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

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.

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

10.1101/677120 ◽

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

mBio ◽

10.1128/mbio.00877-19 ◽

2019 ◽

Vol 10 (3) ◽

Cited By ~ 5

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.

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

PLoS Pathogens ◽

10.1371/journal.ppat.1009932 ◽

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.

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

10.1101/2021.05.27.445799 ◽

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.

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

10.1101/605865 ◽

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.

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Chaperone-Assisted Secretion in Bacteria: Protein and DNA Transport via Cell Membranes

Current Proteomics ◽

10.2174/1570164615666180820154821 ◽

2018 ◽

Vol 16 (1) ◽

pp. 54-63

Author(s):

Lilian Goulart Schultz ◽

Ljubica Tasic ◽

Juliana Fattori

Keyword(s):

Bacterial Infections ◽

Cell Membranes ◽

Protein Complexes ◽

Host Cells ◽

Secretion Systems ◽

Dna Transport ◽

Virulence Proteins ◽

Macromolecular Transport ◽

Pass Through ◽

Type 3

Bacteria use an impressive arsenal of secretion systems (1-7) to infect their host cells by exporting proteins, DNA and DNA-protein complexes via cell membranes. They use chaperone-usher pathways for host colonization as well. To be targeted for transportation across one (Gram-positive) or two membranes (Gram-negative), clients must be selected, guided and unfolded to pass through type 3 (T3SS) or type 4 (T4SS) secretion systems. For these processes, bacteria count on secretory chaperones that guide macromolecular transport via membranes. Moreover, if we know how these processes occur, we might be able to stop them and avoid bacterial infections. Thus, structural and functional characterizations of secretory chaperones become interesting, as these proteins are the perfect targets for blocking bacteria action. Therefore, this review focuses on a story of known mechanisms of chaperone- secretion assisted transport with special attention on virulence proteins and DNA transport in bacteria.

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Identification of Human Junctional Adhesion Molecule 1 as a Functional Receptor for the Hom-1 Calicivirus on Human Cells

mBio ◽

10.1128/mbio.00031-17 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 20

Author(s):

Stanislav V. Sosnovtsev ◽

Carlos Sandoval-Jaime ◽

Gabriel I. Parra ◽

Christine M. Tin ◽

Ronald W. Jones ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Cell Lines ◽

Adhesion Molecule ◽

Human Cell ◽

Cho Cells ◽

Human Cells ◽

Human Cell Lines ◽

Sea Lion ◽

Functional Receptor

ABSTRACTThe Hom-1 vesivirus was reported in 1998 following the inadvertent transmission of the animal calicivirus San Miguel sea lion virus to a human host in a laboratory. We characterized the Hom-1 strain and investigated the mechanism by which human cells could be infected. An expression library of 3,559 human plasma membrane proteins was screened for reactivity with Hom-1 virus-like particles, and a single interacting protein, human junctional adhesion molecule 1 (hJAM1), was identified. Transient expression of hJAM1 conferred susceptibility to Hom-1 infection on nonpermissive Chinese hamster ovary (CHO) cells. Virus infection was markedly inhibited when CHO cells stably expressing hJAM were pretreated with anti-hJAM1 monoclonal antibodies. Cell lines of human origin were tested for growth of Hom-1, and efficient replication was observed in HepG2, HuH7, and SK-CO15 cells. The three cell lines (of hepatic or intestinal origin) were confirmed to express hJAM1 on their surface, and clustered regularly interspaced short palindromic repeats/Cas9-mediated knockout of the hJAM1 gene in each line abolished Hom-1 propagation. Taken together, our data indicate that entry of the Hom-1 vesivirus into these permissive human cell lines is mediated by the plasma membrane protein hJAM1 as a functional receptor.IMPORTANCEVesiviruses, such as San Miguel sea lion virus and feline calicivirus, are typically associated with infection in animal hosts. Following the accidental infection of a laboratory worker with San Miguel sea lion virus, a related virus was isolated in cell culture and named Hom-1. In this study, we found that Hom-1 could be propagated in a number of human cell lines, making it the first calicivirus to replicate efficiently in cultured human cells. Screening of a library of human cell surface membrane proteins showed that the virus could utilize human junctional adhesion molecule 1 as a receptor to enter cells and initiate replication. The Hom-1 virus presents a new system for the study of calicivirus biology and species specificity.

