Recombinant Enterococcus faecium expressing porcine lactoferricin exerts bactericidal effects and protects against enterotoxigenic Escherichia coli in mice

Porcine lactoferricin functions as an antibacterial peptide as well as an antiviral, antifungal, tumor-growth inhibiting, and immune-enhancing agent. Herein, the optimized and non-optimized amino-acid coding sequences for porcine lactoferricin were cloned into pNZ8112 expression vector, generating the recombinant plasmids pNZ8112-pLFcin (pNZ8112 cin) and pNZ8112-pLFcin' (pNZ8112 cin'), respectively. The plasmids were then electroporated into Enterococcus faecium cells, resulting in recombinant strains pNZ8112 cin/E. faecium and pNZ8112 cin'/E. faecium, respectively. Subsequent studies revealed that the recombinant strains exerted antibacterial effects against 12 types of pathogenic bacteria, with strongest activity against Staphylococcus aureus. The studies involving oral immunization of 4-6 weeks old female BALB/c mice with the two recombinant strains revealed an increased average daily weight gain, feed intake, and level of lactic acid bacteria and decreased feed to-weight ratio. Furthermore, these effects were concomitant with a significant increase in the level of serum IgG, intestinal mucus sIgA, and interleukin-2 in immunized mice as compared to those in non-immunized mice. Moreover, the challenge experiment indicated that pre-immunization with the two strains could provide 83.3% protection against enterotoxigenic Escherichia coli in mice, which implicates the potential application of the recombinant E. faecium strains in the development of antibiotic-free domestic livestock feed additives.