Vibrio splendidus Flagellin C-Induced Extracellular Trap Release Relies on AjTLR2 Recognition in Apostichopus japonicus

Abstract

Extracellular traps (ETs) are a novel host defense mechanism used to immobilize and eliminate invading microorganisms, and their formation depends on the recognition of foreign pathogens by membrane receptors. Previous studies have demonstrated that Vibrio splendidus flagellin can induce the release of ETs in coelomocytes of the sea cucumber Apostichopus japonicus, yet the underlying regulatory mechanism remains unclear. Here, we identify another Toll-like receptor (TLR) homolog, AjTLR2, in Apostichopus japonicus, which is composed of an extracellular LRR domain, a transmembrane domain, and an intracellular TIR domain. As a membrane receptor, AjTLR2 is upregulated upon infection with Vibrio splendidus AJ01, which is isolated from diseased Apostichopus japonicus. The extracellular LRR domain exhibits binding activity toward LPS, PGN, and MAN. In addition to these ligands, AjTLR2 recognizes flagellin C of AJ01 (AJ01-FliC), whereas other AjTLRs, such as AjToll and AjTLR3, do not. Further functional analysis reveals that knockdown of AjTLR2 results in a reduction in the typical weblike DNA structures of ETs, accompanied by a significant decrease in the expression of the ET-associated antimicrobial proteins H2A, H2B, and lysozyme. Furthermore, AjTLR2 knockdown similarly inhibits ET formation induced by recombinant AJ01-FliC protein. Mechanistically, the Apostichopus japonicus proto-oncogene tyrosine-protein kinase Src homolog (AjSRC), previously identified in our laboratory, is a downstream signaling molecule of AjTLR2 and is recruited via the TIR domain of AjTLR2. Knockdown of AjSRC also suppresses AJ01-FliC-induced ET formation. Collectively, our results indicate that the recruitment of AjSRC by AjTLR2 represents a potential regulatory pathway for AJ01-FliC induced ET generation.