Abstract
The type VI secretion system (T6SS) is a spear-like nanomachine found in gram-negative pathogens for delivery of toxic effectors to neighboring bacterial and host cells. Its assembly requires a tip spike complex consisting of a VgrG-trimer, a PAAR protein, and the interacting effectors. However, how the spike controls T6SS assembly remains elusive. Here we investigated the role of three VgrG-effector pairs in Aeromonas dhakensis strain SSU, a clinical isolate with a constitutively active T6SS. By swapping VgrG tail sequences, we demonstrate that the C-terminal ~30 amino-acid tail dictates effector specificity. Double deletion of vgrG1&2 genes (VgrG3+) abolished T6SS secretion, which can be rescued by ectopically expressing chimeric VgrG3 with a VgrG1/2-tail but not the wild type VgrG3. In addition, deletion of effector-specific chaperones also severely impaired T6SS secretion, despite the presence of intact VgrG and effector proteins, in both SSU and Vibrio cholerae V52. We further show that SSU could deliver a V. cholerae effector VasX when expressing a plasmid-borne chimeric VgrG with VasX-specific VgrG tail and chaperone sequences. Pull-down analyses show that two SSU effectors, TseP and TseC, could interact with their cognate VgrGs, the baseplate protein TssK, and the key assembly chaperone TssA. Effectors TseL and VasX could interact with TssF, TssK and TssA in V. cholerae. Collectively, we demonstrate that chimeric VgrG-effector pairs could bypass the requirement of heterologous VgrG complex and propose that effector-stuffing inside the baseplate complex, facilitated by chaperones and the interaction with structural proteins, serves as a crucial structural determinant for T6SS assembly.
Highlights
Host-pathogen interaction often involves the translocation of virulence factors by specific protein secretion systems, each considered as a delicate nanomachinery [1]
By constructing a series of combinatorial vgrG deletion mutants, we found that none of the double or triple vgrG deletion mutants was able to kill a competing E. coli prey or to secrete Hcp (Fig 3A and 3B), suggesting that a VgrG homotrimer comprising only one VgrG protein is insufficient for T6SS secretion
Why certain VgrG proteins are more critical than others despite of near identical conserved sequences [13,33,49] and why VgrG-dependent effectors are required for T6SS assembly [34,50]?
Summary
Host-pathogen interaction often involves the translocation of virulence factors by specific protein secretion systems, each considered as a delicate nanomachinery [1]. Their substrate selectivity is a key question for understanding how each system works. Of the six major secretion systems in gram-negative bacteria, the type VI secretion system (T6SS) is of particular interest in host-pathogen interaction for its capability in translocating effectors into a broad range of cell types including bacteria, fungi, and eukaryotic cells [2,3,4,5]. Some Hcp, VgrG and PAAR proteins with extended functional domains may directly act as effectors [12,13,22]. A large number of effectors have been identified, the molecular details of effector selection and loading remain elusive
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