A Novel Molecular Mechanism for Effector Protein Wrapping and Delivery by the Bacterial Type VI Secretion System

Pathogenic bacteria deploy sophisticated strategies to endure hostile environments and outcompete host microbiota or immune cells. Up to 30% of Gram-negative bacteria, including Pseudomonas aeruginosa, harbor a Type VI secretion system (T6SS), a supramolecular nanomachine that operates like a crossbow, for firing effectors into prokaryotic and eukaryotic prey cells. Despite its widespread distribution in nature, the mechanism by which effectors are loaded into ca 120 Hcp ring assemblies that form the T6SS injection tube, and the diversity of effectors delivered per firing event, remain undefined. Here, we reveal this mechanism by solving the cryo-electron microscopy structure of the Tce1 cargo effector loaded into a hexameric Hcp ring. Our structure reveals that a single cargo is enclosed by multiple rings and interacts asymmetrically with individual Hcp protomers. Our data delineate a conceptually novel mode of effector recognition and a stepwise loading mechanism, whereby an initial heterodimeric Hcp-cargo complex forms prior to ring formation occurring around the effector. We showed that another effector, Tce2, which exhibits anti-fungal properties, is similarly a Hcp3 cargo. We thus propose a novel and foundational mechanism by which distinct cargos are wrapped and simultaneously loaded into a single T6SS molecular device, enabling the coordinated delivery of a broad and potent payload into target cells. *Patricia Paracuellos & Ambre Bexter contributed equally.