Study of the Antimicrobial Activity of Tilapia Piscidin 3 (TP3) and TP4 and Their Effects on Immune Functions in Hybrid Tilapia (Oreochromis spp.).

To address the growing concern over antibiotic-resistant microbial infections in aquatic animals, we tested several promising alternative agents that have emerged as new drug candidates. Specifically, the tilapia piscidins are a group of peptides that possess antimicrobial, wound-healing, and antitumor functions. In this study, we focused on tilapia piscidin 3 (TP3) and TP4, which are peptides derived from Oreochromis niloticus, and investigated their inhibition of acute bacterial infections by infecting hybrid tilapia (Oreochromis spp.) with Vibrio vulnificus and evaluating the protective effects of pre-treating, co-treating, and post-treating fish with TP3 and TP4. In vivo experiments showed that co-treatment with V. vulnificus and TP3 (20 µg/fish) or TP4 (20 µg/fish) achieved 95.3% and 88.9% survival rates, respectively, after seven days. When we co-injected TP3 or TP4 and V. vulnificus into tilapia and then re-challenged the fish with V. vulnificus after 28 days, the tilapia exhibited survival rates of 35.6% and 42.2%, respectively. Pre-treatment with TP3 (30 µg/fish) or TP4 (20 µg/fish) for 30 minutes prior to V. vulnificus infection resulted in high survival rates of 28.9% and 37.8%, respectively, while post-treatment with TP3 (20 µg/fish or 30 µg/fish) or TP4 (20 µg/fish) 30 minutes after V. vulnificus infection yielded high survival rates of 33.3% and 48.9%. In summary, pre-treating, co-treating, and post-treating fish with TP3 or TP4 all effectively decreased the number of V. vulnificus bacteria and promoted significantly lower mortality rates in tilapia. The minimum inhibitory concentrations (MICs) of TP3 and TP4 that were effective for treating fish infected with V. vulnificus were 7.8 and 62.5 µg/ml, respectively, whereas the MICs of kanamycin and ampicillin were 31.2 and 3.91 µg/ml. The antimicrobial activity of these peptides was confirmed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM), both of which showed that V. vulnificus disrupted the outer membranes of cells, resulting in the loss of cell shape and integrity. We examined whether TP3 and TP4 increased the membrane permeability of V. vulnificus by measuring the fluorescence resulting from the uptake of 1-N-phenyl-naphthylamine (NPN). Treating fish with TP3 and TP4 under different pH and temperature conditions did not significantly increase MIC values, suggesting that temperature and the acid-base environment do not affect AMP function. In addition, the qPCR results showed that TP3 and TP4 influence the expression of immune-responsive genes, including interleukin (IL)-1ß, IL-6, and IL-8. In this study, we demonstrate that TP3 and TP4 show potential for development as drugs to combat fish bacterial infections in aquaculture.

Anti-apoptotic genes Bcl-2 and Bcl-xL overexpression can block iridovirus serine/threonine kinase-induced Bax/mitochondria-mediated cell death in GF-1 cells.

Although serine/threonine (ST) kinase is known to induce host cell death in GF-1 cells, it remains unclear how ST kinase induces mitochondrial function loss. In the present study, we addressed the issue of mitochondrial function loss by determining whether the Bcl-2 family members Bcl-2 and Bcl-xL can prevent ST kinase-induced cell death activity via interacting with the pro-apoptotic gene Bax. Grouper fin cells (GF-1) carrying EGFP-Bal-xL and EGFP-Bcl-2 fused genes were selected, established in cell culture, and used to examine the involvement of Bcl-2 and Bcl-xL overexpression in protection of GF-1 cells from the effects of the giant sea perch iridovirus (GSIV) ST kinase gene. Using the TUNEL assay, we found that EGFP-Bcl-2 and EGFP-Bcl-xL reduced GSIV ST kinase-induced apoptosis to 20% all at 24 h and 48 h post-transfection (pt). Also, Bcl-2 and Bcl-xL substantially reduced the percentage of cells with GSIV ST kinase-induced loss of mitochondrial membrane potential (Δψps) at 24 and 48 hpt, respectively, and this reduction correlated with a 30% and 50% enhancement of host cell viability at 24 and 48 hpt as compared with vector control. Moreover, analysis of the effect of Bcl-2 and Bcl-xL interaction with Bax targeted to mitochondria during ST kinase expression at 48 hpt found that Bcl-2 and Bcl-xL also interacted with Bax to block cytochrome c release. Finally, Bcl-2 and Bcl-xL overexpression caused blockage of ST kinase function at 48 hpt, which was correlated with preventing caspase-9 and -3 cleavage and activation, thereby blocking downstream death signaling events. Taken together, our results suggest that the ST kinase-induced Bax/mitochondria-mediated cell death pathway can be blocked by the interaction of Bcl-2 and Bcl-xL with Bax to inhibit cytochrome c release during MMP loss. This rescue activity also correlated with inhibition of caspase-9 and -3 activation, thereby enhancing cell viability.

