Structural and mechanistic study of bat NLRP6 inflammasome in detecting RNA viruses

Abstract Bats harbor the unique ability to host a wide array of emerging viruses, such as Ebola virus, Nipah virus, Hendra virus, and severe acute respiratory syndrome coronavirus (SARS-CoV). These RNA viruses are highly pathogenic and often lethal to humans and animals. Intriguingly, bats develop no/minimal signs of diseases in both natural and experimental infections. Significant progress has been made to suggest the altered immunological networks and dampened inflammatory signaling in bats. However, the direct viral sensing mechanisms in bats and the unique immunological features that distinguish bats from other mammals remain poorly studied. Inflammasomes are multi-protein signaling platforms that form in epithelial cells and myeloid cells upon stimulation by pathogen and damage signals. Their primary function is to active the inflammatory caspases such as caspase-1. Canonical inflammasome sensors consist mainly of nucleotide-binding domain (NBD), leucine-rich repeat (LRR)-containing (NLR) family proteins. Among these NLR proteins, NLRP6 is a unique pattern recognition receptor that is predominantly expressed in intestinal and liver system. The inflammasome function of NLRP6 has been reported to directly detect the RNA viruses (rotavirus and mouse hepatitis virus) that infect the gastrointestinal (GI) tract. On the other hand, the excessive activation of NLRP6 inflammasome may exacerbate the tissue damage and cause the autoinflammatory diseases. In bats, the GI tract represents one major organ for viral infection, while infections rarely cause symptoms. The long-term goal of our project is to understand the specific inflammasome sensing mechanisms in detecting RNA viruses in the intestinal epithelium of bats and gain the insights of how bats protect themselves from the pathogenesis of inflammation-induced intestinal barrier dysfunction. In this application, we propose to pursue the following specific aims: 1) Determine the cryo- EM structures of bat NLRP6 monomer, elucidate the biochemical foundation of bat NLRP6- dsRNA interaction, determine the cryo-EM structures of bat NLRP6 with viral dsRNA and compare the structural mechanisms of dsRNA sensing and inflammasome signaling among bat, mouse and human NLRP6; 2) Elucidate the RNA virus-induced bat NLRP6 inflammasome signaling in reconstituted intestinal epithelial cells (IECs), analyze the bat inflammasome signaling in Eonycteris spelaea (Es) in response to bat-borne RNA viruses, study the genetic role of bat NLRP6 in regulating inflammasome signaling in bat primary IECs/bat intestinal organoids. The proposed studies will guide the development of therapeutics to target GI inflammatory disorders in human based on the molecular details of bat NLRP6 inflammasome.