Joel Bozue USAMRIID, Fort Detrick, MD Office: 301-619-4934 FAX: E-mail: joel.bozue@det.amedd.army.mil Job Title: Principal Investigator Ph.D. in Biology from the University of Iowa

Speaker: Joel Bozue, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, MD

Topic: Contribution of Bacillus Anthracis Spore Coat Proteins to Virulence

Place: Building 549, Auditorium, NCI at Frederick, Frederick, MD

Time: Tuesday, November 9, 2004, at 2:00 PM

Abstract: All bacterial spores possess a protective proteinaceous structure known as the coat. This structure plays critical roles in spore protection and germination in Bacillus subtilis and, probably, most sporulating species. Its formation during sporulation constitutes a fundamental model for the study of macromolecular assembly. The spore coat of B. anthracis, the causative agent of anthrax, is of particular interest because the dormant spore is the infective particle. We investigated the program of B. anthracis spore coat morphogenesis in two major strains: the avirulent Sterne strain and the virulent Ames strain, and investigated the roles of key spore coat proteins in virulence. We first mutated the B. anthracis orthologues of several B. subtilis coat protein genes, including two genes with roles in B. subtilis coat assembly, cotE and cotH. In B. subtilis, CotE directs assembly of several coat proteins. One of these is CotH, which guides assembly of a significant subset of the coat proteins under CotE control. We found that, in contrast to B. subtilis, CotE has only a modest affect on spore coat composition in B. anthracis. However, it is necessary for proper assembly of the exosporium, an outer structure that encases B. anthracis spores but is not found in B. subtilis. CotE is predominantly, if not exclusively, in the coat. We hypothesize that CotE is involved in attaching the exosporium to the coat surface. We also found that cotE spores are partially defective in carrying out an early stage of germination. Strikingly, in spite of these physiological and morphological impairments, cotE mutant spores are fully infectious in a guinea pig intramuscular challenge model. This indicates that the exosporium is not required for virulence. We found that disruption of cotH in B. anthracis alters coat protein composition and other spore properties, but less severely than the corresponding mutation in B. subtilis. In addition, mutation of cotH affects spore surface morphology but not exosporium formation or the assembly of coat layers, also in contrast to what was seen in B. subtilis. These results allow us to construct an outline view of the coat assembly program in B. anthracis. The program of B. anthracis spore coat assembly turns out to differ from that of B. subtilis in several important steps, indicating that Bacilli can use a conserved core of morphogenetic coat proteins in surprisingly different ways. Our results also argue that the exosporium is not a critical virulence factor. This has implications both for the pathophysiology of the disease, as well as next generation therapeutics.