scholarly journals The type 3 secretion system requires actin polymerization to open translocon pores

2021 ◽  
Author(s):  
Brian C. Russo ◽  
Jeffrey K. Duncan-Lowey ◽  
Poyin Chen ◽  
Marcia B. Goldberg
Keyword(s):  
Plasma Membrane ◽  
Actin Polymerization ◽  
Shigella Flexneri ◽  
Coiled Coil ◽  
Model Organism ◽  
Secretion System ◽  
Pore Channel ◽  
Type 3 Secretion System ◽  
Type 3 ◽  
Pore Protein

Many bacterial pathogens require a type 3 secretion system (T3SS) to establish a niche. Host contact activates bacterial T3SS assembly of a translocon pore in the host plasma membrane. Following pore formation, the T3SS docks onto the translocon pore. Docking establishes a continuous passage that enables the translocation of virulence proteins, effectors, into the host cytosol. Here we investigate the contribution of actin polymerization to T3SS-mediated translocation. Using the T3SS model organism Shigella flexneri, we show that actin polymerization is required for assembling the translocon pore in an open conformation, thereby enabling effector translocation. Opening of the pore channel is associated with a conformational change to the pore, which is dependent upon actin polymerization and a coiled-coil domain in the pore protein IpaC. An IpaC mutant is identified that shows actin polymerization-dependent pore opening is distinct from the previously described actin polymerization-dependent ruffles that are required for bacterial internalization. Moreover, actin polymerization is not required for other pore functions, including docking or pore protein insertion into the plasma membrane. Thus, activation of the T3SS is a multilayered process in which host signals are sensed by the translocon pore leading to the activation of effector translocation.

scholarly journals The type 3 secretion system requires actin polymerization to open translocon pores

2021 ◽  
Vol 17 (9) ◽  
pp. e1009932
Author(s):  
Brian C. Russo ◽  
Jeffrey K. Duncan-Lowey ◽  
Poyin Chen ◽  
Marcia B. Goldberg
Keyword(s):  
Plasma Membrane ◽  
Actin Polymerization ◽  
Shigella Flexneri ◽  
Coiled Coil ◽  
Model Organism ◽  
Secretion System ◽  
Pore Channel ◽  
Type 3 Secretion System ◽  
Type 3 ◽  
Pore Protein

Many bacterial pathogens require a type 3 secretion system (T3SS) to establish a niche. Host contact activates bacterial T3SS assembly of a translocon pore in the host plasma membrane. Following pore formation, the T3SS docks onto the translocon pore. Docking establishes a continuous passage that enables the translocation of virulence proteins, effectors, into the host cytosol. Here we investigate the contribution of actin polymerization to T3SS-mediated translocation. Using the T3SS model organism Shigella flexneri, we show that actin polymerization is required for assembling the translocon pore in an open conformation, thereby enabling effector translocation. Opening of the pore channel is associated with a conformational change to the pore, which is dependent upon actin polymerization and a coiled-coil domain in the pore protein IpaC. Analysis of an IpaC mutant that is defective in ruffle formation shows that actin polymerization-dependent pore opening is distinct from the previously described actin polymerization-dependent ruffles that are required for bacterial internalization. Moreover, actin polymerization is not required for other pore functions, including docking or pore protein insertion into the plasma membrane. Thus, activation of the T3SS is a multilayered process in which host signals are sensed by the translocon pore leading to the activation of effector translocation.

scholarly journals Topological Analysis of the Type 3 Secretion System Translocon Pore Protein IpaC following Its Native Delivery to the Plasma Membrane during Infection

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Brian C. Russo ◽  
Jeffrey K. Duncan ◽  
Marcia B. Goldberg
Keyword(s):  
Plasma Membrane ◽  
Protein Secretion ◽  
Secretion System ◽  
Bacterial Virulence ◽  
Eukaryotic Cells ◽  
Effector Protein ◽  
Virulence Proteins ◽  
Type 3 Secretion System ◽  
Type 3 ◽  
Pore Protein

