Abstract:

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 (EspHwt), but not the EspHK106R 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 EspHK106R induces higher cytotoxicity than translocated
EspHwt. EspHwt translocated into MG132-pretreated cells also displayed higher cytotoxicity
levels than EspHwt 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.