Exosomes derived from olive flounders infected with Streptococcus parauberis: Proteomic analysis, immunomodulation, and disease resistance capacity.

Multidrug-resistant Streptococcus parauberis causes high fish mortality in aquaculture, necessitating an urgent need for innovative control strategies. This study aimed to develop an immunizing agent against S. parauberis using exosomes isolated from the plasma of olive flounders infected experimentally with S. parauberis (Sp-Exo). Initially, we tested the in vitro immunomodulatory effect of Sp-Exo in murine macrophage RAW264.7 cells and compared it to that of exosomes isolated from naïve fish (PBS-Exo-treated). Notably, Sp-Exo treatment significantly (p < 0.05) upregulated pro-and anti-inflammatory cytokines (Il1ß, Tnfα, and Il10), antimicrobial peptide, defensin isoforms (Def-rs2 and Def-ps1), and antiviral (Ifnß1 and Isg15) genes. In vivo studies in larval and adult zebrafish revealed similar patterns of immunomodulation. Furthermore, larval and adult zebrafish exhibited significantly (p < 0.05) enhanced resistance to S. parauberis infection following treatment with Sp-Exo compared to that with PBS-Exo. Proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) approach revealed the presence of 77 upregulated and 94 downregulated differentially expressed proteins (DEPs) in Sp-Exo, with 22 and 37 significantly (p < 0.05) upregulated and downregulated DEPs, respectively. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Search Tool for the Retrieval of Interacting Genes/Proteins analyses revealed that these genes are associated with key pathways, such as innate immune responses, complement system, acute phase responses, phospholipid efflux, and chylomicron remodeling. In conclusion, Sp-Exo demonstrated superior immunomodulatory activity and significant resistance against S. parauberis infection relative to that on treatment with PBS-Exo. Proteomic analysis further verified that most DEPs in Sp-Exo were associated with immune induction or modulation. These findings highlight the potential of Sp-Exo as a promising vaccine candidate against S. parauberis and other bacterial infections in olive flounder.

Immunomodulatory responses of extracellular vesicles released by gram-positive fish pathogen Streptococcus parauberis.

Bacterial extracellular vesicles (BEVs) are nanosized structures that play a role in intercellular communication and transport of bioactive molecules. Streptococcus parauberis is a Gram-positive pathogenic bacterium that causes "Streptococcosis" in fish. In this study, we isolated S. parauberis-derived extracellular vesicles (SpEVs), and then physicochemical and immunomodulatory properties were determined to elucidate their biological functions. Initially, the biogenesis of SpEVs was detected using field emission scanning electron microscopy, which revealed that secretory phase SpEVs attached to the outer surface of S. parauberis. SpEVs had an average particle diameter and zeta potential of 168.3 ± 6.5 nm and -17.96 ± 2.11 mV, respectively. Field emission transmission electron microscopy analysis confirmed the presence of round or oval-shaped SpEVs with clear membrane margins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results showed three sharp protein bands when SpEVs were stained with Coomassie blue. In vitro toxicity of SpEVs was assayed using the murine macrophage RAW 264.7 cells and we observed no significant (p < 0.05) viability reduction up to 50 µg/mL qRT-PCR results revealed that SpEVs-treated (5 and 10 µg/mL) RAW 264.7 cells significantly (p < 0.05) induced the mRNA of proinflammatory (Il1ß, Il6, and Tnfα) and anti-inflammatory (Il10) cytokines in a concentration-dependent manner. In vivo immunomodulatory effects of SpEVs were investigated by injecting SpEVs (5 and 10 µg/fish) into adult zebrafish. Transcriptional analysis based on qRT-PCR indicates significant (p < 0.05) upregulation of proinflammatory (il1ß, il6, and tnfα) and anti-inflammatory (il10) genes in a concentration-dependent manner in zebrafish kidney. Further, protein expression results in zebrafish spleen tissue confirmed the immunomodulatory activity of SpEVs. In conclusion, SpEVs display the characteristics of BEVs and immunomodulatory activities, suggesting their potential application as vaccine candidate.

In-vitro immunomodulatory responses and antiviral activities of antimicrobial peptide octominin against fish pathogenic viruses.

