鲍曼不动杆菌CRISPR/Cas系统的进化特征及适应性免疫功能研究

A novel and potential baterial defense systems, consist of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated sequences (Cas), is recently identified and named CRISPR/Cas system. This system can provide immunity defense against the invasions of foreign DNA, such as bacteriophages and plasmids. Nowadays, Acinetobacter baumannii has become an important pathogen causing nosocomial infections and its increasing resistance has turned into a global health problem. This study aims to analyze for the first time the evolutionary features of CRISPR/Cas system in A.baumannii isolates with different origins, compare the response of CRISPR/Cas positive and dificient strains against plasmids carrying drug resistant genes, explore the adaptive immunity of CRISPR/Cas system in inhibiting horizontal transmission of drug resistance genes and find out the potential regulatory factors in this pathway. This study initiates a brand new idea for confronting with the increasing resistance of A.baumannii.

由成簇的规律间隔的短回文重复序列（clustered regularly interspaced short palindromic repeats，CRISPR）及其相关序列（CRISPR-associated sequences，Cas）组成的CRISPR/Cas系统是近期发现的、极具潜力的、能抵抗噬菌体和质粒等外源DNA入侵的新型细菌免疫防御系统。鲍曼不动杆菌（Acinetobacter baumannii，Ab）现已成为引起医院感染的重要病原菌，其耐药性日益增加，已成为全球关注的公共卫生问题。本研究率先提出分析不同来源Ab菌株中CRISPR/Cas系统的进化特征，比较CRISPR/Cas阳性株和缺陷株对携带耐药基因的质粒的应答差异，探索CRISPR/Cas系统阻断耐药基因水平传递的适应性免疫功能，并寻找相关通路的潜在调节靶点，为应对Ab日益严峻的耐药问题开创崭新思路。

由成簇的规律间隔的短回文重复序列（clustered regularly interspaced short palindromic repeats，CRISPR）及其相关序列（CRISPR-associated sequences，Cas）组成的CRISPR/Cas系统是近期发现的、极具潜力的、能抵抗噬菌体和质粒等外源DNA入侵的新型细菌免疫防御系统。鲍曼不动杆菌（Acinetobacter baumannii，Ab）现已成为引起医院感染的重要病原菌，其耐药性日益增加，已成为全球关注的公共卫生问题。本研究收集从住院患者呼吸道分泌物、脑脊液、全血、中央静脉导管、导管血、伤口分泌物、尿液、医院污水中分离的不动杆菌225株，进行菌种鉴定、耐药表型和基因型分析以及接合性质粒筛选后，运用多位点序列分型（Multilocus sequence typing）技术完成菌株遗传进化分析找到优势克隆，然后分析其CRISPR/Cas系统的特征，运用基因敲除技术构建CRISPR/Cas缺陷株，对比敲除前后菌株对携带耐药基因接合性质粒的应答差异和蛋白组学差异。结果发现：Ab分离株占不动杆菌分离株的95.5%，其亚胺培南耐药性与ISAba1-blaOXA-23的存在有关；含有ISAba1-blaOXA-23的接合性质粒pAbhx1能将亚胺培南耐药性水平传递到敏感株中；215株Ab菌株分为20种序列型（sequence type，ST），其中16种ST归于国际流行的克隆复合体92 (clonal complex 92，CC92)；选取16株不同ST菌株进行CRISPR/Cas分析，结果显示并不是所有菌株都具有CRISPR/Cas系统，阳性菌株中CRISPR序列长度和spacer也不同，提示不同来源菌株的遗传环境多样性；通过自杀质粒pCVD442敲除BAA1710菌株的CRISPR序列构建CRISPR/Cas缺陷株BAA1710/ΔCRISPR，BAA1710/ΔCRISPR的质粒干扰突变株未能抑制接合性质粒pAbhx1的水平传递，CRISPR/Cas阳性株、缺陷株及突变株的比较蛋白组学分析数据复杂，近期将进一步分析完善并在追加成果中呈现。本研究了解了Ab分离株中CRISPR/Cas特征，并初步探索了该系统阻断耐药基因水平传递的适应性免疫功能，为应对Ab日益严峻的耐药问题开创崭新思路。