B-231. Bacillus anthracis Edema Toxin Suppresses Human Macrophage Phagocytosis and Cytoskeletal Remodeling via the PKA and Epac Pathways

L. A. Yeager, A. K. Chopra, J. W. Peterson;
Univ. of Texas Med. Branch, Galveston, TX.

Background: Bacillus anthracis, the cause of anthrax, is a Gram-positive, spore-forming bacterium that is classified as a Category A select agent. It produces edema toxin (ET), a powerful adenylate cyclase that increases cAMP levels of various target cells. Because other cAMP-increasing agents inhibit key macrophage (MØ) tasks, such as phagocytosis and oxidative burst, it was hypothesized that ET would exhibit similar suppressive activities. Our previous data showed that ET down-regulated genes involved in actin cytoskeleton remodeling in murine MØ’s, including protein kinase A (PKA). To further examine the role of ET during anthrax pathogenesis in humans, we explored the hypothesis that ET-treatment leads to deregulation of the cAMP-dependent PKA and/or Epac system, resulting in impaired cytoskeletal functions essential for MØ activity. Methods: HL-60 cells were differentiated into MØ’s with PMA. ET-treated MØ’s were infected with Ames spores for 1h before lysing and plating to evaluate phagocytosis. Immunofluorescence assays that labeled ET-treated MØ’s with phalloidin allowed measurement of MØ spreading and F-actin content. Western Blot analysis was performed to monitor PKA and Epac levels in response to ET treatment, as well as a fluorescent PKA activity assay. Chemical cAMP analogues with specificity toward PKA or Epac were included in some assays to determine which pathway was involved. Results: ET significantly suppressed phagocytosis of Ames spores as early as 6h post-toxin treatment. However, ET enhanced the respiratory burst response of undifferentiated HL-60 cells. Cytoskeletal changes such as decreased cell spreading, reduced filopodia, and lowered F-actin content were observed for toxin-treated MØ’s. ET also altered expression of PKA and Epac, and changed PKA activity in a time-dependent manner. Finally, both the Epac and PKA pathways were targeted by EdTx-generated cAMP. Conclusion: These results provide new evidence that ET weakens the host immune response by increasing cAMP levels, which then acts via PKA and Epac to impair MØ phagocytosis and interfere with cytoskeletal remodeling.