The Architecture of the Cytoplasmic Region of Type III Secretion Systems

Makino, Fumiaki; Shen, Danhong; Kajimura, Naoko; Kawamoto, Akihiro; Pissaridou, Panayiota; Oswin, Henry; Pain, Maria; Murillo, Isabel; Namba, Keiichi; Blocker, Ariel

doi:10.1038/srep33341

Cited by 33 publications

(28 citation statements)

References 42 publications

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“…It is also important to consider that Spa47 is currently the only T3SS ATPase for which an active subhexameric oligomer has been identified, and although it is admittedly unclear what role (if any) this putative trimeric complex plays in vivo, it has provided an invaluable tool for discerning the mechanistic role of Spa47 oligomerization in enzyme activation and T3SS function. The observed increase in activity levels by the oligomer and the AUC assignment of the Spa47 oligomer geometry as consistent with a triangular trimer (15) suggest that the active site protomer interfaces within the trimeric complex likely mimic those of the hexamer observed by electron microscopy (34,57) and are modeled based on our recent Spa47 structures (Figs. 2 and 3).…”

Section: ⌬1-79supporting

confidence: 56%

Structural and Biochemical Characterization of Spa47 Provides Mechanistic Insight into Type III Secretion System ATPase Activation and Shigella Virulence Regulation

Burgess¹,

Burgess²,

Morales

et al. 2016

Journal of Biological Chemistry

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Edited by Patrick SungLike many Gram-negative pathogens, Shigella rely on a complex type III secretion system (T3SS) to inject effector proteins into host cells, take over host functions, and ultimately establish infection. Despite these critical roles, the energetics and regulatory mechanisms controlling the T3SS and pathogen virulence remain largely unclear. In this study, we present a series of high resolution crystal structures of Spa47 and use the structures to model an activated Spa47 oligomer, finding that ATP hydrolysis may be supported by specific side chain contributions from adjacent protomers within the complex. Follow-up mutagenesis experiments targeting the predicted active site residues validate the oligomeric model and determined that each of the tested residues are essential for Spa47 ATPase activity, although they are not directly responsible for stable oligomer formation. Although N-terminal domain truncation was necessary for crystal formation, it resulted in strictly monomeric Spa47 that is unable to hydrolyze ATP, despite maintaining the canonical ATPase core structure and active site residues. Coupled with studies of ATPase inactive full-length Spa47 point mutants, we find that Spa47 oligomerization and ATP hydrolysis are needed for complete T3SS apparatus formation, a proper translocator secretion profile, and Shigella virulence. This work represents the first structure-function characterization of Spa47, uniquely complementing the multitude of included Shigella T3SS phenotype assays and providing a more complete understanding of T3SS ATPase-mediated pathogen virulence. Additionally, these findings provide a strong platform for follow-up studies evaluating regulation of Spa47 oligomerization in vivo as a much needed means of treating and perhaps preventing shigellosis.

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Section: ⌬1-79supporting

confidence: 56%

Structural and Biochemical Characterization of Spa47 Provides Mechanistic Insight into Type III Secretion System ATPase Activation and Shigella Virulence Regulation

Burgess¹,

Burgess²,

Morales

et al. 2016

Journal of Biological Chemistry

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show abstract

“…However, detailed analysis showed that the number of spots per bacterium was slightly (7–20%) lower for the soluble components, compared to the IM ring protein SctD/YscD, which raises the possibility that not all components are present in all injectisomes. In support of this hypothesis, a recent structural study found a lower electron density in the cytosolic complex of a subset of injectisomes38.…”

Section: Discussionmentioning

confidence: 79%

A dynamic and adaptive network of cytosolic interactions governs protein export by the T3SS injectisome

et al. 2017

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Many bacteria use a type III secretion system (T3SS) to inject effector proteins into host cells. Selection and export of the effectors is controlled by a set of soluble proteins at the cytosolic interface of the membrane spanning type III secretion ‘injectisome’. Combining fluorescence microscopy, biochemical interaction studies and fluorescence correlation spectroscopy, we show that in live Yersinia enterocolitica bacteria these soluble proteins form complexes both at the injectisome and in the cytosol. Binding to the injectisome stabilizes these cytosolic complexes, whereas the free cytosolic complexes, which include the type III secretion ATPase, constitute a highly dynamic and adaptive network. The extracellular calcium concentration, which triggers activation of the T3SS, directly influences the cytosolic complexes, possibly through the essential component SctK/YscK, revealing a potential mechanism involved in the regulation of type III secretion.

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“…The organization of the cytoplasmic platform is poorly understood because it disassociates from the NC after purification and therefore is not accessible to single particle cryo-EM analysis (Lara-Tejero et al, 2011). Recent cryo electron tomography (cryo-ET) studies have been able to visualize this structure in various pathogens (Hu et al, 2015; Kawamoto et al, 2013b; Kudryashev et al, 2013; Makino et al, 2016; Nans et al, 2015). In Shigella the cytoplasmic sorting platform appears as a six-pod structure, which is capped at one end by a central hub (Hu et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

In Situ Molecular Architecture of the Salmonella Type III Secretion Machine

Hu¹,

Lara‐Tejero

Kong

et al. 2017

Cell

190

323

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Type III protein secretion systems have specifically evolved to deliver bacterially encoded proteins into target eukaryotic cells. The core elements of this multi-protein machine are the envelope-associated needle complex, the inner membrane export apparatus, and a large cytoplasmic sorting platform. Here we report a high-resolution in situ structure of the Salmonella Typhimurium type III secretion machine obtained by high-throughput cryo-electron tomography and sub-tomogram averaging. Through molecular modeling and comparative analysis of machines assembled with protein-tagged components or from different deletion mutants we determined the molecular architecture of the secretion machine in situ and localized its structural components. We also show that docking of the sorting platform results in significant conformational changes in the needle complex to provide the symmetry adaptation required for the assembly of the entire secretion machine. These studies provide major insight into the structure and assembly of a broadly distributed protein secretion machine.

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The Architecture of the Cytoplasmic Region of Type III Secretion Systems

Cited by 33 publications

References 42 publications

Structural and Biochemical Characterization of Spa47 Provides Mechanistic Insight into Type III Secretion System ATPase Activation and Shigella Virulence Regulation

Structural and Biochemical Characterization of Spa47 Provides Mechanistic Insight into Type III Secretion System ATPase Activation and Shigella Virulence Regulation

A dynamic and adaptive network of cytosolic interactions governs protein export by the T3SS injectisome

In Situ Molecular Architecture of the Salmonella Type III Secretion Machine

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