scholarly journals Interactions of the Transmembrane Polymeric Rings of the Salmonella enterica Serovar Typhimurium Type III Secretion System

mBio ◽  
2010 ◽  
Vol 1 (3) ◽  
Author(s):  
Sarah Sanowar ◽  
Pragya Singh ◽  
Richard A. Pfuetzner ◽  
Ingemar André ◽  
Hongjin Zheng ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Effector Proteins ◽  
Carboxyl Terminus ◽  
Gram Negative Bacteria ◽  
Secretion Systems ◽  
Gram Negative ◽  
Type Iii ◽  
Serovar Typhimurium ◽  
Cross Links ◽  
And Function

ABSTRACT The type III secretion system (T3SS) is an interspecies protein transport machine that plays a major role in interactions of Gram-negative bacteria with animals and plants by delivering bacterial effector proteins into host cells. T3SSs span both membranes of Gram-negative bacteria by forming a structure of connected oligomeric rings termed the needle complex (NC). Here, the localization of subunits in the Salmonella enterica serovar Typhimurium T3SS NC were probed via mass spectrometry-assisted identification of chemical cross-links in intact NC preparations. Cross-links between amino acids near the amino terminus of the outer membrane ring component InvG and the carboxyl terminus of the inner membrane ring component PrgH and between the two inner membrane components PrgH and PrgK allowed for spatial localization of the three ring components within the electron density map structures of NCs. Mutational and biochemical analysis demonstrated that the amino terminus of InvG and the carboxyl terminus of PrgH play a critical role in the assembly and function of the T3SS apparatus. Analysis of an InvG mutant indicates that the structure of the InvG oligomer can affect the switching of the T3SS substrate to translocon and effector components. This study provides insights into how structural organization of needle complex base components promotes T3SS assembly and function. IMPORTANCE Many biological macromolecular complexes are composed of symmetrical homomers, which assemble into larger structures. Some complexes, such as secretion systems, span biological membranes, forming hydrophilic domains to move substrates across lipid bilayers. Type III secretion systems (T3SSs) deliver bacterial effector proteins directly to the host cell cytoplasm and allow for critical interactions between many Gram-negative pathogenic bacterial species and their hosts. Progress has been made towards the goal of determining the three-dimensional structure of the secretion apparatus by determination of high-resolution crystal structures of individual protein subunits and low-resolution models of the assembly, using reconstructions of cryoelectron microscopy images. However, a more refined picture of the localization of periplasmic ring structures within the assembly and their interactions has only recently begun to emerge. This work localizes T3SS transmembrane rings and identifies structural elements that affect substrate switching and are essential to the assembly of components that are inserted into host cell membranes.

scholarly journals Role of Autocleavage in the Function of a Type III Secretion Specificity Switch Protein in Salmonella enterica Serovar Typhimurium

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
Julia V. Monjarás Feria ◽  
Matthew D. Lefebre ◽  
York-Dieter Stierhof ◽  
Jorge E. Galán ◽  
Samuel Wagner
Keyword(s):  
Host Cell ◽  
Type Iii Secretion ◽  
Effector Proteins ◽  
Switching Function ◽  
Gram Negative Bacteria ◽  
Wild Type ◽  
Gram Negative ◽  
Type Iii ◽  
Autocatalytic Cleavage ◽  
Serovar Typhimurium

