Bacterial motility and its driving force, the flagellar motor, are important virulence factors of B. burgdorferi (the causative agent of Lyme disease) and many other bacteria. The flagellar motor is a remarkable nano-machine, powered by the proton (or sodium) gradient across the cytoplasmic membrane. The coupling of an electrochemical gradient to mechanical rotation is one of the most fascinating features of this molecular machine. The flagellar motor is composed of two major components: the stator and the rotor. Although prior structural studies have revealed the stunning complexity of the flagellar rotor, the mechanism of energy coupling in the flagellar motor remains poorly understood at the molecular level, mainly because of the lack of structural information about the membrane-bound stator and the rotor- stator interactions involved in flagellar rotation. The central hypothesis is that a high- resolution structure of the rotor-stator-C ring interface will provide vital structural information needed to propose models on the mechanisms of rotation and reversal. The objective of this application is to determine the structure/function relationship of the intact flagellar motor in situ by combining novel high throughput Cryo-Electron Tomography (Cryo-ET) approaches with genetic analysis to study the model system, B. burgdorferi. By collaborating with Drs. Steven Norris, Nyles Charon. MD Motaleb, Chunhao Li and Hanspeter Winkler, we propose to focus on two specific aims:
Specific Aim 1 - Determine the detailed structure of the torque-generating unit by analyzing the 3-D structures of the complete flagellar motor and the purified flagellar rotor at 2 nm resolution.
Specific Aim 2 - Determine the structural and functional roles of individual flagellar proteins by comparative analysis of wild-type organisms and flagellar gene mutants. We believe that the detailed analysis of the rotor/stator assembly in situ may provide the clearest avenue yet available to understanding of the mechanism of flagellar rotation and bacterial motility, which will in turn be applicable to the pathogenesis of all spirochetes and other motile bacteria. In addition, the further development of high-throughput Cryo-ET as part of this project will be readily applied to gain new insights into the structural and functional relationship of macromolecular machines related to pathogenesis of a variety of human pathogens, and offer a wide spectrum of important biomedical information at molecular resolution in living organisms.

Public Health Relevance

Project Narrative/Relevance B. burgdorferi is a highly motile and invasive pathogen causing Lyme disease, the most common vector-borne infection in the United States. The bacterial motility and its driving force, the flagellar motor, are important virulence factors of B. burgdorferi and many other bacteria. By elucidating the molecular architecture of flagellar motor and the structural basis of mechanochemical coupling in flagellar rotation, we could gain insight into how the flagellar motor works and contributes to bacterial pathogenesis. The information resulting from these studies can be readily applied to understanding the motility of many pathogenic bacteria that depend on flagella-based movement to colonize, establish infection, and disseminate in humans and other hosts.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI087946-04
Application #
8423780
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Breen, Joseph J
Project Start
2010-02-15
Project End
2015-01-31
Budget Start
2013-02-01
Budget End
2014-01-31
Support Year
4
Fiscal Year
2013
Total Cost
$320,969
Indirect Cost
$74,859
Name
University of Texas Health Science Center Houston
Department
Pathology
Type
Schools of Medicine
DUNS #
800771594
City
Houston
State
TX
Country
United States
Zip Code
77225
Miller, Kelly A; Motaleb, Md A; Liu, Jun et al. (2014) Initial characterization of the FlgE hook high molecular weight complex of Borrelia burgdorferi. PLoS One 9:e98338
Zhao, Xiaowei; Norris, Steven J; Liu, Jun (2014) Molecular architecture of the bacterial flagellar motor in cells. Biochemistry 53:4323-33
Zhao, Xiaowei; Zhang, Kai; Boquoi, Tristan et al. (2013) Cryoelectron tomography reveals the sequential assembly of bacterial flagella in Borrelia burgdorferi. Proc Natl Acad Sci U S A 110:14390-5
Hu, Bo; Margolin, William; Molineux, Ian J et al. (2013) The bacteriophage t7 virion undergoes extensive structural remodeling during infection. Science 339:576-9
Zhang, Kai; Tong, Brian A; Liu, Jun et al. (2012) A single-domain FlgJ contributes to flagellar hook and filament formation in the Lyme disease spirochete Borrelia burgdorferi. J Bacteriol 194:866-74
Raddi, Gianmarco; Morado, Dustin R; Yan, Jie et al. (2012) Three-dimensional structures of pathogenic and saprophytic Leptospira species revealed by cryo-electron tomography. J Bacteriol 194:1299-306
Li, Chen; Kurniyati; Hu, Bo et al. (2012) Abrogation of neuraminidase reduces biofilm formation, capsule biosynthesis, and virulence of Porphyromonas gingivalis. Infect Immun 80:3-13
Liu, Jun; Chen, Cheng-Yen; Shiomi, Daisuke et al. (2011) Visualization of bacteriophage P1 infection by cryo-electron tomography of tiny Escherichia coli. Virology 417:304-11
Xu, Hongbin; Raddi, Gianmarco; Liu, Jun et al. (2011) Chemoreceptors and flagellar motors are subterminally located in close proximity at the two cell poles in spirochetes. J Bacteriol 193:2652-6
Motaleb, M A; Pitzer, Joshua E; Sultan, Syed Z et al. (2011) A novel gene inactivation system reveals altered periplasmic flagellar orientation in a Borrelia burgdorferi fliL mutant. J Bacteriol 193:3324-31

Showing the most recent 10 out of 14 publications