Plectasin

The potassium channels were recently found to be inhibited by animal toxin-like human β-defensin 2 (hBD2), the first defensin blocker of potassium channels. Whether there are other defensin blockers from different organisms remains an open question. Here, we reported the potassium channel-blocking plectasin, the first defensin blocker from a fungus. Based on the similar cysteine-stabilized alpha-beta (CSαβ) structure between plectasin and scorpion toxins acting on potassium channels, we found that plectasin could dose-dependently block Kv1.3 channel currents through electrophysiological experiments. Besides Kv1.3 channel, plectasin could less inhibit Kv1.1, Kv1.2, IKCa, SKCa3, hERG and KCNQ channels at the concentration of 1 μΜ. Using mutagenesis and channel activation experiments, we found that outer pore region of Kv1.3 channel was the binding site of plectasin, which is similar to the interacting site of Kv1.3 channel recognized by animal toxin blockers. Together, these findings not only highlight the novel function of plectasin as a potassium channel inhibitor, but also imply that defensins from different organisms functionally evolve to be a novel kind of potassium channel inhibitors.

The present study was aimed to determine the immunomodulatory effects of dietary supplementation of the antimicrobial peptide (AMP) plectasin on broiler chickens. The experiment involved 300-day-old Ross chicks reared in a conventional housing system and subjected to ambient temperature and relative humidity. The birds were randomly allocated to five treatment groups: the non-supplemented negative control group (T1), enramycin-supplemented group (T2), and groups supplemented with varying doses of plectasin at 150 ppm, 300 ppm, and 450 ppm (T3, T4, and T5, respectively) from day 1 to 35. The results indicated that plectasin supplementation increased jejunal and ileal goblet cell (GC) counts, serum interferon-gamma (IFN-γ) levels at neonatal age, and serum immunoglobulin Y (IgY) titer on days 7, 21, 28, and 35. These findings confirmed that plectasin induces positive immunomodulatory responses by specifically enhancing gut mucosal barriers, early innate immunity, and humoral immune response. Specifically, supplementation at 150 ppm may be considered as the optimal dose for inclusion in broiler chicken feeds.

Plectasin is a promising and potent antimicrobial peptide isolated from the fungus Pseudoplectania nigrella which has been heterologously expressed in various hosts. In this study, a four-copy cassette of plectasin was constructed via 2A peptide assembly to further increase its expression level in recombinant Pichia pastoris. The yeast transformant 4Ple-61 harboring four-copy cassette of plectasin could secrete 183.2 mg/L total protein containing 60.8% of plectasin at the flask level within 120 h, which was 2.3 times higher than that of the yeast transformant Ple-6 carrying one-copy cassette of plectasin. Western blot confirmed the significant peptide expression level in the transformant 4Ple-61. Furthermore, it yielded as high as 426.3 mg/L total protein within 120 h during a 5-L fermentation. The purified plectasin shows superior stability and good antimicrobial activity against conventional Staphylococcus aureus ATCC 26,001 and some food-borne antibiotic-resistant S. aureus strains with the MICs ranging from 8 to 32 μg/mL. Therefore, the strategy based on 2A peptide assembly can enhance the expression of plectasin and further expand its application prospect. KEY POINTS: · A yeast transformant 4Ple-61 with four-copy cassette of plectasin was constructed. · The plectasin level yield by the transformant 4Ple-61 was boosted by 2.3 times. · The plectasin showed good activity against food-borne antibiotic-resistant S. aureus.