ABSTRACTType III secretion systems (T3SSs) are multiprotein machines employed by many Gram-negative bacteria to inject bacterial effector proteins into eukaryotic host cells to promote bacterial survival and colonization. The core unit of T3SSs is the needle complex, a supramolecular structure that mediates the passage of the secreted proteins through the bacterial envelope. A distinct feature of the T3SS is that protein export occurs in a strictly hierarchical manner in which proteins destined to form the needle complex filament and associated structures are secreted first, followed by the secretion of effectors and the proteins that will facilitate their translocation through the target host cell membrane. The secretion hierarchy is established by complex mechanisms that involve several T3SS-associated components, including the “switch protein,” a highly conserved, inner membrane protease that undergoes autocatalytic cleavage. It has been proposed that the autocleavage of the switch protein is the trigger for substrate switching. We show here that autocleavage of theSalmonella entericaserovar Typhimurium switch protein SpaS is an unregulated process that occurs after its folding and before its incorporation into the needle complex. Needle complexes assembled with a precleaved form of SpaS function in a manner indistinguishable from that of the wild-type form. Furthermore, an engineered mutant of SpaS that is processed by an external protease also displays wild-type function. These results demonstrate that the cleavage eventper sedoes not provide a signal for substrate switching but support the hypothesis that cleavage allows the proper conformation of SpaS to render it competent for its switching function.IMPORTANCEBacterial interaction with eukaryotic hosts often involves complex molecular machines for targeted delivery of bacterial effector proteins. One such machine, the type III secretion system of some Gram-negative bacteria, serves to inject a multitude of structurally diverse bacterial proteins into the host cell. Critical to the function of these systems is their ability to secrete proteins in a strict hierarchical order, but it is unclear how the mechanism of switching works. Central to the switching mechanism is a highly conserved inner membrane protease that undergoes autocatalytic cleavage. Although it has been suggested previously that the autocleavage event is the trigger for substrate switching, we show here that this is not the case. Rather, our results show that cleavage allows the proper conformation of the protein to render it competent for its switching function. These findings may help develop inhibitors of type III secretion machines that offer novel therapeutic avenues to treat various infectious diseases.

Type III Secretion: More Systems Than You Think

Physiology ◽  
2005 ◽  
Vol 20 (5) ◽  
pp. 326-339 ◽  
Author(s):  
Paul Troisfontaines ◽  
Guy R. Cornelis
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Type Iii Secretion ◽  
Plant Cells ◽  
Effector Proteins ◽  
Eukaryotic Cells ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Type Iii ◽  
Target Animal

The type III secretion (T3S) pathway allows bacteria to inject effector proteins into the cytosol of target animal or plant cells. T3S systems evolved into seven families that were distributed among Gram-negative bacteria by horizontal gene transfer. There are probably a few hundred effectors interfering with control and signaling in eukaryotic cells and offering a wealth of new tools to cell biologists.

scholarly journals The First 45 Amino Acids of SopA Are Necessary for InvB Binding and SPI-1 Secretion

2006 ◽  
Vol 188 (7) ◽  
pp. 2411-2420 ◽  
Author(s):  
Wendy Higashide ◽  
Daoguo Zhou
Keyword(s):  
Type Iii Secretion ◽  
Catalytic Domain ◽  
Effector Proteins ◽  
Host Cells ◽  
Amino Acid Residues ◽  
Secretion Systems ◽  
Reporter System ◽  
Type Iii ◽  
Serovar Typhimurium ◽  
Type Iii Protein Secretion

ABSTRACT Salmonella enterica serovar Typhimurium encodes two type III secretion systems (TTSSs) within pathogenicity island 1 (SPI-1) and island 2 (SPI-2). These type III protein secretion and translocation systems transport a panel of bacterial effector proteins across both the bacterial and the host cell membranes to promote bacterial entry and subsequent survival inside host cells. Effector proteins contain secretion and translocation signals that are often located at their N termini. We have developed a ruffling-based translocation reporter system that uses the secretion- and translocation-deficient catalytic domain of SopE, SopE78-240, as a reporter. Using this assay, we determined that the N-terminal 45 amino acid residues of Salmonella SopA are necessary and sufficient for directing its secretion and translocation through the SPI-1 TTSS. SopA1-45, but not SopA1-44, is also able to bind to its chaperone, InvB, indicating that SPI-1 type III secretion and translocation of SopA require its chaperone.

scholarly journals Identification of New Secreted Effectors in Salmonella enterica Serovar Typhimurium

2005 ◽  
Vol 73 (10) ◽  
pp. 6260-6271 ◽  
Author(s):  
Kaoru Geddes ◽  
Micah Worley ◽  
George Niemann ◽  
Fred Heffron
Keyword(s):  
Type Iii Secretion ◽  
Salmonella Enterica ◽  
Bacterial Species ◽  
Effector Proteins ◽  
Host Cells ◽  
Secretion Systems ◽  
Cell Cytoplasm ◽  
Type Iii ◽  
Serovar Typhimurium ◽  
Infected Cells

