scholarly journals Functional Analysis of HrpF, a Putative Type III Translocon Protein from Xanthomonas campestris pv. vesicatoria

2002 ◽  
Vol 184 (9) ◽  
pp. 2389-2398 ◽  
Author(s):  
Daniela Büttner ◽  
Dirk Nennstiel ◽  
Birgit Klüsener ◽  
Ulla Bonas
Keyword(s):  
Host Cell ◽  
Lipid Bilayers ◽  
Type Iii Secretion ◽  
Xanthomonas Campestris ◽  
Lipid Binding ◽  
Effector Proteins ◽  
Transport Systems ◽  
Secretion Systems ◽  
Type Iii ◽  
N Terminus

ABSTRACT Type III secretion systems (TTSSs) are specialized protein transport systems in gram-negative bacteria which target effector proteins into the host cell. The TTSS of the plant pathogen Xanthomonas campestris pv. vesicatoria, encoded by the hrp (hypersensitive reaction and pathogenicity) gene cluster, is essential for the interaction with the plant. One of the secreted proteins is HrpF, which is required for pathogenicity but dispensable for type III secretion of effector proteins in vitro, suggesting a role in translocation. In this study, complementation analyses of an hrpF null mutant strain using various deletion derivatives revealed the functional importance of the C-terminal hydrophobic protein region. Deletion of the N terminus abolished type III secretion of HrpF. Employing the type III effector AvrBs3 as a reporter, we show that the N terminus of HrpF contains a signal for secretion but not a functional translocation signal. Experiments with lipid bilayers revealed a lipid-binding activity of HrpF as well as HrpF-dependent pore formation. These data indicate that HrpF presumably plays a role at the bacterial-plant interface as part of a bacterial translocon which mediates effector protein delivery across the host cell membrane.

scholarly journals Identification of a Specific Chaperone for SptP, a Substrate of the Centisome 63 Type III Secretion System ofSalmonella typhimurium

1998 ◽  
Vol 180 (13) ◽  
pp. 3393-3399 ◽  
Author(s):  
Yixin Fu ◽  
Jorge E. Galán
Keyword(s):  
Actin Cytoskeleton ◽  
Host Cell ◽  
Type Iii Secretion ◽  
Tyrosine Phosphatase ◽  
Secretion System ◽  
Effector Proteins ◽  
Loss Of Function ◽  
Secretion Systems ◽  
Type Iii ◽  
Type Iii Protein Secretion

ABSTRACT Salmonella typhimurium uses of a type III protein secretion system encoded at centisome 63 of its chromosome to deliver effector molecules into the host cell. These proteins stimulate host cell responses such as reorganization of the actin cytoskeleton and activation of transcription factors. One of these effector proteins is SptP, a tyrosine phosphatase that causes disruption of the host cell actin cytoskeleton. A characteristic feature of many substrates of type III secretion systems is their association with specific cytoplasmic chaperones which appears to be required for secretion and/or translocation of these proteins into the host cell. We report here the identification of SicP, a 13-kDa acidic polypeptide that is encoded immediately upstream of sptP. A loss-of-function mutation in sicP resulted in drastically reduced levels of SptP but did not affect sptP expression, indicating that SicP exerts its effect posttranscriptionally. Pulse-chase experiments demonstrated that the loss of SicP leads to increased degradation of SptP. In addition, we show that SicP binds to SptP directly and that the binding site is located between residues 15 and 100 of the tyrosine phosphatase. Taken together, these results indicate that SicP acts as a specific chaperone for SptP.

scholarly journals Identification of Novel Host Interactors of Effectors Secreted by Salmonella and Citrobacter

mSystems ◽  
2016 ◽  
Vol 1 (4) ◽  
Author(s):  
Ryan L. Sontag ◽  
Ernesto S. Nakayasu ◽  
Roslyn N. Brown ◽  
George S. Niemann ◽  
Michael A. Sydor ◽  
...  
Keyword(s):  
Host Cell ◽  
Type Iii Secretion ◽  
Defense Mechanisms ◽  
Pathogenic Bacteria ◽  
Effector Proteins ◽  
Host Cells ◽  
Secretion Systems ◽  
Type Iii ◽  
Cell Cytosol ◽  
Host Cell Cytosol

