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

ABSTRACT: 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.

SUBMITTER: Qiu W

PROVIDER: S-EPMC8062930 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Antimicrobial peptides (AMPs) may be the most promising substitute for antibiotics due to their effective bactericidal activity and multiple antimicrobial modes against pathogenic bacteria. In this study, a new functional gene named Spgillcin was identified in Scylla paramamosain, which encoded 216 amino acids of mature peptide. In vivo, Spgillcin was dominantly expressed in the gills of male and female crabs, offering the highest expression level among all tested organs or tissues. The expression pattern of Spgillcin was significantly altered when challenged by Staphylococcus aureus, indicating a positive immune response. In vitro, a functional truncated peptide Spgillcin177-189 derived from the amino acid sequence of Spgillcin was synthesized and showed a broad-spectrum and potent antibacterial activity against several bacterial strains, including the clinical isolates of multidrug-resistant (MDR) strains, with a range of minimum inhibitory concentrations from 1.5 to 48 μM. Spgillcin177-189 also showed rapid bactericidal kinetics for S. aureus and Pseudomonas aeruginosa but did not display any cytotoxicity to mammalian cells and maintained its antimicrobial activity in different conditions. Mechanistic studies indicated that Spgillcin177-189 was mainly involved in the disruption of cell membrane integrity where the membrane components lipoteichoic acid and lipopolysaccharide could significantly inhibit the antimicrobial activity in a dose-dependent manner. In addition, Spgillcin177-189 could change the membrane permeability and cause the accumulation of intracellular reactive oxygen species. No resistance was generated to Spgillcin177-189 when the clinical isolates of methicillin-resistant S. aureus and MDR P. aeruginosa were treated with Spgillcin177-189 and then subjected to a long term of continuous culturing for 50 days. In addition, Spgillcin177-189 exerted a strong anti-biofilm activity by inhibiting biofilm formation and was also effective at killing extracellular S. aureus in the cultural supernatant of RAW 264.7 cells. Taken together, Spgillcin177-189 has strong potential as a substitute for antibiotics in future aquaculture and medical applications.

Project description:BackgroundInfection of bacterial Vibrio parahaemolyticus is common in mud crab farms. However, the mechanisms of the crab's response to pathogenic V. parahaemolyticus infection are not fully understood. MicroRNAs (miRNAs) are a class of small noncoding RNAs that function as regulators of gene expression and play essential roles in various biological processes. To understand the underlying mechanisms of the molecular immune response of the crab to the pathogens, high-throughput Illumina/Solexa deep sequencing technology was used to investigate the expression profiles of miRNAs in S. paramamosain under V. parahaemolyticus infection.Methodology/principal findingsTwo mixed RNA pools of 7 tissues (intestine, heart, liver, gill, brain, muscle and blood) were obtained from V. parahaemolyticus infected crabs and the control groups, respectively. By aligning the sequencing data with known miRNAs, we characterized 421 miRNA families, and 133 conserved miRNA families in mud crab S. paramamosain were either identical or very similar to existing miRNAs in miRBase. Stem-loop qRT-PCRs were used to scan the expression levels of four randomly chosen differentially expressed miRNAs and tissue distribution. Eight novel potential miRNAs were confirmed by qRT-PCR analysis and the precursors of these novel miRNAs were verified by PCR amplification, cloning and sequencing in S. paramamosain. 161 miRNAs (106 of which up-regulated and 55 down-regulated) were significantly differentially expressed during the challenge and the potential targets of these differentially expressed miRNAs were predicted. Furthermore, we demonstrated evolutionary conservation of mud crab miRNAs in the animal evolution process.Conclusions/significanceIn this study, a large number of miRNAs were identified in S. paramamosain when challenged with V. parahaemolyticus, some of which were differentially expressed. The results show that miRNAs might play some important roles in regulating gene expression in mud crab under V. parahaemolyticus infection, providing a basis for further investigation of miRNA-modulating networks in innate immunity of mud crab.

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

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