Defensin-like peptide

Streptococcus dysgalactiae, considered one of the main pathogens that causes bovine mastitis, is a serious threat to humans and animals. However, the excessive use of antibiotics and the characteristic of S. dysgalactiae forming biofilms in mastitic teat canal have serious clinical implications. In this study, in vivo and in vitro multiple mechanisms of action of P2, a mutant of fungal defensin plectasin, against S. dysgalactiae were systematically and comprehensively investigated for the first time. P2 showed potent antibacterial activity against S. dysgalactiae (minimum inhibitory concentration, MIC = 0.23-0.46 μM) and rapid bactericidal action by 3.0 lg units reduction in 2-4 h. No resistant mutants appeared after 30-d serial passage of S. dysgalactiae in the presence of P2. The results of electron microscopy and flow cytometer showed that P2 induced membrane damage of S. dysgalactiae, causing the leakage of cellular content and eventually cell death. Besides, P2 effectively inhibited early biofilm formation, eradicated mature biofilms, and killed 99.9% persisters which were resistant to 100 × MIC vancomycin; and confocal laser scanning microscopy (CLSM) also revealed the potent antibacterial and antibiofilm activity of P2 (the thickness of biofilm reduced from 18.82 to 7.94 μm). The in vivo therapeutic effect of P2 in mouse mastitis model showed that it decreased the number of mammary bacteria and alleviated breast inflammation by regulating cytokines and inhibiting bacterial proliferation, which were superior to vancomycin. These data indicated that P2 maybe a potential candidate peptide for mastitis treatment of S. dysgalactiae infections. KEY POINTS: ·P2 showed potential in vitro antibacterial characteristics towards S. dysgalactiae. ·P2 eradicated biofilms, killed persisters, and induced cell death of S. dysgalactiae. ·P2 could effectively protect mice from S. dysgalactiae infection in gland.

Defensins are the most diverse groups of antimicrobial peptides in invertebrate animals. In ticks, defensins show great potential as targets for tick control, and display future prospect for therapeutic drug development. In the present study, a novel defensin-like gene (Ds-defensin) contributing to the antimicrobial and antioxidant capacity of the tick Dermacentor silvarum was characterized. The full-length of the Ds-defensin gene was 382 bp, which displayed tissue-specific expression and was highly abundant in the salivary glands and carcasses of the adults. It encodes a 71-amino acid defensin-like protein, and the protein precursor is characterized by a 22-amino acid signal peptide and a 34-amino acid mature peptide. The peptide displayed potent activity against most of the tested gram-positive bacteria, including Staphylococcus aureus, S. carnosus and Nocardia asteroides, and one tested gram-negative bacterium, Psychrobacter faecalis. Scanning electron microscopy revealed that the cell wall and surface of treated bacteria became rough and gradually formed pores after a 30-min exposure to the Ds-defensin peptide. Additionally, the peptide also showed significant antioxidant capacity. The above results implied that the defensin-like peptide may play an important role in tick defense and the interaction with microorganisms.

Defensins are one of the major families of antimicrobial peptides (AMPs), and have been reported from prokaryotic to eukaryotic kingdoms. But defensins are seldom found in amphibian which is a major resource of novel AMPs. A novel defensin-like AMP (defensin-TK) was isolated and characterized from skin secretions of the tree frog Theloderma kwangsiensis. The cDNA encoding defensin-TK precursor was cloned from the skin cDNA library of T. kwangsiensis. The deduced precursor of defensin-TK was composed of three domains, a signal peptide of 16 residues, a spacer peptide of 1 residues and a mature peptide of 42 residues. The mature peptide of defensin-TK shared the highest identity with the salamander (Cynops fudingensis) defensin CFBD-1. The six conserved cysteines which formed intramolecular disulfide bonds of defensins also exist in defensin-TK. Phylogenetic analysis indicated that defensin-TK was closely related to fish β-defensins. Defensin-TK showed potent and broad-spectrum antimicrobial activity. In addition, defensin-TK exerted a low hemolytic activity on human red cells. These results suggested defensin-TK might play an important role in defense the skin pathogenic microbes of tree frog T. kwangsiensis, and might be a promising candidate for development of novel antimicrobial agents.