In Situ Molecular Architecture of the Salmonella Type III Secretion Machine

Hu, Bo; Lara‐Tejero, María; Kong, Qingke; Galán, Jorge E.; Li, Jun

doi:10.1016/j.cell.2017.02.022

Cited by 188 publications

(347 citation statements)

References 56 publications

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“…Because the sorting platform dissociates from the needle complex during isolation, its organization and structure are not as well understood (22)(23)(24). Recent cryo-electron tomography studies have provided unique insight into the molecular architecture of the sorting platform although the precise stoichiometry of some of its components is not known (25,26). Although emphasis has been placed on obtaining the highest possible resolution view of the type III secretion machine and its components, there are important questions that require visualization of individual machines in the context of the entire live bacterium.…”

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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.

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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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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.

Self Cite

103

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“…This complex is constituted by a central ATPase oligomeric cylinder formed by InvC (SctN in the unified nomenclature), which is linked through its negative regulator OrgB (SctL) to the flagellar C‐ring orthologue SpaO (SctQ) and the accessory protein OrgA (SctK). Additionally, InvC interacts through its central pore to the stalk protein InvI (SctO) …”

Section: Introductionmentioning

confidence: 99%

Structural analysis of ligand‐bound states of the Salmonella type III secretion system ATPase InvC

et al. 2019

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Translocation of virulence effector proteins through the type III secretion system (T3SS) is essential for the virulence of many medically relevant Gram‐negative bacteria. The T3SS ATPases are conserved components that specifically recognize chaperone–effector complexes and energize effector secretion through the system. It is thought that functional T3SS ATPases assemble into a cylindrical structure maintained by their N‐terminal domains. Using size‐exclusion chromatography coupled to multi‐angle light scattering and native mass spectrometry, we show that in the absence of the N‐terminal oligomerization domain the Salmonella T3SS ATPase InvC can form monomers and dimers in solution. We also present for the first time a 2.05 å resolution crystal structure of InvC lacking the oligomerization domain (InvCΔ79) and map the amino acids suggested for ATPase intersubunit interaction, binding to other T3SS proteins and chaperone–effector recognition. Furthermore, we validate the InvC ATP‐binding site by co‐crystallization of InvCΔ79 with ATPγS (2.65 å) and ADP (2.80 å). Upon ATP‐analogue recognition, these structures reveal remodeling of the ATP‐binding site and conformational changes of two loops located outside of the catalytic site. Both loops face the central pore of the predicted InvC cylinder and are essential for the function of the T3SS ATPase. Our results present a fine functional and structural correlation of InvC and provide further details of the homo‐oligomerization process and ATP‐dependent conformational changes underlying the T3SS ATPase activity.

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“…The integral membrane domain of this protein would therefore be predicted to lie in the inner membrane around the base of our PQR complex. We note that in situ tomograms of the Salmonella injectisome show a distinct density in this location but that cells deficient in A not only lack this density but also demonstrate a distorted membrane leaflet that reaches up to the base of the Q subunits 19 (Fig. S11).…”

Section: Cc-by 40 International License Peer-reviewed) Is the Authormentioning

confidence: 99%

“…We therefore sought to determine the location of the PQR complex in the assembled T3SS. Inspection of earlier single particle reconstructions of isolated basal bodies 17 and in vivo tomograms from both flagellar 18 and injectisome 19 T3SSs demonstrated that the PQR complex forms the structure previously described as the rod "cup and socket" 8,20 . This is particularly striking when fitting our model within the highest resolution structure of a basal body determined to date, where the height, diameter and shape of the density seen in the basal body are an excellent match (Fig.…”

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Structure of the Core of the Type Three Secretion System Export Apparatus

Kuhlen

Abrusci

Johnson

et al. 2018

Preprint

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SummaryExport of proteins through type three secretion systems is critical for bacterial motility and virulence of many major bacterial pathogens. Three putative integral membrane proteins (FliP/FliQ/FliR) are suggested to form the core of an export gate in the inner membrane, but their structure, assembly and location within the final nanomachine remain unclear. We here present the structure of this complex at 4.2 Å by cryo-electron microscopy. None of the subunits adopt canonical integral membrane protein topologies and common helix-turn-helix structural elements allow them to form a helical assembly with 5:4:1 stoichiometry. Fitting of the structure into reconstructions of intact secretion systems localize the export gate as a core component of the periplasmic portion of the machinery, and cross-linking experiments confirm this observation. This study thereby identifies the export gate as a key element of the secretion channel and implies that it primes the helical architecture of the components assembling downstream.One Sentence SummaryThe core of the T3SS export gate forms a supra-membrane helical assembly

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In Situ Molecular Architecture of the Salmonella Type III Secretion Machine

Cited by 188 publications

References 56 publications

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

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

Structural analysis of ligand‐bound states of the Salmonella type III secretion system ATPase InvC

Structure of the Core of the Type Three Secretion System Export Apparatus

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