Quantitative Proteomics Analysis of PknG Functions in <italic>Mycobacterium</italic>

Abstract

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-mc2155, 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-mc2155 and mc2155 strains. We have identified 176 differentially expressed proteins, in which 152 proteins are upregulated and 24 are downregulated in the PknG-mc2155 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-mc2155 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-mc2155 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.