Identification and function analysis of Toll-like receptor 4 (TLR4) from Manila clam (Ruditapes philippinarum)

Toll-like receptor 4 (TLR4) is a pattern recognition receptor that activates innate immunity in response to pathogen infection. However, the role of TLR4 in pathogen-induced apoptosis and host immunity in mollusks remains largely unknown. In this study, the TLR4 of the Manila clam Ruditapes philippinarum (RpTLR4) was cloned. The open reading frame of RpTLR4 encodes a protein of 734 amino acids, containing a conserved TIR domain. Phylogenetic analysis revealed that RpTLR4 clusters closely with TLR4s from mollusks. RpTLR4 mRNA was detected in all tested tissues, with notably high expression in hemocytes (428-fold) and gills (657-fold). Subcellular localization showed that RpTLR4 is expressed on the cell membrane. qRT-PCR and western blot analyses demonstrated that RpTLR4 expression was induced in Manila clams after treatment with Vibrio parahaemolyticus. Overexpression of RpTLR4 significantly increased apoptosis levels and the expression of apoptosis-related genes. Conversely, silencing RpTLR4 markedly reduced the apoptosis rate in hemocytes induced by V. parahaemolyticus, indicating that V. parahaemolyticus-induced hemocyte apoptosis depends on RpTLR4 expression. Overall, these findings confirm that RpTLR4 plays a pro-apoptotic role in the response of Manila clams to V. parahaemolyticus infection. This study provides a theoretical foundation for understanding the molecular mechanisms underlying mollusk responses to pathogen infection.