Alternative Splicing of Toll-Like Receptor 9 Transcript in Teleost Fish Grouper Is Regulated by NF-κB Signaling via Phosphorylation of the C-Terminal Domain of the RPB1 Subunit of RNA Polymerase II.

Similar to its mammalian counterparts, teleost Toll-like receptor 9 (TLR9) recognizes unmethylated CpG DNA presented in the genome of bacteria or DNA viruses and initiates signaling pathway(s) for immune responses. We have previously shown that the TLR9 pathway in grouper, an economically important teleost, can be debilitated by an inhibitory gTLR9B isoform, whose production is mediated by RNA alternative splicing. However, how does grouper TLR9 (gTLR9) signaling impinge on the RNA splicing machinery to produce gTlr9B is unknown. Here we show that the gTlr9 alternative splicing is regulated through ligand-induced phosphorylation of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II). We first observed that ligand-activated NF- κB pathway biased the production of the gTlr9B isoform. Because NF- κB is known to recruit p-TEFb kinase, which phosphorylates the Pol II CTD at Ser2 residues, we examined p-TEFb's role in alternative splicing. We found that promoting p-TEFb kinase activity significantly favored the production of the gTlr9B isoform, whereas inhibiting p-TEFb yielded an opposite result. We further showed that p-TEFb-mediated production of the gTlr9B isoform down-regulates its own immune responses, suggesting a self-limiting mechanism. Taken together, our data indicate a feedback mechanism of the gTLR9 signaling pathway to regulate the alternative splicing machinery, which in turn produces an inhibitor to the pathway.

Transcriptome analysis of the effect of Vibrio alginolyticus infection on the innate immunity-related TLR5-mediated induction of cytokines in Epinephelus lanceolatus.

Epinephelus lanceolatus, considered to be an aquaculture fish species of high economic value in East Asia, is one of the largest groupers in the Epinephelus genus. Vibrio alginolyticus is a bacterial species that causes high morbidity in marine fish; infection can cause exophthalmia, ulcers, septicemia, and corneal opaqueness in fish. Epinephelus lanceolatus larvae infected with Vibrio alginolyticus were subjected to transcriptome analysis to study the immune regulation pathway. Grouper larvae were injected with 2.6 × 10(4) CFU/fish in 20 µl of V. alginolyticus and control larvae were injected with TSB; RNA samples were then collected at 4, 6, 8, 10, 12, 16, 24, and 48 h after infection. Extracted RNA was subjected to reverse transcription, and used to examine the immune gene response of E. lanceolatus by Real-time PCR. Samples taken at 6 h were subjected to next-generation sequencing, resulting in a total read value of 28,705,411 and total base number of 2,152,905,850. The unigene number was 100,848, and 5913 unigenes were filtered using FPKM>0.3, 2FC, p < 0.05. Gene Ontology (GO) analysis of the filtered genes revealed a total of 30 GO numbers in the cellular component, and 58 GO numbers for both biological processes and molecular functions. Of the GO group related to immune pathways, 27 unigenes related to biological processes involving the immune response, 31 related to the immune system, 9 related to the inflammatory response, and 43 related to the response to stress were identified. KEGG pathway analysis only detected 1 to 4 genes, and as such, we selected the GO analysis results for further analysis using GeneSpring. This demonstrated that V. alginolyticus probably stimulates TLR5 activity via the bacterial flagellum, through an MyD88-dependent pathway; the resulting production of IL-1ß and IL-8 through the NFκB pathway induces pro-inflammatory and/or chemotactic effects. Alternatively, serum amyloid A may stimulate neutrophils that induce the secretion of MMP9 from infected tissues, resulting in the cleavage and activation of IL-8. IL-8, in turn, would enhance neutrophil chemotaxis. Infection also induced expression of genes encoding C3, C6, C7, C8, and C9, which induce the complement system and form the membrane attack complex to lyse the bacteria membrane. The qPCR results indicated that TLR5 is significantly increased between 10 and 16 h, IL-1ß between 8 and 16 h, IL-8 between 8 and 12 h, and C6 between 4 and 16 h, as compared to levels in the control. One antimicrobial peptide, hepcidin, was also strongly expressed between 4 and 10 h in infected fish. The results indicate that V. alginolyticus infection probably induces an immune response via TLR5-mediated regulation of down-stream cytokine gene expression. A second possibility is that the complement system and hepcidin may be involved in the immune response. These results may be applied by examining the immune effects of feeding E. lanceolatus larvae on a recombinant protein mixture based on the up-regulated genes.

