定量蛋白质组学分析PknG在分枝杆菌中的功能

丝氨酸苏氨酸蛋白激酶G(PknG)是分枝杆菌中一个类似于真核生物蛋白激酶C的蛋白质,对结核分枝杆菌的生长和新陈代谢等生理过程,以及结核分枝杆菌的耐药和在宿主细胞中的存活都起着重要的调节作用.本文在耻垢分枝杆菌(Mycobacterium smegmatis)mc2155中构建了过表达结核分枝杆菌PknG的重组菌株PknG-mc2155,并发现PknG-mc2155的生长速度慢于mc2155.应用化学修饰结合LC-LC-MS/MS的定量蛋白质组学方法,在mc2155和PknG-mc2155中鉴定到了176种有差异表达的蛋白,其中152种蛋白在PknG-mc2155中表达下调,24种蛋白表达上调.这些差异表达的蛋白参与了多个细胞过程,包括代谢、蛋白翻译等.基于这些结果,我们推测PknG-mc2155生长速度慢的原因是因为代谢相关酶如GlpK,ALD和DesA1等蛋白表达的下调;而Ag85A,Ag85C,SecA2等蛋白的上调则增强细菌的感染性;另外KatG蛋白的下调提示PknG的过表达增强了菌株的抗药性.代谢组学分析发现谷氨酸和谷氨酰胺在PknG-mc2155中的水平低于在mc2155中水平,证实了PknG影响谷氨酰胺的稳态平衡.利用蛋白质磷酸化分析,我们发现PknG的苏氨酸残基T-320上有一个自磷酸化修饰,而且在PknG-mc2155菌株中,也鉴定到gltA和glmM上的磷酸化修饰,显示gltA和glmM是PknG的底物.本研究为理解PknG的功能和作用机制提供了新的依据和解释,为深入研究PknG在结核分枝杆菌中的功能奠定了基础,我们的结果也表明蛋白质组学技术是系统研究细菌蛋白质功能的重要工具.

Quantitative Proteomics Analysis of PknG Functions in Mycobacterium

The eukaryotic like serine/threonine protein kinase G （PknG） from pathogenic mycobacteria plays an important role in inhibiting phagosome-lysosome fusion and mediating intracellular survival of mycobacteria in macrophages. Furthermore, PknG has broad functions in regulation of bacterial metabolism, survival, virulence, and drug-resistance in mycobacteria. In the present work, we construct the PknG-overexpression Mycobacterium smegmatis strain PknG-mc^2155, and show that overexpression of PknG arrests bacterial growth and decreases the cellular glutamate and glutamine levels. Using quantitative proteomic technology, we systematically analyze the differentially expressed proteins between PknG-mc^2155 and mc^2155 strains. We have identified 176 differentially expressed proteins, in which 152 proteins are upregulated and 24 are downregulated in the PknG-mc^2155 strain. Our data suggest that the decrease of bacterial growth rate is associated with the down-regulation of metabolic enzymes such as GlpK, ALD, and DesA in PknG-mc^2155 cells, and the up-regulation of Ag85A, Ag85C, and SecA2 contributes to enhanced bacterial virulence. From our proteomic data, we identify a new phosphorylation site on Thr-329 residue of PknG. Analysis also shows that gltA and glmM are phosphorylated in PknG-mc^2155 cells, indicating that gltA and glmM are potential substrates of PknG. Based on the proteomic data, the working mechanisms of PknG are proposed that provide new evidence for understanding functions of PknG in mycobacteria. Our data also demonstrates that quantitative proteomic analysis is an important tool for understanding functions and working mechanisms of bacteria proteins.