Molecular characterization, antibacterial and immunoregulatory activities of liver-expressed antimicrobial peptide 2 in black rockfish, Sebastes schlegelii.

LEAP2 (liver expression antimicrobial peptide 2), is an antimicrobial peptide widely found in vertebrates and mainly expressed in liver. LEAP2 plays a vital role in host innate immunity. In teleosts, a number of LEAP2 homologs have been reported, but their in vivo effects on host defense are still limited. In this study, a LEAP2 homolog (SsLEAP2) was identified from black rockfish, Sebastes schlegelii, and its structure, expression as well as biological functions were analyzed. The results showed that the open reading frame of SsLEAP2 is 300 bp, with a 5'- untranslated region (UTR) of 375 bp and a 3' - UTR of 238 bp. The deduced amino acid sequence of SsLEAP2 shares the highest overall identity (96.97%) with LEAP2 of Sebastes umbrosus. SsLEAP2 possesses conserved LEAP2 features, including a signal peptide sequence, a prodomain and a mature peptide, in which four well-conserved cysteines formed two intrachain disulphide domain. The expression of SsLEAP2 was highest in liver and could be induced by experimental infection with Listonella anguillarum, Edwardsiealla piscicida and Rock bream iridovirus C1 (RBIV-C1). Recombinant SsLEAP2 (rSsLEAP2) purified from Escherichia coli was able to bind with various Gram-positive and Gram-negative bacteria. Further analysis showed that rSsLEAP2 could enhance the respiratory burst activity, and induce the expression of immune genes including interleukin 1-ß (IL-1ß) and serum amyloid A (SAA) in macrophages; additionally, rSsLEAP2 could also promote the proliferation and chemotactic of peripheral blood lymphocytes (PBLs). In vivo experiments indicated that overexpression of SsLEAP2 could inhibit bacterial infection, and increase the expression level of immune genes including IL-1ß, tumor necrosis factor ligand superfamily member 13B (TNF13B) and haptoglobin (HP); conversely, knock down of SsLEAP2 promoted bacterial infection and decreased the expression level of above genes. Taken together, these results suggest that SsLEAP2 is a novel LEAP2 homolog that possesses apparent antibacterial activity and immunoregulatory property, thus plays a critical role in host defense against pathogens invasion.

NKHs27, a sevenband grouper NK-Lysin peptide that possesses immunoregulatory and antimicrobial activity.

As an effective and broad-spectrum antimicrobial peptide, NK-Lysin is attracted more and more attention at present. However, the functions and action mechanism of NK-Lysin peptides are still not comprehensive enough at present. In this study, a sevenband grouper (Hyporthodus septemfasciatus) NK-Lysin peptide, NKHs27, was identified and synthesized, and its biological functions were studied. The results indicated that NKHs27 shares 44.44%∼88.89% overall sequence identities with other teleost NK-Lysin peptides. The following antibacterial activity assay exhibited that NKHs27 was active against both Gram-negative and Gram-positive bacteria, including Staphylococcus aureus, Listonella anguillarum, Vibrio parahaemolyticus and Vibrio vulnificus. Additionally, NKHs27 showed a synergistic effect when it was combined with rifampicin or erythromycin. In the process of interaction with the L. anguillarum cells, NKHs27 changed the cell membrane permeability and retained its morphological integrity, then penetrated into the cytoplasm to act on genomic DNA or total RNA. Then, in vitro studies showed that NKHs27 could enhance the respiratory burst ability of macrophages and upregulate immune-related genes expression in it. Moreover, NKHs27 incubation improved the proliferation of peripheral blood leukocytes significantly. Finally, in vivo studies showed that administration of NKHs27 prior to bacterial infection significantly reduced pathogen dissemination and replication in tissues. In summary, these results provide new insights into the function of NK-Lysin peptides in teleost and support that NKHs27, as a novel broad-spectrum antibacterial peptide, has potential applications in aquaculture against pathogenic infections.

Antimicrobial and immunoregulatory activities of the derived peptide of a natural killer lysin from black rockfish (Sebastes schlegelii).

Natural killer lysin (NK-lysin) is a small molecule antimicrobial peptide secreted by natural killer cells and T lymphocytes. In this study, we characterized a cDNA sequence encoding an NK-lysin homologue (SsNKL1) from black rockfish, Sebastes schlegelii. The open reading frame (ORF) of SsNKL1 encodes a putative protein of 149 amino acids and shares 44%-87% overall sequence identities with other teleost NK-lysins. SsNKL1 possesses conserved NK-lysin family features, including a signal sequence and a surfactant-associated protein B (SapB) domain, sequence analysis revealed that SsNKL1 is most closely related to false kelpfish (Sebastiscus marmoratus) NK-lysin (with 87% sequence identity). SsNKL1 transcripts were detected in all the tested tissues, with the highest level in the kidney, followed by the spleen and gills. Upon Listonella anguillarum infection, the mRNA expression of SsNKL1 in the black rockfish was significantly up-regulated in the liver and kidney. The derived peptide SsNKLP27 from SsNKL1 was synthesized, and its biological function was studied. SsNKLP27 showed direct antibacterial activity against Gram-negative and Gram-positive bacteria, including Staphylococcus aureus, Bacillus subtilis, L. anguillarum, Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio vulnificus. SsNKLP27 treatment facilitated the bactericidal process of erythromycin by enhancing the permeability of the outer membrane. In the process of interaction with the target bacterial cells, SsNKLP27 changed the permeability and retained the morphological integrity of the cell membrane, then penetrated into the cytoplasm, and induced the degradation of genomic DNA and total RNA. In vivo studies showed that administration of SsNKLP27 before bacterial and viral infection significantly reduced the transmission and replication of pathogens in tissues. In vitro analysis showed that SsNKLP27 could enhance the respiratory burst ability and regulate the expression of some immune-related genes of macrophages. In summary, these results provided new insights into the function of NK-lysins in teleost fish and support that SsNKLP27 is a new broad-spectrum antimicrobial peptide that has a potential application prospect in aquaculture against pathogenic infection.