ABSTRACTA common theme in bacterial pathogenesis is the secretion of bacterial products that modify cellular functions to overcome host defenses. Gram-negative bacterial pathogens use type III secretion systems (TTSSs) to inject effector proteins into host cells. The genes encoding the structural components of the type III secretion apparatus are conserved among bacterial species and can be identified by sequence homology. In contrast, the sequences of secreted effector proteins are less conserved and are therefore difficult to identify. A strategy was developed to identify virulence factors secreted bySalmonella entericaserovar Typhimurium into the host cell cytoplasm. We constructed a transposon, which we refer to as mini-Tn5-cycler, to generate translational fusions betweenSalmonellachromosomal genes and a fragment of the calmodulin-dependent adenylate cyclase gene derived fromBordetella pertussis(cyaA′). In-frame fusions to bacterial proteins that are secreted into the eukaryotic cell cytoplasm were identified by high levels of cyclic AMP in infected cells. The assay was sufficiently sensitive that a single secreted fusion could be identified among several hundred that were not secreted. This approach identified three new effectors as well as seven that have been previously characterized. A deletion of one of the new effectors,steA(Salmonellatranslocated effector A), attenuated virulence. In addition, SteA localizes to thetrans-Golgi network in both transfected and infected cells. This approach has identified new secreted effector proteins inSalmonellaand will likely be useful for other organisms, even those in which genetic manipulation is more difficult.

scholarly journals Discovery of Novel Secreted Virulence Factors fromSalmonella entericaSerovar Typhimurium by Proteomic Analysis of Culture Supernatants

2010 ◽  
Vol 79 (1) ◽  
pp. 33-43 ◽  
Author(s):  
George S. Niemann ◽  
Roslyn N. Brown ◽  
Jean K. Gustin ◽  
Afke Stufkens ◽  
Afshan S. Shaikh-Kidwai ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Type Iii Secretion ◽  
Learning Algorithm ◽  
Effector Proteins ◽  
Secretion Systems ◽  
Type Iii Effectors ◽  
Type Iii ◽  
Type Iii Secretion Systems ◽  
Serovar Typhimurium ◽  
Proteomic Study

ABSTRACTSalmonella entericaserovar Typhimurium is a leading cause of acute gastroenteritis throughout the world. This pathogen has two type III secretion systems (TTSS) encoded inSalmonellapathogenicity islands 1 and 2 (SPI-1 and SPI-2) that deliver virulence factors (effectors) to the host cell cytoplasm and are required for virulence. While many effectors have been identified and at least partially characterized, the full repertoire of effectors has not been catalogued. In this proteomic study, we identified effector proteins secreted into defined minimal medium designed to induce expression of the SPI-2 TTSS and its effectors. We compared the secretomes of the parent strain to those of strains missing essential (ssaK::cat) or regulatory (ΔssaL) components of the SPI-2 TTSS. We identified 20 known SPI-2 effectors. Excluding the translocon components SseBCD, all SPI-2 effectors were biased for identification in the ΔssaLmutant, substantiating the regulatory role of SsaL in TTS. To identify novel effector proteins, we coupled our secretome data with a machine learning algorithm (SIEVE,SVM-basedidentification andevaluation ofvirulenceeffectors) and selected 12 candidate proteins for further characterization. Using CyaA′ reporter fusions, we identified six novel type III effectors and two additional proteins that were secreted into J774 macrophages independently of a TTSS. To assess their roles in virulence, we constructed nonpolar deletions and performed a competitive index analysis from intraperitoneally infected 129/SvJ mice. Six mutants were significantly attenuated for spleen colonization. Our results also suggest that non-type III secretion mechanisms are required for fullSalmonellavirulence.

