Generating STEC-Specific Ackermannviridae Bacteriophages Through Tailspike Protein Chimerization

Shiga toxin-producing <i>Escherichia coli</i> (STEC) pose a significant threat to public health and effective methods of detection are needed. The use of naturally occurring bacteriophages (phages) to detect <i>E. coli</i> has been well documented. However, detecting multiple serotypes at the same time often required multiple phages specific to individual serotypes. To limit the burden of complex cocktails, this study aimed to engineer phages with an expanded host range that allows each phage to contribute to detection across multiple STEC serogroups. <i>Kutterviruses</i>, in the <i>Ackermannviridae</i> family, contain four tailspike proteins (TSPs), each of which confers tropism to a different bacterial strain. The modular nature of TSPs allows for mixing receptor-binding domains from diverse phage types. The host range of the <i>Kuttervirus</i> CBA120 was modified by replacing its native tailspike proteins (TSPs) with chimeric versions incorporating receptor-binding domains from related and unrelated phages. A structure-guided approach was utilized to overcome minimal sequence similarity between donor and recipient phages and achieve novel functional TSP chimeras. Two engineered phage variants were created that collectively detect five STEC serogroups: O26, O45, O103, O111, and O157. Spotting and luciferase assays confirmed that the replacement TSPs were functional and the phages had acquired new host ranges. This study demonstrates the feasibility of engineering <i>Ackermannviridae</i> phages with customized host ranges for detecting multiple STEC strains. This approach has potential applications in developing improved phage-based bacterial detection, therapy, and biocontrol.