利用比较基因组学快速鉴定大肠埃希菌噬菌体耐受基因

Rapid identification of Escherichia coli phage resistance gene by comparative genomics

噬菌体是解决致病菌多重耐药性问题的最佳选择之一,然而噬菌体的宿主特异性使得噬菌体的裂解谱有限,因此研究噬菌体宿主特异性对于噬菌体治疗应用具有重要意义。本研究通过筛选大肠埃希菌宿主菌BL21噬菌体IME253的耐受菌,结合对敏感菌和耐受菌的高通量测序,分析基因组差异,预测噬菌体耐受相关基因,发现耐受与外膜受体蛋白FepA有关。然后利用向导CRISPR/Cas9切开敏感菌fepA基因,同时利用Red同源重组系统无痕敲除fepA基因,证明fepA基因敲除可以导致细菌BL21对噬菌体IME253耐受。本实验利用比较基因组学分析平台,建立了一种宿主耐受噬菌体的分析方法,并且利用CRIPSR无痕敲除技术来验证耐受相关基因,为噬菌体治疗提供理论基础。

Bacteriophage is one of the best choices to solve the problem of multiple drug resistance of pathogenic bacteria. However, the host specificity of phage makes the phage lytic spectrum limited. Therefore, it is of great significance to study the specificity of phage hosts for phage therapy. This study screened phage IME253 resistant Escherichia coli BL21. By sequencing genomes of phage sensitive and resistant bacteria strains, fepA gene was predicted to be related with phage resistance. To prove that thefepA gene was the cause of phage resistance, thefepA gene was cut by using CRISPR/Cas9, and the Red homologous recombination system was used to knock out fepA gene seamlessly. The results demonstrated thatfepA gene knockout can lead to the resistance of bacterial BL21 to phage IME253. This experiment used the comparative genomic analysis to establish an analytical method of host resistance of bacteriophages, and used the CRIPSR technology to verify resistance related genes, which provide supports for phage therapy.