Suppression of the host innate immune response plays an important role in the virulence of B. anthracis. Previous studies have documented that anthrax lethal toxin (LeTx) and edema toxin (EdTx) inhibit phagocytosis and oxidative killing via the NADPH oxidase by leukocytes. However, during lysis and release of vegetative bacilli from macrophages, a critical step in the pathogenesis of B. anthracis, formation of reactive oxygen species (ROS) is paradoxically enhanced. Leukocyte chemotactic responses have been variably reported to be enhanced or inhibited as well. We propose to investigate the signal transduction events that underlie regulation by anthrax toxins of innate immune responsiveness in human leukocytes. The ability of LeTx and EdTx to modulate leukocyte chemotactic responses and motility will be assessed using microscopic time-lapse imaging. The molecular basis for the effects of toxins on motility will be established through biochemical analyses of signaling pathways involved in chemotaxis. We will relate our observation that Rac GTPase activation is induced by LeTx to possible chemotactic defects. Similarly, the ability of LeTx and EdTx to modulate NADPH oxidase-mediated formation of ROS will be evaluated. The biochemical basis for the effects of LeTx and EdTx on NADPH oxidase will be established at the level of known oxidase regulatory components using broken cell assay systems. The molecular mechanisms through which anthrax toxins modulate NADPH oxidase component function will be determined. The biochemical basis for LeTx-induced Rac GTPase activation will be investigated. We will determine whether LeTx activates Rac through proteolytic means, modulation of MAP kinase signaling, or via other signaling mechanisms. Finally, additional molecular targets of LeTx and EdTx will be investigated in the context of human leukocyte immune responses. These studies will require close collaborative interactions with other components of this Program Project. An understanding of the molecular basis for the suppression of human innate immunity by B. anthracis will lead to novel strategies to combat this potential agent of bioterrorism.