A novel C-type lectin, Nattectin-like protein, with the function of regulating AMP expression and phenoloxidase activity in Procambarus clarkii.

As a crucial member of pattern recognition receptors (PRRs), C-type lectins (CTLs) play a pivotal role in innate immunity by recognizing pathogen-associated molecular patterns. In this study, we identified and characterized a novel CTL, Nattectin-like protein (PcNTC), from Procambarus clarkii and conducted a comprehensive functional analysis. PcNTC possesses a carbohydrate recognition domain and a signal peptide. The open reading frame of PcNTC spans 570 bp and encodes a protein consisting of 189 amino acids. Quantitative polymerase chain reaction analysis revealed the ubiquitous expression of PcNTC across various crayfish tissues, with the highest expression observed in hemocytes. Following Aeromonas hydrophila challenge, the relative expression level of PcNTC in hemocytes and gills was considerably up-regulated. Enzyme-linked immunosorbent assay showed that the recombinant PcNTC (rPcNTC) can bind to polysaccharides (lipopolysaccharide and peptidoglycan). Bacterial agglutination assay revealed that rPcNTC can bind to Gram-positive (Staphylococcus aureus, Bacillus subtilis, and Bacillus thuringiensis) and Gram-negative bacteria (Escherichia coli, Vibrio parahaemolyticus, and A. hydrophila). However, the agglutination test results indicated that rPcNTC only exhibited agglutination activity against S. aureus. RNA interference experiments revealed that knockdown of PcNTC resulted in substantial decreases in the transcription levels of immune-related genes (Relish and Dorsal) and antimicrobial peptides (ALF5, ALF6, ALF8, ALF9, CRU1, CRU2, CRU3, CRU4, and Lys-i1) in hemocytes and gills. In addition, the prophenoloxidase gene expression (ppo1 and ppo2) and activity of phenoloxidase decreased considerably in the PcNTC-dsRNA group. Importantly, PcNTC knockdown substantially reduced the survival rate of P. clarkii after A. hydrophila challenge. Collectively, these findings indicate that PcNTC, functioning as a PRR, is integral to the antibacterial defense mechanisms of P. clarkii and plays a crucial role in the immune regulatory system.