Antimicrobial peptides (AMPs) are considered a novel approach to stimulate fish antiviral mechanisms for defense against a broad range of viral infections by enhancing immunomodulatory activities. Octominin is an AMP derived from the defense proteins of Octopus minor. In this study, preliminary screening of octominin against viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), and infectious pancreatic necrosis virus (IPNV) was carried out. Moreover, immune responses upon octominin treatment and IHNV challenge were investigated using fathead minnow (FHM) cells. The CC50s of octominin for FHM and Chinook salmon embryo-214 (CHSE-214) cells were 2146.2 and 1865.2 µg/mL, respectively. With octominin treatment, EC50 resulted in 732.8, 435.1, and 925.9 µg/mL for VHSV, IHNV, and IPNV, respectively. The selectivity indices were 2.9, 4.9, and 2.0, respectively. The transcriptional analysis results demonstrated the induced transcription factors (Irf3; 143-fold, Irf7; 105-fold, and NF-κB; 8-fold), stress response gene (HspB8; 2-fold), and apoptosis functional gene (p53; 3-fold) in octominin treated (500 µg/mL) FHM cells for 48 h. Moreover, IHNV viral copy number was slightly decreased with the octominin treatment (500 µg/mL) in FHM cells. Overall results suggest that octominin could be a potential antiviral agent, although further studies are necessary to understand its mode of action and the mechanism of its antiviral activity.

The Aqueous Leaf Extract of the Medicinal Herb Costus speciosus Suppresses Influenza A H1N1 Viral Activity under In Vitro and In Vivo Conditions.

This study investigated the antiviral activity of aqueous leaf extract of Costus speciosus (TB100) against influenza A. Pretreatment of TB100 in RAW264.7 cells enhanced antiviral activity in an assay using the green fluorescence-expressing influenza A/Puerto Rico/8/1934 (H1N1) virus. The fifty percent effective concentration (EC50) and fifty percent cytotoxic concentration (CC50) were determined to be 15.19 ± 0.61 and 117.12 ± 18.31 µg/mL, respectively, for RAW264.7 cells. Based on fluorescent microscopy, green fluorescence protein (GFP) expression and viral copy number reduction confirmed that TB100 inhibited viral replication in murine RAW264.7 and human A549 and HEp2 cells. In vitro pretreatment with TB100 induced the phosphorylation of transcriptional activators TBK1, IRF3, STAT1, IKB-α, and p65 associated with interferon pathways, indicating the activation of antiviral defenses. The safety and protective efficacy of TB100 were assessed in BALB/c mice as an oral treatment and the results confirmed that it was safe and effective against influenza A/Puerto Rico/8/1934 (H1N1), A/Philippines/2/2008 (H3N2), and A/Chicken/Korea/116/2004 (H9N2). High-performance liquid chromatography of aqueous extracts led to the identification of cinnamic, caffeic, and chlorogenic acids as potential chemicals for antiviral responses. Further confirmatory studies using these acids revealed that each of them confers significant antiviral effects against influenza when used as pretreatment and enhances the antiviral response in a time-dependent manner. These findings suggest that TB100 has the potential to be developed into an antiviral agent that is effective against seasonal influenza.

Exosomes from bacteria (Streptococcus parauberis) challenged olive flounder (Paralichthys olivaceus): Isolation, molecular characterization, wound healing, and regeneration activities.

Exosomes are a group of extracellular vesicles carrying membrane proteins, lipids, RNAs, and, cytosolic proteins, which play key role in intercellular communication and homeostasis. This study describes the isolation, physicochemical, morphological and molecular characterization, toxicity, wound healing, and regeneration properties of plasma derived exosomes from naive (phosphate-buffered saline [PBS]-injected; PBS-Exo) and Streptococcus parauberis-challenged (Sp-Exo) olive flounder (Paralichthys olivaceus). The average diameters of PBS-Exo and Sp-Exo were 120.5 ± 6.1 and 113.1 ± 9.3 nm, respectively, and they presented unique cup shape morphologies. Both exosomes exhibited classical tetraspanin surface markers (CD81, CD9, and CD63) and were enriched with acetylcholinesterase. High-throughput miRNA profiling revealed differentially expressed miRNAs (log2 fold change ≥1; P < 0.05), including 14 known and 22 novel miRNAs, in Sp-Exo. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that the target genes of the miRNAs contribute towards various physiological and immunological functions, including wound healing and fin regeneration. Sp-Exo exhibited a rapid wound healing (cell migration) capacity in human fibroblast cells, and its mRNA and protein expression patterns corroborated its activity. Higher larval fin regeneration was more prevalent in Sp-Exo than in PBS-Exo, which further confirmed its functional significance. Our study provides the first basic physiochemical, morphometric, molecular (miRNA profiling), and wound healing evidences of Sp-Exo in olive flounder and highlights important miRNA cargoes in exosomes that may be potential therapeutic candidates in wound healing.

Antimicrobial Peptide Octominin-Encapsulated Chitosan Nanoparticles Enhanced Antifungal and Antibacterial Activities.

