A Novel Antimicrobial Peptide Sp-LECin with Broad-Spectrum Antimicrobial Activity and Anti-Pseudomonas aeruginosa Infection in Zebrafish.

New antimicrobial agents are urgently needed to address the increasing emergence and dissemination of multidrug-resistant bacteria. In the study, a chemically synthesized truncated peptide containing 22-amino acids derived from a C-type lectin homolog SpCTL6 of Scylla paramamosain was screened and found to exhibit broad-spectrum antimicrobial activity, indicating that it is an antimicrobial peptide (AMP), named Sp-LECin. Sp-LECin possessed the basic characteristics of most cationic AMPs, such as positive charge (+4) and a relatively high hydrophobicity (45%). After treatment with Sp-LECin, the disruption of microbial membrane integrity and even leakage of cellular contents was observed by scanning electron microscopy (SEM). In addition, Sp-LECin could bind lipopolysaccharide (LPS), increase the outer and inner membrane permeability and induce reactive oxygen species (ROS) production, ultimately leading to the death of Pseudomonas aeruginosa. Furthermore, Sp-LECin exhibited potent anti-biofilm activity against P. aeruginosa during both biofilm formation and maturation. Notably, Sp-LECin had no obvious cytotoxicity and could greatly improve the survival of P. aeruginosa-infected zebrafish, by approximately 40% over the control group after 72 h of treatment. This study indicated that Sp-LECin is a promising antibacterial agent with the potential to be used against devastating global pathogen infections such as P. aeruginosa.

A New C-Type Lectin Homolog SpCTL6 Exerting Immunoprotective Effect and Regulatory Role in Mud Crab Scylla paramamosain.

C-type lectin (CTL), a well-known immune-related molecule, has received more and more attention due to its diverse functions, especially its important role in development and host defense of vertebrate and invertebrate. Since the research on crab CTLs is still lack, we screened a new CTL homolog, named SpCTL6 from mud crab Scylla paramamosain. The full-length cDNA sequence of SpCTL6 was 738 bp with a 486 bp of ORF, and the deduced amino acids were 161 aa. SpCTL6 was predicted to have a 17 aa signal peptide and its mature peptide was 144 aa (MW 16.7 kDa) with pI value of 5.22. It had typical CTL structural characteristics, such as a single C-type lectin-like domain, 4 conserved cysteines, similar tertiary structure to that of vertebrate CTLs and a mutated Ca2+ binding motif Gln-Pro-Thr (QPT), clustering into the same branch as the crustacean CTLs. SpCTL6 was highly expressed in the entire zoeal larval stages and widely distributed in adult crab tissues with the highest transcription level in testis. During the molting process of juvenile crabs, the expression level of SpCTL6 was remarkably increased after molting. SpCTL6 could be significantly upregulated in two larval stages (Z1 and megalopa) and adult crab testis under immune challenges. Recombinant SpCTL6 (rSpCTL6) was successfully obtained from eukaryotic expression system. rSpCTL6 exhibited binding activity with PAMPs (LPS, lipoteichoic acid, peptidoglycan, and glucan) and had a broad spectrum bacterial agglutination activity in a Ca2+-dependent manner. In addition, rSpCTL6 could enhance the encapsulation activity of hemocytes and has no cytotoxic effect on hemocytes. Although rSpCTL6 had no bactericidal activity on Vibrio alginolyticus, rSpCTL6 treatment could significantly reduce the bacterial endotoxin level in vitro and greatly improved the survival of S. paramamosain under V. alginolyticus infection in vivo. The immunoprotective effect of rSpCTL6 might be due to the regulatory role of rSpCTL6 in immune-related genes and immunological parameters. Our study provides new information for understanding the immune defense of mud crabs and would facilitate the development of effective strategies for mud crab aquaculture disease control.