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.
Highlights
The type VI secretion system (T6SS) is a multifaceted protein export pathway that is widely distributed in Gram-negative Proteobacteria [1,2]
type VI secretion immunity 2 (Tsi2) protects against type VI secretion exported 2 (Tse2)-induced stasis We have reported that P. aeruginosa donor cells capable of delivering Tse2 by an active H1-T6SS to P. aeruginosa recipient bacteria lacking tsi2 have a pronounced competitive fitness advantage [13]
Absolute colony forming units (CFU) of competing bacteria were not determined in these experiments, which precluded defining whether Tse2 causes cell death or stasis in recipient cells when delivered by the T6SS
Summary
The type VI secretion system (T6SS) is a multifaceted protein export pathway that is widely distributed in Gram-negative Proteobacteria [1,2]. Among the conserved components of the T6SS are a AAA+ family ATPase, ClpV, two proteins with sequence similarity to the type IVB secretion proteins IcmF and DotU, TssM and TssL, and several proteins with sequence or structural similarity to nonfilamentous phage proteins [5]. The latter group of proteins includes Haemolysin co-regulated protein (Hcp) and Valine glycine repeat protein G (VgrG), which bear structural similarity to the tail protein of lambda phage (gpV) and the puncturing device of T4 phage (gp27/gp5), respectively [6,7,8]. These observations have led to a prominent structure-function model in which the T6S apparatus resembles outward facing phage on the bacterial cell surface [9]
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