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Sample records for iii secretion effector

  1. Computational prediction of type III and IV secreted effectors in Gram-negative bacteria

    SciTech Connect

    McDermott, Jason E.; Corrigan, Abigail L.; Peterson, Elena S.; Oehmen, Christopher S.; Niemann, George; Cambronne, Eric; Sharp, Danna; Adkins, Joshua N.; Samudrala, Ram; Heffron, Fred

    2011-01-01

    In this review, we provide an overview of the methods employed by four recent papers that described novel methods for computational prediction of secreted effectors from type III and IV secretion systems in Gram-negative bacteria. The results of the studies in terms of performance at accurately predicting secreted effectors and similarities found between secretion signals that may reflect biologically relevant features for recognition. We discuss the web-based tools for secreted effector prediction described in these studies and announce the availability of our tool, the SIEVEserver (http://www.biopilot.org). Finally, we assess the accuracy of the three type III effector prediction methods on a small set of proteins not known prior to the development of these tools that we have recently discovered and validated using both experimental and computational approaches. Our comparison shows that all methods use similar approaches and, in general arrive at similar conclusions. We discuss the possibility of an order-dependent motif in the secretion signal, which was a point of disagreement in the studies. Our results show that there may be classes of effectors in which the signal has a loosely defined motif, and others in which secretion is dependent only on compositional biases. Computational prediction of secreted effectors from protein sequences represents an important step toward better understanding the interaction between pathogens and hosts.

  2. Die another day: molecular mechanisms of effector-triggered immunity elicited by type III secreted effector proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacterial pathogens inject type III secreted effector (T3SE) proteins into their hosts where they display dual roles depending on the host genotype. T3SEs promote bacterial virulence in susceptible hosts, and elicit immunity in resistant hosts. T3SEs are typically recognized when they modify a host ...

  3. Pseudomonas syringae type III secretion system effectors: repertoires in search of functions.

    PubMed

    Cunnac, Sébastien; Lindeberg, Magdalen; Collmer, Alan

    2009-02-01

    The ability of Pseudomonas syringae to grow and cause diseases in plants is dependent on the injection of multiple effector proteins into plant cells via the type III secretion system (T3SS). Genome-enabled bioinformatic/experimental methods have comprehensively identified the repertoires of effectors and related T3SS substrates for P. syringae pv. tomato DC3000 and three other sequenced strains. The effector repertoires are diverse and internally redundant. Insights into effector functions are being gained through the construction of mutants lacking one or more effector genes, which may be reduced in growth in planta, and through gain-of-function assays for the ability of single effectors to suppress plant innate immune defenses, manipulate hormone signaling, elicit cell death, and/or display biochemical activities on plant protein targets. PMID:19168384

  4. A survey of the Pseudomonas syringae pv. tomato DC3000 type III secretion system effector repertoire reveals several effectors that are deleterious when expressed in Saccharomyces cerevisiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The injection of nearly 30 effector proteins by the type III secretion system underlies the ability of Pseudomonas syringae pv. tomato strain DC3000 to cause disease in tomato and other host plants. The search for effector functions is complicated by redundancy within the repertoire and by plant R-g...

  5. Deletions in the repertoire of Pseudomonas syringae pv. tomato DC3000 type III secretion effector genes reveal functional overlap among effectors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many bacterial pathogens of plants and animals disarm and remodel host cells by injecting large repertoires of effectors via the type III secretion system (T3SS). The repertoires of individual strains appear to function as robust systems that can tolerate loss of individual effectors with little or ...

  6. Identification of a Family of Effectors Secreted by the Type III Secretion System That Are Conserved in Pathogenic Chlamydiae▿

    PubMed Central

    Muschiol, Sandra; Boncompain, Gaelle; Vromman, François; Dehoux, Pierre; Normark, Staffan; Henriques-Normark, Birgitta; Subtil, Agathe

    2011-01-01

    Chlamydiae are Gram-negative, obligate intracellular pathogens that replicate within a membrane-bounded compartment termed an inclusion. Throughout their development, they actively modify the eukaryotic environment. The type III secretion (TTS) system is the main process by which the bacteria translocate effector proteins into the inclusion membrane and the host cell cytoplasm. Here we describe a family of type III secreted effectors that are present in all pathogenic chlamydiae and absent in the environment-related species. It is defined by a common domain of unknown function, DUF582, that is present in four or five proteins in each Chlamydiaceae species. We show that the amino-terminal extremity of DUF582 proteins functions as a TTS signal. DUF582 proteins from C. trachomatis CT620, CT621, and CT711 are expressed at the middle and late phases of the infectious cycle. Immunolocalization further revealed that CT620 and CT621 are secreted into the host cell cytoplasm, as well as within the lumen of the inclusion, where they do not associate with bacterial markers. Finally, we show that DUF582 proteins are present in nuclei of infected cells, suggesting that members of the DUF582 family of effector proteins may target nuclear cell functions. The expansion of this family of proteins in pathogenic chlamydiae and their conservation among the different species suggest that they play important roles in the infectious cycle. PMID:21078856

  7. A bacterial type III secretion assay for delivery of fungal effector proteins into wheat.

    PubMed

    Upadhyaya, Narayana M; Mago, Rohit; Staskawicz, Brian J; Ayliffe, Michael A; Ellis, Jeffrey G; Dodds, Peter N

    2014-03-01

    Large numbers of candidate effectors from fungal pathogens are being identified through whole-genome sequencing and in planta expression studies. Although Agrobacterium-mediated transient expression has enabled high-throughput functional analysis of effectors in dicot plants, this assay is not effective in cereal leaves. Here, we show that a nonpathogenic Pseudomonas fluorescens engineered to express the type III secretion system (T3SS) of P. syringae and the wheat pathogen Xanthomonas translucens can deliver fusion proteins containing T3SS signals from P. syringae (AvrRpm1) and X. campestris (AvrBs2) avirulence (Avr) proteins, respectively, into wheat leaf cells. A calmodulin-dependent adenylate cyclase reporter protein was delivered effectively into wheat and barley by both bacteria. Absence of any disease symptoms with P. fluorescens makes it more suitable than X. translucens for detecting a hypersensitive response (HR) induced by an effector protein with avirulence activity. We further modified the delivery system by removal of the myristoylation site from the AvrRpm1 fusion to prevent its localization to the plasma membrane which could inhibit recognition of an Avr protein. Delivery of the flax rust AvrM protein by the modified delivery system into transgenic tobacco leaves expressing the corresponding M resistance protein induced a strong HR, indicating that the system is capable of delivering a functional rust Avr protein. In a preliminary screen of effectors from the stem rust fungus Puccinia graminis f. sp. tritici, we identified one effector that induced a host genotype-specific HR in wheat. Thus, the modified AvrRpm1:effector-Pseudomonas fluorescens system is an effective tool for large-scale screening of pathogen effectors for recognition in wheat. PMID:24156769

  8. Evaluation of Salmonella enterica Type III Secretion System Effector Proteins as Carriers for Heterologous Vaccine Antigens

    PubMed Central

    Hegazy, Wael Abdel Halim; Xu, Xin; Metelitsa, Leonid

    2012-01-01

    Live attenuated strains of Salmonella enterica have a high potential as carriers of recombinant vaccines. The type III secretion system (T3SS)-dependent translocation of S. enterica can be deployed for delivery of heterologous antigens to antigen-presenting cells. Here we investigated the efficacy of various effector proteins of the Salmonella pathogenicity island (SPI2)-encoded T3SS for the translocation of model antigens and elicitation of immune responses. The SPI2 T3SS effector proteins SifA, SteC, SseL, SseJ, and SseF share an endosomal membrane-associated subcellular localization after translocation. We observed that all effector proteins could be used to translocate fusion proteins with the model antigens ovalbumin and listeriolysin into the cytosol of host cells. Under in vitro conditions, fusion proteins with SseJ and SteC stimulated T-cell responses that were superior to those triggered by fusion proteins with SseF. However, in mice vaccinated with Salmonella carrier strains, only fusion proteins based on SseJ or SifA elicited potent T-cell responses. These data demonstrate that the selection of an optimal SPI2 effector protein for T3SS-mediated translocation is a critical parameter for the rational design of effective Salmonella-based recombinant vaccines. PMID:22252866

  9. Identification and Characterization of Putative Translocated Effector Proteins of the Edwardsiella ictaluri Type III Secretion System.

    PubMed

    Dubytska, Lidiya P; Rogge, Matthew L; Thune, Ronald L

    2016-01-01

    Edwardsiella ictaluri, a major pathogen in channel catfish aquaculture, encodes a type III secretion system (T3SS) that is essential for intracellular replication and virulence. Previous work identified three putative T3SS effectors in E. ictaluri, and in silico analysis of the E. ictaluri genome identified six additional putative effectors, all located on the chromosome outside the T3SS pathogenicity island. To establish active translocation by the T3SS, we constructed translational fusions of each effector to the amino-terminal adenylate cyclase (AC) domain of the Bordetella pertussis adenylate cyclase toxin CyaA. When translocated through the membrane of the Edwardsiella-containing vacuole (ECV), the cyclic AMP produced by the AC domain in the presence of calmodulin in the host cell cytoplasm can be measured. Results showed that all nine effectors were translocated from E. ictaluri in the ECV to the cytoplasm of the host cells in the wild-type strain but not in a T3SS mutant, indicating that translocation is dependent on the T3SS machinery. This confirms that the E. ictaluri T3SS is similar to the Salmonella pathogenicity island 2 T3SS in that it translocates effectors through the membrane of the bacterial vacuole directly into the host cell cytoplasm. Additional work demonstrated that both initial acidification and subsequent neutralization of the ECV were necessary for effector translocation, except for two of them that did not require neutralization. Single-gene mutants constructed for seven of the individual effectors were all attenuated for replication in CCO cells, but only three were replication deficient in head kidney-derived macrophages (HKDM). IMPORTANCE The bacterial pathogen Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), an economically significant disease of farm-raised channel catfish. Commercial catfish production accounts for the majority of the total fin fish aquaculture in the United States, with almost 300,000

  10. Identification and Characterization of Putative Translocated Effector Proteins of the Edwardsiella ictaluri Type III Secretion System

    PubMed Central

    Dubytska, Lidiya P.; Rogge, Matthew L.

    2016-01-01

    ABSTRACT Edwardsiella ictaluri, a major pathogen in channel catfish aquaculture, encodes a type III secretion system (T3SS) that is essential for intracellular replication and virulence. Previous work identified three putative T3SS effectors in E. ictaluri, and in silico analysis of the E. ictaluri genome identified six additional putative effectors, all located on the chromosome outside the T3SS pathogenicity island. To establish active translocation by the T3SS, we constructed translational fusions of each effector to the amino-terminal adenylate cyclase (AC) domain of the Bordetella pertussis adenylate cyclase toxin CyaA. When translocated through the membrane of the Edwardsiella-containing vacuole (ECV), the cyclic AMP produced by the AC domain in the presence of calmodulin in the host cell cytoplasm can be measured. Results showed that all nine effectors were translocated from E. ictaluri in the ECV to the cytoplasm of the host cells in the wild-type strain but not in a T3SS mutant, indicating that translocation is dependent on the T3SS machinery. This confirms that the E. ictaluri T3SS is similar to the Salmonella pathogenicity island 2 T3SS in that it translocates effectors through the membrane of the bacterial vacuole directly into the host cell cytoplasm. Additional work demonstrated that both initial acidification and subsequent neutralization of the ECV were necessary for effector translocation, except for two of them that did not require neutralization. Single-gene mutants constructed for seven of the individual effectors were all attenuated for replication in CCO cells, but only three were replication deficient in head kidney-derived macrophages (HKDM). IMPORTANCE The bacterial pathogen Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), an economically significant disease of farm-raised channel catfish. Commercial catfish production accounts for the majority of the total fin fish aquaculture in the United States, with almost 300,000

  11. Global impact of Salmonella type III secretion effector SteA on host cells

    SciTech Connect

    Cardenal-Muñoz, Elena Gutiérrez, Gabriel Ramos-Morales, Francisco

    2014-07-11

    Highlights: • We analyzed HeLa cells transcriptome in response to Salmonella SteA. • Significant differential expression was detected for 58 human genes. • They are involved in ECM organization and regulation of some signaling pathways. • Cell death, cell adhesion and cell migration were decreased in SteA-expressing cells. • These results contribute to understand the role of SteA during infections. - Abstract: Salmonella enterica is a Gram-negative bacterium that causes gastroenteritis, bacteremia and typhoid fever in several animal species including humans. Its virulence is greatly dependent on two type III secretion systems, encoded in pathogenicity islands 1 and 2. These systems translocate proteins called effectors into eukaryotic host cell. Effectors interfere with host signal transduction pathways to allow the internalization of pathogens and their survival and proliferation inside vacuoles. SteA is one of the few Salmonella effectors that are substrates of both type III secretion systems. Here, we used gene arrays and bioinformatics analysis to study the genetic response of human epithelial cells to SteA. We found that constitutive synthesis of SteA in HeLa cells leads to induction of genes related to extracellular matrix organization and regulation of cell proliferation and serine/threonine kinase signaling pathways. SteA also causes repression of genes related to immune processes and regulation of purine nucleotide synthesis and pathway-restricted SMAD protein phosphorylation. In addition, a cell biology approach revealed that epithelial cells expressing steA show altered cell morphology, and decreased cytotoxicity, cell–cell adhesion and migration.

  12. Quantitative Proteomic Analysis of Burkholderia pseudomallei Bsa Type III Secretion System Effectors Using Hypersecreting Mutants

    PubMed Central

    Vander Broek, Charles W.; Chalmers, Kevin J.; Stevens, Mark P.; Stevens, Joanne M.

    2015-01-01

    Burkholderia pseudomallei is an intracellular pathogen and the causative agent of melioidosis, a severe disease of humans and animals. One of the virulence factors critical for early stages of infection is the Burkholderia secretion apparatus (Bsa) Type 3 Secretion System (T3SS), a molecular syringe that injects bacterial proteins, called effectors, into eukaryotic cells where they subvert cellular functions to the benefit of the bacteria. Although the Bsa T3SS itself is known to be important for invasion, intracellular replication, and virulence, only a few genuine effector proteins have been identified and the complete repertoire of proteins secreted by the system has not yet been fully characterized. We constructed a mutant lacking bsaP, a homolog of the T3SS “gatekeeper” family of proteins that exert control over the timing and magnitude of effector protein secretion. Mutants lacking BsaP, or the T3SS translocon protein BipD, were observed to hypersecrete the known Bsa effector protein BopE, providing evidence of their role in post-translational control of the Bsa T3SS and representing key reagents for the identification of its secreted substrates. Isobaric Tags for Relative and Absolute Quantification (iTRAQ), a gel-free quantitative proteomics technique, was used to compare the secreted protein profiles of the Bsa T3SS hypersecreting mutants of B. pseudomallei with the isogenic parent strain and a bsaZ mutant incapable of effector protein secretion. Our study provides one of the most comprehensive core secretomes of B. pseudomallei described to date and identified 26 putative Bsa-dependent secreted proteins that may be considered candidate effectors. Two of these proteins, BprD and BapA, were validated as novel effector proteins secreted by the Bsa T3SS of B. pseudomallei. PMID:25635268

  13. Structure of the Type III Secretion Effector Protein ExoU in Complex with Its Chaperone SpcU

    PubMed Central

    Halavaty, Andrei S.; Borek, Dominika; Tyson, Gregory H.; Veesenmeyer, Jeff L.; Shuvalova, Ludmilla; Minasov, George; Otwinowski, Zbyszek

    2012-01-01

    Disease causing bacteria often manipulate host cells in a way that facilitates the infectious process. Many pathogenic gram-negative bacteria accomplish this by using type III secretion systems. In these complex secretion pathways, bacterial chaperones direct effector proteins to a needle-like secretion apparatus, which then delivers the effector protein into the host cell cytosol. The effector protein ExoU and its chaperone SpcU are components of the Pseudomonas aeruginosa type III secretion system. Secretion of ExoU has been associated with more severe infections in both humans and animal models. Here we describe the 1.92 Å X-ray structure of the ExoU–SpcU complex, a full-length type III effector in complex with its full-length cognate chaperone. Our crystallographic data allow a better understanding of the mechanism by which ExoU kills host cells and provides a foundation for future studies aimed at designing inhibitors of this potent toxin. PMID:23166655

  14. The Salmonella type III secretion system virulence effector forms a new hexameric chaperone assembly for export of effector/chaperone complexes

    DOE PAGESBeta

    Tsai, Chi -Lin; Burkinshaw, Brianne J.; Strynadka, Natalie C. J.; Tainer, John A.

    2014-12-08

    Bacteria hijack eukaryotic cells by injecting virulence effectors into host cytosol with a type III secretion system (T3SS). Effectors are targeted with their cognate chaperones to hexameric T3SS ATPase at the bacterial membrane's cytosolic face. In this issue of the Journal of Bacteriology, Roblin et al. (P. Roblin, F. Dewitte, V. Villeret, E. G. Biondi, and C. Bompard, J Bacteriol 197:688–698, 2015, http://dx.doi.org/10.1128/JB.02294-14) show that the T3SS chaperone SigE of Salmonella can form hexameric rings rather than dimers when bound to its cognate effector, SopB, implying a novel multimeric association for chaperone/effector complexes with their ATPase.

  15. The Salmonella type III secretion system virulence effector forms a new hexameric chaperone assembly for export of effector/chaperone complexes

    SciTech Connect

    Tsai, Chi -Lin; Burkinshaw, Brianne J.; Strynadka, Natalie C. J.; Tainer, John A.

    2014-12-08

    Bacteria hijack eukaryotic cells by injecting virulence effectors into host cytosol with a type III secretion system (T3SS). Effectors are targeted with their cognate chaperones to hexameric T3SS ATPase at the bacterial membrane's cytosolic face. In this issue of the Journal of Bacteriology, Roblin et al. (P. Roblin, F. Dewitte, V. Villeret, E. G. Biondi, and C. Bompard, J Bacteriol 197:688–698, 2015, http://dx.doi.org/10.1128/JB.02294-14) show that the T3SS chaperone SigE of Salmonella can form hexameric rings rather than dimers when bound to its cognate effector, SopB, implying a novel multimeric association for chaperone/effector complexes with their ATPase.

  16. A bacterial type III secretion-based delivery system for functional assays of fungal effectors in cereals.

    PubMed

    Upadhyaya, Narayana M; Ellis, Jeffery G; Dodds, Peter N

    2014-01-01

    Large numbers of candidate effectors are being identified by genome sequencing of fungal pathogens and in planta expression studies. These effectors are both a boon and a curse for pathogens as they modulate the host cellular environment or suppress defense response to allow fungal growth as well as become targets of plant resistance (R) proteins. Recognition of a fungal effector by a plant R protein triggers a hypersensitive reaction (HR) leading to death of plant cells in and around the infection site, thus preventing further proliferation of the pathogen. Such HR induction has been used as an indicator of effector activity in functional assays of candidate effectors in dicots based on Agrobacterium-mediated transient expression. However, the Agrobacterium assay is not functional in cereal leaves. We therefore have adapted an alternative assay based on effector protein delivery using the type III secretion system (T3SS) of a non-pathogenic Pseudomonas spp. for use in wheat and other cereals. Here, we describe protocols for delivery of effector proteins into wheat and barley cells using the AvrRpm1 T3SS signal in the engineered non-pathogenic Pseudomonas fluorescens strain Effector-to-Host Analyzer (EtHAn). For ease of making expression clones we have generated the GATEWAY cloning compatible vectors. A calmodulin-dependent adenylate cyclase (Cya) reporter protein can be used as an effective marker for fusion protein delivery into wheat and barley by this system. PMID:24643568

  17. Quantitative Mass Spectrometry Identifies Novel Host Binding Partners for Pathogenic Escherichia coli Type III Secretion System Effectors.

    PubMed

    Law, Robyn J; Law, Hong T; Scurll, Joshua M; Scholz, Roland; Santos, Andrew S; Shames, Stephanie R; Deng, Wanyin; Croxen, Matthew A; Li, Yuling; de Hoog, Carmen L; van der Heijden, Joris; Foster, Leonard J; Guttman, Julian A; Finlay, B Brett

    2016-05-01

    Enteropathogenic and enterohemorrhagic Escherichia coli cause enteric diseases resulting in significant morbidity and mortality worldwide. These pathogens remain extracellular and translocate a set of type III secreted effector proteins into host cells to promote bacterial virulence. Effectors manipulate host cell pathways to facilitate infection by interacting with a variety of host targets, yet the binding partners and mechanism of action of many effectors remain elusive. We performed a mass spectrometry screen to identify host targets for a library of effectors. We found five known effector targets and discovered four novel interactions. Interestingly, we identified multiple effectors that interacted with the microtubule associated protein, ensconsin. Using co-immunoprecipitations, we confirmed that NleB1 and EspL interacted with ensconsin in a region that corresponded to its microtubule binding domain. Ensconsin is an essential cofactor of kinesin-1 that is required for intracellular trafficking, and we demonstrated that intracellular trafficking was severely disrupted during wild type EPEC infections but not during infections with ΔnleB1 or ΔespL mutants. Our findings demonstrate the efficacy of quantitative proteomics for identifying effector-host protein interactions and suggest that vesicular trafficking is a crucial cellular process that may be targeted by NleB1 and EspL through their interaction with ensconsin. PMID:27018634

  18. Bioinformatics-enabled identification of the HrpL regulon and type III secretion system effector proteins of Pseudomonas syringae pv. phaseolicola 1448A

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ability of Pseudomonas syringae pv. phaseolicola to cause halo blight of bean is dependent on its ability to translocate effector proteins into host cells via the Hrp type III secretion system (T3SS). To identity genes encoding type III effectors and other potential virulence factors that are r...

  19. The SdiA-regulated gene srgE encodes a type III secreted effector.

    PubMed

    Habyarimana, Fabien; Sabag-Daigle, Anice; Ahmer, Brian M M

    2014-06-01

    Salmonella enterica serovar Typhimurium is a food-borne pathogen that causes severe gastroenteritis. The ability of Salmonella to cause disease depends on two type III secretion systems (T3SSs) encoded in two distinct Salmonella pathogenicity islands, 1 and 2 (SPI1 and SPI2, respectively). S. Typhimurium encodes a solo LuxR homolog, SdiA, which can detect the acyl-homoserine lactones (AHLs) produced by other bacteria and upregulate the rck operon and the srgE gene. SrgE is predicted to encode a protein of 488 residues with a coiled-coil domain between residues 345 and 382. In silico studies have provided conflicting predictions as to whether SrgE is a T3SS substrate. Therefore, in this work, we tested the hypothesis that SrgE is a T3SS effector by two methods, a β-lactamase activity assay and a split green fluorescent protein (GFP) complementation assay. SrgE with β-lactamase fused to residue 40, 100, 150, or 300 was indeed expressed and translocated into host cells, but SrgE with β-lactamase fused to residue 400 or 488 was not expressed, suggesting interference by the coiled-coil domain. Similarly, SrgE with GFP S11 fused to residue 300, but not to residue 488, was expressed and translocated into host cells. With both systems, translocation into host cells was dependent upon SPI2. A phylogenetic analysis indicated that srgE is found only within Salmonella enterica subspecies. It is found sporadically within both typhoidal and nontyphoidal serovars, although the SrgE protein sequences found within typhoidal serovars tend to cluster separately from those found in nontyphoidal serovars, suggesting functional diversification. PMID:24727228

  20. The SdiA-Regulated Gene srgE Encodes a Type III Secreted Effector

    PubMed Central

    Habyarimana, Fabien; Sabag-Daigle, Anice

    2014-01-01

    Salmonella enterica serovar Typhimurium is a food-borne pathogen that causes severe gastroenteritis. The ability of Salmonella to cause disease depends on two type III secretion systems (T3SSs) encoded in two distinct Salmonella pathogenicity islands, 1 and 2 (SPI1 and SPI2, respectively). S. Typhimurium encodes a solo LuxR homolog, SdiA, which can detect the acyl-homoserine lactones (AHLs) produced by other bacteria and upregulate the rck operon and the srgE gene. SrgE is predicted to encode a protein of 488 residues with a coiled-coil domain between residues 345 and 382. In silico studies have provided conflicting predictions as to whether SrgE is a T3SS substrate. Therefore, in this work, we tested the hypothesis that SrgE is a T3SS effector by two methods, a β-lactamase activity assay and a split green fluorescent protein (GFP) complementation assay. SrgE with β-lactamase fused to residue 40, 100, 150, or 300 was indeed expressed and translocated into host cells, but SrgE with β-lactamase fused to residue 400 or 488 was not expressed, suggesting interference by the coiled-coil domain. Similarly, SrgE with GFP S11 fused to residue 300, but not to residue 488, was expressed and translocated into host cells. With both systems, translocation into host cells was dependent upon SPI2. A phylogenetic analysis indicated that srgE is found only within Salmonella enterica subspecies. It is found sporadically within both typhoidal and nontyphoidal serovars, although the SrgE protein sequences found within typhoidal serovars tend to cluster separately from those found in nontyphoidal serovars, suggesting functional diversification. PMID:24727228

  1. Closing the circle on the discovery of genes encoding Hrp regulon members and type III secretion system effectors in the genomes of three model Pseudomonas syringae strains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pseudomonas syringae strains translocate large and distinct collections of effector proteins into plant cells via the type III secretion system (T3SS). P. syringae effectors are designated Hop (Hrp outer protein) or Avr (avirulence) proteins. Some Hop proteins are considered to be extracellular T3...

  2. Pseudomonas syringae pv. Tomato DC3000 Type III secretion effector polymutants reveal an interplay between hopAD1 and AvrPtoB

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The model pathogen Pseudomonas syringae pv. tomato DC3000 suppresses the two-tiered innate immune system of plants by injecting a complex repertoire of effector proteins into host cells via the type III secretion system. The model effector AvrPtoB has multiple domains and plant protein interactors i...

  3. Novel fold of VirA, a type III secretion system effector protein from Shigella flexneri

    SciTech Connect

    Davis, Jamaine; Wang, Jiawei; Tropea, Joseph E.; Zhang, Di; Dauter, Zbigniew; Waugh, David S.; Wlodawer, Alexander

    2009-01-28

    VirA, a secreted effector protein from Shigella sp., has been shown to be necessary for its virulence. It was also reported that VirA might be related to papain-like cysteine proteases and cleave {alpha}-tubulin, thus facilitating intracellular spreading. We have now determined the crystal structure of VirA at 3.0 {angstrom} resolution. The shape of the molecule resembles the letter 'V,' with the residues in the N-terminal third of the 45-kDa molecule (some of which are disordered) forming one clearly identifiable domain, and the remainder of the molecule completing the V-like structure. The fold of VirA is unique and does not resemble that of any known protein, including papain, although its N-terminal domain is topologically similar to cysteine protease inhibitors such as stefin B. Analysis of the sequence conservation between VirA and its Escherichia coli homologs EspG and EspG2 did not result in identification of any putative protease-like active site, leaving open a possibility that the biological function of VirA in Shigella virulence may not involve direct proteolytic activity.

  4. Intracellular translocation and localization of Edwardsiella tarda type III secretion system effector EseG in host cells.

    PubMed

    Fang, Shan; Zhang, Lingzhi; Lou, Ying; Yang, Dahai; Wang, Qiyao; Zhang, Yuanxing; Liu, Qin

    2016-08-01

    Edwardsiella tarda, an important fish pathogenic bacterium, could utilize type III secretion system (T3SS) to transfer multiple effector proteins into host cells during infection. EseG was identified to be an E. tarda T3SS effector, which could be injected by T3SS into non-phagocytic cells. Since E. tarda is a facultative intracellular pathogen that resides and replicates in macrophage, it is interesting to expand our knowledge about EseG translocation and localization within phagocytic cells. Here utilizing murine macrophage cell line J774A.1 as the cell model, we demonstrated that EseG could be transported into J774A.1 via T3SS only after E. tarda was internalized into macrophage cells, indicating that extracellular E. tarda could not inject EseG into host cells. Subcellular fractionation analysis gave the evidence that EseG was specifically localized in the membrane fraction of infected host cells. Furthermore, immunofluorescence detection indicated that EseG specifically targeted the E. tarda-containing vacuoles (ECVs) within macrophage cells. Finally the unique features for EseG were also confirmed in non-phagocytic cells. In summarize, this work illuminates internalization-depending translocation and ECV-targeting localization of E. tarda T3SS effector in both non-phagocytic and phagocytic cells, which might be important to interpret the interaction of EseG with host cells upon infection. PMID:27208750

  5. Shigella enterotoxin-2 is a type III effector that participates in Shigella-induced interleukin 8 secretion by epithelial cells

    PubMed Central

    Farfán, Mauricio J.; Toro, Cecilia S.; Barry, Eileen M.; Nataro, James P.

