细菌耐受噬菌体的多样性机理研究

Antibiotic therapy is obviously the most important weapon against bacterial infections for human bing. However, antibiotic resistance is becoming very rigorous problem as antibiotics are widely used in clinic practice. Almost no antibiotics can be chosed for treatment for some bacterial infections caused by superbugs or multi-drug resisitant pathogens. So, humans have to keep seeking for other new weapons against bacterial infections. Bacteriophages are viruses of bacteria and phage therapy is potentially application perspective. But it can be imagined that phage resistance of bacteria will be another tough problem as phage therapy is widely used in practice. Therefore, prospective investigations of phage resistance of bacteria are obviously significant. We had reported that phage receptor of Pseudomonas aeruginosa is chemically LPS and P. aeruginosa can become resistant to phage PaP1 infection by deleting a big DNA fragment (219.6 Kb) and hence losing the gene galU which is a key gene for LPS synthesis. We call this mechanism of bacterium resistance to phage as “cutting an arm for survival”. Based on above mentions, this project is focus on investigation of alterative mechanism of another phage-resistant mutant PA1ar which has been confirmed no deletion of big DNA fragment and no loss of gene galU. Obviously, this phage resistant mutant must have different resistant mechanism. The purpose of the project is trying to validate deletion or mutation of what gene among many genes related to LPS synthesis pathways will cause loss of LPS and phage receptor of host bacteria. For different strain of host bacteria, although deletion or mutation of different gene probably occur but all lead to same consequence, that is resistance of host bacteria to phages. Completion of this project will lay theory foundation for phage therapy. Although using P.aeruginosa as research object, completion of this project is also significant reference to study of phage resistant mechanism for other different bacteria.

人类抗微生物感染的主要武器是抗生素。但随着抗生素的广泛应用，细菌耐药性成为了严峻挑战。人类迫切需要寻找更多有效的抗感染手段。噬菌体是细菌的病毒，有望成为又一种抗感染手段。但可以想象，随着噬菌体治疗在实践中的应用，噬菌体耐受又会成为严峻问题。及早研究噬菌体耐受有着极为重要的意义。我们已经发现铜绿假单胞菌PA1的噬菌体受体为LPS；细菌可通过丢失大片段DNA,从而丢失噬菌体受体这样一种“断臂求生”机制建立噬菌体耐受。本项目旨在探讨另一种没有DNA大片段缺失的菌株PA1ar耐受噬菌体的另样机制，全面考察众多LPS合成相关基因中，哪些基因的缺失或突变有可能导致细菌LPS变异，进而导致噬菌体耐受。勾画出细菌经过不同基因缺失或突变，殊途同归，最终导致LPS及其受体改变，建立噬菌体耐受的新机制，为未来的噬菌体治疗奠定理论基础。项目虽以铜绿假单胞菌为研究对象，但对于探讨其他细菌的噬菌体耐受具有借鉴意义。

本项目的研究目标是探讨“铜绿假单胞菌耐受噬菌体感染的多样性耐受机制”。当初的目标聚焦在研究“白色耐受菌落”（非大片段丢失菌株）的耐受机制。实际执行中，我们通过单分子测序技术发现了耐受菌株发生了8个点突变，进而确定了突变株基因组1794539位置处的wzy 基因中发生了C→T的突变，使得原密码子CAG突变为TAG（成为终止密码），导致该基因翻译提前终止，使Wzy蛋白成为一个“截短蛋白”(氨基酸序列由417个变为198个）。Wzy蛋白为LPS的O-抗原聚合酶，它变为截短蛋白后导致LPS合成被阻断，从而使得菌株的噬菌体结合受体缺失，故变得对噬菌体感染耐受。该成果发表于Front Microbiol, 2018, 9: 1170（IF=4.019）。.我们在完成了阐明“白色耐受菌落的耐受机制”的同时，还超额完成了“棕色耐受菌落”丢失大片段的分子机制：通过MutL介导了DNA的双链断裂，随后被非同源末端连接关键酶Ku和LigD修复，从而导致基因组的大片段缺失。该研究不仅完整揭示了细菌通过丢失基因组片段从而耐受噬菌体的全新机制，更颠覆了人们对MutL功能的认识：MutL是错配修复系统的关键酶，具有DNA单链剪切功能，被认为是维持基因组稳定的因素。但是，该研究意外的发现，MutL还可以导致DNA双链的断裂和基因组大片段丢失。这一发现使得科研人员将重新审视细菌MutL的生理功能及其在进化中的作用。该成果于2018年发表于国际权威学术期刊Nucleic Acids Res, 2018, 46(9): 4505-4514（IF=10.162）。.此外，利用剩余经费，我们还鉴定了一个可水平转移的缺陷型前噬菌体pp3（发表于PLoS One, 2017, 12(3): e0174429. IF= 2.766）；还完成了HicAB毒素-抗毒素系统的鉴定及功能研究（发表于Toxins, 2016, 8(4): 113-124. IF=3.03）。