Molecular insights into the frontline of bacterial warfare
The associated manuscript in Nature Microbiology is here: https://go.nature.com/2QaguVs
Microbial populations are ubiquitous. Within their diverse communities, bacteria are engaged in a constant battle for resources to ensure their own survival. In order to outcompete rivaling cells, they have acquired a vast arsenal of molecular weaponry. For example, many species of Gram-negative bacteria can directly inject toxic proteins, also called effectors, into competing cells. For this purpose, they utilize a sophisticated molecular machine called the type VI secretion system (T6SS), which shares a high structural similarity to the contractile bacteriophage. Unsurprisingly, this syringe-like injection apparatus uses a mechanism similar to that of bacteriophages, where it first punctures the membrane of contacting cells and then releases its toxic payload.
In the past decade, structural and biochemical findings have contributed to a better understanding of how the T6SS functions on a mechanistic level. The core of a T6SS is an inner tube formed by stacks of the hexameric Hcp protein, which is in turn surrounded by an outer sheath complex. This sheath can mechanically contract and hence propel the inner tube outwards from the cell. The outward facing end of the Hcp nanotube is decorated with an arrowhead-like tip, consisting of the trimeric protein VgrG in complex with a proline-alanine-alanine-arginine (PAAR) domain-containing effector.
Although this spike complex likely mediates the first point of contact with recipient cells, structural knowledge about VgrG proteins loaded with T6SS effector proteins is limited. Furthermore, the exact role of chaperone proteins, which are found to be involved in the association of certain effectors with VgrG, is also poorly understood.
We previously determined the overall architecture of a P. aeruginosa T6SS ‘pre-firing’ complex (PFC), which consists of the NAD(P) glycohydrolase effector Tse6 loaded onto the VgrG1 protein, as well as the immune protein Tsi6, the chaperone EagT6 and – unexpectedly – the ubiquitous elongation factor EF-Tu. The low resolution of this negative-stain structure, however, limited our understanding of how the single components are arranged within the PFC and how they interact with each other.
In our current work, we provide a more detailed picture of this elusive spike complex by using electron cryomicroscopy. To our surprise, the obtained structure revealed that two copies of the dimeric chaperone are responsible for shielding the two hydrophobic transmembrane domains (TMDs) of Tse6. Our structural findings made us wonder whether these TMDs are also involved in the loading of Tse6 onto VgrG1. Using biochemical experiments with single and double TMD deletion mutants, we not only further delineated the interaction between EagT6 and the TMDs, but also demonstrated that both of these interactions are crucial for the proper loading of Tse6.
An ongoing debate within the T6SS community is whether effectors are delivered into the peri- or the cytoplasm of target cells. Therefore, we also decided to take a closer look at the delivery of Tse6. We suspected that the TMDs might contribute not only to the loading but also be implicated in the translocation of the toxin domain of Tse6. To test our theory, we devised an in vitro assay with NAD-filled liposomes, with which we demonstrated the ability of Tse6 to spontaneously enter membranes and self-translocate its enzymatic domain across lipid bilayers. These above findings provide support for the understanding that Tse6 is delivered by the T6SS into the periplasm of recipient cells, and its toxin domain transits the inner membrane to access the cytoplasm, which is its target compartment.
In conclusion, our study provides insight into the unique molecular architecture and loading mechanism of the T6SS needle tip, which is at the very frontline of bacterial warfare. Since similar domain arrangements and chaperone/effector gene pairs can be found in diverse members of the Proteobacteria family, we anticipate that our findings will be generalizable to numerous other bacteria and membrane-associated effectors that are exported by the T6SS.