替加环素触发的肺炎克雷伯菌sRNA差异表达及其参与耐药调控作用

Tigecycline is a novel glycylcycline group of antibiotics, and was approved for treating serious infections in 2005. But Klebsiella pneumoniae and other clinical strains were reduced susceptibility to tigecycline in the world. Although tigecycline resistance results from regulation of transcriptional regulators that causes the overexpression of multidrug efflux system, but their resistance mechanism and regulatory pathway are unclear. Despite mounting evidence for the importance of post-transcriptional network constituted by bacterial small RNA (sRNA) in stress response and cascades regulation, but their response and biological effects upon antibiotic exposure remains unknown. For the purpose of uncovering the change of sRNA profiles triggered by tigecycline and their contributions in the resistance, ①sRNA expression profiles related to tigecycline resistance would be determined by sRNA high-throughput sequencing; ②sRNA changes in clinical isolates of Klebsiella pneumoniae would also be verified; ③function and contributions to tigecycline resistance of the sRNA would be studied in mutant strains with target sRNA deleted and complemented. This study would explore the novel antimicrobial targets to provide new strategy for preventing antibiotics resistance.

替加环素是2005年初批准使用的新型甘氨酰环素类抗生素，用于治疗复杂性细菌感染。但上市不久，在世界范围内，肺炎克雷伯菌等临床菌株对其敏感性呈不同程度下降。尽管已发现替加环素不敏感菌株基因组外排系统表达升高，并受相关转录调控因子调节，但其耐药机制及调控通路仍不清。已知由sRNA组成的细菌转录后调控网络，在感受环境压力、启动调控级联中发挥重要作用。但细菌在抗生素压力下sRNA应答机制及其生物学作用研究甚少。为揭示细菌在替加环素触发下sRNA的表达变化及参与耐药调控作用，本项目拟通过：①小RNA高通量测序技术筛选与肺炎克雷伯菌替加环素耐药相关的sRNA类别；②在临床分离菌株中验证靶标sRNA的表达水平，及其与细菌耐药性的关联；③构建靶sRNA基因缺失突变株及基因缺失回补株，进一步分析药物触发下靶标sRNA在细菌耐药形成中的作用与机制，以阐明新型抗菌药物作用的潜在靶点，为防控抗生素耐药提供新策略。

替加环素（Tigecycline）是首个被美国FDA批准使用的甘氨酰环素类抗生素，用来治疗腹腔内感染、皮肤和皮肤结构感染以及社区获得性肺炎等。目前在世界范围内的肺炎克雷伯菌等临床菌株，对替加环素的敏感性有不同程度的下降，给临床控制复杂性感染制造了难题。由sRNA（small regulatory RNA，sRNA）组成的细菌转录后水平调控网络，对感受环境压力、表达细菌毒力基因等具有重要的调控作用。最新研究发现的几种与肠杆菌科细菌耐药性相关的sRNA，其中有些与细菌的多药外排系统及整体调控网络相关联。但肺炎克雷伯菌中仅有少量被鉴定且序列明确的sRNA，并且对其耐药功能和调控网络研究并不清晰。. 本项目通过转录组高通量测序技术，检测和分析了替加环素触发前后肺炎克雷伯菌ATCC13883的sRNA表达水平变化，结合生物信息学分析手段，初步筛选到候选sRNA共972条，其中差异表达的sRNA为61条，与细菌抗生素应答相关的sRNA为4条；进而在临床收集的214株肺炎克雷伯菌中检测到13株替加环素不敏感株，其外排泵acrB及转录调控因子ramA表达水平升高，分别为对照株的3.03~57.98倍和1.18~35.62倍，筛选的4条sRNA在临床替加环素敏感株和耐药株中存在差异表达现象；另外，构建1株靶标sRNA基因缺失突变株，为进一步分析突变株的生长曲线、药物敏感性及耐药基因表达水平，分析靶sRNA在替加环素耐药中作用奠定实验基础。. 本研究探索了sRNA表达与肺炎克雷伯菌替加环素耐药水平增强的关系，以肺炎克雷伯菌为生物学模型，初步分析小RNA耐药调控网络关键节点及生物学作用，为防止新型抗菌药物耐药提供理论依据。