Virulence of Bacillus anthracis, the causative agent of anthrax, is associated with the activities of its secreted toxin. Anthrax toxin is comprised of a single cell-binding moiety, protective antigen (PA), and two catalytic moieties, lethal factor (LF) and edema factor (EF). LF is a protease that cleaves members of the MAP kinase kinase family of signaling proteins, and causes lysis of macrophages. EF is an adenylate cyclase that increases cellular levels of cAMP, disrupting water homeostasis and neutrophil function. PA binds the host cellular anthrax toxin receptor (ATR) and subsequently binds and translocates LF and EF into the host cytosol where they are active. Thus, the ability of anthrax toxin to exert its toxic effects on mammalian cells depends on the ability of PA to bind ATR. Very little is known about the natural function of ATR, and no natural ligands for this receptor have been reported. However, PA binds ATR through a structurally conserved integrin-like inserted (I) domain. I domains function as protein-protein interaction modules, and thus it is likely that host proteins that normally interact with ATR do so via the same domain to which PA binds. Identifying the non-toxin interactions that ATR is capable of engaging in and defining the binding requirements for both toxin and natural ligands will provide valuable information for the development of antitoxins that block toxin-receptor interaction while leaving normal ATR interactions intact. Therefore, the primary goal of these proposed studies is to elucidate the molecular requirements for binding to ATR. ATR ligands will be identified and ATR-PA and ATR-ligand interactions will be characterized. Comparative analysis between toxin and ligand interactions will be used to determine if the requirements for binding overlap or are distinct. Additionally phage display libraries will be screened for peptides that bind specifically to ATR. The information learned from these studies wilt be used to develop distinct classes of antitoxins based on soluble receptor, toxin or ligand mimetics, or small peptides that specifically block PA-ATR interactions. ? ?
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