Giant seaperch iridovirus infection upregulates Bas and Bak expression, leading to apoptotic death of fish cells.

The giant seaperch iridovirus (GSIV) induces host cell apoptosis by a poorly-understood process. In this study, GSIV is shown to upregulate the pro-apoptotic death genes Bax and Bak at the middle replication stage, and factors in the grouper fin cell line (GF-1) are shown to modulate this process. Studying the mechanism of cell death, we found that upregulated, de novo-synthesized Bax and Bak proteins formed heterodimers. This up-regulation process correlated with mitochondrial membrane potential (MMP) loss, increased caspase-3 activity, and increased apoptotic cell death. All effects were diminished by treatment of infected GF-1 cells with the protein synthesis inhibitor cycloheximide. Interestingly, overexpression of the anti-apoptotic gene Bcl-xL also diminished GSIV-induced mitochondria-mediated cell death, increasing host cell viability and decreasing MMP loss at the early replication stage. Our data suggest that GSIV induces GF-1 apoptotic cell death through up-regulation of the pro-apoptotic genes Bax and Bak, which are regulated by Bcl-xL overexpression on mitochondria in GF-1 cells.

Use of tilapia piscidin 3 (TP3) to protect against MRSA infection in mice with skin injuries.

Antimicrobial peptides (AMPs), represent promising agents for new therapeutic approaches of infected wound treatment, on account of their antimicrobial and wound closure activities, and low potential for inducing resistance. However, therapeutic applications of these AMPs are limited by their toxicity and low stability in vivo. Previously, we reported that the 23 amino-acid designer peptide TP3 possessed antimicrobial activities. Here, we analyzed the wound closure activities of TP3 both and in vivo. TP3 at doses of up to 40 µg/ml did not affect the viability of baby hamster kidney cells. Furthermore, TP3 was found to be highly effective at combating peritonitis and wound infection caused by MRSA in mouse models, without inducing adverse behavioral effects or liver or kidney toxicity. TP3 treatment increased survival by 100% at 8 days after infection, and accelerated the progression of proliferation, remodeling, and maturation of infected wounds. Taken together, our results indicate that TP3 enhances the rate of survival of mice infected with the bacterial pathogen MRSA through both antimicrobial and immunomodulatory effects. Overall, these results suggest that TP3 may be suitable for development as a novel topical agent for treatment of infected wounds.

Piscidin is highly active against carbapenem-resistant Acinetobacter baumannii and NDM-1-producing Klebsiella pneumonia in a systemic Septicaemia infection mouse model.

