Lawsonia intracellularis LI0666 is a new EPIYA effector exported by the Yersinia enterocolitica type III secretion system

Lawsonia intracellularis is the causative agent of proliferative enteropathy. While it harbors genes encoding the entire apparatus required for the type III secretion system (T3SS) and the expression of some of these components has been detected during experimental infection, the identification of L. intracellularis T3SS substrates (effector proteins) has been hampered. The Yersinia T3SS and yeast growth inhibition assays are two important heterologous systems used for the characterization of effector proteins. Bacterial EPIYA effectors are a distinct class of bacterial effectors defined by the presence of EPIYA or the EPIYA-related motif. When delivered into host cells via a T3SS or type IV secretion system, these effectors undergo tyrosine phosphorylation of the EPIYA motif, which enables them to manipulate host cell signaling by promiscuously interacting with multiple SH2 domain-containing proteins. A previous study showed that L. intracellularis LI0666 contains two EPIYA motifs and speculated that this protein could be a T3SS effector. In this study, we show that LI0666 is secreted by Yersinia in a T3SS-dependent manner and inhibits yeast growth. LI0666 is phosphorylated at tyrosine residues in porcine intestinal epithelial cells and in human epithelial cells. Like the archetypal EPIYA effector CagA, the EPIYA-containing region is not required for LI0666 association with yeast and mammalian cell membranes. Our results indicate that LI0666 is an authentic bacterial EPIYA effector. Identification of the tyrosine kinases that are responsible for LI0666 phosphorylation and the SH2 domain-containing host proteins that LI0666 interacts with will help to explore the molecular mechanisms of LI0666 in disease development.