Study of Legionella effector domains revealed novel and prevalent phosphatidylinositol 3-phosphate binding domains

Legionella pneumophila and other Legionella species replicate intracellu-larly using the Icm/Dot type IV secretion system. In L. pneumophila this system translocates 300 effectors into host cells and in the Legionella genus thousands of effectors were identified, the function of most of which is unknown. Fourteen L. pneumophila effectors were previously shown to specifically bind phosphoinositides (PIs) using dedicated domains. We found that PI-binding domains of effectors are usually not homologous to one another; they are relatively small and located at the effectors’ C termini. We used the previously identified Legionella effector domains (LEDs) with unknown function and the above characteristics of effector PI-binding domains to discover novel PI-binding LEDs. We identified three predicted PI-binding LEDs that are present in 14 L. pneumophila effectors and in 200 effectors in the Legionella genus. Using an in vitro protein-lipid overlay assay, we found that 11 of these L. pneumophila effectors specifically bind phosphatidylinositol 3-phosphate (PI3P), almost doubling the number of L. pneumophila effectors known to bind PIs. Further, we identified in each of these newly discovered PI3P-binding LEDs conserved, mainly positively charged, amino acids that are essential for PI3P binding. Our results indicate that Legionella effectors harbor unique domains, shared by many effectors, which directly mediate PI3P binding.