scholarly journals The flagellar substrate specificity switch protein FlhB assembles onto the extra-membrane export gate to regulate type three secretion

2019 ◽  
Author(s):  
Lucas Kuhlen ◽  
Steven Johnson ◽  
Andreas Zeitler ◽  
Sandra Bäurle ◽  
Justin C. Deme ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Conformational Changes ◽  
Complex Structure ◽  
Transmembrane Helices ◽  
Secretion Systems ◽  
Cryo Electron Microscopy ◽  
Type 3 Secretion System ◽  
Membrane Location ◽  
Type 3 ◽  
Type Three Secretion

AbstractExport of proteins through type three secretion systems (T3SS) is critical for motility and virulence of many major bacterial pathogens. Proteins are transported through an export gate complex consisting of three proteins (FliPQR in flagellar systems, SctRST in virulence systems) that were initially annotated as membrane proteins, but which we have recently shown assemble into an extra-membranous helical assembly. A fourth putative membrane protein (FlhB/SctU) is essential to the export process, and also functions to “switch” secretion substrate specificity once the growing hook/needle structures reach their determined length. Here we present the structure of an export gate containing the switch protein from a Vibrio polar flagellar system at 3.2 Å resolution by cryo-electron microscopy. The structure reveals that the FlhB/SctU further extends the helical export gate assembly with its four putative transmembrane helices adopting an out-of-membrane location, wrapped around the other export gate components at the base of the structure. The unusual topology of the switch protein helices creates a loop that wraps around the bottom of the closed export gate complex. Structure-informed mutagenesis suggests that this loop is critical in gating secretion and we propose that a series of conformational changes in the type 3 secretion system trigger opening of the export gate through the interactions between FlhB/SctU and FliPQR/SctRST.

scholarly journals A Primed Subpopulation of Bacteria Enables Rapid Expression of the Type 3 Secretion System in Pseudomonas aeruginosa

mBio ◽  
2021 ◽  
Author(s):  
Christina K. Lin ◽  
Daniel S. W. Lee ◽  
Saria McKeithen-Mead ◽  
Thierry Emonet ◽  
Barbara Kazmierczak
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Secretion System ◽  
Innate Immune ◽  
Secretion Systems ◽  
Content Type ◽  
Acute Infections ◽  
A Cell ◽  
Type 3 Secretion System ◽  
Type 3 ◽  
Specific Virulence

The expression of specific virulence traits is strongly associated with Pseudomonas aeruginosa ’s success in establishing acute infections but is thought to carry a cost for bacteria. Producing multiprotein secretion systems or motility organelles is metabolically expensive and can target a cell for recognition by innate immune system receptors that recognize structural components of the type 3 secretion system (T3SS) or flagellum.

scholarly journals The structure of an injectisome export gate demonstrates conservation of architecture in the core export gate between flagellar and virulence type three secretion systems

2019 ◽  
Author(s):  
Steven Johnson ◽  
Lucas Kuhlen ◽  
Justin C. Deme ◽  
Patrizia Abrusci ◽  
Susan M. Lea
Keyword(s):  
Electron Microscopy ◽  
Membrane Proteins ◽  
Complex Analysis ◽  
Secretion Systems ◽  
Core Complex ◽  
The Core ◽  
Cryo Electron Microscopy ◽  
Equivalent Complex ◽  
Type Three Secretion ◽  
Resolution Structure

AbstractExport of proteins through type three secretion systems (T3SS) is critical for motility and virulence of many major bacterial pathogens. Proteins are exported though a genetically defined export gate complex consisting of three proteins. We have recently shown at 4.2 Å that the flagellar complex of these three putative membrane proteins (FliPQR in flagellar systems, SctRST in virulence systems) assemble into an extra-membrane helical assembly that likely seeds correct assembly of the rod above. Here we present the structure of an equivalent complex from the more fragile Shigella virulence system at 3.5 Å by cryo-electron microscopy. This higher resolution structure reveals further detail and confirms the prediction of structural conservation in this core complex. Analysis of particle heterogeneity also reveals details of how the SctS/FliQ subunits sequentially assemble in the complex.