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

10.1101/2021.10.07.463542 ◽

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.

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Small-Molecule Type III Secretion System Inhibitors Block Assembly of the Shigella Type III Secreton

Journal of Bacteriology ◽

10.1128/jb.01004-08 ◽

2008 ◽

Vol 191 (2) ◽

pp. 563-570 ◽

Cited By ~ 71

Author(s):

Andreas K. J. Veenendaal ◽

Charlotta Sundin ◽

Ariel J. Blocker

Keyword(s):

Type Iii Secretion ◽

Bacterial Infections ◽

Shigella Flexneri ◽

Effector Proteins ◽

Host Cells ◽

Incomplete Penetrance ◽

Secretion Systems ◽

Bacterial Surface ◽

Type Iii ◽

Type Iii Secretion Systems

ABSTRACT Type III secretion systems (T3SSs) are essential virulence devices for many gram-negative bacteria that are pathogenic for plants, animals, and humans. They serve to translocate virulence effector proteins directly into eukaryotic host cells. T3SSs are composed of a large cytoplasmic bulb and a transmembrane region into which a needle is embedded, protruding above the bacterial surface. The emerging antibiotic resistance of bacterial pathogens urges the development of novel strategies to fight bacterial infections. Therapeutics that rather than kill bacteria only attenuate their virulence may reduce the frequency or progress of resistance emergence. Recently, a group of salicylidene acylhydrazides were identified as inhibitors of T3SSs in Yersinia, Chlamydia, and Salmonella species. Here we show that these are also effective on the T3SS of Shigella flexneri, where they block all related forms of protein secretion so far known, as well as the epithelial cell invasion and induction of macrophage apoptosis usually demonstrated by this bacterium. Furthermore, we show the first evidence for the detrimental effect of these compounds on T3SS needle assembly, as demonstrated by increased numbers of T3S apparatuses without needles or with shorter needles. Therefore, the compounds generate a phenocopy of T3SS export apparatus mutants but with incomplete penetrance. We discuss why this would be sufficient to almost completely block the later secretion of effector proteins and how this begins to narrow the search for the molecular target of these compounds.

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Shigella flexneri Phagosomal Escape Is Independent of Invasion

Infection and Immunity ◽

10.1128/iai.00454-07 ◽

2007 ◽

Vol 75 (10) ◽

pp. 4826-4830 ◽

Cited By ~ 24

Author(s):

Susanne Paetzold ◽

Sebastian Lourido ◽

Bärbel Raupach ◽

Arturo Zychlinsky

Keyword(s):

Cell Invasion ◽

Type Iii Secretion ◽

Shigella Flexneri ◽

Mucosal Inflammation ◽

Virulence Plasmid ◽

Secretion Systems ◽

Type Iii Secretion Systems ◽

Macrophage Cytotoxicity ◽

Serovar Typhimurium ◽

Epithelial Cell Invasion

ABSTRACT Infections with Salmonella enterica serovar Typhimurium and Shigella flexneri result in mucosal inflammation in response to epithelial cell invasion and macrophage cytotoxicity. These processes are mediated by type III secretion systems encoded in homologous virulence loci in the two species, namely, Salmonella pathogenicity island 1 (SPI-1), carried in the genome, and the Shigella entry region (SER), carried in a large virulence plasmid. Here we show that SPI-1 can functionally complement a deletion of SER in S. flexneri, restoring invasion of epithelial cells, macrophage cytotoxicity, and phagosomal escape. Furthermore, S. flexneri phagosomal escape requires the SER and another gene(s) carried on the large virulence plasmid. We demonstrate that the processes of invasion and phagosomal escape can be uncoupled in S. flexneri.

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