    2011-01-01

    We have previously described a protein termed Shigella enterotoxin 2 (ShET-2), which induces rises in short circuit current in rabbit ileum mounted in the Ussing chamber. Published reports have postulated that ShET-2 may be secreted by the Shigella type III secretion system (T3SS). In this study we show that ShET-2 secretion into the extracellular space requires the T3SS in S. flexneri 2a strain 2457T and a ShET-2-TEM fusion was translocated into epithelial cells in a T3SS-dependent manner. The ShET-2 gene, sen, is encoded downstream of the ospC1 gene of S. flexneri, and we show that sen is co-transcribed with this T3SS-secreted product. Considering that T3SS effectors have diverse roles in Shigella infection and that vaccine constructs lacking ShET-2 are attenuated in volunteers, we asked whether ShET-2 has a function other than its enterotoxic activity. We constructed a ShET-2 mutant in 2457T and tested its effect on epithelial cell invasion, plaque formation, guinea pig keratoconjunctivitis and interleukin 8 (IL-8) secretion from infected monolayers. Though other phenotypes were not different compared to the wild-type parent, we found that HEp-2 and T84 cells infected with the ShET-2 mutant exhibited significantly reduced IL-8 secretion into the basolateral compartment, suggesting that ShET-2 might participate in the Shigella-induced inflammation of epithelial cells. PMID:21219446

  6. Edwardsiella tarda EscE (Orf13 Protein) Is a Type III Secretion System-Secreted Protein That Is Required for the Injection of Effectors, Secretion of Translocators, and Pathogenesis in Fish.

    PubMed

    Lu, Jin Fang; Wang, Wei Na; Wang, Gai Ling; Zhang, He; Zhou, Ying; Gao, Zhi Peng; Nie, Pin; Xie, Hai Xia

    2016-01-01

    The type III secretion system (T3SS) of Edwardsiella tarda is crucial for its intracellular survival and pathogenesis in fish. The orf13 gene (escE) of E. tarda is located 84 nucleotides (nt) upstream of esrC in the T3SS gene cluster. We found that EscE is secreted and translocated in a T3SS-dependent manner and that amino acids 2 to 15 in the N terminus were required for a completely functional T3SS in E. tarda. Deletion of escE abolished the secretion of T3SS translocators, as well as the secretion and translocation of T3SS effectors, but did not influence their intracellular protein levels in E. tarda. Complementation of the escE mutant with a secretion-incompetent EscE derivative restored the secretion of translocators and effectors. Interestingly, the effectors that were secreted and translocated were positively correlated with the EscE protein level in E. tarda. The escE mutant was attenuated in the blue gourami fish infection model, as its 50% lethal dose (LD50) increased to 4 times that of the wild type. The survival rate of the escE mutant-strain-infected fish was 69%, which was much higher than that of the fish infected with the wild-type bacteria (6%). Overall, EscE represents a secreted T3SS regulator that controls effector injection and translocator secretion, thus contributing to E. tarda pathogenesis in fish. The homology of EscE within the T3SSs of other bacterial species suggests that the mechanism of secretion and translocation control used by E. tarda may be commonly used by other bacterial pathogens. PMID:26459509

  7. Edwardsiella tarda EscE (Orf13 Protein) Is a Type III Secretion System-Secreted Protein That Is Required for the Injection of Effectors, Secretion of Translocators, and Pathogenesis in Fish

    PubMed Central

    Lu, Jin Fang; Wang, Wei Na; Wang, Gai Ling; Zhang, He; Zhou, Ying; Gao, Zhi Peng

    2015-01-01

    The type III secretion system (T3SS) of Edwardsiella tarda is crucial for its intracellular survival and pathogenesis in fish. The orf13 gene (escE) of E. tarda is located 84 nucleotides (nt) upstream of esrC in the T3SS gene cluster. We found that EscE is secreted and translocated in a T3SS-dependent manner and that amino acids 2 to 15 in the N terminus were required for a completely functional T3SS in E. tarda. Deletion of escE abolished the secretion of T3SS translocators, as well as the secretion and translocation of T3SS effectors, but did not influence their intracellular protein levels in E. tarda. Complementation of the escE mutant with a secretion-incompetent EscE derivative restored the secretion of translocators and effectors. Interestingly, the effectors that were secreted and translocated were positively correlated with the EscE protein level in E. tarda. The escE mutant was attenuated in the blue gourami fish infection model, as its 50% lethal dose (LD50) increased to 4 times that of the wild type. The survival rate of the escE mutant-strain-infected fish was 69%, which was much higher than that of the fish infected with the wild-type bacteria (6%). Overall, EscE represents a secreted T3SS regulator that controls effector injection and translocator secretion, thus contributing to E. tarda pathogenesis in fish. The homology of EscE within the T3SSs of other bacterial species suggests that the mechanism of secretion and translocation control used by E. tarda may be commonly used by other bacterial pathogens. PMID:26459509

  8. A multi-pronged search for a common structural motif in the secretion signal of Salmonella enterica serovar Typhimurium type III effector proteins

    SciTech Connect

    Buchko, Garry W.; Niemann, George; Baker, Erin Shammel; Belov, Mikhail E.; Smith, Richard D.; Heffron, Fred; Adkins, Joshua N.; McDermott, Jason E.

    2010-11-08

    Many pathogenic Gram-negative bacteria use a type III secretion system (T3SS) to deliver effector proteins into the host cell where they reprogram host defenses and facilitate pathogenesis. While it has been determined that the first 20 - 30 N-terminal residues usually contain the ‘secretion signal’ that targets effector proteins for translocation, the molecular basis for recognition of this signal is not understood. Recent machine-learning approaches, such as SVM-based Identification and Evaluation of Virulence Effectors (SIEVE), have improved the ability to identify effector proteins from genomics sequence information. While these methods all suggest that the T3SS secretion signal has a characteristic amino acid composition bias, it is still unclear if the amino acid pattern is important and if there are any unifying structural properties that direct recognition. To address these issues a peptide corresponding to the secretion signal for Salmonella enterica serovar Typhimurium effector SseJ was synthesized (residues 1-30, SseJ) along with scrambled peptides of the same amino acid composition that produced high (SseJ-H) and low (SseJ-L) SIEVE scores. The secretion properties of these three peptides were tested using a secretion signal-CyaA fusion assay and their structures systematically probed using circular dichroism, nuclear magnetic resonance, and ion mobility spectrometry-mass spectrometry. The signal-CyaA fusion assay showed that the native and SseJ-H fusion constructs were secreted into J774 macrophage at similar levels via the SPI-2 secretion pathway while secretion of the SseJ-L fusion construct was substantially retarded, suggesting that the SseJ secretion signal was sequence order dependent. The structural studies showed that the SseJ, SseJ-H, and SseJ-L peptides were intrinsically disordered in aqueous solution with only a small predisposition to adopt nascent helical structure in the presence of the powerful structure stabilizing agent, 1

  9. BEAN 2.0: an integrated web resource for the identification and functional analysis of type III secreted effectors

    PubMed Central

    Dong, Xiaobao; Lu, Xiaotian; Zhang, Ziding

    2015-01-01

    Gram-negative pathogenic bacteria inject type III secreted effectors (T3SEs) into host cells to sabotage their immune signaling networks. Because T3SEs constitute a meeting-point of pathogen virulence and host defense, they are of keen interest to host–pathogen interaction research community. To accelerate the identification and functional understanding of T3SEs, we present BEAN 2.0 as an integrated web resource to predict, analyse and store T3SEs. BEAN 2.0 includes three major components. First, it provides an accurate T3SE predictor based on a hybrid approach. Using independent testing data, we show that BEAN 2.0 achieves a sensitivity of 86.05% and a specificity of 100%. Second, it integrates a set of online sequence analysis tools. Users can further perform functional analysis of putative T3SEs in a seamless way, such as subcellular location prediction, functional domain scan and disorder region annotation. Third, it compiles a database covering 1215 experimentally verified T3SEs and constructs two T3SE-related networks that can be used to explore the relationships among T3SEs. Taken together, by presenting a one-stop T3SE bioinformatics resource, we hope BEAN 2.0 can promote comprehensive understanding of the function and evolution of T3SEs. Database URL: http://systbio.cau.edu.cn/bean/ PMID:26120140

  10. BEAN 2.0: an integrated web resource for the identification and functional analysis of type III secreted effectors.

    PubMed

    Dong, Xiaobao; Lu, Xiaotian; Zhang, Ziding

    2015-01-01

    Gram-negative pathogenic bacteria inject type III secreted effectors (T3SEs) into host cells to sabotage their immune signaling networks. Because T3SEs constitute a meeting-point of pathogen virulence and host defense, they are of keen interest to host-pathogen interaction research community. To accelerate the identification and functional understanding of T3SEs, we present BEAN 2.0 as an integrated web resource to predict, analyse and store T3SEs. BEAN 2.0 includes three major components. First, it provides an accurate T3SE predictor based on a hybrid approach. Using independent testing data, we show that BEAN 2.0 achieves a sensitivity of 86.05% and a specificity of 100%. Second, it integrates a set of online sequence analysis tools. Users can further perform functional analysis of putative T3SEs in a seamless way, such as subcellular location prediction, functional domain scan and disorder region annotation. Third, it compiles a database covering 1215 experimentally verified T3SEs and constructs two T3SE-related networks that can be used to explore the relationships among T3SEs. Taken together, by presenting a one-stop T3SE bioinformatics resource, we hope BEAN 2.0 can promote comprehensive understanding of the function and evolution of T3SEs. PMID:26120140

  11. A multi-pronged search for a common structural motif in the secretion signal of Salmonella enterica serovar Typhimurium type III effector proteins

    PubMed Central

    Buchko, Garry W.; Niemann, George; Baker, Erin S.; Belov, Mikhail E.; Smith, Richard D.; Heffron, Fred; Adkins, Joshua N.; McDermott, Jason E.

    2012-01-01

    Many pathogenic Gram-negative bacteria use a type III secretion system (T3SS) to deliver effector proteins into the host cell where they reprogram host defenses and facilitate pathogenesis. The first 20–30 N-terminal residues usually contain the ‘secretion signal’ that targets effector proteins for translocation, however, a consensus sequence motif has never been discerned. Recent machine-learning approaches, such as support vector machine (SVM)-based Identification and Evaluation of Virulence Effectors (SIEVE), have improved the ability to identify effector proteins from genomics sequence information. While these methods all suggest that the T3SS secretion signal has a characteristic amino acid composition bias, it is still unclear if the amino acid pattern is important and if there are any unifying structural properties that direct recognition. To address these issues a peptide corresponding to the secretion signal for Salmonella enterica serovar Typhimurium effector SseJ was synthesized (residues 1–30, SseJ) along with scrambled peptides of the same amino acid composition that produced high (SseJ-H) and low (SseJ-L) SIEVE scores. The secretion properties of these three peptides were tested using a secretion signal–CyaA fusion assay and their structural properties probed using circular dichroism, nuclear magnetic resonance, and ion mobility spectrometry–mass spectrometry. The secretion predictions from SIEVE matched signal–CyaA fusion experimental results with J774 macrophages suggesting that the SseJ secretion signal has some sequence order dependence. The structural studies showed that the SseJ, SseJ-H, and SseJ-L peptides were intrinsically disordered in aqueous solution with a small predisposition to adopt nascent helical structure only in the presence of structure stabilizing agents such as 1,1,1,3,3,3-hexafluoroisopropanol. Intrinsic disorder may be a universal feature of effector secretion signals as similar conclusions were reached following

  12. Characterization of the N-Terminal Domain of BteA: A Bordetella Type III Secreted Cytotoxic Effector

    PubMed Central

    Guttman, Chen; Davidov, Geula; Shaked, Hadassa; Kolusheva, Sofiya; Bitton, Ronit; Ganguly, Atish; Miller, Jeff F.; Chill, Jordan H.; Zarivach, Raz

    2013-01-01

    BteA, a 69-kDa cytotoxic protein, is a type III secretion system (T3SS) effector in the classical Bordetella, the etiological agents of pertussis and related mammalian respiratory diseases. Currently there is limited information regarding the structure of BteA or its subdomains, and no insight as to the identity of its eukaryotic partners(s) and their modes of interaction with BteA. The mechanisms that lead to BteA dependent cell death also remain elusive. The N-terminal domain of BteA is multifunctional, acting as a docking platform for its cognate chaperone (BtcA) in the bacterium, and targeting the protein to lipid raft microdomains within the eukaryotic host cell. In this study we describe the biochemical and biophysical characteristics of this domain (BteA287) and determine its architecture. We characterize BteA287 as being a soluble and highly stable domain which is rich in alpha helical content. Nuclear magnetic resonance (NMR) experiments combined with size exclusion and analytical ultracentrifugation measurements confirm these observations and reveal BteA287 to be monomeric in nature with a tendency to oligomerize at concentrations above 200 µM. Furthermore, diffusion-NMR demonstrated that the first 31 residues of BteA287 are responsible for the apparent aggregation behavior of BteA287. Light scattering analyses and small angle X-ray scattering experiments reveal a prolate ellipsoidal bi-pyramidal dumb-bell shape. Thus, our biophysical characterization is a first step towards structure determination of the BteA N-terminal domain. PMID:23383256

  13. Fueling type III secretion

    PubMed Central

    Lee, Pei-Chung

    2015-01-01

    Type III secretion systems are complex nanomachines that export proteins from the bacterial cytoplasm across the cell envelope in a single step. They are at the core of the machinery used to assemble the bacterial flagellum, and the needle complex many Gram-negative pathogens use to inject effector proteins into host cells and cause disease. Several models have been put forward to explain how this export is energized, and the mechanism has been the subject of considerable debate. Here we present an overview of these models and discuss their relative merits. Recent evidence suggests that the proton motive force is the primary energy source for type III secretion, although contribution from refolding of secreted proteins has not been ruled out. The mechanism, by which the proton motive force is converted to protein export, remains enigmatic. PMID:25701111

  14. A Family of Salmonella Type III Secretion Effector Proteins Selectively Targets the NF-κB Signaling Pathway to Preserve Host Homeostasis

    PubMed Central

    Sun, Hui; Kamanova, Jana; Lara-Tejero, Maria; Galán, Jorge E.

    2016-01-01

    Microbial infections usually lead to host innate immune responses and inflammation. These responses most often limit pathogen replication although they can also result in host-tissue damage. The enteropathogenic bacteria Salmonella Typhimurium utilizes a type III secretion system to induce intestinal inflammation by delivering specific effector proteins that stimulate signal transduction pathways resulting in the production of pro-inflammatory cytokines. We show here that a family of related Salmonella Typhimurium effector proteins PipA, GogA and GtgA redundantly target components of the NF-κB signaling pathway to inhibit transcriptional responses leading to inflammation. We show that these effector proteins are proteases that cleave both the RelA (p65) and RelB transcription factors but do not target p100 (NF-κB2) or p105 (NF-κB1). A Salmonella Typhimurium strain lacking these effectors showed increased ability to stimulate NF-κB and increased virulence in an animal model of infection. These results indicate that bacterial pathogens can evolve determinants to preserve host homeostasis and that those determinants can reduce the pathogen’s virulence. PMID:26933955

  15. A Family of Salmonella Type III Secretion Effector Proteins Selectively Targets the NF-κB Signaling Pathway to Preserve Host Homeostasis.

    PubMed

    Sun, Hui; Kamanova, Jana; Lara-Tejero, Maria; Galán, Jorge E

    2016-03-01

    Microbial infections usually lead to host innate immune responses and inflammation. These responses most often limit pathogen replication although they can also result in host-tissue damage. The enteropathogenic bacteria Salmonella Typhimurium utilizes a type III secretion system to induce intestinal inflammation by delivering specific effector proteins that stimulate signal transduction pathways resulting in the production of pro-inflammatory cytokines. We show here that a family of related Salmonella Typhimurium effector proteins PipA, GogA and GtgA redundantly target components of the NF-κB signaling pathway to inhibit transcriptional responses leading to inflammation. We show that these effector proteins are proteases that cleave both the RelA (p65) and RelB transcription factors but do not target p100 (NF-κB2) or p105 (NF-κB1). A Salmonella Typhimurium strain lacking these effectors showed increased ability to stimulate NF-κB and increased virulence in an animal model of infection. These results indicate that bacterial pathogens can evolve determinants to preserve host homeostasis and that those determinants can reduce the pathogen's virulence. PMID:26933955

  16. Dissection of Bacterial Wilt on Medicago truncatula Revealed Two Type III Secretion System Effectors Acting on Root Infection Process and Disease Development[C][W][OA

    PubMed Central

    Turner, Marie; Jauneau, Alain; Genin, Stéphane; Tavella, Marie-José; Vailleau, Fabienne; Gentzbittel, Laurent; Jardinaud, Marie-Françoise

    2009-01-01

    Ralstonia solanacearum is the causal agent of the devastating bacterial wilt disease, which colonizes susceptible Medicago truncatula via the intact root tip. Infection involves four steps: appearance of root tip symptoms, root tip cortical cell invasion, vessel colonization, and foliar wilting. We examined this pathosystem by in vitro inoculation of intact roots of susceptible or resistant M. truncatula with the pathogenic strain GMI1000. The infection process was type III secretion system dependent and required two type III effectors, Gala7 and AvrA, which were shown to be involved at different stages of infection. Both effectors were involved in development of root tip symptoms, and Gala7 was the main determinant for bacterial invasion of cortical cells. Vessel invasion depended on the host genetic background and was never observed in the resistant line. The invasion of the root tip vasculature in the susceptible line caused foliar wilting. The avrA mutant showed reduced aggressiveness in all steps of the infection process, suggesting a global role in R. solanacearum pathogenicity. The roles of these two effectors in subsequent stages were studied using an assay that bypassed the penetration step; with this assay, the avrA mutant showed no effect compared with the GMI1000 strain, indicating that AvrA is important in early stages of infection. However, later disease symptoms were reduced in the gala7 mutant, indicating a key role in later stages of infection. PMID:19493968

  17. A Putative Type III Secretion System Effector Encoded by the MA20_12780 Gene in Bradyrhizobium japonicum Is-34 Causes Incompatibility with Rj4 Genotype Soybeans

    PubMed Central

    Hashimoto, Syougo; Okizaki, Kouhei; Kanesaki, Yu; Yoshikawa, Hirofumi; Yamakawa, Takeo

    2015-01-01

    The nodulation of Bradyrhizobium japonicum Is-34 is restricted by Rj4 genotype soybeans (Glycine max). To identify the genes responsible for this incompatibility, Tn5 mutants of B. japonicum Is-34 that were able to overcome this nodulation restriction were obtained. Analysis of the Tn5 mutants revealed that Tn5 was inserted into a region containing the MA20_12780 gene. In addition, direct disruption of this gene using marker exchange overcame the nodulation restriction by Rj4 genotype soybeans. The MA20_12780 gene has a tts box motif in its upstream region, indicating a possibility that this gene encodes a type III secretion system (T3SS) effector protein. Bioinformatic characterization revealed that the MA20_12780 protein contains the small ubiquitin-like modifier (SUMO) protease domain of the C48 peptidase (ubiquitin-like protease 1 [Ulp1]) family. The results of the present study indicate that a putative T3SS effector encoded by the MA20_12780 gene causes the incompatibility with Rj4 genotype soybeans, and they suggest the possibility that the nodulation restriction of B. japonicum Is-34 may be due to Rj4 genotype soybeans recognizing the putative T3SS effector (MA20_12780 protein) as a virulence factor. PMID:26092458

  18. A Putative Type III Secretion System Effector Encoded by the MA20_12780 Gene in Bradyrhizobium japonicum Is-34 Causes Incompatibility with Rj4 Genotype Soybeans.

    PubMed

    Tsurumaru, Hirohito; Hashimoto, Syougo; Okizaki, Kouhei; Kanesaki, Yu; Yoshikawa, Hirofumi; Yamakawa, Takeo

    2015-09-01

    The nodulation of Bradyrhizobium japonicum Is-34 is restricted by Rj4 genotype soybeans (Glycine max). To identify the genes responsible for this incompatibility, Tn5 mutants of B. japonicum Is-34 that were able to overcome this nodulation restriction were obtained. Analysis of the Tn5 mutants revealed that Tn5 was inserted into a region containing the MA20_12780 gene. In addition, direct disruption of this gene using marker exchange overcame the nodulation restriction by Rj4 genotype soybeans. The MA20_12780 gene has a tts box motif in its upstream region, indicating a possibility that this gene encodes a type III secretion system (T3SS) effector protein. Bioinformatic characterization revealed that the MA20_12780 protein contains the small ubiquitin-like modifier (SUMO) protease domain of the C48 peptidase (ubiquitin-like protease 1 [Ulp1]) family. The results of the present study indicate that a putative T3SS effector encoded by the MA20_12780 gene causes the incompatibility with Rj4 genotype soybeans, and they suggest the possibility that the nodulation restriction of B. japonicum Is-34 may be due to Rj4 genotype soybeans recognizing the putative T3SS effector (MA20_12780 protein) as a virulence factor. PMID:26092458

  19. Pseudomonas syringae pv. tomato DC3000 Type III Secretion Effector Polymutants Reveal an Interplay between HopAD1 and AvrPtoB.

    PubMed

    Wei, Hai-Lei; Chakravarthy, Suma; Mathieu, Johannes; Helmann, Tyler C; Stodghill, Paul; Swingle, Bryan; Martin, Gregory B; Collmer, Alan

    2015-06-10

    The bacterial pathogen Pseudomonas syringae pv. tomato DC3000 suppresses the two-tiered plant innate immune system by injecting a complex repertoire of type III secretion effector (T3E) proteins. Beyond redundancy and interplay, individual T3Es may interact with multiple immunity-associated proteins, rendering their analysis challenging. We constructed a Pst DC3000 polymutant lacking all 36 T3Es and restored individual T3Es or their mutants to explore the interplay among T3Es. The weakly expressed T3E HopAD1 was sufficient to elicit immunity-associated cell death in Nicotiana benthamiana. HopAD1-induced cell death was suppressed partially by native AvrPtoB and completely by AvrPtoBM3, which has mutations disrupting its E3 ubiquitin ligase domain and two known domains for interacting with immunity-associated kinases. AvrPtoBM3 also gained the ability to interact with the immunity-kinase MKK2, which is required for HopAD1-dependent cell death. Thus, AvrPtoB has alternative, competing mechanisms for suppressing effector-triggered plant immunity. This approach allows the deconvolution of individual T3E activities. PMID:26067603

  20. Inhibition of Nuclear Transport of NF-ĸB p65 by the Salmonella Type III Secretion System Effector SpvD.

    PubMed

    Rolhion, Nathalie; Furniss, R Christopher D; Grabe, Grzegorz; Ryan, Aindrias; Liu, Mei; Matthews, Sophie A; Holden, David W

    2016-05-01

    Salmonella enterica replicates in macrophages through the action of effector proteins translocated across the vacuolar membrane by a type III secretion system (T3SS). Here we show that the SPI-2 T3SS effector SpvD suppresses proinflammatory immune responses. SpvD prevented activation of an NF-ĸB-dependent promoter and caused nuclear accumulation of importin-α, which is required for nuclear import of p65. SpvD interacted specifically with the exportin Xpo2, which mediates nuclear-cytoplasmic recycling of importins. We propose that interaction between SpvD and Xpo2 disrupts the normal recycling of importin-α from the nucleus, leading to a defect in nuclear translocation of p65 and inhibition of activation of NF-ĸB regulated promoters. SpvD down-regulated pro-inflammatory responses and contributed to systemic growth of bacteria in mice. This work shows that a bacterial pathogen can manipulate host cell immune responses by interfering with the nuclear transport machinery. PMID:27232334

  1. Inhibition of Nuclear Transport of NF-ĸB p65 by the Salmonella Type III Secretion System Effector SpvD

    PubMed Central

    Rolhion, Nathalie; Furniss, R. Christopher D.; Grabe, Grzegorz; Ryan, Aindrias; Liu, Mei; Matthews, Sophie A.; Holden, David W.

    2016-01-01

    Salmonella enterica replicates in macrophages through the action of effector proteins translocated across the vacuolar membrane by a type III secretion system (T3SS). Here we show that the SPI-2 T3SS effector SpvD suppresses proinflammatory immune responses. SpvD prevented activation of an NF-ĸB-dependent promoter and caused nuclear accumulation of importin-α, which is required for nuclear import of p65. SpvD interacted specifically with the exportin Xpo2, which mediates nuclear-cytoplasmic recycling of importins. We propose that interaction between SpvD and Xpo2 disrupts the normal recycling of importin-α from the nucleus, leading to a defect in nuclear translocation of p65 and inhibition of activation of NF-ĸB regulated promoters. SpvD down-regulated pro-inflammatory responses and contributed to systemic growth of bacteria in mice. This work shows that a bacterial pathogen can manipulate host cell immune responses by interfering with the nuclear transport machinery. PMID:27232334

  2. Pseudomonas syringae lytic transglycosylases coregulated with the type III secretion system contribute to the translocation of effector proteins into plant cells.

    PubMed

    Oh, Hye-Sook; Kvitko, Brian H; Morello, Joanne E; Collmer, Alan

    2007-11-01

    Pseudomonas syringae translocates virulence effector proteins into plant cells via a type III secretion system (T3SS) encoded by hrp (for hypersensitive response and pathogenicity) genes. Three genes coregulated with the Hrp T3SS system in P. syringae pv. tomato DC3000 have predicted lytic transglycosylase domains: PSPTO1378 (here designated hrpH), PSPTO2678 (hopP1), and PSPTO852 (hopAJ1). hrpH is located between hrpR and avrE1 in the Hrp pathogenicity island and is carried in the functional cluster of P. syringae pv. syringae 61 hrp genes cloned in cosmid pHIR11. Strong expression of DC3000 hrpH in Escherichia coli inhibits bacterial growth unless the predicted catalytic glutamate at position 148 is mutated. Translocation tests involving C-terminal fusions with a Cya (Bordetella pertussis adenylate cyclase) reporter indicate that HrpH and HopP1, but not HopAJ1, are T3SS substrates. Pseudomonas fluorescens carrying a pHIR11 derivative lacking hrpH is poorly able to translocate effector HopA1, and this deficiency can be restored by HopP1 and HopAJ1, but not by HrpH(E148A) or HrpH(1-241). DC3000 mutants lacking hrpH or hrpH, hopP1, and hopAJ1 combined are variously reduced in effector translocation, elicitation of the hypersensitive response, and virulence. However, the mutants are not reduced in secretion of T3SS substrates in culture. When produced in wild-type DC3000, the HrpH(E148A) and HrpH(1-241) variants have a dominant-negative effect on the ability of DC3000 to elicit the hypersensitive response in nonhost tobacco and to grow and cause disease in host tomato. The three Hrp-associated lytic transglycosylases in DC3000 appear to have overlapping functions in contributing to T3SS functions during infection. PMID:17827286

  3. Variable Suites of Non-effector Genes Are Co-regulated in the Type III Secretion Virulence Regulon across the Pseudomonas syringae Phylogeny

    PubMed Central

    Mucyn, Tatiana S.; Yourstone, Scott; Lind, Abigail L.; Biswas, Surojit; Nishimura, Marc T.; Baltrus, David A.; Cumbie, Jason S.; Chang, Jeff H.; Jones, Corbin D.; Dangl, Jeffery L.; Grant, Sarah R.

    2014-01-01

    Pseudomonas syringae is a phylogenetically diverse species of Gram-negative bacterial plant pathogens responsible for crop diseases around the world. The HrpL sigma factor drives expression of the major P. syringae virulence regulon. HrpL controls expression of the genes encoding the structural and functional components of the type III secretion system (T3SS) and the type three secreted effector proteins (T3E) that are collectively essential for virulence. HrpL also regulates expression of an under-explored suite of non-type III effector genes (non-T3E), including toxin production systems and operons not previously associated with virulence. We implemented and refined genome-wide transcriptional analysis methods using cDNA-derived high-throughput sequencing (RNA-seq) data to characterize the HrpL regulon from six isolates of P. syringae spanning the diversity of the species. Our transcriptomes, mapped onto both complete and draft genomes, significantly extend earlier studies. We confirmed HrpL-regulation for a majority of previously defined T3E genes in these six strains. We identified two new T3E families from P. syringae pv. oryzae 1_6, a strain within the relatively underexplored phylogenetic Multi-Locus Sequence Typing (MLST) group IV. The HrpL regulons varied among strains in gene number and content across both their T3E and non-T3E gene suites. Strains within MLST group II consistently express the lowest number of HrpL-regulated genes. We identified events leading to recruitment into, and loss from, the HrpL regulon. These included gene gain and loss, and loss of HrpL regulation caused by group-specific cis element mutations in otherwise conserved genes. Novel non-T3E HrpL-regulated genes include an operon that we show is required for full virulence of P. syringae pv. phaseolicola 1448A on French bean. We highlight the power of integrating genomic, transcriptomic, and phylogenetic information to drive concise functional experimentation and to derive better

  4. Expression of enteropathogenic Escherichia coli map is significantly different than that of other type III secreted effectors in vivo.

