Synergistic Effect, Improved Cell Selectivity, and Elucidating the Action Mechanism of Antimicrobial Peptide YS12.

Antimicrobial peptides (AMPs) have attracted considerable attention as potential substitutes for traditional antibiotics. In our previous research, a novel antimicrobial peptide YS12 derived from the Bacillus velezensis strain showed broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria. In this study, the fractional inhibitory concentration index (FICI) indicated that combining YS12 with commercial antibiotics produced a synergistic effect. Following these findings, the combination of YS12 with an antibiotic resulted in a faster killing effect against bacterial strains compared to the treatment with the peptide YS12 or antibiotic alone. The peptide YS12 maintained its antimicrobial activity under different physiological salts (Na+, Mg2+, and Fe3+). Most importantly, YS12 exhibited no cytotoxicity towards Raw 264.7 cells and showed low hemolytic activity, whereas positive control melittin indicated extremely high toxicity. In terms of mode of action, we found that peptide YS12 was able to bind with LPS through electrostatic interaction. The results from fluorescent measurement revealed that peptide YS12 damaged the integrity of the bacterial membrane. Confocal laser microscopy further confirmed that the localization of peptide YS12 was almost in the cytoplasm of the cells. Peptide YS12 also exhibited anti-inflammatory activity by reducing the release of LPS-induced pro-inflammatory mediators such as TNF-α, IL-1ß, and NO. Collectively, these properties strongly suggest that the antimicrobial peptide YS12 may be a promising candidate for treating microbial infections and inflammation.

Amyloid ß cytotoxicity is enhanced or reduced depending on formation of amyloid ß oligomeric forms.

OBJECTIVES: We explored the underlying mechanisms that facilitate reducing and enhancing effects of exogenous proteins on cytotoxicity of amyloid ß (Aß), a main pathogen of Alzheimer's disease, by using an Escherichia coli chaperonin DnaK. RESULTS: DnaK was chosen as a tool, because it, easily available and functionally stable, reduced or enhanced Aß cytotoxicity depending on its concentration. Cytotoxicity was enhanced when the molar ratio of DnaK to Aß42, at 20 µM Aß42, was 0.01-0.5, while reduced cytotoxicity was observed at higher ratios (> 1) at 1 µM Aß42. Significant amounts of oligomeric Aß42 species accumulated concomitantly with enhanced cytotoxicity, whereas the oligomers appeared to form complexes with DnaK in conditions of reduced cytotoxicity. CONCLUSIONS: The difference in cytotoxicity was due to variations in the toxic oligomeric Aß species and DnaK is a useful tool for the study of the Aß ultrastructure formation and toxicity of Aß peptide.

Evidence for a Strong Relationship between the Cytotoxicity and Intracellular Location of ß-Amyloid.

ß-Amyloid (Aß) is a hallmark peptide of Alzheimer's disease (AD). Herein, we explored the mechanism underlying the cytotoxicity of this peptide. Double treatment with oligomeric 42-amino-acid Aß (Aß42) species, which are more cytotoxic than other conformers such as monomers and fibrils, resulted in increased cytotoxicity. Under this treatment condition, an increase in intracellular localization of the peptide was observed, which indicated that the peptide administered extracellularly entered the cells. The cell-permeable peptide TAT-tagged Aß42 (tAß42), which was newly prepared for the study and found to be highly cell-permeable and soluble, induced Aß-specific lamin protein cleavage, caspase-3/7-like DEVDase activation, and high cytotoxicity (5-10-fold higher than that induced by the wild-type oligomeric preparations). Oligomeric species enrichment and double treatment were not necessary for enhancing the cytotoxicity and intracellular location of the fusion peptide. Taiwaniaflavone, an inhibitor of the cytotoxicity of wild-type Aß42 and tAß42, strongly blocked the internalization of the peptides into the cells. These data imply a strong relationship between the cytotoxicity and intracellular location of the Aß peptide. Based on these results, we suggest that agents that can reduce the cell permeability of Aß42 are potential AD therapeutics.

Reduction of Oxidative Stress through Activating the Nrf2 mediated HO-1 Antioxidant Efficacy Signaling Pathway by MS15, an Antimicrobial Peptide from Bacillus velezensis.

The efficient culture and purification of antimicrobial peptides (AMPs), along with intense antioxidant activity, have drawn the interest to study antioxidant activity mechanism. We report the culture conditions optimization, efficient biosynthesis, and purification of an antioxidant peptide MS15 from Bacillus velezensis obtained from fermented food that would generate heme oxygenase-1 (HO-1) expression and lead to nuclear factor erythroid 2-related factor-2 (Nrf2) nuclear translocation. We explored the ability of kinetics and potency for the bacterial killing to work against various pathogenic bacteria. A bioassay showed the lysis zone of MS15 by tricine SDS-PAGE near at 6 kDa. MALDI-TOF/MS verified molecular weight, and the existence of a molecular mass of 6091 Da was reported by purity. The MIC of MS15 ranged from 2.5-160 µg/mL for many pathogenic bacteria, showing greater potency. In macrophage RAW 264.7 cells, MS15 was exposed to assess its inhibitory effect against the generation of reactive oxygen species (ROS) in oxidative stress. In the sample treated group, the translation, and transcriptional levels of CAT (catalase), GPx (glutathione peroxidase), and SOD (superoxide dismutase) were significantly greater. In short, MS15 has significant antioxidant properties, reducing ROS production in RAW 264.7 cells, and raising the translation and transcriptional rates of antioxidant enzymes with stimulating HO-1 induction facilitated by Nrf2.