A Model Method for the Rapid Identification of Biological Warfare Agents
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1 A Model Method for the Rapid Identification of Biological Warfare Agents Lawrence Shieh Manhasset Science Research In cooperation with Polytechnic University Dr.Olga Tarasenko and Prof.Kalle Levon
2 Anthrax in Biological Warfare Bacillus anthracis spores Highly resistant to environmental effects- heat, drying, and sunlight (Eitzen, 1997). Cheap to produce- $50 per kilogram. Easily grown in quantity. Stable agent for dissemination (National Academy, Washington D.C., 1999).
3 Bacillus Spores B. subtilis Difference in Morphology (Mizuki et al., 1998; Driks, 2002). No appendages B. cereus Phylogenetically and Morphologically similar. Appendages Similarity verified through VNTR analysis and BOX-PCR genomic fingerprinting (Kim et al., 2002).
4 Detection Technologies Metabolic assays (National Academy, Washintgon, D.C., 1999). Nucleic acid-based probes (Lytwyn et al., 2000; Hamels et al., 2001). Molecular typing (Boyd et al., 1996). Unfeasible for real-world use- Slow Unstable Non-specific
5 Glycoconjugate Polymers Study of Carbohydrate Interactions (Bovin et al., 1993). High affinity and specificity of interactions achieved by polyvalency. Predetermined Properties Type of Ligands attached Medicinal Use
6 Purpose The purpose of this project was to develop a rapid method of identifying B. anthracis spores by analyzing the monosaccharide compositions of closely related Bacillus spores and determining their binding specificities with glycoconjugate polymers.
7 Methodology Bacterial Spore Samples B. cereus ATCC B. subtilis ATCC 9372 Multivalent Fluoresceinated Polymers Disaccharide-PAA-flu ELISA Glycoconjugate assay Isolation of Spore Outer Layer/Appendages Monosaccharide Compositions of Spores FACE Monosaccharide Composition Kit
8 Results- FACE Electrophoresis Figure 1. Monosaccharides composition of (1-5) spores inner shell: Lane 1- neutral B. cereus; Lane 2- amino B. cereus; Lane 3- neutral B. subtilis; Lane 4- amino B. subtilis; Lane 5- AMAC labeled monosaccharides standard.
9 Neutral Monosaccharide Composition of Inner Shells of Bacillus Spores Monosaccharides Spores GalNAc Mannose Fucose Glucose Galactose GlcNAc B. cereus B. subtilis Amine Monosaccharide Composition of Inner Shells of Bacillus Spores Monosaccharides Spores GalNAc Mannose Fucose Glucose Galactose GlcNAc B. cereus B. subtilis
10 Figure 2. Monosaccharides composition of appendages of B. cereus and outer layer of B. subtilis: Lane 1, 6- AMAC labeled monosaccharides standard; Lane 2 neutral B. cereus; Lane 3- amino B. cereus; Lane 4- neutral B. subtilis; Lane 5- amino B. subtilis.
11 Amine Monosaccharide Composition of Inner Shells of Bacillus Spores Monosaccharides Spores GalNAc Mannose Fucose Glucose Galactose GlcNAc B. cereus B. subtilis Amine Monosaccharide Composition of Inner Shells of Bacillus Spores Monosaccharides Spores GalNAc Mannose Fucose Glucose Galactose GlcNAc B. cereus B. subtilis
12 Results- ELISA (Polymer A) ELISA value (OD=492nm) B. cereus B. subtilis A. Bacterial spores
13 Results- ELISA (Polymer B) ELISA value (OD=492nm) B. cereus B. subtilis B. Bacterial spores
14 Discussion Unique monosaccharides present in Bacillus spores. Monosaccharide composition of outer layer/appendages and inner shells are different. Monosaccharides serve as specific receptors.
15 Conclusion Glycoconjugate polymers may be used to rapidly identify Bacillus spores due to their ability to bind to glycan receptors present on the cell surfaces of the spores. This method may also be applied to the detection of B. anthracis spores due to their similarity with B. cereus spores.
16 References Bentivegna. C.S Advancing monosaccharides as biomarkers: part I. Development of fluorophore-assisted carbohydrate-electrophoresis in Chironomus riparius. Aquatic Toxicology. 61: Bovin, N.V Polyacrylamide-Based Glyococonjugates as Tools in Glycobiology. Glycoconjugate Journal. 15: Bovin, N.V., Gabius, H.-J Polymer-immobilized carbohydrate ligands: versatile chemical tools for biochemistry and medical sciences. Chemical Society Reviews. 24: Bovin, N.V., Korchagina, E.Y., Zemlyanukhina, T.V., Byramova, N.E., Galanina, O.E., Zemlyakov, A.E., Ivanov, A.E., Zubov, V.P., Mochalova, L.V Synthesis of polymeric neoglycoconjugates based on N-substituted polyacrylamides. Glycoconjugate Journal. 10: Boyd, E.F., Wang, F.S., Whittam, T.S., Selander, R.K Molecular genetic relationship of the salmonellae. Applied Environmental Microbiology. 62: Carr, S.A., Okafo, G., Camilleri, P A coordinated high-performance liquid chromatographic, capillary electrophoresis, and mass spectrometric approach for the analysis of oligosaccharide mixtures derivatized with 2-aminoacridone. Analytical Chemistry. 68: Center for Disease Control and Prevention. FAQ s About Anthrax. December, 12, URL Dove, A The bittersweet promise of glycobiology. Nature Biotechnology. 19: Driks, A Maximum shields: the assembly and function of the bacterial spore coat. Trends in Microbiology. 10(6): Drobniewski, F.A Bacillus and related species. Clinical Microbiological Review. 6: Dwek, R.A Glycobiology: Toward understanding the function of sugars. Chemical Review. 96: Eitzen, E.M Use of biological weapons, p In F.R. Sidell, E.T. Takfuji and D.R. Franz (ed.), Medical aspects of chemical and biological warfare. Office of the Surgeon General, Washington, D.C. Freidlander, A.M Anthrax, p In F.R. Sidell, E.T. Takfuji and D.R. Franz (ed.), Medical aspects of chemical and biological warfare. Office of the Surgeon General, Washington, D.C. Furuike, T., Nishi, N., Tokura, S., Nishimura, S.-I Synthetic glycoconjugates. 6. Preparation and biochemical evaluation of novel cluster type glycopolymers containing Galβ(1 4) GlcNAc (Nacetyllactosamine) residue. Macromolecules. 28:
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