    PubMed

    Nguyen, Mai; Rizvi, Jason; Hecht, Gail

    2015-01-01

    The enteropathogenic Escherichia coli (EPEC) locus of enterocyte effacement (LEE)-encoded effectors EspF and Map are multifunctional and have an impact on the tight junction barrier while the non-LEE-encoded proteins NleH1 and NleH2 possess significant anti-inflammatory activity. In order to address the temporal expression of these important genes in vivo, their promoters were cloned upstream of the luxCDABE operon, and luciferase expression was measured in EPEC-infected mice by bioluminescence using an in vivo imaging system (IVIS). Bioluminescent images of living mice, of excised whole intestines, and of whole intestines longitudinally opened and washed were assessed. The majority of bioluminescent bacteria localized in the cecum by 3 h postinfection, indicating that the cecum is not only a major colonization site of EPEC but also a site of EPEC effector gene expression in mice. espF, nleH1, and nleH2 were abundantly expressed over the course of infection. In contrast, map expression was suppressed at 2 days postinfection, and at 4 days postinfection it was totally abolished. After 2 to 4 days postinfection, when map is suppressed, EPEC colonization is significantly reduced, indicating that map may be one of the factors required to maintain EPEC colonization. This was confirmed in a competitive colonization study and in two models of chronic infection, repeated exposure to ketamine and Citrobacter rodentium infection. Our data suggest that map expression contributes to the maintenance of EPEC colonization. PMID:25312947

  5. GENOMIC MINING TYPE III SECRETION SYSTEM EFFECTORS IN PSEUDOMONAS SYRINGAE YIELDS NEW PICKS FOR ALL TTSS PROSPECTORS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A pre-closure draft sequence of Pseudomonas syringae pv. tomato DC3000, a pathogen of tomato and Arabidopsis, has recently supported five complementary studies which, collectively, identify 36 TTSS-secreted proteins and many more candidates in this strain. These studies demonstrate the advantages o...

  6. Functional Differences and Interactions between the Escherichia coli Type III Secretion System Effectors NleH1 and NleH2

    PubMed Central

    Pham, Thanh H.; Gao, Xiaofei; Tsai, Karen; Olsen, Rachel; Wan, Fengyi

    2012-01-01

    The human pathogens enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC), as well as the related mouse pathogen Citrobacter rodentium, utilize a type III secretion system (T3SS) to inject multiple effector proteins into host cells. The E. coli O157:H7 strain EDL933 carries two copies of non-locus of enterocyte effacement (LEE)-encoded protein H, designated NleH1 and NleH2, both of which bind to the human ribosomal protein S3 (RPS3), a subunit of NF-κB transcriptional complexes. In this study, we describe significant functional differences between NleH1 and NleH2 in their ability to regulate the host NF-κB pathway. We show that the EHEC and EPEC NleH effectors are functionally equivalent in their ability to affect RPS3 nuclear translocation. NleH1, but not NleH2, inhibited NF-κB activity without altering the kinetics of IκBα phosphorylation/degradation. We also determined that the class I PSD-95/Disc Large/ZO-1 (PDZ)-binding domain of NleH was important for its activity in the NF-κB pathway. In addition to binding RPS3, we found that NleH1 and NleH2 are able to bind to each other in vitro and in vivo, suggesting an additional mechanism by which the E. coli NleH effectors may regulate the extent and duration of NF-κB activation after their T3SS-dependent translocation. We also performed mouse infection experiments and established that mouse mortality and Citrobacter colonization were reduced in mice infected with ΔnleH. Complementing ΔnleH with NleH1 restored Citrobacter virulence and colonization to wild-type levels, whereas complementing with NleH2 reduced them. Taken together, our data show that NleH1 and NleH2 have pronounced functional differences in their ability to alter host transcriptional responses to bacterial infection. PMID:22451523

  7. AvrRxo1 Is a Bifunctional Type III Secreted Effector and Toxin-Antitoxin System Component with Homologs in Diverse Environmental Contexts.

    PubMed

    Triplett, Lindsay R; Shidore, Teja; Long, John; Miao, Jiamin; Wu, Shuchi; Han, Qian; Zhou, Changhe; Ishihara, Hiromichi; Li, Jianyong; Zhao, Bingyu; Leach, Jan E

    2016-01-01

    Toxin-antitoxin (TA) systems are ubiquitous bacterial systems that may function in genome maintenance and metabolic stress management, but are also thought to play a role in virulence by helping pathogens survive stress. We previously demonstrated that the Xanthomonas oryzae pv. oryzicola protein AvrRxo1 is a type III-secreted virulence factor that has structural similarities to the zeta family of TA toxins, and is toxic to plants and bacteria in the absence of its predicted chaperone Arc1. In this work, we confirm that AvrRxo1 and its binding partner Arc1 function as a TA system when expressed in Escherichia coli. Sequences of avrRxo1 homologs were culled from published and newly generated phytopathogen genomes, revealing that avrRxo1:arc1 modules are rare or frequently inactivated in some species and highly conserved in others. Cloning and functional analysis of avrRxo1 from Acidovorax avenae, A. citrulli, Burkholderia andropogonis, Xanthomonas translucens, and Xanthomonas euvesicatoria showed that some AvrRxo1 homologs share the bacteriostatic and Rxo1-mediated cell death triggering activities of AvrRxo1 from X. oryzae. Additional distant putative homologs of avrRxo1 and arc1 were identified in genomic or metagenomic sequence of environmental bacteria with no known pathogenic role. One of these distant homologs was cloned from the filamentous soil bacterium Cystobacter fuscus. avrRxo1 from C. fuscus caused watersoaking and triggered Rxo1-dependent cell collapse in Nicotiana benthamiana, but no growth suppression in E. coli was observed. This work confirms that a type III effector can function as a TA system toxin, and illustrates the potential of microbiome data to reveal new environmental origins or reservoirs of pathogen virulence factors. PMID:27391081

  8. AvrRxo1 Is a Bifunctional Type III Secreted Effector and Toxin-Antitoxin System Component with Homologs in Diverse Environmental Contexts

    PubMed Central

    Triplett, Lindsay R.; Shidore, Teja; Long, John; Miao, Jiamin; Wu, Shuchi; Han, Qian; Zhou, Changhe; Ishihara, Hiromichi; Li, Jianyong; Zhao, Bingyu; Leach, Jan E.

    2016-01-01

    Toxin-antitoxin (TA) systems are ubiquitous bacterial systems that may function in genome maintenance and metabolic stress management, but are also thought to play a role in virulence by helping pathogens survive stress. We previously demonstrated that the Xanthomonas oryzae pv. oryzicola protein AvrRxo1 is a type III-secreted virulence factor that has structural similarities to the zeta family of TA toxins, and is toxic to plants and bacteria in the absence of its predicted chaperone Arc1. In this work, we confirm that AvrRxo1 and its binding partner Arc1 function as a TA system when expressed in Escherichia coli. Sequences of avrRxo1 homologs were culled from published and newly generated phytopathogen genomes, revealing that avrRxo1:arc1 modules are rare or frequently inactivated in some species and highly conserved in others. Cloning and functional analysis of avrRxo1 from Acidovorax avenae, A. citrulli, Burkholderia andropogonis, Xanthomonas translucens, and Xanthomonas euvesicatoria showed that some AvrRxo1 homologs share the bacteriostatic and Rxo1-mediated cell death triggering activities of AvrRxo1 from X. oryzae. Additional distant putative homologs of avrRxo1 and arc1 were identified in genomic or metagenomic sequence of environmental bacteria with no known pathogenic role. One of these distant homologs was cloned from the filamentous soil bacterium Cystobacter fuscus. avrRxo1 from C. fuscus caused watersoaking and triggered Rxo1-dependent cell collapse in Nicotiana benthamiana, but no growth suppression in E. coli was observed. This work confirms that a type III effector can function as a TA system toxin, and illustrates the potential of microbiome data to reveal new environmental origins or reservoirs of pathogen virulence factors. PMID:27391081

  9. Identification of Anaplasma marginale Type IV Secretion System Effector Proteins

    PubMed Central

    Brayton, Kelly A.; Beare, Paul A.; Brown, Wendy C.; Heinzen, Robert A.; Broschat, Shira L.

    2011-01-01

    Background Anaplasma marginale, an obligate intracellular alphaproteobacterium in the order Rickettsiales, is a tick-borne pathogen and the leading cause of anaplasmosis in cattle worldwide. Complete genome sequencing of A. marginale revealed that it has a type IV secretion system (T4SS). The T4SS is one of seven known types of secretion systems utilized by bacteria, with the type III and IV secretion systems particularly prevalent among pathogenic Gram-negative bacteria. The T4SS is predicted to play an important role in the invasion and pathogenesis of A. marginale by translocating effector proteins across its membrane into eukaryotic target cells. However, T4SS effector proteins have not been identified and tested in the laboratory until now. Results By combining computational methods with phylogenetic analysis and sequence identity searches, we identified a subset of potential T4SS effectors in A. marginale strain St. Maries and chose six for laboratory testing. Four (AM185, AM470, AM705 [AnkA], and AM1141) of these six proteins were translocated in a T4SS-dependent manner using Legionella pneumophila as a reporter system. Conclusions The algorithm employed to find T4SS effector proteins in A. marginale identified four such proteins that were verified by laboratory testing. L. pneumophila was shown to work as a model system for A. marginale and thus can be used as a screening tool for A. marginale effector proteins. The first T4SS effector proteins for A. marginale have been identified in this work. PMID:22140462

  10. The type three secreted effector SipC regulates the trafficking of PERP during Salmonella infection

    PubMed Central

    Hallstrom, Kelly N.; McCormick, Beth A.

    2016-01-01

    abstract Salmonella enterica Typhimurium employs type III secreted effectors to induce cellular invasion and pathogenesis. We previously reported the secreted effector SipA is in part responsible for inducing the apical accumulation of the host membrane protein PERP, a host factor we have shown is key to the inflammatory response induced by Salmonella. We now report that the S. Typhimurium type III secreted effector SipC significantly contributes to PERP redistribution to the apical membrane surface. To our knowledge, this is the first report demonstrating a role for SipC in directing the trafficking of a host membrane protein to the cell surface. In sum, facilitation of PERP trafficking appears to be a result of type III secreted effector-mediated recruitment of vesicles to the apical surface. Our study therefore reveals a new role for SipC, and builds upon previous reports suggesting recruitment of vesicles to the cell surface is important for Salmonella invasion. PMID:27078059

  11. Further Characterization of a Type III Secretion System (T3SS) and of a New Effector Protein from a Clinical Isolate of Aeromonas Hydrophila - Part I

    EPA Science Inventory

    A type III secretion system (T3SS)-associated cytotoxin, AexT, with ADP-ribosyltransferase activity and homology to Pseudomonas aeruginosa bifuncational toxins ExoT/S, was recently identified from a fish pathogen Aeromonas salmonicida. In this study, we reported the molecular cha...

  12. The Chlamydia trachomatis Type III Secretion Chaperone Slc1 Engages Multiple Early Effectors, Including TepP, a Tyrosine-phosphorylated Protein Required for the Recruitment of CrkI-II to Nascent Inclusions and Innate Immune Signaling

    PubMed Central

    Chen, Yi-Shan; Bastidas, Robert J.; Saka, Hector A.; Carpenter, Victoria K.; Richards, Kristian L.; Plano, Gregory V.; Valdivia, Raphael H.

    2014-01-01

    Chlamydia trachomatis, the causative agent of trachoma and sexually transmitted infections, employs a type III secretion (T3S) system to deliver effector proteins into host epithelial cells to establish a replicative vacuole. Aside from the phosphoprotein TARP, a Chlamydia effector that promotes actin re-arrangements, very few factors mediating bacterial entry and early inclusion establishment have been characterized. Like many T3S effectors, TARP requires a chaperone (Slc1) for efficient translocation into host cells. In this study, we defined proteins that associate with Slc1 in invasive C. trachomatis elementary bodies (EB) by immunoprecipitation coupled with mass spectrometry. We identified Ct875, a new Slc1 client protein and T3S effector, which we renamed TepP (Translocated early phosphoprotein). We provide evidence that T3S effectors form large molecular weight complexes with Scl1 in vitro and that Slc1 enhances their T3S-dependent secretion in a heterologous Yersinia T3S system. We demonstrate that TepP is translocated early during bacterial entry into epithelial cells and is phosphorylated at tyrosine residues by host kinases. However, TepP phosphorylation occurs later than TARP, which together with the finding that Slc1 preferentially engages TARP in EBs leads us to postulate that these effectors are translocated into the host cell at different stages during C. trachomatis invasion. TepP co-immunoprecipitated with the scaffolding proteins CrkI-II during infection and Crk was recruited to EBs at entry sites where it remained associated with nascent inclusions. Importantly, C. trachomatis mutants lacking TepP failed to recruit CrkI-II to inclusions, providing genetic confirmation of a direct role for this effector in the recruitment of a host factor. Finally, endocervical epithelial cells infected with a tepP mutant showed altered expression of a subset of genes associated with innate immune responses. We propose a model wherein TepP acts downstream of TARP

  13. Accurate prediction of secreted substrates and identification of a conserved putative secretion signal for type III secretion systems

    SciTech Connect

    Samudrala, Ram; Heffron, Fred; McDermott, Jason E.

    2009-04-24

    The type III secretion system is an essential component for virulence in many Gram-negative bacteria. Though components of the secretion system apparatus are conserved, its substrates, effector proteins, are not. We have used a machine learning approach to identify new secreted effectors. The method integrates evolutionary measures, such as the pattern of homologs in a range of other organisms, and sequence-based features, such as G+C content, amino acid composition and the N-terminal 30 residues of the protein sequence. The method was trained on known effectors from Salmonella typhimurium and validated on a corresponding set of effectors from Pseudomonas syringae, after eliminating effectors with detectable sequence similarity. The method was able to identify all of the known effectors in P. syringae with a specificity of 84% and sensitivity of 82%. The reciprocal validation, training on P. syringae and validating on S. typhimurium, gave similar results with a specificity of 86% when the sensitivity level was 87%. These results show that type III effectors in disparate organisms share common features. We found that maximal performance is attained by including an N-terminal sequence of only 30 residues, which agrees with previous studies indicating that this region contains the secretion signal. We then used the method to define the most important residues in this putative secretion signal. Finally, we present novel predictions of secreted effectors in S. typhimurium, some of which have been experimentally validated, and apply the method to predict secreted effectors in the genetically intractable human pathogen Chlamydia trachomatis. This approach is a novel and effective way to identify secreted effectors in a broad range of pathogenic bacteria for further experimental characterization and provides insight into the nature of the type III secretion signal.

  14. Energy source of flagellar type III secretion.

    PubMed

    Paul, Koushik; Erhardt, Marc; Hirano, Takanori; Blair, David F; Hughes, Kelly T

    2008-01-24

    Bacterial flagella contain a specialized secretion apparatus that functions to deliver the protein subunits that form the filament and other structures to outside the membrane. This apparatus is related to the injectisome used by many gram-negative pathogens and symbionts to transfer effector proteins into host cells; in both systems this export mechanism is termed 'type III' secretion. The flagellar secretion apparatus comprises a membrane-embedded complex of about five proteins, and soluble factors, which include export-dedicated chaperones and an ATPase, FliI, that was thought to provide the energy for export. Here we show that flagellar secretion in Salmonella enterica requires the proton motive force (PMF) and does not require ATP hydrolysis by FliI. The export of several flagellar export substrates was prevented by treatment with the protonophore CCCP, with no accompanying decrease in cellular ATP levels. Weak swarming motility and rare flagella were observed in a mutant deleted for FliI and for the non-flagellar type-III secretion ATPases InvJ and SsaN. These findings show that the flagellar secretion apparatus functions as a proton-driven protein exporter and that ATP hydrolysis is not essential for type III secretion. PMID:18216859

  15. Yersinia type III effectors perturb host innate immune responses.

    PubMed

    Pha, Khavong; Navarro, Lorena

    2016-02-26

    The innate immune system is the first line of defense against invading pathogens. Innate immune cells recognize molecular patterns from the pathogen and mount a response to resolve the infection. The production of proinflammatory cytokines and reactive oxygen species, phagocytosis, and induced programmed cell death are processes initiated by innate immune cells in order to combat invading pathogens. However, pathogens have evolved various virulence mechanisms to subvert these responses. One strategy utilized by Gram-negative bacterial pathogens is the deployment of a complex machine termed the type III secretion system (T3SS). The T3SS is composed of a syringe-like needle structure and the effector proteins that are injected directly into a target host cell to disrupt a cellular response. The three human pathogenic Yersinia spp. (Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis) are Gram-negative bacteria that share in common a 70 kb virulence plasmid which encodes the T3SS. Translocation of the Yersinia effector proteins (YopE, YopH, YopT, YopM, YpkA/YopO, and YopP/J) into the target host cell results in disruption of the actin cytoskeleton to inhibit phagocytosis, downregulation of proinflammatory cytokine/chemokine production, and induction of cellular apoptosis of the target cell. Over the past 25 years, studies on the Yersinia effector proteins have unveiled tremendous knowledge of how the effectors enhance Yersinia virulence. Recently, the long awaited crystal structure of YpkA has been solved providing further insights into the activation of the YpkA kinase domain. Multisite autophosphorylation by YpkA to activate its kinase domain was also shown and postulated to serve as a mechanism to bypass regulation by host phosphatases. In addition, novel Yersinia effector protein targets, such as caspase-1, and signaling pathways including activation of the inflammasome were identified. In this review, we summarize the recent discoveries made on Yersinia

  16. Yersinia type III effectors perturb host innate immune responses

    PubMed Central

    Pha, Khavong; Navarro, Lorena

    2016-01-01

    The innate immune system is the first line of defense against invading pathogens. Innate immune cells recognize molecular patterns from the pathogen and mount a response to resolve the infection. The production of proinflammatory cytokines and reactive oxygen species, phagocytosis, and induced programmed cell death are processes initiated by innate immune cells in order to combat invading pathogens. However, pathogens have evolved various virulence mechanisms to subvert these responses. One strategy utilized by Gram-negative bacterial pathogens is the deployment of a complex machine termed the type III secretion system (T3SS). The T3SS is composed of a syringe-like needle structure and the effector proteins that are injected directly into a target host cell to disrupt a cellular response. The three human pathogenic Yersinia spp. (Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis) are Gram-negative bacteria that share in common a 70 kb virulence plasmid which encodes the T3SS. Translocation of the Yersinia effector proteins (YopE, YopH, YopT, YopM, YpkA/YopO, and YopP/J) into the target host cell results in disruption of the actin cytoskeleton to inhibit phagocytosis, downregulation of proinflammatory cytokine/chemokine production, and induction of cellular apoptosis of the target cell. Over the past 25 years, studies on the Yersinia effector proteins have unveiled tremendous knowledge of how the effectors enhance Yersinia virulence. Recently, the long awaited crystal structure of YpkA has been solved providing further insights into the activation of the YpkA kinase domain. Multisite autophosphorylation by YpkA to activate its kinase domain was also shown and postulated to serve as a mechanism to bypass regulation by host phosphatases. In addition, novel Yersinia effector protein targets, such as caspase-1, and signaling pathways including activation of the inflammasome were identified. In this review, we summarize the recent discoveries made on Yersinia

  17. Immunomodulation by the Pseudomonas syringae HopZ Type III Effector Family in Aribidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pseudomonas syringae employs a type III secretion system to inject 20-30 different type III effector (T3SE) proteins into plant host cells. A major role of T3SEs is to suppress plant immune responses and promote bacterial infection. The YopJ/HopZ acetyltransferases are a superfamily of T3SEs found i...

  18. Using hierarchical clustering of secreted protein families to classify and rank candidate effectors of rust fungi.

    PubMed

    Saunders, Diane G O; Win, Joe; Cano, Liliana M; Szabo, Les J; Kamoun, Sophien; Raffaele, Sylvain

    2012-01-01

    Rust fungi are obligate biotrophic pathogens that cause considerable damage on crop plants. Puccinia graminis f. sp. tritici, the causal agent of wheat stem rust, and Melampsora larici-populina, the poplar leaf rust pathogen, have strong deleterious impacts on wheat and poplar wood production, respectively. Filamentous pathogens such as rust fungi secrete molecules called disease effectors that act as modulators of host cell physiology and can suppress or trigger host immunity. Current knowledge on effectors from other filamentous plant pathogens can be exploited for the characterisation of effectors in the genome of recently sequenced rust fungi. We designed a comprehensive in silico analysis pipeline to identify the putative effector repertoire from the genome of two plant pathogenic rust fungi. The pipeline is based on the observation that known effector proteins from filamentous pathogens have at least one of the following properties: (i) contain a secretion signal, (ii) are encoded by in planta induced genes, (iii) have similarity to haustorial proteins, (iv) are small and cysteine rich, (v) contain a known effector motif or a nuclear localization signal, (vi) are encoded by genes with long intergenic regions, (vii) contain internal repeats, and (viii) do not contain PFAM domains, except those associated with pathogenicity. We used Markov clustering and hierarchical clustering to classify protein families of rust pathogens and rank them according to their likelihood of being effectors. Using this approach, we identified eight families of candidate effectors that we consider of high value for functional characterization. This study revealed a diverse set of candidate effectors, including families of haustorial expressed secreted proteins and small cysteine-rich proteins. This comprehensive classification of candidate effectors from these devastating rust pathogens is an initial step towards probing plant germplasm for novel resistance components. PMID:22238666

  19. Diverse Secreted Effectors Are Required for Salmonella Persistence in a Mouse Infection Model

    SciTech Connect

    Kidwai, Afshan S.; Mushamiri, Ivy T.; Niemann, George; Brown, Roslyn N.; Adkins, Joshua N.; Heffron, Fred

    2013-08-12

    Salmonella enterica serovar Typhimurium causes typhoid-like disease in mice and is a model of typhoid fever in humans. One of the hallmarks of typhoid is persistence, the ability of the bacteria to survive in the host weeks after infection. Virulence factors called effectors facilitate this process by direct transfer to the cytoplasm of infected cells thereby subverting cellular processes. Secretion of effectors to the cell cytoplasm takes place through multiple routes, including two separate type III secretion (T3SS) apparati as well as outer membrane vesicles. The two T3SS are encoded on separate pathogenicity islands, SPI-1 and -2, with SPI-1 more strongly associated with the intestinal phase of infection, and SPI-2 with the systemic phase. Both T3SS are required for persistence, but the effectors required have not been systematically evaluated. In this study, mutations in 48 described effectors were tested for persistence. We replaced each effector with a specific DNA barcode sequence by allelic exchange and co-infected with a wild-type reference to calculate the ratio of wild-type parent to mutant at different times after infection. The competitive index (CI) was determined by quantitative PCR in which primers that correspond to the barcode were used for amplification. Mutations in all but seven effectors reduced persistence demonstrating that most effectors were required. One exception was CigR, a recently discovered effector that is widely conserved throughout enteric bacteria. Deletion of cigR increased lethality, suggesting that it may be an anti-virulence factor. The fact that almost all Salmonella effectors are required for persistence argues against redundant functions. This is different from effector repertoires in other intracellular pathogens such as Legionella.

  20. Genome Sequencing of Xanthomonas vasicola Pathovar vasculorum Reveals Variation in Plasmids and Genes Encoding Lipopolysaccharide Synthesis, Type-IV Pilus and Type-III Secretion Effectors.

    PubMed

    Wasukira, Arthur; Coulter, Max; Al-Sowayeh, Noorah; Thwaites, Richard; Paszkiewicz, Konrad; Kubiriba, Jerome; Smith, Julian; Grant, Murray; Studholme, David J

    2014-01-01

    Xanthomonas vasicola pathovar vasculorum (Xvv) is the bacterial agent causing gumming disease in sugarcane. Here, we compare complete genome sequences for five isolates of Xvv originating from sugarcane and one from maize. This identified two distinct types of lipopolysaccharide synthesis gene clusters among Xvv isolates: one is similar to that of Xanthomonas axonopodis pathovar citri (Xac) and is probably the ancestral type, while the other is similar to those of the sugarcane-inhabiting species, Xanthomonas sacchari. Four of six Xvv isolates harboured sequences similar to the Xac plasmid, pXAC47, and showed a distinct Type-IV pilus (T4P) sequence type, whereas the T4P locus of the other two isolates resembled that of the closely related banana pathogen, Xanthomonas campestris pathovar musacearum (Xcm). The Xvv isolate from maize has lost a gene encoding a homologue of the virulence effector, xopAF, which was present in all five of the sugarcane isolates, while xopL contained a premature stop codon in four out of six isolates. These findings shed new light on evolutionary events since the divergence of Xvv and Xcm, as well as further elucidating the relationships between the two closely related pathogens. PMID:25437615

  1. Genome Sequencing of Xanthomonas vasicola Pathovar vasculorum Reveals Variation in Plasmids and Genes Encoding Lipopolysaccharide Synthesis, Type-IV Pilus and Type-III Secretion Effectors

    PubMed Central

    Wasukira, Arthur; Coulter, Max; Al-Sowayeh, Noorah; Thwaites, Richard; Paszkiewicz, Konrad; Kubiriba, Jerome; Smith, Julian; Grant, Murray; Studholme, David J.

    2014-01-01

    Xanthomonas vasicola pathovar vasculorum (Xvv) is the bacterial agent causing gumming disease in sugarcane. Here, we compare complete genome sequences for five isolates of Xvv originating from sugarcane and one from maize. This identified two distinct types of lipopolysaccharide synthesis gene clusters among Xvv isolates: one is similar to that of Xanthomonas axonopodis pathovar citri (Xac) and is probably the ancestral type, while the other is similar to those of the sugarcane-inhabiting species, Xanthomonas sacchari. Four of six Xvv isolates harboured sequences similar to the Xac plasmid, pXAC47, and showed a distinct Type-IV pilus (T4P) sequence type, whereas the T4P locus of the other two isolates resembled that of the closely related banana pathogen, Xanthomonas campestris pathovar musacearum (Xcm). The Xvv isolate from maize has lost a gene encoding a homologue of the virulence effector, xopAF, which was present in all five of the sugarcane isolates, while xopL contained a premature stop codon in four out of six isolates. These findings shed new light on evolutionary events since the divergence of Xvv and Xcm, as well as further elucidating the relationships between the two closely related pathogens. PMID:25437615

  2. Bacterial secreted effectors and caspase-3 interactions

    PubMed Central

    Wall, Daniel M; McCormick, Beth A

    2014-01-01

    Apoptosis is a critical process that intrinsically links organism survival to its ability to induce controlled death. Thus, functional apoptosis allows organisms to remove perceived threats to their survival by targeting those cells that it determines pose a direct risk. Central to this process are apoptotic caspases, enzymes that form a signalling cascade, converting danger signals via initiator caspases into activation of the executioner caspase, caspase-3. This enzyme begins disassembly of the cell by activating DNA degrading enzymes and degrading the cellular architecture. Interaction of pathogenic bacteria with caspases, and in particular, caspase-3, can therefore impact both host cell and bacterial survival. With roles outside cell death such as cell differentiation, control of signalling pathways and immunomodulation also being described for caspase-3, bacterial interactions with caspase-3 may be of far more significance in infection than previously recognized. In this review, we highlight the ways in which bacterial pathogens have evolved to subvert caspase-3 both through effector proteins that directly interact with the enzyme or by modulating pathways that influence its activation and activity. PMID:25262664

  3. SepD/SepL-Dependent Secretion Signals of the Type III Secretion System Translocator Proteins in Enteropathogenic Escherichia coli

    PubMed Central

    Deng, Wanyin; Yu, Hong B.; Li, Yuling

    2015-01-01

    ABSTRACT The type III protein secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE) is essential for the pathogenesis of attaching/effacing bacterial pathogens, including enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and Citrobacter rodentium. These pathogens use the T3SS to sequentially secrete three categories of proteins: the T3SS needle and inner rod protein components; the EspA, EspB, and EspD translocators; and many LEE- and non-LEE-encoded effectors. SepD and SepL are essential for translocator secretion, and mutations in either lead to hypersecretion of effectors. However, how SepD and SepL control translocator secretion and secretion hierarchy between translocators and effectors is poorly understood. In this report, we show that the secreted T3SS components, the translocators, and both LEE- and non-LEE-encoded effectors all carry N-terminal type III secretion and translocation signals. These signals all behave like those of the effectors and are sufficient for mediating type III secretion and translocation by wild-type EPEC and hypersecretion by the sepD and sepL mutants. Our results extended previous observations and suggest that the secretion hierarchy of the different substrates is determined by a signal other than the N-terminal secretion signal. We identified a domain located immediately downstream of the N-terminal secretion signal in the translocator EspB that is required for SepD/SepL-dependent secretion. We further demonstrated that this EspB domain confers SepD/SepL- and CesAB-dependent secretion on the secretion signal of effector EspZ. Our results thus suggest that SepD and SepL control and regulate secretion hierarchy between translocators and effectors by recognizing translocator-specific export signals. IMPORTANCE Many bacterial pathogens use a syringe-like protein secretion apparatus, termed the type III protein secretion system (T3SS), to secrete and inject numerous proteins directly into

  4. Preliminary Analysis of Soybean Gene Expression Response to a Bradyrhizobium japonicum Type III Secretion System Mutant

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant pathogens deliver proteinaceous effector molecules into their host via complex secretion systems, such as the type III secretion system (T3SS). Some of these T3SS effectors have been shown to function as suppressors of host defense responses. The role of the T3SS during plant interactions wit...

