Interplay of Bacillus anthracis spore proteins in the assembly process of the exosporium
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Spores are the infectious form of the zoonotic pathogen, Bacillus anthracis. The outermost layer of these spores is the exosporium. It consists of a basal layer (major structural proteins are ExsY, CotY, BxpB, and ExsFB) and an outermost nap layer containing, mainly, the BclA glycoprotein. The major structural proteins of the inner basal layer are CotY (at the bottlecap) and ExsY. Outer basal layer proteins, BxpB and ExsFB, are needed for BclA incorporation to the spore surface. We investigated the role of sequence differences between these proteins in localization during exosporium assembly. We found that the sequence differences between CotY and ExsY were less important for their localization than timing of expression of the respective genes. The timing of expression of the bxpB and exsFB genes, and the N-terminal sequences of their protein products, are important for proper assembly into the basal layer. Fluorescent fusion constructs with fluorophore at the C-terminus of CotY or ExsY resulted in good incorporation but the fusion hybrid proteins were not fully functional. BxpB and ExsFB assembly appears not to be completely abolished in mutants lacking ExsY and CotY, despite these spores lacking a visible exosporium. Bacterial two hybrid studies were used to examine interaction strength with full-length and truncated proteins. Interactions were not only observed between ExsY and CotY, but between these two basal layer structural proteins and CotE and CotO. These latter proteins are part of the polypeptide chain that anchors the exosporium to the spore coat during the exosporium assembly process. Using transmission electron and fluorescent microscopy, western blotting, and immunofluorescence we examined the impact of single, double, and triple mutants of basal layer proteins on spore morphology, protein content and distribution. BxpB and to a lesser extent ExsFB can self-assemble in vitro into higher molecular weight forms resistant to heat and reducing conditions. This self-assembly capacity is shared by other exosporium proteins, as others have demonstrated for ExsY and CotY. However, the mechanisms may be different with BxpB and ExsFB as disulfide bonds do not play a significant role in this process.
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Ph. D.