Controlling and Patterning Retractable, Micron-Scale, Membrane-Breaking Protein Needles

R bodies are needle-like bacterial protein machines that function as toxin delivery devices. At low pH, R bodies extend from a coiled ribbon to form a hollow tube up to 20um long. Importantly, R bodies are made of just two structural proteins each ~100 amino acids long, rendering the sequence space of this machine small enough to be explored and manipulated. Ultimately, we wish to engineer this operon to produce force-generating structures of varied size, stiffness, and sensitivity to varied inputs. These structures can serve as patterned materials, intracellular delivery devices, and as control elements in cellular programming.

Using R bodies purified from heterologous expression in E. coli, we employed a high-throughput visual screen to produce mutants that require different pH levels to extend. We also generated extendable fluorescent fusions of R bodies, providing a proof of concept that they can be functionalized. In addition, we demonstrated that R bodies can rupture foreign membranes, triggering the release of encapsulated cargo.

In order to design R bodies with specific geometries and surface patterns, we need to understand their assembly at multiple scales. We first identified residues crucial for a covalent assembly step with Tandem Mass Tag Mass Spec (TMT-MS), suggesting that this assembly process relies on isopeptide bond formation. Furthermore, electron microscopy implicates a filamentous non-structural component, RebC, in producing long-range order. Finally, timelapse Structured Illumination Microscopy (SIM) reveals an assembly mechanism that temporally separates one end of the polymer from the other, enabling the generation of multifunctional R bodies with pulse-chase approaches. This would permit their arrangement into complex devices and higher-order polymers capable of expanding and contracting in response to chemical signals.

Further uses of R bodies – to enhance cytoplasmic delivery of molecules from the phagosome, as a remodeling scaffold, as a smart material that can cause reversible hemostasis, and as a micron-scale switch – are also explored.