金黄色葡萄球菌转录因子NWMN_0037调节持留菌形成、毒力和代谢的机制研究

Staphylococcus aureus is a prominent human pathogen and responses for common bacterial pyogenic infections and nosocomial infections. The virulence factors and persisiter formation of S. aureus bear the primary responsibility of chronic and recurrent infections. Our pre-project study had obtained a NWMN_0037 mutant by screening S. aureus Newman strain mutant library using ampicillin. The mutant was more sensitive to antibiotics and various external pressures, and had higher LD50 to BALB/c mice than did the wild type strain. The pathogenicity measured by mice subcutaneous pyogenic test of the NWMN_0037 mutant was severely weakened than in the wild type strain. These defects were restored after complementation. RNAseq discovered that the transcription level of the NWMN_0037 mutant partial virulence genes were down-regulated while partial metabolisms related genes were up-regulated against wild type strain. Although NWMN_0037 protein is the transcription factor of S. aureus Newman strain, the mechanism of action is still unclear. In this project, sobaric tag for relative and absolute quantitation (iTRAQ) labeled technique for proteomics research will be used to explore the impacts of NWMN_0037 transcription factor on regulating the protein of S. aureus Newman strain expression. ELISA will be used to detect the membrane damage toxins expression difference between the NWMN_0037 mutant and wild type strain. Liquid chromatography-mass spectrum (LC-MS) method for metabonomics research will be used to analyze the metabolic products difference between the NWMN_0037 mutant and wild type strain. The monoclonal antibody (McAb) of NWMN_0037 protein will be obtained by hybridoma technique and the McAb will be used in chromatin immunoprecipitation-sequencing (ChIP-seq). The subsequent sequence against the enriched nucleic acid fragments will help to explore sites of NWMN_0037 transcription factor action on S. aureus Newman strain DNA. Bioinformatics will be used to analyze the pathways of NWMN_0037 transcription factor function. The key genes included in the pathways will be knocked out and the variation of the bacterial virulence and the ability of persister formation will be verified. These works will help us to explore the molecular mechanisms of NWMN_0037 transcription factor on regulating S. aureus Newman strain persister formation, virulence, and metabolism and to find effective drug targets to defend S. aureus persister formation and reduce bacterial virulence.

金黄色葡萄球菌为引起化脓性感染和院内感染的重要致病菌，毒力因子和持留菌形成影响其感染。NWMN_0037为金葡菌未知功能转录因子。项目组前期筛选金葡菌Newman株突变文库得到不易形成持留菌的NWMN_0037突变株，表现为对抗生素和外界压力敏感性增高，LD50增大，抗吞噬和致小鼠皮肤化脓能力减弱，基因互补后恢复野生株特性。转录组测序显示突变株部分毒力基因下调，代谢基因上调。本项目拟通过蛋白组学iTRAQ标记法探讨NWMN_0037对金葡菌蛋白表达影响；ELISA法测定突变株和野生株膜损伤毒素表达差异；代谢组学LC-MS法研究代谢产物差异；制备单克隆抗体，用ChIP-seq探索NWMN_0037与DNA作用位点；生物信息学分析NWMN_0037作用通路；敲除相关基因并验证毒力和持留菌变化。探讨金葡菌转录因子NWMN_0037调节持留菌形成、毒力及代谢机制，寻找金葡菌减毒和抗持留菌防治靶点。

金黄色葡萄球菌（金葡菌）NWMN_0037突变后显著影响其毒力和持留菌形成，本研究探讨调控机制。采用同源建模预测金葡菌NWMN_0037有两个结构域和一活性中心。NWMN_0037与hla、hlg、crt启动子区结合，上调hla、hlgB、crtI、crtM和crtN表达；NWMN_0037突变后可导致hla、hlgA、hlgB、hlgC、lukF、lukS和lukD表达下调，抑制金葡菌毒力。NWMN_0037可抑制金葡菌糖酵解和三羧酸循环；促进磷酸二羟基丙酮磷酸酯积累；可调控磷酸胆碱和甘油磷酸胆碱合成而影响细胞膜合成；调控L-色氨酸、L-精氨酸、L-谷氨酸等多种氨基酸以及肽聚糖合成；促进黄素合成发挥抗氧化作用。通过上述机制调控细菌的代谢包括碳水化合物代谢、蛋白质和氨基酸代谢、能量代谢、脂质代谢、核苷酸代谢等而调控持留菌形成。1:1000稀释的金葡菌在培养5小时后开始大量形成持留菌，细菌代谢、合成、核糖体、信号转导及膜转运等通路参与形成过程；purN敲除后影响金葡菌持留菌形成和毒力。小檗碱在中性环境中具有抗金葡菌持留菌作用。诱导建立小檗碱金葡菌耐药株，基因组测序证实有41个单核苷酸多态性位点突变和11个小片段插入和缺失序列，分布在膜蛋白、离子运输、药物外排泵、细胞分裂、DNA损伤修复、RNA代谢、脂质代谢、糖代谢、氨基酸代谢等通路。金葡菌L-型表面不规则且硬度增加；“油煎蛋样”菌落的核心嵌入琼脂生长，且核心及周围细菌均具有形成L-型的能力；L-型携带zeta电位低，与聚集生长有关。起泡、出芽及内生囊泡是L-型增殖的主要形式；在高渗环境下，金葡菌L-型的溶血能力高于野生型；L-型可致小鼠皮肤化脓性感染。能量代谢、压力响应、蛋白质合成、RNA代谢和毒力因子表达等在金葡菌L-型形成中有重要作用。荧光标记有助于金葡菌L-型菌落观察和毒力研究。项目发表论文4篇，获专利1项，培养硕士生9名。