Bacteriophage Sf6 Tailspike Protein for Detection of Shigella flexneri Pathogens
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials and Bacterial Strains
2.2. Cloning and Protein Purification
2.3. Oligo- and Polysaccharide Preparation
2.4. ELISA Like Tailspike Adsorption (ELITA) Assay
2.5. Fluorescence Labeling
2.6. Fluorescence Spectroscopy
2.7. Surface Plasmon Resonance
3. Results
3.1. Sf6TSP Binds and Enzymatically Cleaves the S. flexneri Serotype 2a O-Antigen
3.2. Binding of Sf6TSP to S. flexneri O-Antigens with O-Acetyl and Glucosyl Modifications
3.3. ELITA: ELISA Like Tailspike Adsorption Assay for Detection of S. flexneri
3.4. Construction of an Sf6TSP Probe with O-Antigen Specific Fluorescence Amplitude Increase
4. Discussion
4.1. Binding Specificity of the Sf6TSP Endorhamnosidase
4.2. Sf6TSP as a Pathogen Sensor for S. flexneri
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Bacterial Isolate 1 | Anti-S. flexneri Monoclonal Antibody | PCR (wzx) 2 | |
---|---|---|---|
Group 3,4 (Y) | Type 2 | ||
S. flexneri Y 99-2001 | + | − | + |
S. flexneri Y 03-650 | + | − | + |
S. flexneri 2a 03-6557 | + | + | + |
S. flexneri 2a 08-7230 | + | + | - |
Sf6 TSP E366A/D399A Cysteine Mutant | Labeling Efficiency % | NBD Fluorescence Amplitude Increase at 540 nm 1 % |
---|---|---|
V204C | 19.4 | −10 |
S246C | 42.8 | 53 |
T315C | 43.2 | −15 |
N340C | 66.7 | 78 |
Y400C | 48.3 | 25 |
T443C | 63.3 | 12 |
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Kunstmann, S.; Scheidt, T.; Buchwald, S.; Helm, A.; Mulard, L.A.; Fruth, A.; Barbirz, S. Bacteriophage Sf6 Tailspike Protein for Detection of Shigella flexneri Pathogens. Viruses 2018, 10, 431. https://doi.org/10.3390/v10080431
Kunstmann S, Scheidt T, Buchwald S, Helm A, Mulard LA, Fruth A, Barbirz S. Bacteriophage Sf6 Tailspike Protein for Detection of Shigella flexneri Pathogens. Viruses. 2018; 10(8):431. https://doi.org/10.3390/v10080431
Chicago/Turabian StyleKunstmann, Sonja, Tom Scheidt, Saskia Buchwald, Alexandra Helm, Laurence A. Mulard, Angelika Fruth, and Stefanie Barbirz. 2018. "Bacteriophage Sf6 Tailspike Protein for Detection of Shigella flexneri Pathogens" Viruses 10, no. 8: 431. https://doi.org/10.3390/v10080431