Stepwise assembly and release of Tc toxins from Yersinia entomophaga.

Tc toxins are virulence factors of bacterial pathogens. Although their structure and intoxication mechanism are well understood, it remains elusive where this large macromolecular complex is assembled and how it is released. Here we show by an integrative multiscale imaging approach that Yersinia entomophaga Tc (YenTc) toxin components are expressed only in a subpopulation of cells that are 'primed' with several other potential virulence factors, including filaments of the protease M66/StcE. A phage-like lysis cassette is required for YenTc release; however, before resulting in complete cell lysis, the lysis cassette generates intermediate 'ghost' cells, which may serve as assembly compartments and become packed with assembled YenTc holotoxins. We hypothesize that this stepwise mechanism evolved to minimize the number of cells that need to be killed. The occurrence of similar lysis cassettes in diverse organisms indicates a conserved mechanism for Tc toxin release that may apply to other extracellular macromolecular machines.

Identification and structure of an extracellular contractile injection system from the marine bacterium Algoriphagus machipongonensis.

Contractile injection systems (CISs) are phage tail-like nanomachines, mediating bacterial cell-cell interactions as either type VI secretion systems (T6SSs) or extracellular CISs (eCISs). Bioinformatic studies uncovered a phylogenetic group of hundreds of putative CIS gene clusters that are highly diverse and widespread; however, only four systems have been characterized. Here we studied a putative CIS gene cluster in the marine bacterium Algoriphagus machipongonensis. Using an integrative approach, we show that the system is compatible with an eCIS mode of action. Our cryo-electron microscopy structure revealed several features that differ from those seen in other CISs: a 'cap adaptor' located at the distal end, a 'plug' exposed to the tube lumen, and a 'cage' formed by massive extensions of the baseplate. These elements are conserved in other CISs, and our genetic tools identified that they are required for assembly, cargo loading and function. Furthermore, our atomic model highlights specific evolutionary hotspots and will serve as a framework for understanding and re-engineering CISs.

A contractile injection system stimulates tubeworm metamorphosis by translocating a proteinaceous effector.

The swimming larvae of many marine animals identify a location on the sea floor to undergo metamorphosis based on the presence of specific bacteria. Although this microbe-animal interaction is critical for the life cycles of diverse marine animals, what types of biochemical cues from bacteria that induce metamorphosis has been a mystery. Metamorphosis of larvae of the tubeworm Hydroides elegans is induced by arrays of phage tail-like contractile injection systems, which are released by the bacterium Pseudoalteromonas luteoviolacea. Here we identify the novel effector protein Mif1. By cryo-electron tomography imaging and functional assays, we observe Mif1 as cargo inside the tube lumen of the contractile injection system and show that the mif1 gene is required for inducing metamorphosis. Purified Mif1 is sufficient for triggering metamorphosis when electroporated into tubeworm larvae. Our results indicate that the delivery of protein effectors by contractile injection systems may orchestrate microbe-animal interactions in diverse contexts.

A Bacterial Phage Tail-like Structure Kills Eukaryotic Cells by Injecting a Nuclease Effector.

Many bacteria interact with target organisms using syringe-like structures called contractile injection systems (CISs). CISs structurally resemble headless bacteriophages and share evolutionarily related proteins such as the tail tube, sheath, and baseplate complex. In many cases, CISs mediate trans-kingdom interactions between bacteria and eukaryotes by delivering effectors to target cells. However, the specific effectors and their modes of action are often unknown. Here, we establish an ex vivo model to study an extracellular CIS (eCIS) called metamorphosis-associated contractile structures (MACs) that target eukaryotic cells. MACs kill two eukaryotic cell lines, fall armyworm Sf9 cells and J774A.1 murine macrophage cells, by translocating an effector termed Pne1. Before the identification of Pne1, no CIS effector exhibiting nuclease activity against eukaryotic cells had been described. Our results define a new mechanism of CIS-mediated bacteria-eukaryote interaction and are a step toward developing CISs as novel delivery systems for eukaryotic hosts.