  5. The type III secreted effector DspE is required early in Solanum tuberosum leaf infection by Pectobacterium carotovorum to elicit cell death, and requires Wx(3-6)D/E motifs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pectobacterium species are enterobacterial plant-pathogens that cause soft rot disease in diverse plant species. Unlike hemi-biotrophic plant pathogenic bacteria, the type III secretion system (T3SS) of Pectobacterium carotovorum subsp. carotovorum (P. carotovorum) appears to secrete only one effect...

  6. Type IV secretion system of Brucella spp. and its effectors

    PubMed Central

    Ke, Yuehua; Wang, Yufei; Li, Wengfeng; Chen, Zeliang

    2015-01-01

    Brucella spp. are intracellular bacterial pathogens that cause infection in domestic and wild animals. They are often used as model organisms to study intracellular bacterial infections. Brucella VirB T4SS is a key virulence factor that plays important roles in mediating intracellular survival and manipulating host immune response to infection. In this review, we discuss the roles of Brucella VirB T4SS and 15 effectors that are proposed to be crucial for Brucella pathogenesis. VirB T4SS regulates the inflammation response and manipulates vesicle trafficking inside host cells. VirB T4SS also plays crucial roles in the inhibition of the host immune response and intracellular survival during infection. Here, we list the key molecular events in the intracellular life cycle of Brucella that are potentially targeted by the VirB T4SS effectors. Elucidating the functions of these effectors will help clarify the molecular role of T4SS during infection. Furthermore, studying the effectors secreted by Brucella spp. might provide insights into the mechanisms used by the bacteria to hijack the host signaling pathways and aid in the development of better vaccines and therapies against brucellosis. PMID:26528442

  7. Bacteriophage-encoded type III effectors in Salmonella enterica subspecies 1 serovar Typhimurium.

    PubMed

    Ehrbar, Kristin; Hardt, Wolf-Dietrich

    2005-01-01

    Salmonella spp. are Gram-negative bacteria which cause infections ranging from mild, self-limiting enterocolitis to systemic (typhoid) disease. Recent work has established that the genetic makeup varies considerably between different Salmonella strains. Phages play an important role in this diversity. In fact, Salmonella has emerged as a prime example for the involvement of virulence factor encoding phages in the emergence of new epidemic strains. Among other virulence factors, Salmonella enterica utilizes two specialized protein secretion systems termed type III secretion systems (TTSS) to deliver effector proteins into host cells which manipulate host cell signaling cascades. These two TTSS and several effectors are encoded within Salmonella pathogenicity islands 1 and 2. Some effectors including SopE, SspH1, SseI and SopE2 are encoded by phages or phage remnants. These phage-encoded effectors seem to be transferred between different Salmonella strains. They have attracted much interest because they might contribute to the evolution of Salmonella spp. Here we will focus on SopEPhi which encodes the SPI-1 effector SopE. It provides an excellent example to illustrate how horizontally transferred effector proteins are integrated into the complex regulatory network of a TTSS in a recipient bacterium. Additional data supporting the hypothesis are presented. This is a prerequisite to allow optimization of the bacterium host cell interaction by reassortment of the phage-encoded effector protein repertoire. PMID:15567133

  8. Effective Identification of Bacterial Type III Secretion Signals Using Joint Element Features

    PubMed Central

    Wang, Yejun; Sun, Ming’an; Bao, Hongxia; Zhang, Qing; Guo, Dianjing

    2013-01-01

    Type III secretion system (T3SS) plays important roles in bacteria and host cell interactions by specifically translocating type III effectors into the cytoplasm of the host cells. The N-terminal amino acid sequences of the bacterial type III effectors determine their specific secretion via type III secretion conduits. It is still unclear as to how the N-terminal sequences guide this specificity. In this work, the amino acid composition, secondary structure, and solvent accessibility in the N-termini of type III and non-type III secreted proteins were compared and contrasted. A high-efficacy mathematical model based on these joint features was developed to distinguish the type III proteins from the non-type III ones. The results indicate that secondary structure and solvent accessibility may make important contribution to the specific recognition of type III secretion signals. Analysis also showed that the joint feature of the N-terminal 6th–10th amino acids are especially important for guiding specific type III secretion. Furthermore, a genome-wide screening was performed to predict Salmonella type III secreted proteins, and 8 new candidates were experimentally validated. Interestingly, type III secretion signals were also predicted in gram-positive bacteria and yeasts. Experimental validation showed that two candidates from yeast can indeed be secreted through Salmonella type III secretion conduit. This research provides the first line of direct evidence that secondary structure and solvent accessibility contain important features for guiding specific type III secretion. The new software based on these joint features ensures a high accuracy (general cross-validation sensitivity of ∼96% at a specificity of ∼98%) in silico identification of new type III secreted proteins, which may facilitate our understanding about the specificity of type III secretion and the evolution of type III secreted proteins. PMID:23593149

  9. Multiple Xanthomonas euvesicatoria Type III Effectors Inhibit flg22-Triggered Immunity.

    PubMed

    Popov, Georgy; Fraiture, Malou; Brunner, Frederic; Sessa, Guido

    2016-08-01

    Xanthomonas euvesicatoria is the causal agent of bacterial spot disease in pepper and tomato. X. euvesicatoria bacteria interfere with plant cellular processes by injecting effector proteins into host cells through the type III secretion (T3S) system. About 35 T3S effectors have been identified in X. euvesicatoria 85-10, and a few of them were implicated in suppression of pattern-triggered immunity (PTI). We used an Arabidopsis thaliana pathogen-free protoplast-based assay to identify X. euvesicatoria 85-10 effectors that interfere with PTI signaling induced by the bacterial peptide flg22. Of 33 tested effectors, 17 inhibited activation of a PTI-inducible promoter. Among them, nine effectors also interfered with activation of an abscisic acid-inducible promoter. However, effectors that inhibited flg22-induced signaling did not affect phosphorylation of mitogen-activated protein (MAP) kinases acting downstream of flg22 perception. Further investigation of selected effectors revealed that XopAJ, XopE2, and XopF2 inhibited activation of a PTI-inducible promoter by the bacterial peptide elf18 in Arabidopsis protoplasts and by flg22 in tomato protoplasts. The effectors XopF2, XopE2, XopAP, XopAE, XopH, and XopAJ inhibited flg22-induced callose deposition in planta and enhanced disease symptoms caused by attenuated Pseudomonas syringae bacteria. Finally, selected effectors were found to localize to various plant subcellular compartments. These results indicate that X. euvesicatoria bacteria utilize multiple T3S effectors to suppress flg22-induced signaling acting downstream or in parallel to MAP kinase cascades and suggest they act through different molecular mechanisms. PMID:27529660

  10. Type VI secretion effectors: poisons with a purpose

    PubMed Central

    Russell, Alistair B.; Peterson, S. Brook; Mougous, Joseph D.

    2014-01-01

    The type VI secretion system (T6SS) mediates interactions between a diverse range of Gram-negative bacterial species. Recent studies have led to a drastic increase in the number of characterized T6SS effector proteins and produced a more complete and nuanced view of the adaptive significance of the system. While the system is most often implicated in antagonism, in this review we consider the case for its involvement in both antagonistic and non-antagonistic behaviors. Clarifying the roles that T6S plays in microbial communities will contribute to broader efforts to understand the importance of microbial interactions in maintaining human and environmental health, and will inform efforts to manipulate these interactions for therapeutic or environmental benefit. PMID:24384601

  11. Five Xanthomonas type III effectors suppress cell death induced by components of immunity-associated MAP kinase cascades

    PubMed Central

    Teper, Doron; Sunitha, Sukumaran; Martin, Gregory B; Sessa, Guido

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades play a fundamental role in signaling of plant immunity and mediate elicitation of cell death. Xanthomonas spp. manipulate plant signaling by using a type III secretion system to deliver effector proteins into host cells. We examined the ability of 33 Xanthomonas effectors to inhibit cell death induced by overexpression of components of MAPK cascades in Nicotiana benthamiana plants. Five effectors inhibited cell death induced by overexpression of MAPKKKα and MEK2, but not of MAP3Kϵ. In addition, expression of AvrBs1 in yeast suppressed activation of the high osmolarity glycerol MAPK pathway, suggesting that the target of this effector is conserved in eukaryotic organisms. These results indicate that Xanthomonas employs several type III effectors to suppress immunity-associated cell death mediated by MAPK cascades. PMID:26237448

  12. Molecular weaponry: diverse effectors delivered by the Type VI secretion system

    PubMed Central

    Alcoforado Diniz, Juliana; Liu, Yi‐Chia

    2015-01-01

    Summary The Type VI secretion system is a widespread bacterial nanomachine, used to deliver toxins directly into eukaryotic or prokaryotic target cells. These secreted toxins, or effectors, act on diverse cellular targets, and their action provides the attacking bacterial cell with a significant fitness advantage, either against rival bacteria or eukaryotic host organisms. In this review, we discuss the delivery of diverse effectors by the Type VI secretion system, the modes of action of the so‐called ‘anti‐bacterial’ and ‘anti‐eukaryotic’ effectors, the mechanism of self‐resistance against anti‐bacterial effectors and the evolutionary implications of horizontal transfer of Type VI secretion system‐associated toxins. Whilst it is likely that many more effectors remain to be identified, it is already clear that toxins delivered by this secretion system represent efficient weapons against both bacteria and eukaryotes. PMID:26432982

  13. Real-time imaging of type III secretion: Salmonella SipA injection into host cells.

    PubMed

    Schlumberger, Markus C; Müller, Andreas J; Ehrbar, Kristin; Winnen, Brit; Duss, Iwan; Stecher, Bärbel; Hardt, Wolf-Dietrich

    2005-08-30

    Many pathogenic and symbiotic Gram-negative bacteria employ type III secretion systems to inject "effector" proteins into eukaryotic host cells. These effectors manipulate signaling pathways to initiate symbiosis or disease. By using time-lapse microscopy, we have imaged delivery of the Salmonella type III effector protein SipA/SspA into animal cells in real time. SipA delivery mostly began 10-90 sec after docking and proceeded for 100-600 sec until the bacterial SipA pool (6 +/- 3 x 10(3) molecules) was exhausted. Similar observations were made for the effector protein SopE. This visualization of type III secretion in real time explains the efficiency of host cell manipulation by means of this virulence system. PMID:16107539

  14. Evaluation of Secretion Prediction Highlights Differing Approaches Needed for Oomycete and Fungal Effectors

    PubMed Central

    Sperschneider, Jana; Williams, Angela H.; Hane, James K.; Singh, Karam B.; Taylor, Jennifer M.

    2015-01-01

    The steadily increasing number of sequenced fungal and oomycete genomes has enabled detailed studies of how these eukaryotic microbes infect plants and cause devastating losses in food crops. During infection, fungal and oomycete pathogens secrete effector molecules which manipulate host plant cell processes to the pathogen's advantage. Proteinaceous effectors are synthesized intracellularly and must be externalized to interact with host cells. Computational prediction of secreted proteins from genomic sequences is an important technique to narrow down the candidate effector repertoire for subsequent experimental validation. In this study, we benchmark secretion prediction tools on experimentally validated fungal and oomycete effectors. We observe that for a set of fungal SwissProt protein sequences, SignalP 4 and the neural network predictors of SignalP 3 (D-score) and SignalP 2 perform best. For effector prediction in particular, the use of a sensitive method can be desirable to obtain the most complete candidate effector set. We show that the neural network predictors of SignalP 2 and 3, as well as TargetP were the most sensitive tools for fungal effector secretion prediction, whereas the hidden Markov model predictors of SignalP 2 and 3 were the most sensitive tools for oomycete effectors. Thus, previous versions of SignalP retain value for oomycete effector prediction, as the current version, SignalP 4, was unable to reliably predict the signal peptide of the oomycete Crinkler effectors in the test set. Our assessment of subcellular localization predictors shows that cytoplasmic effectors are often predicted as not extracellular. This limits the reliability of secretion predictions that depend on these tools. We present our assessment with a view to informing future pathogenomics studies and suggest revised pipelines for secretion prediction to obtain optimal effector predictions in fungi and oomycetes. PMID:26779196

  15. The Type III Secretion System of Bradyrhizobium japonicum USDA122 Mediates Symbiotic Incompatibility with Rj2 Soybean Plants

    PubMed Central

    Tsukui, Takahiro; Eda, Shima; Kaneko, Takakazu; Sato, Shusei; Okazaki, Shin; Kakizaki-Chiba, Kaori; Itakura, Manabu; Mitsui, Hisayuki; Yamashita, Akifumi; Terasawa, Kimihiro

    2013-01-01

    The rhcJ and ttsI mutants of Bradyrhizobium japonicum USDA122 for the type III protein secretion system (T3SS) failed to secrete typical effector proteins and gained the ability to nodulate Rj2 soybean plants (Hardee), which are symbiotically incompatible with wild-type USDA122. This suggests that effectors secreted via the T3SS trigger incompatibility between these two partners. PMID:23204412

  16. A genetic screen to isolate type III effectors translocated into pepper cells during Xanthomonas infection

    SciTech Connect

    Julie Anne Roden, Branids Belt, Jason Barzel Ross, Thomas Tachibana, Joe Vargas, Mary Beth Mudgett

    2004-11-23

    The bacterial pathogen Xanthomonas campestris pv. vesicatoria (Xcv) uses a type III secretion system (TTSS) to translocate effector proteins into host plant cells. The TTSS is required for Xcv colonization, yet the identity of many proteins translocated through this apparatus is not known. We used a genetic screen to functionally identify Xcv TTSS effectors. A transposon 5 (Tn5)-based transposon construct including the coding sequence for the Xcv AvrBs2 effector devoid of its TTSS signal was randomly inserted into the Xcv genome. Insertion of the avrBs2 reporter gene into Xcv genes coding for proteins containing a functional TTSS signal peptide resulted in the creation of chimeric TTSS effector::AvrBs2 fusion proteins. Xcv strains containing these fusions translocated the AvrBs2 reporter in a TTSS-dependent manner into resistant BS2 pepper cells during infection, activating the avrBs2-dependent hypersensitive response (HR). We isolated seven chimeric fusion proteins and designated the identified TTSS effectors as Xanthomonas outer proteins (Xops). Translocation of each Xop was confirmed by using the calmodulin-dependent adenylate cydase reporter assay. Three xop genes are Xanthomonas spp.-specific, whereas homologs for the rest are found in other phytopathogenic bacteria. XopF1 and XopF2 define an effector gene family in Xcv. XopN contains a eukaryotic protein fold repeat and is required for full Xcv pathogenicity in pepper and tomato. The translocated effectors identified in this work expand our knowledge of the diversity of proteins that Xcv uses to manipulate its hosts.

  17. Application of β-Lactamase Reporter Fusions as an Indicator of Effector Protein Secretion during Infections with the Obligate Intracellular Pathogen Chlamydia trachomatis

    PubMed Central

    Mueller, Konrad E.; Fields, Kenneth A.

    2015-01-01

    Chlamydia spp. utilize multiple secretion systems, including the type III secretion system (T3SS), to deploy host-interactive effector proteins into infected host cells. Elucidation of secreted proteins has traditionally required ectopic expression in a surrogate T3SS followed by immunolocalization of endogenous candidate effectors to confirm secretion by chlamydiae. The ability to transform Chlamydia and achieve stable expression of recombinant gene products has enabled a more direct assessment of secretion. We adapted TEM-1 β-lactamase as a reporter system for assessment of chlamydial protein secretion. We provide evidence that this system facilitates visualization of secretion in the context of infection. Specifically, our findings provide definitive evidence that C. trachomatis CT695 is secreted during infection. Follow-up indirect immunofluorescence studies confirmed CT695 secretion and indicate that this effector can be secreted at multiple points during the chlamydial developmental cycle. Our results indicate that the BlaM-fusion reporter assay will allow efficacious identification of novel secreted proteins. Moreover, this approach can easily be adapted to enable more sophisticated studies of the secretion process in Chlamydia. PMID:26258949

  18. A bacterial pathogen uses distinct type III secretion systems to alternate between host kingdoms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant and animal-pathogenic bacteria utilize phylogenetically distinct type III secretion systems (T3SS) that produce needle-like injectisomes or pili for the delivery of effector proteins into host cells. Pantoea stewartii subsp. stewartii (Pnss), the causative agent of Stewart’s bacterial wilt and...

  19. Contribution of Bordetella bronchiseptica Type III secretion system to respiratory disease in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: The type III secretion system (TTSS) of gram negative bacteria allows injection of effector proteins directly into the cytosol of eukaryotic cells. Previous studies have demonstrated that the B. bronchiseptica TTSS plays a role in the persistent bacterial colonization of the trachea of m...

  20. Unconventionally secreted effectors of two filamentous pathogens target plant salicylate biosynthesis.

    PubMed

    Liu, Tingli; Song, Tianqiao; Zhang, Xiong; Yuan, Hongbo; Su, Liming; Li, Wanlin; Xu, Jing; Liu, Shiheng; Chen, Linlin; Chen, Tianzi; Zhang, Meixiang; Gu, Lichuan; Zhang, Baolong; Dou, Daolong

    2014-01-01

    Plant diseases caused by fungi and oomycetes pose an increasing threat to food security and ecosystem health worldwide. These filamentous pathogens, while taxonomically distinct, modulate host defense responses by secreting effectors, which are typically identified based on the presence of signal peptides. Here we show that Phytophthora sojae and Verticillium dahliae secrete isochorismatases (PsIsc1 and VdIsc1, respectively) that are required for full pathogenesis. PsIsc1 and VdIsc1 can suppress salicylate-mediated innate immunity in planta and hydrolyse isochorismate in vitro. A conserved triad of catalytic residues is essential for both functions. Thus, the two proteins are isochorismatase effectors that disrupt the plant salicylate metabolism pathway by suppressing its precursor. Furthermore, these proteins lack signal peptides, but exhibit characteristics that lead to unconventional secretion. Therefore, this secretion pathway is a novel mechanism for delivering effectors and might play an important role in host-pathogen interactions. PMID:25156390

  1. Prediction of bacterial type IV secreted effectors by C-terminal features

    PubMed Central

    2014-01-01

    Background Many bacteria can deliver pathogenic proteins (effectors) through type IV secretion systems (T4SSs) to eukaryotic cytoplasm, causing host diseases. The inherent property, such as sequence diversity and global scattering throughout the whole genome, makes it a big challenge to effectively identify the full set of T4SS effectors. Therefore, an effective inter-species T4SS effector prediction tool is urgently needed to help discover new effectors in a variety of bacterial species, especially those with few known effectors, e.g., Helicobacter pylori. Results In this research, we first manually annotated a full list of validated T4SS effectors from different bacteria and then carefully compared their C-terminal sequential and position-specific amino acid compositions, possible motifs and structural features. Based on the observed features, we set up several models to automatically recognize T4SS effectors. Three of the models performed strikingly better than the others and T4SEpre_Joint had the best performance, which could distinguish the T4SS effectors from non-effectors with a 5-fold cross-validation sensitivity of 89% at a specificity of 97%, based on the training datasets. An inter-species cross prediction showed that T4SEpre_Joint could recall most known effectors from a variety of species. The inter-species prediction tool package, T4SEpre, was further used to predict new T4SS effectors from H. pylori, an important human pathogen associated with gastritis, ulcer and cancer. In total, 24 new highly possible H. pylori T4S effector genes were computationally identified. Conclusions We conclude that T4SEpre, as an effective inter-species T4SS effector prediction software package, will help find new pathogenic T4SS effectors efficiently in a variety of pathogenic bacteria. PMID:24447430

  2. The Type III Secretion Translocation Pore Senses Host Cell Contact

    PubMed Central

    Armentrout, Erin I.; Rietsch, Arne

    2016-01-01

    Type III secretion systems (T3SS) are nano-syringes used by a wide range of Gram-negative pathogens to promote infection by directly injecting effector proteins into targeted host cells. Translocation of effectors is triggered by host-cell contact and requires assembly of a pore in the host-cell plasma membrane, which consists of two translocator proteins. Our understanding of the translocation pore, how it is assembled in the host cell membrane and its precise role in effector translocation, is extremely limited. Here we use a genetic technique to identify protein-protein contacts between pore-forming translocator proteins, as well as the T3SS needle-tip, that are critical for translocon function. The data help establish the orientation of the translocator proteins in the host cell membrane. Analysis of translocon function in mutants that break these contacts demonstrates that an interaction between the pore-forming translocator PopD and the needle-tip is required for sensing host cell contact. Moreover, tethering PopD at a dimer interface also specifically prevents host-cell sensing, arguing that the translocation pore is actively involved in detecting host cell contact. The work presented here therefore establishes a signal transduction pathway for sensing host cell contact that is initiated by a conformational change in the translocation pore, and is subsequently transmitted to the base of the apparatus via a specific contact between the pore and the T3SS needle-tip. PMID:27022930

  3. The Vibrio cholerae type VI secretion system employs diverse effector modules for intraspecific competition

    PubMed Central

    Unterweger, Daniel; Miyata, Sarah T.; Bachmann, Verena; Brooks, Teresa M.; Mullins, Travis; Kostiuk, Benjamin; Provenzano, Daniele; Pukatzki, Stefan

    2014-01-01

    Vibrio cholerae is a Gram-negative bacterial pathogen that consists of over 200 serogroups with differing pathogenic potential. Only strains that express the virulence factors cholera toxin (CT) and toxin-coregulated pilus (TCP) are capable of pandemic spread of cholera diarrhoea. Regardless, all V. cholerae strains sequenced to date harbour genes for the type VI secretion system (T6SS) that translocates effectors into neighbouring eukaryotic and prokaryotic cells. Here we report that the effectors encoded within these conserved gene clusters differ widely among V. cholerae strains, and that immunity proteins encoded immediately downstream from the effector genes protect their host from neighbouring bacteria producing corresponding effectors. As a consequence, strains with matching effector-immunity gene sets can coexist, while strains with different sets compete against each other. Thus, the V. cholerae T6SS contributes to the competitive behaviour of this species. PMID:24686479

  4. A Salmonella Type Three Secretion Effector/Chaperone Complex Adopts a Hexameric Ring-Like Structure

    PubMed Central

    Roblin, Pierre; Dewitte, Frédérique; Villeret, Vincent; Biondi, Emanuele G.

    2014-01-01

    Many bacterial pathogens use type three secretion systems (T3SS) to inject virulence factors, named effectors, directly into the cytoplasm of target eukaryotic cells. Most of the T3SS components are conserved among plant and animal pathogens, suggesting a common mechanism of recognition and secretion of effectors. However, no common motif has yet been identified for effectors allowing T3SS recognition. In this work, we performed a biochemical and structural characterization of the Salmonella SopB/SigE chaperone/effector complex by small-angle X-ray scattering (SAXS). Our results showed that the SopB/SigE complex is assembled in dynamic homohexameric-ring-shaped structures with an internal tunnel. In this ring, the chaperone maintains a disordered N-terminal end of SopB molecules, in a good position to be reached and processed by the T3SS. This ring dimensionally fits the ring-organized molecules of the injectisome, including ATPase hexameric rings; this organization suggests that this structural feature is important for ATPase recognition by T3SS. Our work constitutes the first evidence of the oligomerization of an effector, analogous to the organization of the secretion machinery, obtained in solution. As effectors share neither sequence nor structural identity, the quaternary oligomeric structure could constitute a strategy evolved to promote the specificity and efficiency of T3SS recognition. PMID:25404693

  5. A Pathogen Type III Effector with a Novel E3 Ubiquitin Ligase Architecture

    PubMed Central

    Skarina, Tatiana; Xu, Xiaohui; Cui, Hong; Eschen-Lippold, Lennart; Egler, Monique; Srikumar, Tharan; Raught, Brian; Lee, Justin; Scheel, Dierk; Savchenko, Alexei; Bonas, Ulla

    2013-01-01

    Type III effectors are virulence factors of Gram-negative bacterial pathogens delivered directly into host cells by the type III secretion nanomachine where they manipulate host cell processes such as the innate immunity and gene expression. Here, we show that the novel type III effector XopL from the model plant pathogen Xanthomonas campestris pv. vesicatoria exhibits E3 ubiquitin ligase activity in vitro and in planta, induces plant cell death and subverts plant immunity. E3 ligase activity is associated with the C-terminal region of XopL, which specifically interacts with plant E2 ubiquitin conjugating enzymes and mediates formation of predominantly K11-linked polyubiquitin chains. The crystal structure of the XopL C-terminal domain revealed a single domain with a novel fold, termed XL-box, not present in any previously characterized E3 ligase. Mutation of amino acids in the central cavity of the XL-box disrupts E3 ligase activity and prevents XopL-induced plant cell death. The lack of cysteine residues in the XL-box suggests the absence of thioester-linked ubiquitin-E3 ligase intermediates and a non-catalytic mechanism for XopL-mediated ubiquitination. The crystal structure of the N-terminal region of XopL confirmed the presence of a leucine-rich repeat (LRR) domain, which may serve as a protein-protein interaction module for ubiquitination target recognition. While the E3 ligase activity is required to provoke plant cell death, suppression of PAMP responses solely depends on the N-terminal LRR domain. Taken together, the unique structural fold of the E3 ubiquitin ligase domain within the Xanthomonas XopL is unprecedented and highlights the variation in bacterial pathogen effectors mimicking this eukaryote-specific activity. PMID:23359647

  6. Genetically distinct pathways guide effector export through the type VI secretion system

    PubMed Central

    Whitney, John C.; Beck, Christina M.; Goo, Young Ah; Russell, Alistair B.; Harding, Brittany; De Leon, Justin A.; Cunningham, David A.; Tran, Bao Q.; Low, David A.; Goodlett, David R.; Hayes, Christopher S.; Mougous, Joseph D.

    2014-01-01

    Summary Bacterial secretion systems often employ molecular chaperones to recognize and facilitate export of their substrates. Recent work demonstrated that a secreted component of the type VI secretion system (T6SS), hemolysin co-regulated protein (Hcp), binds directly to effectors, enhancing their stability in the bacterial cytoplasm. Herein, we describe a quantitative cellular proteomics screen for T6S substrates that exploits this chaperone-like quality of Hcp. Application of this approach to the Hcp secretion island I-encoded T6SS (H1-T6SS) of Pseudomonas aeruginosa led to the identification of a novel effector protein, termed Tse4 (type VI secretion exported 4), subsequently shown to act as a potent intra-specific H1-T6SS-delivered antibacterial toxin. Interestingly, our screen failed to identify two predicted H1-T6SS effectors, Tse5 and Tse6, which differ from Hcp-stabilized substrates by the presence of toxin-associated PAAR-repeat motifs and genetic linkage to members of the valine-glycine repeat protein G (vgrG) genes. Genetic studies further distinguished these two groups of effectors: Hcp-stabilized effectors were found to display redundancy in interbacterial competition with respect to the requirement for the two H1-T6SS-exported VgrG proteins, whereas Tse5 and Tse6 delivery strictly required a cognate VgrG. Together, we propose that interaction with either VgrG or Hcp defines distinct pathways for T6S effector export. PMID:24589350

  7. InvB is a type III secretion chaperone specific for SspA.

    PubMed

    Bronstein, P A; Miao, E A; Miller, S I

    2000-12-01

    A wide variety of gram-negative bacteria utilize a specialized apparatus called the type III secretion system (TTSS) to translocate virulence factors directly into the cytoplasm of eukaryotic cells. These translocated effectors contribute to the pathogen's ability to infect and replicate within plant and animal hosts. The amino terminus of effector proteins contains sequences that are necessary and sufficient for both secretion and translocation by TTSS. Portions of these sequences contain binding sites for type III chaperones, which facilitate efficient secretion and translocation of specific effectors through TTSS. In this study, we have utilized the yeast two-hybrid assay to identify protein-protein interactions between effector and chaperone proteins encoded within Salmonella pathogenicity island 1 (SPI-1). Several interactions were identified including a novel interaction between the effector protein, SspA (SipA), and a putative chaperone, InvB. InvB was demonstrated to bind to the amino terminus of SspA in the bacterial cytoplasm. Furthermore, InvB acts as a type III chaperone for the efficient secretion and translocation of SspA by SPI-1. InvB also permitted translocation of SspA through the SPI-2 TTSS, indicating that it is an important regulator in the recognition of SspA as a target of TTSS. Finally, it was determined that InvB does not alter the transcription of sspA but that its absence results in reduced SspA protein levels in Salmonella enterica serovar Typhimurium. PMID:11073906

  8. SpiC Is Required for Translocation of Salmonella Pathogenicity Island 2 Effectors and Secretion of Translocon Proteins SseB and SseC

    PubMed Central

    Freeman, Jeremy A.; Rappl, Catherine; Kuhle, Volker; Hensel, Michael; Miller, Samuel I.

