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

ABSTRACT: 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.

INSTRUMENT(S): impact II

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

SUBMITTER: Liam Worrall

LAB HEAD: Natalie Strynadka

PROVIDER: PXD005133 | Pride | 2016-12-19

REPOSITORIES: Pride

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Near-atomic-resolution cryo-EM analysis of the Salmonella T3S injectisome basal body.

Worrall L J LJ Hong C C Vuckovic M M Deng W W Bergeron J R C JRC Majewski D D DD Huang R K RK Spreter T T Finlay B B BB Yu Z Z Strynadka N C J NCJ

Nature 20161214 7634

The type III secretion (T3S) injectisome is a specialized protein nanomachine that is critical for the pathogenicity of many Gram-negative bacteria, including purveyors of plague, typhoid fever, whooping cough, sexually transmitted infections and major nosocomial infections. This syringe-shaped 3.5-MDa macromolecular assembly spans both bacterial membranes and that of the infected host cell. The internal channel formed by the injectisome allows for the direct delivery of partially unfolded virul ...[more]

PMID: 27974800

Similar Datasets

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

Project description: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.

2018-10-16 | PXD010239 | Pride

FliP subcomplex analysis - A flagellum-specific chaperone facilitates assembly of the core type III export apparatus of the bacterial flagellum

Project description:Biosynthesis of the bacterial flagellum depends on a flagellar-specific type III secretion system (T3SS), a protein export machine homologous to the export machinery of the virulence-associated injectisome. Six cytoplasmic (FliH/I/J/G/M/N) and seven integral membrane proteins (FlhA/B FliF/O/P/Q/R) form the flagellar basal body and are involved in transport of flagellar building blocks across the inner membrane in a protein motive force-dependent manner. FlhA/B and FliP/Q/R are conserved in all T3SS and form the export gate complex. FliO is absent in some T3SS and was presumed to regulate FliP during flagellum assembly. However, the molecular function of FliO and how the large, multi-component transmembrane export gate complex assembles in a coordinated manner remained enigmatic. Here, we demonstrate that FliO protects FliP from proteolytic degradation and promotes formation of FliP-containing subcomplexes during the first step of core export apparatus assembly. We further reveal the sub-cellular localization of FliO by dSTORM superresolution microscopy and show that FliO is not part of the assembled flagellar basal body. In summary, our results suggest that FliO functions as a novel, flagellar T3SS-specific chaperone, which facilitates quality control and productive assembly of the core T3SS export machinery.

2017-08-22 | PXD005597 | Pride

Transcriptional profile of Salmonella enterica serovar Typhimurium 4/74 wild-type and mutants using strand-specific RNA-seq

Project description:New tools for studying bacterial transcripts at the single nucleotide level offer an unparalleled opportunity to understand the bacterial transcriptome. For the model pathogen Salmonella enterica serovar Typhimurium, it is necessary to identify the regulatory inputs for all RNA transcripts, including small RNAs (sRNAs) and coding genes. Here, we use RNA-seq to define the transcriptomes of mutants lacking 18 global regulatory systems that, among other functions, modulate the expression of the SPI1 and SPI2 Type Three secretion systems in S. Typhimurium strain 4/74. We directly compared the roles of the major regulators of transcription, and reported the effects of the regulatory mutations on expression of sRNAs. We also use this method to describe the impact of the RNA chaperone Hfq upon the steady state levels of 280 sRNA transcripts. Transcriptome analysis of S. Typhimurium 4/74 using RNA isolated wild-type and mutants grown under infection-relevant conditions.

2016-08-09 | E-GEOD-79314 | biostudies-arrayexpress

Reduced basal insulin secretion from islets isolated from tafazzin deficient mice is associated with reduced mitochondrial function

Project description:Tafazzin is a transacylase which causes the content and molecular structure of cardiolipin (CL) to be altered in the inner mitochondrial membrane. The enzymatic machinery responsible for oxidative respiration are localized to the inner mitochondrial membrane and require CL for activity. As a result, tafazzin is critical for maintaining mitochondrial function. In beta-cells, which are the insulin secreting cells in pancreatic islets, mitochondrial function is linked to insulin secretion. We previously established that tafazzin knock-down mice were lean and maintained insulin sensitivity compared to the obese insulin resistant control litter mates. However, it was unknown whether tafazzin deficiency also influenced insulin secretion in the establishment of the lean phenotype. Using X week-old mice, we ascertained that the number of beta-cells was similar between genotypes. Ex vivo insulin secretion under low glucose conditions was reduced (52%) from islets isolated from tafazzin knock-down mice. Consistent with this tafazzin knock-down mice exhibited reduced fasting insulin plasma insulin levels. Measurement of mitochondrial oxygen consumption revealed that basal oxygen consumption was reduced (58%) in islets from tafazzin-deficient animals. The addition of etomoxir, an inhibitor of mitochondrial fatty acid translocation, significantly elevated basal mitochondrial respiration (5-fold) in islets from tafazzin knockdown mice, indicating a significant fatty acid-mediated inhibitory effect. In contrast, etomoxir lacked any measurable effect on control wild-type islets. The altered mitochondrial function identified in taffazin knock-down islets was not associated with reduced CL content or elevated oxyCL species. Our experiments indicate that tafazzin plays a key role in regulating normal islet beta-cell function.

