# Porcine β-Defensin 2 Expressed in Pichia pastoris Alleviates Enterotoxigenic Escherichia coli-Induced Intestinal Injury and Inflammatory Response in Mice

## TL;DR

A pig-derived antimicrobial peptide, porcine β-defensin 2, shows promise in fighting intestinal infections in mice caused by a harmful E. coli strain.

## Contribution

The study demonstrates that porcine β-defensin 2 produced in Pichia pastoris is effective against ETEC-induced intestinal damage and inflammation in mice.

## Key findings

- Crude recombinant porcine β-defensin 2 showed broad-spectrum antibacterial activity against Gram-positive and -negative bacteria.
- Oral administration of porcine β-defensin 2 reduced diarrhea, intestinal damage, and inflammation in ETEC-infected mice.
- The peptide was stable under various pH, temperature, and protease conditions, making it suitable for oral delivery.

## Abstract

Porcine β-defensin 2 enhances immunity and protects the host from bacterial infection. In this study, we evaluated the in vitro antibacterial activity of crude recombinant porcine β-defensin 2 expressed in Pichia pastoris and the in vivo antibacterial activity in an enterotoxigenic Escherichia coli-induced mouse model. The results indicated that crude recombinant porcine β-defensin 2 had broad-spectrum antibacterial activity against Gram-positive and -negative bacteria. Crude recombinant porcine β-defensin 2 also showed high resistance to pH, proteases, salts, and temperatures ranging from 20 to 60 °C. In addition, the oral administration of crude recombinant porcine β-defensin 2 alleviated clinical symptoms, intestinal damage, and inflammatory response and decreased pathogen loads in stools and the colon. Our results indicate that porcine β-defensin 2 expressed in Pichia pastoris is an attractive alternative to traditional antibiotics that can be used to combat enterotoxigenic Escherichia coli-induced infection.

Enterotoxigenic Escherichia coli (ETEC), a common intestinal pathogen, can colonize the intestines and induce diarrhea in piglets, which brings great economic losses to the swine industry. Antibiotics are recommended to the treatment for diarrhea caused by ETEC in weaned piglets. However, with the emergence and spread of multidrug-resistant ETEC, there is an urgent need to develop alternatives to antibiotics. Due to the unique antibacterial mechanism of targeting bacterial membranes, antimicrobial peptides (AMPs) are promising candidates. In this study, the activity of crude recombinant porcine β-defensin 2 (rPBD2) expressed in Pichia pastoris (P. pastoris) was measured in vitro. Mice infected with ETEC were orally administered 16, 8, and 4 AU crude rPBD2 for 7 consecutive days to evaluate its anti-infective activity in vivo. The results showed that in addition to broad antibacterial activity against Gram-positive and -negative bacteria, crude rPBD2 displayed high tolerance to temperatures ranging from 20 to 60 °C, a broad range of pH, trypsin, pepsin, and physiological concentrations of salts. In an ETEC-induced mouse model, the oral administration of crude rPBD2 decreased diarrhea scores and the intestinal/carcass ratio and alleviated body weight loss. Additionally, crude rPBD2 decreased bacterial loads in stools and the colon (HP group), and the levels of serum pro-inflammatory cytokines IL-6 (HP group) and TNF-α (HP and MP groups), and increased the villus height and the ratio of villus height to crypt depth (VH/CD) in the ileum (HP and MP groups). Our study provides a cost-effective way for PBD2 production and identifies it as a promising candidate to combat ETEC-induced infection.