金黄色葡萄球菌临床菌株III-A CRISPR-Cas 系统的功能与机制研究

Bacterial CRISPR-Cas (clustered regularly interspaced short palindromic repeats [CRISPR]-CRISPR associated proteins [Cas]) systems can act with defense ability against invading nucleotide acids. The immunity functions and mechanisms have been recognized, but other functions and relative regulatory mechanisms are largely unknown. How the CRISPR-Cas system in Staphylococcus aureus, an important human pathogen, modulates metabolism and virulence has not been reported. We have identified six S. aureus clinical strains containing III-A CRISPR-Cas system by PCR screening, and further performed whole genome sequencing and RNA-Seq analyses. Our data indicate that the sequences of cas genes and repeats in III-A CRISPR-Cas systems are highly conserved, while the spacers vary from each other, and that CRISPR-Cas system is involved in the regulation of nitrogen metabolism and virulence gene expression. In this research proposal, we will elucidate the molecular mechanisms of how the CRISPR-Cas system modulates immunity, nitrogen metabolism, and virulence gene expression in S. aureus clinical strains. This study should add our knowledge of how CRISPR-Cas system regulates physiology and pathogenicity of S. aureus, and provide novel strategy for the treatment of S. aureus infection.

细菌的CRISPR-Cas系统具有针对外源入侵核酸的防御能力，其免疫功能和机制正逐渐被解析，但免疫以外的功能及调控机制研究甚少。金黄色葡萄球菌作为重要的人类病原体，其CRISPR-Cas系统对代谢和毒力的影响还未见报道。我们经PCR筛选获得6个含有III-A CRISPR-Cas系统的金黄色葡萄球菌临床菌株，并进行全基因组测序及RNA-Seq分析。结果显示III-A CRISPR-Cas系统的cas基因和repeat序列非常保守，而spacer具有多样性；CRISPR-Cas系统还参与金黄色葡萄球菌氮代谢及毒力基因表达的调节。我们期望阐释金黄色葡萄球菌临床菌株III-A CRISPR-Cas系统行使免疫功能，以及调控金黄色葡萄球菌氮代谢和毒力基因表达的分子机制。本项研究将加深关于金黄色葡萄球菌CRISPR-Cas系统对生理调节和致病性机制的认识，并可能为金黄色葡萄球菌的感染治疗提供新策略。

本研究阐释了金黄色葡萄球菌CRISPR-Cas系统在细菌免疫及维持基因组稳定性中的功能。我们经PCR筛选获得6株含有III-A型CRISPR-Cas系统的金黄色葡萄球菌临床菌株，并进行全基因组测序及RNA-Seq分析。结果显示III-A型CRISPR-Cas系统的cas基因和repeat序列非常保守，而spacer具有多样性，进一步的研究确定了金黄色葡萄球菌CRISPR-Cas系统的免疫功能，及在此过程中靶标识别的基本特征及所必需的Cas蛋白。此外，我们还研究了MRSA临床菌株中III-A型CRISPR-Cas系统自我靶向攻击可移动遗传元件SCCmec上的抗性基因mecA对宿主基因组稳定性和耐药性的影响。基于以上研究，我们还构建了一套简单高效的金黄色葡萄球菌基因敲低系统CRISPRi，通过共表达dCas9和一段引导RNA（sgRNA）实现对目标基因的特异性敲低。上述研究拓展了对于金黄色葡萄球菌III-A型CRISPR-Cas系统的认识，相关结果发表于International Journal of Medical Microbiology, Applied and Environmental Microbiology，mSphere等期刊。本课题的进展按照原计划的进度顺利进行，完成了各年度预定的任务。