UuCTL6, a novel C-type lectin of Urechis unicinctus, agglutinates various bacteria and promotes immune defense against bacterial infection.

C-type lectins (CTLs) serve as pivotal pattern recognition receptors (PRRs) within the innate immune system of invertebrates. In this study, we identified and characterized a novel CTL from the echiuran worm (Urechis unicinctus), termed UuCTL6, to elucidate its function in antimicrobial defense. Sequence analysis revealed that the open reading frame (ORF) of UuCTL6 comprises 1074 bp, encoding a polypeptide of 358-amino acid, which includes a conserved carbohydrate recognition domain (CRD) stabilized by two disulfide bonds. Sequence identity analysis showed that UuCTL6 shares 26.7%-31.6% amino acid sequence identity with CTLs from other species. Quantitative real-time PCR (qPCR) analysis demonstrated constitutive expression of UuCTL6 across all examined tissues in healthy U. unicinctus, with the highest expression level observed in skin and the lowest level in respiratory gut. Upon challenge with Vibrio anguillarum, UuCTL6 expression was peaked at 12-24 h post-infection (hpi) in the skin, renal tubule, coelomocytes, and crop. The recombinant UuCTL6 (rUuCTL6) exhibited calcium-dependent, broad-spectrum binding to both Gram-positive and Gram-negative bacteria, and ELISA assays further showed its dose-dependent binding to four microbial polysaccharides, with the highest affinity for PGN followed by LPS, LTA, and mannan. Functional analyses indicated that rUuCTL6 significantly enhanced the production of reactive oxygen species (ROS) in hemocytes and expedited the clearance of V. anguillarum from infected U. unicinctus, thereby confirming its role in pathogen clearance. Collectively, these findings suggest that UuCTL6 functions as a critical PRR in U. unicinctus, facilitating bacterial recognition and agglutination to enhance innate immune responses against pathogenic infections.