Characterization and functional analysis of a novel C-type lectin in blunt snout bream (Megalobrama amblycephala)

Abstract

C-type lectins, one of the pattern recognition receptors (PRRs), play significant roles in innate immune responses through binding to the pathogen-associated molecular patterns (PAMPs) presented on surfaces of microorganisms. Here, a novel C-type lectin (named as MaCTL) from blunt snout bream (Megalobrama amblycephala) was cloned and characterized. The open reading frame (ORF) of MaCTL is 573 bp long encoding a putative protein of 190 amino acids (aa), which contains a typical feature of signal peptide at 1-23 aa, a characteristic CRD domain at 45-178 aa and a WND/EPN motif that is required for carbohydrates-binding specificity. Phylogenetic analysis indicated that MaCTL is a novel member of CTL family and possessed the highest similarity to that of grass carp (92.11%). The qRT-PCR analysis revealed that MaCTL expressed widely in all examined normal tissues, including heart, liver, spleen, kidney, head-kidney, gill, intestine and muscle, with the higher expression in the spleen, liver and muscle. The expression of MaCTL in spleen was significantly elevated, peaking at 9 h and 6 h after LPS stimulation and Aeromonas hydrophila challenge, respectively, suggesting its association with involvement in innate immune response. The recombinant MaCTL protein (rMaCTL) agglutinated markedly both Gram-positive (Staphylococcus aureus) and Gram-negative bacteria, including Escherichia coli, Vibrio anguillarum, Vibrio vulnificus and Aeromonas hydrophila, in a Ca²⁺-dependent manner. Meanwhile, rMaCTL showed the binding effects on the five bacteria and four carbohydrates, such as glucose, surose, LPS and PGN. Moreover, rMaCTL could remarkably inhibit the growth of three types of bacteria in vitro. Overall, the results obtained above demonstrated firmly that MaCTL binds to carbohydrates on the surface of diverse pathogens as a PRR and elicits antimicrobial responses, which shed new light on a better understanding of antibacterial functions of CTLs in teleost fish.

Keywords: Antibacterial response; C-type lectin; Megalobrama amblycephala; Pattern recognition receptor.