Structural Studies of Straight and Supercoiled Flagellar Filaments from Campylobacter jejuni

Abstract

Thousands of angstroms long, the flagellar filament serves as the propeller of the bacterial flagellum. In the two-state switching model, protofilaments of the flagella switch between one of two states which allows for changing between different flagellar waveforms, associated with different modes of motility. Many pathogenic bacteria have flagellar-based motility and thus produce thousands of copies of the flagellar filament subunit, the flagellin. A small region of about 10 amino acids in domain D1 in most bacterial flagellins such as those from Salmonella typhimurium and Bacillus subtilis are recognized by toll-like receptor 5 (TLR5) which then activates innate immune response. ε Proteobacteria such as Campylobacter jejuni and H. pylori escape detection by TLR5 due to sequence changes in this 10 AA region of their flagellin sequence. When mutated onto the S. typhimurium flagellin the H. pylori sequence impairs flagellar filament formation, thus motility. The similar C. jejuni sequence is thought to have the same destabilizing interactions. This leads to the question “How do ε Proteobacteria compensate for these destabilizing mutations in their D1 domains?”. With a 3.5 Å resolution cryoEM structure of straight C. jejuni G508A flagellar filaments we are able to show a unique and extensive network of interactions between the outer domains of adjacent flagellins which compensates for weakened interactions in D1. These interactions are further stabilized by glycosylation of specific residues with pseudaminic acid. Lastly, recent high-resolution structures of the wild type flagellar filament and flagellar hook have called into question the validity of the two-state switching model for flagella. Using single particle and helical cryoEM reconstruction techniques, as well as cryo-electron tomography and fluorescence light microscopy we investigate the structure of wildtype supercoiled C. jejuni flagellar filaments.