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

2018 ◽  
Author(s):  
Lucas Kuhlen ◽  
Patrizia Abrusci ◽  
Steven Johnson ◽  
Joseph Gault ◽  
Justin Deme ◽  
...  
Keyword(s):  
Integral Membrane Protein ◽  
Integral Membrane Proteins ◽  
Structural Elements ◽  
Secretion Systems ◽  
Core Component ◽  
The Core ◽  
Cryo Electron Microscopy ◽  
Type Three Secretion System ◽  
Structure Assembly ◽  
Type Three Secretion

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

scholarly journals Staying out or Going in? The Interplay between Type 3 and Type 5 Secretion Systems in Adhesion and Invasion of Enterobacterial Pathogens

2020 ◽  
Vol 21 (11) ◽  
pp. 4102
Author(s):  
Rachel Whelan ◽  
Gareth McVicker ◽  
Jack C. Leo
Keyword(s):  
Host Cell ◽  
Effector Proteins ◽  
Enterohemorrhagic Escherichia Coli ◽  
Secretion Systems ◽  
Bacterial Membranes ◽  
Cell Plasma ◽  
Cytoskeletal Dynamics ◽  
Shigella Spp ◽  
Type 3 Secretion System ◽  
Type 3

Enteric pathogens rely on a variety of toxins, adhesins and other virulence factors to cause infections. Some of the best studied pathogens belong to the Enterobacterales order; these include enteropathogenic and enterohemorrhagic Escherichia coli, Shigella spp., and the enteropathogenic Yersiniae. The pathogenesis of these organisms involves two different secretion systems, a type 3 secretion system (T3SS) and type 5 secretion systems (T5SSs). The T3SS forms a syringe-like structure spanning both bacterial membranes and the host cell plasma membrane that translocates toxic effector proteins into the cytoplasm of the host cell. T5SSs are also known as autotransporters, and they export part of their own polypeptide to the bacterial cell surface where it exerts its function, such as adhesion to host cell receptors. During infection with these enteropathogens, the T3SS and T5SS act in concert to bring about rearrangements of the host cell cytoskeleton, either to invade the cell, confer intracellular motility, evade phagocytosis or produce novel structures to shelter the bacteria. Thus, in these bacteria, not only the T3SS effectors but also T5SS proteins could be considered “cytoskeletoxins” that bring about profound alterations in host cell cytoskeletal dynamics and lead to pathogenic outcomes.

scholarly journals Activation ofShigella flexneritype 3 secretion requires a host-induced conformational change to the translocon pore

2019 ◽  
Author(s):  
Brian C. Russo ◽  
Jeffrey K. Duncan ◽  
Alexandra L. Wiscovitch ◽  
Austin C. Hachey ◽  
Marcia B. Goldberg
Keyword(s):  
Intermediate Filaments ◽  
Secretion System ◽  
Human Cells ◽  
Bacterial Virulence ◽  
Secretion Systems ◽  
Virulence Proteins ◽  
Type 3 Secretion System ◽  
Type 3 ◽  
Effector Secretion ◽  
Pore Protein

AbstractType 3 secretion systems (T3SSs) are conserved bacterial nanomachines that inject virulence proteins (effectors) into eukaryotic cells during infection. Due to their ability to introduce heterologous protein into human cells, these systems are being developed as therapeutic delivery devices. The T3SS assembles a translocon pore in the plasma membrane and then docks onto the pore. Docking activates effector secretion through the pore and into the host cytosol. Here, usingShigella flexneri, a model pathogen for the study of type 3 secretion, we determined the molecular mechanisms by which host intermediate filaments trigger docking and enable effector secretion. We show that the interaction of intermediate filaments with the translocon pore protein IpaC changed the pore’s conformation in a manner that was required for docking. Intermediate filaments repositioned residues of theShigellapore protein IpaC that are located on the surface of the pore and in the pore channel. Restricting these conformational changes blocked docking in an intermediate filament-dependent manner. These data demonstrate that a host-induced conformational change to the pore enables T3SS docking and effector secretion, providing new mechanistic insight into the regulation of type 3 secretion.Author summaryThe movement of bacterial proteins across membranes is essential for bacterial physiology and bacterial virulence. The type 3 secretion system moves bacterial virulence proteins from the inside of bacterial pathogens into human cells. To do so, the type 3 secretion system forms a pore in the plasma membrane of the target cell, attaches (docks) onto the pore, and delivers virulence proteins through the pore. Docking is essential for establishing a continuous channel from the inside of the bacteria to the inside of the human cell. What enables the type 3 secretion system to dock onto pores is not understood. We show that structural proteins in human cells, intermediate filament proteins, induce structural rearrangements to the type 3 secretion pore that trigger docking and that enable the subsequent delivery of virulence proteins into human cells. Due to the wide prevalence of type 3 secretion systems among human pathogens, these findings are likely to broadly enhance our understanding of type 3 secretion.