Antimicrobial peptides (AMPs) have become a key solution for controlling multi-drug-resistant (MDR) pathogens, and the nanoencapsulation of AMPs has been used as a strategy to overcome challenges, such as poor stability, adverse interactions, and toxicity. In previous studies, we have shown the potent antimicrobial activity of Octominin against Candida albicans and Acinetobacter baumannii. This study is focused on the nanoencapsulation of Octominin with chitosan (CS) and carboxymethyl chitosan (CMC) as a drug delivery system using the ionotropic gelation technique. Octominin-encapsulated CS nanoparticles (Octominin-CNPs) had an average diameter and zeta potential of 372.80 ± 2.31 nm and +51.23 ± 0.38 mV, respectively, while encapsulation efficiency and loading capacity were 96.49 and 40.20%, respectively. Furthermore, Octominin-CNPs showed an initial rapid and later sustained biphasic release profile, and up to 88.26 ± 3.26% of the total Octominin release until 96 h. Transmission electron microscopy data showed the irregular shape of the Octominin-CNPs with aggregations. In vitro and in vivo toxicity of Octominin-CNPs was significantly lower than the Octominin at higher concentrations. The antifungal and antibacterial activities of Octominin-CNPs were slightly higher than those of Octominin in both the time-kill kinetic and microbial viability assays against C. albicans and A. baumannii, respectively. Mode of action assessments of Octominin-CNPs revealed that morphological alterations, cell membrane permeability alterations, and reactive oxygen species generation were slightly higher than those of Octominin at the tested concentrations against both C. albicans and A. baumannii. In antibiofilm activity assays, Octominin-CNPs showed slightly higher biofilm inhibition and biofilm eradication activities compared to that of Octominin. In conclusion, Octominin was successfully encapsulated into CS, and Octominin-CNPs showed lower toxicity and greater antimicrobial activity against C. albicans and A. baumannii compared to Octominin.

Novel Antimicrobial Peptide "Octoprohibitin" against Multidrug Resistant Acinetobacter baumannii.

Octoprohibitin is a synthetic antimicrobial peptide (AMP), derived from the prohibitin-2 gene of Octopus minor. It showed substantial activity against multidrug resistant (MDR) Acinetobacter baumannii with a minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 200 and 400 µg/mL, respectively. Time-kill kinetics and bacterial viability assays confirmed the concentration-dependent antibacterial activity of octoprohibitin against A. baumannii. The morphology and ultrastructure of A. baumannii were altered by treatment with octoprohibitin at the MIC and MBC levels. Furthermore, propidium iodide-fluorescein diacetate (PI-FDA) staining and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) staining of octoprohibitin-treated A. baumannii revealed membrane permeability alterations and reactive oxygen species (ROS) generation, respectively. Agarose gel retardation results confirmed the DNA-binding ability of octoprohibitin to the genomic DNA of A. baumannii. Furthermore, octoprohibitin showed concentration-dependent inhibition of biofilm formation and eradication. The minimum biofilm inhibition concentration (MBIC) and minimum biofilm eradication concentration (MBEC) of octoprohibitin were 1000 and 1460 µg/mL, respectively. Octoprohibitin produced no significant cytotoxicity up to 800 µg/mL, and no hemolysis was observed up to 400 µg/mL. Furthermore, in vivo analysis in an A. baumannii-infected zebrafish model confirmed the effective bactericidal activity of octoprohibitin with higher cumulative survival percent (46.6%) and fewer pathological signs. Histological analysis showed reduced alterations in the gut, kidney, and gill tissues in the octoprohibitin-treated group compared with those in the phosphate-buffered saline (PBS)-treated group. In conclusion, our results suggest that octoprohibitin is a potential antibacterial and antibiofilm agent against MDR A. baumannii.

Isolation and characterization of plasma-derived exosomes from olive flounder (Paralichthys olivaceus) and their wound healing and regeneration activities.

Exosomes have garnered enormous interest for their role in physiological and pathological processes and their potential for therapeutic and diagnostic applications. In this study, exosomes were isolated from plasma of olive flounder (Paralichthys olivaceus) and their physiochemical and morphological characteristics, as well as wound healing and regeneration activities were determined. Isolated exosomes had typical characteristics, including average particle diameter (151.82 ± 9.17 nm), concentration (6.31 × 1010 particles/mL) with a membrane-bound, cup-shaped morphology. Exosome marker proteins, tetraspanins (CD63, CD9, and CD81), and acetylcholinesterase were detected, indicating the presence of exosomes in olive flounder plasma. Exosomes exhibited no toxicity in in vitro and in vivo studies, even at the highest treatment concentrations (100 and 400 µg/mL, respectively), confirming their suitability for further functional studies. Following exosome treatment (50 and 100 µg/mL), substantial cell migration with rapid closure of the open wound area in in vitro scratch wound healing assay and faster zebrafish larvae fin regeneration rate was observed compared to that of the vehicle. Moreover, exosomes exhibited immunomodulatory properties associated with wound healing, based on mRNA expression patterns in fathead minnow (FHM) cells. In conclusion, exosomes isolated from olive flounder plasma using ultracentrifugation exhibited minimal toxicity and enhanced wound healing and tissue regeneration activities. Identification and in-depth investigation of olive flounder plasma-derived exosome constituents will support the development of exosomes as an efficient therapeutic carrier system for fish medicine in the future.

