MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, and Center for Tuberculosis Research, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
Laboratory of Molecular Biology, Beijing Key Laboratory for Drug Resistance Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing, China.
Microbiol Spectr. 2023 Jun 15;11(3):e0501522. doi: 10.1128/spectrum.05015-22. Epub 2023 May 8.
Mycobacteriophages are viruses that specifically infect mycobacteria and which, due to their diversity, represent a large gene pool. Characterization of the function of these genes should provide useful insights into host-phage interactions. Here, we describe a next-generation sequencing (NGS)-based, high-throughput screening approach for the identification of mycobacteriophage-encoded proteins that are toxic to mycobacteria. A plasmid-derived library representing the mycobacteriophage TM4 genome was constructed and transformed into Mycobacterium smegmatis. NGS and growth assays showed that the expression of TM4 gp43, gp77, -78, and -79, or gp85 was toxic to M. smegmatis. Although the genes associated with bacterial toxicity were expressed during phage infection, they were not required for lytic replication of mycobacteriophage TM4. In conclusion, we describe here an NGS-based approach which required significantly less time and resources than traditional methods and allowed the identification of novel mycobacteriophage gene products that are toxic to mycobacteria. The wide spread of drug-resistant Mycobacterium tuberculosis has brought an urgent need for new drug development. Mycobacteriophages are natural killers of M. tuberculosis, and their toxic gene products might provide potential anti-M. tuberculosis candidates. However, the enormous genetic diversity of mycobacteriophages poses challenges for the identification of these genes. Here, we used a simple and convenient screening method, based on next-generation sequencing, to identify mycobacteriophage genes encoding toxic products for mycobacteria. Using this approach, we screened and validated several toxic products encoded by mycobacteriophage TM4. In addition, we also found that the genes encoding these toxic products are nonessential for lytic replication of TM4. Our work describes a promising method for the identification of phage genes that encode proteins that are toxic to mycobacteria and which might facilitate the identification of novel antimicrobial molecules.
分枝杆菌噬菌体是专门感染分枝杆菌的病毒，由于其多样性，它们代表了一个庞大的基因库。这些基因功能的表征应该能深入了解宿主-噬菌体相互作用。在这里，我们描述了一种基于下一代测序（NGS）的高通量筛选方法，用于鉴定对分枝杆菌有毒的分枝杆菌噬菌体编码蛋白。构建了一个代表分枝杆菌噬菌体 TM4 基因组的质粒衍生文库，并转化到耻垢分枝杆菌中。NGS 和生长测定表明，TM4 gp43、gp77、-78 和-79 或 gp85 的表达对耻垢分枝杆菌有毒。尽管与细菌毒性相关的基因在噬菌体感染期间表达，但它们不是分枝杆菌噬菌体 TM4 裂解复制所必需的。总之，我们在这里描述了一种基于 NGS 的方法，与传统方法相比，该方法需要的时间和资源要少得多，并允许鉴定对分枝杆菌有毒的新型分枝杆菌噬菌体基因产物。广泛传播的耐多药结核分枝杆菌迫切需要新的药物开发。分枝杆菌噬菌体是结核分枝杆菌的天然杀伤者，其毒性基因产物可能为抗结核分枝杆菌提供潜在的候选药物。然而，分枝杆菌噬菌体巨大的遗传多样性给这些基因的鉴定带来了挑战。在这里，我们使用了一种简单方便的基于下一代测序的筛选方法，来鉴定编码对分枝杆菌有毒的产物的分枝杆菌噬菌体基因。使用这种方法，我们筛选并验证了分枝杆菌噬菌体 TM4 编码的几种毒性产物。此外，我们还发现编码这些毒性产物的基因对 TM4 的裂解复制不是必需的。我们的工作描述了一种很有前途的方法，可以鉴定编码对分枝杆菌有毒的蛋白的噬菌体基因，这可能有助于鉴定新的抗菌分子。
