Design, heterologous expression, and functional characterization of the Salmonella-specific bacteriocin SalE1a.

Abstract

Salmocin E1a (SalE1a) is a colicin-like bacteriocin with targeted antibacterial activity against Salmonella spp. Salmocins have been reported as Generally Recognized as Safe (GRAS) and approved for certain food applications, highlighting their potential for food safety interventions. In this study, the SalE1a gene was codon-optimized and heterologously expressed in Escherichia coli BL21(DE3) pLysS, yielding approximately 2.8 mg/l of soluble recombinant protein. Purified SalE1a exhibited potent and specific antibacterial activity against various Salmonella serovars without affecting probiotic bacteria and showed bacteriostatic effects on the E. coli host. MIC and MBC assays confirmed its strong anti-Salmonella efficacy. SalE1a remained active over a broad range of pH conditions and in the presence of different salts and metal ions but showed reduced activity after exposure to temperatures above 50 °C, SDS, and proteolytic enzymes. Circular dichroism analysis revealed a predominantly α-helical structure. Mechanistic studies using flow cytometry, live/dead cell imaging, and scanning electron microscopy demonstrated that SalE1a exerts its antibacterial activity primarily through membrane disruption and depolarization, accompanied by reactive oxygen species generation. Furthermore, SalE1a exhibited negligible hemolytic and cytotoxic effects toward normal human epithelial and intestinal cell lines. Overall, this study establishes heterologous production of SalE1a in an E. coli expression system and provides expanded structural, biochemical, and mechanistic insights into this Salmonella-specific bacteriocin, supporting its potential as a targeted antimicrobial for food safety applications.