Evaluation of the efficacy of a novel Vibrio vulnificus vaccine based on antibacterial peptide inactivation in turbot, Scophthalmus maximus.

Tongue sole tissue factor pathway inhibitor 2 (TFPI-2) C-terminus derived peptide, TC38, has previously been shown to kill Vibrio vulnificus cells without lysing the cell membrane; thus, the remaining bacterial shell has potential application as an inactivated vaccine. Therefore, this study aimed to evaluate the immune response induced by the novel V. vulnificus vaccine. The protective potential of TC38-killed V. vulnificus cells (TKC) was examined in a turbot model. Fish were intramuscularly vaccinated with TKC or FKC (formalin-killed V. vulnificus cells) and challenged with a lethal-dose of V. vulnificus. The results showed that compared with FKC, TKC was effective in protecting fish against V. vulnificus infection, with relative percent of survival (RPS) rates of 53.29% and 63.64%, respectively. The immunological analysis revealed that compared with the FKC and control groups, the TKC group exhibited: 1) significantly higher respiratory burst ability and bactericidal activity of macrophages at 7 d post-vaccination; 2) increased alkaline phosphatase, acid phosphatase, lysozyme, and total superoxide dismutase levels post-vaccination; 3) higher serum agglutinating antibody titer with corresponding higher serum bactericidal ability, and a more potent serum agglutination effect, as well as an increased IgM expression level; 4) higher expression of immune relevant genes, which were involved in both innate and adaptive immunity. Taken together, this is the first study to develop a novel V. vulnificus inactivated vaccine based on AMP inactivation, and TKC is an effective vaccine against V. vulnificus infection for aquaculture.

A teleost bactericidal permeability-increasing protein-derived peptide that possesses a broad antibacterial spectrum and inhibits bacterial infection as well as human colon cancer cells growth.

The bactericidal permeability-increasing protein (BPI) is a multifunctional cationic protein produced by neutrophils with antibacterial, antitumor, and LPS-neutralizing properties. In teleost, a number of BPIs have been reported, but their functions are very limited. In this study, an N-terminal peptide, BO18 (with 18 amino acids), derived from rock bream (Oplegnathus fasciatus) BPI, was synthesized and investigated for its antibacterial spectrum, action mechanism, immunoregulatory property as well as the inhibition effects on bacterial invasion and human colon cancer cells growth. The results showed that BO18 was active against Gram-positive bacteria Bscillus subiilis, Micrococcus luteus, and Staphylococcus aureus, as well as Gram-negative bacteria Vibrio alginolyticus, Vibrio litoralis, Vibrio parahaemolyticus and Vibrio vulnificus. BO18 treatment facilitated the bactericidal process of erythromycin and rifampicin by enhancing the permeability of the outer membrane. During its interaction with V. alginolyticus, BO18 exerted its antibacterial activity by destroying cell membrane integrity, penetrating into the cytoplasm and binding to genomic DNA and total RNA. In vitro analysis indicated BO18 could enhance the respiratory burst ability and regulate the expression of immune related genes of macrophages. In vivo detection showed the administration of fish with BO18 before bacterial infection significantly reduced pathogen dissemination and replication in tissues. In addition, BO18 exerted a cytotoxic effect on the growth of human colon cancer cells HT-29. Together, these results add new insights into the function of teleost BPIs, and support that BO18 is a novel and broad-spectrum antibacterial peptide with potential to apply in fighting pathogenic infection in aquaculture.

Genome-wide sequence characterization and expression analysis of major intrinsic proteins in soybean (Glycine max L.).

Water is essential for all living organisms. Aquaporin proteins are the major facilitator of water transport activity through cell membranes of plants including soybean. These proteins are diverse in plants and belong to a large major intrinsic (MIP) protein family. In higher plants, MIPs are classified into five subfamilies including plasma membrane intrinsic proteins (PIP), tonoplast intrinsic proteins (TIP), NOD26-like intrinsic proteins (NIP), small basic intrinsic proteins (SIP), and the recently discovered X intrinsic proteins (XIP). This paper reports genome wide assembly of soybean MIPs, their functional prediction and expression analysis. Using a bioinformatic homology search, 66 GmMIPs were identified in the soybean genome. Phylogenetic analysis of amino acid sequences of GmMIPs divided the large and highly similar multi-gene family into 5 subfamilies: GmPIPs, GmTIPs, GmNIPs, GmSIPs and GmXIPs. GmPIPs consisted of 22 genes and GmTIPs 23, which showed high sequence similarity within subfamilies. GmNIPs contained 13 and GmSIPs 6 members which were diverse. In addition, we also identified a two member GmXIP, a distinct 5(th) subfamily. GmMIPs were further classified into twelve subgroups based on substrate selectivity filter analysis. Expression analyses were performed for a selected set of GmMIPs using semi-quantitative reverse transcription (semi-RT-qPCR) and qPCR. Our results suggested that many GmMIPs have high sequence similarity but diverse roles as evidenced by analysis of sequences and their expression. It can be speculated that GmMIPs contains true aquaporins, glyceroporins, aquaglyceroporins and mixed transport facilitators.