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

Abstract

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