Outer Membrane Integrity-Dependent Fluorescence of the Japanese Eel UnaG Protein in Live Escherichia coli Cells.

Reporter genes are important tools in many biological disciplines. The discovery of novel reporter genes is relatively rare. However, known reporter genes are constantly applied to novel applications. This study reports the performance of the bilirubin-dependent fluorescent protein UnaG from the Japanese eel Anguilla japonicas in live Escherichia coli cells in response to the disruption of outer membrane (OM) integrity at low bilirubin (BR) concentrations. Using the E. coli wild-type strain MC4100, its isogenic OM-deficient mutant strain NR698, and different OM-active compounds, we show that BR uptake and UnaG fluorescence depend on a leaky OM at concentrations of 10 µM BR and below, while fluorescence is mostly OM integrity-independent at concentrations above 50 µM BR. We suggest that these properties of the UnaG-BR couple might be applied as a biosensor as an alternative to the OM integrity assays currently in use.

Antimicrobial Activity of Small Synthetic Peptides Based on the Marine Peptide Turgencin A: Prediction of Antimicrobial Peptide Sequences in a Natural Peptide and Strategy for Optimization of Potency.

Turgencin A, a potent antimicrobial peptide isolated from the Arctic sea squirt Synoicum turgens, consists of 36 amino acid residues and three disulfide bridges, making it challenging to synthesize. The aim of the present study was to develop a truncated peptide with an antimicrobial drug lead potential based on turgencin A. The experiments consisted of: (1) sequence analysis and prediction of antimicrobial potential of truncated 10-mer sequences; (2) synthesis and antimicrobial screening of a lead peptide devoid of the cysteine residues; (3) optimization of in vitro antimicrobial activity of the lead peptide using an amino acid replacement strategy; and (4) screening the synthesized peptides for cytotoxic activities. In silico analysis of turgencin A using various prediction software indicated an internal, cationic 10-mer sequence to be putatively antimicrobial. The synthesized truncated lead peptide displayed weak antimicrobial activity. However, by following a systematic amino acid replacement strategy, a modified peptide was developed that retained the potency of the original peptide. The optimized peptide StAMP-9 displayed bactericidal activity, with minimal inhibitory concentrations of 7.8 µg/mL against Staphylococcus aureus and 3.9 µg/mL against Escherichia coli, and no cytotoxic effects against mammalian cells. Preliminary experiments indicate the bacterial membranes as immediate and primary targets.

Isolation and Characterization of Antimicrobial Peptides with Unusual Disulfide Connectivity from the Colonial Ascidian Synoicum turgens.

This study reports the isolation of two novel cysteine-rich antibacterial peptides, turgencin A and turgencin B, along with their oxidized derivatives, from the Arctic marine colonial ascidian Synoicum turgens. The peptides are post-translationally modified, containing six cysteines with an unusual disulfide connectivity of Cys1-Cys6, Cys2-Cys5, and Cys3-Cys4 and an amidated C-terminus. Furthermore, the peptides contain methionine residues resulting in the isolation of peptides with different degrees of oxidation. The most potent peptide, turgencin AMox1 with one oxidized methionine, displayed antimicrobial activity against both Gram-negative and Gram-positive bacteria with a minimum inhibitory concentration (MIC) as low as 0.4 µM against selected bacterial strains. In addition, the peptide inhibited the growth of the melanoma cancer cell line A2058 (IC50 = 1.4 µM) and the human fibroblast cell line MRC-5 (IC50 = 4.8 µM). The results from this study show that natural peptides isolated from marine tunicates have the potential to be promising drug leads.