scholarly journals The blueprint of the type-3 injectisome

2012 ◽  
Vol 367 (1592) ◽  
pp. 1140-1154 ◽  
Author(s):  
Agata Kosarewicz ◽  
Lisa Königsmaier ◽  
Thomas C. Marlovits
Keyword(s):  
Protein Delivery ◽  
Effector Proteins ◽  
Central Component ◽  
Small Subset ◽  
Secretion Systems ◽  
Bacterial Membranes ◽  
Secretion Pathway ◽  
Specific Pathogen ◽  
Multiple Copies ◽  
Type 3

Type-3 secretion systems are sophisticated syringe-like nanomachines present in many animal and plant Gram-negative pathogens. They are capable of translocating an arsenal of specific bacterial toxins (effector proteins) from the prokaryotic cytoplasm across the three biological membranes directly into the eukaryotic cytosol, some of which modulate host cell mechanisms for the benefit of the pathogen. They populate a particular biological niche, which is maintained by specific, pathogen-dependent effectors. In contrast, the needle complex, which is the central component of this specialized protein delivery machine, is structurally well-conserved. It is a large supramolecular cylindrical structure composed of multiple copies of a relatively small subset of proteins, is embedded in the bacterial membranes and protrudes from the pathogen's surface with a needle filament. A central channel traverses the entire needle complex, and serves as a hollow conduit for proteins destined to travel this secretion pathway. In the past few years, there has been a tremendous increase in an understanding on both the structural and the mechanistic level. This review will thus focus on new insights of this remarkable molecular machine.

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 Salmonella Pathogenicity Island 2-Mediated Overexpression of Chimeric SspH2 Proteins for Simultaneous Induction of Antigen-Specific CD4 and CD8 T Cells

2005 ◽  
Vol 73 (1) ◽  
pp. 334-341 ◽  
Author(s):  
Klaus Panthel ◽  
Katrin M. Meinel ◽  
Victòria E. Sevil Domènech ◽  
Heike Retzbach ◽  
Emeka I. Igwe ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Protein Delivery ◽  
Effector Proteins ◽  
Foreign Protein ◽  
Secretion Systems ◽  
Heterologous Antigen ◽  
Type Iii ◽  
Serovar Typhimurium ◽  
Carrier Molecule

ABSTRACT Salmonella enterica serovar Typhimurium employs two different type III secretion systems (TTSS) encoded within Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) for targeting of effector proteins into the cytosol of eukaryotic cells during different stages of the infection cycle. The SPI1 TTSS translocates virulence factors across the plasma membrane when the bacterium initially contacts the host cell. In contrast, the SPI2 TTSS functions to translocate proteins across the membrane of the Salmonella-containing vacuole and promotes intracellular survival and replication. The aim of the present study was to directly compare the potentials of SPI1 and SPI2 type III effector proteins to act as carrier molecules for a heterologous antigen. The p60 protein of Listeria monocytogenes was used as a model antigen to construct chimeric SopE2 (SPI1), SifA (SPI2), and SspH2 (SPI2) proteins. SPI1- and SPI2-dependent up- and down-regulation of hybrid gene expression led to sequential translocation of p60 fusion proteins into the cytosol of Salmonella-infected macrophages. Mice orally immunized with recombinant Salmonella strains expressing these hybrid proteins revealed comparable numbers of p60-specific CD8 T cells. However, only overexpression of translocated SspH2/p60 from a medium-copy-number vector induced simultaneous antigen-specific CD4 and CD8 T-cell responses, suggesting that SspH2 is an attractive carrier molecule for foreign-protein delivery.

scholarly journals Substrate-engaged type III secretion system structures reveal gating mechanism for unfolded protein translocation

2020 ◽  
Author(s):  
Sean Miletic ◽  
Dirk Fahrenkamp ◽  
Nikolaus Goessweiner-Mohr ◽  
Jiri Wald ◽  
Maurice Pantel ◽  
...  
Keyword(s):  
Host Cell ◽  
Type Iii Secretion ◽  
Bacterial Infections ◽  
Biological Membrane ◽  
Critical Role ◽  
Effector Proteins ◽  
Central Component ◽  
Secretion Systems ◽  
Type Iii ◽  
Gating Mechanism

