Characterization and Potentiating Effects of the Ethanolic Extracts of the Red Seaweed Gracillaria sp. on the Activity of Carbenicillin against Vibrios.

ß-lactam-resistant Vibrio strains are a significant clinical problem, and ß-lactamase inhibitors are generally coadministered with ß-lactam drugs to control drug-resistant bacteria. Seaweed is a rich source of natural bioactive compounds; however, their potential as ß-lactamase inhibitors against bacterial pathogens remains unknown. Herein, we evaluated the potential ß-lactamase inhibitory effect of the ethanolic extracts of the red seaweed Gracilaria sp. (GE) against four Vibrio strains. The minimum inhibitory concentration, half-maximal inhibitory concentration, checkerboard assay results, and time-kill study results indicate that GE has limited antibacterial activity but can potentiate the activity of the ß-lactam antibiotic carbenicillin against Vibrio parahemolyticus and V. cholerae. We overexpressed and purified recombinant metallo-ß-lactamase, VarG, from V. cholerae for in vitro studies and observed that adding GE reduced the carbenicillin and nitrocefin degradation by VarG by 20% and 60%, respectively. Angiotensin I-converting enzyme inhibition studies demonstrated that GE did not inhibit VarG via metal chelation. Toxicity assays indicated that GE exhibited mild toxicity against human cells. Through gas chromatography and mass spectrometry, we showed that GE comprises alkaloids, phenolic compounds, terpenoids, terpenes, and halogenated aromatic compounds. This study revealed that extracts of the red seaweed Gracillaria sp. can potentially inhibit ß-lactamase activity.

Secretory Production of Functional Grouper Type I Interferon from Epinephelus septemfasciatus in Escherichia coli and Bacillus subtilis.

Nervous necrosis virus (NNV) results in high mortality rates of infected marine fish worldwide. Interferons (IFNs) are cytokines in vertebrates that suppress viral replication and regulate immune responses. Heterologous overexpression of fish IFN in bacteria could be problematic because of protein solubility and loss of function due to protein misfolding. In this study, a protein model of the IFN-α of Epinephelus septemfasciatus was built based on comparative modeling. In addition, PelB and SacB signal peptides were fused to the N-terminus of E. septemfasciatus IFN-α for overexpression of soluble, secreted IFN in Escherichia coli (E-IFN) and Bacillus subtilis (B-IFN). Cytotoxicity tests indicated that neither recombinant grouper IFN-α were cytotoxic to a grouper head kidney cell line (GK). The GK cells stimulated with E-IFN and B-IFN exhibited elevated expression of antiviral Mx genes when compared with the control group. The NNV challenge experiments demonstrated that GK cells pretreated or co-treated with E-IFN and B-IFN individually had three times the cell survival rates of untreated cells, indicating the cytoprotective ability of our recombinant IFNs. These data provide a protocol for the production of soluble, secreted, and functional grouper IFN of high purity, which may be applied to aquaculture fisheries for antiviral infection.