Expression of recombination antimicrobial protein PIL22-PBD-2 in Pichia pastoris and verification of its biological function in vitro

Abstract

Finding suitable alternatives to antibiotics as feed additives is challenging for the livestock industry. Porcine beta-defensin 2 (PBD-2) is an endogenous antimicrobial peptide produced by pigs. Due to its broad-spectrum antibacterial activity against various microorganisms and its low tendency for drug resistance, it is considered a potential substitute for antibiotics. Additionally, given its strong ability to repair intestinal epithelial damage and maintain intestinal mucosal barrier function, porcine interleukin-22 (PIL-22) is a potential feed additive to combat intestinal damage caused by intestinal pathogens in piglets. In this study, the amino acid sequences of PBD-2 and PIL-22 were combined to express the fusion protein in Pichia pastoris, and its biological activity was evaluated in vitro. Our results showed that the PIL22-PBD-2 exhibits broad-spectrum antibacterial activity against multidrug-resistant enterotoxigenic Escherichia coli O8 (ETEC O8), Escherichia coli (E. coli), Salmonella typhimurium, and Staphylococcus aureus (S. aureus). PIL22-PBD-2 demonstrated wound repair capability through a healing assay in the intestinal porcine epithelial cell line-J2 (IPEC-J2). Furthermore, PIL22-PBD-2 significantly enhanced the expression of the major intercellular junction-associated proteins ZO-1 and E-cadherin in IPEC-J2. It is important to note that PIL22-PBD-2 reduced intestinal epithelial cell apoptosis (p < 0.05) considerably and decreased bacterial adhesion (p < 0.05) in ETEC O8-challenged IPEC-J2. We also found that the PIL22-PBD-2 treatment attenuated ETEC O8-induced inflammatory responses in IPEC-J2 by exerting antibacterial activity, increasing the expression of endogenous antimicrobial peptides, and significantly decreasing the mRNA expression levels of IL-6 and TNF-α (p < 0.05). In conclusion, our studies demonstrate that PIL22-PBD-2 has a positive effect on inhibiting pathogenic bacteria and repairing intestinal damage.

Keywords: Antibiotic-resistant; eukaryotic expression; feed additive; intestinal repair; recombinant antimicrobial protein.

Figure 1

Construction and expression of recombinant PIL22-PBD-2 protein in P. pastoris. A Schematic diagram of recombinant proteins design. B Identification of the plasmids pPIC9k-PIL22-PBD-2 by double enzyme digestion. M: DL10000 DNA marker; Lane 1: plasmid p9k-PIL22-PBD-2 digested with EcoR I-Not I; Lane 2: p9k-PIL22-PBD-2 without EcoR I-Not I. C Colony PCR identification of recombinant P. pastoris containing PIL22-PBD-2 gene. Lane M: DL2000 DNA marker; Lane N: negative control; Lane 1 and 2: P. pastoris GS115; Lane 3 and 4: P. pastoris GS115 containing PIL22-PBD-2 gene; SDS-PAGE (D) and WB (E) analyses of PIL22-PBD-2 protein. Lane M: protein marker. Lane 1: the fermentation supernatant of PIL22-PBD-2. Lane 2: the fermentation supernatant of blank vector as negative control; Lane 3: purified PIL22-PBD-2 protein.

Figure 2

The Antibacterial effect of the PIL22-PBD-2. A Representative bacteriostasis plates of sterile water (1), kanamycin (100 μg/mL) (2), and PIL22-PBD-2 (100 μg/mL) (3) were tested for their effects on ETEC O8, E. coli ATCC25922, Salmonella typhimurium ATCC19659, and S. aureus ATCC25923. MIC of PIL22-PBD-2 on ETEC O8 (B), E. coli ATCC25922 (C), Salmonella typhimurium ATCC19659 (D) and S. aureus ATCC25923 (E) was determined. Each test was performed in duplicate and repeated three times. F The representative field of view for observing the damage caused by PIL22-PBD-2 on pathogenic bacteria was observed under TEM, and the data on bacterial damage rate were statistically analysed. Blue and green arrows are used to indicate bacterial damage. Blue arrows represent holes, blurred borders, and irregular shapes of bacteria, whereas green arrows indicate bacterial cavitation and widened gaps in the cell membrane (wall). *p < 0.05, and **p < 0.01 were compared by t-test.

Figure 3

The PIL22-PBD-2 promotes the proliferation and healing of IPEC-J2 cells. The effects of PBD-2 (A), PIL-22 (B) and PIL22-PBD-2 (C) on the proliferation of IPEC-J2 cells within 24 h. D For wound healing assay, the recombinant proteins of PBD-2, PIL22 and PIL22-PBD-2 treated IPEC-J2 in 12 h. PBS treatment was used as control. E Results of statistical analysis of the wound healing areas of IPEC-J treated by PBD-2, PIL22 and PIL22-PBD-2. Error bars indicate standard deviations. *p < 0.05 and **p < 0.01 were compared by two-way ANOVA followed by Bonferroni’s multiple comparisons test.

Figure 4

The PIL22-PBD-2 promotes the expression of intercellular junction proteins of IPEC-J2. qPCR analysis for ZO-1 (A) and E-cadherin (B). C Western blot analysis for intercellular junction protein ZO-1 and E-cadherin. D Immunofluorescence analysis for ZO-1 and E-cadherin. PBS treatment was used as control. Error bars indicate standard deviations. *p < 0.05 and **p < 0.01 were compared by two-way ANOVA followed by Bonferroni’s multiple comparisons test.

Figure 5

The effect of PIL22-PBD-2 on apoptosis in ETEC O8-infected cells was detected using flow cytometry. Frames were divided into four quadrants: Q1 represents necrotic cells; Q2 represents late-stage apoptotic cells; Q3 represents early-stage apoptotic cells; Q4 represents normal cells; Control, sterile PBS; and error bars indicate standard deviations. *p < 0.05 and **p < 0.01 were compared by two-way ANOVA followed by Bonferroni’s multiple comparisons test.

Figure 6

The PIL22-PBD-2 alleviates the effect of ETEC O8 infection on IPEC-J2. A PIL22-PBD-2 decreased ETEC O8 adhesion. Cells pre-treated with PIL22-PBD-2, PIL-22 and PBD-2 were incubated with equal numbers of ETEC O8 for 4 h, and the number of adherent bacteria (CFU) was determined. Real-time PCR analysis of PBD-1 (B) and (C) PBD-2 mRNA abundance in IPEC-J2 infected by ETEC O8. Real-time PCR analysis of IL-6 (D) and TNF-α (E) mRNA abundance in IPEC-J2 infected by ETEC O8. Error bars indicate standard deviations. *p < 0.05 and **p < 0.01 were compared by two-way ANOVA followed by Bonferroni’s multiple comparisons test.