Regulation of Drosophila innate immune signaling by amyloids and phospholipids. Anni Kleino¹, Jixi Li², Johanna Napetschnig², Lucy Chai¹, Kingsley Essien¹, Hao Wu², Neal Silverman¹. 1) Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA; 2) Program in Cellular and Molecular Medicine, Children's Hospital Boston, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.

   In Drosophila, innate immune responses against Gram-negative bacteria are mainly mediated via the IMD signaling pathway. The transmembrane receptor PGRP-LC, and the intracellular receptor PGRP-LE are required for the recognition of DAP-type peptidoglycan, and they both utilize RIP-homotypic interaction motif (RHIM)-like motifs to drive signal transduction. In mammals, the RHIM-motifs of RIP1 and RIP3 are involved in TNF-induced programmed necrosis, and were recently reported to form amyloid fibrils upon death signaling. Our aim is to clarify the mechanism of the PGRP-LC/LE RHIM-like motifs in the IMD signaling and to investigate the role of lipid-protein interactions in the regulation of the IMD signaling. We find that RHIM-like motifs from PGRP-LC and PGRP-LE, as well as their interaction partners IMD and Pirk, form amyloids in vitro. Furthermore, the treatment of S2* cells with a known inhibitor of amyloid formation, Thioflavin T, blocked the IMD pathway activity, suggesting that amyloid formation is required for immune signaling. However, Toll signaling was unaffected by Thioflavin T. Additionally, we find that the receptor-proximal adaptor protein IMD and the negative regulator Pirk bind negatively charged phospholipids, indicating association with cellular membranes. Further experiments are required to elucidate the mechanism by which amyloid fibrils and lipid-interactions regulate the IMD signaling pathway.