Multidisciplinary Methods for Screening Toxic Proteins from Phages and Their Potential Molecular Targets

Area of Science:

• Microbiology
• Bioinformatics
• Structural Biology

Background:

• Phages encode numerous hypothetical proteins of unknown function (HPUFs).
• Some HPUFs may possess toxic properties, impacting bacterial hosts.
• A systematic approach is needed to identify and characterize these potentially toxic proteins.

Purpose of the Study:

• To present a comprehensive methodology for identifying and characterizing potentially toxic hypothetical proteins of unknown function (toxHPUFs) in phages.
• To provide a pipeline for analyzing toxHPUFs and their potential targets.
• To facilitate future experimental validation of identified toxHPUFs.

Main Methods:

• In vivo toxicity verification using drop tests and growth curves in bacterial hosts.
• Computational structure and function prediction using Phyre2, HHpred, and AlphaFold2.
• Comparative genomics for target identification via sequence alignment and functional annotation.
• Protein-protein interaction prediction and visualization using PyMOL.

Main Results:

• Established a multi-step methodology for toxHPUF identification and characterization.
• Integrated in vivo, in silico, and comparative genomic approaches.
• Developed a pipeline for analyzing toxHPUFs and predicting their interactions and targets.

Conclusions:

• The presented methodology offers an effective pipeline for identifying and analyzing phage-encoded toxHPUFs.
• This approach lays the foundation for future experimental studies to confirm the toxicity and function of these proteins.
• The pipeline aids in understanding phage-host interactions and potential applications in biotechnology.