Dissecting how phages modulate bacterial immunity - structural basis of dNTPase inhibition by T7-like phages

噬菌体是可以感染和杀死细菌的病毒。针对多药耐药细菌的噬菌体疗法（目前在临床试验中）利用了这一特性。然而，细菌已经进化出了自我防御机制。CRISPR-Cas通常用作基因编辑的工具，形成“适应性”免疫系统，而其他细菌蛋白质如dNTR酶形成“先天性”免疫系统。细菌的dNTPs，这个建议的焦点，通过耗尽噬菌体复制所需的DNA构建块（dNTPs）的供应来阻断噬菌体复制。这种重要的机制在脊椎动物中也是保守的，人类dNTR同源物SAMHD 1限制了致病病毒如艾滋病病原体HIV的复制。由于dNTPases在细菌和人类先天免疫中的重要性，感染人类的病毒和病原体已经进化出它们自己对宿主dNTPases的“反击”。T7样噬菌体是噬菌体治疗中使用的一个超家族，它产生一种称为gp1.2的蛋白质，抑制dNTPs以增加dNTPs的供应，使噬菌体能够复制其DNA基因组。然而，虽然所有的细菌都编码dNTR，但并不是所有的细菌都进化出dNTPase-targeting gp1.2蛋白。该提案旨在提高噬菌体疗法对人类机会致病菌铜绿假单胞菌（P. aeruginosa）的疗效。首先，该项目旨在使用X射线晶体学，冷冻电子显微镜（cryoEM）和互补的生物物理方法来研究铜绿假单胞菌dNTR如何被T7样噬菌体gp1.2蛋白抑制。其次，将使用结构指导的方法突变gp1.2氨基酸序列以提高其沉默铜绿假单胞菌dNTR的能力。总之，我们将在分子水平上确定T7样噬菌体gp1.2蛋白如何关闭铜绿假单胞菌先天免疫蛋白dNTR。这可以提高未来噬菌体疗法对这种细菌和其他人类病原体的疗效。

Bacteriophages are viruses that can infect and kill bacteria. Phage Therapy against multi-drug resistant bacteria (currently in clinical trials) exploits this property. However, bacteria have evolved self-defence mechanisms. CRISPR-Cas, which is routinely used as a tool for gene editing, forms the "adaptive" immune system, while other bacterial proteins such as the dNTPase enzyme form the "innate" immune system. The bacterial dNTPase, the focus of this proposal, blocks phage replication by depleting the supply of DNA building blocks (dNTPs) that the phage needs to replicate. This important mechanism is also conserved in vertebrates, with the human dNTPase homologue, SAMHD1, restricting the replication of pathogenic viruses such as HIV, the causative agent of AIDS. Due to the importance of dNTPases in bacterial and human innate immunity, human-infecting viruses and phages have evolved their own "counter-attack" against the host dNTPase. T7-like phages, one superfamily used in Phage Therapy, produce a protein called gp1.2 that inhibits the dNTPase to increase the supply of dNTPs to enable phages to replicate their DNA genome. However, while all bacteria encode a dNTPase, not all phages have evolved a dNTPase-targeting gp1.2 protein. This proposal aims to improve the efficacy of Phage Therapy against the opportunistic human pathogen Pseudomonas aeruginosa (P. aeruginosa). First, the project aims to characterise how P. aeruginosa dNTPase is inhibited by T7-like phage gp1.2 protein using X-ray crystallography, cryo-electron microscopy (cryoEM) and complementary biophysical methods. Secondly, a structure-guided approach will be used to mutate gp1.2 amino acid sequence to improve its ability to silence the P. aeruginosa dNTPase. Overall, we will determine at the molecular level how the T7-like phage gp1.2 protein shuts down P. aeruginosa innate immunity protein dNTPase. This could improve the efficacy of future Phage Therapy against this bacterium and other human pathogens.