ML1 suppresses bacterial infection by recognizing LPS and regulating AMP expression in shrimp.

Toll and Toll-like receptors (TLRs) play essential roles in the innate immunity of and mammals. Recent studies have revealed the presence of Toll-mediated immune signaling pathways in shrimp. However, the recognition and activation mechanism of Toll signaling pathways in crustaceans remain poorly understood due to the absence of key recognition molecules, such as peptidoglycan recognition proteins. Here, a novel MD2-related lipid-recognition (ML) member named ML1 was characterized in . We found that ML1 shared a similar 3D structure with human MD2 that could specifically recognize lipopolysaccharides (LPS) participating in LPS-mediated TLR4 signaling. was highly expressed in hemocytes and remarkably upregulated after challenge. Furthermore, the binding and agglutinating assays showed that ML1 possessed strong binding activities to LPS and its key portion lipid A as well as cells, and the binding of ML1 with bacterial cells led to the agglutination of bacteria, suggesting ML1 may act as a potential pathogen recognition protein upon interaction with LPS. Besides, coating with recombinant ML1 promoted bacterial clearance and increased the survival rate of bacterium-challenged shrimp. This result was further confirmed by RNAi experiments. The knockdown of remarkably suppressed the clearance of bacteria in hemolymph and decreased the survival rate of infected shrimp. Meanwhile, the silencing of severely impaired the expression of a few antimicrobial peptides (AMPs). These results demonstrated the significant correlation of bacterial clearance mediated by ML1 with the AMP expression. Interestingly, we found that ML1 interacted with the extracellular region of Toll2, which had been previously shown to participate in bacterial clearance by regulating AMP expression. Taken together, the proposed antibacterial model mediated by ML1 might be described as follows. ML1 acted as a potential recognition receptor for Gram-negative bacteria by binding to LPS, and then it activated Toll2-mediated signaling pathway by interacting with Toll2 to eliminate invading bacteria through producing specific AMPs. This study provided new insights into the recognition and activation mechanism of Toll signaling pathways of invertebrates and the defense functions of ML members.