Harding Kate R, Malone Lucia M, Kyte Natalie A P, Jackson Simon A, Smith Leah M, Fineran Peter C
Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9054, New Zealand.
Genetics Otago, University of Otago, PO Box 56, Dunedin 9054, New Zealand.
Nucleic Acids Res. 2025 Jan 24;53(3). doi: 10.1093/nar/gkae1194.
The Chimalliviridae family of bacteriophages (phages) form a proteinaceous nucleus-like structure during infection of their bacterial hosts. This phage 'nucleus' compartmentalises phage DNA replication and transcription, and shields the phage genome from DNA-targeting defence systems such as CRISPR-Cas and restriction-modification. Their insensitivity to DNA-targeting defences makes nucleus-forming jumbo phages attractive for phage therapy. However, little is known about the bacterial gene requirements during the infectious cycle of nucleus-forming phages or how phage resistance may emerge. To address this, we used the Serratia nucleus-forming jumbo phage PCH45 and exploited a combination of high-throughput transposon mutagenesis and deep sequencing (Tn-seq), and CRISPR interference (CRISPRi). We identified over 90 host genes involved in nucleus-forming phage infection, the majority of which were either involved in the biosynthesis of the primary receptor, flagella, or influenced swimming motility. In addition, the bacterial outer membrane lipopolysaccharide contributed to PCH45 adsorption. Other unrelated Serratia-flagellotropic phages used similar host genes as the nucleus-forming phage, indicating that phage resistance can lead to cross-resistance against diverse phages. Our findings demonstrate that resistance to nucleus-forming jumbo phages can readily emerge via bacterial surface receptor mutation and this should be a major factor when designing strategies for their use in phage therapy.
噬菌体的奇马利病毒科（Chimalliviridae）在感染其细菌宿主的过程中会形成一种类似蛋白质核的结构。这种噬菌体“核”将噬菌体DNA复制和转录分隔开来，并保护噬菌体基因组免受诸如CRISPR-Cas和限制修饰等靶向DNA的防御系统的影响。它们对靶向DNA防御的不敏感性使得形成核的巨型噬菌体在噬菌体治疗中具有吸引力。然而，对于形成核的噬菌体感染周期中的细菌基因需求或噬菌体抗性如何产生，我们知之甚少。为了解决这个问题，我们使用了粘质沙雷氏菌形成核的巨型噬菌体PCH45，并采用了高通量转座子诱变和深度测序（Tn-seq）以及CRISPR干扰（CRISPRi）相结合的方法。我们鉴定出了90多个参与形成核的噬菌体感染的宿主基因，其中大多数基因要么参与主要受体鞭毛的生物合成，要么影响游动性。此外，细菌外膜脂多糖有助于PCH45的吸附。其他不相关的嗜粘质沙雷氏菌鞭毛噬菌体使用与形成核的噬菌体类似的宿主基因，这表明噬菌体抗性可导致对多种噬菌体的交叉抗性。我们的研究结果表明，通过细菌表面受体突变很容易产生对形成核的巨型噬菌体的抗性，而这在设计将其用于噬菌体治疗的策略时应是一个主要因素。
