Scallop Toll-like Receptor Engages in Complex Interactions with Diverse MyD88 Proteins and Orchestrates Host Innate Immune Responses.

Abstract

Toll-like receptor (TLR) family proteins act as pattern recognition receptors with pivotal roles in immune defense; however, the function of TLRs in marine mollusks requires further investigation. In this study, we identified a novel TLR gene (CfTLR-Like-1 [CfTLR-L1]) in Chlamys farreri and analyzed its immune functions. CfTLR-L1 has a coding sequence of 1,785 bp and encodes 594 amino acids. It includes typical protein domains; the amino acid sequences of the protein domains are conserved. Phylogenetic analyses demonstrated a close evolutionary relationship among proteins in the mollusk TLR subfamily. CfTLR-L1 is predominantly located on the cell membrane. CfTLR-L1 mRNA is expressed in all tissues, particularly in the hepatopancreas and mantle. Pathogenic stressors, including lipopolysaccharide, peptidoglycan, polyinosinic-polycytidylic acid, Vibrio anguillarum, Vibrio alginolyticus, Vibrio splendidus, and acute viral necrosis virus, significantly increase the levels of CfTLR-L1 mRNA transcripts. The Toll/interleukin 1 receptor (TIR) domain of CfTLR-L1 interacts with the MyD88 family proteins of scallops (including CfMyD88-1, CfMyD88-3, and CfMyD88-4), implying that multiple unique TLR signal transduction mechanisms exist in marine mollusks. CfTLR-L1-TIR interacts with CfMyD88-1-P3, which contains only the TIR structural domain. Overexpression of CfTLR-L1-TIR in HEK293T cells activated reporter genes, including IFNβ, IFNγ, and STAT3. Similarly, overexpression of CfTLR-L1 significantly affected mitogen-activated protein kinase phosphorylation, specifically of p38 and c-Jun N-terminal kinase. Our findings expand the knowledge of the role of TLR in mollusk immunity and may provide evidence for selecting disease-resistant scallops for cultivation.