ABSTRACTMany Gram-negative bacterial pathogens require a type 3 secretion system (T3SS) to deliver effector proteins into eukaryotic cells. Contact of the tip complex of the T3SS with a target eukaryotic cell initiates secretion of the two bacterial proteins that assemble into the translocon pore in the plasma membrane. The translocon pore functions to regulate effector protein secretion and is the conduit for effector protein translocation across the plasma membrane. To generate insights into how the translocon pore regulates effector protein secretion, we defined the topology of theShigellatranslocon pore protein IpaC in the plasma membrane following its native delivery by the T3SS. Using single cysteine substitution mutagenesis and site-directed labeling with a membrane-impermeant chemical probe, we mapped residues accessible from the extracellular surface of the cell. Our data support a model in which the N terminus of IpaC is extracellular and the C terminus of IpaC is intracellular. These findings resolve previously conflicting data on IpaC topology that were based on nonnative delivery of IpaC to membranes.Salmonella entericaserovar Typhimurium also requires the T3SS for effector protein delivery into eukaryotic cells. Although the sequence of IpaC is closely related to theSalmonellatranslocon pore protein SipC, the two proteins have unique functional attributes during infection. We showed a similar overall topology for SipC and IpaC and identified subtle topological differences between their transmembrane α-helixes and C-terminal regions. Together, our data suggest that topological differences among distinct translocon pore proteins may dictate organism-specific functional differences of the T3SSs during infection.IMPORTANCEThe type 3 secretion system (T3SS) is a nanomachine required for virulence of many bacterial pathogens that infect humans. The system delivers bacterial virulence proteins into the cytosol of human cells, where the virulence proteins promote bacterial infection. The T3SS forms a translocon pore in the membranes of target cells. This pore is the portal through which bacterial virulence proteins are delivered by the T3SS into the eukaryotic cytosol. The pore also regulates secretion of these virulence proteins. Our work defines the topology of translocon pore proteins in their native context during infection, resolves previously conflicting reports about the topology of theShigellatranslocon pore protein IpaC, and provides new insights into how interactions of the pore with the T3SS likely produce signals that activate secretion of virulence proteins.

scholarly journals Topological analysis of the type 3 secretion system translocon pore protein IpaC following its native delivery to the plasma membrane during infection

2019 ◽  
Author(s):  
Brian C. Russo ◽  
Jeffrey K. Duncan ◽  
Marcia B. Goldberg
Keyword(s):  
Plasma Membrane ◽  
Protein Secretion ◽  
Secretion System ◽  
Bacterial Virulence ◽  
Eukaryotic Cells ◽  
Effector Protein ◽  
Virulence Proteins ◽  
Type 3 Secretion System ◽  
Type 3 ◽  
Pore Protein

AbstractMany Gram-negative bacterial pathogens require a type 3 secretion system (T3SS) to deliver effector proteins into eukaryotic cells. Contact of the tip complex of the T3SS with a target eukaryotic cell initiates the secretion of the two bacterial proteins that assemble into the translocon pore in the plasma membrane. The translocon pore functions to regulate effector protein secretion and is the conduit for effector protein translocation across the plasma membrane. To generate insights into how the translocon pore regulates effector protein secretion, we defined the topology of theShigellatranslocon pore protein IpaC in the plasma membrane following its native delivery by the T3SS. Using single-cysteine substitution mutagenesis and site-directed labeling with a membrane-impermeant chemical probe, we mapped residues accessible from the extracellular surface of the cell. Our data support a model in which the N-terminus of IpaC is extracellular and the C-terminus of IpaC is intracellular. These findings resolve previously conflicting data on IpaC topology that were based on non-native delivery of IpaC to membranes.Salmonella entericaserovar Typhimurium also requires the T3SS for effector protein delivery into eukaryotic cells. Although the sequence of IpaC is closely related to theSalmonellatranslocon pore protein SipC, the two proteins have unique functional attributes during infection. We showed a similar overall topology for SipC and IpaC and identified subtle topological differences between their transmembrane α-helixes and C-terminal regions. Together, our data suggest that topological differences among distinct translocon pore proteins may dictate organism-specific functional differences of the T3SSs during infection.ImportanceThe type 3 secretion system (T3SS) is a nanomachine required for virulence of many bacterial pathogens that infect humans. The system delivers bacterial virulence proteins into the cytosol of human cells, where the virulence proteins promote bacterial infection. The T3SS forms a translocon pore in the membrane of target cells. This pore is the portal through which bacterial virulence proteins are delivered by the T3SS into the eukaryotic cytosol. The pore also regulates the secretion of these virulence proteins. Our work defines the topology of translocon pore proteins in their native context during infection, resolves previously conflicting reports about the topology of theShigellatranslocon pore protein IpaC, and provides new insights into how interactions of the pore with the T3SS likely produce signals that activate secretion of virulence proteins.

