Wendy E. Kaman

Peptide-based fluorescence resonance energy transfer protease substrates for the detection and diagnosis of Bacillus species

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.

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

ABSTRACT Porphyromonas gingivalis is associated with the development of periodontitis. Here we describe the development of a highly specific protease-based diagnostic method for the detection of P. gingivalis in gingival crevicular fluid. Screening of a proteolytic peptide substrate library, including fluorogenic dipeptides that contain d-amino acids, led to the discovery of five P. gingivalis-specific substrates. Due to the presence of lysine and arginine residues in these substrates, it was hypothesized that the cleavage was mediated by the gingipains, a group of P. gingivalis-specific proteases. This hypothesis was confirmed by the observation that P. gingivalis gingipain knockout strains demonstrated clearly impaired substrate cleavage efficacy. Further, proteolytic activity on the substrates was increased by the addition of the gingipain stimulators dithiothreitol and l-cysteine and decreased by the inhibitors leupeptin and N-ethylmaleimide. Screening of saliva and gingival crevicular fluid of periodontitis patients and healthy controls showed the potential of the substrates to diagnose the presence of P. gingivalis proteases. By using paper points, a sensitivity of approximately 105 CFU/ml was achieved. P. gingivalis-reactive substrates fully composed of l-amino acids and Bz-l-Arg-NHPhNO2 showed a relatively low specificity (44 to 85%). However, the five P. gingivalis-specific substrates that each contained a single d-amino acid showed high specificity (96 to 100%). This observation underlines the importance of the presence of d-amino acids in substrates used for the detection of bacterial proteases. We envisage that these substrates may improve the specificity of the current enzyme-based diagnosis of periodontitis associated with P. gingivalis.

Comparing culture, real-time PCR and fluorescence resonance energy transfer technology for detection of Porphyromonas gingivalis in patients with or without peri-implant infections

BACKGROUND AND OBJECTIVE The aim of the study was to compare the detection of Porphyromonas gingivalis using a fluorescence resonance energy transfer (FRET) technology with commonly used diagnostic methods in salivary and subgingival plaque samples from subjects with dental implants. P. gingivalis was considered as a marker for a pathogenic microbiota. MATERIAL AND METHODS Ninety-seven adult subjects were recruited, including periodontally healthy controls with no dental implants, implant controls with no peri-implant disease and patients with peri-implant disease. Saliva and subgingival/submucosal plaque samples were collected from all subjects and were analyzed using culture, real-time PCR and FRET technology employing P. gingivalis-specific substrates. RESULTS It was found that the P. gingivalis-specific substrates were highly suitable for detecting the presence of P. gingivalis in saliva and in subgingival plaque samples, showing comparable specificity to culture and real-time PCR. CONCLUSION We applied the FRET technology to detect P. gingivalis in implant patients with or without an implant condition and in controls without implants. The technique seems suitable for detection of P. gingivalis in both plaque and saliva samples. However, with all three techniques, P. gingivalis was not very specific for peri-implantitis cases. Future work includes fine-tuning the FRET technology and also includes the development of a chair-side application.

Nepenthesin protease activity indicates digestive fluid dynamics in carnivorous Nepenthes plants

Carnivorous plants use different morphological features to attract, trap and digest prey, mainly insects. Plants from the genus Nepenthes possess specialized leaves called pitchers that function as pitfall-traps. These pitchers are filled with a digestive fluid that is generated by the plants themselves. In order to digest caught prey in their pitchers, Nepenthes plants produce various hydrolytic enzymes including aspartic proteases, nepenthesins (Nep). Knowledge about the generation and induction of these proteases is limited. Here, by employing a FRET (fluorescent resonance energy transfer)-based technique that uses a synthetic fluorescent substrate an easy and rapid detection of protease activities in the digestive fluids of various Nepenthes species was feasible. Biochemical studies and the heterologously expressed Nep II from Nepenthes mirabilis proved that the proteolytic activity relied on aspartic proteases, however an acid-mediated auto-activation mechanism was necessary. Employing the FRET-based approach, the induction and dynamics of nepenthesin in the digestive pitcher fluid of various Nepenthes plants could be studied directly with insect (Drosophila melanogaster) prey or plant material. Moreover, we observed that proteolytic activity was induced by the phytohormone jasmonic acid but not by salicylic acid suggesting that jasmonate-dependent signaling pathways are involved in plant carnivory.

