The <i>Escherichia coli</i> Phosphotyrosine Proteome Relates to Core Pathways and Virulence

<div><p>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 <i>Escherichia coli</i>, 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 <i>E. coli</i> K12 and the human pathogen enterohemorrhagic <i>E. coli</i> (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 <i>etk wzc</i> double mutant strongly indicated the presence of hitherto unknown tyrosine kinases in <i>E. coli</i>. 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.</p></div

BY kinases Etk and Wzc are dispensable for the A/E lesion phenotype of EHEC O157:H7.

<p>HeLa cells were co-cultured with wild type EHEC O157:H7, <i>etk</i>, <i>wzc</i> and <i>etk wzc</i> double mutant derivatives for 5 h followed by actin staining of infected cells using FITC-phalloidin to visualize A/E lesion formation. Representative images of FITC-phalloidin stained actin of infected HeLa cells are shown with arrowheads indicating A/E lesions.</p

Tyrosine phosphorylation of SspA Tyr92 affects virulence phenotypes of EHEC O157:H7.

<p>(A) Location of Tyr92 in dimeric SspA (PDB 1YY7 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003403#ppat.1003403-Hansen1" target="_blank">[42]</a>). The structure of dimeric SspA is shown as blue ribbon diagrams. The hydrophobic residues Tyr92 and His85 of the functionally important surface-exposed pocket are shown in green and orange, respectively. The hydroxyl group of Tyr92 that is subject to phosphorylation is shown in red. The SspA structure was visualized using PyMOL (Schrödinger LLC). (B) SspA Tyr92 positively affects expression and secretion of T3SS proteins. The abundance of LEE-encoded proteins in whole cell lysates (lanes 1–4) and their abundance in culture supernatants (lanes 5–8) from cultures of wild type EHEC O157:H7 and isogenic <i>sspA</i> mutants were determined by western analyses as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003403#s4" target="_blank"><i>Material and Methods</i></a>. Strains tested included the <i>sspA</i> mutant containing the vector control pSec10*, the <i>sspA</i> mutant expressing wild type SspA from pSspA and the SspA Y92F mutant from pSspAY92F. EspA, EspB, Tir, SspA and GroEL were detected using polyclonal antisera against the respective proteins. GroEL served as an internal control for the total amount of protein in cell samples, and for the precipitation of proteins in culture supernatants to which 100 ng of GroEL were added. (C) SspA Tyr92 positively affects the A/E phenotype of EHEC O157:H7. A/E lesion formation was assessed using the FAS test as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003403#s4" target="_blank"><i>Material and Methods</i></a>. HeLa cells were co-cultured for 5 h with wild type EHEC O157:H7, an <i>sspA</i> mutant and the <i>sspA</i> mutant harboring the vector pSec10*, pSspA (SspA) and pSspAY92F (SspAY92F). The actin cytoskeleton of HeLa cells was stained with FITC-phalloidin for visualization of the A/E lesions. Representative images of fluorescence stained actin of infected HeLa cells are shown. Arrows indicate examples of A/E lesions.</p

Definition of <i>E. coli</i> tyrosine phosphorylation site motifs.

<p>(A–E) Probability logos of significantly enriched phosphotyrosine site motifs extracted from 512 unique pTyr sites by aligning peptide sequences comprising 12 residues surrounding the phosphorylated tyrosine residue using Motif-X (<i>p value</i><0.001). Site motif consensus sequences with variable residues indicated as x and the number of unique sites comprising each motif are indicated. (F) Sequence logos of the general residue representation surrounding the phosphorylated tyrosine residue from the 512 unique sites with residues above the midline being overrepresented and those below underrepresented constructed using Phophosite logo generator.</p

Phosphotyrosine proteins are central in the metabolic network.

<p>Protein-centric network representations of <i>E. coli</i> K12 (A) and EHEC O157:H7 (B) metabolism with pTyr (red nodes) and non-pTyr (yellow nodes) proteins indicated. Nodes represent proteins and edges represent compounds produced by one protein and consumed by other protein. Only names for the phosphotyrosine proteins are shown. Density plots showing the distribution of centrality closeness of pTyr (red) and non-pTyr (yellow) proteins in the metabolic network are shown. The higher the closeness centrality of a given protein (node), the shorter is its geodesic distances to other nodes in the graph.</p

Rapid Characterization of Candidate Biomarkers for Pancreatic Cancer Using Cell Microarrays (CMAs)

Tissue microarrays have become a valuable tool for high-throughput analysis using immunohistochemical labeling. However, the large majority of biochemical studies are carried out in cell lines to further characterize candidate biomarkers or therapeutic targets with subsequent studies in animals or using primary tissues. Thus, cell line-based microarrays could be a useful screening tool in some situations. Here, we constructed a cell microarray (CMA) containing a panel of 40 pancreatic cancer cell lines available from American Type Culture Collection in addition to those locally available at Johns Hopkins. As proof of principle, we performed immunocytochemical labeling of an epithelial cell adhesion molecule (Ep-CAM), a molecule generally expressed in the epithelium, on this pancreatic cancer CMA. In addition, selected molecules that have been previously shown to be differentially expressed in pancreatic cancer in the literature were validated. For example, we observed strong labeling of CA19-9 antigen, a prognostic and predictive marker for pancreatic cancer. We also carried out a bioinformatics analysis of a literature curated catalog of pancreatic cancer biomarkers developed previously by our group and identified two candidate biomarkers, HLA class I and transmembrane protease, serine 4 (TMPRSS4), and examined their expression in the cell lines represented on the pancreatic cancer CMAs. Our results demonstrate the utility of CMAs as a useful resource for rapid screening of molecules of interest and suggest that CMAs can become a universal standard platform in cancer research

Proteomics approach used for phosphotyrosine profiling of <i>E. coli</i>.

<p>EHEC O157:H7 strain TUV93-0 and <i>E. coli</i> K12 strain MG1655 were grown to stationary phase in DMEM. Total protein was trypsin-digested, peptides purified using reversed-phased chromatography and subjected to immunoaffinity (IP)-based phosphotyrosine enrichment. Tyrosine phosphorylated peptides were identified by high resolution LC-MS/MS analysis using LTQ Orbitrap XL and Velos instruments.</p

Rapid Characterization of Candidate Biomarkers for Pancreatic Cancer Using Cell Microarrays (CMAs)

Tissue microarrays have become a valuable tool for high-throughput analysis using immunohistochemical labeling. However, the large majority of biochemical studies are carried out in cell lines to further characterize candidate biomarkers or therapeutic targets with subsequent studies in animals or using primary tissues. Thus, cell line-based microarrays could be a useful screening tool in some situations. Here, we constructed a cell microarray (CMA) containing a panel of 40 pancreatic cancer cell lines available from American Type Culture Collection in addition to those locally available at Johns Hopkins. As proof of principle, we performed immunocytochemical labeling of an epithelial cell adhesion molecule (Ep-CAM), a molecule generally expressed in the epithelium, on this pancreatic cancer CMA. In addition, selected molecules that have been previously shown to be differentially expressed in pancreatic cancer in the literature were validated. For example, we observed strong labeling of CA19-9 antigen, a prognostic and predictive marker for pancreatic cancer. We also carried out a bioinformatics analysis of a literature curated catalog of pancreatic cancer biomarkers developed previously by our group and identified two candidate biomarkers, HLA class I and transmembrane protease, serine 4 (TMPRSS4), and examined their expression in the cell lines represented on the pancreatic cancer CMAs. Our results demonstrate the utility of CMAs as a useful resource for rapid screening of molecules of interest and suggest that CMAs can become a universal standard platform in cancer research