Cryo-EM structure of the needle filament tip complex of the Salmonella type III secretion injectisome

2021 ◽  
Vol 118 (44) ◽  
pp. e2114552118
Author(s):  
Emily Z. Guo ◽  
Jorge E. Galán
Keyword(s):  
Type Iii Secretion ◽  
Central Element ◽  
Eukaryotic Cell ◽  
Effector Proteins ◽  
Target Cells ◽  
Secretion Systems ◽  
Bacterial Surface ◽  
Type Iii ◽  
Cell Plasma ◽  
And Function

Type III secretion systems are multiprotein molecular machines required for the virulence of several important bacterial pathogens. The central element of these machines is the injectisome, a ∼5-Md multiprotein structure that mediates the delivery of bacterially encoded proteins into eukaryotic target cells. The injectisome is composed of a cytoplasmic sorting platform, and a membrane-embedded needle complex, which is made up of a multiring base and a needle-like filament that extends several nanometers from the bacterial surface. The needle filament is capped at its distal end by another substructure known as the tip complex, which is crucial for the translocation of effector proteins through the eukaryotic cell plasma membrane. Here we report the cryo-EM structure of the Salmonella Typhimurium needle tip complex docked onto the needle filament tip. Combined with a detailed analysis of structurally guided mutants, this study provides major insight into the assembly and function of this essential component of the type III secretion protein injection machine.

scholarly journals Characterization of the Ruler Protein Interaction Interface on the Substrate Specificity Switch Protein in the Yersinia Type III Secretion System

2016 ◽  
Vol 292 (8) ◽  
pp. 3299-3311 ◽  
Author(s):  
Oanh Ho ◽  
Per Rogne ◽  
Tomas Edgren ◽  
Hans Wolf-Watz ◽  
Frédéric H. Login ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Type Iii ◽  
Binding Interface ◽  
Interaction Interface

Many pathogenic Gram-negative bacteria use the type III secretion system (T3SS) to deliver effector proteins into eukaryotic host cells. In Yersinia, the switch to secretion of effector proteins is induced first after intimate contact between the bacterium and its eukaryotic target cell has been established, and the T3SS proteins YscP and YscU play a central role in this process. Here we identify the molecular details of the YscP binding site on YscU by means of nuclear magnetic resonance (NMR) spectroscopy. The binding interface is centered on the C-terminal domain of YscU. Disrupting the YscU-YscP interaction by introducing point mutations at the interaction interface significantly reduced the secretion of effector proteins and HeLa cell cytotoxicity. Interestingly, the binding of YscP to the slowly self-cleaving YscU variant P264A conferred significant protection against autoproteolysis. The YscP-mediated inhibition of YscU autoproteolysis suggests that the cleavage event may act as a timing switch in the regulation of early versus late T3SS substrates. We also show that YscUC binds to the inner rod protein YscI with a dissociation constant (Kd) of 3.8 μm and with 1:1 stoichiometry. The significant similarity among different members of the YscU, YscP, and YscI families suggests that the protein-protein interactions discussed in this study are also relevant for other T3SS-containing Gram-negative bacteria.

scholarly journals Inhibition of Type III Secretion in Salmonella enterica Serovar Typhimurium by Small-Molecule Inhibitors

2007 ◽  
Vol 51 (7) ◽  
pp. 2631-2635 ◽  
Author(s):  
Debra L. Hudson ◽  
Abigail N. Layton ◽  
Terry R. Field ◽  
Alison J. Bowen ◽  
Hans Wolf-Watz ◽  
...  
Keyword(s):  
Small Molecules ◽  
Type Iii Secretion ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Gram Negative Bacteria ◽  
Secretion Systems ◽  
Type Iii ◽  
Type Iii Secretion Systems ◽  
Cultured Epithelial Cells ◽  
Serovar Typhimurium

ABSTRACT Type III secretion systems (T3SS) are conserved in many pathogenic gram-negative bacteria. Small molecules that specifically target T3SS in Yersinia and Chlamydia spp. have recently been identified. Here we show that two such compounds inhibit Salmonella T3SS-1, preventing secretion of T3SS-1 effectors, invasion of cultured epithelial cells, and enteritis in vivo.

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.

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