Abstract
In many Gram-negative bacteria, the type 2 secretion system (T2SS) plays an important role in virulence because of its capacity to deliver a large amount of fully folded protein effectors to the extracellular milieu. Despite our knowledge of most T2SS components, the mechanisms underlying effector recruitment and secretion by the T2SS remain enigmatic. Using complementary biophysical and biochemical approaches, we identified here two direct interactions between the secreted effector CbpD and two components, XcpYL and XcpZM, of the T2SS assembly platform (AP) in the opportunistic pathogen Pseudomonas aeruginosa Competition experiments indicated that CbpD binding to XcpYL is XcpZM-dependent, suggesting sequential recruitment of the effector by the periplasmic domains of these AP components. Using a bacterial two-hybrid system, we then tested the influence of the effector on the AP protein-protein interaction network. Our findings revealed that the presence of the effector modifies the AP interactome and, in particular, induces XcpZM homodimerization and increases the affinity between XcpYL and XcpZM The observed direct relationship between effector binding and T2SS dynamics suggests an additional synchronizing step during the type 2 secretion process, where the activation of the AP of the T2SS nanomachine is triggered by effector binding.
Highlights
In many Gram-negative bacteria, the type 2 secretion system (T2SS) plays an important role in virulence because of its capacity to deliver a large amount of fully folded protein effectors to the extracellular milieu
Because protein letters are specific to the Pseudomonas genus, we will systematically refer to the general Gsp nomenclature using a subscript; i.e. the GspL homolog in P. aeruginosa is named XcpYL
We previously showed by surface plasmon resonance that the purified periplasmic domains of the secretin XcpQD, the assembly platform (AP) component XcpPC, and the pseudopilus tip quaternary complex (XcpUHVIWJXK) directly bind secreted effectors, allowing us to propose an integrated model of effector recognition and transport by the T2SS (34)
Summary
In many Gram-negative bacteria, the type 2 secretion system (T2SS) plays an important role in virulence because of its capacity to deliver a large amount of fully folded protein effectors to the extracellular milieu. In contrast to GspM and GspC, GspL harbors an additional N-terminal cytoplasmic domain presenting structural homology with actin-like ATPases (20). This domain is, together with the integral inner membrane protein GspF, involved in the recruitment of the ATPase GspE at the secretion site (21–26). We report that in addition to interacting with GspC, the secretin, and the pseudopilus tip, the T2SS secreted effector interacts with the periplasmic domains of GspM and GspL inner membrane components of the AP. We propose that these newly discovered interactions constitute an additional step of the T2SS secretion process, synchronizing effector loading and pseudopilus assembly
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