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Identification of proteins in the Salmonella SPI-1 type III secretion injectisome needle complex

ABSTRACT: We determined the cryo-EM structure of the type III secretion injectisome needle complex. We used LC-MS/MS to idensitfy the constituent proteins showing the predominant presence of the major inner- and outer basal body proteins (PrgH+PrgK and InvG respectively), export apparatus protein SpaP and SpaR, and the rod and needle filament proteins PrgJ and PrgI.

INSTRUMENT(S): impact II

ORGANISM(S): Salmonella Typhimurium (strain Lt2 / Sgsc1412 / Atcc 700720)

SUBMITTER: Liam Worrall

LAB HEAD: Natalie Strynadka

PROVIDER: PXD010239 | Pride | 2018-10-16

REPOSITORIES: pride

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			Action	DRS
	analysis.baf	Other
	ceba4fee-0a00-4522-95d2-db0cd4dbc59d_1.mcf	Other

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Publications

Cryo-EM analysis of the T3S injectisome reveals the structure of the needle and open secretin.

Hu J J Worrall L J LJ Hong C C Vuckovic M M Atkinson C E CE Caveney N N Yu Z Z Strynadka N C J NCJ

Nature communications 20180921 1

The bacterial type III secretion system, or injectisome, is a syringe shaped nanomachine essential for the virulence of many disease causing Gram-negative bacteria. At the core of the injectisome structure is the needle complex, a continuous channel formed by the highly oligomerized inner and outer membrane hollow rings and a polymerized helical needle filament which spans through and projects into the infected host cell. Here we present the near-atomic resolution structure of a needle complex f ...[more]

PMID: 30242280

Similar Datasets

Identification of proteins in a Salmonella SPI-1 type III secretion injectisome basal body mutant

Project description:We determined the cryo-EM structure of a secretion incompetent PrgH130-392 type III secretion injectisome basal body mutant at 6.3 Å. The map shows no density attributable to the inner rod or needle and the periplasmic gate in a closed conformation. We used LC-MS/MS to idensitfy the constituent proteins showing the predominant presence of the major inner- and outer basal body proteins (PrgH+PrgK and InvG respectively) and export apparatus protein SpaP.

2016-12-19 | PXD005133 | Pride

Stoichiometry of the bacterial type III secretion export apparatus

Project description:Bacterial type III secretion systems are cell envelope-spanning effector protein-delivery machines essential for colonization and survival of many Gram-negative pathogens and symbionts. The membrane-embedded core unit of these secretion systems is termed needle complex. The needle complex is composed of a base that anchors the machinery to the inner and outer membranes, a hollow filament formed by inner rod and needle subunits that serves as conduit for substrate proteins, and a membrane-embedded export apparatus facilitating substrate translocation. While the stoichiometry of the base and of the major export apparatus protein have been revealed by structural analyses, the stoichiometries of the other export apparatus components and of the inner rod remain unknown. We employed peptide concatenated standard and absolute quantification-based strategies to analyze the stoichiometry of the entire needle complex by mass spectrometry. Here we provide evidence that the export apparatus of the type III secretion system encoded on Salmonella pathogenicity island 1 contains 5 SpaP, 1 SpaQ, 1 SpaR, and 1 SpaS. We have corroborated the previously suggested stoichiometry of 9 InvA per needle complex and describe a loose association of InvA with other needle complex components that may reflect its function. These numbers indicate that the inner membrane patch of the needle complex base houses 104 transmembrane domains in total, a dense assembly whose function in the secretion process we merely understand. Furthermore, we present evidence that not more than 6 PrgJ form the inner rod of the needle complex.

