Xingjun Feng

Identification of a novel cathelicidin antimicrobial peptide from ducks and determination of its functional activity and antibacterial mechanism

The family of antimicrobial peptide, cathelicidins, which plays important roles against infections in animals, has been identified from many species. Here, we identified a novel avian cathelicidin ortholog from ducks and named dCATH. The cDNA sequence of dCATH encodes a predicted 146-amino-acid polypeptide composed of a 17-residue signal peptide, a 109-residue conserved cathelin domain and a 20-residue mature peptide. Phylogenetic analysis demonstrated that dCATH is highly divergent from other avian peptides. The α-helical structure of the peptide exerted strong antimicrobial activity against a broad range of bacteria in vitro, with most minimum inhibitory concentrations in the range of 2 to 4 μM. Moreover, dCATH also showed cytotoxicity, lysing 50% of mammalian erythrocytes in the presence or absence of 10% fetal calf serum at concentrations of 32 μM or 20 μM and killing 50% HaCaT cells at a concentration of 10 μM. The effects on bacterial outer and inner membranes, as examined by scanning electron microscope and transmission electron microscopy, indicate that dCATH kills microbial cells by increasing permeability, causing a loss of membrane integrity.

The Central Hinge Link Truncation of the Antimicrobial Peptide Fowlicidin-3 Enhances Its Cell Selectivity without Antibacterial Activity Loss

ABSTRACT Antimicrobial peptides (AMPs) have been paid considerable attention because of their broad-spectrum antimicrobial activity and a reduced possibility of the development of bacterial drug resistance. Fowlicidin-3 (Fow-3) is an identified type of chicken cathelicidin AMP that has exhibited considerable antimicrobial activity and cytotoxicity. To reduce cell toxicity and improve cell selectivity, several truncated peptides of fowlicidin-3, Fow-3(1-15), Fow-3(1-19), Fow-3(1-15-20-27), and Fow-3(20-27), were synthesized. Our results indicated that neither the N- nor C-terminal segment alone [Fow-3(1-15), Fow-3(1-19), Fow-3(20-27)] was sufficient to confer antibacterial activity. However, Fow-3(1-19) with the inclusion of the central hinge link (-AGIN-) retained substantial cell toxicity, which other analogs lost. Fow-3(1-15-20-27) displayed potent antimicrobial activity for a wide range of Gram-negative and Gram-positive bacteria and no obvious hemolytic activity or cytotoxicity. The central link region was shown to be critically important in the function of cell toxicity but was not relevant to antibacterial activity. Fow-3(1-15-20-27) maintained antibacterial activity in the presence of physiological concentrations of salts. The results from fluorescence spectroscopy, scanning electron microcopy, and transmission electron microcopy showed that Fow-3(1-15-20-27) as well as fowlicidin-3 killed bacterial cells by increasing membrane permeability and damaging the membrane envelope integrity. Fow-3(1-15-20-27) could be a promising antimicrobial agent for clinical application.

High-yield recombinant expression of the chicken antimicrobial peptide fowlicidin-2 in Escherichia coli

The antimicrobial peptide fowlicidin‐2 identified in chicken is a member of the cathelicidins family. The mature fowlicidin‐2 possesses high antibacterial efficacy and lipopolysaccharide (LPS) neutralizing activity, and also represents an excellent candidate as an antimicrobial agent. In the present study, the recombinant fowlicidin‐2 was successfully produced by Escherichia coli (E. coli) recombinant expression system. The gene encoding fowlicidin‐2 with the codon preference of E. coli was designed through codon optimization and synthesized in vitro. The gene was then ligated into the plasmid pET‐32a(+), which features fusion protein thioredoxin at the N‐terminal. The recombinant plasmid was transformed into E. coli BL21(DE3) and cultured in Luria‐Bertani (LB) medium. After isopropyl‐β‐D‐thiogalactopyranoside (IPTG) induction, the fowlicidin‐2 fusion protein was successfully expressed as inclusion bodies. The inclusion bodies were dissolved and successfully released the peptide in 70% formic acid solution containing cyanogen bromide (CNBr) in a single step. After purification by reverse‐phase high‐performance liquid chromatography (RP‐HPLC), ∼6.0 mg of fowlicidin‐2 with purity more than 97% was obtained from 1 litre of bacteria culture. The recombinant peptide exhibited high antibacterial activity against the Gram‐positive and Gram‐negative bacteria, and even drug‐resistant strains. This system could be used to rapidly and efficiently produce milligram quantities of a battery of recombinant antimicrobial peptides as well as for large‐scale production.

Deleting the first disulfide bond in a arenicin derivative enhances its expression in Pichia pastoris

The marine antimicrobial peptide NZ17074, a variant of arenicin‐3 from Arenicola marina that has broad antimicrobial activity and high bioavailability, can be designed to treat bacterial and fungal diseases. To reduce the toxicity of NZ17074, N6 was designed by replacing a cysteine in positions 3 and 20 with alanine, fused to the C‐terminus of the small ubiquitin‐like modifier tag (SUMO), and expressed in yeast. SUMO‐N6 yielded as much as 921 mg l−1 at 72 h after induction in a fermentor and increased 1·8–fold over SUMO‐NZ17074. After cleavage with 30% formic acid and purification by a Sephadex G‐25 column, 9·7 mg of the recombinant peptide N6 (rN6) was obtained from one‐litre fermentation broth, increasing 1·4−fold over NZ17074. Compared to NZ17074, rN6 displayed almost identical antimicrobial activity with a minimal inhibitory concentration of 0·5, 0·25–0·5, 4, 0·25–16 and 16 μg ml−1 against Escherichia, Salmonella, Pseudomonas, Staphylococcus and Streptococcus strains. Our results indicate that the first disulphide bond, Cys3‐Cys20, in NZ17074 is not necessary for antimicrobial activity and that its deletion might reduce toxicity to host cells. These findings may help design new antimicrobial peptides harbouring fewer disulphide bridges and may have more potent activity.