AbstractMany bacterial pathogens strictly rely on the activity of type III secretion systems (T3SSs) to secrete and translocate effector proteins in order to establish infection. The central component of T3SSs is the needle complex, a supramolecular machine which assembles a continuous conduit crossing the bacterial envelope and the host cell membrane to allow bacterial effectors to gain entry into the host cell cytoplasm to modulate signal transduction processes. Disruption of this process impairs pathogenicity, providing an avenue for antimicrobial design. However, the molecular principles underlying T3 secretion remain elusive. Here, we report the first structure of an active Salmonella enterica sv. Typhimurium needle complex engaged with the late effector protein SptP in two functional states, revealing the complete 800Å-long secretion conduit and unravelling the critical role of the export apparatus (EA) subcomplex in T3 secretion. Unfolded substrates enter the EA through a hydrophilic constriction formed by SpaQ proteins, which enables side chain-independent transport, explaining heterogeneity and structural disorder of signal sequences in T3SS effector proteins. Above, a methionine gasket formed by SpaP proteins functions as a gate that dilates to accommodate substrates but prevents leaky pore formation to maintain the physical boundaries of compartments separated by a biological membrane. Following gate penetration, a moveable SpaR loop first folds up to then act akin to a linear ratchet to steer substrates through the needle complex. Together, these findings establish the molecular basis for substrate translocation through T3SSs, improving our understanding of bacterial pathogenicity and motility of flagellated bacteria, and paves the way for the development of novel concepts combating bacterial infections.

scholarly journals Visualization of bacterial type 3 secretion system components down to the molecular level by MINFLUX nanoscopy

2021 ◽  
Author(s):  
Alexander Carsten ◽  
Maren Rudolph ◽  
Tobias Weihs ◽  
Roman Schmidt ◽  
Christian A. Wurm ◽  
...  
Keyword(s):  
Super Resolution ◽  
Infection Process ◽  
Effector Proteins ◽  
Host Cells ◽  
Human Pathogens ◽  
Secretion Systems ◽  
Spatially Resolved ◽  
Treatment And Prevention ◽  
And Function ◽  
Type 3

AbstractType 3 secretion systems (T3SS) are essential virulence factors of numerous bacterial pathogens and inject effector proteins into host cells. The needle-like T3SS machinery consists of more than 20 components, has a length of around 100 nm and a diameter of up to 30 nm according to EM studies. Its intrabacterial components are highly dynamic and in permanent exchange with other bacterial structures. Therefore, a temporally and spatially resolved visualization of the T3SS using fluorescence microscopy techniques has been challenging. In the present study, novel labeling strategies were combined with super-resolution microscopy such as STED, STORM and MINFLUX. MINFLUX nanoscopy allowed to visualize components of the T3SS machinery such as the dynamic sorting platform component YscL and the extrabacterial pore protein YopD at unprecedented resolutions. The presented results represent the basis for an in depth investigation of T3SS structure and function and therefore gain new insights into the infection process of human pathogens in order to develop novel treatment and prevention strategies.

scholarly journals Bacterial Effector Kinases

2014 ◽  
Vol 70 (a1) ◽  
pp. C428-C428
Author(s):  
Andrey Grishin ◽  
Maia Cherney ◽  
Tara Condos ◽  
Kathryn Barber ◽  
Deborah Anderson ◽  
...  
Keyword(s):  
Host Cell ◽  
Pathogenic Bacteria ◽  
Regulatory Element ◽  
Kinase Domain ◽  
Effector Proteins ◽  
Activation Mechanism ◽  
Secretion Systems ◽  
Human Kinase ◽  
Ubiquitin Conjugating Enzyme ◽  
Type 3

Protein phosphorylation is one of the main signaling mechanisms in eukaryotic cells. Not surprisingly, pathogenic adopted this mechanism to interfere with signaling processes in the host cell. To this end pathogens evolved kinases that, in addition to other bacterial effector proteins, are injected into the host cell via a syringe-like type 3 (T3SS) or type 4 (T4SS) secretion systems. Kinases NleH1 and NleH2 from pathogenic E. coli, OspG from Shigella, SteC and SboH from Salmonella, LegK1-4 from Legionella and YspK and YpkA from Yersinia represent currently known effector kinases. Some of these kinases were likely derived from eukaryotes via horizontal gene transfer (SteC, LegK1-4, YpkA). Other kinases (NleH, OspG, SboH and YspK) have been so far identified only in the pathogenic bacteria. The structures of NleH and OspG proved that these kinases, which are half the size of an average human kinase, contain only a core kinase fold. These kinases lack the main regulatory element – the activation loop. The structure of NleH suggests that it has no activation mechanism since the apo-kinase domain adopts an active conformation and no change is observed on nucleotide binding. The OspG kinase, which also contains only the core kinase fold, is stimulated by its binding partner, the ubiquitin-conjugating enzyme E2-ubiquitin complex. The structure of OspG:UbcH7-Ub complex shows that OspG binds the E2 and ubiquitin (Ub) at two distinct sites on its surface. In this complex the OspG active site is unobstructed and primed for catalysis. However the mechanism of OspG activation remains presently unknown. Both NleH and OspG were found to inhibit the NF-kB pathway, however the substrates forOspG and NleH kinase activities are not yet known.

