A 14-amino acid cationic peptide Bolespleenin334-347 from the marine fish mudskipper Boleophthalmus pectinirostris exhibiting potent antimicrobial activity and therapeutic potential.

Antimicrobial peptides (AMPs) are an important component of innate immunity in both vertebrates and invertebrates, and some of the unique characteristics of AMPs are usually associated with their living environment. The marine fish, mudskipper Boleophthalmus pectinirostris, usually live amphibiously in intertidal environments that are quite different from other fish species, which would be an exceptional source of new AMPs. In the study, an AMP named Bolespleenin334-347 was identified, which was a truncated peptide derived from a new functional gene found in B. pectinirostris, that was up-regulated in response to bacterial challenge. Bolespleenin334-347 had only 14 amino acid residues, including five consecutive arginine residues. It was found that the peptide had broad-spectrum antibacterial activity, good thermal stability and sodium ion tolerance. Bolespleenin334-347 killed Acinetobacter baumannii and Staphylococcus aureus by disrupting the structural integrity of the bacterial membrane, leading to leakage of the cellular contents, and inducing accumulation of bacterial endogenous reactive oxygen species (ROS). In addition, Bolespleenin334-347 effectively inhibited biofilm formation of A. baumannii and S. aureus and long-term treatment did not lead to the development of resistance. Importantly, Bolespleenin334-347 maintained stable activity against clinically multi-drug resistant bacterial strains. In addition, it was noteworthy that Bolespleenin334-347 showed superior efficacy to LL-37 and vancomycin in a constructed mouse model of MRSA-induced superficial skin infections, as evidenced by a significant reduction in bacterial load and more favorable wound healing. This study provides an effective antimicrobial agent for topical skin infections with potential therapeutic efficacy for infections with drug-resistant bacteria, including MRSA.

Characterization of the intestinal digesta and mucosal microbiome of the grass carp (Ctenopharyngodon idella).

The intestinal microbiome plays a pivotal role in the nutritional digestion and metabolism of the grass carp (Ctenopharyngodon idella). Here, we characterized the digesta and mucosal microbiome of the anterior, middle, and posterior intestine of the grass carp, using 16S rRNA next-generation sequencing. Based on 16S rRNA amplicon data, Proteobacteria, Firmicutes and Bacteroides were the dominant phyla in the intestine of grass carp. Our results also showed that microbial communities of the middle intestine exhibited higher alpha diversity indices compared with the anterior and posterior intestine. The clustering of microbial communities that had either colonized in the digesta or were attached to the mucosa, were significantly tighter in the posterior intestine, based on average unweighted Unifrac distances (P < 0.05). The digesta or mucosa of the anterior and middle intestines were similar in microbial composition, but were significantly different to the posterior intestine (P < 0.05). In digesta and mucosa samples from the posterior intestine, we observed a significantly increased abundance of cellulose-degrading microbiomes, such as Bacteroides, Clostridiales and Spirochaetia (P < 0.05). Our results suggested that the microbiomes of the posterior intestine, either attached to the mucosa or colonized in the digesta, were distinct from the microbiomes of the anterior and middle intestine in grass carp.

Identification, characterization, and immunological analysis of complement component 4 from grass carp (Ctenopharyngodon idella).

Complement component 4 (C4) has critical immunological functions in vertebrates. In the current study, a C4 homolog (gcC4) was identified in grass carp (Ctenopharyngodon idella). The full-length 5458 bp gcC4 cDNA contained a 5148 bp open reading frame (ORF) encoding a protein of 1715 amino acids with a signal peptide and eight conservative domains. The gcC4 protein has a high level of identity with other fish C4 counterparts and is phylogenetically clustered with cyprinid fish C4. The gcC4 transcript shows wide tissue distribution and is inducible by Aeromonas hydrophila in vivo and in vitro. Furthermore, its expression also fluctuates upon lipopolysaccharide or flagellin stimulation in vitro. During infection, the gcC4 protein level decreases or increases to varying degrees, and the intrahepatic C4 expression location changes. With gcC4 overexpression, interleukin 1 beta, tumor necrosis factor alpha, and interferon transcripts are all upregulated by A. hydrophila infection. Meanwhile, overexpression of gcC4 reduces bacterial invasion or proliferation. Moreover, gcC4 may activate the NF-κB signaling pathway. These findings demonstrate the vital role of gcC4 in the innate immunity of grass carp.

16S rRNA sequencing analysis of the correlation between the intestinal microbiota and body-mass of grass carp (Ctenopharyngodon idella).

