Redefining the role of chaperones for Dot/Icm T4BSS-mediated substrate translocation by Legionella pneumophila

Legionella pneumophila, the etiological agent of legionnaires' disease, establishes its intracellular niche through the Dot/Icm type IVB secretion system, which translocates ∼300 effector proteins into host cells. Efficient secretion of many effectors requires the IcmS and IcmW chaperones, which interact with both substrates and the type IV coupling protein DotL. A prior model proposed that IcmSW are released from substrates during translocation by transfer to the C terminus of DotL and then recycled to the cytoplasm. This model predicts an excess cytoplasmic pool of IcmSW and their dissociation from DotL. We tested these predictions using quantitative stoichiometric analysis of Dot/Icm components and DotL-chaperone fusion constructs. IcmSW were present at levels comparable to DotL, and a DotL-IcmW-IcmS fusion fully complemented an ΔicmS ΔicmW mutant for intracellular replication. These findings refute the recycling model and support an updated "anchored chaperone" model, wherein IcmSW remain stably associated with DotL during substrate translocation.