scholarly journals Structures of the Shigella flexneri Type 3 Secretion System Protein MxiC Reveal Conformational Variability Amongst Homologues

2008 ◽  
Vol 377 (4) ◽  
pp. 985-992 ◽  
Author(s):  
Janet E. Deane ◽  
Pietro Roversi ◽  
Carole King ◽  
Steven Johnson ◽  
Susan M. Lea
Keyword(s):  
Shigella Flexneri ◽  
Secretion System ◽  
Conformational Variability ◽  
Type 3 Secretion System ◽  
Type 3

scholarly journals Invasion of epithelial cells are correlated with secretion of Biosurfactant via the type 3 secretion system (SST3) of Shigella flexneri

2020 ◽  
Author(s):  
Duchel Jeanedvi Kinouani Kinavouidi ◽  
Christian Aimé Kayath ◽  
Etienne NGuimbi
Keyword(s):  
Epithelial Cells ◽  
Type Iii Secretion ◽  
Shigella Flexneri ◽  
Secretion System ◽  
Extracellular Medium ◽  
Diesel Fuels ◽  
Membrane Interfaces ◽  
Type 3 Secretion System ◽  
Type 3 ◽  
Hydrophobic Areas

AbstractBiosurfactants are amphipathic molecules produced by many microorganisms, usually bacteria, fungi and yeasts. They possess the property of reducing the tension of the membrane interfaces. No studies have been conducted on Shigella species showing their involvement of biosurfactant like molecules (BLM) in pathogenicity. This study aims to show that environmental and clinical strains of Shigella are able to produce BLM by emulsifying gasoline and diesel fuels. Our study has shown that BLM are secreted in the extracellular medium with EI24 ranging from 80 to 100%. The secretion is depending on the type III secretion system (T3SS). We did show that S. flexneri, S. boydii and S. sonnei are able to interact with hydrophobic areas with respectively 17.64%, 21.42% and 22.22% of hydrophobicity. 100 mM Benzoic and 1.5mg/mL Salycilic acids have been inhibited T3SS and this totally stops the BLM secretion. Pseudomonas aeruginosa which has T3SS is able to produce 100% of BLM in the presence or in the absence of both T3SS inhibitors. The secreted BLM is extractable with an organic solvent such as chloroform and could entirely be considered like lipopeptide or polypeptidic compound. By secreting BLM, Shigella is able to perform multicellular phenomena like “swarming” allowing to invade and disseminate inside epithelial cells.

scholarly journals Invasion of Epithelial Cells Is Correlated with Secretion of Biosurfactant via the Type 3 Secretion System (T3SS) of Shigella flexneri

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Duchel Jeanedvi Kinouani Kinavouidi ◽  
Christian Aimé Kayath ◽  
Etienne Nguimbi
Keyword(s):  
Organic Solvent ◽  
Type Iii Secretion ◽  
Shigella Flexneri ◽  
Secretion System ◽  
Extracellular Medium ◽  
Membrane Interfaces ◽  
Type 3 Secretion System ◽  
Type 3 ◽  
Hydrophobic Areas