scholarly journals Structural basis for activation and gating of IP3 receptors

2021 ◽  
Author(s):  
Emily A Schmitz ◽  
Hirohide Takahashi ◽  
Erkan Karakas
Keyword(s):  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Receptor Activation ◽  
Structural Basis ◽  
Ip3 Receptors ◽  
Cellular Processes ◽  
Cryo Electron Microscopy ◽  
Channel Opening ◽  
High Concentrations ◽  
Type 3

Calcium (Ca2+) is a universal and versatile cellular messenger used to regulate numerous cellular processes in response to external or internal stimuli. A pivotal component of the Ca2+ signaling toolbox in cells is the inositol 1,4,5-triphosphate (IP3) receptors (IP3Rs), which mediate Ca2+ release from the endoplasmic reticulum (ER), controlling cytoplasmic and organellar Ca2+ concentrations. IP3Rs are activated by IP3 and Ca2+, inhibited by Ca2+ at high concentrations, and potentiated by ATP1-3. However, the underlying molecular mechanisms are unclear due to the lack of structures in the active conformation. Here we report cryo-electron microscopy (cryo-EM) structures of human type-3 IP3R in multiple gating conformations; IP3-ATP bound pre-active states with closed channels, IP3-ATP-Ca2+ bound active state with an open channel, and IP3-ATP-Ca2+ bound inactive state with a closed channel. The structures demonstrate how IP3-induced conformational changes prime the receptor for activation by Ca2+, how Ca2+ binding leads to channel opening, and how ATP modulates the activity, providing insights into the long-sought questions regarding the molecular mechanism of the receptor activation and gating.

Computer processing of high-resolution images of periodic specimens

1986 ◽  
Vol 44 ◽  
pp. 2-5
Author(s):  
W. Chiu ◽  
M.F. Schmid ◽  
T.-W. Jeng
Keyword(s):  
High Resolution ◽  
Fourier Transforms ◽  
Complex Structure ◽  
Distribution Functions ◽  
Multiple Image ◽  
Cryo Electron Microscopy ◽  
Structure Factors ◽  
Unit Cells ◽  
High Resolution Images ◽  
Phase Probability Distribution

Cryo-electron microscopy has been developed to the point where one can image thin protein crystals to 3.5 Å resolution. In our study of the crotoxin complex crystal, we can confirm this structural resolution from optical diffractograms of the low dose images. To retrieve high resolution phases from images, we have to include as many unit cells as possible in order to detect the weak signals in the Fourier transforms of the image. Hayward and Stroud proposed to superimpose multiple image areas by combining phase probability distribution functions for each reflection. The reliability of their phase determination was evaluated in terms of a crystallographic “figure of merit”. Grant and co-workers used a different procedure to enhance the signals from multiple image areas by vector summation of the complex structure factors in reciprocal space.

