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Proc Natl Acad Sci U S A. 2017 Jun 6;114(23):6098-6103. doi: 10.1073/pnas.1705823114. Epub 2017 May 22.

Visualization and characterization of individual type III protein secretion machines in live bacteria.

Author information

1
Department of Cell Biology, School of Medicine, Yale University, New Haven, CT 06520.
2
Department of Microbial Pathogenesis, School of Medicine, Yale University, New Haven, CT 06536.
3
Department of Cell Biology, School of Medicine, Yale University, New Haven, CT 06520; jorge.galan@yale.edu joerg.bewersdorf@yale.edu.
4
Department of Biomedical Engineering, School of Medicine, Yale University, New Haven, CT 06520.
5
Department of Microbial Pathogenesis, School of Medicine, Yale University, New Haven, CT 06536; jorge.galan@yale.edu joerg.bewersdorf@yale.edu.

Abstract

Type III protein secretion machines have evolved to deliver bacterially encoded effector proteins into eukaryotic cells. Although electron microscopy has provided a detailed view of these machines in isolation or fixed samples, little is known about their organization in live bacteria. Here we report the visualization and characterization of the Salmonella type III secretion machine in live bacteria by 2D and 3D single-molecule switching superresolution microscopy. This approach provided access to transient components of this machine, which previously could not be analyzed. We determined the subcellular distribution of individual machines, the stoichiometry of the different components of this machine in situ, and the spatial distribution of the substrates of this machine before secretion. Furthermore, by visualizing this machine in Salmonella mutants we obtained major insights into the machine's assembly. This study bridges a major resolution gap in the visualization of this nanomachine and may serve as a paradigm for the examination of other bacterially encoded molecular machines.

KEYWORDS:

Salmonella virulence; bacterial nanomachines; bacterial pathogenesis; protein secretion; superresolution microscopy

PMID:
28533372
PMCID:
PMC5468683
DOI:
10.1073/pnas.1705823114
[Indexed for MEDLINE]
Free PMC Article

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