Identification of Toxic Proteins Encoded by Mycobacteriophage TM4 Using a Next-Generation Sequencing-Based Method.

Area of Science:

• Microbiology
• Virology
• Genetics

Background:

• Mycobacteriophages are viruses infecting mycobacteria, possessing diverse gene pools.
• Identifying mycobacteriophage genes encoding toxic proteins is crucial for understanding host-phage interactions and developing new antimicrobials.
• Drug-resistant Mycobacterium tuberculosis necessitates novel therapeutic strategies, with mycobacteriophages as potential sources of anti-TB agents.

Purpose of the Study:

• To develop and implement a high-throughput screening approach using next-generation sequencing (NGS) for identifying mycobacteriophage-encoded proteins toxic to mycobacteria.
• To identify specific genes from mycobacteriophage TM4 that encode proteins exhibiting toxicity to Mycobacterium smegmatis.
• To assess the essentiality of these toxic genes for the lytic replication of mycobacteriophage TM4.

Main Methods:

• Construction of a plasmid-derived library representing the mycobacteriophage TM4 genome.
• Transformation of the library into Mycobacterium smegmatis.
• Application of NGS and growth assays to screen for toxic gene expression and identify specific toxic proteins.

Main Results:

• The expression of TM4 phage genes encoding proteins gp43, gp77, gp78, gp79, or gp85 was found to be toxic to Mycobacterium smegmatis.
• These identified toxic genes were expressed during natural phage infection but were not essential for the lytic replication cycle of mycobacteriophage TM4.
• The NGS-based approach proved significantly faster and more resource-efficient than traditional methods for identifying toxic mycobacteriophage proteins.

Conclusions:

• A novel, efficient NGS-based screening method has been established for identifying mycobacteriophage genes encoding proteins toxic to mycobacteria.
• Several TM4 mycobacteriophage gene products were identified as toxic to M. smegmatis, representing potential candidates for novel antimicrobial drug development.
• The identified toxic genes are nonessential for TM4 lytic replication, highlighting their potential as targeted therapeutic agents without compromising phage viability.