Octopromycin: Antibacterial and antibiofilm functions of a novel peptide derived from Octopus minor against multidrug-resistant Acinetobacter baumannii.

The emergence of carbapenem-resistant Acinetobacter baumannii has increased the risk of nosocomial infections, which pose a huge health threat. There is an urgent need to develop alternative therapies, including broad-spectrum antimicrobial peptides. In this study, we designed, characterized, and studied the antibacterial, antibiofilm effects and possible mode of actions of a novel synthetic peptide Octopromycin, derived from the proline-rich protein 5 of Octopus minor. Octopromycin consists of 38 amino acids, (+5) net positive charge, high hydrophobic residue ratio (36%), and two α-helix secondary structures. The minimum inhibitory concentration and minimum bactericidal concentration against A. baumannii were 50 and 200 µg/mL, respectively. Time-kill kinetics and bacterial viability assays confirmed the concentration-dependent antibacterial activity of Octopromycin. Field emission scanning electron microscopy images clearly showed ultrastructural alterations in Octopromycin-treated A. baumannii cells. Propidium iodide penetrated into Octopromycin-treated A. baumannii cells, demonstrating the loss of cell membrane integrity. Octopromycin treatment increased the production of reactive oxygen species in a concentration-dependent manner, and it inhibited the biofilm formation and showed biofilm eradication activity against A. baumannii. In vitro and in vivo safety evaluation revealed that Octopromycin was nontoxic to HEK293T and Raw 264.7 cells (<400 µg/mL), as well as mice red blood cells (<300 µg/mL), and zebrafish embryos (<4 µg/mL). An in vivo study results revealed that the A. baumannii-infected fish treated with Octopromycin exhibited a significantly higher relative percent survival (37.5%) than the infected mock-treated fish with PBS (16.6%). Furthermore, a decreased bacterial load and fewer alterations in histological analysis confirmed the successful control of A. baumannii by Octopromycin in vivo. Collectively, the results indicate that the antibacterial peptide Octopromycin may achieve rapid control of A. baumannii through multi-target interactions; it presents a desirable therapeutic option for the prevention and control of the infections.

Antimicrobial and Anti-Biofilm Peptide Octominin for Controlling Multidrug-Resistant Acinetobacter baumannii.

Acinetobacter baumannii is a serious nosocomial pathogen with multiple drug resistance (MDR), the control of which has become challenging due to the currently used antibiotics. Our main objective in this study is to determine the antibacterial and antibiofilm activities of the antimicrobial peptide, Octominin, against MDR A. baumannii and derive its possible modes of actions. Octominin showed significant bactericidal effects at a low minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of 5 and 10 µg/mL, respectively. Time-kill kinetic analysis and bacterial viability tests revealed that Octominin showed a concentration-dependent antibacterial activity. Field-emission scanning electron microscopy (FE-SEM) analysis revealed that Octominin treatment altered the morphology and membrane structure of A. baumannii. Propidium iodide (PI) and reactive oxygen species (ROS) generation assays showed that Octominin increased the membrane permeability and ROS generation in A. baumannii, thereby causing bacterial cell death. Further, a lipopolysaccharides (LPS) binding assay showed an Octominin concentration-dependent LPS neutralization ability. Biofilm formation inhibition and eradication assays further revealed that Octominin inhibited biofilm formation and showed a high biofilm eradication activity against A. baumannii. Furthermore, up to a concentration of 100 µg/mL, Octominin caused no hemolysis and cell viability changes in mammalian cells. An in vivo study in zebrafish showed that the Octominin-treated group had a significantly higher relative percentage survival (54.1%) than the untreated group (16.6%). Additionally, a reduced bacterial load and fewer alterations in histological analysis confirmed the successful control of A. baumannii by Octominin in vivo. Collectively, these data suggest that Octominin exhibits significant antibacterial and antibiofilm activities against the multidrug-resistant A. baumannii, and this AMP can be developed further as a potent AMP for the control of antibiotic resistance.