    2002-01-01

    The Salmonella pathogenicity island 2 (SPI2) type III secretion system (TTSS) promotes Salmonella enterica serovar Typhimurium virulence for mice and increased survival and replication within eukaryotic cells. After phagocytosis, Salmonella serovar Typhimurium assembles the SPI2 TTSS to translocate over a dozen effector proteins across the phagosome membrane. SpiC has been previously shown to be a translocated effector with a large contribution to virulence (K. Uchiya, M. A. Barbieri, K. Funato, A. H. Shah, P. D. Stahl, and E. A. Groisman, EMBO J. 18:3924-3933, 1999). This report demonstrates by competitive index that the virulence phenotype of a spiC mutant is equivalent to that of a secretion component mutant. In addition, translocation of SPI2 effector proteins was shown to require SpiC. Thus, the severe virulence phenotype resulting from deletion of spiC is likely due to the inability to translocate all SPI2 effectors. SpiC was also required to secrete translocon proteins SseB and SseC but not translocated effector SseJ, indicating that lack of assembly of the translocon explains the spiC mutant phenotype. PMID:12193612

  9. The Structure and Function of Type III Secretion Systems

    PubMed Central

    Notti, Ryan Q.; Stebbins, C. Erec

    2015-01-01

    ARTICLE SUMMARY Type III secretion systems (T3SS) afford gram-negative bacteria a most intimate means of altering the biology of their eukaryotic hosts — the direct delivery of effector proteins from the bacterial cytoplasm to that of the eukaryote. This incredible biophysical feat is accomplished by nanosyringe “injectisomes,” which form a conduit across the three plasma membranes, peptidoglycan layer and extracellular space that form a barrier to the direct delivery of proteins from bacterium to host. The focus of this chapter is T3SS function at the structural level; we will summarize the core findings that have shaped our understanding of the structure and function of these systems and highlight recent developments in the field. In turn, we describe the T3SS secretory apparatus, consider its engagement with secretion substrates, and discuss the post-translational regulation of secretory function. Lastly, we close with a discussion of the future prospects for the interrogation of structure-function relationships in the T3SS. PMID:26999392

  10. Visualizing the Translocation and Localization of Bacterial Type III Effector Proteins by Using a Genetically Encoded Reporter System

    PubMed Central

    Gawthorne, Jayde A.; Audry, Laurent; McQuitty, Claire; Dean, Paul; Christie, John M.; Enninga, Jost

    2016-01-01

    Bacterial type III secretion system (T3SS) effector proteins are critical determinants of infection for many animal and plant pathogens. However, monitoring of the translocation and delivery of these important virulence determinants has proved to be technically challenging. Here, we used a genetically engineered LOV (light-oxygen-voltage) sensing domain derivative to monitor the expression, translocation, and localization of bacterial T3SS effectors. We found the Escherichia coli O157:H7 bacterial effector fusion Tir-LOV was functional following its translocation and localized to the host cell membrane in discrete foci, demonstrating that LOV-based reporters can be used to visualize the effector translocation with minimal manipulation and interference. Further evidence for the versatility of the reporter was demonstrated by fusing LOV to the C terminus of the Shigella flexneri effector IpaB. IpaB-LOV localized preferentially at bacterial poles before translocation. We observed the rapid translocation of IpaB-LOV in a T3SS-dependent manner into host cells, where it localized at the bacterial entry site within membrane ruffles. PMID:26921426

  11. The Burkholderia pseudomallei Proteins BapA and BapC Are Secreted TTSS3 Effectors and BapB Levels Modulate Expression of BopE

    PubMed Central

    Treerat, Puthayalai; Alwis, Priyangi; D’Cruze, Tanya; Cullinane, Meabh; Vadivelu, Jamunarani; Devenish, Rodney J.; Prescott, Mark; Adler, Ben; Boyce, John D.

    2015-01-01

    Many Gram-negative pathogens use a type III secretion system (TTSS) for the injection of bacterial effector proteins into host cells. The injected effector proteins play direct roles in modulation of host cell pathways for bacterial benefit. Burkholderia pseudomallei, the causative agent of melioidosis, expresses three different TTSSs. One of these systems, the TTSS3, is essential for escape from host endosomes and therefore intracellular survival and replication. Here we have characterized three putative TTSS3 proteins; namely BapA, BapB and BapC. By employing a tetracysteine (TC)-FlAsH™ labelling technique to monitor the secretion of TC-tagged fusion proteins, BapA and BapC were shown to be secreted during in vitro growth in a TTSS3-dependant manner, suggesting a role as TTSS3 effectors. Furthermore, we constructed B. pseudomallei bapA, bapB and bapC mutants and used the well-characterized TTSS3 effector BopE as a marker of secretion to show that BapA, BapB and BapC are not essential for the secretion process. However, BopE transcription and secretion were significantly increased in the bapB mutant, suggesting that BapB levels modulate BopE expression. In a BALB/c mouse model of acute melioidosis, the bapA, bapB and bapC mutants showed a minor reduction of in vivo fitness. Thus, this study defines BapA and BapC as novel TTSS3 effectors, BapB as a regulator of BopE production, and all three as necessary for full B. pseudomallei in vivo fitness. PMID:26624293

  12. The Burkholderia pseudomallei Proteins BapA and BapC Are Secreted TTSS3 Effectors and BapB Levels Modulate Expression of BopE.

    PubMed

    Treerat, Puthayalai; Alwis, Priyangi; D'Cruze, Tanya; Cullinane, Meabh; Vadivelu, Jamunarani; Devenish, Rodney J; Prescott, Mark; Adler, Ben; Boyce, John D

    2015-01-01

    Many Gram-negative pathogens use a type III secretion system (TTSS) for the injection of bacterial effector proteins into host cells. The injected effector proteins play direct roles in modulation of host cell pathways for bacterial benefit. Burkholderia pseudomallei, the causative agent of melioidosis, expresses three different TTSSs. One of these systems, the TTSS3, is essential for escape from host endosomes and therefore intracellular survival and replication. Here we have characterized three putative TTSS3 proteins; namely BapA, BapB and BapC. By employing a tetracysteine (TC)-FlAsH™ labelling technique to monitor the secretion of TC-tagged fusion proteins, BapA and BapC were shown to be secreted during in vitro growth in a TTSS3-dependant manner, suggesting a role as TTSS3 effectors. Furthermore, we constructed B. pseudomallei bapA, bapB and bapC mutants and used the well-characterized TTSS3 effector BopE as a marker of secretion to show that BapA, BapB and BapC are not essential for the secretion process. However, BopE transcription and secretion were significantly increased in the bapB mutant, suggesting that BapB levels modulate BopE expression. In a BALB/c mouse model of acute melioidosis, the bapA, bapB and bapC mutants showed a minor reduction of in vivo fitness. Thus, this study defines BapA and BapC as novel TTSS3 effectors, BapB as a regulator of BopE production, and all three as necessary for full B. pseudomallei in vivo fitness. PMID:26624293

  13. NopC Is a Rhizobium-Specific Type 3 Secretion System Effector Secreted by Sinorhizobium (Ensifer) fredii HH103

    PubMed Central

    Medina, Carlos; Ollero, Francisco Javier; López-Baena, Francisco Javier

    2015-01-01

    Sinorhizobium (Ensifer) fredii HH103 is a broad host-range nitrogen-fixing bacterium able to nodulate many legumes, including soybean. In several rhizobia, root nodulation is influenced by proteins secreted through the type 3 secretion system (T3SS). This specialized secretion apparatus is a common virulence mechanism of many plant and animal pathogenic bacteria that delivers proteins, called effectors, directly into the eukaryotic host cells where they interfere with signal transduction pathways and promote infection by suppressing host defenses. In rhizobia, secreted proteins, called nodulation outer proteins (Nops), are involved in host-range determination and symbiotic efficiency. S. fredii HH103 secretes at least eight Nops through the T3SS. Interestingly, there are Rhizobium-specific Nops, such as NopC, which do not have homologues in pathogenic bacteria. In this work we studied the S. fredii HH103 nopC gene and confirmed that its expression was regulated in a flavonoid-, NodD1- and TtsI-dependent manner. Besides, in vivo bioluminescent studies indicated that the S. fredii HH103 T3SS was expressed in young soybean nodules and adenylate cyclase assays confirmed that NopC was delivered directly into soybean root cells by means of the T3SS machinery. Finally, nodulation assays showed that NopC exerted a positive effect on symbiosis with Glycine max cv. Williams 82 and Vigna unguiculata. All these results indicate that NopC can be considered a Rhizobium-specific effector secreted by S. fredii HH103. PMID:26569401

  14. NopC Is a Rhizobium-Specific Type 3 Secretion System Effector Secreted by Sinorhizobium (Ensifer) fredii HH103.

    PubMed

    Jiménez-Guerrero, Irene; Pérez-Montaño, Francisco; Medina, Carlos; Ollero, Francisco Javier; López-Baena, Francisco Javier

    2015-01-01

    Sinorhizobium (Ensifer) fredii HH103 is a broad host-range nitrogen-fixing bacterium able to nodulate many legumes, including soybean. In several rhizobia, root nodulation is influenced by proteins secreted through the type 3 secretion system (T3SS). This specialized secretion apparatus is a common virulence mechanism of many plant and animal pathogenic bacteria that delivers proteins, called effectors, directly into the eukaryotic host cells where they interfere with signal transduction pathways and promote infection by suppressing host defenses. In rhizobia, secreted proteins, called nodulation outer proteins (Nops), are involved in host-range determination and symbiotic efficiency. S. fredii HH103 secretes at least eight Nops through the T3SS. Interestingly, there are Rhizobium-specific Nops, such as NopC, which do not have homologues in pathogenic bacteria. In this work we studied the S. fredii HH103 nopC gene and confirmed that its expression was regulated in a flavonoid-, NodD1- and TtsI-dependent manner. Besides, in vivo bioluminescent studies indicated that the S. fredii HH103 T3SS was expressed in young soybean nodules and adenylate cyclase assays confirmed that NopC was delivered directly into soybean root cells by means of the T3SS machinery. Finally, nodulation assays showed that NopC exerted a positive effect on symbiosis with Glycine max cv. Williams 82 and Vigna unguiculata. All these results indicate that NopC can be considered a Rhizobium-specific effector secreted by S. fredii HH103. PMID:26569401

  15. Secretion of the housekeeping protein glyceraldehyde-3-phosphate dehydrogenase by the LEE-encoded type III secretion system in enteropathogenic Escherichia coli.

    PubMed

    Aguilera, Laura; Ferreira, Elaine; Giménez, Rosa; Fernández, Francisco José; Taulés, Marta; Aguilar, Juan; Vega, M Cristina; Badia, Josefa; Baldomà, Laura

    2012-06-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional housekeeping protein secreted by pathogens and involved in adhesion and/or virulence. Previously we reported that enterohemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli secrete GAPDH into the culture medium. This bacterial protein binds human plasminogen and fibrinogen and remains associated with Caco-2 cells upon infection. In these pathogens, GAPDH secretion is not linked to outer membrane vesicles and depends on growth conditions, although the secretion mechanism is still unknown. EPEC is an attaching and effacing pathogen able to secrete and translocate multiple effector proteins into infected cells through a type III secretion system (T3SS). The secretion process is often dependent on a bacterial chaperone. The chaperone CesT displays broad substrate specificity and plays a central role in the recruitment of multiple type III effectors to the T3SS apparatus. Here we provide genetic evidences on GAPDH secretion through T3SS by EPEC grown in DMEM. Secretion of GAPDH is increased in ΔsepD mutants and abolished in mutants defective in the type III ATPase EscN. Complementation with escN gene restores GAPDH secretion. In addition, we prove by means of pull down experiments, overlay immunoblotting and biolayer interferometry a novel interaction between GAPDH and the chaperone CesT. This interaction, which is strong and slow dissociating, may stabilize a population of GAPDH molecules in a secretion competent-state and target them to the type III secretion apparatus. This is the first description of CesT interaction with a housekeeping protein and its export through T3SS. PMID:22433988

  16. Functional and computational analysis of amino acid patterns predictive of type III secretion system substrates in Pseudomonas syringae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacterial type III secretion systems (T3SSs) deliver proteins called effectors into eukaryotic cells. Although N-terminal amino acid sequences are required for translocation, the mechanism of substrate recognition by the T3SS is unknown. Almost all actively deployed T3SS substrates in the plant path...

  17. Phytophthora infestans effector AVRblb2 prevents secretion of a plant immune protease at the haustorial interface

    PubMed Central

    Bozkurt, Tolga O.; Schornack, Sebastian; Win, Joe; Shindo, Takayuki; Ilyas, Muhammad; Oliva, Ricardo; Cano, Liliana M.; Jones, Alexandra M. E.; Huitema, Edgar; van der Hoorn, Renier A. L.; Kamoun, Sophien

    2011-01-01

    In response to pathogen attack, plant cells secrete antimicrobial molecules at the site of infection. However, how plant pathogens interfere with defense-related focal secretion remains poorly known. Here we show that the host-translocated RXLR-type effector protein AVRblb2 of the Irish potato famine pathogen Phytophthora infestans focally accumulates around haustoria, specialized infection structures that form inside plant cells, and promotes virulence by interfering with the execution of host defenses. AVRblb2 significantly enhances susceptibility of host plants to P. infestans by targeting the host papain-like cysteine protease C14 and specifically preventing its secretion into the apoplast. Plants altered in C14 expression were significantly affected in susceptibility to P. infestans in a manner consistent with a positive role of C14 in plant immunity. Our findings point to a unique counterdefense strategy that plant pathogens use to neutralize secreted host defense proteases. Effectors, such as AVRblb2, can be used as molecular probes to dissect focal immune responses at pathogen penetration sites. PMID:22143776

  18. Visualizing the Translocation and Localization of Bacterial Type III Effector Proteins by Using a Genetically Encoded Reporter System.

    PubMed

    Gawthorne, Jayde A; Audry, Laurent; McQuitty, Claire; Dean, Paul; Christie, John M; Enninga, Jost; Roe, Andrew J

    2016-05-01

    Bacterial type III secretion system (T3SS) effector proteins are critical determinants of infection for many animal and plant pathogens. However, monitoring of the translocation and delivery of these important virulence determinants has proved to be technically challenging. Here, we used a genetically engineered LOV (light-oxygen-voltage) sensing domain derivative to monitor the expression, translocation, and localization of bacterial T3SS effectors. We found theEscherichia coliO157:H7 bacterial effector fusion Tir-LOV was functional following its translocation and localized to the host cell membrane in discrete foci, demonstrating that LOV-based reporters can be used to visualize the effector translocation with minimal manipulation and interference. Further evidence for the versatility of the reporter was demonstrated by fusing LOV to the C terminus of theShigella flexnerieffector IpaB. IpaB-LOV localized preferentially at bacterial poles before translocation. We observed the rapid translocation of IpaB-LOV in a T3SS-dependent manner into host cells, where it localized at the bacterial entry site within membrane ruffles. PMID:26921426

  19. Conserved type III secretion system exerts important roles in Chlamydia trachomatis

    PubMed Central

    Dai, Wenting; Li, Zhongyu

    2014-01-01

    Upon infection, Chlamydiae alter host cellular functions in a variety of ways. Chlamydial infection prevents host cell apoptosis, induces re-organization of the actin cytoskeleton and alters host cellular signaling mechanisms. Chlamydia is among the many pathogenic Gram-negative bacteria that employ the type III secretion system (T3SS) to overcome host defenses and exploit available resources. T3SS are used by many Gram-negative bacterial pathogens to manipulate eukaryotic host cells through the delivery of effector proteins into their cytosol and membranes. T3SS is an evolutionarily refined, virulence determinant of Gram-negative bacteria where more than 20 proteins form an apparatus, generally termed injectisome, to achieve the vectorial secretion and translocation of anti-host effector proteins. This review describes challenges and recent advances that have revealed how Chlamydia trachomatis utilizes diversification to produce a conserved T3SS that exerts an important role in Chlamydia trachomatis. PMID:25337183

  20. Aeromonas salmonicida Ati2 is an effector protein of the type three secretion system.

    PubMed

    Dallaire-Dufresne, Stéphanie; Barbeau, Xavier; Sarty, Darren; Tanaka, Katherine H; Denoncourt, Alix M; Lagüe, Patrick; Reith, Michael E; Charette, Steve J

    2013-09-01

    The bacterium Aeromonas salmonicida, a fish pathogen, uses the type three secretion system (TTSS) to inject effector proteins into host cells to promote the infection. The study of the genome of A. salmonicida has revealed the existence of Ati2, a potential TTSS effector protein. In the present study, a structure-function analysis of Ati2 has been done to determine its role in the virulence of A. salmonicida. Biochemical assays revealed that Ati2 is secreted into the medium in a TTSS-dependent manner. Protein sequence analyses, molecular modelling and biochemical assays demonstrated that Ati2 is an inositol polyphosphate 5-phosphatase, which hydrolyses PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in a way similar to VPA0450, a protein from Vibrio parahaemolyticus having high sequence similarity with Ati2. Mutants of Ati2 with altered amino acids at two different locations in the catalytic site displayed no phosphatase activity. Wild-type and mutant forms of Ati2 were cloned into expression systems for Dictyostelium discoideum, a soil amoeba used as an alternative host to study A. salmonicida virulence. Expression tests allowed us to demonstrate that Ati2 is toxic for the host cell in a catalytic-dependent manner. Finally, this study demonstrated the existence of a new TTSS effector protein in A. salmonicida. PMID:23832001

  1. Actin Cytoskeleton Manipulation by Effector Proteins Secreted by Diarrheagenic Escherichia coli Pathotypes

    PubMed Central

    Navarro-Garcia, Fernando; Serapio-Palacios, Antonio; Ugalde-Silva, Paul; Tapia-Pastrana, Gabriela; Chavez-Dueñas, Lucia

    2013-01-01

    The actin cytoskeleton is a dynamic structure necessary for cell and tissue organization, including the maintenance of epithelial barriers. Disruption of the epithelial barrier coincides with alterations of the actin cytoskeleton in several disease states. These disruptions primarily affect the paracellular space, which is normally regulated by tight junctions. Thereby, the actin cytoskeleton is a common and recurring target of bacterial virulence factors. In order to manipulate the actin cytoskeleton, bacteria secrete and inject toxins and effectors to hijack the host cell machinery, which interferes with host-cell pathways and with a number of actin binding proteins. An interesting model to study actin manipulation by bacterial effectors is Escherichia coli since due to its genome plasticity it has acquired diverse genetic mobile elements, which allow having different E. coli varieties in one bacterial species. These E. coli pathotypes, including intracellular and extracellular bacteria, interact with epithelial cells, and their interactions depend on a specific combination of virulence factors. In this paper we focus on E. coli effectors that mimic host cell proteins to manipulate the actin cytoskeleton. The study of bacterial effector-cytoskeleton interaction will contribute not only to the comprehension of the molecular causes of infectious diseases but also to increase our knowledge of cell biology. PMID:23509714

  2. A Novel Periplasmic Protein, VrpA, Contributes to Efficient Protein Secretion by the Type III Secretion System in Xanthomonas spp.

    PubMed

    Zhou, Xiaofeng; Hu, Xiufang; Li, Jinyun; Wang, Nian

    2015-02-01

    Efficient secretion of type III effector proteins from the bacterial cytoplasm to host cell cytosol via a type III secretion system (T3SS) is crucial for virulence of plant-pathogenic bacterium. Our previous study revealed a conserved hypothetical protein, virulence-related periplasm protein A (VrpA), which was identified as a critical virulence factor for Xanthomonas citri subsp. citri. In this study, we demonstrate that mutation of vrpA compromises X. citri subsp. citri virulence and hypersensitive response induction. This deficiency is also observed in the X. campestris pv. campestris strain, suggesting a functional conservation of VrpA in Xanthomonas spp. Our study indicates that VrpA is required for efficient protein secretion via T3SS, which is supported by multiple lines of evidence. A CyaA reporter assay shows that VrpA is involved in type III effector secretion; quantitative reverse-transcription polymerase chain reaction analysis suggests that the vrpA mutant fails to activate citrus-canker-susceptible gene CsLOB1, which is transcriptionally activated by transcription activator-like effector PthA4; in vitro secretion study reveals that VrpA plays an important role in secretion of T3SS pilus, translocon, and effector proteins. Our data also indicate that VrpA in X. citri subsp. citri localizes to bacterial periplasmic space and the periplasmic localization is required for full function of VrpA and X. citri subsp. citri virulence. Protein-protein interaction studies show that VrpA physically interacts with periplasmic T3SS components HrcJ and HrcC. However, the mutation of VrpA does not affect T3SS gene expression. Additionally, VrpA is involved in X. citri subsp. citri tolerance of oxidative stress. Our data contribute to the mechanical understanding of an important periplasmic protein VrpA in Xanthomonas spp. PMID:25338144

  3. SseBCD Proteins Are Secreted by the Type III Secretion System of Salmonella Pathogenicity Island 2 and Function as a Translocon

    PubMed Central

    Nikolaus, Thomas; Deiwick, Jörg; Rappl, Catherine; Freeman, Jeremy A.; Schröder, Werner; Miller, Samuel I.; Hensel, Michael

    2001-01-01

    The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI2) is required for systemic infections and intracellular accumulation of Salmonella enterica. This system is induced by intracellular Salmonella and subsequently transfers effector proteins into the host cell. Growth conditions either inducing expression of the type III secretion system or the secretion of substrate proteins were defined. Here we report the identification of a set of substrate proteins consisting of SseB, SseC, and SseD that are secreted by the SPI2 system in vitro. Secretion was observed if bacterial cells were exposed to acidic pH after growth in minimal medium with limitation of Mg2+ or phosphate. SseB, -C, and -D were isolated in a fraction detached from the bacterial cell surface by mechanical shearing, indicating that these proteins are predominantly assembled into complexes on the bacterial cell surface. The three proteins were required for the translocation of SPI2 effector proteins SspH1 and SspH2 into infected host cells. Thus, SseB, SseC, and SseD function as the translocon for effector proteins by intracellular Salmonella. PMID:11567004

  4. Metabolic Effectors Secreted by Bacterial Pathogens: Essential Facilitators of Plastid Endosymbiosis?[W][OA

    PubMed Central

    Ball, Steven G.; Subtil, Agathe; Bhattacharya, Debashish; Moustafa, Ahmed; Weber, Andreas P.M.; Gehre, Lena; Colleoni, Christophe; Arias, Maria-Cecilia; Cenci, Ugo; Dauvillée, David

    2013-01-01

    Under the endosymbiont hypothesis, over a billion years ago a heterotrophic eukaryote entered into a symbiotic relationship with a cyanobacterium (the cyanobiont). This partnership culminated in the plastid that has spread to forms as diverse as plants and diatoms. However, why primary plastid acquisition has not been repeated multiple times remains unclear. Here, we report a possible answer to this question by showing that primary plastid endosymbiosis was likely to have been primed by the secretion in the host cytosol of effector proteins from intracellular Chlamydiales pathogens. We provide evidence suggesting that the cyanobiont might have rescued its afflicted host by feeding photosynthetic carbon into a chlamydia-controlled assimilation pathway. PMID:23371946

  5. RNA Type III Secretion Signals that require Hfq

    SciTech Connect

    Niemann, George; Brown, Roslyn N.; Mushamiri, Ivy T.; Nguyen, Nhu T.; Taiwo, Rukayat; Stufkens, Afke; Smith, Richard D.; Adkins, Joshua N.; McDermott, Jason E.; Heffron, Fred

    2013-05-01

    effector proteins from the bacterium to a host cell; however, the secretion signal is poorly defined. Effector N-termini are thought to contain the signal, but they lack homology, possess no identifiable motif, and adopt intrinsically disordered structures. We identified a panel of RNA secretion signals that facilitated reporter translocation into host cells via a mechanism dependent upon the RNA chaperone Hfq. Each of these signals was localized to an RNA leader sequence preceding the translational start codon. To obtain this panel of RNA signals, we fused untranslated leader sequences from 42 different Salmonella effector proteins to the adenylate cyclase reporter (CyaA'), and tested each of them for translocation into J774 macrophages. RNA sequences derived from five effectors, gtgA, cigR, gogB, sseL, and steD were sufficient for CyaA' injection into host cells. The gtgA RNA also directed translocation of the β-lactamase reporter. To determine the mechanism of signal recognition, we identified proteins that bound specifically to the gtgA RNA. One of the unique proteins identified was Hfq. Translocation of all five UTR fusions was abolished in the Hfq mutant, confirming the importance of Hfq. Our results suggest that Hfq may direct a subset of RNA transcripts to the T3S apparatus for translation and secretion. Signal diversity may explain why the T3S signal has been difficult to define.

  6. Dynamics of the Type III Secretion System Activity of Enteropathogenic Escherichia coli

    PubMed Central

    Mills, Erez; Baruch, Kobi; Aviv, Gili; Nitzan, Mor; Rosenshine, Ilan

    2013-01-01

    ABSTRACT Type III secretion systems (TTSSs) are employed by pathogens to translocate host cells with effector proteins, which are crucial for virulence. The dynamics of effector translocation, behavior of the translocating bacteria, translocation temporal order, and relative amounts of each of the translocated effectors are all poorly characterized. To address these issues, we developed a microscopy-based assay that tracks effector translocation. We used this assay alongside a previously described real-time population-based translocation assay, focusing mainly on enteropathogenic Escherichia coli (EPEC) and partly comparing it to Salmonella. We found that the two pathogens exhibit different translocation behaviors: in EPEC, a subpopulation that formed microcolonies carried out most of the translocation activity, while Salmonella executed protein translocation as planktonic bacteria. We also noted variability in host cell susceptibility, with some cells highly resistant to translocation. We next extended the study to determine the translocation dynamics of twenty EPEC effectors and found that all exhibited distinct levels of translocation efficiency. Further, we mapped the global effects of key TTSS-related components on TTSS activity. Our results provide a comprehensive description of the dynamics of the TTSS activity of EPEC and new insights into the mechanisms that control the dynamics. PMID:23900171

  7. Hfq negatively regulates type III secretion in EHEC and several other pathogens

    PubMed Central

    Shakhnovich, Elizabeth A.; Davis, Brigid M.; Waldor, Matthew K.

