Abstract
The Type VI secretion system (T6SS) is widespread among bacterial pathogens and acts as an effective weapon against competitor bacteria and eukaryotic hosts by delivering toxic effector proteins directly into target cells. The T6SS utilises a bacteriophage-like contractile machinery to expel a puncturing device based on a tube of Hcp topped with a VgrG spike, which can be extended by a final tip from a PAAR domain-containing protein. Effector proteins are believed to be delivered by specifically associating with particular Hcp, VgrG or PAAR proteins, either covalently (‘specialised’) or non-covalently (‘cargo’ effectors). Here we used the T6SS of the opportunistic pathogen Serratia marcescens, together with integratecd genetic, proteomic and biochemical approaches, to elucidate the role of specific VgrG and PAAR homologues in T6SS function and effector specificity, revealing new aspects and unexpected subtleties in effector delivery by the T6SS. We identified effectors, both cargo and specialised, absolutely dependent on a particular VgrG for delivery to target cells, and discovered that other cargo effectors can show a preference for a particular VgrG. The presence of at least one PAAR protein was found to be essential for T6SS function, consistent with designation as a ‘core’ T6SS component. We showed that specific VgrG-PAAR combinations are required to assemble a functional T6SS and that the three distinct VgrG-PAAR assemblies in S. marcescens exhibit distinct effector specificity and efficiency. Unexpectedly, we discovered that two different PAAR-containing Rhs proteins can functionally pair with the same VgrG protein. Showing that accessory EagR proteins are involved in these interactions, native VgrG-Rhs-EagR complexes were isolated and specific interactions between EagR and cognate Rhs proteins identified. This study defines an essential yet flexible role for PAAR proteins in the T6SS and highlights the existence of distinct versions of the machinery with differential effector specificity and efficiency of target cell delivery.
Highlights
Bacteria utilise a variety of mechanisms to deliver specific proteins to the extracellular environment or directly into a target cell, processes collectively termed protein secretion
This work aimed to learn more about how the machinery is able to recruit effectors and fire them from the bacterial cell into the target cell
We used a T6SS in Serratia marcescens, a pathogen causing hospital-acquired infections, which kills competitor bacteria very efficiently and is a good model for other T6SSs
Summary
Bacteria utilise a variety of mechanisms to deliver specific proteins to the extracellular environment or directly into a target cell, processes collectively termed protein secretion. The Type VI secretion system (T6SS) is widely distributed and has been linked with pathogenicity and/or interaction with eukaryotic cells in a number of important bacterial pathogens These include Pseudomonas aeruginosa, Vibrio cholerae and Burkholderia species, where anti-eukaryotic effector proteins delivered into target host cells by the T6SS have been described [2,3,4,5,6,7,8]. It is becoming clear that many, probably most, T6SSs are used as highly efficient weapons in competition against rival bacteria Such ‘anti-bacterial’ T6SSs have been described in varied species, including P. aeruginosa, V. cholerae, Serratia marcescens and Acinetobacter baumannii [9,10,11,12]. Anti-bacterial effectors identified to date include cell wall-degrading peptidoglycan hydrolases [14,15,16,17], cytoplasmic acting DNases [18,19,20] and membrane targeting toxins [21, 22]
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