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Principal Investigator: MARCUS AARON HORWITZ
Organization: UNIVERSITY OF CALIFORNIA LOS ANGELES
Fiscal Year: 2020
Award: $549,664
Funding agency: National Institute of Allergy and Infectious Diseases
Project Summary/Abstract
Francisella tularensis is a bacterium that causes tularemia, a disease which, when in its pneumonic form, can
be fatal even with appropriate treatment. Due to its low infectious dose, ease of spread by aerosol, and high
virulence, F. tularensis is classified as a Tier 1 Select Agent by the U.S. federal government. This R01 project
builds on our earlier identification (by contact PI Horwitz's group) of the Francisella Type VI Secretion System
(T6SS) and our subsequent determination (by Horwitz's and MPI Zhou's group) of the first atomic models of its
sheath and its uniquely endowed central spike complex through cryo electron microscopy (cryoEM). T6SSs are
large, complex, multi-protein nanomachines that Gram-negative bacteria use to sense environmental cues and
deliver toxins into other bacteria or into eukaryotic hosts; in Francisella, they mediate phagosome escape and
intracytoplasmic replication. They are important virulence determinants, present in 25% of Gram-negative
bacteria and in an even higher percentage of those that are human pathogens. However, without knowing
T6SS composition and structure, we cannot fully understand its mechanisms of pathogenesis nor effectively
design countermeasures against a myriad of bacterial diseases. The T6SS of Francisella is both significant
and attractive to study because of the high infectivity and lethality of Francisella species and its relative
simplicity compared with other T6SSs. However, significant knowledge gaps remain, including the following:
(1) an atomic model of the structure of the pre-contraction outer sheath; (2) the composition and an atomic
model of the baseplate and membrane complex; and (3) the composition of the Francisella central spike and
secreted effector protein complex and an atomic model of its interaction with the sheath, baseplate, and
membrane complex in the pre-contraction state and during the contraction process.
To fill these gaps, we propose to carry out three major structure-function studies on T6SS using
Francisella novicida [and its closely related F. tularensis live vaccine strain (LVS)] as a model. First, we shall
obtain the atomic model of the sheath and tube complex in purified T6SS in its pre-contraction state with
cryoEM, and elucidate the energetics and mechanism of T6SS contraction by structural comparison with the
contracted sheath and structure-guided mutagenesis. Second, using proximity labeling, crosslinking, affinity
pull-down, immunoblotting, proteomics, and bacterial 2-hybrid analyses, we shall determine the composition
and protein interactions of the baseplate and membrane core complex. This information will be used in
conjunction with cryo electron tomography of T6SS-containing mini-cells to determine the composition and
structure of the T6SS baseplate and membrane complex in their pre- and post-contraction states. Third, we
shall determine the composition and structure of the Francisella T6SS central spike and secreted effector
complex. The results will form the foundation for future function studies and the development of new strategies
for treating and preventing diseases caused by the numerous important pathogenic bacteria that have a T6SS.
Terms: <Aerosols><Affinity><Attenuated Vaccines><B pseudomallei><B. pseudomallei><Bacteria><Bacterial Infections><Biochemical Genetics><Biological Terrorism><Bioterrorism><Burkholderia pseudomallei><Cell Body><Cells><Chemicals><Complex><Contracting Opportunities><Contracts><Cryo-electron Microscopy><Cryo-electron tomography><Cryoelectron Microscopy><Cues><Cytosol><Development><Disease><Disorder><Dose><E coli><E. coli><Early identification><Electron Cryomicroscopy><Electrons><Escherichia coli><Eukaryotic Cell><Exhibits><F tularensis><F tularensis infection><F. tularensis><F. tularensis infection><Federal Government><Foundations><Francisella><Francisella infection><Francisella tularensis><Francisella tularensis infection><Free Energy><Freezing><Future><Genes><Genetics-Mutagenesis><Goals><Gram-Negative Bacteria><Human><Hybrids><Immunoblotting><In Situ><Infection><Infection prevention><Injections><Knowledge><Label><Life Style><Lifestyle><Macromolecular Structure><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Mediating><Membrane><Methods><Modeling><Modern Man><Molecular Configuration><Molecular Conformation><Molecular Stereochemistry><Molecular Structure><Morbidity><Morbidity - disease rate><Mutagenesis><Mutagenesis Molecular Biology><National Government><Negative Beta Particle><Negatrons><P pseudomallei><P. pseudomallei><Pasteurella tularensis><Pathogenesis><Pathogenicity><Pathogenicity Island><Phagosomes><Prevent infection><Process><Prokaryotae><Prokaryotic Cells><Proteins><Proteomics><Pseudomonas pseudomallei><Rest><Role><Rotation><Sequence Homology><Structural Models><Structure><System><Technology><Testing><Toxin><Tube><Tularemia><V cholerae><V. cholerae><Vibrio cholerae><Vibrio comma><Virulence><Virulent><bacteria infection><bacterial disease><bacterial pathogen><combat><conformation><conformational state><cross-link><crosslink><cryo-EM><cryo-EM tomography><cryoEM><cryoEM tomography><cryoelectron tomography><design><designing><developmental><electron cryo-tomography><gene manipulation><genetic manipulation><genetically manipulate><genetically perturb><human disease><human pathogen><infected with F tularensis><infected with F. tularensis><infected with Francisella tularensis><live vaccine><macrophage><membrane structure><mortality><nano machine><nanomachine><particle><pathogenic bacteria><prevent><preventing><prokaryote><protein complex><reconstruction><social role>