Disarming Salmonella virulence with 32H-hilE anti-virulence bacteriophage

As the development of new antibiotics struggles with the continuous emergence of drug-resistant bacteria, targeting bacterial virulence factors instead of viability has been proposed as one of the alternative approaches to combat the pathogens. Here, we genetically engineered the temperate phage SPC32H to serve as a delivery vehicle for key negative regulators of Salmonella virulence to inhibit Salmonella infection. Negative regulator genes associated with Salmonella pathogenicity island 1 (SPI1), hilE, csrA, and lrp, were inserted into the phage SPC32H genome and expressed under a strong constitutive promoter. When Salmonella cells lysogenized by the engineered phages were exposed to the virulence-inducing condition, the expression of key positive regulators and major virulence factors associated with SPI1 was significantly reduced. Furthermore, in a murine early-intervention model, oral administration of the engineered phage 32H-hilE shortly after a lethal Salmonella challenge led to a significant increase in survival. No noticeable side effects were observed in mice treated with the engineered phage alone. These results suggest the relevance of the engineered phages that suppress the Salmonella virulence network as alternative anti-Salmonella agents without resistance concerns. This proof-of-concept study of anti-virulence phages could open a new avenue for controlling pathogenic bacteria using engineered temperate phages as vectors of anti-virulence factors.