Abstract
The bacterial pathogen Pseudomonas aeruginosa uses three type VI secretion systems (T6SSs) to drive a multitude of effector proteins into eukaryotic or prokaryotic target cells. The T6SS is a supramolecular nanomachine, involving a set of 13 core proteins, which resembles the contractile tail of bacteriophages and whose tip is considered as a puncturing device helping to cross membranes. Effectors can attach directly to the T6SS spike which is composed of a VgrG (valine-glycine-rich proteins) trimer, of which P. aeruginosa produces several. We have previously shown that the master regulator RsmA controls the expression of all three T6SS gene clusters (H1-, H2- and H3-T6SS) and a range of remote vgrG and effector genes. We also demonstrated that specific interactions between VgrGs and various T6SS effectors are prerequisite for effector delivery in a process we called “à la carte delivery.” Here, we provide an in-depth description on how the two H2-T6SS-dependent effectors PldA and PldB are delivered via their cognate VgrGs, VgrG4b and VgrG5, respectively. We show that specific recognition of the VgrG C terminus is required and effector specificity can be swapped by exchanging these C-terminal domains. Importantly, we established that effector recognition by a cognate VgrG is not always sufficient to achieve successful secretion, but it is crucial to provide effector stability. This study highlights the complexity of effector adaptation to the T6SS nanomachine and shows how the VgrG tip can possibly be manipulated to achieve effector delivery.
Highlights
The type VI secretion system (T6SS) is a sophisticated protein secretion system that is found in about 25% of Gram-negative bacteria (Bingle et al, 2008)
The tandemly organized genes encoding the T6SS effector phospholipase PldA, known as Tle5a (Russell et al, 2013), and the structural component VgrG4b are upregulated in an rsmA mutant and both proteins are secreted by the H2-T6SS in P. aeruginosa PAO1 (Supplementary Figure S1; Russell et al, 2013; Allsopp et al, 2017)
Notably PldA and PldB, which are described in this study and which are considered as trans-kingdom effectors since they challenge the survival of eukaryotic cells as much as bacterial cells
Summary
The type VI secretion system (T6SS) is a sophisticated protein secretion system that is found in about 25% of Gram-negative bacteria (Bingle et al, 2008). The T6SS is placed onto the bacterial cytoplasmic membrane via a membrane complex consisting of TssL, TssM and TssJ proteins (Durand et al, 2012, 2015). The membrane complex is connected to a socalled T6SS baseplate, sitting at the cytosolic side of the inner membrane and made of TssA, TssE, TssF, TssG, TssK, and VgrG (Brunet et al, 2015; Planamente et al, 2016). Composed of TssB and TssC subunits (Leiman et al, 2009), this helical contractile sheath envelops an inner tube, that consists of stacked hexamers of Hcp proteins (Brunet et al, 2014) which are topped by the VgrG spike (Renault et al, 2018). Contraction of the sheath toward the baseplate leads to extracellular release of the VgrG spike and the Hcp tube, where presence of Hcp in the supernatant fraction of bacterial cultures is a standard readout for T6SS activity, since it is a direct measure of a functional T6SS machinery (Pukatzki et al, 2006)
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