The role of histones in the immune responses of aquatic invertebrates

Histones are primary components of eukaryotic chromatin and highly abundant in all animal cells. In addition to their important role in chromatin structure and transcriptional regulation, histones contribute to innate immune responses. In several aquatic invertebrate species, as well as in many other invertebrate and vertebrate species, the transcripts for core histones are upregulated in response to immune challenge and exposure to environmental stressors. Histones show antimicrobial activity against bacteria and parasites in vitro and in vivo and have the ability to bind bacterial lipopolysaccharide and other pathogen-associated molecules. Several mechanisms regulating and facilitating the antimicrobial action of histones against pathogens have been described in vertebrate and some invertebrate species, including the production of Extracellular Traps (ETs) and the accumulation of histones in lipid droplets that can be selectively released in response to immune stimuli. Further studies are needed to determine the mechanisms of action of histones in immune responses in aquatic invertebrates and investigate the potential use of histones in the treatment of infectious diseases in aquaculture

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

Identification of potential general markers of disease resistance in American oysters, Crassostrea virginica through gene expression studies

Several diseases have a significant impact on American oyster populations in the Atlantic coasts of North America. Knowledge about the responses of oysters to pathogenic challenge could help in identifying potential markers of disease resistance and biomarkers of the health status of an oyster population. A previous analysis of the transcriptome of resistant and susceptible American oysters in response to challenge with the bacterial pathogen Roseovarius crassostreae, as well as sequencing of suppression subtractive hybridization libraries from oysters challenged with the protozoan parasite Perkinsus marinus, provided a list of genes potentially involved in disease resistance or susceptibility. We investigated the patterns of inducible gene expression of several of these genes in response to experimental challenge with the oyster pathogens R. crassostreae, Vibrio tubiashii, and P. marinus. Oysters showing differential susceptibility to R. crassostreae demonstrated differential patterns of expression of genes coding for immune (serine protease inhibitor-1, SPI1) and stress-related (heat shock protein 70, HSP70; arginine kinase) proteins 30 days after challenge with this bacterial pathogen. Differential patterns of expression of immune (spi1, galectin and a matrix metalloproteinase) and stress-related (hsp70, histone H4, and arginine kinase) genes was observed in hemocytes from adult oysters challenged with P. marinus, but not with V. tubiashii. While levels of spi1 expression in hemocytes collected 8 and 21 days after P. marinus challenge were negatively correlated with parasite load in oysters tissues at the end of the challenge (62 days), levels of expression of hsp70 in hemocytes collected 1-day after challenge were positively correlated with oyster parasite load at 62 days. Our results confirm previous research on the role of serine protease inhibitor-1 in immunity and disease resistance in oysters. They also suggest that HSP70 and histone H4 could be used as a markers of health status or disease susceptibility in oysters

Transcriptome of American oysters, Crassostrea virginica, in response to bacterial challenge: insights into potential mechanisms of disease resistance.

The American oyster Crassostrea virginica, an ecologically and economically important estuarine organism, can suffer high mortalities in areas in the Northeast United States due to Roseovarius Oyster Disease (ROD), caused by the gram-negative bacterial pathogen Roseovarius crassostreae. The goals of this research were to provide insights into: 1) the responses of American oysters to R. crassostreae, and 2) potential mechanisms of resistance or susceptibility to ROD. The responses of oysters to bacterial challenge were characterized by exposing oysters from ROD-resistant and susceptible families to R. crassostreae, followed by high-throughput sequencing of cDNA samples from various timepoints after disease challenge. Sequence data was assembled into a reference transcriptome and analyzed through differential gene expression and functional enrichment to uncover genes and processes potentially involved in responses to ROD in the American oyster. While susceptible oysters experienced constant levels of mortality when challenged with R. crassostreae, resistant oysters showed levels of mortality similar to non-challenged oysters. Oysters exposed to R. crassostreae showed differential expression of transcripts involved in immune recognition, signaling, protease inhibition, detoxification, and apoptosis. Transcripts involved in metabolism were enriched in susceptible oysters, suggesting that bacterial infection places a large metabolic demand on these oysters. Transcripts differentially expressed in resistant oysters in response to infection included the immune modulators IL-17 and arginase, as well as several genes involved in extracellular matrix remodeling. The identification of potential genes and processes responsible for defense against R. crassostreae in the American oyster provides insights into potential mechanisms of disease resistance

Novel Antimicrobial Peptide “Octoprohibitin” against Multidrug Resistant <i>Acinetobacter baumannii</i>

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.</i

Silver nanoparticles enhance wound healing in zebrafish (Danio rerio)

