Peptide-Based Fluorescence Resonance Energy Transfer Protease Substrates for the Detection and Diagnosis of Bacillus Species

Department of Medical Microbiology and Infectious Diseases, Erasmus MC, 's-Gravendijkwal 230, 3015 CE Rotterdam, The Netherlands.
Analytical Chemistry (Impact Factor: 5.64). 03/2011; 83(7):2511-7. DOI: 10.1021/ac102764v
Source: PubMed


We describe the development of a highly specific enzyme-based fluorescence resonance energy transfer (FRET) assay for easy and rapid detection both in vitro and in vivo of Bacillus spp., among which are the members of the B. cereus group. Synthetic substrates for B. anthracis proteases were designed and exposed to secreted enzymes of a broad spectrum of bacterial species. 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. With the use of 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, with the use of 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.

Download full-text


Available from: Wendy E Kaman,
  • Source
    • "showed very active proteolytic activity against several of the LL, LD, and DD substrates. Furthermore, the D-amino acid substrates containing k-k, l-l, and r-r were cleaved by proteases from Bacillus spp., in agreement with previous data [14]. P. gingivalis culture supernatant showed a relative broad proteolytic activity on the LD-amino-acid-containing substrates, with 12% of the D-amino acid substrates being cleaved. "
    [Show abstract] [Hide abstract]
    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 utilized 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 which 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. Though 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.
    Analytical Biochemistry 07/2013; DOI:10.1016/j.ab.2013.06.015 · 2.22 Impact Factor
  • Source
    • "The use of reporter molecules for which the mass changes when in contact with microbial virulence factors has been described to help assess the putative invasive potential of bacterial species. Signal peptides that are specifically cut by known proteases have shown diagnostic value for the identification of anthrax and periodontitis [49, 50]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Clinical microbiology has always been a slowly evolving and conservative science. The sub-field of bacteriology has been and still is dominated for over a century by culture-based technologies. The integration of serological and molecular methodologies during the seventies and eighties of the previous century took place relatively slowly and in a cumbersome fashion. When nucleic acid amplification technologies became available in the early nineties, the predicted "revolution" was again slow but in the end a real paradigm shift did take place. Several of the culture-based technologies were successfully replaced by tests aimed at nucleic acid detection. More recently a second revolution occurred. Mass spectrometry was introduced and broadly accepted as a new diagnostic gold standard for microbial species identification. Apparently, the diagnostic landscape is changing, albeit slowly, and the combination of newly identified infectious etiologies and the availability of innovative technologies has now opened new avenues for modernizing clinical microbiology. However, the improvement of microbial antibiotic susceptibility testing is still lagging behind. In this review we aim to sketch the most recent developments in laboratory-based clinical bacteriology and to provide an overview of emerging novel diagnostic approaches.
    Annals of Laboratory Medicine 01/2013; 33(1):14-27. DOI:10.3343/alm.2013.33.1.14 · 1.48 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Organic molecules are a significant and highly varied component of atmospheric aerosols. Measurement of aerosol composition and improvements in our understanding of the complex chemistry involved in their formation and aging are being aided by innovations in soft ionization aerosol MS. (To listen to a podcast about this feature, please go to the Analytical Chemistry multimedia page at
    Analytical Chemistry 04/2011; 83(7):2409-15. DOI:10.1021/ac102737k · 5.64 Impact Factor
Show more