Visualization of Engineered M13 Phages Bound to Bacterial Targets by Transmission Electron Microscopy.

Yanxi Yang, Irene A Chen

Methods in molecular biology (Clifton, N.J.) 2024

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The filamentous phage M13 is one of the most well-studied and characterized phages, particularly since it was introduced as a scaffold for phage display, a technique to express and evolve fusion proteins on the M13 phage's coat to study protein or peptide binding interactions. Since phages can be engineered or evolved to specifically bind to a variety of targets, engineered M13 phages have been explored for applications such as drug delivery, biosensing, and cancer therapy, among others. Specifically, with the rising challenge of antimicrobial resistance among bacteria, chimeric M13 phages have been explored both as detection and therapeutic agents due to the flexibility in tuning target specificity. Transmission electron microscopy (TEM) is a powerful tool enabling researchers to directly visualize and characterize binding of phages to bacterial surfaces. However, the filamentous phage structure poses a challenge for this technique, as the phages have similar morphology to bacterial structures such as pili. In order to differentiate between bacterial structures and the filamentous phages, here we describe a protocol to prepare TEM samples of engineered M13 phages bound to bacterial cells, in which the phage virions have been specifically labeled by decoration of the major capsid proteins with gold nanoparticles. This protocol enables clear visualization and unambiguous identification of attached filamentous phages within the context of bacterial cells expressing numerous pili. © 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Citation

Yanxi Yang, Irene A Chen. Visualization of Engineered M13 Phages Bound to Bacterial Targets by Transmission Electron Microscopy. Methods in molecular biology (Clifton, N.J.). 2024;2793:175-183