Holliday Junction解离酶RuvA在分枝杆菌噬菌体抗性中的作用与分子机理

耐药分枝杆菌感染是重大临床难题。分枝杆菌噬菌体已经用于治疗耐药脓肿分枝杆菌感染患者。但是噬菌体抗性非常突出。分枝杆菌噬菌体抗性的机理缺乏研究。我们实验室在对分枝杆菌噬菌体及其与宿主分枝杆菌的相互作用10多年的研究中发现：RuvA在分枝杆菌噬菌体感染过程中持续升高，到裂解时达到峰值。这种表达模式与其他温和噬菌体或者药物处理均不同。我们假设：RuvA表达降低可能有助于产生噬菌体抗性。为此，我们将①构建RuvA缺失菌株和CRISPRi敲降分枝杆菌；②筛选野生型、缺失菌株和敲降菌株对分枝杆菌噬菌体SWU1抗性的菌株；③比较野生型、缺失菌株和敲降菌株三者噬菌体抗性菌株出现频率。④研究三种菌株产生的噬菌体抗性菌株的机理。这将揭示RuvA在分枝杆菌噬菌体抗性中的作用和机理，为治疗耐药和耐噬菌体的结核病和非结核分枝杆菌感染导致的疾病提供基础。

Multidrug resistant Mycobacterium infection is a critical clinical problem. Mycobacteriophage has been successfully applied in treating the infection of drug resistant Mycobacterium abscessus.However, resistance in mycobacteriophage is very frequent, with few mechanistic insights. During more than one decade studies in the mycobacteriophage and its interaction with Mycobacterium host, we observed a unique expression pattern elicited by Mycobacteriophage SWU1, namely persistent upregulation of RuvA, a crucial component of the Holliday Junction resolvase complex. This pattern is in sharp contrast with RuvA trends in Mycobacterium upon antibiotics or temparate phage exposure. We speculated that the lytic mycobacteriophage upregulated the RuvA to prevent the occurrence of phage resistance. To test this hypothesis, we will perform the following experiments:1.construction of RuvA deletion mutants and CRISPRi knockdown RuvA mutants;2. screen phage resistant Mycobacterium strains from different the above genetic backgrounds, as well as wild type strains;3.compare the frequency of phage resistant mycobacteria upon 3 genetic backgrounds;4.explore the molecular basis underlying the phage resistant Mycobacteria strains. The study will unveil the role of RuvA in mycobacterium phage resistance and molecular mechanisms,which will inform better design of phage therapy for mycobacteria infection, as well as the synergistic small peptides for phage therapy.