Construction of Bacillus subtilis strain engineered for expression of porcine β-defensin-2/cecropin P1 fusion antimicrobial peptides and its growth-promoting effect and antimicrobial activity

Abstract

Objective: To generate recombinant Bacillus subtilis (B. subtilis) engineered for expression of porcine β-defensin-2 (pBD-2) and cecropin P1 (CP1) fusion antimicrobial peptide and investigate their anti-bacterial activity in vitro and their growth-promoting and disease resisting activity in vivo.

Methods: The pBD-2 and CP1 fused gene was synthesized using the main codons of B. subtilis and inserted into plasmid pMK4 vector to construct their expression vector. The fusion peptide-expressing B. subtilis was constructed by transformation with the vector. The expressed fusion peptide was detected with Western blot. The antimicrobial activity of the expressed fusion peptide and the recovered pBD-2 and CP1 by enterokinase digestion in vitro was analyzed by the bacterial growth-inhibitory activity assay. To analyze the engineered B. subtilis on growth promotion and disease resistance, the weaned piglets were fed with basic diet supplemented with the recombinant B. subtilis. Then the piglets were challenged by enteropathogenic Escherichia coli (E. coli). The weight gain and diarrhea incidence of piglets were measured after challenge.

Results: The recombinant B. subtilis engineered for expression of pBD-2/CP1 fusion peptide was successfully constructed using the main codons of the B. subtilis. Both expressed pBD-2/CP1 fusion peptide and their individual peptides recovered from parental fusion peptide by enterokinase digestion possessed the antimicrobial activities to a variety of the bacteria, including gram-negative bacteria (E. coli, Salmonella typhimurium, and Haemophilus parasuis) and gram-positive bacteria (Staphylococcus aureus). Supplementing the engineered B. subtilis to the pig feed could significantly promote the piglet growth and reduced diarrhea incidence of the piglets.

Conclusion: The generated B. subtilis strain can efficiently express pBD-2/CP1 fusion antimicrobial peptide, the recovered pBD-2 and CP1 peptides possess potent antimicrobial activities to a variety of bacterial species in vitro. Supplementation of the engineered B. subtilis in pig feed obviously promote piglet growth and resistance to the colibacillosis.

Keywords: Antimicrobial Activity; Cecropin P1; Engineered Bacillus subtilis; Piglets; Porcine β-defensin-2.

Figure 1

Codon optimization and expression vector construction of pBD-2/cecropin P1 fusion gene. (A) Sequence comparison between wild-type and codon optimized pBD-2/cecropin P1 fusion gene. Opt, codon-optimized; WT, wild-type; AA, animo acids; AP leader, signal peptide of alkaline protease; ASASA, a linker; DDDDK, enterokinase site; His-tag, 6-histidine tag. (B) Prokaryotic expression vector of pBD-2/cecropin P1 fusion gene. The fusion gene (465 bp) was controlled by Lac promoter. BD-CP1, pBD-2/cecropin P1 fusion gene. (C) Restriction analysis of the pMK4-BD/CP1/His plasmid. M, DL 2000DNA Marker; Lane 1, the pMK4-BD/CP/His plasmid; Lane 2, digested fragments of the pMK4-BD/CP/His plasmid by Nde I and BamH I; Lane 3, digested fragments by BamH I and EcoR I.

Figure 2

pBD-2/cecropin P1 fusion peptide expression and some factors affecting the expression. (A) The fusion peptides in the culture media detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. M, protein markers; Lane 1, pMK4 empty vector-transformed sample; Lane 2, pMK4-pBD/CP/His vector-tramsformed sample; Lane 3, pMK4-pBD/ CP/His vector-tramsformed sample after purification with Ni-NTA-agarose beads. (B) The different fractions of the fusion peptides eluted from Ni-NTA-agarose beads. M, protein markers; Lane 1–4, elute 4-1. (C) The purified fusion peptide detected by Western blot with mouse anti-His-tag antibody and goat anti-mouse IgG-AP. M, prestained protein markers; Lane 1, sample from the culture medium of pMK-4 empty vector-transformed Bacillus subtilis (B. subtilis); Lane 2, sample from the culture medium of pMK4-pBD/CP/His vector-transformed B. subtilis. (D) The fusion peptide and its cleaved forms by enterokinase. M, protein markers; Lane 1, pBD-2/cecropin P1 fusion peptide; Lane 2, pBD-2 (6 kDa) and cecropin P1 (8 kDa) peptides generated from the fusion peptide by enterokinase digestion. Lane 3, cecropin P1 (8 kDa) isolated from pBD-2 and cecropin P1 mixture. (E) and (F), Effects of expression time and temperature on fusion peptide expression. (G) Comparison of bacterial growth between wide-type and engineered B. subtilis at 37°C for 48 h. The data are shown as means±standard deviation, and representative of three independent experiments. * p<0.05; ** p<0.01. NS, no significance.

Figure 3

Antimicrobial activities of different antibacterial peptides to the different bacteria. The different species of bacteria, including Escherichia coli (A), Salmonella typhimurium (B), Haemophilus parasuis (C) and Staphylococcus aureus (D), were used to test the antimicrobial activities of different antibacterial peptides. The optical density (OD₆₀₀) values of the bacterial cultures reflect the antimicrobial activities of the antimicrobial peptides. The data are shown as means±standard deviation, and representative of three independent experiments. * p<0.05; ** p<0.01.