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An extensively glycosylated archaeal pilus survives extreme conditions

Abstract

Pili on the surface of Sulfolobus islandicus are used for many functions, and serve as receptors for certain archaeal viruses. The cells grow optimally at pH 3 and ~80 °C, exposing these extracellular appendages to a very harsh environment. The pili, when removed from cells, resist digestion by trypsin or pepsin, and survive boiling in sodium dodecyl sulfate or 5 M guanidine hydrochloride. We used electron cryo-microscopy to determine the structure of these filaments at 4.1 Å resolution. An atomic model was built by combining the electron density map with bioinformatics without previous knowledge of the pilin sequence—an approach that should prove useful for assemblies where all of the components are not known. The atomic structure of the pilus was unusual, with almost one-third of the residues being either threonine or serine, and with many hydrophobic surface residues. While the map showed extra density consistent with glycosylation for only three residues, mass measurements suggested extensive glycosylation. We propose that this extensive glycosylation renders these filaments soluble and provides the remarkable structural stability. We also show that the overall fold of the archaeal pilin is remarkably similar to that of archaeal flagellin, establishing common evolutionary origins.

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Fig. 1: Cryo-EM of the LAL14/1 pilus.
Fig. 2: De novo atomic model building of LAL14/1 pilin.
Fig. 3: The LAL14/1 pilus contains an unusually high percentage of hydrophobic residues.
Fig. 4: O-linked sugar modifications of the LAL14/1 pilus.
Fig. 5: Comparison of the LAL14/1 pilus with bacterial T4P and the archaeal flagellar filament.

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Data availability

The three-dimensional reconstruction has been deposited in the Electron Microscopy Data Bank with accession code EMD-0397. The atomic model has been deposited in the Protein Data Bank with accession code 6NAV. The mass spectrometry data have been deposited in PRIDE with accession code PXD012799.

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Acknowledgements

This work was supported by NIH grants GM122510 (to E.H.E.) and GM123089 (to F.D.), as well as l’Agence Nationale de la Recherche project ENVIRA (ANR-17-CE15-0005-01 to M.K.). M.A.B.K. was supported by NIH grant T32 GM080186. The cryo-EM imaging conducted at the Molecular Electron Microscopy Core facility at the University of Virginia was supported by the School of Medicine and built with NIH grant G20-RR31199. The Titan Krios and Falcon II direct electron detectors were obtained with NIH grants S10-RR025067 and S10-OD018149, respectively. We thank V. Conticello for the suggestion of TFMS. We are also grateful to the Ultrastructural BioImaging (UTechS UBI) unit of Institut Pasteur for access to electron microscopes.

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V.C.-K. isolated and purified the pili. J.S.W. performed the STEM analysis. N.S. performed the mass spectrometry. G.A.P.d.O. performed the amyloid assays. F.D. performed the interfacial analysis. F.W. performed the cryo-EM, three-dimensional reconstruction and model building, with assistance from T.O. and E.H.E. M.K. and Z.S. performed the bioinformatics analysis. M.A.B.K. performed the TFMS deglycosylation. D.P., M.K. and E.H.E. designed the project. F.W., D.P., M.K. and E.H.E. wrote the paper.

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Correspondence to Mart Krupovic or Edward H. Egelman.

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Wang, F., Cvirkaite-Krupovic, V., Kreutzberger, M.A.B. et al. An extensively glycosylated archaeal pilus survives extreme conditions. Nat Microbiol 4, 1401–1410 (2019). https://doi.org/10.1038/s41564-019-0458-x

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