Three-Dimensional Model of
            <i>Salmonella</i>
            ’s Needle Complex at Subnanometer Resolution

Schraidt, Oliver; Marlovits, Thomas C.

doi:10.1126/science.1199358

Cited by 190 publications

(275 citation statements)

References 26 publications

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“…Their connections with the basal body appear to be delicate, potentially explaining why they typically are not retained during purification of basal bodies (9,10). The pods also were not resolved in a recent in situ structure from whole Yersinia cells (11).…”

Section: Cryoelectron Tomography Of Shigella Minicells Reveals Intactmentioning

confidence: 99%

“…3D reconstructions of purified injectisomes from Salmonella and Shigella, together with the atomic structures of major basal body proteins, have provided a detailed view of basal body architecture (9,10). Recent in situ structures of injectisomes from Shigella flexneri, Salmonella enterica, and Yersinia enterocolitica revealed an export gate and the structural flexibility of the basal body (11,12).…”

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confidence: 99%

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Visualization of the type III secretion sorting platform of Shigella flexneri

Hu¹,

Morado²,

Margolin³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

204

300

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Bacterial type III secretion machines are widely used to inject virulence proteins into eukaryotic host cells. These secretion machines are evolutionarily related to bacterial flagella and consist of a large cytoplasmic complex, a transmembrane basal body, and an extracellular needle. The cytoplasmic complex forms a sorting platform essential for effector selection and needle assembly, but it remains largely uncharacterized. Here we use high-throughput cryoelectron tomography (cryo-ET) to visualize intact machines in a virulent Shigella flexneri strain genetically modified to produce minicells capable of interaction with host cells. A high-resolution in situ structure of the intact machine determined by subtomogram averaging reveals the cytoplasmic sorting platform, which consists of a central hub and six spokes, with a pod-like structure at the terminus of each spoke. Molecular modeling of wild-type and mutant machines allowed us to propose a model of the sorting platform in which the hub consists mainly of a hexamer of the Spa47 ATPase, whereas the MxiN protein comprises the spokes and the Spa33 protein forms the pods. Multiple contacts among those components are essential to align the Spa47 ATPase with the central channel of the MxiA protein export gate to form a unique nanomachine. The molecular architecture of the Shigella type III secretion machine and its sorting platform provide the structural foundation for further dissecting the mechanisms underlying type III secretion and pathogenesis and also highlight the major structural distinctions from bacterial flagella.nanomachine | injectisome | pathogen-host interaction | cryo-electron tomography | protein secretion

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Section: Cryoelectron Tomography Of Shigella Minicells Reveals Intactmentioning

confidence: 99%

mentioning

confidence: 99%

Visualization of the type III secretion sorting platform of Shigella flexneri

Hu¹,

Morado²,

Margolin³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

204

300

View full text Add to dashboard Cite

show abstract

“…The needle is capped at its end by the tip complex, which is thought to coordinate the activation of the secretion machine upon contact with target cells (9,10). Because the needle complex can be isolated in a manner suitable for single-particle cryo-electron microscopy, its organization at the quasi-atomic level is known (11)(12)(13)(14)(15)(16)(17)(18). Passage of the secreted proteins through the inner membrane requires the export apparatus, which is composed of a group of poly-topic inner membrane proteins located at the center of the needle complex base (19).…”

mentioning

confidence: 99%

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

Zhang

Lara‐Tejero

Bewersdorf

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

103

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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.bacterial nanomachines | protein secretion | superresolution microscopy | bacterial pathogenesis | Salmonella virulence T ype III secretion systems are remarkable molecular machines with the capacity to inject multiple bacterial proteins into eukaryotic cells (1, 2). These machines play an essential role in the pathogenic or symbiotic relationships between the bacteria that encode them and their respective hosts. Consequently, type III secretion systems are rapidly emerging as targets for the development of novel therapeutic and prevention strategies with the potential to combat several important infectious diseases (3-5). The components of type III secretion machines are highly conserved across bacterial species although the effector proteins that they deliver are customized for the specific bacteria that harbor them (1, 6, 7). The entire type III secretion machine or injectisome is ∼40 nm in width and ∼150 nm in length and is organized in defined substructures (Fig. 1A). The bestcharacterized substructure is the needle complex, which is a multi-ring cylindrical structure embedded in the bacterial envelope with a needle-like extension that projects several nanometers from the bacterial surface. The needle complex is traversed by a narrow channel that serves as the conduit for the proteins that travel this secretion pathway (8). The needle is capped at its end by the tip complex, which is thought to coordinate the activation of the secretion machine upon contact with target cells (9, 10). Because the needle complex can be isolated in a manner suitable for single-particle cryo-electron microscopy, its organization at the quasi-atomic level is known (11-18). Passage of the secreted proteins through the inner membrane requires the export apparatus, which is composed of a group of poly-topic inner membrane proteins located at the center of the needle complex base (19). Finally, several cytoplasmic proteins form a large complex known as the...

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“…Because of recent outstanding improvements in structural biology and particularly in cryo-electron microscopy (cryo-EM), acknowledged by the 2017 Nobel prize for chemistry, the pace at which larger secretion systems has dramatically increased. We can now, for example, visualize the whole T3SS (2,3) or exquisite details of the T4SS (4). In the latter case, visualization came with a surprise in that what composes the outer membrane channel has no resemblance to our standard belief; in particular, the portion of the protein which plugs the system into the outer membrane is not a ␤-barrel but a series of amphipathic ␣-helices.…”

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confidence: 99%

Multiple Structures Disclose the Secretins' Secrets

Filloux

Voulhoux

2018

J Bacteriol

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Bacterial secretins are outer membrane proteins that provide a path for secreted proteins to access the cell exterior/surface. They are one of the core components of secretion machines and are found in type II and type III secretion systems (T2SS and T3SS, respectively). The secretins comprise giant ring-shaped homooligomers whose precise atomic organization was only recently deciphered thanks to spectacular developments in cryo-electron microscopy (cryo-EM) imaging techniques.

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Three-Dimensional Model of Salmonella ’s Needle Complex at Subnanometer Resolution

Abstract: A cryo–electron microscopy structure of a bacterial secretion complex reveals threefold symmetry.

Cited by 190 publications

References 26 publications

Visualization of the type III secretion sorting platform of Shigella flexneri

Visualization of the type III secretion sorting platform of Shigella flexneri

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

Multiple Structures Disclose the Secretins' Secrets

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