This study was designed to investigate the antimicrobial activity of two synthetic antimicrobial peptides from an aquatic organism, tilapia piscidin 3 (TP3) and tilapia piscidin 4 (TP4), in vitro and in a murine sepsis model, as compared with ampicillin, tigecycline, and imipenem. Mice were infected with (NDM-1)-producing K. pneumonia and multi-drug resistant Acinetobacter baumannii, and subsequently treated with TP3, TP4, or antibiotics for different periods of time (up to 168 h). Mouse survival and bacterial colony forming units (CFU) in various organs were measured after each treatment. Toxicity was determined based on observation of behavior and measurement of biochemical parameters. TP3 and TP4 exhibited strong activity against K. pneumonia and A. baumannii in vitro. Administration of TP3 (150 µg/mouse) or TP4 (50 µg/mouse) 30 min after infection with K. pneumonia or A. baumannii significantly increased survival in mice. TP4 was more effective than tigecycline at reducing CFU counts in several organs. TP3 and TP4 were shown to be non-toxic, and did not affect mouse behavior. TP3 and TP4 are able at potentiate anti-Acinetobacter baumannii or anti-Klebsiella pneumonia drug activity, reduce bacterial load, and prevent drug resistance, indicating their potential for use in combating multidrug-resistant bacteria.

Zebrafish fed on recombinant Artemia expressing epinecidin-1 exhibit increased survival and altered expression of immunomodulatory genes upon Vibrio vulnificus infection.

Artemia has been used extensively in aquaculture as fodder for larval fish, shrimp, and shellfish. Epinecidin-1, an antimicrobial peptide, was isolated from grouper (Epinephelus coioides) in 2005. Epinecidin-1 has been previously reported to possess antimicrobial activity against several Gram-positive and Gram-negative bacterial species, including Staphylococcus coagulase, Pseudomonas aeruginosa, Streptococcus pyogenes, and Vibrio vulnificus. In this study, we used electroporation to introduce plasmid DNA encoding a green fluorescent protein (EGFP)-epinecidin-1 fusion protein under the control of the cytomegalovirus (CMV) promoter into decapsulated Artemia cysts. Optimization of various properties (including cyst weight (0.2 g), plasmid concentration (50 µg/100 µl), and pulse voltage (150 V), length (10 ms), and number (2)) resulted in a hatching rate of 41.15%, a transfection efficiency of 49.81%, and a fluorescence intensity (A.U.) of 47.46. The expression of EGFP-epinecidin-1 was first detected by quantitative RT-PCR at 120 h post-electroporation, and protein was identified by Western blot at the same time. Furthermore, the EGFP-epinecidin-1 protein inhibited V. vulnificus (204) growth, as demonstrated by zone of inhibition studies. Zebrafish fed on transgenic Artemia expressing CMV-gfp-epi combined with commercial fodder were more resistant to infection by V. vulnificus (204): survival rate was enhanced by over 70% at 7, 14, and 21 days post-infection, and bacterial numbers in the liver and intestine were reduced. In addition, feeding of transgenic Artemia to zebrafish affected the immunomodulatory response to V. vulnificus (204) infection; expression of immune-responsive genes, including hepcidin and defbl2, was altered, as shown by qPCR. These findings suggest that feeding transgenic Artemia expressing CMV-gfp-epi to larval fish has antimicrobial effects, without the drawbacks of introducing drug residues or inducing bacterial drug resistance.

The activity of carp muscle-specific creatine kinase at low temperature is enhanced by decreased hydrophobicity of residue 268.

Abstract The muscle-specific forms of creatine kinase in rabbit (RM-CK) and carp (M1-CK) exhibit different temperature-dependent functional properties. Replacing the glycine at residue 268 of RM-CK with asparagine increases the enzyme's activity at 10°C. In this study, we investigated how hydrophobicity of residue 268 affects the biochemical properties of RM-CK and M1-CK at low temperature. We generated three mutants of both RM-CK and M1-CK: Asp268, Lys268, and Leu268. The secondary structures of these mutants were similar, as revealed by their circular dichroism spectra. Similar to the Asn268 mutants, the Asp268 and Lys268 mutants of RM-CK and M1-CK exhibited higher specific activities at 10°C and pH 8.0. However, no such effect was observed for the RM-CK and M1-CK Leu268 mutants. While in the presence of cryoprotectant (sucrose or trehalose), the activities of wild-type RM-CK and M1-CK mutant enzymes with a hydrophobic residue at 268 were higher, and the effect was more profound at pH 8.0. It may be inferred that water molecules affect protein conformation around residue 268, thereby influencing protein stability at low temperature.

