Our laboratory has recently reported the structure of a Bacillus anthracis species-specific polysaccharide. This polysaccharide (termed HF-PS) has been classified a 'non-classical'secondary cell wall polymer. It is isolated from the cell wall of vegetative cells by treatment with aqueous hydrogen fluoride (HF), and was previously reported to function in anchoring/exporting the S-layer proteins, Sap and EA1, to the cell surface. Our work strongly supports that the HF-PS is structurally specific to B. anthracis strains, is immunogenic in that animals injected with live or dead spores of B. anthracis Sterne (34F2) produce antibodies against HF-PS, and is immunogenically specific with regard to the binding of these antibodies to the HF-PSs of even closely related B. cereus strains. Further, we have shown that sera from Rhesus macaques that survive exposure to B. anthracis spores contain IgG anti-HF-PS antibodies. Preliminary data and results in the literature support that these polysaccharides could be crucial for important functions with regard to the growth and virulence of pathogenic Bacillus species, including B. anthracis. However, to date direct empirical evidence is still missing. The aims of this proposal are to engineer mutants that are impaired in the biosynthesis of this HF-PS polysaccharide and to characterize the phenotype of these mutants with regard to their growth, sporulation, HF-PS structure, HF-PS interaction with S-layer proteins and various phage endolysins, and to study the effect of each mutation on the virulence in a mouse model. This research will provide us with necessary insight regarding the HF-PS's pathogenic functions, and its usefulness for the development of vaccines, diagnostics and therapeutics. PUBLIC HEALTH RELEVANCE: The bacterium Bacillus anthracis causes anthrax in humans;as a bioterrorism agent the bacteria are a threat to public health. Our laboratory has recently reported a cell wall polysaccharide of a B. anthracis (termed HF- PS) with a structure specific to these bacteria. We will create B. anthracis mutants impaired in the biosynthesis of this cell wall polysaccharide and investigate whether these mutants are impaired in important bacterial growth functions and disease development. This investigation will lay the groundwork for future studies into improved vaccines, diagnostics, and therapeutics, and into new anthrax-related public health practices.