Generating STEC-Specific Ackermannviridae Bacteriophages Through Tailspike Protein Chimerization.

Area of Science:

• Microbiology and Virology
• Bacteriophage Engineering
• Food Safety and Public Health

Background:

• Shiga toxin-producing Escherichia coli (STEC) represent a significant public health concern, necessitating advanced detection methods.
• Current phage-based detection often requires multiple phages for individual STEC serotypes, leading to complex cocktails.
• Bacteriophages, particularly Kutterviruses within the Ackermannviridae family, possess tailspike proteins (TSPs) that determine bacterial host specificity.

Purpose of the Study:

• To engineer Kutterviruses with expanded host ranges for simultaneous detection of multiple STEC serogroups.
• To overcome limitations of complex phage cocktails by creating single phages capable of targeting diverse STEC strains.
• To demonstrate the feasibility of modifying phage TSPs for customized bacterial detection.

Main Methods:

• Engineered the Kuttervirus CBA120 by replacing native tailspike proteins (TSPs) with chimeric versions.
• Utilized a structure-guided approach to create functional TSP chimeras from diverse phage types, overcoming sequence dissimilarities.
• Assessed the host range expansion and functionality of engineered phages using spotting and luciferase assays.

Main Results:

• Successfully created two engineered phage variants with collectively expanded host ranges.
• The engineered phages collectively detect five key STEC serogroups: O26, O45, O103, O111, and O157.
• Confirmed functionality of the replacement TSPs and demonstrated successful acquisition of new host ranges.

Conclusions:

• Demonstrated the feasibility of engineering Ackermannviridae phages with customized host ranges for multi-serotype STEC detection.
• This approach offers a promising strategy for developing improved phage-based detection systems.
• Potential applications include enhanced bacterial detection, phage therapy, and biocontrol strategies against STEC.