Electrotransfer of the epinecidin-1 gene into skeletal muscle enhances the antibacterial and immunomodulatory functions of a marine fish, grouper (Epinephelus coioides).

Electrotransfer of plasmid DNA into skeletal muscle is a common non-viral delivery system for the study of gene function and for gene therapy. However, the effects of epinecidin-1 (epi) on bacterial growth and immune system modulation following its electrotransfer into the muscle of grouper (Epinephelus coioides), a marine fish species, have not been addressed. In this study, pCMV-gfp-epi plasmid was electroporated into grouper muscle, and its effect on subsequent infection with Vibrio vulnificus was examined. Over-expression of epi efficiently reduced bacterial numbers at 24 and 48 h after infection, and augmented the expression of immune-related genes in muscle and liver, inducing a moderate innate immune response associated with pro-inflammatory infiltration. Furthermore, electroporation of pCMV-gfp-epi plasmid without V. vulnificus infection induced moderate expression of certain immune-related genes, particularly innate immune genes. These data suggest that electroporation-mediated gene transfer of epi into the muscle of grouper may hold potential as an antimicrobial therapy for pathogen infection in marine fish.

Truncated antimicrobial peptides from marine organisms retain anticancer activity and antibacterial activity against multidrug-resistant Staphylococcus aureus.

Antimicrobial peptides (AMPs) were recently determined to be potential candidates for treating drug-resistant bacterial infections. The aim of this study was to develop shorter AMP fragments that combine maximal bactericidal effect with minimal synthesis cost. We first synthesized a series of truncated forms of AMPs (anti-lipopolysaccharide factor from shrimp, epinecidin from grouper, and pardaxin from Pardachirus marmoratus). The minimum inhibitory concentrations (MICs) of modified AMPs against ten bacterial species were determined. We also examined the synergy between peptide and non-peptide antibiotics. In addition, we measured the inhibitory rate of cancer cells treated with AMPs by MTS assay. We found that two modified antibacterial peptides (epinecidin-8 and pardaxin-6) had a broad range of action against both gram-positive and gram-negative bacteria. Furthermore, epinecidin and pardaxin were demonstrated to have high antibacterial and anticancer activities, and both AMPs resulted in a significant synergistic improvement in the potencies of streptomycin and kanamycin against methicillin-resistant Staphylococcus aureus. Neither AMP induced significant hemolysis at their MICs. In addition, both AMPs inhibited human epithelial carcinoma (HeLa) and fibrosarcoma (HT-1080) cell growth. The functions of these truncated AMPs were similar to those of their full-length equivalents. In conclusion, we have successfully identified shorter, inexpensive fragments with maximal bactericidal activity. This study also provides an excellent basis for the investigation of potential synergies between peptide and non-peptide antibiotics, for a broad range of antimicrobial and anticancer activities.

Characterization of tilapia (Oreochromis niloticus) viperin expression, and inhibition of bacterial growth and modulation of immune-related gene expression by electrotransfer of viperin DNA into zebrafish muscle.

Viperin is an anti-viral protein, induced by viral infection. In this study, we examined whether over-expression of viperin in fish muscle could inhibit bacterial growth. We first obtained the cDNA sequence of tilapia viperin, through RT-PCR-mediated cloning and sequencing. The cDNA sequence was similar to those of several fish viperins in GenBank, and it was predicted to encode the conserved domain of radical S-adenosylmethionine superfamily proteins. Phylogenetic analysis revealed that tilapia viperin was most closely related to viperin of Sciaenops ocellatus, Coreoperca kawamebari, and C. whiteheadi. Expression of tilapia viperin was significantly up-regulated in the kidney, liver, spleen, and gills upon challenge with lipopolysaccharide (LPS) and poly(I:C) in a time- and dose-dependent manner. Injection of Vibrio vulnificus (204) and Streptococcus agalactiae (SA47) bacteria into tilapia resulted in significant induction of viperin expression in the whole body, kidney, liver, and spleen. Electrotransfer of a viperin-expressing plasmid into zebrafish muscles decreased bacterial numbers and altered expression of immune-related genes. These data indicate that such altered expression may account for the improvement in bacterial clearance following electroporation of viperin, suggesting that fish viperin has antiviral and antibacterial activities.