There appears to be a close correlation between intestinal microbiotas and obesity. Still, our understanding of the relationship between the intestinal microbiota and body-mass in grass carp (Ctenopharyngodon idella) remains limited. Herein, we explored this association in the anterior, middle, and posterior intestine of cohabitating grass carp by using next-generation sequencing of the 16S rRNA gene. The results showed that alpha diversity indices of the low-weight-gain (LWG) groups were higher than that of the high-weight-gain (HWG) groups. HWG groups possessed the decreased ratio of Bacteroidetes to Firmicutes compared with that in the LWG groups. Principal coordinate analysis (PCoA) and analysis of similarities (ANOSIM) revealed that there were significant differences between the HWG and LWG groups. Furthermore, linear discriminant analysis (LDA) coupled with effect size (LEfSe) showed that the order Clostridiales were significantly abundant in the HWG groups. Phylogenetic molecular ecology networks (pMENs) showed a lower average path distance (GD), higher average clustering coefficient (avgCC), and higher average degree (avgK) in the HWG group. Our results suggested that there appeared to be a tight correlation between the intestinal microbiota and body-mass in grass carp. The study provides a referable resource for establishing the relationship between intestinal microbiotas and economic traits, which also lays a foundation for the progress of new fish probiotic in the future.

Role of mannose-binding lectin in regulating monocytes/macrophages functions during Aeromonas hydrophila infection in grass carp, Ctenopharyngodon idella.

Mannose-binding lectin (MBL) is a vital component in host's innate immune system and the initiator of the lectin pathway of complement cascade. However, its opsonic role has rarely been reported. In this study, we revealed the biological function of Ctenopharyngodon idella MBL (CiMBL) in regulating monocytes/macrophages (MO/MФ) in the grass carp (C. idella). Flow cytometry results indicated that recombinant CiMBL (rCiMBL) significantly enhanced the phagocytotic activity of MO/MФ. Recombinant CiMBL also enhanced bactericidal activity and respiratory burst capacity in Aeromonas hydrophila-infected MO/MФ, regulated A. hydrophila-induced polarization of MO/MФ including down- and up-regulated pro- and anti-inflammatory cytokines, respectively, suppressed the inducible nitric oxide synthase activity, and enhanced the arginase activity. In addition, rCiMBL suppressed the bacteria burden in tissues and blood in vivo and enhanced the survival rate of juvenile A. hydrophila-infected grass carp. We provide evidence that CiMBL was synthesized by MO/MФ, regulating the biological function of MO/MФ against A. hydrophila infection.

Complement component 3 (C3): An important role in grass carp (Ctenopharyngodon idella) experimentally exposed to Aeromonas hydrophila.

Complement is traditionally recognized as part of the innate immune system, defending the host against the invasion of foreign pathogens. In complement system, C3 (complement component 3) is a central component. Therefore, research into C3 can help us better understand the functions of fish complement system. In this study, we detected the grass carp C3 (gcC3) mRNA expression in all sample tissues from healthy grass carp, which was highest in the liver, followed by the heart and the spleen, and lowest in the muscle, head kidney, trunk kidney, blood and intestine. After infection with Aeromonas hydrophila, gcC3 mRNA expression levels were significantly upregulated in the gill, liver, spleen, intestine, trunk kidney and head kidney. Interestingly, C3 protein levels were downregulated and subsequently upregulated in the liver and serum. Histologically, C3 protein at 24 h pi was over expressed in necrotic liver sites, and the liver index (LI) at this point was significantly higher than that of the control. These findings are indicated that C3 plays an important role in the immune response of grass carp after A. hydrophila infection, and C3 protein may play an assistant role in repairing liver tissues from A. hydrophila injury.

Complement component Bf/C2b gene mediates immune responses against Aeromonas hydrophila in grass carp Ctenopharyngodon idella.

The complement system is a significant component of innate immunity. Here, we identified a Bf/C2 homolog (gcBf/C2b) in grass carp. gcBf/C2b shares a high similarity with Bf/C2b counterparts in other teleosts. gcBf/C2b transcription was widely distributed in different tissues, induced by Aeromonas hydrophila in vivo and in vitro, and affected by lipopolysaccharide and flagellin stimulation in vitro. In cells over-expressing gcBf/C2b, transcript levels of all components except gcC5 were significantly enhanced, and gcBf/C2b, gcIL1ß, gcTNF-α, gcIFN, gcCD59, gcC5aR1, and gcITGß-2 were significantly upregulated after A. hydrophila challenge or stimulation with bacterial pathogen-associated molecular patterns (PAMPs). However, gcBf/C2b in interference cells down-regulated the transcript levels after A. hydrophila challenge, and gcBf/C2b induced NF-κB signaling. These findings indicate the vital role of gcBf/C2b in innate immunity in grass carp.