Silver nanoparticles (AgNPs) were successfully synthesized by a chemical reduction method, physicochemically characterized and their effect on wound-healing activity in zebrafish was investigated. The prepared AgNPs were circular-shaped, water soluble with average diameter and zeta potential of 72.66 nm and -0.45 mv, respectively. Following the creation of a laser skin wound on zebrafish, the effect of AgNPs on wound-healing activity was tested by two methods, direct skin application (2 μg/wound) and immersion in a solution of AgNPs and water (50 μg/L). The zebrafish were followed for 20 days post-wounding (dpw) by visual observation of wound size, calculating wound healing percentage (WHP), and histological examination. Visually, both direct skin application and immersion AgNPs treatments displayed clear and faster wound closure at 5, 10 and 20 dpw compared to the controls, which was confirmed by 5 dpw histology data. At 5 dpw, WHP was highest in the AgNPs immersion group (36.6%) > AgNPs direct application group (23.7%) > controls (18.2%), showing that WHP was most effective in fish immersed in AgNPs solution. In general, exposure to AgNPs induced gene expression of selected wound-healing-related genes, namely, transforming growth factor (TGF-β), matrix metalloproteinase (MMP) -9 and -13, pro-inflammatory cytokines (IL-1β and TNF-α) and antioxidant enzymes (superoxide dismutase and catalase), which observed differentiation at 12 and 24 h against the control; but the results were not consistently significant, and many either reached basal levels or were down regulated at 5 dpw in the wounded muscle. These results suggest that AgNPs are effective in acceleration of wound healing and altered the expression of some wound-healing-related genes. However, the detailed mechanism of enhanced wound healing remains to be investigated in fish

Principal components (PC) analysis of gene expression in resistant and susceptible oysters experimentally challenged with <i>Roseovarius crassostreae:</i> Spatial projection of PC1 and PC2.

<p>The Z-score centered log2-transformed RPKM for each transcript in challenged susceptible oysters at days 1, 5, 15, and 30 after challenge (F3L_1 to F3L_30), challenged resistant oysters at days 1 to 30 (GX_1 to GX_30), and unchallenged resistant oysters at days 15 and 30 (CGX_15, CGX_30) was used in the PCA. Data from unchallenged resistant oysters at days 1 and 5 were not included in the analysis due to the potential confounding effect of an unrelated mortality event observed before day 7. Gene expression in unchallenged susceptible oysters (CF3L) was not studied.</p

Isolation and Characterization of Plasma-Derived Exosomes from the Marine Fish Rock Bream (<i>Oplegnathus fasciatus</i>) by Two Isolation Techniques

Exosomes are important mediators of intercellular communication and modulate many physiological and pathological processes. Knowledge of secretion, content, and biological functions of fish exosomes during pathological infection is still scarce due to lack of suitable standardized isolation techniques. In this study, we aimed to isolate exosomes from the plasma of marine fish, rock bream (Oplegnathus fasciatus), by two isolation methods: differential ultracentrifugation (UC) and a commercial membrane affinity spin column technique (kit). Morphological and physicochemical characteristics of the isolated exosomes were determined by these two methods, and the efficiencies of the two methods were compared. Exosomes isolated by both methods were in the expected size range (30–200 nm) and had a characteristic cup-shape in transmission electron microscopy observation. Moreover, more intact exosomes were identified using the kit-based method than UC. Nanoparticle tracking analysis demonstrated a heterogeneous population of exosomes with a mean particle diameter of 114.6 ± 4.6 and 111.2 ± 2.2 nm by UC and a kit-based method, respectively. The particle concentration obtained by the kit method (1.05 × 1011 ± 1.23 × 1010 particles/mL) was 10-fold higher than that obtained by UC (4.90 × 1010 ± 2.91 × 109 particles/mL). The kit method had a comparatively higher total protein yield (1.86 mg) and exosome protein recovery (0.55 mg/mL plasma). Immunoblotting analysis showed the presence of exosome marker proteins (CD81, CD63, and HSP90) in the exosomes isolated by both methods and suggests the existence of exosomes. However, the absence of cytotoxicity or adverse immune responses to fish and mammalian cells by the exosomes isolated by the UC procedure indicates its suitability for functional studies in vitro. Overall, our basic characterization results indicate that the kit-based method is more suitable for isolating high-purity exosomes from fish plasma, whereas UC has higher safety in terms of yielding exosomes with low toxicity. This study provides evidence for the existence of typical exosomes in rock beam plasma and facilitates the selection of an efficient exosome isolation procedure for future applications in disease diagnosis and exosome therapy as fish medicine