Tuning retractable, microscale, membrane-breaking protein needles

10 The refractile (R) bodies found in Caedibacter taeniospiralis, a bacterial endosymbiont of Paramecium 11 tetraurelia, are large, polymeric protein structures that can switch between two conformations. At 12 cytoplasmic pH, they resemble coiled ribbons of protein 500nm in diameter. At low pH, they extend to 13 form hollow needles up to 20 microns long. They can be expressed heterologously from an operon 14 containing four short open reading frames and can function in vitro in diverse buffer conditions. 15 In this study, R bodies purified from Escherichia coli were found to be capable of undergoing many 16 consecutive extension-contraction cycles. Furthermore, the solubility of R bodies, which can easily be 17 interpreted by eye, was found to correlate with their extension state. This macroscopic phenotype was 18 used to develop a quantitative, high-throughput assay for R body state, enabling a visual screen of R 19 body mutants defective in extension. The role of specific amino acids in extension was determined, and 20 this information was used to construct rationally-designed mutants tailored to extend at higher pH. 21 Furthermore, R bodies were able to rupture E. coli spheroplasts to release soluble proteins across lipid 22 bilayers. Taken together, these results show that R bodies act as tunable, pH-actuated pistons suitable 23 for a variety of membrane-breaking applications. 24 Importance 25 R bodies are natural toxin delivery machines made by bacteria that live inside of single-celled 26 eukaryotes. Under normal conditions, they resemble large, coiled protein ribbons. However, under 27 acidic conditions (such as those encountered when ingested by a eukaryotic cell), they dramatically 28 extend into a long, hollow tube that can disrupt membranes. R bodies are made from only four small 29 proteins, function independently of cells, and can withstand harsh conditions. As such, they hold 30 promise as tools to facilitate gene or drug delivery. 31 Here we show the R body extension process is reversible over many cycles and that R bodies are capable 32 of releasing E. coli cell contents into the environment. Furthermore, we generated a panel of mutant R 33 bodies that extend at varying pH values. These mutants demonstrate that R bodies can be tuned to 34 function in specific applications. 35 Introduction 36 R bodies (Type 51 refractile bodies) are ribbon-like protein polymers that are naturally expressed in the 37 cytoplasm of Caedibacter taeniospiralis, an endosymbiont of “killer” strains of Paramecium tetraurelia 38 . CC-BY-NC 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/029587 doi: bioRxiv preprint first posted online Oct. 22, 2015;