Anthrax Toxins Impair Phagocyte Actin-based Motility

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DESCRIPTION (provided by applicant): Neutrophils and macrophages are often overwhelmed by systemic anthrax infection. The anthrax toxins, protective antigen (PA) combined with lethal factor (LF) and edema factor (EF) are able to enter the cytoplasm of phagocytic cells and impair their function. Loss of innate immunity allows the bacillus to grow unchecked within the host, leading to rapid death. We have found that PA + LF (50 ng/ml), called lethal toxin (LT), markedly impairs human neutrophil chemotaxis and chemoattractant-induced actin assembly. In this way anthrax toxins can weaken innate immunity by blocking phagocyte actin-based motility. We propose to: 1. Explore anthrax toxins effects on phagocyte actin-based motile processes. A. Compare the effects of LT on formyl-methionly-leucyl-phenylalanine (FMLP), platelet activating factor (PAF) and IgG-immune complex induction of neutrophil actin assembly using Alexa-phalloidin staining and FACs analysis. B. Examine the effects of PA + EF, and PA + LF + EF on neutrophil chemotaxis, phagocytosis, and actin assembly. C. Study the ability of anthrax toxins to block monocyte, macrophage and dendritic actin-based motility. D. Explore anthrax toxins ability to impair platelet actin assembly and spreading. 2. Determine how anthrax toxins directly or indirectly alter the function of one or more specific contractile proteins. A. Compare the proteomic signatures of LT and identify the up-regulated and down-regulated proteins in toxin-treated cells. B. Explore the effects of LT on PI-3 kinase signaling using PH-GFP constructs, examine Rac signaling using a GFP- probe that binds active Rac, and investigate p38 MAPK signaling using specific antibodies and inhibitors. C. Examine the effects of LT on Listeria and Shigella actin-based motility in infected cells, as well as cytoplasmic and reconstituted extracts, using rhodamine actin and time lapse video microscopy. D. Examine LT's effects on the in vitro function of the actin-regulatory protein Hsp27. LT blocks phosphorylation of the actin-regulatory protein Hsp27. Therefore, we will compare the function of phosphorylated to dephosphorylated Hsp27 by assaying the effects of purified recombinant wild-type and mutant, Hsp27 on the assembly and disassembly of pyrene-conjugated actin. These investigations promise to provide new insights into the mechanisms by which anthrax toxins mediate paralysis of the immune system, and are likely to provide new strategies for the diagnosis and treatment of systemic anthrax infections.