Research Article: The Escherichia coli Phosphotyrosine Proteome Relates to Core Pathways and Virulence

Date Published: June 13, 2013

Publisher: Public Library of Science

Author(s): Anne-Marie Hansen, Raghothama Chaerkady, Jyoti Sharma, J. Javier Díaz-Mejía, Nidhi Tyagi, Santosh Renuse, Harrys K. C. Jacob, Sneha M. Pinto, Nandini A. Sahasrabuddhe, Min-Sik Kim, Bernard Delanghe, Narayanaswamy Srinivasan, Andrew Emili, James B. Kaper, Akhilesh Pandey, Guy Tran Van Nhieu.


While phosphotyrosine modification is an established regulatory mechanism in eukaryotes, it is less well characterized in bacteria due to low prevalence. To gain insight into the extent and biological importance of tyrosine phosphorylation in Escherichia coli, we used immunoaffinity-based phosphotyrosine peptide enrichment combined with high resolution mass spectrometry analysis to comprehensively identify tyrosine phosphorylated proteins and accurately map phosphotyrosine sites. We identified a total of 512 unique phosphotyrosine sites on 342 proteins in E. coli K12 and the human pathogen enterohemorrhagic E. coli (EHEC) O157:H7, representing the largest phosphotyrosine proteome reported to date in bacteria. This large number of tyrosine phosphorylation sites allowed us to define five phosphotyrosine site motifs. Tyrosine phosphorylated proteins belong to various functional classes such as metabolism, gene expression and virulence. We demonstrate for the first time that proteins of a type III secretion system (T3SS), required for the attaching and effacing (A/E) lesion phenotype characteristic for intestinal colonization by certain EHEC strains, are tyrosine phosphorylated by bacterial kinases. Yet, A/E lesion and metabolic phenotypes were unaffected by the mutation of the two currently known tyrosine kinases, Etk and Wzc. Substantial residual tyrosine phosphorylation present in an etk wzc double mutant strongly indicated the presence of hitherto unknown tyrosine kinases in E. coli. We assess the functional importance of tyrosine phosphorylation and demonstrate that the phosphorylated tyrosine residue of the regulator SspA positively affects expression and secretion of T3SS proteins and formation of A/E lesions. Altogether, our study reveals that tyrosine phosphorylation in bacteria is more prevalent than previously recognized, and suggests the involvement of phosphotyrosine-mediated signaling in a broad range of cellular functions and virulence.

Partial Text

Protein phosphorylation is an evolutionarily highly conserved post-translational modification important for signal transduction in living organisms. The ability of bacteria to rapidly adapt to changing environments, crucial for survival and successful infection of the host by bacterial pathogens, relies on an extensive regulatory network also involving protein phosphorylation. Reversible protein phosphorylation targeting arginine, aspartate, histidine, serine, threonine and tyrosine residues is highly integrated in regulatory networks of bacteria. Among these, phosphorylation-mediated signaling through histidine and aspartate in bacterial two-component systems is the best characterized [1]. Phosphorylation on serine/threonine/tyrosine (Ser/Thr/Tyr) residues was initially associated with signaling in eukaryotes; however, during the past two decades it has emerged as an important regulatory function in prokaryotes as well. Recent high resolution mass spectrometry-based phosphoproteomic studies have unambiguously identified phosphorylation events in bacteria on Ser, Thr and less frequently on Tyr residues [2], significantly expanding the repertoire especially of Ser and Thr phosphorylated proteins. Notably, a comprehensive phosphoproteomic analysis of Mycobacterium tuberculosis revealed more than 500 phosphorylation events on Thr/Ser but none on Tyr residues [3]. About 121 phosphotyrosine (pTyr) sites have so far been reported on 114 proteins in 11 bacterial species by phosphoproteomics studies [4]. While the role of pTyr modification in eukaryotes is well established in cell growth, proliferation and differentiation [5], its role is less clear in bacteria.

Although tyrosine phosphorylation has become a recognized protein modification in bacteria, the extent of this modification remains uncertain. Using a combined immunoaffinity enrichment and mass spectrometry-based phosphoproteomic approach we established tyrosine phosphorylation in bacteria as far more prevalent than previously known with 512 unique pTyr sites identified on 342 proteins from E. coli. Our results indicate that tyrosine phosphorylation targets a broad range of fundamental cellular processes ranging from control of gene expression to metabolic pathways, a range that is indicative of a global regulatory network that likely affects various aspects of bacterial cell physiology and virulence. In E. coli phosphotyrosine-based regulation appears highly integrated as reflected by our identification of phosphorylated proteins involved in the synthesis, turnover and modification of DNA, RNA and proteins. The complexity of phosphotyrosine-mediated signaling at multiple levels of regulation is exemplified by the identification of tyrosine phosphorylation of Cra, a global transcription regulator controlling the expression of enzymes involved in major carbon and energy metabolism pathways [62], as well as more than half of the Cra-controlled enzymes involved in these metabolic processes. Additional phosphotyrosine profiling studies of cells grown under different conditions could help unveil the biological role of tyrosine phosphorylation, such as whether, it like acetylation in Salmonella enterica, coordinates carbon source utilization and metabolic flux of the central carbon metabolism [63].




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