Shrimp anti-lipopolysaccharide factor (SALF), an antimicrobial peptide, inhibits proinflammatory cytokine expressions through the MAPK and NF-κB pathways in LPS-induced HeLa cells.

Recently, an antimicrobial peptide (AMP), the shrimp anti-lipopolysaccharide factor (SALF), was shown to act against vaginal pathogens as demonstrated by a minimum inhibitory concentration (MIC) assay and suggested that the SALF might play a protective role in orchestrating various defensive responses. The demonstration of a protective role of the SALF in cervical cancer epithelial cells (HeLa cells) led us to investigate the anti-inflammatory effects of the SALF by determining its inhibitory effects on proinflammatory markers in LPS-stimulated cervical cancer HeLa cells. The SALF was shown to inhibit the production of inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, IL-1α, and monocyte chemoattractant protein (MCP)-1 according to an ELISA analysis. The SALF also suppressed mRNA levels of il-6, il-8, il-1α, and mcp-1 according to an RT-PCR. We also found that the SALF might regulate vaginal epithelial cell immune responses through the MAPK and NF-κB pathways. These findings suggest that the SALF is a potential drug candidate for treating chronic inflammatory diseases, such as urethritis, vaginitis, cervicitis, and pelvic inflammatory diseases.

The antimicrobial peptide, shrimp anti-lipopolysaccharide factor (SALF), inhibits proinflammatory cytokine expressions through the MAPK and NF-κB pathways in Trichomonas vaginalis adherent to HeLa cells.

Trichomonas vaginalis is a parasitic protozoan that causes sexually transmitted infections (STIs) worldwide. The infection is dangerous and easily spreads within a community. Also, some cases of drug resistance were reported. Previously, we reported that the shrimp anti-lipopolysaccharide factor (SALF), an antimicrobial peptide of 24 amino acids, modulates inflammatory responses and inhibits T. vaginalis growth. To date, there is no report on the mechanism of SALF's actions in T. vaginalis' adherence to HeLa cells. In this research using an ELISA, we found that the SALF downregulated the release of proinflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1α, IL-6, IL-8, and monocyte chemoattractant protein (MCP)-1) secreted by T. vaginalis which was adhering to HeLa cells. We also performed real-time PCR experiments to determine the roles of the SALF in the expressions of several proinflammatory genes. Through a Western blot analysis, we determined that SALF treatment inhibited T. vaginalis-treated HeLa cells through the p38 and NF-κB pathways. Furthermore, we used different inhibitors to confirm the pathway by ELISA and Western blotting. Taken together, it is apparent that the SALF suppresses T. vaginalis-induced secretion of proinflammatory cytokines by HeLa cells by acting through the p38 and NF-κB pathways.

Pardaxin, a fish antimicrobial peptide, exhibits antitumor activity toward murine fibrosarcoma in vitro and in vivo.

The antitumor activity of pardaxin, a fish antimicrobial peptide, has not been previously examined in in vitro and in vivo systems for treating murine fibrosarcoma. In this study, the antitumor activity of synthetic pardaxin was tested using murine MN-11 tumor cells as the study model. We show that pardaxin inhibits the proliferation of MN-11 cells and reduces colony formation in a soft agar assay. Transmission electron microscopy (TEM) showed that pardaxin altered the membrane structure similar to what a lytic peptide does, and also produced apoptotic features, such as hollow mitochondria, nuclear condensation, and disrupted cell membranes. A qRT-PCR and ELISA showed that pardaxin induced apoptosis, activated caspase-7 and interleukin (IL)-7r, and downregulated caspase-9, ATF 3, SOCS3, STAT3, cathelicidin, p65, and interferon (IFN)-γ suggesting that pardaxin induces apoptosis through the death receptor/nuclear factor (NF)-κB signaling pathway after 14 days of treatment in tumor-bearing mice. An antitumor effect was observed when pardaxin (25 mg/kg; 0.5 mg/day) was used to treat mice for 14 days, which caused significant inhibition of MN-11 cell growth in mice. Overall, these results indicate that pardaxin has the potential to be a novel therapeutic agent to treat fibrosarcomas.

