Detecting the Unseen Enemies - Bio-warfareDetecting the Unseen Enemies - Bio-warfare

The overall goal of this project is to use mass spectrometry (MS) to develop 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 rapid, real-time, and facile methods for remote detection of biological

warfare agents in the atmosphere . To facilitate these aims, studies are warfare agents in the atmosphere . To facilitate these aims, studies are conducted using simulants of Bacillus anthracis (our model organism and conducted using simulants of Bacillus anthracis (our model organism and

the causative agent of anthrax). The major spore coat proteins of the the causative agent of anthrax). The major spore coat proteins of the simulants are isolated and characterized and then analyzed by MS . simulants are isolated and characterized and then analyzed by MS .

Results would provide unique molecular signatures enabling the Results would provide unique molecular signatures enabling the identification of similar biological warfare agents in sample aerosol.identification of similar biological warfare agents in sample aerosol.

Adebomi Omikunle Adebomi Omikunle Mississippi State UniversitMississippi State Universityy

Joanne Horn Ph.D., Earth & Environmental Sciences Joanne Horn Ph.D., Earth & Environmental Sciences

Directorate, Lawrence Livermore National Laboratory,Directorate, Lawrence Livermore National Laboratory,Office of Defense Programs, U.S. Department of EnergyOffice of Defense Programs, U.S. Department of Energy

Results:Results:

Discussion:Discussion:

IntroductionIntroduction Methods:Methods:

The aim of these studies is to determine The aim of these studies is to determine whether different biological particles yield whether different biological particles yield different mass spectrometry signatures. If so, different mass spectrometry signatures. If so, this will be the basis for the development of a this will be the basis for the development of a spectrometer that will, in real time, detect and spectrometer that will, in real time, detect and distinguish between different organisms. distinguish between different organisms.

The analogs of The analogs of Bacillus anthracisBacillus anthracis used for used for this study are: this study are: Bacillus subtilis, Bacillus Bacillus subtilis, Bacillus subtilis var. niger, Bacillus cereus, subtilis var. niger, Bacillus cereus, and and Bacillus thuringiensis. Bacillus thuringiensis. This study aims to This study aims to isolate and characterize the spore coat isolate and characterize the spore coat proteins of these simulants using sodium proteins of these simulants using sodium dodecyl sulfate (SDS)-Polyacrylamide gel dodecyl sulfate (SDS)-Polyacrylamide gel electrophoresis (PAGE), ion - exchange electrophoresis (PAGE), ion - exchange chromatography, electroelution techniques chromatography, electroelution techniques and mass spectrometry. and mass spectrometry.

The organisms were cultured separately in The organisms were cultured separately in 1/4X tryptone-yeast sporulation media1/4X tryptone-yeast sporulation media11. . The spores produced were harvested by The spores produced were harvested by centrifugation, and the coat proteins centrifugation, and the coat proteins extracted by incubating at 37extracted by incubating at 37°°C. for one C. for one hour in 50mM TRIS, pH 8, 8M urea, 1% hour in 50mM TRIS, pH 8, 8M urea, 1% SDS, 50mM dithiothreitol. The resulting SDS, 50mM dithiothreitol. The resulting extract was dialyzed against 50mM TRIS, extract was dialyzed against 50mM TRIS, pH 8, 1mM phenylmethyl-sulfonyl fluoride . pH 8, 1mM phenylmethyl-sulfonyl fluoride . The crude extracts were then either The crude extracts were then either partially purified using gel elutionpartially purified using gel elution22 or ion- or ion-exchange chromatography on SP exchange chromatography on SP sepharose using 50mM NaPOsepharose using 50mM NaPO44 buffer, buffer, pH7.5 and a linear gradient of 0 - 1M NaCl. pH7.5 and a linear gradient of 0 - 1M NaCl. Results were analyzed using SDS-PAGEResults were analyzed using SDS-PAGE22..References:References:1)1) Lazzarini, Robert A. and E. Santangelo. 1967. Lazzarini, Robert A. and E. Santangelo. 1967. “Medium-dependent Alteration of Lysine Transfer “Medium-dependent Alteration of Lysine Transfer Ribonucleic Acid in Sporulating Ribonucleic Acid in Sporulating Bacillus subtilisBacillus subtilis Cells.” Journal of Bacteriol. 94: 125-130.Cells.” Journal of Bacteriol. 94: 125-130.2)2) Jacobs, E. and A. Clad. 1986. “Electroelution of Jacobs, E. and A. Clad. 1986. “Electroelution of fixed and stained membrane proteins from fixed and stained membrane proteins from preparative SDS-polyacrylamide gels into preparative SDS-polyacrylamide gels into membrane trap.” Anal. Biochem. 154: 583-589.membrane trap.” Anal. Biochem. 154: 583-589.

