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A syringe-like injection mechanism in Photorhabdus luminescens toxins

Abstract

Photorhabdus luminescens is an insect pathogenic bacterium that is symbiotic with entomopathogenic nematodes1. On invasion of insect larvae, P. luminescens is released from the nematodes and kills the insect through the action of a variety of virulence factors including large tripartite ABC-type toxin complexes2 (Tcs). Tcs are typically composed of TcA, TcB and TcC proteins and are biologically active only when complete3,4,5. Functioning as ADP-ribosyltransferases, TcC proteins were identified as the actual functional components that induce actin-clustering, defects in phagocytosis and cell death5,6,7. However, little is known about the translocation of TcC into the cell by the TcA and TcB components. Here we show that TcA in P. luminescens (TcdA1) forms a transmembrane pore and report its structure in the prepore and pore state determined by cryoelectron microscopy. We find that the TcdA1 prepore assembles as a pentamer forming an α-helical, vuvuzela-shaped channel less than 1.5 nanometres in diameter surrounded by a large outer shell. Membrane insertion is triggered not only at low pH as expected, but also at high pH, explaining Tc action directly through the midgut of insects8. Comparisons with structures of the TcdA1 pore inserted into a membrane and in complex with TcdB2 and TccC3 reveal large conformational changes during membrane insertion, suggesting a novel syringe-like mechanism of protein translocation. Our results demonstrate how ABC-type toxin complexes bridge a membrane to insert their lethal components into the cytoplasm of the host cell. We believe that the proposed mechanism is characteristic of the whole ABC-type toxin family. This explanation of toxin translocation is a step towards understanding the host–pathogen interaction and the complex life cycle of P. luminescens and other pathogens, including human pathogenic bacteria, and serves as a strong foundation for the development of biopesticides.

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Figure 1: Cryoelectron microscopy structure of the TcdA1 prepore complex.
Figure 2: Cryoelectron microscopy structure of the TcdA1 pore and comparison with the prepore complex.
Figure 3: Structure of the PTC3 holotoxin complex.
Figure 4: Syringe-like mechanism for membrane insertion.

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Data deposits

The coordinates for the electron microscopy structures have been deposited in EMDataBank under accession codes EMD-2297 to EMD-2301.

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Acknowledgements

We are grateful to R. S. Goody for continuous support and for comments on the manuscript. We thank I. Vetter for stimulating discussions. This work was supported by the Deutsche Forschungsgemeinschaft grants RA 1781/1-1 (S.R.) and AK 6/22-1 (K.A.) and by Max Planck Society (S.R.).

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Authors

Contributions

A.E.L. and V.P. cloned constructs and purified proteins; D.M. performed pH studies; A.E.L. and R.B. performed and analysed black lipid bilayer assays; C.G. screened samples and collected negative-stain electron microscopy data; C.G. and O.H. collected cryoelectron microscopy data; C.G. processed and refined electron microscopy data; C.G. and S.R. analysed electron microscopy data; K.A. and S.R. designed the study. C.G. and S.R. wrote the paper. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Stefan Raunser.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-14, Supplementary Methods, Supplementary Table 1 and Supplementary References. (PDF 1909 kb)

Cryo-EM structure of the TcdA1 prepore complex

The video shows a simple linear interpolation between the negative stain and cryo-EM reconstruction of TcdA1. The two domains of the outer shell and the central pore are highlighted in green, khaki and yellow, respectively. In the end, the video focuses on the central pore and subsequently on a protomer, depicting the fitted α-helices. (MOV 5964 kb)

Morph between the negative stain EM reconstructions of TcdA1 in prepore and pore state

The video shows the two states in side and cut-away view and a simple linear interpolation between them. (MOV 4966 kb)

Syringe-like mechanism for membrane insertion

(Animated version of Fig. 4). The two domains of the outer shell and the central pore of TcdA1 are depicted in green, khaki and yellow, respectively. The dimeric complex of TcdB2 and TccC3 is depicted in orange. A brown bar indicates the membrane bilayer. (MOV 845 kb)

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Gatsogiannis, C., Lang, A., Meusch, D. et al. A syringe-like injection mechanism in Photorhabdus luminescens toxins. Nature 495, 520–523 (2013). https://doi.org/10.1038/nature11987

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