Pathogenic bacteria use specialized ?nanomachines? to identify and interact with host cells. These machines are attractive drug targets because they are surface-exposed, widespread, and vital for pathogenicity. While one of these machines, the Type III Secretion System, has been well studied, other systems remain relatively poorly understood. Here, we propose to use electron cryotomography (ECT) to dissect the structures and functions of multiple pathogenic nanomachines. ECT is a powerful technique to image intact structures with macromolecular (2-5 nm) resolution inside cells. Previously, we applied ECT to the Type VI Secretion System (T6SS), where our images immediately revealed its spring-loaded contractile mechanism. Going a step further, we realized that we could combine ECT and high-resolution subtomogram averaging with available knowledge from other techniques to produce a complete architectural model of the Myxococcus xanthus Type IVa Pilus (T4aP). This produced a flood of new mechanistic insights and inspired us to apply the same approach to pathogenic secretion systems. In this project, we propose to use ECT to reveal the structure and function of pathogenic Type IV Pilus (T4P), Type VI Secretion (T6SS), and Type IV Secretion System (T4SS) machineries. For each system, we will image the entire, intact structure in situ. In most cases, this will be the first high-resolution imaging of these structures. We will then combine subtomogram averaging with difference analysis of mutants in which individual components are knocked out or tagged in order to produce architectural models of the structures. In cases where crystal structures of components (or homologs) are available, we will dock them into our maps to produce pseudo-atomic models of each machine. By comparing these structures with those of non-pathogenic relatives (solved previously or in the current study), we aim to identify adaptations underlying virulence functions. We will also apply state-of-the-art cryogenic-focused ion beam milling and correlated cryogenic fluorescence light microscopy and ECT to image secretion structures in action ? in bacterial cells infecting eukaryotic hosts. This will provide the first such images of the critical human pathogens Helicobacter pylori and Legionella pneumophila, which we expect to provide invaluable insights into the operation of their pathogenic machinery in vivo. Together, we expect this project to produce a detailed mechanistic picture of the T4P, T6SS, and T4SS nanomachines that mediate pathogenesis, an important first step in identifying therapeutic targets in the future.

Public Health Relevance

Pathogenic bacteria have a devastating effect on public health, and the rise of antibiotic-resistant strains means new drug targets are desperately needed. Bacterial secretion systems are critical virulence factors and represent attractive therapeutic targets, but their structures and mechanisms remain largely unclear. In the proposed work, we will use state-of-the-art imaging techniques to reveal the native molecular structures of the secretion systems that pathogenic bacteria use to infect humans, leading to functional insights and potential drug targets for the future.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI127401-04
Application #
9765150
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Mills, Melody
Project Start
2016-09-23
Project End
2021-08-31
Budget Start
2019-09-01
Budget End
2020-08-31
Support Year
4
Fiscal Year
2019
Total Cost
Indirect Cost
Name
California Institute of Technology
Department
Type
Schools of Arts and Sciences
DUNS #
009584210
City
Pasadena
State
CA
Country
United States
Zip Code
91125
Chang, Yi-Wei; Shaffer, Carrie L; Rettberg, Lee A et al. (2018) In Vivo Structures of the Helicobacter pylori cag Type IV Secretion System. Cell Rep 23:673-681
Oikonomou, Catherine M; Jensen, Grant J (2017) Cellular Electron Cryotomography: Toward Structural Biology In Situ. Annu Rev Biochem 86:873-896
Dobro, Megan J; Oikonomou, Catherine M; Piper, Aidan et al. (2017) Uncharacterized bacterial structures revealed by electron cryotomography. J Bacteriol :
Ghosal, Debnath; Chang, Yi-Wei; Jeong, Kwangcheol C et al. (2017) In situ structure of the Legionella Dot/Icm type IV secretion system by electron cryotomography. EMBO Rep 18:726-732
Jeong, Kwangcheol C; Ghosal, Debnath; Chang, Yi-Wei et al. (2017) Polar delivery of Legionella type IV secretion system substrates is essential for virulence. Proc Natl Acad Sci U S A 114:8077-8082
Briegel, Ariane; Oikonomou, Catherine M; Chang, Yi-Wei et al. (2017) Morphology of the archaellar motor and associated cytoplasmic cone in Thermococcus kodakaraensis. EMBO Rep 18:1660-1670
Chang, Yi-Wei; Rettberg, Lee A; Ortega, Davi R et al. (2017) In vivo structures of an intact type VI secretion system revealed by electron cryotomography. EMBO Rep 18:1090-1099
Chang, Yi-Wei; Kjær, Andreas; Ortega, Davi R et al. (2017) Architecture of the Vibrio cholerae toxin-coregulated pilus machine revealed by electron cryotomography. Nat Microbiol 2:16269