CRISPR-Cas系统和宿主调控IncFII质粒接合的相关因子在产KPC酶肺炎克雷伯菌大规模流行中的作用和机制研究

The blaKPC, the most prevalence carbapenemase gene in Klebsiella pneumoniae, become one of the most disturbing problems for clinical anti-infective therapy. K. pneumoniae clonal complex (CC) 258 is the most notorious lineage mediating the global dissemination of blaKPC, and IncFII account for majority of plasmids associated with blaKPC. However, the mechanism behind this phenomenon is still unclear. Based on the previous findings that all IncFII blaKPC-haboring (IncFII-blaKPC) plasmids possess protospacer corresponding to CRISPR-Cas system in K. pneumoniae while CRISPR-Cas system only exists in non-CC258 isolates, and that several host factors regulating plasmid conjugation in CC258 are apparently different with that in non-CC258, we proposed hypothesis that the interaction between host factors and IncFII plasmid is the key factor leading to the global dissemination of CC258 with blaKPC; additionally, CRISPR-Cas systems interfering the uptake and survival of IncFII-blaKPC plasmids within non-CC258 and promoting dissemination in CC258 also contribute in blaKPC spreading in CC258. We will concentrate on the interaction between host factors and IncFII plasmid and the function of CRISPR-Cas system to study the mechanism of CC258 dissemination, providing scientific evidence and data for establishment of effective control system to prevent hospital infection.

blaKPC是肺炎克雷伯菌中流行最广的碳青霉烯酶基因，是困扰当前抗感染治疗最严重的耐药基因之一。产KPC酶肺炎克雷伯菌的大规模流行仅见于CC258型，IncFII质粒是最常见blaKPC播散质粒，但播撒机制不明。本项目组基于前期发现：携blaKPC的IncFII质粒含多种能被细菌适应性免疫系统CRISPR-Cas识别的Protospacer；该系统在CC258中缺失；CC258调控IncFII质粒与宿主接合的因子与非CC258显著不同，提出：CC258调控因子与IncFII质粒相互作用，是携blaKPC的CC258广泛播撒的主因；CRISPR-Cas限制IncFII-blaKPC质粒进入含此系统的宿主，促使其向无该系统的CC258传播，是辅因。本项目拟从CC258调控IncF质粒接合因子及CRISPR-Cas两方面入手，研究CC258广泛播散的机制，为建立有效的院感控制措施提供理论基础。

blaKPC 介导的 KPC-KP在全球形成了长时间、大范围、 高阳性率的传播，成为临床抗感染治疗的严峻挑战。众多研究均发现，blaKPC 多位于高移动性 IncFII（Incompatibility group FII）型质粒（包括 IncFIIK、IncFIIY、 IncFIIS、IncFII 等）上。肺炎克雷伯菌所携带的 CRISPR系统，是该细菌中常见的免疫系统，可通过对外来入侵核酸进行剪切来阻止其对自身基因组的干扰，本研究发现 KPC-KP 大多是“免疫缺陷”型菌株，缺乏 CRISPR系统，且 blaKPC 基因的流行分布与CRISPER系统呈现明显负相关，通过一系列功能实验和表型实验探究，本研究进一步发现 IncFII 型 blaKPC 质粒是肺炎克雷伯菌所携带的 CRISPR 系统的良好靶标，该系统在影响该质粒传播和宿主选择中发挥了重要作用。最后，本研究深度分析了肺炎克雷伯菌所携带 的 IncFII 型高危质粒的结构和序列特点，并构建了携带 CRISPR 系统的可移动性工具小质粒来靶向 IncFII 质粒。