    2009-01-01

    Summary Hfq is a conserved RNA-binding protein that regulates diverse cellular processes through post-transcriptional control of gene expression, often by functioning as a chaperone for regulatory sRNAs. Here, we explored the role of Hfq in enterohaemorrhagic E. coli (EHEC), a group of non-invasive intestinal pathogens. EHEC virulence is dependent on a Type III secretion system encoded in the LEE pathogenicity island. The abundance of transcripts for all 41 LEE genes and more than half of confirmed non-LEE-encoded T3 effectors were elevated in an EHEC hfq deletion mutant. Thus, Hfq promotes coordinated expression of the LEE-encoded T3S apparatus and both LEE- and non-LEE-encoded effectors. Increased transcript levels led to the formation of functional secretion complexes capable of secreting high quantities of effectors into the supernatant. The increase in LEE-derived transcripts and proteins was dependent on Ler, the LEE-encoded transcriptional activator, and the ler transcript appears to be a direct target of Hfq-mediated negative regulation. Finally, we found that Hfq contributes to the negative regulation of T3SSs in several other pathogens, suggesting that Hfq, potentially along with species-specific sRNAs, underlies a common means to prevent unfettered expression of T3SSs. PMID:19703108

  8. Secreted effectors in Toxoplasma gondii and related species: determinants of host range and pathogenesis?

    PubMed Central

    English, E D; Adomako-Ankomah, Y; Boyle, J P

    2015-01-01

    Recent years have witnessed the discovery of a number of secreted proteins in Toxoplasma gondii that play important roles in host–pathogen interactions and parasite virulence, particularly in the mouse model. However, the role that these proteins play in driving the unique features of T. gondii compared to some of its nearest apicomplexan relatives (Hammondia hammondi and Neospora caninum) is unknown. These unique features include distinct dissemination characteristics in vivo and a vast host range. In this review we comprehensively survey what is known about disease outcome, the host response and host range for T. gondii, H. hammondi, and N. caninum. We then review what is presently known about recently identified secreted virulence effectors in these three genetically related, but phenotypically distinct, species. Finally we exploit the existence of genome sequences for these three organisms and discuss what is known about the presence, and functionality, of key T. gondii effectors in these three species. PMID:25655311

  9. Secretome Analysis of Vibrio cholerae Type VI Secretion System Reveals a New Effector-Immunity Pair

    PubMed Central

    Altindis, Emrah; Dong, Tao; Catalano, Christy

    2015-01-01

    ABSTRACT The type VI secretion system (T6SS) is a dynamic macromolecular organelle that many Gram-negative bacteria use to inhibit or kill other prokaryotic or eukaryotic cells. The toxic effectors of T6SS are delivered to the prey cells in a contact-dependent manner. In Vibrio cholerae, the etiologic agent of cholera, T6SS is active during intestinal infection. Here, we describe the use of comparative proteomics coupled with bioinformatics to identify a new T6SS effector-immunity pair. This analysis was able to identify all previously identified secreted substrates of T6SS except PAAR (proline, alanine, alanine, arginine) motif-containing proteins. Additionally, this approach led to the identification of a new secreted protein encoded by VCA0285 (TseH) that carries a predicted hydrolase domain. We confirmed that TseH is toxic when expressed in the periplasm of Escherichia coli and V. cholerae cells. The toxicity observed in V. cholerae was suppressed by coexpression of the protein encoded by VCA0286 (TsiH), indicating that this protein is the cognate immunity protein of TseH. Furthermore, exogenous addition of purified recombinant TseH to permeabilized E. coli cells caused cell lysis. Bioinformatics analysis of the TseH protein sequence suggest that it is a member of a new family of cell wall-degrading enzymes that include proteins belonging to the YD repeat and Rhs superfamilies and that orthologs of TseH are likely expressed by species belonging to phyla as diverse as Bacteroidetes and Proteobacteria. PMID:25759499

  10. [The lethiferous journey of a bacterium--the research progress of secretion systems and effectors in Legionella pneumophila].

    PubMed

    Lu, Yong-Jun; Li, Xiang-Hui; Zeng, Yong-Lun

    2011-10-01

    Legionella pneumophila is the intracellular bacterial pathogen that causes severe Legionnaires' disease and flu-like Pontiac fever. To accomplish successful aggression against hosts, L. pneumophila secrets more than 150 kinds of substrate effector proteins into host cells via its Type IVB secretion system. With the multiple functions of effectors, L. pneumophila evades effectively the defense systems of hosts, converts or adjusts intracellular vesicular transport of hosts, modifies or disguises its Legionella containing vacuole (LCV), modulates the cell cycle program and inhibits the apoptosis of host cells, and finally gains the comfortable intracellular replicative niche. Effectors can also help L. pneumophila escape from hosts cells after completing the proliferation.. L. pneumophila has became the distinct model for pathogen-host interaction research, and its secretion systems as well as the substrate effectors are attracting more and more attentions. Researching on T4BSS and effectors could not only help investigate the pathogenesis of intracellular bacterial pathogens, but also promote the comprehension about innate immune responses of hosts. This article reviews the progresses of L. pneumophila T4BSS and effectors, trying to demonstrate to the readers the cunning survival strategy and the delicate virulent machine of L. pneumophila. PMID:21993284

  11. Mutualistic Co-evolution of Type III Effector Genes in Sinorhizobium fredii and Bradyrhizobium japonicum

    PubMed Central

    Jiang, Yuan; Creason, Allison L.; Thireault, Caitlin A.; Sachs, Joel L.; Chang, Jeff H.

    2013-01-01

    Two diametric paradigms have been proposed to model the molecular co-evolution of microbial mutualists and their eukaryotic hosts. In one, mutualist and host exhibit an antagonistic arms race and each partner evolves rapidly to maximize their own fitness from the interaction at potential expense of the other. In the opposing model, conflicts between mutualist and host are largely resolved and the interaction is characterized by evolutionary stasis. We tested these opposing frameworks in two lineages of mutualistic rhizobia, Sinorhizobium fredii and Bradyrhizobium japonicum. To examine genes demonstrably important for host-interactions we coupled the mining of genome sequences to a comprehensive functional screen for type III effector genes, which are necessary for many Gram-negative pathogens to infect their hosts. We demonstrate that the rhizobial type III effector genes exhibit a surprisingly high degree of conservation in content and sequence that is in contrast to those of a well characterized plant pathogenic species. This type III effector gene conservation is particularly striking in the context of the relatively high genome-wide diversity of rhizobia. The evolution of rhizobial type III effectors is inconsistent with the molecular arms race paradigm. Instead, our results reveal that these loci are relatively static in rhizobial lineages and suggest that fitness conflicts between rhizobia mutualists and their host plants have been largely resolved. PMID:23468637

  12. The Rab11 Effector Protein FIP1 Regulates Adiponectin Trafficking and Secretion

    PubMed Central

    Moreno-Navarrete, Jose Maria; Fernandez-Real, Jose Manuel; Mora, Silvia

    2013-01-01

    Adiponectin is an adipokine secreted by white adipocytes involved in regulating insulin sensitivity in peripheral tissues. Secretion of adiponectin in adipocytes relies on the endosomal system, however, the intracellular machinery involved in mediating adiponectin release is unknown. We have previously reported that intracellular adiponectin partially compartmentalizes with rab 5 and rab11, markers for the early/sorting and recycling compartments respectively. Here we have examined the role of several rab11 downstream effector proteins (rab11 FIPs) in regulating adiponectin trafficking and secretion. Overexpression of wild type rab11 FIP1, FIP3 and FIP5 decreased the amount of secreted adiponectin expressed in HEK293 cells, whereas overexpression of rab11 FIP2 or FIP4 had no effect. Furthermore shRNA-mediated depletion of FIP1 enhanced adiponectin release whereas knock down of FIP5 decreased adiponectin secretion. Knock down of FIP3 had no effect. In 3T3L1 adipocytes, endogenous FIP1 co-distributed intracellularly with endogenous adiponectin and FIP1 depletion enhanced adiponectin release without altering insulin-mediated trafficking of the glucose transporter Glut4. While adiponectin receptors internalized with transferrin receptors, there were no differences in transferrin receptor recycling between wild type and FIP1 depleted adipocytes. Consistent with its inhibitory role, FIP1 expression was decreased during adipocyte differentiation, by treatment with thiazolidinediones, and with increased BMI in humans. In contrast, FIP1 expression increased upon exposure of adipocytes to TNFα. In all, our findings identify FIP1 as a novel protein involved in the regulation of adiponectin trafficking and release. PMID:24040321

  13. Prophage-Encoded Peroxidase in 'Candidatus Liberibacter asiaticus' Is a Secreted Effector That Suppresses Plant Defenses.

    PubMed

    Jain, Mukesh; Fleites, Laura A; Gabriel, Dean W

    2015-12-01

    'Candidatus Liberibacter asiaticus' is transmitted by psyllids and causes huanglongbing (HLB), a lethal disease of citrus. Most pathogenic 'Ca. L. asiaticus' strains carry two nearly identical prophages similar to SC1 and SC2 in strain UF506. SC2 was observed to replicate as a moderately high-copy excision plasmid encoding a reactive oxygen species-scavenging peroxidase (SC2_gp095), a predicted lysogenic conversion factor. SC2_gp095 was expressed at significantly higher levels in periwinkle than in citrus and was suppressed in psyllids. SC2_gp095 was cloned in a shuttle vector and transformed into Escherichia coli and Liberibacter crescens, a culturable proxy for 'Ca. L. asiaticus'. Transformed L. crescens cells showed 20 to 25% enhanced resistance to H₂O₂on agar plates, 47% greater enzymatic activity, and enhanced growth in liquid cultures. A nonclassical secretion potential was predicted for SC2_gp095 and secretion from L. crescens was confirmed by enzymatic and Western blot analyses. Transient expression of SC2_gp095 in planta resulted in strong transcriptional downregulation of RbohB, the key gatekeeper of the H₂O₂-mediated defense signaling in plants, helping explain the surprisingly long incubation period (years) before HLB symptoms appear in 'Ca. L. asiaticus'-infected citrus. 'Ca. L. asiaticus' peroxidase is likely a secreted, horizontally acquired effector that suppresses host symptom development, a tactic used by most biotrophic plant pathogens. PMID:26313412

  14. a Computational Approach to Explore Protein Translocation Through Type III Secretion Apparatus

    NASA Astrophysics Data System (ADS)

    Rathinavelan, Thenmalarchelvi; Im, Wonpil

    2010-01-01

    Many Gram-negative bacteria initiate infections by injecting effector proteins into host cells through the type III secretion apparatus (TTSA) that is comprised of a basal body, a needle, and a tip. The needle channel is formed by the assembly of a single needle protein. To explore the export mechanisms of MxiH needle protein through the needle of Shigella flexneri, an essential step during needle assembly, we have performed steered molecular dynamics simulations in implicit solvent. Interestingly, the electronegative channel interior creates an energy barrier for MxiH to enter the channel, while the same may facilitate the ejection of the effectors into host cells. Structurally-known basal regions and ATPase underneath the basal region have also such electronegative interior, while effector proteins have considerable electronegative patches on their surfaces. Based on these observations, we propose a repulsive electrostatic mechanism for protein translocation through the TTSA. This mechanism is supported by the suggestion that an ATPase is required for protein translocation through these nanomachines, which may provide the energy to overcome the initial electrostatic energy barrier. A similar mechanism may be applicable to macromolecular channels in other secretion systems or viruses through which proteins or nucleic acids are transported.

  15. Type III secretion: a bacterial device for close combat with cells of their eukaryotic host.

    PubMed

    Cornelis, G R

    2000-05-29

    Salmonella, Shigella, Yersinia, Pseudomonas aeruginosa, enteropathogenic Escherichia coli and several plant-pathogenic Gram-negative bacteria use a new type of systems called 'type III secretion' to attack their host. These systems are activated by contact with a eukaryotic cell membrane and they allow bacteria to inject bacterial proteins across the two bacterial membranes and the eukaryotic cell membrane to reach a given compartment and destroy or subvert the target cell. These systems consist of a secretion apparatus made up of about 25 individual proteins and a set of proteins released by this apparatus. Some of these released proteins are 'effectors' that are delivered by extracellular bacteria into the cytosol of the target cell while the others are 'translocators' that help the 'effectors' to cross the membrane of the eukaryotic cell. Most of the 'effectors' act on the cytoskeleton or on intracellular signalling cascades. One of the proteins injected by the enteropathogenic E. coli serves as a membrane receptor for the docking of the bacterium itself at the surface of the cell. PMID:10874740

  16. Interactions of Xanthomonas type-III effector proteins with the plant ubiquitin and ubiquitin-like pathways

    PubMed Central

    Üstün, Suayib; Börnke, Frederik

    2014-01-01

    In eukaryotes, regulated protein turnover is required during many cellular processes, including defense against pathogens. Ubiquitination and degradation of ubiquitinated proteins via the ubiquitin–proteasome system (UPS) is the main pathway for the turnover of intracellular proteins in eukaryotes. The extensive utilization of the UPS in host cells makes it an ideal pivot for the manipulation of cellular processes by pathogens. Like many other Gram-negative bacteria, Xanthomonas species secrete a suite of type-III effector proteins (T3Es) into their host cells to promote virulence. Some of these T3Es exploit the plant UPS to interfere with immunity. This review summarizes T3E examples from the genus Xanthomonas with a proven or suggested interaction with the host UPS or UPS-like systems and also discusses the apparent paradox that arises from the presence of T3Es that inhibit the UPS in general while others rely on its activity for their function. PMID:25566304

  17. Secretion of Rhoptry and Dense Granule Effector Proteins by Nonreplicating Toxoplasma gondii Uracil Auxotrophs Controls the Development of Antitumor Immunity.

    PubMed

    Fox, Barbara A; Sanders, Kiah L; Rommereim, Leah M; Guevara, Rebekah B; Bzik, David J

    2016-07-01

    Nonreplicating type I uracil auxotrophic mutants of Toxoplasma gondii possess a potent ability to activate therapeutic immunity to established solid tumors by reversing immune suppression in the tumor microenvironment. Here we engineered targeted deletions of parasite secreted effector proteins using a genetically tractable Δku80 vaccine strain to show that the secretion of specific rhoptry (ROP) and dense granule (GRA) proteins by uracil auxotrophic mutants of T. gondii in conjunction with host cell invasion activates antitumor immunity through host responses involving CD8α+ dendritic cells, the IL-12/interferon-gamma (IFN-γ) TH1 axis, as well as CD4+ and CD8+ T cells. Deletion of parasitophorous vacuole membrane (PVM) associated proteins ROP5, ROP17, ROP18, ROP35 or ROP38, intravacuolar network associated dense granule proteins GRA2 or GRA12, and GRA24 which traffics past the PVM to the host cell nucleus severely abrogated the antitumor response. In contrast, deletion of other secreted effector molecules such as GRA15, GRA16, or ROP16 that manipulate host cell signaling and transcriptional pathways, or deletion of PVM associated ROP21 or GRA3 molecules did not affect the antitumor activity. Association of ROP18 with the PVM was found to be essential for the development of the antitumor responses. Surprisingly, the ROP18 kinase activity required for resistance to IFN-γ activated host innate immunity related GTPases and virulence was not essential for the antitumor response. These data show that PVM functions of parasite secreted effector molecules, including ROP18, manipulate host cell responses through ROP18 kinase virulence independent mechanisms to activate potent antitumor responses. Our results demonstrate that PVM associated rhoptry effector proteins secreted prior to host cell invasion and dense granule effector proteins localized to the intravacuolar network and host nucleus that are secreted after host cell invasion coordinately control the

  18. Secretion of Rhoptry and Dense Granule Effector Proteins by Nonreplicating Toxoplasma gondii Uracil Auxotrophs Controls the Development of Antitumor Immunity

    PubMed Central

    Fox, Barbara A.; Sanders, Kiah L.; Rommereim, Leah M.; Bzik, David J.

    2016-01-01

    Nonreplicating type I uracil auxotrophic mutants of Toxoplasma gondii possess a potent ability to activate therapeutic immunity to established solid tumors by reversing immune suppression in the tumor microenvironment. Here we engineered targeted deletions of parasite secreted effector proteins using a genetically tractable Δku80 vaccine strain to show that the secretion of specific rhoptry (ROP) and dense granule (GRA) proteins by uracil auxotrophic mutants of T. gondii in conjunction with host cell invasion activates antitumor immunity through host responses involving CD8α+ dendritic cells, the IL-12/interferon-gamma (IFN-γ) TH1 axis, as well as CD4+ and CD8+ T cells. Deletion of parasitophorous vacuole membrane (PVM) associated proteins ROP5, ROP17, ROP18, ROP35 or ROP38, intravacuolar network associated dense granule proteins GRA2 or GRA12, and GRA24 which traffics past the PVM to the host cell nucleus severely abrogated the antitumor response. In contrast, deletion of other secreted effector molecules such as GRA15, GRA16, or ROP16 that manipulate host cell signaling and transcriptional pathways, or deletion of PVM associated ROP21 or GRA3 molecules did not affect the antitumor activity. Association of ROP18 with the PVM was found to be essential for the development of the antitumor responses. Surprisingly, the ROP18 kinase activity required for resistance to IFN-γ activated host innate immunity related GTPases and virulence was not essential for the antitumor response. These data show that PVM functions of parasite secreted effector molecules, including ROP18, manipulate host cell responses through ROP18 kinase virulence independent mechanisms to activate potent antitumor responses. Our results demonstrate that PVM associated rhoptry effector proteins secreted prior to host cell invasion and dense granule effector proteins localized to the intravacuolar network and host nucleus that are secreted after host cell invasion coordinately control the

  19. Intracellular Growth of Bacterial Pathogens: The Role of Secreted Effector Proteins in the Control of Phagocytosed Microorganisms.

    PubMed

    Poirier, Valérie; Av-Gay, Yossef

    2015-12-01

    The ability of intracellular pathogens to subvert the host response, to facilitate invasion and subsequent infection, is the hallmark of microbial pathogenesis. Bacterial pathogens produce and secrete a variety of effector proteins, which are the primary means by which they exert control over the host cell. Secreted effectors work independently, yet in concert with each other, to facilitate microbial invasion, replication, and intracellular survival in host cells. In this review we focus on defined host cell processes targeted by bacterial pathogens. These include phagosome maturation and its subprocesses: phagosome-endosome and phagosome-lysosome fusion events, as well as phagosomal acidification, cytoskeleton remodeling, and lysis of the phagosomal membrane. We further describe the mode of action for selected effectors from six pathogens: the Gram-negative Legionella, Salmonella, Shigella, and Yersinia, the Gram-positive Listeria, and the acid-fast actinomycete Mycobacterium. PMID:27337278

  20. Inhibition of inflammasome activation by Coxiella burnetii type IV secretion system effector IcaA

    PubMed Central

    Cunha, Larissa D.; Ribeiro, Juliana M.; Fernandes, Talita D.; Massis, Liliana M.; Khoo, Chen Ai; Moffatt, Jennifer H.; Newton, Hayley J.; Roy, Craig R.; Zamboni, Dario S.

    2015-01-01

    Coxiella burnetii is a highly infectious bacterium that promotes its own replication in macrophages by inhibiting several host cell responses. Here, we show that C. burnetii inhibits caspase-1 activation in primary mouse macrophages. By using co-infection experiments, we determine that the infection of macrophages with C. burnetii inhibits the caspase-11-mediated non-canonical activation of the NLRP3 inflammasome induced by subsequent infection with Escherichia coli or Legionella pneumophila. Genetic screening using flagellin mutants of L. pneumophila as a surrogate host, reveals a novel C. burnetii gene (IcaA) involved in the inhibition of caspase activation. Expression of IcaA in L. pneumophila inhibited the caspase-11 activation in macrophages. Moreover, icaA- mutants of C. burnetii failed to suppress the caspase-11-mediated inflammasome activation induced by L. pneumophila. Our data reveal IcaA as a novel C. burnetii effector protein that is secreted by the Dot/Icm type IV secretion system and interferes with the caspase-11-induced, non-canonical activation of the inflammasome. PMID:26687278

  1. Searching algorithm for type IV secretion system effectors 1.0: a tool for predicting type IV effectors and exploring their genomic context.

    PubMed

    Meyer, Damien F; Noroy, Christophe; Moumène, Amal; Raffaele, Sylvain; Albina, Emmanuel; Vachiéry, Nathalie

    2013-11-01

    Type IV effectors (T4Es) are proteins produced by pathogenic bacteria to manipulate host cell gene expression and processes, divert the cell machinery for their own profit and circumvent the immune responses. T4Es have been characterized for some bacteria but many remain to be discovered. To help biologists identify putative T4Es from the complete genome of α- and γ-proteobacteria, we developed a Perl-based command line bioinformatics tool called S4TE (searching algorithm for type-IV secretion system effectors). The tool predicts and ranks T4E candidates by using a combination of 13 sequence characteristics, including homology to known effectors, homology to eukaryotic domains, presence of subcellular localization signals or secretion signals, etc. S4TE software is modular, and specific motif searches are run independently before ultimate combination of the outputs to generate a score and sort the strongest T4Es candidates. The user keeps the possibility to adjust various searching parameters such as the weight of each module, the selection threshold or the input databases. The algorithm also provides a GC% and local gene density analysis, which strengthen the selection of T4E candidates. S4TE is a unique predicting tool for T4Es, finding its utility upstream from experimental biology. PMID:23945940

  2. The Type III secretion system of Gram-negative bacteria: a potential therapeutic target?

    PubMed

    Müller, Simone; Feldman, Mario F; Cornelis, Guy R

    2001-06-01

    Several pathogenic Gram-negative bacteria, including Salmonella, Shigella, Yersinia, Pseudomonas aeruginosa and enteropathogenic Escherichia coli harbour a complex attack system called 'Type III secretion' which is, in every case, an essential virulence determinant. This system, activated by contact with an eukaryotic cell membrane, allows bacteria to inject bacterial proteins across the two bacterial membranes and the eukaryotic cell membrane, to reach the cell's cytosol and destroy or subvert the host cell. The Type III virulence mechanism consists of a secretion apparatus, made up of about 25 proteins, and a set of effector proteins released by this apparatus. The mechanism of protein secretion is highly conserved among the different bacteria, although they cause a variety of diseases with different symptoms and severities, from fatal septicaemia to mild diarrhoea or from fulgurant diarrhoea to chronic infection of the lung. This review focuses on the proteins that make up the secretion machinery and examine if it could be a potential target for novel antimicrobials. PMID:12540268

  3. RNA-activated DNA cleavage by the Type III-B CRISPR-Cas effector complex.

    PubMed

    Estrella, Michael A; Kuo, Fang-Ting; Bailey, Scott

    2016-02-15

    The CRISPR (clustered regularly interspaced short palindromic repeat) system is an RNA-guided immune system that protects prokaryotes from invading genetic elements. This system represents an inheritable and adaptable immune system that is mediated by multisubunit effector complexes. In the Type III-B system, the Cmr effector complex has been found to cleave ssRNA in vitro. However, in vivo, it has been implicated in transcription-dependent DNA targeting. We show here that the Cmr complex from Thermotoga maritima can cleave an ssRNA target that is complementary to the CRISPR RNA. We also show that binding of a complementary ssRNA target activates an ssDNA-specific nuclease activity in the histidine-aspartate (HD) domain of the Cmr2 subunit of the complex. These data suggest a mechanism for transcription-coupled DNA targeting by the Cmr complex and provide a unifying mechanism for all Type III systems. PMID:26848046

  4. The type III secretion system apparatus determines the intracellular niche of bacterial pathogens.

    PubMed

    Du, Juan; Reeves, Analise Z; Klein, Jessica A; Twedt, Donna J; Knodler, Leigh A; Lesser, Cammie F

    2016-04-26

    Upon entry into host cells, intracellular bacterial pathogens establish a variety of replicative niches. Although some remodel phagosomes, others rapidly escape into the cytosol of infected cells. Little is currently known regarding how professional intracytoplasmic pathogens, including Shigella, mediate phagosomal escape. Shigella, like many other Gram-negative bacterial pathogens, uses a type III secretion system to deliver multiple proteins, referred to as effectors, into host cells. Here, using an innovative reductionist-based approach, we demonstrate that the introduction of a functional Shigella type III secretion system, but none of its effectors, into a laboratory strain of Escherichia coli is sufficient to promote the efficient vacuole lysis and escape of the modified bacteria into the cytosol of epithelial cells. This establishes for the first time, to our knowledge, a direct physiologic role for the Shigella type III secretion apparatus (T3SA) in mediating phagosomal escape. Furthermore, although protein components of the T3SA share a moderate degree of structural and functional conservation across bacterial species, we show that vacuole lysis is not a common feature of T3SA, as an effectorless strain of Yersinia remains confined to phagosomes. Additionally, by exploiting the functional interchangeability of the translocator components of the T3SA of Shigella, Salmonella, and Chromobacterium, we demonstrate that a single protein component of the T3SA translocon-Shigella IpaC, Salmonella SipC, or Chromobacterium CipC-determines the fate of intracellular pathogens within both epithelial cells and macrophages. Thus, these findings have identified a likely paradigm by which the replicative niche of many intracellular bacterial pathogens is established. PMID:27078095

  5. Using Transcriptional Control To Increase Titers of Secreted Heterologous Proteins by the Type III Secretion System

    PubMed Central

    Metcalf, Kevin J.; Finnerty, Casey; Azam, Anum; Valdivia, Elias

    2014-01-01

    The type III secretion system (T3SS) encoded at the Salmonella pathogenicity island 1 (SPI-1) locus secretes protein directly from the cytosol to the culture media in a concerted, one-step process, bypassing the periplasm. While this approach is attractive for heterologous protein production, product titers are too low for many applications. In addition, the expression of the SPI-1 gene cluster is subject to native regulation, which requires culturing conditions that are not ideal for high-density growth. We used transcriptional control to increase the amount of protein that is secreted into the extracellular space by the T3SS of Salmonella enterica. The controlled expression of the gene encoding SPI-1 transcription factor HilA circumvents the requirement of endogenous induction conditions and allows for synthetic induction of the secretion system. This strategy increases the number of cells that express SPI-1 genes, as measured by promoter activity. In addition, protein secretion titer is sensitive to the time of addition and the concentration of inducer for the protein to be secreted and SPI-1 gene cluster. Overexpression of hilA increases secreted protein titer by >10-fold and enables recovery of up to 28 ± 9 mg/liter of secreted protein from an 8-h culture. We also demonstrate that the protein beta-lactamase is able to adopt an active conformation after secretion, and the increase in secreted titer from hilA overexpression also correlates to increased enzyme activity in the culture supernatant. PMID:25038096

  6. Colletotrichum orbiculare Secretes Virulence Effectors to a Biotrophic Interface at the Primary Hyphal Neck via Exocytosis Coupled with SEC22-Mediated Traffic[W

    PubMed Central

    Irieda, Hiroki; Maeda, Hitomi; Akiyama, Kaoru; Hagiwara, Asuka; Saitoh, Hiromasa; Uemura, Aiko; Terauchi, Ryohei; Takano, Yoshitaka

    2014-01-01

    The hemibiotrophic pathogen Colletotrichum orbiculare develops biotrophic hyphae inside cucumber (Cucumis sativus) cells via appressorial penetration; later, the pathogen switches to necrotrophy. C. orbiculare also expresses specific effectors at different stages. Here, we found that virulence-related effectors of C. orbiculare accumulate in a pathogen–host biotrophic interface. Fluorescence-tagged effectors accumulated in a ring-like region around the neck of the biotrophic primary hyphae. Fluorescence imaging of cellular components and transmission electron microscopy showed that the ring-like signals of the effectors localized at the pathogen–plant interface. Effector accumulation at the interface required induction of its expression during the early biotrophic phase, suggesting that transcriptional regulation may link to effector localization. We also investigated the route of effector secretion to the interface. An exocytosis-related component, the Rab GTPase SEC4, localized to the necks of biotrophic primary hyphae adjacent to the interface, thereby suggesting focal effector secretion. Disruption of SEC4 in C. orbiculare reduced virulence and impaired effector delivery to the ring signal interface. Disruption of the v-SNARE SEC22 also reduced effector delivery. These findings suggest that biotrophy-expressed effectors are secreted, via the endoplasmic reticulum-to-Golgi route and subsequent exocytosis, toward the interface generated between C. orbiculare and the host cell. PMID:24850852

  7. Structure of a bacterial type III secretion system in contact with a host membrane in situ

    NASA Astrophysics Data System (ADS)

    Nans, Andrea; Kudryashev, Mikhail; Saibil, Helen R.; Hayward, Richard D.

    2015-12-01

    Many bacterial pathogens of animals and plants use a conserved type III secretion system (T3SS) to inject virulence effector proteins directly into eukaryotic cells to subvert host functions. Contact with host membranes is critical for T3SS activation, yet little is known about T3SS architecture in this state or the conformational changes that drive effector translocation. Here we use cryo-electron tomography and sub-tomogram averaging to derive the intact structure of the primordial Chlamydia trachomatis T3SS in the presence and absence of host membrane contact. Comparison of the averaged structures demonstrates a marked compaction of the basal body (4 nm) occurs when the needle tip contacts the host cell membrane. This compaction is coupled to a stabilization of the cytosolic sorting platform-ATPase. Our findings reveal the first structure of a bacterial T3SS from a major human pathogen engaged with a eukaryotic host, and reveal striking `pump-action' conformational changes that underpin effector injection.

  8. Structure of a bacterial type III secretion system in contact with a host membrane in situ

    PubMed Central

    Nans, Andrea; Kudryashev, Mikhail; Saibil, Helen R.; Hayward, Richard D.