Crystal structures of Magnaporthe oryzae trehalose-6-phosphate synthase (MoTps1) suggest a model for catalytic process of Tps1

2019 ◽  
Vol 476 (21) ◽  
pp. 3227-3240 ◽  
Author(s):  
Shanshan Wang ◽  
Yanxiang Zhao ◽  
Long Yi ◽  
Minghe Shen ◽  
Chao Wang ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Magnaporthe Oryzae ◽  
Structural Information ◽  
Complex Structure ◽  
Critical Role ◽  
Catalytic Process ◽  
Structural Basis ◽  
Salt Bridges ◽  
Terminal Domain

Trehalose-6-phosphate (T6P) synthase (Tps1) catalyzes the formation of T6P from UDP-glucose (UDPG) (or GDPG, etc.) and glucose-6-phosphate (G6P), and structural basis of this process has not been well studied. MoTps1 (Magnaporthe oryzae Tps1) plays a critical role in carbon and nitrogen metabolism, but its structural information is unknown. Here we present the crystal structures of MoTps1 apo, binary (with UDPG) and ternary (with UDPG/G6P or UDP/T6P) complexes. MoTps1 consists of two modified Rossmann-fold domains and a catalytic center in-between. Unlike Escherichia coli OtsA (EcOtsA, the Tps1 of E. coli), MoTps1 exists as a mixture of monomer, dimer, and oligomer in solution. Inter-chain salt bridges, which are not fully conserved in EcOtsA, play primary roles in MoTps1 oligomerization. Binding of UDPG by MoTps1 C-terminal domain modifies the substrate pocket of MoTps1. In the MoTps1 ternary complex structure, UDP and T6P, the products of UDPG and G6P, are detected, and substantial conformational rearrangements of N-terminal domain, including structural reshuffling (β3–β4 loop to α0 helix) and movement of a ‘shift region' towards the catalytic centre, are observed. These conformational changes render MoTps1 to a ‘closed' state compared with its ‘open' state in apo or UDPG complex structures. By solving the EcOtsA apo structure, we confirmed that similar ligand binding induced conformational changes also exist in EcOtsA, although no structural reshuffling involved. Based on our research and previous studies, we present a model for the catalytic process of Tps1. Our research provides novel information on MoTps1, Tps1 family, and structure-based antifungal drug design.

scholarly journals Comparative Genomics of Xanthomonaseuroxanthea and Xanthomonas arboricola pv. juglandis Strains Isolated from a Single Walnut Host Tree

2021 ◽  
Vol 9 (3) ◽  
pp. 624
Author(s):  
Camila Fernandes ◽  
Leonor Martins ◽  
Miguel Teixeira ◽  
Jochen Blom ◽  
Joël F. Pothier ◽  
...  
Keyword(s):  
Extracellular Enzymes ◽  
Walnut Tree ◽  
Size Number ◽  
A Genome ◽  
Type 3 Secretion System ◽  
Xanthomonas Arboricola ◽  
Chromosomal Sequences ◽  
Related Proteins ◽  
Type 3 ◽  
Genome Comparisons

The recent report of distinct Xanthomonas lineages of Xanthomonas arboricola pv. juglandis and Xanthomonas euroxanthea within the same walnut tree revealed that this consortium of walnut-associated Xanthomonas includes both pathogenic and nonpathogenic strains. As the implications of this co-colonization are still poorly understood, in order to unveil niche-specific adaptations, the genomes of three X. euroxanthea strains (CPBF 367, CPBF 424T, and CPBF 426) and of an X. arboricola pv. juglandis strain (CPBF 427) isolated from a single walnut tree in Loures (Portugal) were sequenced with two different technologies, Illumina and Nanopore, to provide consistent single scaffold chromosomal sequences. General genomic features showed that CPBF 427 has a genome similar to other X. arboricola pv. juglandis strains, regarding its size, number, and content of CDSs, while X. euroxanthea strains show a reduction regarding these features comparatively to X. arboricola pv. juglandis strains. Whole genome comparisons revealed remarkable genomic differences between X. arboricola pv. juglandis and X. euroxanthea strains, which translates into different pathogenicity and virulence features, namely regarding type 3 secretion system and its effectors and other secretory systems, chemotaxis-related proteins, and extracellular enzymes. Altogether, the distinct genomic repertoire of X. euroxanthea may be particularly useful to address pathogenicity emergence and evolution in walnut-associated Xanthomonas.

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