The ubiquitin system targets translocated EspH to proteasomal degradation

EspH is an effector protein secreted by the type III secretion system of various pathogenic Escherichia coli strains, including enteropathogenic E. coli (EPEC). The ability of EspH to inhibit host RhoGTPases, disrupt the actin cytoskeleton, and induce host cell cytotoxicity has been well-documented. Mass spectrometry analysis of EspH translocated into EPEC-infected cells revealed that a lysine at position 106 (K106) is modified with ubiquitin. Immunoblotting using the FK2 anti-ubiquitin antibodies has confirmed these results, suggesting that EspH undergoes polyubiquitylation. Prediction algorithms have identified a single ubiquitylation site at K106 and a phosphodegron in EspH. Moreover, we show that wild-type (EspH_wt), but not the EspH_K106R mutant, is subjected to degradation following translocation in an MG132-sensitive manner, indicating that the proteasome degrades the polyubiquitylated effector following translocation. Finally, we show that translocated EspH_K106R induces higher cytotoxicity than translocated EspH_wt. EspH_wt translocated into MG132-pretreated cells also displayed higher cytotoxicity levels than EspH_wt in untreated cells. These data reinforce the idea that EspH is polyubiquitylated and that the host proteasome degrades the translocated effector, possibly limiting its ability to toxicate the host cells. Additional implications of these effects on bacterial-host interactions are discussed.