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Microscopic Phage Adsorption Assay: High-throughput quantification of virus particle attachment to host bacterial
Jyot D Antani1,2,3, Timothy Ward1, Thierry Emonet3,4,5
1Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT 06520, USA.
Abstract:
Phages, viruses of bacteria, play a pivotal role in Earth's biosphere and hold great promise as therapeutic and diagnostic tools in combating infectious diseases. Attachment of phages to bacterial cells is a crucial initial step of the interaction. The classic assay to quantify the dynamics of phage attachment involves co-culturing and enumeration of bacteria and phages, which is laborious, lengthy, hence low-throughput, and only provides ensemble estimates of model-based adsorption rate constants. Here, we utilized fluorescence microscopy and particle tracking to obtain trajectories of individual virus particles interacting with cells. The trajectory durations quantified the heterogeneity in dwell time, the time that each phage spends interacting with a bacterium. The average dwell time strongly correlated with the classically-measured adsorption rate constant. We successfully applied this technique to quantify host-attachment dynamics of several phages including those targeting key bacterial pathogens. This approach should benefit the field of phage biology by providing highly quantitative, model-free readouts at single-virus resolution, helping to uncover single-virus phenomena missed by traditional measurements. Owing to significant reduction in manual effort, our method should enable rapid, high-throughput screening of a phage library against a target bacterial strain for applications such as therapy or diagnosis.
Insights
We developed a new method using fluorescence microscopy to track individual virus particles attaching to bacteria. This technique offers a faster, more detailed way to study phage-host interactions for potential therapeutic applications.
Area of Science:
- Microbiology
- Virology
- Biophysics
Background:
- Bacteriophages (phages) are crucial in microbial ecosystems and have therapeutic potential.
- Phage-bacteria attachment is a key interaction, traditionally measured with low-throughput methods.
- Existing methods for quantifying phage attachment are laborious and provide only average values.
Purpose of the Study:
- To develop a high-throughput, single-virus resolution method for quantifying phage-bacteria attachment dynamics.
- To overcome limitations of traditional ensemble-based assays.
- To enable rapid screening of phage libraries for therapeutic applications.
Main Methods:
- Utilized fluorescence microscopy and particle tracking to monitor individual phage-bacteria interactions.
- Quantified dwell times (time phages interact with bacteria) from particle trajectories.
- Correlated single-virus dwell time measurements with classical adsorption rate constants.
Main Results:
- The average dwell time measured by particle tracking strongly correlated with classical adsorption rate constants.
- Successfully quantified host-attachment dynamics for multiple phages, including those targeting pathogens.
- Demonstrated the ability to uncover single-virus phenomena missed by traditional methods.
Conclusions:
- This novel single-virus tracking method provides highly quantitative, model-free readouts of phage-bacteria interactions.
- The technique significantly reduces manual effort, enabling rapid, high-throughput screening.
- This approach advances phage biology research and facilitates the development of phage-based therapies and diagnostics.