ABSTRACT During infection, pathogenic bacteria face an adverse environment of factors driven by both cellular and humoral defense mechanisms. To help evade the immune response and ultimately proliferate inside the host, many bacteria evolved specialized secretion systems to deliver effector proteins directly into host cells. Translocated effector proteins function to subvert host defense mechanisms. Numerous pathogenic bacteria use a specialized secretion system called type III secretion to deliver effectors into the host cell cytosol. Here, we identified 75 new host targets of Salmonella and Citrobacter effectors, which will help elucidate their mechanisms of action. Many pathogenic bacteria of the family Enterobacteriaceae use type III secretion systems to inject virulence proteins, termed “effectors,” into the host cell cytosol. Although host-cellular activities of several effectors have been demonstrated, the function and host-targeted pathways of most of the effectors identified to date are largely undetermined. To gain insight into host proteins targeted by bacterial effectors, we performed coaffinity purification of host proteins from cell lysates using recombinant effectors from the Enterobacteriaceae intracellular pathogens Salmonella enterica serovar Typhimurium and Citrobacter rodentium. We identified 54 high-confidence host interactors for the Salmonella effectors GogA, GtgA, GtgE, SpvC, SrfH, SseL, SspH1, and SssB collectively and 21 interactors for the Citrobacter effectors EspT, NleA, NleG1, and NleK. We biochemically validated the interaction between the SrfH Salmonella protein and the extracellular signal-regulated kinase 2 (ERK2) host protein kinase, which revealed a role for this effector in regulating phosphorylation levels of this enzyme, which plays a central role in signal transduction. IMPORTANCE During infection, pathogenic bacteria face an adverse environment of factors driven by both cellular and humoral defense mechanisms. To help evade the immune response and ultimately proliferate inside the host, many bacteria evolved specialized secretion systems to deliver effector proteins directly into host cells. Translocated effector proteins function to subvert host defense mechanisms. Numerous pathogenic bacteria use a specialized secretion system called type III secretion to deliver effectors into the host cell cytosol. Here, we identified 75 new host targets of Salmonella and Citrobacter effectors, which will help elucidate their mechanisms of action.

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 Type III Secretion Systems and Disease

2007 ◽  
Vol 20 (4) ◽  
pp. 535-549 ◽  
Author(s):  
Bryan Coburn ◽  
Inna Sekirov ◽  
B. Brett Finlay
Keyword(s):  
Host Cell ◽  
Type Iii Secretion ◽  
Human Infection ◽  
Effector Proteins ◽  
Secretion Systems ◽  
Barrier Dysfunction ◽  
Type Iii ◽  
Extracellular Milieu ◽  
Host Cell Cytoplasm ◽  
Type Iii Secretion Systems

SUMMARY Type III secretion systems (T3SSs) are complex bacterial structures that provide gram-negative pathogens with a unique virulence mechanism enabling them to inject bacterial effector proteins directly into the host cell cytoplasm, bypassing the extracellular milieu. Although the effector proteins vary among different T3SS pathogens, common pathogenic mechanisms emerge, including interference with the host cell cytoskeleton to promote attachment and invasion, interference with cellular trafficking processes, cytotoxicity and barrier dysfunction, and immune system subversion. The activity of the T3SSs correlates closely with infection progression and outcome, both in animal models and in human infection. Therefore, to facilitate patient care and improve outcomes, it is important to understand the T3SS-mediated virulence processes and to target T3SSs in therapeutic and prophylactic development efforts.

Characterization of the type III secretion associated low calcium response genes of Vibrio parahaemolyticus RIMD2210633

2012 ◽  
Vol 58 (11) ◽  
pp. 1306-1315 ◽  
Author(s):  
Darren Sarty ◽  
Noel T. Baker ◽  
Euan L. Thomson ◽  
Cheryl Rafuse ◽  
Roger O. Ebanks ◽  
...  
Keyword(s):  
Host Cell ◽  
Type Iii Secretion ◽  
Vibrio Parahaemolyticus ◽  
Effector Proteins ◽  
Cell Cytotoxicity ◽  
Secretion Systems ◽  
Calcium Response ◽  
Type Iii ◽  
Low Calcium ◽  
Biochemical Analyses