Mannose-binding lectin and its roles in immune responses in grass carp (Ctenopharyngodon idella) against Aeromonas hydrophila.

The complement system is a crucial component of the innate immune system that links innate and adaptive immunity via four pathways. Mannose-binding lectin (MBL), the initiating molecule of the lectin pathway, plays a significant role in the innate immune system in mammals and fish. Herein, we identified an MBL homolog (gcMBL) in grass carp (Ctenopharyngodon idella). The full-length 948 bp gcMBL cDNA includes a 741 bp open reading frame encoding a 246 amino acid protein with a signal peptide, collagen triple helix repeat domain, and a C-type lectin-like/link domain. The gcMBL protein shares low similarity with MBL counterparts in other species, and is most closely related to Cyprinus carpio MBL. Transcription of gcMBL was widely distributed in different tissues, and was induced by Aeromonas hydrophila in vivo and in vitro. Expression of gcMBL was also affected by LPS and flagellin stimulation in vitro. In cells over-expressing gcMBL, transcripts of almost all components except gcC5 were up-regulated, and gcMBL, gcIL1ß, gcTNF-α, gcIFN, gcCD59, gcC5aR and gcITGß-2 were significantly up-regulated following exposure to A. hydrophila or stimulation by bacterial PAMPs. Meanwhile, gcMBL deficiency achieved by RNAi down-regulated transcript levels following A. hydrophila challenge, and gcMBL induced NF-κB signalling. These findings indicate a vital role of gcMBL in innate immunity in grass carp.

Biological parameters, immune enzymes, and histological alterations in the livers of grass carp infected with Aeromonas hydrophila.

Aeromonas hydrophila is the causative agent of bacterial septicemia that is frequently observed in grass carp, Ctenopharyngodon idellus. In this study, we evaluated the biological parameters and immune enzymes in the liver of grass carp following A. hydrophila infection and quantified the alterations in liver histology using a semi-quantitative system. For the biological parameters, we found that the liver somatic index (LSI) was more sensitive than Fulton's condition factor (CF) and was significantly decreased at three days post-injection (DPI). At the immune enzyme level, the level of peroxidase (POD) in the liver significantly increased at 1 and 3 DPI. The activity of alkaline phosphatase (ALP) significantly increased at 3 DPI. Similarly, acid phosphatase (ACP) activity significantly increased at 1, 3, and 5 DPI. Histologically, the results indicated that the liver index at 3, 5, and 7 DPI was significantly higher than that of control groups. The regressive alterations as the highly variable reactions patterns and its index at 5 DPI was significantly higher than that of 1, 21 DPI, and the control groups. Based on our results, we suggest that grass carp resist A. hydrophila infection via an innate immune mechanism in the liver. The findings of this study will help elucidate the underlying mechanisms of resistance to A. hydrophila infection.

Mannan-binding lectin-associated serine protease-1 (MASP-1) mediates immune responses against Aeromonas hydrophila in vitro and in vivo in grass carp.

The mannan-binding lectin-associated serine protease-1 (MASP-1) gene is a crucial component of the lectin pathway in the complement and coagulation cascade. Although MASP-1 has been found in the immune system of teleosts, its immune functions in response to bacterial infection are unclear. In this study, we identified a MASP-1 homolog (gcMASP-1) in the grass carp (Ctenopharyngodon idella). The full-length 3308-bp gcMASP-1 cDNA includes a 2160-bp open reading frame encoding a protein composed of 719 amino acids with epidermal growth factor-like, complement control protein, and trypsin-like domains. gcMASP-1 shares a high similarity with MASP-1 counterparts in other species, and it is most closely related to Cyprinus carpio MASP-1 and Sinocyclocheilus anshuiensis MASP-1. Transcription of gcMASP-1 was widely distributed in different tissues and induced by Aeromonas hydrophila in vivo and in vitro. Expression of gcMASP-1 was also affected by lipopolysaccharide and flagellin stimulation in vitro. In cells over-expressing gcMASP-1, transcript levels of almost all components, except gcMBL and gcC5, were significantly enhanced, and gcIL1ß, gcTNF-α, gcIFN, gcCD59, gcC5aR1, and gcITGß-2 were significantly upregulated after exposure to A. hydrophila; gcMASP-1 interference downregulated the transcript levels after A. hydrophila challenge. In addition, gcMASP-1 activated NF-κB signaling. These findings indicate the vital role of gcMASP-1 in innate immunity in C. idella.