Broad-Spectrum Regulation of Nonreceptor Tyrosine Kinases by the Bacterial ADP-Ribosyltransferase EspJ

Tyrosine phosphorylation is key for signal transduction from exogenous stimuli, including the defense against pathogens. Conversely, pathogens can subvert protein phosphorylation to control host immune responses and facilitate invasion and dissemination. The bacterial effectors EspJ and SeoC are injected into host cells through a type III secretion system by enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively), Citrobacter rodentium, and Salmonella enterica, where they inhibit Src kinase by coupled amidation and ADP-ribosylation. C. rodentium, which is used to model EPEC and EHEC infections in humans, is a mouse pathogen triggering colonic crypt hyperplasia (CCH) and colitis. Enumeration of bacterial shedding and CCH confirmed that EspJ affects neither tolerance nor resistance to infection. However, comparison of the proteomes of intestinal epithelial cells isolated from mice infected with wild-type C. rodentium or C. rodentium encoding catalytically inactive EspJ revealed that EspJ-induced ADP-ribosylation regulates multiple nonreceptor tyrosine kinases in vivo. Investigation of the substrate repertoire of EspJ revealed that in HeLa and A549 cells, Src and Csk were significantly targeted; in polarized Caco2 cells, EspJ targeted Src and Csk and the Src family kinase (SFK) Yes1, while in differentiated Thp1 cells, EspJ modified Csk, the SFKs Hck and Lyn, the Tec family kinases Tec and Btk, and the adapter tyrosine kinase Syk. Furthermore, Abl (HeLa and Caco2) and Lyn (Caco2) were enriched specifically in the EspJ-containing samples. Biochemical assays revealed that EspJ, the only bacterial ADP-ribosyltransferase that targets mammalian kinases, controls immune responses and the Src/Csk signaling axis.