Oral administration of recombinant epinecidin-1 protected grouper (Epinephelus coioides) and zebrafish (Danio rerio) from Vibrio vulnificus infection and enhanced immune-related gene expressions.

Immunostimulatory effects of the oral administration of the recombinant epinecidin-1 protein from BL21 Escherichia coli (containing the pET28a-epinecidin-1-dsRed plasmid) were studied in grouper (Epinephelus coioides) and zebrafish (Danio rerio). For this purpose, fish were fed diets for 30 days containing the recombinant epinecidin-1 protein from BL21 E. coli (containing the pET28a-epinecidin-1-dsRed plasmid) at different bacterial numbers (10(4), 10(6), 10(8), and 10(10) colony-forming units (cfu) of BL21 E. coli in 50 ml of LB medium) mixed with 50 g of eel powder as fodder. After 30 days of feeding, immune-related gene expressions for bacterial-infection responses and disease resistance against Vibrio vulnificus (204) were determined. The V. vulnificus (204) injected into the fish abdominal cavity mimicked gram-negative bacterial infections in culture ponds. Experimental results assessed whether the recombinant epinecidin-1 protein from BL21 E. coli (containing the pET28a-epinecidin-1-dsRed plasmid) has up- (or down-) regulation immune-related genes expression. Results indicated that the recombinant epinecidin-1 protein from BL21 E. coli administered as a feed supplement significantly enhanced expressions several immune-related genes such as tumor necrosis factor (TNF)-1 in grouper and Toll-like receptor (TLR)4, interleukin (IL)-1ß, nitric oxide synthase (NOS)2, and nuclear factor (NF)-κB in zebrafish. After being challenged with V. vulnificus (204) for 24, 48, 72, or 96 h, the percentage mortality was significantly reduced in treated fish, which indicated that the recombinant epinecidin-1 protein from BL21 E. coli administered as a feed supplement could bring about downregulation of TNF-1 expression and functioned like an antagonist for binding TLR4, which reduced the signal transduction pathway for inhibiting TNF and IL-1ß expressions while reducing binding of the transcription factor, NF-κB, to TNF and the IL-1ß promoter region. The experimental results indicated that dietary intake of the recombinant epinecidin-1 protein from BL21 E. coli modulated immune-related gene expressions and disease resistance of grouper and zebrafish after a V. vulnificus (204) infection.

In vivo screening of zebrafish microRNA responses to bacterial infection and their possible roles in regulating immune response genes after lipopolysaccharide stimulation.

Micro (mi)RNAs are abundant small noncoding RNAs found in plants and animals, the regulatory functions of which are not fully understood in fish. To identify potential miRNAs, we screened an miRNA microarray with total RNA from zebrafish infected with Vibrio harveyi and another from uninfected zebrafish. Six miRNAs were obtained from the microarray screening. We studied miRNA expression patterns of 2 miRNAs (miR-122 and miR-194) after bacterial infection of transgenic zebrafish (containing tilapia hepcidin (TH)2-3) and non-transgenic zebrafish from which the 2 miRNAs were obtained from the microarray experiment. The results indicated that miR-122 and miR-194 were higher in PBS-injected zebrafish compared with TH2-3 zebrafish or wild-type (WT) zebrafish after V. harveyi infection. Overexpression of miRNAs (miR-122, miR-192, and miR-194a) was seen in zebrafish liver (ZFL) cells after lipopolysaccharide (LPS) treatment and in untreated fish. Our results showed that after 24 h of doxycycline treatment without LPS stimulation, interleukin (IL)-22, lysozyme, toll-like receptor (TLR)1, TLR3, TLR4a, and tumor necrosis factor (TNF)-α gene expressions were, respectively, upregulated by ~14-, 22-, 2.2-, 13-, 200-, and 38-fold in miR-122-transfected compared with non-transfected (WT) ZFL cells. In cells transfected with miR-192 and treated with LPS after 8-12 h, IL-22, lysozyme, TLR1, TLR3, TLR4a, and TNF-α expressions significantly differed between WT and miR-192-overexpressing ZFL cells. However, we observed significantly higher IL-22 expression levels after 12 h of LPS treatment in miR-192-transfected ZFL cells compared with non-transfected cells. In contrast, IL-22, lysozyme, and TNF-α were markedly upregulated (>100-fold) after miR-194a transfection and overexpression in ZFL cells and treatment with LPS. Our cloning and expression analyses indicated that miR-122, miR-192, and miR-194a play important roles in zebrafish immunology.