2016-02-25 | PXD003113 | Pride

SpaP and SpaR interaction partner identification

Project description:Bacterial type III protein secretion systems inject effector proteins into eukaryotic host cells in order to promote survival and colonization of Gram-negative pathogens and symbionts. Secretion across the bacterial cell envelope and injection into host cells is facilitated by a so-called needle complex. Its small hydrophobic export apparatus components SpaP and SpaR were shown to nucleate assembly of the needle complex and to form the central “cup” substructure of a Salmonella Typhimurium secretion system, however, the in vivo placement of these components in the needle complex and their function during the secretion process remained poorly defined. Here we show that a SpaP pentamer forms a 15 Å wide pore and present a detailed map of SpaP interactions with the export apparatus components SpaQ, SpaR, and SpaS. We further demonstrate the formation of a continuous conduit for substrate translocation and injection by intimate interactions of the periplasmic domains of SpaP and SpaR with the inner rod protein PrgJ, refine the current view of export apparatus assembly and consolidate transmembrane topology models for SpaP and SpaR.

2016-11-29 | PXD005028 | Pride

Transcriptomic analysis of Lysinibacillus varians reveal the genetic foundation of its cell cycle

Project description:Lysinibacillus varians GY32 was isolated from river sediment of electronic waste recycling site. Its invariably filament-to-rod cell cycle represents a novel bacteria morphogenesis that is crucial in understanding cell division coordination with lifecycle and environmental bacteria adaptation. A description of genes and biological processes involved in the special filament-to-rod cell cycle of L. varians GY32 is within reach.

2017-08-15 | GSE99804 | GEO

Lamin A dimer and tetramer cross-links help explain disease mutations and polymer plasticity

Project description:Lamins are intermediate filament proteins responsible for nuclear mechanical integrity. Though linked to multiple heritable diseases, lamin structure and that of other intermediate filaments remains elusive. We employed cross-linking mass spectrometry to gain structural insights into lamin A dimer and tetramer structure. While confirming the parallel coiled-coil rod, we report that, contrary to prediction, rod linker regions are highly flexible and compressible. This explains the recently reported rod shortening in the assembled polymer and aspects of intermediate filament stretch properties. We further elucidate an extended interface in lamin A tetramers where both head and tail unstructured regions flanking the rod of each dimer act as polar bridges to stabilize lamin head-to-tail assembly. Furthermore, changes in these regions between dimer and tetramer forms suggest an ordered polymer assembly. Importantly, several residues mutated in laminopathies disrupt this interface and impair assembly, potentially explaining their role in disease.

2019-07-12 | PXD008337 | Pride

Lamin A molecular compression and sliding as mechanisms behind nucleoskeleton elasticity (complementary data of PXD008337)

Project description:Lamin A is a nuclear intermediate filament protein critical for nuclear architecture and mechanics and mutated in a wide range of human diseases. Yet little is known about the molecular architecture of lamins and mechanisms of their assembly. Here we use cross-linking SILAC mass spectrometry to determine interactions within lamin dimers and between dimers in higher-order polymers. We find evidence for a compression mechanism where coiled coils in the lamin A rod can slide onto each other to contract rod length, likely driven by a wide range of electrostatic interactions with the flexible linkers between coiled coils. Similar interactions occur with unstructured regions flanking the rod domain during oligomeric assembly. Mutations linked to human disease block these interactions, suggesting that this spring-like contraction can explain in part the dynamic mechanical stretch and flexibility properties of the lamin polymer and other intermediate filament networks.

2019-07-12 | PXD014009 | Pride

Lamin A upregulation reorganizes the genome during rod photoreceptor degeneration

Project description:Two proteins have previously been shown to be necessary and sufficient to tether heterochromatin at the nuclear envelope. The lamin B receptor (Lbr) is expressed during development, but downregulates upon rod differentiation. A second tether is the intermediate filament lamin A (LA), which is not normally expressed in murine rods.To determine how heterochromatin tethering affects the genome, we used in vivo electroporation to misexpress LA or Lbr in mature rods in the absence of degeneration, resulting in the restoration of conventional nuclear architecture. Using scRNA-seq, we show that reorganizing the nucleus via LA/Lbr misexpression has only minor effects on rod gene expression. Next, using ATAC-seq, we show that LA and Lbr similarly increase genome accessibility. Novel ATAC-seq peaks tended to distal to genes, but some peaks were associated with stress-responsive genes. Together, our data reveal that heterochromatin tethers have a global effect on genome accessibility, and suggest that LA –dependent genome reconfiguration might contribute to the stress response of rod photoreceptors.

