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. These pili, when removed from cells, resist digestion by trypsin or pepsin, and survive boiling in SDS or 5M guanidinium-HCl. We have used cryo-EM to determine the structure of these filaments at 4.1 Å resolution. An atomic model was built by combining the map with bioinformatics without prior 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 a 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 archaeal flagellin, establishing common evolutionary origins.

Escape of TLR5 Recognition by Leptospira spp.: A Rationale for Atypical Endoflagella

“…Interestingly, we also showed a very high stability of the leptospiral filaments and FlaB proteins that resist heating to 100 • C for 30 min and 85 • C for 3 h. This unusual thermoresistance of the leptospiral flagella is reminiscent of the hydrophobic and very highly glycosylated pili of hyperthermophilic Archaea (53). Glycosylations also occur in bacteria.…”

The structures of two archaeal type IV pili illuminate evolutionary relationships

“…Thus, to identify the exact pilin genes encoding receptors of SSRV1 and PFV2, we have sequenced the complete genomes of S. solfataricus POZ149 and P. arsenaticum 2GA (see Methods), and used a combination of mass spectrometry, bioinformatics, and cryo-EM to build de novo the atomic models ( Fig. 2a, b), as was done for the pili from S. islandicus 26 . The result in each case was finding a single protein sequence that could be threaded through the maps, which is HC235_07175 for P. arsenaticum (Par_PilA) and HFC64_06120 for S. solfataricus (Sso_PilA) ( Supplementary Figs.…”

“…Remarkably, the S. islandicus pilus was found to be extremely resilient under various harsh conditions, including extended treatment with different proteases and even boiling in 5 M guanidine hydrochloride. The reason for the robustness of the pilus was attributed to a combination of profound hydrophobicity and extensive surface glycosylation of the filament 26 . Notably, the resilience of the S. islandicus pilus precluded the identification of the constituent pilin protein using conventional approaches, such as gel electrophoresis and mass spectrometry.…”

“…Notably, the resilience of the S. islandicus pilus precluded the identification of the constituent pilin protein using conventional approaches, such as gel electrophoresis and mass spectrometry. Instead, the identity of the protein was determined using a combination of cryo-EM and bioinformatics, whereby candidate protein sequences were directly threaded through the density maps, which allowed building an atomic model de novo, exemplifying the power of cryo-EM for protein identification 26 .…”

Surface resistance to SSVs and SIRVs in pilin deletions of Sulfolobus islandicus

“…Minimally, all type IV pili including the archaellum are comprised of biosynthesis machinery including an ATPase (PilB), multi‐spanning membrane protein (PilC) and the pilin subunits themselves (PilA). Archaea generally carry multiple type IV pili loci in each genome (Wang et al, ) with these minimal components in a single operon (Albers & Pohlschröder, ; Esquivel et al, ; Makarova, Koonin, & Albers, ; Zolghadr, Klingl, Rachel, Driessen, & Albers, ). Pili structure and functions are all dependent on the enzyme PibD which is responsible for the maturation of the type IV prepilin subunits for the assembly of the pilus (Albers, Szabó, & Driessen, ; Szabó, Sani, et al, ; Szabó, Stahl, et al, ).…”