scholarly journals Tandem Translation Generates a Chaperone for the Salmonella Type III Secretion System Protein SsaQ

2011 ◽  
Vol 286 (41) ◽  
pp. 36098-36107 ◽  
Author(s):  
Xiu-Jun Yu ◽  
Mei Liu ◽  
Steve Matthews ◽  
David W. Holden
Keyword(s):  
Type Iii Secretion ◽  
Ex Vivo ◽  
Eukaryotic Cell ◽  
Effector Proteins ◽  
Secretion Systems ◽  
Type Iii ◽  
Type Iii Secretion Systems ◽  
Function Loss ◽  
Bacterial Cytoplasm

Type III secretion systems (T3SSs) of bacterial pathogens involve the assembly of a surface-localized needle complex, through which translocon proteins are secreted to form a pore in the eukaryotic cell membrane. This enables the transfer of effector proteins from the bacterial cytoplasm to the host cell. A structure known as the C-ring is thought to have a crucial role in secretion by acting as a cytoplasmic sorting platform at the base of the T3SS. Here, we studied SsaQ, an FliN-like putative C-ring protein of the Salmonella pathogenicity island 2 (SPI-2)-encoded T3SS. ssaQ produces two proteins by tandem translation: a long form (SsaQL) composed of 322 amino acids and a shorter protein (SsaQS) comprising the C-terminal 106 residues of SsaQL. SsaQL is essential for SPI-2 T3SS function. Loss of SsaQS impairs the function of the T3SS both ex vivo and in vivo. SsaQS binds to its corresponding region within SsaQL and stabilizes the larger protein. Therefore, SsaQL function is optimized by a novel chaperone-like protein, produced by tandem translation from its own mRNA species.

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

2008 ◽  
Vol 191 (2) ◽  
pp. 563-570 ◽  
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.

scholarly journals Peptidomimetic Small Molecules Disrupt Type IV Secretion System Activity in Diverse Bacterial Pathogens

mBio ◽  
2016 ◽  
Vol 7 (2) ◽  
Author(s):  
Carrie L. Shaffer ◽  
James A. D. Good ◽  
Santosh Kumar ◽  
K. Syam Krishnan ◽  
Jennifer A. Gaddy ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Small Molecules ◽  
Bacterial Pathogens ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Target Cells ◽  
Effector Protein ◽  
Secretion Systems ◽  
Type Iv ◽  
H Pylori

ABSTRACT Bacteria utilize complex type IV secretion systems (T4SSs) to translocate diverse effector proteins or DNA into target cells. Despite the importance of T4SSs in bacterial pathogenesis, the mechanism by which these translocation machineries deliver cargo across the bacterial envelope remains poorly understood, and very few studies have investigated the use of synthetic molecules to disrupt T4SS-mediated transport. Here, we describe two synthetic small molecules (C10 and KSK85) that disrupt T4SS-dependent processes in multiple bacterial pathogens. Helicobacter pylori exploits a pilus appendage associated with the cag T4SS to inject an oncogenic effector protein (CagA) and peptidoglycan into gastric epithelial cells. In H. pylori , KSK85 impedes biogenesis of the pilus appendage associated with the cag T4SS, while C10 disrupts cag T4SS activity without perturbing pilus assembly. In addition to the effects in H. pylori , we demonstrate that these compounds disrupt interbacterial DNA transfer by conjugative T4SSs in Escherichia coli and impede vir T4SS-mediated DNA delivery by Agrobacterium tumefaciens in a plant model of infection. Of note, C10 effectively disarmed dissemination of a derepressed IncF plasmid into a recipient bacterial population, thus demonstrating the potential of these compounds in mitigating the spread of antibiotic resistance determinants driven by conjugation. To our knowledge, this study is the first report of synthetic small molecules that impair delivery of both effector protein and DNA cargos by diverse T4SSs. IMPORTANCE Many human and plant pathogens utilize complex nanomachines called type IV secretion systems (T4SSs) to transport proteins and DNA to target cells. In addition to delivery of harmful effector proteins into target cells, T4SSs can disseminate genetic determinants that confer antibiotic resistance among bacterial populations. In this study, we sought to identify compounds that disrupt T4SS-mediated processes. Using the human gastric pathogen H. pylori as a model system, we identified and characterized two small molecules that prevent transfer of an oncogenic effector protein to host cells. We discovered that these small molecules also prevented the spread of antibiotic resistance plasmids in E. coli populations and diminished the transfer of tumor-inducing DNA from the plant pathogen A. tumefaciens to target cells. Thus, these compounds are versatile molecular tools that can be used to study and disarm these important bacterial machines.

