VgrG-dependent effectors and chaperones modulate the assembly of the type VI secretion system

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.

A DNase T6SS effector requires its MIX domain for secretion

“…Indeed, we show here that the MIX-effector, VPA1263, interacts with VgrG and not with two other T6SS structural components, Hcp and PAAR. The loading of MIX-effectors onto VgrG is further supported by a recent report that showed direct interaction between VasX, a MIX-effector from V. cholerae (Miyata et al, 2011), and its cognate VgrG (Liang et al, 2021). Nevertheless, although we expected to find that MIX plays a major role in mediating interaction with VgrG, we did not observe a dramatic difference in the VgrG binding ability between the full-length effector and a variant lacking the MIX domain.…”

“…We speculate that MIX contributes toward a stable or desirable effector conformation that is required for correct loading or positioning on the T6SS spike. Alternatively, following recent observations by Liang et al, who suggested that effectors interact with T6SS components, such as TssK and TssF, to fulfill the requirement for the "stuffing model" (Liang et al, 2021), it is possible that MIX plays a role in mediating the interaction of a spike-loaded effector with the T6SS base plate. Inconclusive results obtained during repeated attempts to detect a MIX-mediated interaction between VPA1263 and TssK prevent us from currently confirming or ruling out this possibility.…”

More Than Just a Spearhead: Diverse Functions of PAAR for Assembly and Delivery of Toxins of the Contractile Injection Systems

“…T6SS secretion in V. cholerae , A. dhakensis , Agrobacterium tumefaciens , and Enterobacter cloacae requires the presence of multiple VgrG-dependent effectors ( 17 , 28 , 31 , 32 ). The previously known T6SS requirement for a heterotrimer VgrG spike in V. cholerae and in A. dhakensis is tightly linked to the specific structural effects of effectors because a pseudohomotrimer VgrG spike made of VgrG chimeras, differing only by the C-terminal effector-loading tail, is functional for T6SS assembly ( 33 ). There is also direct interaction between effectors and the baseplate protein TssK and the assembly chaperone TssA ( 33 ).…”

“…The previously known T6SS requirement for a heterotrimer VgrG spike in V. cholerae and in A. dhakensis is tightly linked to the specific structural effects of effectors because a pseudohomotrimer VgrG spike made of VgrG chimeras, differing only by the C-terminal effector-loading tail, is functional for T6SS assembly ( 33 ). There is also direct interaction between effectors and the baseplate protein TssK and the assembly chaperone TssA ( 33 ). These findings not only suggest that the effector stuffing within the baseplate cavity is crucial for stabilizing T6SS assembly in these species but also lead to the hypothesis that the differential requirement for PAAR in different species may be dependent on PAAR-associated effectors.…”

Multiple T6SSs, mobile auxiliary modules, and effectors revealed in a systematic analysis of theVibrio parahaemolyticuspan-genome

“…Several experimental and computational approaches have been used to identify effector and immunity (E/I) pairs (2,7,11,12,29,(33)(34)(35)(36). Nevertheless, because T6SS effectors employ diverse mechanisms for secretion (13,(37)(38)(39), and therefore lack a canonical secretion signal, it is estimated that many more effectors still await discovery.…”