Bacterial proteases: Targets for diagnostics and therapy

Proteases are essential for the proliferation and growth of bacteria, and are also known to contribute to bacterial virulence. This makes them interesting candidates as diagnostic and therapeutic targets for infectious diseases. In this review, the authors discuss the most recent developments and potential applications for bacterial proteases in the diagnosis and treatment of bacterial infections. Current and future bacterial protease targets are described and their limitations outlined.

Activation of Toll-like receptors and dendritic cells by a broad range of bacterial molecules.

Activation of pattern recognition receptors such as Toll-like receptors (TLRs) by pathogens leads to activation and maturation of dendritic cells (DC), which orchestrate the development of the adaptive immune response. To create an overview of the effects of a broad range of pathogenic bacteria, their capacity to activate TLRs and to affect DC maturation, cytokine production and T cell polarizing capacity were determined. Different bacterial species differed in their potency to affect these parameters. In general, on the DC level differences were found in the maturation-inducing capacity of gram-negative and gram-positive bacteria. Remarkably, these differences did not result in differential polarization of the T cell response. With respect to TLRs, TLR4 activation by pathogens correlated with their ability to induce DC maturation, while for TLR2 and TLR5 such a correlation was absent. Taken together, this study provides insight into qualitative differences and general effects of pathogen-derived molecules on dendritic cells.

Perceptions of point-of-care infectious disease testing among European medical personnel, point-of-care test kit manufacturers, and the general public

Background The proper development and implementation of point-of-care (POC) diagnostics requires knowledge of the perceived requirements and barriers to their implementation. To determine the current requirements and perceived barriers to the introduction of POC diagnostics in the field of medical microbiology (MM)-POC a prospective online survey (TEMPOtest-QC) was established. Methods and results The TEMPOtest-QC survey was online between February 2011 and July 2012 and targeted the medical community, POC test diagnostic manufacturers, general practitioners, and the general public. In total, 293 individuals responded to the survey, including 91 (31%) medical microbiologists, 39 (13%) nonmedical microbiologists, 25 (9%) employees of POC test manufacturers, and 138 (47%) members of the general public. Responses were received from 18 different European countries, with the largest percentage of these living in The Netherlands (52%). The majority (>50%) of medical specialists regarded the development of MM-POC for blood culture and hospital acquired infections as “absolutely necessary”, but were much less favorable towards their use in the home environment. Significant differences in perceptions between medical specialists and the general public included the: (1) Effect on quality of patient care; (2) Ability to better monitor patients; (3) Home testing and the doctor-patient relationship; and (4) MM-POC interpretation. Only 34.7% of the general public is willing to pay more than a€10 (

Evaluation of a D-amino-acid-containing fluorescence resonance energy transfer peptide library for profiling prokaryotic proteases.

13) for a single MM-POC test, with 85.5% preferring to purchase their MM-POC test from a pharmacy. Conclusion The requirements for the proper implementation of MM-POC were found to be generally similar between medical specialists and POC test kit manufacturers. The general public was much more favorable with respect to a perceived improvement in the quality of healthcare that these tests would bring to the hospital and home environment.

The papain inhibitor (SPI) of Streptomyces mobaraensis inhibits bacterial cysteine proteases and is an antagonist of bacterial growth

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.

Current problems associated with the microbiological point-of-care testing of respiratory tract infections in primary care

ABSTRACT A novel papain inhibitory protein (SPI) from Streptomyces mobaraensis was studied to measure its inhibitory effect on bacterial cysteine protease activity (Staphylococcus aureus SspB) and culture supernatants (Porphyromonas gingivalis, Bacillus anthracis). Further, growth of Bacillus anthracis, Staphylococcus aureus, Pseudomonas aeruginosa, and Vibrio cholerae was completely inhibited by 10 μM SPI. At this concentration of SPI, no cytotoxicity was observed. We conclude that SPI inhibits bacterial virulence factors and has the potential to become a novel therapeutic treatment against a range of unrelated pathogenic bacteria.