    2015-01-01

    Many bacterial pathogens of animals and plants use a conserved type III secretion system (T3SS) to inject virulence effector proteins directly into eukaryotic cells to subvert host functions. Contact with host membranes is critical for T3SS activation, yet little is known about T3SS architecture in this state or the conformational changes that drive effector translocation. Here we use cryo-electron tomography and sub-tomogram averaging to derive the intact structure of the primordial Chlamydia trachomatis T3SS in the presence and absence of host membrane contact. Comparison of the averaged structures demonstrates a marked compaction of the basal body (4 nm) occurs when the needle tip contacts the host cell membrane. This compaction is coupled to a stabilization of the cytosolic sorting platform–ATPase. Our findings reveal the first structure of a bacterial T3SS from a major human pathogen engaged with a eukaryotic host, and reveal striking ‘pump-action' conformational changes that underpin effector injection. PMID:26656452

  9. T3DB: an integrated database for bacterial type III secretion system

    PubMed Central

    2012-01-01

    Background Type III Secretion System (T3SS), which plays important roles in pathogenesis or symbiosis, is widely expressed in a variety of gram negative bacteria. However, lack of unique nomenclature for T3SS genes has hindered T3SS related research. It is necessary to set up a knowledgebase integrating T3SS-related research data to facilitate the communication between different research groups interested in different bacteria. Description A T3SS-related Database (T3DB) was developed. T3DB serves as an integrated platform for sequence collection, function annotation, and ortholog classification for T3SS related apparatus, effector, chaperone and regulatory genes. The collection of T3SS-containing bacteria, T3SS-related genes, function annotation, and the ortholog information were all manually curated from literature. BPBAac, a highly efficient T3SS effector prediction tool, was also implemented. Conclusions T3DB is the first systematic platform integrating well-annotated T3SS-related gene and protein information to facilitate T3SS and bacterial pathogenecity related research. The newly constructed T3 ortholog clusters may faciliate effective communication between different research groups and will promote de novo discoveries. Besides, the manually-curated high-quality effector and chaperone data are useful for feature analysis and evolutionary studies of these important proteins. PMID:22545727

  10. EXTRACELLULAR PROTEINS INVOLVED IN SOYBEAN CULTIVAR-SPECIFIC NODULATION ARE ASSOCIATED WITH PILUS-LIKE SURFACE APPENDANGES AND EXPORTED BY A TYPE III PROTEIN SECRETION SYSTEM IN SINORHIZOBIUM FREDII USDA257

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several Gram-negative plant and animal pathogenic bacteria have evolved a type III secretion system (TTSS) to deliver effector proteins directly into the host cell cytosol. Sinorhizobium fredii USDA257, a symbiont of soybean and many other legumes, secretes signal-responsive proteins (SR proteins) ...

  11. Bacterial type III secretion systems: specialized nanomachines for protein delivery into target cells

    PubMed Central

    Galán, Jorge E.; Lara-Tejero, Maria; Marlovits, Thomas C.; Wagner, Samuel

    2015-01-01

    One of the most exciting developments in the field of bacterial pathogenesis in recent years is the discovery that many pathogens utilized complex nanomachines to deliver bacterially encoded effector proteins into target eukaryotic cells. These effector proteins modulate a variety of cellular functions for the pathogen’s benefit. One of these protein-delivery machines is the type III secretion system (T3SS). T3SSs are widespread in nature and are encoded not only by bacteria pathogenic to vertebrates or plants, but also by bacteria that are symbiotic to plants or insects. A central component of T3SSs is the needle complex, a supramolecular structure that mediates the passage of the secreted proteins across the bacterial envelope. Working in conjunction with several cytoplasmic components, the needle complex engages specific substrates in sequential order, moves them across the bacterial envelope, and ultimately delivers them into eukaryotic cells. The central role of T3SSs in pathogenesis makes them great targets for novel antimicrobial strategies. PMID:25002086

  12. Yeast as a Heterologous Model System to Uncover Type III Effector Function

    PubMed Central

    Popa, Crina; Coll, Núria S.; Valls, Marc; Sessa, Guido

    2016-01-01

    Type III effectors (T3E) are key virulence proteins that are injected by bacterial pathogens inside the cells of their host to subvert cellular processes and contribute to disease. The budding yeast Saccharomyces cerevisiae represents an important heterologous system for the functional characterisation of T3E proteins in a eukaryotic environment. Importantly, yeast contains eukaryotic processes with low redundancy and are devoid of immunity mechanisms that counteract T3Es and mask their function. Expression in yeast of effectors from both plant and animal pathogens that perturb conserved cellular processes often resulted in robust phenotypes that were exploited to elucidate effector functions, biochemical properties, and host targets. The genetic tractability of yeast and its amenability for high-throughput functional studies contributed to the success of this system that, in recent years, has been used to study over 100 effectors. Here, we provide a critical view on this body of work and describe advantages and limitations inherent to the use of yeast in T3E research. “Favourite” targets of T3Es in yeast are cytoskeleton components and small GTPases of the Rho family. We describe how mitogen-activated protein kinase (MAPK) signalling, vesicle trafficking, membrane structures, and programmed cell death are also often altered by T3Es in yeast and how this reflects their function in the natural host. We describe how effector structure–function studies and analysis of candidate targeted processes or pathways can be carried out in yeast. We critically analyse technologies that have been used in yeast to assign biochemical functions to T3Es, including transcriptomics and proteomics, as well as suppressor, gain-of-function, or synthetic lethality screens. We also describe how yeast can be used to select for molecules that block T3E function in search of new antibacterial drugs with medical applications. Finally, we provide our opinion on the limitations of S

  13. Inhibition of Plasmodium berghei Development in Mosquitoes by Effector Proteins Secreted from Asaia sp. Bacteria Using a Novel Native Secretion Signal

    PubMed Central

    Bongio, Nicholas J.; Lampe, David J.

    2015-01-01

    Novel interventions are needed to prevent the transmission of the Plasmodium parasites that cause malaria. One possible method is to supply mosquitoes with antiplasmodial effector proteins from bacteria by paratransgenesis. Mosquitoes have a diverse complement of midgut microbiota including the Gram-negative bacteria Asaia bogorensis. This study presents the first use of Asaia sp. bacteria for paratransgenesis against P. berghei. We identified putative secreted proteins from A. bogorensis by a genetic screen using alkaline phosphatase gene fusions. Two were secreted efficiently: a siderophore receptor protein and a YVTN beta-propeller repeat protein. The siderophore receptor gene was fused with antiplasmodial effector genes including the scorpine antimicrobial peptide and an anti-Pbs21 scFv-Shiva1 immunotoxin. Asaia SF2.1 secreting these fusion proteins were fed to mosquitoes and challenged with Plasmodium berghei-infected blood. With each of these effector constructs, significant inhibition of parasite development was observed. These results provide a novel and promising intervention against malaria transmission. PMID:26636338

  14. Cross talk between type III secretion and flagellar assembly systems in Pseudomonas aeruginosa.

    PubMed

    Soscia, Chantal; Hachani, Abderrahman; Bernadac, Alain; Filloux, Alain; Bleves, Sophie

    2007-04-01

    Pseudomonas aeruginosa cytotoxicity is linked to a type III secretion system (T3SS) that delivers effectors into the host cell. We show here that a negative cross-control exists between T3SS and flagellar assembly. We observed that, in a strain lacking flagella, T3SS gene expression, effector secretion, and cytotoxicity were increased. Conversely, we revealed that flagellar-gene expression and motility were decreased in a strain overproducing ExsA, the T3SS master regulator. Interestingly, a nonmotile strain lacking the flagellar filament (DeltafliC) presented a hyperefficient T3SS and a nonmotile strain assembling flagella (DeltamotAB) did not. More intriguingly, a strain lacking motCD genes is a flagellated strain with a slight defect in swimming. However, in this strain, T3SS gene expression was up-regulated. These results suggest that flagellar assembly and/or mobility antagonizes the T3SS and that a negative cross talk exists between these two systems. An illustration of this is the visualization by electron microscopy of T3SS needles in a nonmotile P. aeruginosa strain, needles which otherwise are not detected. The molecular basis of the cross talk is complex and remains to be elucidated, but proteins like MotCD might have a crucial role in signaling between the two processes. In addition, we found that the GacA response regulator negatively affects the T3SS. In a gacA mutant, the T3SS effector ExoS is hypersecreted. Strikingly, GacA was previously reported as a positive regulator for motility. Globally, our data document the idea that some virulence factors are coordinately but inversely regulated, depending on the bacterial colonization phase and infection types. PMID:17307856

  15. Structural and Biochemical Characterization of SrcA, a Multi-cargo Type III Secretion Chaperone in Salmonella Required for Pathogenic Association with a Host

    SciTech Connect

    Cooper, C.; Zhang, K; Andres, S; Fnag, Y; Kaniuk, N; Hannemann, M; Brumell, J; Foster, L; Junop, M; Coombes, B

    2010-01-01

    Many Gram-negative bacteria colonize and exploit host niches using a protein apparatus called a type III secretion system (T3SS) that translocates bacterial effector proteins into host cells where their functions are essential for pathogenesis. A suite of T3SS-associated chaperone proteins bind cargo in the bacterial cytosol, establishing protein interaction networks needed for effector translocation into host cells. In Salmonella enterica serovar Typhimurium, a T3SS encoded in a large genomic island (SPI-2) is required for intracellular infection, but the chaperone complement required for effector translocation by this system is not known. Using a reverse genetics approach, we identified a multi-cargo secretion chaperone that is functionally integrated with the SPI-2-encoded T3SS and required for systemic infection in mice. Crystallographic analysis of SrcA at a resolution of 2.5 {angstrom} revealed a dimer similar to the CesT chaperone from enteropathogenic E. coli but lacking a 17-amino acid extension at the carboxyl terminus. Further biochemical and quantitative proteomics data revealed three protein interactions with SrcA, including two effector cargos (SseL and PipB2) and the type III-associated ATPase, SsaN, that increases the efficiency of effector translocation. Using competitive infections in mice we show that SrcA increases bacterial fitness during host infection, highlighting the in vivo importance of effector chaperones for the SPI-2 T3SS.

  16. Structure and Function of the Type III Secretion System of Pseudomonas aeruginosa

    PubMed Central

    Galle, Marlies; Carpentier, Isabelle; Beyaert, Rudi

    2012-01-01

    Pseudomonas aeruginosa is a dangerous pathogen particularly because it harbors multiple virulence factors. It causes several types of infection, including dermatitis, endocarditis, and infections of the urinary tract, eye, ear, bone, joints and, of particular interest, the respiratory tract. Patients with cystic fibrosis, who are extremely susceptible to Pseudomonas infections, have a bad prognosis and high mortality. An important virulence factor of P. aeruginosa, shared with many other gram-negative bacteria, is the type III secretion system, a hollow molecular needle that transfers effector toxins directly from the bacterium into the host cell cytosol. This complex macromolecular machine works in a highly regulated manner and can manipulate the host cell in many different ways. Here we review the current knowledge of the structure of the P. aeruginosa T3SS, as well as its function and recognition by the immune system. Furthermore, we describe recent progress in the development and use of therapeutic agents targeting the T3SS. PMID:23305368

  17. The Pseudomonas aeruginosa Type VI Secretion PGAP1-like Effector Induces Host Autophagy by Activating Endoplasmic Reticulum Stress.

    PubMed

    Jiang, Feng; Wang, Xia; Wang, Bei; Chen, Lihong; Zhao, Zhendong; Waterfield, Nicholas R; Yang, Guowei; Jin, Qi

    2016-08-01

    Pseudomonas aeruginosa is an opportunistic pathogen that regularly causes nosocomial infections in hospitalized patients. The type VI secretion system (T6SS) is responsible for the secretion of numerous virulence effector proteins that can both interfere with competing microbes and manipulate host cells. Here, we report a detailed investigation of a P. aeruginosa H2-T6SS-dependent phospholipase effector, TplE, which acts as a trans-kingdom toxin. Delivery of TplE to the periplasmic space of rival bacteria leads to growth inhibition. Importantly, TplE, also contains a eukaryotic PGAP1-like domain, which targets the host ER apparatus, ultimately leading to disruption of the ER. TplE activity leads to the activation of the unfolded protein response (UPR) through the IRE1α-XBP1 pathway, enhancing autophagic flux. These findings indicate that this T6SS-delivered phospholipase effector is active against both prokaryotic and eukaryotic cellular targets, highlighting the T6SS as a versatile weapon in the Pseudomonas arsenal. PMID:27477276

  18. A Substrate-Fusion Protein Is Trapped inside the Type III Secretion System Channel in Shigella flexneri

    PubMed Central

    Dohlich, Kim; Zumsteg, Anna Brotcke; Goosmann, Christian; Kolbe, Michael

    2014-01-01

    The Type III Secretion System (T3SS) is a macromolecular complex used by Gram-negative bacteria to secrete effector proteins from the cytoplasm across the bacterial envelope in a single step. For many pathogens, the T3SS is an essential virulence factor that enables the bacteria to interact with and manipulate their respective host. A characteristic structural feature of the T3SS is the needle complex (NC). The NC resembles a syringe with a basal body spanning both bacterial membranes and a long needle-like structure that protrudes from the bacterium. Based on the paradigm of a syringe-like mechanism, it is generally assumed that effectors and translocators are unfolded and secreted from the bacterial cytoplasm through the basal body and needle channel. Despite extensive research on T3SS, this hypothesis lacks experimental evidence and the mechanism of secretion is not fully understood. In order to elucidate details of the T3SS secretion mechanism, we generated fusion proteins consisting of a T3SS substrate and a bulky protein containing a knotted motif. Because the knot cannot be unfolded, these fusions are accepted as T3SS substrates but remain inside the NC channel and obstruct the T3SS. To our knowledge, this is the first time substrate fusions have been visualized together with isolated NCs and we demonstrate that substrate proteins are secreted directly through the channel with their N-terminus first. The channel physically encloses the fusion protein and shields it from a protease and chemical modifications. Our results corroborate an elementary understanding of how the T3SS works and provide a powerful tool for in situ-structural investigations in the future. This approach might also be applicable to other protein secretion systems that require unfolding of their substrates prior to secretion. PMID:24453973

  19. T3_MM: A Markov Model Effectively Classifies Bacterial Type III Secretion Signals

    PubMed Central

    Wang, Yejun; Sun, Ming'an; Bao, Hongxia; White, Aaron P.

    2013-01-01

    Motivation Type III Secretion Systems (T3SSs) play important roles in the interaction between gram-negative bacteria and their hosts. T3SSs function by translocating a group of bacterial effector proteins into the host cytoplasm. The details of specific type III secretion process are yet to be clarified. This research focused on comparing the amino acid composition within the N-terminal 100 amino acids from type III secretion (T3S) signal sequences or non-T3S proteins, specifically whether each residue exerts a constraint on residues found in adjacent positions. We used these comparisons to set up a statistic model to quantitatively model and effectively distinguish T3S effectors. Results In this study, the amino acid composition (Aac) probability profiles conditional on its sequentially preceding position and corresponding amino acids were compared between N-terminal sequences of T3S and non-T3S proteins. The profiles are generally different. A Markov model, namely T3_MM, was consequently designed to calculate the total Aac conditional probability difference, i.e., the likelihood ratio of a sequence being a T3S or a non-T3S protein. With T3_MM, known T3S and non-T3S proteins were found to well approximate two distinct normal distributions. The model could distinguish validated T3S and non-T3S proteins with a 5-fold cross-validation sensitivity of 83.9% at a specificity of 90.3%. T3_MM was also shown to be more robust, accurate, simple, and statistically quantitative, when compared with other T3S protein prediction models. The high effectiveness of T3_MM also indicated the overall Aac difference between N-termini of T3S and non-T3S proteins, and the constraint of Aac exerted by its preceding position and corresponding Aac. Availability An R package for T3_MM is freely downloadable from: http://biocomputer.bio.cuhk.edu.hk/softwares/T3_MM. T3_MM web server: http://biocomputer.bio.cuhk.edu.hk/T3DB/T3_MM.php. PMID:23472154

  20. Supramolecular Structure and Functional Analysis of the Type III Secretion System in Pseudomonas fluorescens 2P24

    PubMed Central

    Liu, Ping; Zhang, Wei; Zhang, Li-Qun; Liu, Xingzhong; Wei, Hai-Lei

    2016-01-01

    The type III secretion system (T3SS) of plant and animal bacterial pathogens directs the secretion and injection of proteins into host cells. Some homologous genes of T3SS were found also in non-pathogenic bacteria, but the organization of its machinery and basic function are still unknown. In this study, we identified a T3SS gene cluster from the plant growth-promoting Pseudomonas fluorescens 2P24 and isolated the corresponding T3SS apparatus. The T3SS gene cluster of strain 2P24 is similar organizationally to that of pathogenic P. syringae, except that it lacks the regulator hrpR and the hrpK1 and hrpH genes, which are involved in translocation of proteins. Electron microscopy revealed that the T3SS supramolecular structure of strain 2P24 was comprised of two distinctive substructures: a long extracellular, filamentous pilus, and a membrane-embedded base. We show that strain 2P24 deploys a harpin homolog protein, RspZ1, to elicit a hypersensitive response when infiltrated into Nicotiana tabacum cv. xanthi leaves with protein that is partially purified, and by complementing the hrpZ1 mutation of pHIR11. The T3SS of strain 2P24 retained ability to secrete effectors, whereas its effector translocation activity appeared to be excessively lost. Mutation of the rscC gene from 2P24 T3SS abolished the secretion of effectors, but the general biocontrol properties were unaffected. Remarkably, strain 2P24 induced functional MAMP-triggered immunity that included a burst of reactive oxygen species, strong suppression of challenge cell death, and disease expansion, while it was not associated with the secretion functional T3SS. PMID:26779224

  1. Tight Junction Disruption Induced by Type 3 Secretion System Effectors Injected by Enteropathogenic and Enterohemorrhagic Escherichia coli

    PubMed Central

    Ugalde-Silva, Paul; Gonzalez-Lugo, Octavio; Navarro-Garcia, Fernando

    2016-01-01

    The intestinal epithelium consists of a single cell layer, which is a critical selectively permeable barrier to both absorb nutrients and avoid the entry of potentially harmful entities, including microorganisms. Epithelial cells are held together by the apical junctional complexes, consisting of adherens junctions, and tight junctions (TJs), and by underlying desmosomes. TJs lay in the apical domain of epithelial cells and are mainly composed by transmembrane proteins such as occludin, claudins, JAMs, and tricellulin, that are associated with the cytoplasmic plaque formed by proteins from the MAGUK family, such as ZO-1/2/3, connecting TJ to the actin cytoskeleton, and cingulin and paracingulin connecting TJ to the microtubule network. Extracellular bacteria such as EPEC and EHEC living in the intestinal lumen inject effectors proteins directly from the bacterial cytoplasm to the host cell cytoplasm, where they play a relevant role in the manipulation of the eukaryotic cell functions by modifying or blocking cell signaling pathways. TJ integrity depends on various cell functions such as actin cytoskeleton, microtubule network for vesicular trafficking, membrane integrity, inflammation, and cell survival. EPEC and EHEC effectors target most of these functions. Effectors encoded inside or outside of locus of enterocyte effacement (LEE) disrupt the TJ strands. EPEC and EHEC exploit the TJ dynamics to open this structure, for causing diarrhea. EPEC and EHEC secrete effectors that mimic host proteins to manipulate the signaling pathways, including those related to TJ dynamics. In this review, we focus on the known mechanisms exploited by EPEC and EHEC effectors for causing TJ disruption. PMID:27606286

  2. Tight Junction Disruption Induced by Type 3 Secretion System Effectors Injected by Enteropathogenic and Enterohemorrhagic Escherichia coli.

    PubMed

    Ugalde-Silva, Paul; Gonzalez-Lugo, Octavio; Navarro-Garcia, Fernando

    2016-01-01

    The intestinal epithelium consists of a single cell layer, which is a critical selectively permeable barrier to both absorb nutrients and avoid the entry of potentially harmful entities, including microorganisms. Epithelial cells are held together by the apical junctional complexes, consisting of adherens junctions, and tight junctions (TJs), and by underlying desmosomes. TJs lay in the apical domain of epithelial cells and are mainly composed by transmembrane proteins such as occludin, claudins, JAMs, and tricellulin, that are associated with the cytoplasmic plaque formed by proteins from the MAGUK family, such as ZO-1/2/3, connecting TJ to the actin cytoskeleton, and cingulin and paracingulin connecting TJ to the microtubule network. Extracellular bacteria such as EPEC and EHEC living in the intestinal lumen inject effectors proteins directly from the bacterial cytoplasm to the host cell cytoplasm, where they play a relevant role in the manipulation of the eukaryotic cell functions by modifying or blocking cell signaling pathways. TJ integrity depends on various cell functions such as actin cytoskeleton, microtubule network for vesicular trafficking, membrane integrity, inflammation, and cell survival. EPEC and EHEC effectors target most of these functions. Effectors encoded inside or outside of locus of enterocyte effacement (LEE) disrupt the TJ strands. EPEC and EHEC exploit the TJ dynamics to open this structure, for causing diarrhea. EPEC and EHEC secrete effectors that mimic host proteins to manipulate the signaling pathways, including those related to TJ dynamics. In this review, we focus on the known mechanisms exploited by EPEC and EHEC effectors for causing TJ disruption. PMID:27606286

  3. The Xanthomonas campestris Type III Effector XopJ Proteolytically Degrades Proteasome Subunit RPT61[OPEN

    PubMed Central

    2015-01-01

    Many animal and plant pathogenic bacteria inject type III effector (T3E) proteins into their eukaryotic host cells to suppress immunity. The Yersinia outer protein J (YopJ) family of T3Es is a widely distributed family of effector proteins found in both animal and plant pathogens, and its members are highly diversified in virulence functions. Some members have been shown to possess acetyltransferase activity; however, whether this is a general feature of YopJ family T3Es is currently unknown. The T3E Xanthomonas outer protein J (XopJ), a YopJ family effector from the plant pathogen Xanthomonas campestris pv vesicatoria, interacts with the proteasomal subunit Regulatory Particle AAA-ATPase6 (RPT6) in planta to suppress proteasome activity, resulting in the inhibition of salicylic acid-related immune responses. Here, we show that XopJ has protease activity to specifically degrade RPT6, leading to reduced proteasome activity in the cytoplasm as well as in the nucleus. Proteolytic degradation of RPT6 was dependent on the localization of XopJ to the plasma membrane as well as on its catalytic triad. Mutation of the Walker B motif of RPT6 prevented XopJ-mediated degradation of the protein but not XopJ interaction. This indicates that the interaction of RPT6 with XopJ is dependent on the ATP-binding activity of RPT6, but proteolytic cleavage additionally requires its ATPase activity. Inhibition of the proteasome impairs the proteasomal turnover of Nonexpressor of Pathogenesis-Related1 (NPR1), the master regulator of salicylic acid responses, leading to the accumulation of ubiquitinated NPR1, which likely interferes with the full induction of NPR1 target genes. Our results show that YopJ family T3Es are not only highly diversified in virulence function but also appear to possess different biochemical activities. PMID:25739698

  4. A Secreted Effector Protein of Ustilago maydis Guides Maize Leaf Cells to Form Tumors

    PubMed Central

    Redkar, Amey; Hoser, Rafal; Schilling, Lena; Zechmann, Bernd; Krzymowska, Magdalena; Walbot, Virginia; Doehlemann, Gunther

    2015-01-01

    The biotrophic smut fungus Ustilago maydis infects all aerial organs of maize (Zea mays) and induces tumors in the plant tissues. U. maydis deploys many effector proteins to manipulate its host. Previously, deletion analysis demonstrated that several effectors have important functions in inducing tumor expansion specifically in maize leaves. Here, we present the functional characterization of the effector See1 (Seedling efficient effector1). See1 is required for the reactivation of plant DNA synthesis, which is crucial for tumor progression in leaf cells. By contrast, See1 does not affect tumor formation in immature tassel floral tissues, where maize cell proliferation occurs independent of fungal infection. See1 interacts with a maize homolog of SGT1 (Suppressor of G2 allele of skp1), a factor acting in cell cycle progression in yeast (Saccharomyces cerevisiae) and an important component of plant and human innate immunity. See1 interferes with the MAPK-triggered phosphorylation of maize SGT1 at a monocot-specific phosphorylation site. We propose that See1 interferes with SGT1 activity, resulting in both modulation of immune responses and reactivation of DNA synthesis in leaf cells. This identifies See1 as a fungal effector that directly and specifically contributes to the formation of leaf tumors in maize. PMID:25888589

  5. Determination of the Stoichiometry of the Complete Bacterial Type III Secretion Needle Complex Using a Combined Quantitative Proteomic Approach.

    PubMed

    Zilkenat, Susann; Franz-Wachtel, Mirita; Stierhof, York-Dieter; Galán, Jorge E; Macek, Boris; Wagner, Samuel

    2016-05-01

    Precisely knowing the stoichiometry of their components is critical for investigating structure, assembly, and function of macromolecular machines. This has remained a technical challenge in particular for large, hydrophobic membrane-spanning protein complexes. Here, we determined the stoichiometry of a type III secretion system of Salmonella enterica serovar Typhimurium using two complementary protocols of gentle complex purification combined with peptide concatenated standard and synthetic stable isotope-labeled peptide-based mass spectrometry. Bacterial type III secretion systems are cell envelope-spanning effector protein-delivery machines essential for colonization and survival of many Gram-negative pathogens and symbionts. The membrane-embedded core unit of these secretion systems, termed the needle complex, is composed of a base that anchors the machinery to the inner and outer membranes, a hollow filament formed by inner rod and needle subunits that serves as conduit for substrate proteins, and a membrane-embedded export apparatus facilitating substrate translocation. Structural analyses have revealed the stoichiometry of the components of the base, but the stoichiometry of the essential hydrophobic export apparatus components and of the inner rod protein remain unknown. Here, we provide evidence that the export apparatus of type III secretion systems contains five SpaP, one SpaQ, one SpaR, and one SpaS. We confirmed that the previously suggested stoichiometry of nine InvA is valid for assembled needle complexes and describe a loose association of InvA with other needle complex components that may reflect its function. Furthermore, we present evidence that not more than six PrgJ form the inner rod of the needle complex. Providing this structural information will facilitate efforts to obtain an atomic view of type III secretion systems and foster our understanding of the function of these and related flagellar machines. Given that other virulence

  6. Structural Features Reminiscent of ATP-Driven Protein Translocases Are Essential for the Function of a Type III Secretion-Associated ATPase

    PubMed Central

    Kato, Junya; Lefebre, Matthew

    2015-01-01

    ABSTRACT Many bacterial pathogens and symbionts utilize type III secretion systems to interact with their hosts. These machines have evolved to deliver bacterial effector proteins into eukaryotic target cells to modulate a variety of cellular functions. One of the most conserved components of these systems is an ATPase, which plays an essential role in the recognition and unfolding of proteins destined for secretion by the type III pathway. Here we show that structural features reminiscent of other ATP-driven protein translocases are essential for the function of InvC, the ATPase associated with a Salmonella enterica serovar Typhimurium type III secretion system. Mutational and functional analyses showed that a two-helix-finger motif and a conserved loop located at the entrance of and within the predicted pore formed by the hexameric ATPase are essential for InvC function. These findings provide mechanistic insight into the function of this highly conserved component of type III secretion machines. IMPORTANCE Type III secretion machines are essential for the virulence or symbiotic relationships of many bacteria. These machines have evolved to deliver bacterial effector proteins into host cells to modulate cellular functions, thus facilitating bacterial colonization and replication. An essential component of these machines is a highly conserved ATPase, which is necessary for the recognition and secretion of proteins destined to be delivered by the type III secretion pathway. Using modeling and structure and function analyses, we have identified structural features of one of these ATPases from Salmonella enterica serovar Typhimurium that help to explain important aspects of its function. PMID:26170413

  7. Functional assignment to positively selected sites in the core type III effector RipG7 from Ralstonia solanacearum.

    PubMed

    Wang, Keke; Remigi, Philippe; Anisimova, Maria; Lonjon, Fabien; Kars, Ilona; Kajava, Andrey; Li, Chien-Hui; Cheng, Chiu-Ping; Vailleau, Fabienne; Genin, Stéphane; Peeters, Nemo

    2016-05-01

    The soil-borne pathogen Ralstonia solanacearum causes bacterial wilt in a broad range of plants. The main virulence determinants of R. solanacearum are the type III secretion system (T3SS) and its associated type III effectors (T3Es), translocated into the host cells. Of the conserved T3Es among R. solanacearum strains, the Fbox protein RipG7 is required for R. solanacearum pathogenesis on Medicago truncatula. In this work, we describe the natural ripG7 variability existing in the R. solanacearum species complex. We show that eight representative ripG7 orthologues have different contributions to pathogenicity on M. truncatula: only ripG7 from Asian or African strains can complement the absence of ripG7 in GMI1000 (Asian reference strain). Nonetheless, RipG7 proteins from American and Indonesian strains can still interact with M. truncatula SKP1-like/MSKa protein, essential for the function of RipG7 in virulence. This indicates that the absence of complementation is most likely a result of the variability in the leucine-rich repeat (LRR) domain of RipG7. We identified 11 sites under positive selection in the LRR domains of RipG7. By studying the functional impact of these 11 sites, we show the contribution of five positively selected sites for the function of RipG7CMR15 in M. truncatula colonization. This work reveals the genetic and functional variation of the essential core T3E RipG7 from R. solanacearum. This analysis is the first of its kind on an essential disease-controlling T3E, and sheds light on the co-evolutionary arms race between the bacterium and its hosts. PMID:26300048

  8. A small-molecule inhibitor of type III secretion inhibits different stages of the infectious cycle of Chlamydia trachomatis

    PubMed Central

    Muschiol, Sandra; Bailey, Leslie; Gylfe, Åsa; Sundin, Charlotta; Hultenby, Kjell; Bergström, Sven; Elofsson, Mikael; Wolf-Watz, Hans; Normark, Staffan; Henriques-Normark, Birgitta

    2006-01-01

    The intracellular pathogen Chlamydia trachomatis possesses a type III secretion (TTS) system believed to deliver a series of effector proteins into the inclusion membrane (Inc-proteins) as well as into the host cytosol with perceived consequences for the pathogenicity of this common venereal pathogen. Recently, small molecules were shown to block the TTS system of Yersinia pseudotuberculosis. Here, we show that one of these compounds, INP0400, inhibits intracellular replication and infectivity of C. trachomatis at micromolar concentrations resulting in small inclusion bodies frequently containing only one or a few reticulate bodies (RBs). INP0400, at high concentration, given at the time of infection, partially blocked entry of elementary bodies into host cells. Early treatment inhibited the localization of the mammalian protein 14-3-3β to the inclusions, indicative of absence of the early induced TTS effector IncG from the inclusion membrane. Treatment with INP0400 during chlamydial mid-cycle prevented secretion of the TTS effector IncA and homotypic vesicular fusions mediated by this protein. INP0400 given during the late phase resulted in the detachment of RBs from the inclusion membrane concomitant with an inhibition of RB to elementary body conversion causing a marked decrease in infectivity. PMID:16973741

  9. The plant phenolic compound p-coumaric acid represses gene expression in the Dickeya dadantii type III secretion system.

