金属蛋白质组学研究锌离子匮乏对肺炎链球菌的影响

Effect of zinc depletion on Streptococcus pneumoniae revealed by metalloproteomics

摘要:

【目的】研究锌离子缺乏对肺炎链球菌的影响，找到其适应性生长机制。【方法】以肺炎链球菌为模型，利用加锌和不加锌的培养基对细菌进行培养，收集细胞蛋白，采用双向凝胶电泳，结合金属亲和层析和质谱技术鉴定差异表达蛋白，进而通过生物信息学分析蛋白质相互关系，从中找到细菌适应锌离子匮乏条件的关键代谢通路和蛋白。【结果】测定了在限制培养条件下肺炎链球菌的最适生长浓度，建立了锌离子调控蛋白双向凝胶电泳图谱，鉴定到了96个差异表达蛋白斑点，共67个差异蛋白，其中32个表达下调，35个表达上调，锌离子调控蛋白的作用可能主要体现在糖代谢、核酸代谢、氧化还原作用、辅助蛋白质翻译、合成及折叠等方面。建立了锌结合蛋白的差异表达图谱，鉴定到了10个差异表达蛋白斑点，共7个差异蛋白，其中1个表达下调，6个表达上调。锌离子结合蛋白的作用可能主要体现在应对压力、蛋白质折叠和转运、氨基酸代谢等方面。【结论】肺炎链球菌主要通过调控碳水化合物代谢和核酸代谢等多个代谢通路来应对宿主锌金属离子匮乏的环境，从而使自身能够存活并对宿主形成感染。本研究为揭示细菌在宿主环境，特别是金属离子匮乏条件下的适应性生长机制提供理论基础。

Abstract:

[Objective] This study is aimed to explore the effects of zinc deficiency and the corresponding adaptive growth of Streptococcus pneumoniae D39. [Methods] We extracted whole proteins from S. pneumoniae D39 cultured with or without zinc, performed two-dimensional electrophoresis (2-DE) combined with immobilized metal affinity chromatography (IMAC) and mass spectra (MS) to identify differentially expressed proteins. [Results] The 2-DE maps of whole cell proteins showed that 32 proteins were down-regulated and 35 up-regulated. These Zn2+ regulated proteins are mainly involved in carbohydrate metabolism, nucleotide metabolism, oxidation, proteins translation, synthesis and folding. The 2-DE maps of Zn2+-binding proteins by using Zn-IMAC separation exhibited 7 differentially expressed proteins, 1 down-regulated and 6 up-regulated. These Zn2+-binding proteins participate in the bacterial response to stress stimuli, proteins folding and amino acid metabolism. [Conclusion] To survive and infect the host, S. pneumoniae regulate many metabolic pathways mainly including carbohydrate metabolism and nucleotide metabolism under zinc deficiency. This study provides a theoretical basis for revealing the adaptive growth mechanism of bacteria in host environment with limited metal ions.