-- Visualization and molecular weight Visualization and molecular weight analysis of the major coat proteins of analysis of the major coat proteins of the four species of Bacillus was the four species of Bacillus was achieved by SDS-PAGE (fig. 2).achieved by SDS-PAGE (fig. 2).-- Visualization of eluted and partially Visualization of eluted and partially purified 22.5Kd & 24Kd proteins from purified 22.5Kd & 24Kd proteins from B. B. cereuscereus and and B. thuringiensisB. thuringiensis by SDS- by SDS- PAGE PAGE electroelution was electroelution was accomplished (fig.4)accomplished (fig.4)-- A single ion-exchange purification A single ion-exchange purification achieved the separation of four achieved the separation of four BacillusBacillus subtilissubtilis spore coat proteins from crude spore coat proteins from crude extracts (fig.3).extracts (fig.3).-- Mass spectrometry signatures of four Mass spectrometry signatures of four types of Bacillus spores illustrates types of Bacillus spores illustrates significant differences (fig. 1), significant differences (fig. 1), demonstrating that mass spectrometry demonstrating that mass spectrometry is is a promising tool for identifying a promising tool for identifying bacteria. bacteria. -- Future additional efforts include: large Future additional efforts include: large scale preparation of spores, use of scale preparation of spores, use of silver staining techniques on gels to silver staining techniques on gels to improve sensitivity, N-terminal analysis improve sensitivity, N-terminal analysis of of the isolated proteins, and simulated the isolated proteins, and simulated weaponization of the analogs.weaponization of the analogs.

fig. 3. Partially Purified fig. 3. Partially Purified B. B. subtilissubtilis Spore Coat Proteins Spore Coat Proteins

Ion Exchange:Sepharose, pH 7.5, 0-0.5M Ion Exchange:Sepharose, pH 7.5, 0-0.5M NaCl, 15% SP SDS-PAGE gelNaCl, 15% SP SDS-PAGE gel

fig. 2. Bacillus Spore Coat fig. 2. Bacillus Spore Coat Proteins-Crude Extracts Proteins-Crude Extracts

Extraction of whole sporesExtraction of whole spores 15% SDS-PAGE15% SDS-PAGE

Fig. 4. Gel-Electroeluted Fig. 4. Gel-Electroeluted Spore Coat Proteins of Spore Coat Proteins of

B. cereus & B. thuringiensisB. cereus & B. thuringiensisElectrophoretic Separation - 15% SDS-PAGE Electrophoretic Separation - 15% SDS-PAGE

(fig. 4)(fig. 4)

Bacillus Bacillus nigerniger

Bacillus Bacillus thuringiensisthuringiensis

Bacillus Bacillus cereuscereus

fig. 1. Time of flight - MS Spectra of fig. 1. Time of flight - MS Spectra of three different Bacillus spore species three different Bacillus spore species

B. thuringiensis 22.5Kd

B. thuringiensis 24Kd

Low MW B. cereus 22.5Kd

B. cereus 24Kd

Polypeptide

UCRL-#####-##-#Work performed under the auspices of the U.S. Department of Energy by the Lawrence Livermore National Laboratory under Contract W-7405-Eng-48.