Gamma-Aminobutyric Acid Production from a Novel Enterococcus avium JS-N6B4 Strain Isolated from Edible Insects.

Gamma-aminobutyric acid (GABA)-producing strains were isolated from four edible insects and subjected to 16S rRNA sequence analysis. Among the four GABA-producing bacteria, Enterococcus avium JS-N6B4 exhibited the highest GABA-production, while cultivation temperature, initial pH, aerobic condition, and mono-sodium glutamate (MSG) feeding were found to be the key factors affecting GABA production rate. The culture condition was optimized in terms of glucose, yeast extract, and MSG concentrations using response surface methodology (RSM). GABA production up to 16.64 g/l was obtained under the conditions of 7 g/l glucose, 45 g/l yeast extract, and 62 g/l MSG through the optimization of medium composition by RSM. Experimental GABA production was 13.68 g/l, which was close to the predicted value (16.64 g/l) calculated from the analysis of variance, and 2.79-fold higher than the production achieved with basic medium. Therefore, GABA-producing strains may help improve the GABA production in edible insects, and provide a new approach to the use of edible insects as effective food biomaterials.

The Silencing of a 14-3-3ɛ Homolog in Tenebrio molitor Leads to Increased Antimicrobial Activity in Hemocyte and Reduces Larval Survivability.

The 14-3-3 family of phosphorylated serine-binding proteins acts as signaling molecules in biological processes such as metabolism, division, differentiation, autophagy, and apoptosis. Herein, we report the requirement of 14-3-3ɛ isoform from Tenebrio molitor (Tm14-3-3ɛ) in the hemocyte antimicrobial activity. The Tm14-3-3ɛ transcript is 771 nucleotides in length and encodes a polypeptide of 256 amino acid residues. The protein has the typical 14-3-3 domain, the nuclear export signal (NES) sequence, and the peptide binding residues. The Tm14-3-3ɛ transcript shows a significant three-fold expression in the hemocyte of T. molitor larvae when infected with Escherichia coli Tm14-3-3ɛ silenced larvae show significantly lower survival rates when infected with E. coli. Under Tm14-3-3ɛ silenced condition, a strong antimicrobial activity is elicited in the hemocyte of the host inoculated with E. coli. This suggests impaired secretion of antimicrobial peptides (AMP) into the hemolymph. Furthermore, a reduction in AMP secretion under Tm14-3-3ɛ silenced condition would be responsible for loss in the capacity to kill bacteria and might explain the reduced survivability of the larvae upon E. coli challenge. This shows that Tm14-3-3ɛ is required to maintain innate immunity in T. molitor by enabling antimicrobial secretion into the hemolymph and explains the functional specialization of the isoform.

Antifungal activity of synthetic peptide derived from halocidin, antimicrobial peptide from the tunicate, Halocynthia aurantium.

Halocidin is an antimicrobial peptide isolated from the hemocytes of the tunicate. Among the several known synthetic halocidin analogues, di-K19Hc has been previously confirmed to have the most profound antibacterial activity against antibiotic-resistant bacteria. This peptide has been considered to be an effective candidate for the development of a new type of antibiotic. In this study, we have assessed the antifungal activity of di-K19Hc, against a panel of fungi including several strains of Aspergillus and Candida. As a result, we determined that the MICs of di-K19Hc against six Candida albicans and two Aspergillus species were below 4 and 16 microg/ml, respectively, thereby indicating that di-K19Hc may be appropriate for the treatment of several fungal diseases. We also conducted an investigation into di-K19Hc's mode of action against Candida albicans. Our colony count assay showed that di-K19Hc killed C. albicans within 30s. Di-K19Hc bound to the surface of C. albicans via a specific interaction with beta-1,3-glucan, which is one of fungal cell wall components. Di-K19Hc also induced the formation of ion channels within the membrane of C. albicans, and eventually observed cell death, which was confirmed via measurements of the K+ released from C. albicans cells which had been treated with di-K19Hc, as well as by monitoring of the uptake of propidium iodide into the C. albicans cells. This membrane-attacking quality of di-K19Hc was also visualized via confocal laser and scanning electron microscopy.

Fungicidal effect of indolicidin and its interaction with phospholipid membranes.

The fungicidal effect and mechanism of a tryptophan-rich 13-mer peptide, indolicidin derived from granules of bovine neutrophils, were investigated. Indolicidin displayed a strong fungicidal activity against various fungi. In order to understand the fungicidal mechanism(s) of indolicidin, we examined the interaction of indolicidin with the pathogenic fungus Trichosporon beigelii. Fluorescence confocal microscopy and flow cytometry analysis revealed that indolicidin acted rapidly on the plasma membrane of the fungal cells in an energy-independent manner. This interaction is also dependent on the ionic environment. Furthermore, indolicidin caused significant morphological changes when tested for the membrane disrupting activity using liposomes (phosphatidylcholine/cholesterol; 10:1, w/w). The results suggest that indolicidin may exert its fungicidal activity by disrupting the structure of cell membranes, via direct interaction with the lipid bilayers, in a salt-dependent and energy-independent manner.