A Bifunctional T3SS-Effector Simultaneously Cleaves Host MAP Kinase and Inhibits PPM1A Phosphatase.

NleD belongs to a family of metalloproteases produced by multiple pathogens and functions as a Type III secretion system (T3SS) effector. NleD inactivates the p38 and JNK MAP kinases by cleaving them at a single site within the conserved threonine-X-tyrosine (TXY) motif. Here, we show that NleD from enteropathogenic E. coli (EPEC) interacts with PPM1A, a host metallophosphatase that targets multiple substrates, including the MAPK TXY motif. Binding of NleD inhibits the phosphatase activity of PPM1A while preserving NleD's proteolytic function. Structural analysis of the NleD-PPM1A complex reveals that NleD suppresses PPM1A activity by blocking phospho-protein substrates from accessing its catalytic pocket. Intriguingly, using a Citrobacter rodentium murine infection model, we found that NleD can enhance intestinal colonization in a manner independent of its protease activity, possibly via interaction with PPM1A. Together, these findings identify NleD as a bifunctional effector, highlighting the sophisticated strategies by which T3SS effectors manipulate key host signaling pathways.