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A Novel Antimicrobial Peptide Sparanegtin Identified in Scylla paramamosain Showing Antimicrobial Activity and Immunoprotective Role In Vitro and Vivo.

ABSTRACT: The abuse of antibiotics in aquaculture and livestock no doubt has exacerbated the increase in antibiotic-resistant bacteria, which imposes serious threats to animal and human health. The exploration of substitutes for antibiotics from marine animals has become a promising area of research, and antimicrobial peptides (AMPs) are worth investigating and considering as potential alternatives to antibiotics. In the study, we identified a novel AMP gene from the mud crab Scylla paramamosain and named it Sparanegtin. Sparanegtin transcripts were most abundant in the testis of male crabs and significantly expressed with the challenge of lipopolysaccharide (LPS) or Vibrio alginolyticus. The recombinant Sparanegtin (rSparanegtin) was expressed in Escherichia coli and purified. rSparanegtin exhibited activity against Gram-positive and Gram-negative bacteria and had potent binding affinity with several polysaccharides. In addition, rSparanegtin exerted damaging activity on the cell walls and surfaces of P. aeruginosa with rougher and fragmented appearance. Interestingly, although rSparanegtin did not show activity against V. alginolyticus in vitro, it played an immunoprotective role in S. paramamosain and exerted an immunomodulatory effect by modulating several immune-related genes against V. alginolyticus infection through significantly reducing the bacterial load in the gills and hepatopancreas and increasing the survival rate of crabs.

SUBMITTER: Zhu X

PROVIDER: S-EPMC8744658 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:Crustins are the most abundant class of antimicrobial peptides in crustaceans and are essential for protecting animals from infection. Among them, type II crustins usually exhibit potent antimicrobial activity. Interestingly, in this study, a newly identified type II crustin gene homolog (named SpCrus8) from mud crab Scylla paramamosain, the recombinant proteins of which (rSpCrus8 and rTrx-SpCrus8) showed no obvious antibacterial effects, but could significantly reduce the bacterial load in crab hemolymph and improve the survival rate of crabs infected with Vibrio alginolyticus. The immune-related function of SpCrus8 and the underlying mechanism deserve further investigation. It was found that the SpCrus8 gene was widely distributed in various tissues of adult crabs. In the hepatopancreas of crabs infected with V. alginolyticus or Staphylococcus aureus, transcripts of the SpCrus8 gene were remarkably induced, indicating that the SpCrus8 gene was involved in the immune response to bacterial infection in vivo. In addition, rSpCrus8 and rTrx-SpCrus8 had strong binding activity not only to microbial surface components (lipopolysaccharide, lipoteichoic acid, peptidoglycan, and glucan), but also to the tested bacteria (S. aureus, Pseudomonas aeruginosa and V. alginolyticus). Notably, rSpCrus8 and rTrx-SpCrus8 could significantly promote hemocyte phagocytosis. After rSpCrus8 and rTrx-SpCrus8 treatment, a large number of fluorescent microspheres were observed to aggregate into clusters and be phagocytosed by multiple hemocytes, while hemocytes in the control group phagocytosed only individual microspheres, indicating that SpCrus8 played an important role in opsonization. When the SpCrus8 gene was knocked down, the expression levels of the key phagocytosis-related genes SpRab5 and SpRab7 were significantly downregulated, as well as the IMD signaling pathway genes SpIKKβ and SpRelish, and another crustin gene SpCrus5. Correspondingly, all the SpIKKβ, SpRelish and SpCrus5 genes were significantly upregulated after rSpCrus8 treatment, suggesting that SpCrus8 might be involved in the immunomodulation of S. paramamosain. Taken together, this study revealed the immune-related functions of the SpCrus8 gene in opsonization and regulation, which will help us further understand the role of the crustin gene family in the immune system of mud crabs and provide new insights into the function of type II crutins.

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A Novel Antimicrobial Peptide Sparanegtin Identified in Scylla paramamosain Showing Antimicrobial Activity and Immunoprotective Role In Vitro and Vivo.

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