scholarly journals Association of a Protective Monoclonal IgA with the O Antigen of Salmonella enterica Serovar Typhimurium Impacts Type 3 Secretion and Outer Membrane Integrity

2012 ◽  
Vol 80 (7) ◽  
pp. 2454-2463 ◽  
Author(s):  
Stephen J. Forbes ◽  
Daniel Martinelli ◽  
Chyongere Hsieh ◽  
Jeffrey G. Ault ◽  
Michael Marko ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Epithelial Cells ◽  
Outer Membrane ◽  
Salmonella Enterica ◽  
Membrane Integrity ◽  
Effector Proteins ◽  
Content Type ◽  
O Antigen ◽  
Serovar Typhimurium ◽  
Type 3

ABSTRACTInvasion of intestinal epithelial cells bySalmonella entericaserovar Typhimurium is an energetically demanding process, involving the transfer of effector proteins from invading bacteria into host cells via a specialized organelle known as theSalmonellapathogenicity island 1 (SPI-1) type 3 secretion system (T3SS). By a mechanism that remains poorly understood, entry ofS. Typhimurium into epithelial cells is inhibited by Sal4, a monoclonal, polymeric IgA antibody that binds an immunodominant epitope within the O-antigen (O-Ag) component of lipopolysaccharide. In this study, we investigated how the binding of Sal4 to the surface ofS. Typhimurium influences T3SS activity, bacterial energetics, and outer membrane integrity. We found that Sal4 treatment impaired T3SS-mediated translocon formation and attenuated the delivery of tagged effector proteins into epithelial cells. Sal4 treatment coincided with a partial reduction in membrane energetics and intracellular ATP levels, possibly explaining the impairment in T3SS activity. Sal4's effects on bacterial secretion and energetics occurred concurrently with an increase in O-Ag levels in culture supernatants, alterations in outer membrane permeability, and changes in surface ultrastructure, as revealed by transmission electron microscopy and cryo-electron microscopy. We propose that Sal4, by virtue of its ability to bind and cross-link the O-Ag, induces a form of outer membrane stress that compromises the integrity of theS. Typhimurium cell envelope and temporarily renders the bacterium avirulent.

scholarly journals A Salmonella type III effector, PipA, works in a different manner than the PipA family effectors GogA and GtgA

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0248975
Author(s):  
Momo Takemura ◽  
Takeshi Haneda ◽  
Hikari Idei ◽  
Tsuyoshi Miki ◽  
Nobuhiko Okada
Keyword(s):  
Hela Cells ◽  
Critical Role ◽  
Effector Proteins ◽  
Host Cells ◽  
Microbial Pathogens ◽  
Secretion Systems ◽  
Type Iii ◽  
Zinc Metalloprotease ◽  
Type Iii Secretion Systems ◽  
Serovar Typhimurium

Nuclear factor-kappa B (NF-κB) plays a critical role in the host defense against microbial pathogens. Many pathogens modulate NF-κB signaling to establish infection in their host. Salmonella enterica serovar Typhimurium (S. Typhimurium) possesses two type III secretion systems (T3SS-1 and T3SS-2) and directly injects many effector proteins into host cells. It has been reported that some effectors block NF-κB signaling, but the molecular mechanism of the inactivation of NF-κB signaling in S. Typhimurium is poorly understood. Here, we identified seven type III effectors—GogA, GtgA, PipA, SseK1, SseK2, SseK3, and SteE—that inhibited NF-κB activation in HeLa cells stimulated with TNF-α. We also determined that only GogA and GtgA are involved in regulation of the activation of NF-κB in HeLa cells infected with S. Typhimurium. GogA, GtgA, and PipA are highly homologous to one another and have the consensus zinc metalloprotease HEXXH motif. Our experiments demonstrated that GogA, GtgA, and PipA each directly cleaved NF-κB p65, whereas GogA and GtgA, but not PipA, inhibited the NF-κB activation in HeLa cells infected with S. Typhimurium. Further, expressions of the gogA or gtgA gene were induced under the SPI-1-and SPI-2-inducing conditions, but expression of the pipA gene was induced only under the SPI-2-inducing condition. We also showed that PipA was secreted into RAW264.7 cells through T3SS-2. Finally, we indicated that PipA elicits bacterial dissemination in the systemic stage of infection of S. Typhimurium via a T3SS-1-independent mechanism. Collectively, our results suggest that PipA, GogA and GtgA contribute to S. Typhimurium pathogenesis in different ways.

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