Role of Ca2+ in B. anthracis exosporium assembly

Bacillus anthracis is a Gram-positive soil bacterium that forms spores when starved for nutrients, and contact with these spores causes anthrax in animals and humans. B. anthracis spores are surrounded by three protective layers, the outermost of which is a loosely fitting exosporium. The exosporium plays key roles in spore survival and infectivity. Over the past two decades, there has been significant progress in identifying the proteins that comprise the exosporium and their functions, however, the assembly process is poorly understood. This proposal explores a previously unrecognized role for Ca2+ in exosporium assembly. The exosporium is a bipartite structure consisting of a paracrystalline basal layer and an external hair- like nap. Each filament of the nap is formed by a trimer of the collagen-like glycoprotein BclA. In contrast, the basal layer contains ~25 different proteins. One of these proteins is called BxpB, which appears to play two critical roles in exosporium assembly. First, BxpB trimers form an array of basal layer surface protrusions to which individual BclA-containing filaments are stably attached. Second, BxpB binds to and stabilizes the structure of an underlying basal layer scaffold formed by protein self-assembly. In the absence of BxpB, the scaffold is structurally disordered and the insertion of other basal layer proteins is aberrant. We recently published the crystal structure of the BxpB trimer. Each monomer folds into a jelly roll structure composed of two antiparallel b-sheets. An unexpected feature of the BxpB monomer was a bound Ca2+ ion hexa-coordinated by oxygen atoms of four residues located on neighboring loops that connect the two b-sheets. The bound Ca2+ appears to stabilize the relative positions of the two b-sheets thereby fixing a major element of BxpB structure. In addition, recent structural studies of the BclA-BxpB complex reveal that the BxpB-bound Ca2+ establishes the structure of a peripheral region of BxpB that closely interacts with BclA. Presumably, the activities of BxpB in exosporium assembly require the Ca2+-dependent structures. The goal of this R03 proposal is to investigate the role for Ca2+ in BxpB structure and function. Aim 1 is to produce Ca2+-free BxpB, determine its structure by X-ray crystallography (or NMR), and compare this structure to that of Ca2+-bound BxpB. Aim 2 is to assess the ability of Ca2+-free BxpB to form stable complexes with BclA and to normally insert into the basal layer scaffold. The expected outcome is the demonstration of a critical role for Ca2+ in BxpB structure and exosporium assembly in B. anthracis spores. This situation is likely to apply to many spore-forming bacterial species, including important pathogens, that possess a BxpB homolog and produce an exosporium that is structurally like that of B. anthracis. The discoveries in this proposal will significantly enhance our understanding of bacterial spore formation and potentially lead to better responses to the adverse effects of disease-causing spores. Project Number: 1R03AI182814-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: CHARLES TURNBOUGH | Institution: UNIVERSITY OF ALABAMA AT BIRMINGHAM, BIRMINGHAM, AL | Award Amount: $148,500 | Activity Code: R03 | Study Section: Prokaryotic Cell and Molecular Biology Study Section[PCMB] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R03AI18281401A1