2023-10-12 | GSE240465 | GEO

The Alk receptor tyrosine kinase regulates Sparkly, a novel activity regulating neuropeptide precursor in the Drosophila CNS

Project description:Numerous roles for the Alk receptor tyrosine kinase have been described in Drosophila, including functions in the CNS, however the molecular details are poorly understood. To gain mechanistic insight, we employed Targeted DamID (TaDa) transcriptional profiling to identify targets of Alk signaling in the larval CNS. TaDa was employed in larval CNS tissues, while genetically manipulating Alk signaling output. The resulting TaDa data were analysed together with larval CNS scRNA-seq datasets performed under similar conditions, identifying a role for Alk in the transcriptional regulation of neuroendocrine gene expression. Further integration with bulk/scRNA-seq and protein datasets from larval brains in which Alk signaling was manipulated, identified a previously uncharacterized Drosophila neuropeptide precursor encoded by CG4577 as an Alk signaling transcriptional target. CG4577, which we named Sparkly (Spar), is expressed in a subset of Alk-positive neuroendocrine cells in the developing larval CNS, including circadian clock neurons. In agreement with our TaDa analysis, overexpression of the Drosophila Alk ligand Jeb resulted in increased levels of Spar protein in the larval CNS. We show that Spar protein is expressed in circadian (Clock) neurons, and Spar mutants exhibit defects in sleep and circadian rhythm control. In summary, we report a novel activity regulating neuropeptide precursor gene that is regulated by Alk signaling in the Drosophila CNS.

2024-03-05 | GSE229518 | GEO

Methylation of Salmonella Typhimurium flagella promotes bacterial adhesion and host cell invasion

Project description:The long external filament of bacterial flagella is composed of several thousand copies of a single protein, flagellin. Here, we explore the role played by lysine methylation of flagellin in Salmonella, which requires the methylase FliB. We show that both flagellins of Salmonella enterica serovar Typhimurium, FliC and FljB, are methylated at surface-exposed lysine residues by FliB. A Salmonella Typhimurium mutant deficient in flagellin methylation is outcompeted for gut colonization in a gastroenteritis mouse model, and methylation of flagellin promotes bacterial invasion of epithelial cells in vitro. Lysine methylation increases the surface hydrophobicity of flagellin and enhances flagella-dependent adhesion of Salmonella to phosphatidylcholine vesicles and epithelial cells.

2020-04-27 | PXD017935 | Pride

A Multiplexed, Next-Generation Sequencing Platform for High-Throughput Detection of SARS-CoV-2 [extended cohort]

Project description:Population scale sweeps of viral pathogens, such as SARS-CoV-2, require high intensity testing for effective management. However, reliable systems affording parallel testing of thousands of patients for pathogen infection have not yet been routinely employed. Here we describe “Systematic Parallel Analysis of RNA coupled to Sequencing for Covid-19 screening” (C19-SPAR-Seq), a multiplexed, readily automated platform for SARS-CoV-2 detection capable of analyzing tens of thousands of patient samples in a single instrument run. To address strict requirements for control of assay parameters and output demanded by clinical diagnostics, we employed a control-based Precision-Recall and Receiver Operator Characteristics (coPR) analysis to assign run-specific quality control metrics. C19-SPAR-Seq coupled to coPR on a trial cohort of several hundred patients performed with a specificity of 100% and sensitivity of 91% on samples with low viral loads. Our study thus establishes the feasibility of employing C19-SPAR-Seq for the large-scale monitoring of SARS-CoV-2 and other pathogens.

2021-01-24 | GSE160034 | GEO

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