Studies Of Salmonella Virulence Protein Regulation By Phosphoproteomics

Protein phosphorylation plays crucial roles in various biological processes such as enzymatic activity,protein conformation,protein-protein interaction,and cellular localization.Our current understanding regarding protein phosphorylation are primarily based on studies of eukaryote systems.In prokaryote cells,however,due to the low phosphorylation content,only limited progresses have been made on phosphoprotein identification and functional studiesIn this study,by combining a TiO2 enrichment method with high resolution mass spectrometry analysis,we identified 293 unique phosphorylation peptides containing 337 unique phosphorylation sites on 224 proteins.The phosphosite distribution is Serine 49.0%,Threonine 38.5%and Tyrosine 12.5%,which differs from the general distribution pattern in eukaryote systems.These phosphoproteome data provide resources and clues for further functional studies of protein phosphorylation in Salmonella virulence processes.Then we used an important Salmonella virulence protein as an example to elucidate the effect of phophosrylation on bacterial protein function.On the phosphorylation protein list we found an interesting protein called H-NS(Histone-like nucleoid structuring protein)which are phosphorylated at two sites,Thr-13 and Tyr-97.H-NS is a global silencer in Salmonella which specifically suppresses horizontally acquired virulence genes by targeting sequences with GC content lower than the resident genome.As consequence,H-NS regulates various biological processes.We constructed a series of amino acid point mutation strains to mimic the dephosphorylated and phosphorylation status on Thr-13 and Tyr-97.Through a series of biochemistry and molecular biology analysis on these phosphomimetic bacterial strains and H-NS proteins,we discovered that the transcriptional repression ability of H-NS was abolished when it was phosphorylated at either Thr-13 or Tyr-97.Further studies revealed that the Thr-13 phosphorylation induced H-NS conformational change and reduced dimer formation which lead to decreased H-NS binding to DNA promoters.While phophorylation at Tyr-97 rendered H-NS protein degradation,which also released transcriptional repression by H-NS.Not only this work provided new insight into H-NS regulation,it also revealed two biochemical mechanisms by which bacterial phosphorylation regulates protein function.Studying bacterial protein network and regulation requires tool which can quantitatively measure large numbers of proteins at the same time.To study the dynamic change of PhoP/PhoQ two-component system regulatory,we built a time-scheduled dimethyl-SRM method that can provide the precise relative quantification of 92 proteins in one run.By applying this method to the Salmonella PhoP/PhoQ two-component system,we found that the expression of selected PhoP/PhoQ-activated proteins in response to Mg2+ concentrations(from 8 ?M to 10 mM)could be divided into two distinct patterns and identified a potential regulatory switch for a subgroup of PhoP/PhoQ-regulated proteins at 50 ?M Mg2+.For the time-course(from 3 h to 24 h)SRM experiment,we found that the dynamics of the selected PhoP/PhoQ-activated proteins could be divided into three distinct patterns.In addition,we found that proteins in several metabolic pathways could be regulated either by Mgig concentrations or different growth time courses.Our study provides new insights into the protein networks regulated by low Mgig conditions,which may provide a new clue regarding PhoP/PhoQ activation and regulation.