2021-07-31 | GSE136242 | GEO

Project description:RNA sequencing of apical and basal inner hair cells

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The ternary complex CEP90, FOPNL and OFD1 specifies the future location of centriolar distal appendages, and promotes their assembly

Project description:Cilia assembly starts with centriole to basal body maturation, migration to the cell surface and docking to the plasma membrane. The basal body docking process involves the interaction of both the distal end of the basal body and the transition fibers (or mature distal appendages), with the plasma membrane. During this process, the transition zone assembles and forms the structural junction between the basal body and the nascent cilium. Mutations in numerous genes involved in basal body docking and transition zone assembly are associated with the most severe ciliopathies, highlighting the importance of these events in cilium biogenesis. The conservation of this sequence of events across phyla is paralleled by a high conservation of the proteins involved. We identified CEP90 by BioID using FOPNL as a bait. Ultrastructure expansion microscopy showed that CEP90, FOPNL and OFD1 localized at the distal end of both centrioles/basal bodies in Paramecium and mammalian cells. These proteins are recruited early after duplication on the procentriole. Finally, functional analysis performed both in Paramecium and mammalian cells demonstrate the requirement of this complex for distal appendage assembly and basal body docking. Altogether, we propose that this ternary complex is required to determine the future position of distal appendages.

2022-07-20 | PXD028740 | Pride

Stress Responses in Bacteria Induced by a Giant Channel

Project description:Pathogenic bacteria export large proteins and protein complexes, including virulence factors, using dedicated trans-envelope multi-protein machineries, collectively called secretion systems. Four of these protein export machines found in Gram-negative bacteria: type 2 and 3 secretion systems, the type 4 pilus biogenesis system and the filamentous phage assembly-secretion system (FFSS), contain large homologous gated channels in the outer membrane, called secretins. These channels are radially symmetrical homomultimers (luminal diameter 6-8 nm) interrupted by an internal septum, which blocks the channel in its resting state. By analyzing the transcriptome of Escherichia coli producing the FFSS secretin pIV and an isogenic leaky-gate variant, we have expanded the known secretin responses to include the Sox, Cpx, Rcs and RyhB regulons, in addition to the known secretin responder, the Psp regulon. Furthermore, we show that responses dependent on transcriptional regulators CpxR, SoxS and RcsB (in addition to PspF) are required for survival of E. coli producing the leaky-gate variant of secretin pIV, while CpxR, SoxS and PspF are required for survival of cells producing wild-type T3SS secretin InvG. Our analysis shows that secretins induce three out of six envelope stress responses and responses to oxidative stress. Identification of the key stress response regulators required for survival of secretin stress suggests pathways that may be of interest in developing strategies for control of secretin-expressing pathogenic bacteria.

2021-01-31 | GSE85194 | GEO

Proteomic analysis of secreted proteins from Salmonella cells deficient in STnc470

Project description:This study has the main goal of studying the impact of the small non coding RNA STnc470 on protein expression and secretion.

2017-05-03 | PXD003590 | Pride

Loss of Macf1 abolishes ciliogenesis and disrupts apicobasal polarity establishment in the retina

Project description:Microtubule actin crosslinking factor 1 (Macf1) plays a role in coordinated actions of actin and microtubules in multiple cellular processes. Here we show that Macf1 is also critical for ciliogenesis in multiple cell types. Ablation of Macf1 in the developing retina abolishes ciliogenesis and basal bodies fail to dock to ciliary vesicles or migrate apically. Photoreceptor polarity is randomized while inner retinal cells laminate correctly, suggesting that photoreceptor maturation is guided by polarity cues provided by cilia. Deletion of Macf1 in adult photoreceptors caused reversal of basal body docking and loss of outer segments, reflecting a continuous requirement for Macf1 function. Macf1 was also shown to interact with ciliary proteins Mkks and Talpid3. We propose that a disruption of trafficking across microtubles to actin filaments underlies the ciliogenesis defect in cells lacking Macf1, and that Mkks and Talpid3 are involved in the coordination of microtubule and actin interactions.

2016-11-01 | GSE86997 | GEO

Inefficient secretion of anti-sigma factor FlgM inhibits bacterial motility at high temperature

Project description:Regulation of gene expression according to the environmental conditions is vitally important for all living organisms. Temperature is one of the key cues for microorganisms that can exist in different environments, and several mechanisms of temperature-dependent re-allocation of cellular resources have been described. Here we compared transcriptome and proteome of E. coli grown at basal (37°C) and febrile temperatures (42°C) of the mammalian host. We demonstrated that the downregulation of Escherichia coli motility at high temperature primarily occurred due to the malfunction of secretion apparatus, apparently related to the disassembly of the flagellar basal body. For proteome analysis we performed data-independent acquisition (DIA) mass spectrometry via Spectronaut. The Spectronaut file can be assessed with the freely available Spectronaut viewer (https://www.biognosys.com/technology/spectronaut-viewer).

2019-08-14 | PXD012611 | Pride

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