pilL基因在肺炎克雷伯菌ST11胃肠道定植中的作用及其机制研究

ST11 is a major epidemic clone of carbapenem-resistant Klebsiella pneumoniae in our country. The colonization of this bacteria in gastrointestinal tract is closely associted with subsequent infection. However, the key factor controbuting the colonization in the gastrointestinal tract and the mechanism is still unclear. Our previous study found that pilL gene encoding type IV-like pilus probably plays a key role in the colonization of K. pneumoniae in human gastrointestinal tract. Therefore, in this study, we intend to construct pilL gene knockout strains using K. pneumoniae ST11 strains, the differences on the bacterial activity, biofilm formation, etc were compared between the wild-type and knockout strains to analyze the biological effect of pilL gene on K. pneumoniae ST11; The effect of pilL gene on bacterial colonization ability will be further verified by cell adhesion and invasion experiments as well as in the mouse colonization experiment; On this basis, the pathological changes of gastrointestinal mucosa colonized by the bacteria will be observed, and the transcription and expression levels of inflammatory factors will be further investigated to analyze the effect and the key mechanism of pilL gene on the colonization of K. pneumoniae ST11 in the gastrointestinal tract. The implementation of this study will provide theoretical basis and new ideas for the prevention and control of carbapenem-resistant Klebsiella pneumoniae.

ST11是我国耐碳青霉烯肺炎克雷伯菌的主要流行克隆菌株，该菌在胃肠道的定植与随后的感染密切相关，然而影响该菌胃肠道定植的关键因子及机制尚不明确。申请人预实验研究发现，pilL基因编码IV型菌毛，极有可能是ST11在胃肠道定植的关键基因。因此，本研究拟利用肺炎克雷伯菌ST11菌株构建pilL基因敲除株，通过对比野生株和敲除株在细菌运动活性、生物膜形成等方面的差异，分析pilL基因在肺炎克雷伯菌ST11中的生物学作用；通过细胞粘附侵袭实验和小鼠定植实验，验证pilL基因对肺炎克雷伯菌ST11在小鼠胃肠道定植能力的影响。在此基础上，通过进一步观察细菌定植部位小鼠胃肠道粘膜的病理组织变化、检测炎症因子转录和表达水平等方法，探讨pilL基因在肺炎克雷伯菌ST11胃肠道定植的关键机制。本研究的实施可为耐碳青霉烯肺炎克雷伯菌的防控提供理论基础与新思路。

碳青霉烯耐药肺炎克雷伯菌（CRKP）在全球范围内不断出现并广泛流行，常造成难治性甚至致死性的感染，已逐渐成为威胁公共卫生安全的严重问题。ST11是CRKP的主要流行克隆菌株，其在感染宿主以前主要定植在患者的胃肠道，而且在胃肠道的定植与随后的感染密切相关，但该菌在胃肠道的定植机制尚不清楚。本项目以细菌的胃肠道定植为出发点探索CRKP-ST11感染流行的原因，前期的研究发现，pilL可能是 K. pneumoniae ST11的核心定植基因，但我们构建pilL基因敲除株后，通过对比野生株和敲除株在细菌的生长曲线、运动活性、生物膜形成能力及肠上皮细胞细胞黏附及侵袭，抗单核吞噬细胞吞噬和抗血清杀伤能力等方面的差异，没有观察到预期的结果。在后续的研究中，我们通过进一步扩大全球K. pneumoniae基因组的数据量，深入挖掘了K. pneumoniae ST11的核心定植基因，发现铁代谢的一系列相关基因（irp1、irp2、ybtT、ybtX、ybtP、ybtU、ybtQ、ybtS、ybtE、ybtA）可能为定植的关键基因。此外，我们通过使用我院CRKP的流行克隆ST11菌株和人肠上皮细胞Int407及小鼠单核巨噬细胞J774A.1的单独及联合作用，发现铁蛋白轻链的过表达可能在其定植中起着重要作用，但这些结果均需要进一步的验证。本研究的实施可为CRKP的防控提供理论基础与新思路。