High-resolution Cryo-EM visualises hydrated type I and IV pilus structures from enterotoxigenic Escherichia coli

Abstract

To efficiently colonise intestinal epithelium, enteric pathogens utilise a wide variety of pilus filaments, including those synthesised by the chaperone-usher or type IV pilus assembly pathway. Despite the importance of these filaments as potential drug and vaccine targets, their large size and dynamic nature present challenges for atomic-resolution structural determination. Here, we used cryo-electron microscopy (cryo-EM) and whole genome sequencing to identify and determine the structures of type I and IV pili expressed in enterotoxigenic Escherichia coli. The well-defined cryo-EM maps at resolutions of 2.20 and 1.78 [A] for Type I and Type IV pilus, respectively, facilitated de novo structural modelling for these filaments, revealing side chain structures in detail. Further, we resolved thousands of hydrated water molecules around and within the inner core of the filaments, functioning as a buffer to stabilise the otherwise metastable subunit assembly. The high-resolution structures offer novel insights into the subunit-subunit interactions, providing important clues to understand pilus assembly, stability, and flexibility.