Abstract
We sought to identify proteins in the Bacillus anthracis spore, conserved in other strains of the closely related Bacillus cereus group, that elicit an immune response in mammals. Two high throughput approaches were used. First, an in silico screening identified 200 conserved putative B. anthracis spore components. A total of 192 of those candidate genes were expressed and purified in vitro, 75 of which reacted with the rabbit immune sera generated against B. anthracis spores. The second approach was to screen for cross-reacting antigens in the spore proteome of 10 diverse B. cereus group strains. Two-dimensional electrophoresis resolved more than 200 protein spots in each spore preparation. About 72% of the protein spots were found in all the strains. 18 of these conserved proteins reacted against anti-B. anthracis spore rabbit immune sera, two of which (alanine racemase, Dal-1 and the methionine transporter, MetN) overlapped the set of proteins identified using the in silico screen. A conserved repeat domain protein (Crd) was the most immunoreactive protein found broadly across B. cereus sensu lato strains. We have established an approach for finding conserved targets across a species using population genomics and proteomics. The results of these screens suggest the possibility of a multiepitope antigen for broad host range diagnostics or therapeutics against Bacillus spore infection.
Highlights
We sought to identify proteins in the Bacillus anthracis spore, conserved in other strains of the closely related Bacillus cereus group, that elicit an immune response in mammals
Bacterial Cultures—For comparative proteomics analysis, 10 strains from the B. cereus group were selected: B. anthracis Sterne 34F2; B. thuringiensis BGSC 4AJ1 and BGSC 4Y1; five strains of B. cereus, ATCC 10987, ATCC 10876, BGSC 6E1, m1293 (Oslo, Norway), and m1550; B. mycoides DSMZ 2048; and B. pseudomycoides DSMZ 12442
The resulting transcriptionally active products were used as a template for cell-free in vitro translation reactions using the Rapid Translational System (RTS) ProteoMaster by Roche Applied Science, and high levels of protein were recovered and purified
Summary
Bacterial Cultures—For comparative proteomics analysis, 10 strains from the B. cereus group were selected: B. anthracis Sterne 34F2 (a pXO2Ϫ laboratory strain); B. thuringiensis BGSC 4AJ1 and BGSC 4Y1; five strains of B. cereus, ATCC 10987, ATCC 10876, BGSC 6E1, m1293 (Oslo, Norway), and m1550; B. mycoides DSMZ 2048; and B. pseudomycoides DSMZ 12442 (sources of the strains are shown in Supplement 1). The heat-treated spore suspensions were centrifuged at 6000 rpm (4,350 ϫ g) at 25 °C for 30 min and washed extensively with cold (4 °C) Milli-Q water. Spore pellets were purified through a 50% renograffin layer (Bracco Diagnostics) (35) prepared in Milli-Q water and centrifuged at 4,350 ϫ g for 30 min at 25 °C to remove cell debris. Spore suspensions were centrifuged at 13,200 rpm (16,100 ϫ g) at 25 °C for 10 min, and the pellets were resuspended in 500 l of lysis buffer containing ZOOM 2D protein solubilizing buffer-1 (Invitrogen) and protease inhibitor mixture (Roche Applied Science). Two-dimensional Gel Electrophoresis Separation of Spore Proteins—The spore protein extracts were mixed with IPG rehydration buffer (8 M urea, 4% (w/v) CHAPS, 0.04 M Tris base, 0.065 M dithiothreitol, and 0.01% (w/v) bromphenol blue) in a final volume of 260 l.
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