Vibrio parahaemolyticus is a significant human pathogen associated with gastroenteritis. Two V. parahaemolyticus type 3 secretion systems, T3SS-1 and T3SS-2, secrete effector proteins and have been implicated in host-cell cytotoxicity and enterotoxicity, respectively. Vibrio parahaemolyticus T3SS-1 substrates have been identified, although many predicted substrates (based on homologies) remain undetected in secreted fractions and therefore uncharacterized. We have experimentally developed and optimized a secretion assay protocol allowing for reliable and reproducible detection of V. parahaemolyticus T3SS-1 secreted proteins within culture supernatants. The presence of magnesium and absence of calcium were critical factors in promoting type III secretion of protein substrates. Proteomic approaches identified known V. parahaemolyticus secreted effectors in addition to previously unidentified proteins. Isogenic mutants in putative low calcium response genes were generated, and experiments further implicated the genes in secretion and V. parahaemolyticus-mediated host-cell cytotoxicity during infection. These approaches should be valuable towards future detailed genetic and biochemical analyses of T3SS-1 in V. parahaemolyticus.

scholarly journals Domains of the Shigella flexneri Type III Secretion System IpaB Protein Involved in Secretion Regulation

2010 ◽  
Vol 78 (12) ◽  
pp. 4999-5010 ◽  
Author(s):  
Da-Kang Shen ◽  
Saroj Saurya ◽  
Carolin Wagner ◽  
Hiroaki Nishioka ◽  
Ariel J. Blocker
Keyword(s):  
Host Cell ◽  
Type Iii Secretion ◽  
Effector Proteins ◽  
Physical Contact ◽  
Host Cells ◽  
Cell Contact ◽  
Secretion Systems ◽  
Host Cell Membrane ◽  
Type Iii ◽  
Secretion Regulation

ABSTRACT Type III secretion systems (T3SSs) are key determinants of virulence in many Gram-negative bacterial pathogens. Upon cell contact, they inject effector proteins directly into eukaryotic cells through a needle protruding from the bacterial surface. Host cell sensing occurs through a distal needle “tip complex,” but how this occurs is not understood. The tip complex of quiescent needles is composed of IpaD, which is topped by IpaB. Physical contact with host cells initiates secretion and leads to assembly of a pore, formed by IpaB and IpaC, in the host cell membrane, through which other virulence effector proteins may be translocated. IpaB is required for regulation of secretion and may be the host cell sensor. It binds needles via its extreme C-terminal coiled coil, thereby likely positioning a large domain containing its hydrophobic regions at the distal tips of needles. In this study, we used short deletion mutants within this domain to search for regions of IpaB involved in secretion regulation. This identified two regions, amino acids 227 to 236 and 297 to 306, the presence of which are required for maintenance of IpaB at the needle tip, secretion regulation, and normal pore formation but not invasion. We therefore propose that removal of either of these regions leads to an inability to block secretion prior to reception of the activation signal and/or a defect in host cell sensing.

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.

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 Bridging the Gap: Type III Secretion Systems in Plant-Beneficial Bacteria

2022 ◽  
Vol 10 (1) ◽  
pp. 187
Author(s):  
Antoine Zboralski ◽  
Adrien Biessy ◽  
Martin Filion
Keyword(s):  
Type Iii Secretion ◽  
Plant Pathogens ◽  
Plant Immunity ◽  
Effector Proteins ◽  
Secretion Systems ◽  
Beneficial Bacteria ◽  
Living Plant ◽  
Pseudomonas Spp ◽  
Type Iii ◽  
Type Iii Secretion Systems

Type III secretion systems (T3SSs) are bacterial membrane-embedded nanomachines translocating effector proteins into the cytoplasm of eukaryotic cells. They have been intensively studied for their important roles in animal and plant bacterial diseases. Over the past two decades, genome sequencing has unveiled their ubiquitous distribution in many taxa of Gram-negative bacteria, including plant-beneficial ones. Here, we discuss the distribution and functions of the T3SS in two agronomically important bacterial groups: the symbiotic nodule-forming nitrogen-fixing rhizobia and the free-living plant-beneficial Pseudomonas spp. In legume-rhizobia symbiosis, T3SSs and their cognate effectors play important roles, including the modulation of the plant immune response and the initiation of the nodulation process in some cases. In plant-beneficial Pseudomonas spp., the roles of T3SSs are not fully understood, but pertain to plant immunity suppression, biocontrol against eukaryotic plant pathogens, mycorrhization facilitation, and possibly resistance against protist predation. The diversity of T3SSs in plant-beneficial bacteria points to their important roles in multifarious interkingdom interactions in the rhizosphere. We argue that the gap in research on T3SSs in plant-beneficial bacteria must be bridged to better understand bacteria/eukaryotes rhizosphere interactions and to support the development of efficient plant-growth promoting microbial inoculants.

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