Liu Yanhua, Liu Qian, Qi Linlu, Ding Tao, Wang Zhen, Fu Jiaqi, Hu Mo, Li Min, Song Jeongmin, Liu Xiaoyun
From the ‡Institute of Analytical Chemistry and Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.
§Department of Laboratory Medicine, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200127, China.
Mol Cell Proteomics. 2017 Sep;16(9):1683-1693. doi: 10.1074/mcp.M117.068296. Epub 2017 Jul 3.
We previously examined proteome within infected host cells and found differential expression of many proteins with defined functional roles such as metabolism or virulence. However, the precise roles of other altered proteins in pathogenesis are largely unknown. A putative transcriptional regulator, YdcR, was highly induced intracellularly whereas barely expressed , implicating potential relevance to bacterial infection. To unveil its physiological functions, we exploited quantitative proteomics of intracellular and found that genetic ablation of resulted in severe repression of SrfN, a known virulence factor. Immunoblotting, qRT-PCR, and β-galactosidase assays further demonstrate YdcR-dependent transcription and expression of Moreover, we found physical interaction of YdcR with the promoter region of , suggesting direct activation of its transcription. Importantly, a mutant lacking was markedly attenuated in a mouse model of infection. Our findings reveal that YdcR temporally regulates the virulence factor SrfN during infection, thus contributing to pathogenesis. Our work also highlights the utility of combining quantitative proteomics and bacterial genetics for uncovering the functional roles of transcription factors and likely other uncharacterized proteins as well.
我们之前研究了受感染宿主细胞内的蛋白质组，发现许多具有明确功能作用（如代谢或毒力）的蛋白质存在差异表达。然而，其他改变的蛋白质在发病机制中的精确作用在很大程度上尚不清楚。一种假定的转录调节因子YdcR在细胞内被高度诱导，而几乎不表达，这暗示其与细菌感染可能相关。为了揭示其生理功能，我们利用细胞内定量蛋白质组学发现，YdcR的基因缺失导致已知毒力因子SrfN的严重抑制。免疫印迹、qRT-PCR和β-半乳糖苷酶分析进一步证明了YdcR依赖的SrfN转录和表达。此外，我们发现YdcR与SrfN的启动子区域存在物理相互作用，表明其直接激活SrfN的转录。重要的是，缺乏YdcR的突变体在感染小鼠模型中明显减毒。我们的研究结果表明，YdcR在感染过程中暂时调节毒力因子SrfN，从而促进发病机制。我们的工作还强调了结合定量蛋白质组学和细菌遗传学来揭示转录因子以及可能其他未表征蛋白质功能作用的实用性。
