The Escherichia coli Phosphotyrosine Proteome Relates to Core Pathways and Virulence
Figure 5
Tyrosine phosphorylation of SspA Tyr92 affects virulence phenotypes of EHEC O157:H7.
(A) Location of Tyr92 in dimeric SspA (PDB 1YY7 [42]). 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 sspA mutants were determined by western analyses as described in Material and Methods. Strains tested included the sspA mutant containing the vector control pSec10*, the sspA 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 Material and Methods. HeLa cells were co-cultured for 5 h with wild type EHEC O157:H7, an sspA mutant and the sspA 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.