Biosurfactants are amphipathic molecules produced by many microorganisms, usually bacteria, fungi, and yeasts. They possess the property of reducing the tension of the membrane interfaces. No studies have been conducted on Shigella species showing the role of biosurfactant-like molecules (BLM) in pathogenicity. The aim of this study is to assess the ability of Shigella environmental and clinical strains to produce BLM and investigate the involvement of biosurfactants in pathogenicity. Our study has shown that BLM are secreted in the extracellular medium with EI24 ranging from 80% to 100%. The secretion is depending on the type III secretion system (T3SS). Moreover, our results have shown that S. flexneri, S. boydii, and S. sonnei are able to interact with hydrophobic areas with 17.64%, 21.42%, and 22.22% hydrophobicity, respectively. BLM secretion is totally prevented due to inhibition of T3SS by 100 mM benzoic and 1.5 mg/ml salicylic acids. P. aeruginosa harboring T3SS is able to produce 100% of BLM in the presence or in the absence of both T3SS inhibitors. The secreted BLM are extractable with an organic solvent such as chloroform, and this could entirely be considered a lipopeptide or polypeptide compound. Secretion of BLM allows some Shigella strains to induce multicellular phenomena like “swarming.”

scholarly journals Topology and Contribution to the Pore Channel Lining of Plasma Membrane-Embedded Shigella flexneri Type 3 Secretion Translocase IpaB

mBio ◽  
2021 ◽  
Author(s):  
Poyin Chen ◽  
Brian C. Russo ◽  
Jeffrey K. Duncan-Lowey ◽  
Natasha Bitar ◽  
Keith T. Egger ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Human Cell ◽  
Shigella Flexneri ◽  
Cellular Function ◽  
Human Cells ◽  
Bacterial Virulence ◽  
Pore Channel ◽  
Secretion Systems ◽  
Virulence Proteins ◽  
Type 3

Type 3 secretion systems are nanomachines employed by many bacteria, including Shigella , which deliver into human cells bacterial virulence proteins that alter cellular function in ways that promote infection. Delivery of Shigella virulence proteins occurs through a pore formed in human cell membranes by the IpaB and IpaC proteins.

scholarly journals Topology and contribution to the pore channel lining of plasma membrane-embedded S. flexneri type 3 secretion translocase IpaB

2021 ◽  
Author(s):  
Poyin Chen ◽  
Brian C Russo ◽  
Jeffrey K Duncan-Lowey ◽  
Natasha Bitar ◽  
Keith Egger ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Polymerization ◽  
Plasma Membranes ◽  
Plant Pathogens ◽  
Transmembrane Domain ◽  
Acyl Carrier Protein ◽  
Pore Channel ◽  
Cell Contact ◽  
Post Translational Modification ◽  
Type 3

Shigella spp. are human bacterial pathogens that cause bacillary dysentery. Virulence depends on a type 3 secretion system (T3SS), a highly conserved structure present in multiple important human and plant pathogens. Upon host cell contact, the T3SS translocon is delivered to the host membrane, facilitates bacterial docking to the membrane, and enables delivery of effector proteins into the host cytosol. The Shigella translocon is composed of two proteins, IpaB and IpaC, which together form this multimeric structure within host plasma membranes. Upon interaction of IpaC with host intermediate filaments, the translocon undergoes a conformational change that allows for bacterial docking onto the translocon and, together with host actin polymerization, enables subsequent effector translocation through the translocon pore. To generate additional insights into the translocon, we mapped the topology of IpaB in plasma membrane-embedded pores using cysteine substitution mutagenesis coupled with site-directed labeling and proximity-enabled crosslinking by membrane permeant sulfhydryl reactants. We demonstrate that IpaB function is dependent on post translational modification by a plasmid-encoded acyl carrier protein. We show that the first transmembrane domain of IpaB lines the interior of the translocon pore channel such that the IpaB portion of the channel forms a funnel-like shape leading into the host cytosol. In addition, we identify regions of IpaB within its cytosolic domain that protrude into and are closely associated with the pore channel. Taken together, these results provide a framework for how IpaB is arranged within translocons natively delivered by Shigella during infection.

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.

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