aureus SrtA is able to recognize and process a variety of exogenously added synthetic SrtA substrates, including K(FITC)LPMTG-amide and K(FITC)-K-vancomycin-LPMTG-amide. These synthetic substrates are covalently incorporated into the bacterial peptidoglycan (PG) of S. aureus with varying efficiencies. In this study, we examined if native and synthetic substrates are processed by SrtA via the same pathway. Therefore, the effect of the lipid II inhibiting antibiotic bacitracin on the incorporation of native and synthetic SrtA substrates was assessed. Treatment of S. aureus with bacitracin resulted in a decreased incorporation of protein A in the bacterial cell wall, whereas incorporation of exogenous synthetic substrates was increased. These results suggest that natural and exogenous synthetic substrates are processed by S. aureus via different pathways.Microbial Biotechnolog

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

Rapid peptide based diagnosis: peptide-based Fluorescence Resonance Energy Transfer (FRET) protease substrates for the detection and diagnosis of bacillus spp

We describe the development of a highly specific protease-based Fluorescence Resonance Energy Transfer (FRET) assay for easy and rapid detection both in vitro and in vivo of Bacillus spp, including Bacillus anthracis. Synthetic substrates for B. anthracis proteases were designed and exposed to secreted enzymes of a broad spectrum of bacterial species, including Bio Warfare Agents. The rational design of the substrates was based on the fact that the presence of D-amino acids in the target is highly specific for bacterial proteases. The designed D-amino acids containing substrates appeared to be specific for B. anthracis, but also for several other Bacillus spp. and for both vegetative cells and spores. Using Mass Spectrometry (MS), cleavage products of the substrates could be detected in sera of B. anthracis infected mice, but not in healthy mice. Due to the presence of mirrored amino acids present in the substrate, the substrates showed high species specificity, and enzyme isolation and purification was redundant. The substrate wherein the D-amino acid was replaced by its L-isomer showed a loss of specificity. In conclusion, using these substrates a rapid tool for detection of B. anthracis spores and diagnosis of anthrax infection is at hand. We are the first who present fluorogenic substrates for detection of bacterial proteolytic enzymes that can be directly applied in situ by the use of D-oriented amino acids. Furthermore the specific characteristic of our substrates makes it a general tool to screen for novel bacteria - substrate interactions. This information can subsequently be used in the design of competitive inhibitors and the development of diagnostic peptides and may be useful in the search for novel bacterial protease inhibitors