title: Structural basis for type VI secretion effector recognition by a cognate immunity protein.
abstract: The type VI secretion system (T6SS) has emerged as an important mediator of interbacterial interactions. A T6SS from Pseudomonas aeruginosa targets at least three effector proteins, type VI secretion exported 1-3 (Tse1-3), to recipient Gram-negative cells. The Tse2 protein is a cytoplasmic effector that acts as a potent inhibitor of target cell proliferation, thus providing a pronounced fitness advantage for P. aeruginosa donor cells. P. aeruginosa utilizes a dedicated immunity protein, type VI secretion immunity 2 (Tsi2), to protect against endogenous and intercellularly-transferred Tse2. Here we show that Tse2 delivered by the T6SS efficiently induces quiescence, not death, within recipient cells. We demonstrate that despite direct interaction of Tsi2 and Tse2 in the cytoplasm, Tsi2 is dispensable for targeting the toxin to the secretory apparatus. To gain insights into the molecular basis of Tse2 immunity, we solved the 1.00 Å X-ray crystal structure of Tsi2. The structure shows that Tsi2 assembles as a dimer that does not resemble previously characterized immunity or antitoxin proteins. A genetic screen for Tsi2 mutants deficient in Tse2 interaction revealed an acidic patch distal to the Tsi2 homodimer interface that mediates toxin interaction and immunity. Consistent with this finding, we observed that destabilization of the Tsi2 dimer does not impact Tse2 interaction. The molecular insights into Tsi2 structure and function garnered from this study shed light on the mechanisms of T6 effector secretion, and indicate that the Tse2-Tsi2 effector-immunity pair has features distinguishing it from previously characterized toxin-immunity and toxin-antitoxin systems.
authors.affiliation: Department of Microbiology, University of Washington, Seattle, Washington, United States of America.
authors.forename: Mo
authors.initials: M
authors.lastname: Li
authors.forename: Isolde
authors.initials: I
authors.lastname: Le Trong
authors.forename: Mike A
authors.initials: MA
authors.lastname: Carl
authors.forename: Eric T
authors.initials: ET
authors.lastname: Larson
authors.forename: Seemay
authors.initials: S
authors.lastname: Chou
authors.forename: Justin A
authors.initials: JA
authors.lastname: De Leon
authors.forename: Simon L
authors.initials: SL
authors.lastname: Dove
authors.forename: Ronald E
authors.initials: RE
authors.lastname: Stenkamp
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Mougous
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D058947:Bacterial Secretion Systems / Q000502:physiology*; D018360:Crystallography, X-Ray; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D058947:Bacterial Secretion Systems