Insights into the antibacterial and immunomodulatory functions of the antimicrobial peptide, epinecidin-1, against Vibrio vulnificus infection in zebrafish.

In the present study, we used Vibrio vulnificus and a zebrafish model system to investigate the inhibitory effect of epinecidin-1 on acute bacterial infection and studied the impacts of pretreatment, co-treatment, and post-treatment with epinecidin-1 on its protective efficacy. In vivo experiments showed that co-treatment with epinecidin-1 and V. vulnificus achieved 78%-97% survival rates after 30 days. When epinecidin-1 and V. vulnificus were co-injected into zebrafish and zebrafish were re-challenged with V. vulnificus after 30 days, zebrafish had survival rates of 22%-47%. Pretreatment and post-treatment with epinecidin-1 obtained respective survival rates of 57% and 60%. In addition, epinecidin-1 modulated the expressions of immune-responsive genes like interleukin (IL)-10, IL-1b, tumor necrosis factor-α, and interferon-γ as analyzed by a microarray and qPCR approach. This study demonstrates the use of epinecidin-1 to develop inactivated material for fish bacterial infections which can provide guidelines for the future design of epinecidin-1-bacterial formulations for various in vivo applications.

Modulation of the immune-related gene responses to protect mice against Japanese encephalitis virus using the antimicrobial peptide, tilapia hepcidin 1-5.

Japanese encephalitis virus (JEV), a neurotropic flavivirus, is one of the major causes of acute encephalitis in humans. After infection, it is commonly associated with inflammatory reactions and neurological disease. There is still no effective antiviral drug available against Japanese encephalitis virus infection. Recently, a number of investigators found that antimicrobial peptide (AMPs) present a broad range of biological activities including antimicrobial and immunomodulatory activities. In this study, we found that an AMP, tilapia hepcidin (TH)1-5, caused no harm to either cells or test animals during the test course and could control JEV viral infection in BHK-21 cells. Mice co-injected with TH1-5/JEV and subsequently subjected to JEV re-challenge survived and behaved normally. The neuroprotective effects were associated with marked decreases in: (i) the viral load and viral replication within the brain, (ii) neuronal death, and (iii) secondary inflammation resulting from microglial activation. TH1-5 was also determined to enhance adaptive immunity by elevating levels of anti-JEV-neutralizing antibodies in the serum. The microarray data also showed that TH1-5 modulated Socs-6, interleukin (IL)-6, Toll-like receptor (TLR)-1, TLR-7, caspase-4, interferon (IFN)-ß1, ATF-3, and several immune-responsive genes to protect mice against JEV infection. In addition, TH1-5 was confirmed to modulate the expressions of several proinflammatory and immune-responsive genes, such as IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, tumor necrosis factor (TNF)-α, IFN-γ and monocyte chemoattractant protein (MCP)-1 at both the transcriptional and translational levels in JEV-infected mice. In conclusion, our findings provide mechanistic insights into the actions of TH1-5 against JEV. Results from our in vivo and in vitro experiments clearly indicate that TH1-5 has antiviral, neuroprotective, anti-inflammatory, and immunomodulatory activities. Furthermore, TH1-5 successfully reduced the severity of disease induced by JEV. Our results point out that TH1-5 is a promising candidate for further development as an antiviral agent against JEV infection.