金黄色葡萄球菌保守蛋白YeaZ对细菌生长的调控机制

YeaZ is essential for Staphylococcus aureus to grow and survive. It is a global regulator to mediate important metabolic pathways, thus is an attractive target for development of novel antibacterial agents. We found that YeaZ is able to bind its paralog Gcp and form a complex, which plays a crucial role in bacterial growth. However，the reason why YeaZ is necessary for bacterial growth remains a mystery. In the proposed studies, we hypothesize that YeaZ interacts with Gcp and coordinately regulates the expression of key target genes, which in turn contributes to bacterial survival. Our objective is to identify the mechanisms underlying the essentiality of the novel target, YeaZ, for bacterial growth in vitro using a combination of well-established genetic and advanced high throughput ChIP-seq technologies. We will identify the target genes that are directly regulated by YeaZ in S. aureus, and identify key residues on YeaZ that are necessary for bacterial growth, binding to target genes, and/or binding to Gcp. The successful completion of these studies will contribute fundamental knowledge about the essential mechanism of the novel target YeaZ for bacterial growth.

YeaZ是金黄色葡萄球菌生长的必需因子，具有全局性调控因子的调节功能，是新型抗菌药物的首选靶位。YeaZ与Gcp结合形成的YeaZ/Gcp复合物对细菌的生长起到关键作用。YeaZ是否通过与Gcp相互作用对靶基因的表达进行调控从而控制细菌生长，是非常值得深入探究的科学问题。本研究利用可控制YeaZ表达的突变菌株，通过高通量ChIP-Seq技术、DNA-蛋白质体外反应等实验筛选鉴定与YeaZ特异结合的启动区相对应的靶基因；通过靶基因启动子-lux报告基因融合系统、凝胶阻滞和DNase I足迹实验阐明YeaZ对其靶基因调控的精细分子机制；利用DNA定点突变技术和YeaZ补偿实验鉴定YeaZ关键氨基酸残基，研究其对结合靶基因启动子DNA能力，并通过细菌双杂合系统探寻YeaZ关键氨基酸残基对YeaZ/Gcp间相互作用的影响。综合分析上述研究结果，阐明YeaZ对细菌生长的调控机制。

金黄色葡萄球菌（Staphylococcus aureus）是引起人及动物感染的重要致病菌之一，多重耐药菌株的出现严重威胁全球公共卫生安全。金黄色葡萄球菌YeaZ是一个全局性调控转录因子，在细菌的多种氨基酸的生物合成、代谢和BAC转运蛋白及群体感应等通路中具有重要的调控作用。本项目对构建的IPTG诱导yeaZ表达的突变菌株进行了转录组和代谢组联合分析，揭示了YeaZ具有调控代谢途径和重要关键基因的潜在作用机制。通过对不同生长时期的样本（yeaZ_02和yeaZ02、yeaZ_05和yeaZ05、yeaZ_10和yeaZ10）进行两两比较，三组差异基因整合并集数量为681个，在多种氨基酸的生物合成、2-氧代羧酸代谢、C5-支链二元酸代谢、ABC转运蛋白、糖代谢、氮代谢及群体感应、泛酸和辅酶A的生物合成等KEGG通路显著富集。通过对差异基因进行STEM 趋势分析，选择160个关键趋势基因（上升基因56个，下降基因104个）进行KEGG通路富集分析，差异基因主要参与的通路与细菌的生长密切相关。蛋白互作网络和共表达分析，发现yeaZ基因可以显著调控37个关键基因，其中TPI、leuC、dapH、yidC 基因与yeaZ基因显著互作，主要参与次级代谢产物的生物合成、氨基酸的生物合成、糖酵解与糖代谢合成、群体感应、蛋白质外排和细菌分泌系统等通路，可以作为潜在的hub基因。不同生长时期yeaZ突变株和野生株共鉴定出468种代谢物，其中yeaZ_05和yeaZ05中存在74种代谢物显著变化，yeaZ_10和yeaZ10中存在42种代谢物显著变化，野生菌株间不同生长时期显著差异代谢物种类较少，差异代谢物主要在嘧啶代谢、赖氨酸降解、核黄素代谢、酮体合成和降解、亚油酸代谢、酪氨酸代谢和精氨酸生物合成等通路富集。通过转录组和代谢组学数据分析挖掘，揭示了YeaZ调控细菌生长的分子网络机制，为基于YeaZ研制新型抗多重耐药金黄色葡萄球菌药物提供科学依据。