Structural Basis of Flagellar Filament Asymmetry and Supercoil Templating by Leptospira Sheath Factors
Date of Award
Doctor of Philosophy (PhD)
Molecular Biophysics and Biochemistry
The spirochete family of bacteria, including pathogens such as Borrelia burgdorferi (Lyme disease), Treponema pallidum (syphilis), and Leptospira interrogans (leptospirosis), generates a unique, corkscrew-like form of motility that is crucial for pathogenicity. This motility is driven by flagella that are periplasmic rather than extracellular in nature and are composed of a FlaB core and a FlaA sheath. Purified Leptospira flagellar filaments form tight coils and contain the coiling proteins FcpA and FcpB, in addition to two FlaA isoforms. Loss of either the FlaA or Fcp proteins results in straighter flagella and bacteria that are non-motile and non-pathogenic. I have used cryo-electron microscopy to solve the structure of three Leptospira flagellar mutants to near-atomic resolution. First, I resolved the structure of an fcpB- mutant, allowing for identification of the FcpB density in the sheath region of the asymmetric wild-type structure. I also developed a method to quantify the curvature of purified flagellar filaments, allowing us to investigate how the flagellar forms are affected by various mutations. Next, I determined the structure of a fcpA- mutant, and found that the FlaA sheath components strictly localize to the inner curvature. We also discovered a previously uncharacterized flagellar sheath protein, which we have named FlaAP (FlaA-associated protein). We found that these sheath proteins interact with the core through interactions with glycans, and that the FlaB core appears to have ten ‘L’ protofilaments and one ‘R’ protofilament, with the ‘R’ protofilament directly underlying the sheath proteins and disrupting the helical symmetry of the core. Third, I solved the structure of an flaA2- mutant. In doing so, I found that whereas the Fcp coiling factors bind solely to the outer curvature of the wild-type filament, forming an extensive helical lattice, this Fcp lattice extends symmetrically around the entire FlaB core when the FlaA sheath factors are missing. These three mutant flagellar structures provide crucial insight into the role of the flagellar sheath factors and highlight the importance of the supercoiled wild-type form for motility and virulence. This work has implications for other spirochetes as well as other flagellated bacteria, where structural work has mainly been focused on flagellar filaments rendered straight by specific mutations, instead of the naturally occurring supercoiled forms as we have described here.
Brady, Megan Rose, "Structural Basis of Flagellar Filament Asymmetry and Supercoil Templating by Leptospira Sheath Factors" (2022). Yale Graduate School of Arts and Sciences Dissertations. 564.