Detecting the Unseen Enemies - Bio-warfare

1. Detecting the Unseen Enemies - Bio-warfare Adebomi Omikunle Mississippi State University Joanne Horn Ph.D., Earth & Environmental Sciences Directorate, Lawrence Livermore National Laboratory, Office of Defense Programs, U.S. Department of Energy The overall goal of this project is to use mass spectrometry (MS) to develop rapid, real-time, and facile methods for remote detection of biological warfare agents in the atmosphere . To facilitate these aims, studies are conducted using simulants of Bacillus anthracis (our model organism and the causative agent of anthrax). The major spore coat proteins of the simulants are isolated and characterized and then analyzed by MS . Results would provide unique molecular signatures enabling the identification of similar biological warfare agents in sample aerosol. Introduction Methods: The organisms were cultured separately in 1/4X tryptone-yeast sporulation media1. The spores produced were harvested by centrifugation, and the coat proteins extracted by incubating at 37°C. for one hour in 50mM TRIS, pH 8, 8M urea, 1% SDS, 50mM dithiothreitol. The resulting extract was dialyzed against 50mM TRIS, pH 8, 1mM phenylmethyl-sulfonyl fluoride . The crude extracts were then either partially purified using gel elution2 or ion-exchange chromatography on SP sepharose using 50mM NaPO4 buffer, pH7.5 and a linear gradient of 0 - 1M NaCl. Results were analyzed using SDS-PAGE2. References: 1) Lazzarini, Robert A. and E. Santangelo. 1967. “Medium-dependent Alteration of Lysine Transfer Ribonucleic Acid in Sporulating Bacillus subtilis Cells.” Journal of Bacteriol. 94: 125-130. 2) Jacobs, E. and A. Clad. 1986. “Electroelution of fixed and stained membrane proteins from preparative SDS-polyacrylamide gels into membrane trap.” Anal. Biochem. 154: 583-589. The aim of these studies is to determine whether different biological particles yield different mass spectrometry signatures. If so, this will be the basis for the development of a spectrometer that will, in real time, detect and distinguish between different organisms. The analogs of Bacillus anthracis used for this study are: Bacillus subtilis, Bacillus subtilis var. niger, Bacillus cereus, and Bacillus thuringiensis. This study aims to isolate and characterize the spore coat proteins of these simulants using sodium dodecyl sulfate (SDS)-Polyacrylamide gel electrophoresis (PAGE), ion - exchange chromatography, electroelution techniques and mass spectrometry. Results: Discussion: Bacillus niger - Visualization and molecular weight analysis of the major coat proteins of the four species of Bacillus was achieved by SDS-PAGE (fig. 2). - Visualization of eluted and partially purified 22.5Kd & 24Kd proteins from B. cereus and B. thuringiensis by SDS- PAGE electroelution was accomplished (fig.4) - A single ion-exchange purification achieved the separation of four Bacillussubtilis spore coat proteins from crude extracts (fig.3). - Mass spectrometry signatures of four types of Bacillus spores illustrates significant differences (fig. 1), demonstrating that mass spectrometry is a promising tool for identifying bacteria. - Future additional efforts include: large scale preparation of spores, use of silver staining techniques on gels to improve sensitivity, N-terminal analysis of the isolated proteins, and simulated weaponization of the analogs. Bacillus thuringiensis fig. 2. Bacillus Spore Coat Proteins-Crude Extracts Extraction of whole spores 15% SDS-PAGE Bacillus cereus fig. 3. Partially Purified B. subtilis Spore Coat Proteins Ion Exchange:Sepharose, pH 7.5, 0-0.5M NaCl, 15% SP SDS-PAGE gel fig. 1. Time of flight - MS Spectra of three different Bacillus spore species Polypeptide B. cereus 22.5Kd B. cereus 24Kd Low MW B. thuringiensis 22.5Kd B. thuringiensis 24Kd Fig. 4. Gel-Electroeluted Spore Coat Proteins of B. cereus & B. thuringiensis Electrophoretic Separation - 15% SDS-PAGE (fig. 4) Work performed under the auspices of the U.S. Department of Energy by the Lawrence Livermore National Laboratory under Contract W-7405-Eng-48. UCRL-#####-##-#