运用基于质谱的组学技术研究胞内病原细菌的代谢重组

Biological processes typically involve the participation of numerous proteins as well as other molecules, thus a deeper and overall understanding of such complex and dynamic processes requires systems-level approaches that enable the detection of hundreds and thousands of molecules from an integrative perspective. In the past decade, emerging mass spectrometry-based high-throughput, high-sensitivity technologies have enjoyed major advances, thereby leading to the rapid growth of proteomics and other omics areas. In the post-genomic era, utilization of large-scale omics tools has become an important trend in studying fundamental biological processes especially with respect to the molecular mechanisms underlying human diseases. Infectious diseases pose a major threat to human health, thus understanding microbial pathogenesis on the molecular level represents an important task. The proposed work aims to apply an integrated proteomics and metabolomics approach to examine, on large-scales, the levels of bacterial protein expression as well as their metabolites throughout the course of infection. With Salmonella being used as a model, emphasis will be placed on understanding the metabolic reprogramming of bacterial pathogens during their adaptation to the intracellular niche within the mammalian host. The expected results are likely to delineate metabolic pathways instrumental to pathogen survival within the host and these findings will aid in the development of novel anti-microbial strategies.

生命过程的发生往往涉及到多个蛋白质或其它分子的共同参与，因而全面、深入的认识复杂的生命现象亟须在整体、网络、动态的水平上对成百上千个分子进行研究。在此背景下，近年来基于质谱的高通量、高灵敏度检测技术的革命性进步极大推动了蛋白质组学及其它组学研究的兴起。运用大规模组学手段已成为后基因组时代探索生命现象特别是人类疾病机理研究中的一大趋势。传染病是威胁人类健康的重大疾病之一，因此研究病原微生物的分子致病机制是一项必须长期坚持的紧迫任务。申请人拟研究的项目将以沙门氏菌为模型、代谢重组与调控为视角，运用最前沿的蛋白质组学和代谢组学方法大规模表征病原细菌在感染宿主过程中蛋白质表达及代谢产物水平的动态变化。研究结果将有助于揭示病原细菌在宿主体内赖以生存的重要代谢过程，进而为开发以特定代谢通路为靶标的新型小分子抗感染药物提供新的设计思路。

本研究以沙门氏菌为模型、代谢重组与调控为视角，运用最前沿的蛋白质组学方法大规模表征病原细菌在感染宿主过程中蛋白质表达水平的动态变化。沙门氏菌是典型的胞内病原细菌，需在特定的囊泡结构中生存与繁殖。为了系统探究沙门氏菌在感染过程中的适应机制，我们首次大规模表征了感染后的沙门氏菌蛋白质组。在感染初期，沙门氏菌会大规模上调与铁吸收相关系统的蛋白质，另外与锰及锌等吸收相关蛋白也发生了显著上调，这表明沙门氏菌在宿主中会面临大范围的金属离子缺乏。此外，与磷酸利用相关的蛋白，PhoPQ二元系统调控的下游蛋白，部分氨基酸合成代谢及生物素合成相关蛋白也发生了上调。反之，沙门氏菌降低对甘油，麦芽糖的需求。至感染后期，数据阐明沙门氏菌的鞭毛系统及趋化系统严重退化，沙门氏菌代谢发生显著重调。总结与碳代谢相关的数据，我们发现在宿主细胞中沙门氏菌优先使用糖酵解，戊糖磷酸化途径，混酸发酵及核酸代谢途径。其三羧酸循环，有氧及无氧相关通路中的蛋白质表达皆显著被抑制。进一步地，通过遗传学手段敲除糖酵解及嘌呤合成通路中的关键基因可导致严重的沙门氏菌胞内生长缺陷，表明这两条代谢通路对于沙门氏菌感染至关重要。以上研究结果表明定量蛋白质组学方法为合理设计后续生物学实验及发现感染过程中的重要代谢通路奠定了坚实的基础。