    PubMed

    Li, Yan; Peng, Quan; Selimi, Dija; Wang, Qi; Charkowski, Amy O; Chen, Xin; Yang, Ching-Hong

    2009-03-01

    The type III secretion system (T3SS) is a major virulence factor in many gram-negative bacterial pathogens. This secretion system translocates effectors directly into the cytosol of eukaryotic host cells, where the effector proteins facilitate bacterial pathogenesis by interfering with host cell signal transduction and other cellular processes. Plants defend themselves against bacterial pathogens by recognizing either the type 3 effectors or their actions and initiating a cascade of defense responses that often results in programmed cell death of the plant cell being attacked. Here we show that a plant phenolic compound, p-coumaric acid (PCA), represses the expression of T3SS genes of the plant pathogen Dickeya dadantii, suggesting that plants can also defend against bacterial pathogens by manipulating the expression of the T3SS. PCA repressed the expression of T3SS regulatory genes through the HrpX/Y two-component system, a core regulator of the T3SS, rather than through the global regulator GacS/A, which indirectly regulates the T3SS. A further analysis of several PCA analogs suggests that the para positioning of the hydroxyl group in the phenyl ring and the double bond of PCA may be important for its biological activity. PMID:19114532

  10. The Plant Phenolic Compound p-Coumaric Acid Represses Gene Expression in the Dickeya dadantii Type III Secretion System▿ †

    PubMed Central

    Li, Yan; Peng, Quan; Selimi, Dija; Wang, Qi; Charkowski, Amy O.; Chen, Xin; Yang, Ching-Hong

    2009-01-01

    The type III secretion system (T3SS) is a major virulence factor in many gram-negative bacterial pathogens. This secretion system translocates effectors directly into the cytosol of eukaryotic host cells, where the effector proteins facilitate bacterial pathogenesis by interfering with host cell signal transduction and other cellular processes. Plants defend themselves against bacterial pathogens by recognizing either the type 3 effectors or their actions and initiating a cascade of defense responses that often results in programmed cell death of the plant cell being attacked. Here we show that a plant phenolic compound, p-coumaric acid (PCA), represses the expression of T3SS genes of the plant pathogen Dickeya dadantii, suggesting that plants can also defend against bacterial pathogens by manipulating the expression of the T3SS. PCA repressed the expression of T3SS regulatory genes through the HrpX/Y two-component system, a core regulator of the T3SS, rather than through the global regulator GacS/A, which indirectly regulates the T3SS. A further analysis of several PCA analogs suggests that the para positioning of the hydroxyl group in the phenyl ring and the double bond of PCA may be important for its biological activity. PMID:19114532

  11. Functional Activation of the Flagellar Type III Secretion Export Apparatus

    PubMed Central

    Phillips, Andrew M.; Calvo, Rebecca A.; Kearns, Daniel B.

    2015-01-01

    Flagella are assembled sequentially from the inside-out with morphogenetic checkpoints that enforce the temporal order of subunit addition. Here we show that flagellar basal bodies fail to proceed to hook assembly at high frequency in the absence of the monotopic protein SwrB of Bacillus subtilis. Genetic suppressor analysis indicates that SwrB activates the flagellar type III secretion export apparatus by the membrane protein FliP. Furthermore, mutants defective in the flagellar C-ring phenocopy the absence of SwrB for reduced hook frequency and C-ring defects may be bypassed either by SwrB overexpression or by a gain-of-function allele in the polymerization domain of FliG. We conclude that SwrB enhances the probability that the flagellar basal body adopts a conformation proficient for secretion to ensure that rod and hook subunits are not secreted in the absence of a suitable platform on which to polymerize. PMID:26244495

  12. Interaction between the Type III Effector VopO and GEF-H1 Activates the RhoA-ROCK Pathway

    PubMed Central

    Hiyoshi, Hirotaka; Okada, Ryu; Matsuda, Shigeaki; Gotoh, Kazuyoshi; Akeda, Yukihiro; Iida, Tetsuya; Kodama, Toshio

    2015-01-01

    Vibrio parahaemolyticus is an important pathogen that causes food-borne gastroenteritis in humans. The type III secretion system encoded on chromosome 2 (T3SS2) plays a critical role in the enterotoxic activity of V. parahaemolyticus. Previous studies have demonstrated that T3SS2 induces actin stress fibers in various epithelial cell lines during infection. This stress fiber formation is strongly related to pathogenicity, but the mechanisms that underlie T3SS2-dependent actin stress fiber formation and the main effector have not been elucidated. In this study, we identified VopO as a critical T3SS2 effector protein that activates the RhoA-ROCK pathway, which is an essential pathway for the induction of the T3SS2-dependent stress fiber formation. We also determined that GEF-H1, a RhoA guanine nucleotide exchange factor (GEF), directly binds VopO and is necessary for T3SS2-dependent stress fiber formation. The GEF-H1-binding activity of VopO via an alpha helix region correlated well with its stress fiber-inducing capacity. Furthermore, we showed that VopO is involved in the T3SS2-dependent disruption of the epithelial barrier. Thus, VopO hijacks the RhoA-ROCK pathway in a different manner compared with previously reported bacterial toxins and effectors that modulate the Rho GTPase signaling pathway. PMID:25738744

  13. Manipulation of intestinal epithelial cell function by the cell contact-dependent type III secretion systems of Vibrio parahaemolyticus.

    PubMed

    O'Boyle, Nicky; Boyd, Aoife

    2014-01-01

    Vibrio parahaemolyticus elicits gastroenteritis by deploying Type III Secretion Systems (TTSS) to deliver effector proteins into epithelial cells of the human intestinal tract. The bacteria must adhere to the human cells to allow colonization and operation of the TTSS translocation apparatus bridging the bacterium and the host cell. This article first reviews recent advances in identifying the molecules responsible for intercellular adherence. V. parahaemolyticus possesses two TTSS, each of which delivers an exclusive set of effectors and mediates unique effects on the host cell. TTSS effectors primarily target and alter the activation status of host cell signaling proteins, thereby bringing about changes in the regulation of cellular behavior. TTSS1 is responsible for the cytotoxicity of V. parahaemolyticus, while TTSS2 is necessary for the enterotoxicity of the pathogen. Recent publications have elucidated the function of several TTSS effectors and their importance in the virulence of the bacterium. This review will explore the ability of the TTSS to manipulate activities of human intestinal cells and how this modification of cell function favors bacterial colonization and persistence of V. parahaemolyticus in the host. PMID:24455490

  14. Crystal structure of Spa40, the specificity switch for the Shigella flexneri type III secretion system

    PubMed Central

    Deane, Janet E; Graham, Stephen C; Mitchell, Edward P; Flot, David; Johnson, Steven; Lea, Susan M

    2008-01-01

    The pathogenic bacterium Shigella flexneri uses a type III secretion system to inject virulence factors from the bacterial cytosol directly into host cells. The machinery that identifies secretion substrates and controls the export of extracellular components and effector proteins consists of several inner-membrane and cytoplasmic proteins. One of the inner membrane components, Spa40, belongs to a family of proteins proposed to regulate the switching of substrate specificity of the export apparatus. We show that Spa40 is cleaved within the strictly conserved amino acid sequence NPTH and substitution of the proposed autocatalytic residue abolishes cleavage. Here we also report the crystal structure of the cytoplasmic complex Spa40C and compare it with the recent structures of the homologues from Escherichia coli and Salmonella typhimurium. These structures reveal the tight association of the cleaved fragments and show that the conserved NPTH sequence lies on a loop which, when cleaved, swings away from the catalytic N257 residue, resulting in different surface features in this region. This structural rearrangement suggests a mechanism by which non-cleaving forms of these proteins interfere with correct substrate switching of the apparatus. PMID:18485071

  15. Bacteroides fragilis type VI secretion systems use novel effector and immunity proteins to antagonize human gut Bacteroidales species.

    PubMed

    Chatzidaki-Livanis, Maria; Geva-Zatorsky, Naama; Comstock, Laurie E

    2016-03-29

    Type VI secretion systems (T6SSs) are multiprotein complexes best studied in Gram-negative pathogens where they have been shown to inhibit or kill prokaryotic or eukaryotic cells and are often important for virulence. We recently showed that T6SS loci are also widespread in symbiotic human gut bacteria of the order Bacteroidales, and that these T6SS loci segregate into three distinct genetic architectures (GA). GA1 and GA2 loci are present on conserved integrative conjugative elements (ICE) and are transferred and shared among diverse human gut Bacteroidales species. GA3 loci are not contained on conserved ICE and are confined toBacteroides fragilis Unlike GA1 and GA2 T6SS loci, most GA3 loci do not encode identifiable effector and immunity proteins. Here, we studied GA3 T6SSs and show that they antagonize most human gut Bacteroidales strains analyzed, except forB. fragilisstrains with the same T6SS locus. A combination of mutation analyses,trans-protection analyses, and in vitro competition assays, allowed us to identify novel effector and immunity proteins of GA3 loci. These proteins are not orthologous to known proteins, do not contain identified motifs, and most have numerous predicted transmembrane domains. Because the genes encoding effector and immunity proteins are contained in two variable regions of GA3 loci, GA3 T6SSs of the speciesB. fragilisare likely the source of numerous novel effector and immunity proteins. Importantly, we show that the GA3 T6SS of strain 638R is functional in the mammalian gut and provides a competitive advantage to this organism. PMID:26951680

  16. Type III Protein Secretion Systems in Bacterial Pathogens of Animals and Plants

    PubMed Central

    Hueck, Christoph J.

    1998-01-01

    Various gram-negative animal and plant pathogens use a novel, sec-independent protein secretion system as a basic virulence mechanism. It is becoming increasingly clear that these so-called type III secretion systems inject (translocate) proteins into the cytosol of eukaryotic cells, where the translocated proteins facilitate bacterial pathogenesis by specifically interfering with host cell signal transduction and other cellular processes. Accordingly, some type III secretion systems are activated by bacterial contact with host cell surfaces. Individual type III secretion systems direct the secretion and translocation of a variety of unrelated proteins, which account for species-specific pathogenesis phenotypes. In contrast to the secreted virulence factors, most of the 15 to 20 membrane-associated proteins which constitute the type III secretion apparatus are conserved among different pathogens. Most of the inner membrane components of the type III secretion apparatus show additional homologies to flagellar biosynthetic proteins, while a conserved outer membrane factor is similar to secretins from type II and other secretion pathways. Structurally conserved chaperones which specifically bind to individual secreted proteins play an important role in type III protein secretion, apparently by preventing premature interactions of the secreted factors with other proteins. The genes encoding type III secretion systems are clustered, and various pieces of evidence suggest that these systems have been acquired by horizontal genetic transfer during evolution. Expression of type III secretion systems is coordinately regulated in response to host environmental stimuli by networks of transcription factors. This review comprises a comparison of the structure, function, regulation, and impact on host cells of the type III secretion systems in the animal pathogens Yersinia spp., Pseudomonas aeruginosa, Shigella flexneri, Salmonella typhimurium, enteropathogenic Escherichia coli

  17. Composition, Formation, and Regulation of the Cytosolic C-ring, a Dynamic Component of the Type III Secretion Injectisome

    PubMed Central

    Diepold, Andreas; Kudryashev, Mikhail; Delalez, Nicolas J.; Berry, Richard M.; Armitage, Judith P.

    2015-01-01

    Many gram-negative pathogens employ a type III secretion injectisome to translocate effector proteins into eukaryotic host cells. While the structure of the distal “needle complex” is well documented, the composition and role of the functionally important cytosolic complex remain less well understood. Using functional fluorescent fusions, we found that the C-ring, an essential and conserved cytosolic component of the system, is composed of ~22 copies of SctQ (YscQ in Yersinia enterocolitica), which require the presence of YscQC, the product of an internal translation initiation site in yscQ, for their cooperative assembly. Photoactivated localization microscopy (PALM) reveals that in vivo, YscQ is present in both a free-moving cytosolic and a stable injectisome-bound state. Notably, fluorescence recovery after photobleaching (FRAP) shows that YscQ exchanges between the injectisome and the cytosol, with a t½ of 68 ± 8 seconds when injectisomes are secreting. In contrast, the secretin SctC (YscC) and the major export apparatus component SctV (YscV) display minimal exchange. Under non-secreting conditions, the exchange rate of YscQ is reduced to t½ = 134 ± 16 seconds, revealing a correlation between C-ring exchange and injectisome activity, which indicates a possible role for C-ring stability in regulation of type III secretion. The stabilization of the C-ring depends on the presence of the functional ATPase SctN (YscN). These data provide new insights into the formation and composition of the injectisome and present a novel aspect of type III secretion, the exchange of C-ring subunits, which is regulated with respect to secretion. PMID:25591178

  18. Comparative Secretome Analysis of Ralstonia solanacearum Type 3 Secretion-Associated Mutants Reveals a Fine Control of Effector Delivery, Essential for Bacterial Pathogenicity.

    PubMed

    Lonjon, Fabien; Turner, Marie; Henry, Céline; Rengel, David; Lohou, David; van de Kerkhove, Quitterie; Cazalé, Anne-Claire; Peeters, Nemo; Genin, Stéphane; Vailleau, Fabienne

    2016-02-01

    Ralstonia solanacearum, the causal agent of bacterial wilt, exerts its pathogenicity through more than a hundred secreted proteins, many of them depending directly on the functionality of a type 3 secretion system. To date, only few type 3 effectors have been identified as required for bacterial pathogenicity, notably because of redundancy among the large R. solanacearum effector repertoire. In order to identify groups of effectors collectively promoting disease on susceptible hosts, we investigated the role of putative post-translational regulators in the control of type 3 secretion. A shotgun secretome analysis with label-free quantification using tandem mass spectrometry was performed on the R. solanacearum GMI1000 strain. There were 228 proteins identified, among which a large proportion of type 3 effectors, called Rip (Ralstonia injected proteins). Thanks to this proteomic approach, RipBJ was identified as a new effector specifically secreted through type 3 secretion system and translocated into plant cells. A focused Rip secretome analysis using hpa (hypersensitive response and pathogenicity associated) mutants revealed a fine secretion regulation and specific subsets of Rips with different secretion patterns. We showed that a set of Rips (RipF1, RipW, RipX, RipAB, and RipAM) are secreted in an Hpa-independent manner. We hypothesize that these Rips could be preferentially involved in the first stages of type 3 secretion. In addition, the secretion of about thirty other Rips is controlled by HpaB and HpaG. HpaB, a candidate chaperone was shown to positively control secretion of numerous Rips, whereas HpaG was shown to act as a negative regulator of secretion. To evaluate the impact of altered type 3 effectors secretion on plant pathogenesis, the hpa mutants were assayed on several host plants. HpaB was required for bacterial pathogenicity on multiple hosts whereas HpaG was found to be specifically required for full R. solanacearum pathogenicity on the legume

  19. How filamentous pathogens co-opt plants; the ins and outs of eukaryotic effectors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Research on effectors secreted by pathogens during host attack has dominated the field of molecular plant-microbe interactions over recent years. Functional analysis of type III secreted effectors that are injected by pathogenic bacteria into host cells has significantly advanced the field and demon...

  20. The bacterium Pantoea stewartii uses two different type III secretion systems to colonize its plant host and insect vector.

    PubMed

    Correa, Valdir R; Majerczak, Doris R; Ammar, El-Desouky; Merighi, Massimo; Pratt, Richard C; Hogenhout, Saskia A; Coplin, David L; Redinbaugh, Margaret G

    2012-09-01

    Plant- and animal-pathogenic bacteria utilize phylogenetically distinct type III secretion systems (T3SS) that produce needle-like injectisomes or pili for the delivery of effector proteins into host cells. Pantoea stewartii subsp. stewartii (herein referred to as P. stewartii), the causative agent of Stewart's bacterial wilt and leaf blight of maize, carries phylogenetically distinct T3SSs. In addition to an Hrc-Hrp T3SS, known to be essential for maize pathogenesis, P. stewartii has a second T3SS (Pantoea secretion island 2 [PSI-2]) that is required for persistence in its flea beetle vector, Chaetocnema pulicaria (Melsh). PSI-2 belongs to the Inv-Mxi-Spa T3SS family, typically found in animal pathogens. Mutagenesis of the PSI-2 psaN gene, which encodes an ATPase essential for secretion of T3SS effectors by the injectisome, greatly reduces both the persistence of P. stewartii in flea beetle guts and the beetle's ability to transmit P. stewartii to maize. Ectopic expression of the psaN gene complements these phenotypes. In addition, the PSI-2 psaN gene is not required for P. stewartii pathogenesis of maize and is transcriptionally upregulated in insects compared to maize tissues. Thus, the Hrp and PSI-2 T3SSs play different roles in the life cycle of P. stewartii as it alternates between its insect vector and plant host. PMID:22773631

  1. Oleanolic Acid Induces the Type III Secretion System of Ralstonia solanacearum

    PubMed Central

    Wu, Dousheng; Ding, Wei; Zhang, Yong; Liu, Xuejiao; Yang, Liang

    2015-01-01

    Ralstonia solanacearum, the causal agent of bacterial wilt, can naturally infect a wide range of host plants. The type III secretion system (T3SS) is a major virulence determinant in this bacterium. Studies have shown that plant-derived compounds are able to inhibit or induce the T3SS in some plant pathogenic bacteria, though no specific T3SS inhibitor or inducer has yet been identified in R. solanacearum. In this study, a total of 50 different compounds were screened and almost half of them (22 of 50) significantly inhibited or induced the T3SS expression of R. solanacearum. Based on the strong induction activity on T3SS, the T3SS inducer oleanolic acid (OA) was chosen for further study. We found that OA induced the expression of T3SS through the HrpG-HrpB pathway. Some type III effector genes were induced in T3SS inducing medium supplemented with OA. In addition, OA targeted only the T3SS and did not affect other virulence determinants. Finally, we observed that induction of T3SS by OA accelerated disease progress on tobacco. Overall our results suggest that plant-derived compounds are an abundant source of R. solanacearum T3SS regulators, which could prove useful as tools to interrogate the regulation of this key virulence pathway. PMID:26732647

  2. Yersinia Type III Secretion System Master Regulator LcrF

    PubMed Central

    Schwiesow, Leah; Lam, Hanh

    2015-01-01

    Many Gram-negative pathogens express a type III secretion (T3SS) system to enable growth and survival within a host. The three human-pathogenic Yersinia species, Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica, encode the Ysc T3SS, whose expression is controlled by an AraC-like master regulator called LcrF. In this review, we discuss LcrF structure and function as well as the environmental cues and pathways known to regulate LcrF expression. Similarities and differences in binding motifs and modes of action between LcrF and the Pseudomonas aeruginosa homolog ExsA are summarized. In addition, we present a new bioinformatics analysis that identifies putative LcrF binding sites within Yersinia target gene promoters. PMID:26644429

  3. Yersinia Type III Secretion System Master Regulator LcrF.

    PubMed

    Schwiesow, Leah; Lam, Hanh; Dersch, Petra; Auerbuch, Victoria

    2016-02-01

    Many Gram-negative pathogens express a type III secretion (T3SS) system to enable growth and survival within a host. The three human-pathogenic Yersinia species, Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica, encode the Ysc T3SS, whose expression is controlled by an AraC-like master regulator called LcrF. In this review, we discuss LcrF structure and function as well as the environmental cues and pathways known to regulate LcrF expression. Similarities and differences in binding motifs and modes of action between LcrF and the Pseudomonas aeruginosa homolog ExsA are summarized. In addition, we present a new bioinformatics analysis that identifies putative LcrF binding sites within Yersinia target gene promoters. PMID:26644429

  4. Broadly protective Shigella vaccine based on type III secretion apparatus proteins.

    PubMed

    Martinez-Becerra, Francisco J; Kissmann, Julian M; Diaz-McNair, Jovita; Choudhari, Shyamal P; Quick, Amy M; Mellado-Sanchez, Gabriela; Clements, John D; Pasetti, Marcela F; Picking, Wendy L

    2012-03-01

    Shigella spp. are food- and waterborne pathogens that cause severe diarrheal and dysenteric disease associated with high morbidity and mortality. Individuals most often affected are children under 5 years of age in the developing world. The existence of multiple Shigella serotypes and the heterogenic distribution of pathogenic strains, as well as emerging antibiotic resistance, require the development of a broadly protective vaccine. All Shigella spp. utilize a type III secretion system (TTSS) to initiate infection. The type III secretion apparatus (TTSA) is the molecular needle and syringe that form the energized conduit between the bacterial cytoplasm and the host cell to transport effector proteins that manipulate cellular processes to benefit the pathogen. IpaB and IpaD form a tip complex atop the TTSA needle and are required for pathogenesis. Because they are common to all virulent Shigella spp., they are ideal candidate antigens for a subunit-based, broad-spectrum vaccine. We examined the immunogenicity and protective efficacy of IpaB and IpaD, alone or combined, coadministered with a double mutant heat-labile toxin (dmLT) from Escherichia coli, used as a mucosal adjuvant, in a mouse model of intranasal immunization and pulmonary challenge. Robust systemic and mucosal antibody- and T cell-mediated immunities were induced against both proteins, particularly IpaB. Mice immunized in the presence of dmLT with IpaB alone or IpaB combined with IpaD were fully protected against lethal pulmonary infection with Shigella flexneri and Shigella sonnei. We provide the first demonstration that the Shigella TTSAs IpaB and IpaD are promising antigens for the development of a cross-protective Shigella vaccine. PMID:22202122

  5. Potassium transport of Salmonella is important for type III secretion and pathogenesis.

    PubMed

    Liu, Yehao; Ho, Katharina Kim; Su, Jing; Gong, Hao; Chang, Alexander C; Lu, Sangwei

    2013-08-01

    Intracellular cations are essential for the physiology of all living organisms including bacteria. Cations such as potassium ion (K(+)), sodium ion (Na(+)) and proton (H(+)) are involved in nearly all aspects of bacterial growth and survival. K(+) is the most abundant cation and its homeostasis in Escherichia coli and Salmonella is regulated by three major K(+) transporters: high affinity transporter Kdp and low affinity transporters Kup and Trk. Previous studies have demonstrated the roles of cations and cation transport in the physiology of Escherichia coli; their roles in the virulence and physiology of pathogenic bacteria are not well characterized. We have previously reported that the Salmonella K(+) transporter Trk is important for the secretion of effector proteins of the type III secretion system (TTSS) of Salmonella pathogenicity island 1 (SPI-1). Here we further explore the role of Salmonella cation transport in virulence in vitro and pathogenesis in animal models. Impairment of K(+) transport through deletion of K(+) transporters or exposure to the chemical modulators of cation transport, gramicidin and valinomycin, results in a severe defect in the TTSS of SPI-1, and this defect in the TTSS was not due to a failure to regulate intrabacterial pH or ATP. Our results also show that K(+) transporters are critical to the pathogenesis of Salmonella in mice and chicks and are involved in multiple growth and virulence characteristics in vitro, including protein secretion, motility and invasion of epithelial cells. These results suggest that cation transport of the pathogenic bacterium Salmonella, especially K(+) transport, contributes to its virulence in addition to previously characterized roles in maintaining homeostasis of bacteria. PMID:23728623

  6. WtsE, an AvrE-family type III effector protein of Pantoea stewartii subsp. stewartii, causes cell death in non-host plants.

    PubMed

    Ham, Jong Hyun; Majerczak, Doris; Ewert, Sophie; Sreerekha, Mysore-Venkatarau; Mackey, David; Coplin, David

    2008-09-01

    Pantoea stewartii subsp. stewartii (Pnss) causes Stewart's bacterial wilt of sweet corn and leaf blight of maize. The pathogenicity of Pnss depends on synthesis of extracellular polysaccharide and an Hrp type III secretion system. WtsE, a type III secreted effector protein, is essential for the virulence of Pnss on corn. It belongs to the AvrE family of effectors, which includes DspA/E from Erwinia amylovora and AvrE1 from Pseudomonas syringae. Previously, WtsE was shown to cause disease-associated cell death in its host plant, sweet corn. Here, we examine the biological activity of WtsE in several non-host plants. WtsE induced cell death in Nicotiana benthamiana, tobacco, beet and Arabidopsis thaliana when it was transiently produced in plant cells following agroinfiltration or translocated into plant cells from Pnss, Escherichia coli or Pseudomonas syringae pv. phaseolicola (Pph). WtsE-induced cell death in N. benthamiana, tobacco and beet resembled a hypersensitive response and in N. benthamiana it was delayed by cycloheximide. Interestingly, WtsE strongly promoted the growth of Pnss in N. benthamiana prior to the onset of cell death. Deletion derivatives of WtsE that failed to induce cell death in N. benthamiana and tobacco also did not complement wtsE mutants of Pnss for virulence in sweet corn, indicating a correlation between the two activities. WtsE also induced cell death in A. thaliana, where it suppressed basal defences induced by Pph. Thus, WtsE has growth-promoting, defence-suppressing and cell death-inducing activities in non-host plants. Expression of WtsE also prevented the growth of yeast, possibly due to an innate toxicity to eukaryotic cells. PMID:19018993

  7. Towards the Identification of Type III Effectors Associated with Ralstonia solanacearum Virulence on Tomato and Eggplant.

    PubMed

    Pensec, Flora; Lebeau, Aurore; Daunay, M C; Chiroleu, Frédéric; Guidot, Alice; Wicker, Emmanuel

    2015-12-01

    For the development of pathogen-informed breeding strategies, identifying the microbial genes involved in interactions with the plant is a critical step. To identify type III effector (T3E) repertoires associated with virulence of the bacterial wilt pathogen Ralstonia solanacearum on Solanaceous crops, we used an original association genetics approach combining DNA microarray data and pathogenicity data on resistant eggplant, pepper, and tomato accessions. From this first screen, 25 T3Es were further full-length polymerase chain reaction-amplified within a 35-strain field collection, to assess their distribution and allelic diversity. Six T3E repertoire groups were identified, within which 11 representative strains were chosen to challenge the bacterial wilt-resistant egg plants 'Dingras multiple Purple' and 'AG91-25', and tomato Hawaii 7996. The virulence or avirulence phenotypes could not be explained by specific T3E repertoires, but rather by individual T3E genes. We identified seven highly avirulence-associated genes, among which ripP2, primarily referenced as conferring avirulence to Arabidopsis thaliana. Interestingly, no T3E was associated with avirulence to both egg-plants. Highly virulence-associated genes were also identified: ripA5_2, ripU, and ripV2. This study should be regarded as a first step toward investigating both avirulence and virulence function of the highlighted genes, but also their evolutionary dynamics in natural R. solanacearum populations. PMID:26368514

  8. Pseudomonas syringae type III effector AvrRpt2 alters Arabidopsis thaliana auxin physiology

    PubMed Central

    Chen, Zhongying; Agnew, Jennifer L.; Cohen, Jerry D.; He, Ping; Shan, Libo; Sheen, Jen; Kunkel, Barbara N.

    2007-01-01

    The Pseudomonas syringae type III effector AvrRpt2 promotes bacterial virulence on Arabidopsis thaliana plants lacking a functional RPS2 gene (rps2 mutant plants). To investigate the mechanisms underlying the virulence activity of AvrRpt2, we examined the phenotypes of transgenic A. thaliana rps2 seedlings constitutively expressing AvrRpt2. These seedlings exhibited phenotypes reminiscent of A. thaliana mutants with altered auxin physiology, including longer primary roots, increased number of lateral roots, and increased sensitivity to exogenous auxin. They also had increased levels of free indole acetic acid (IAA). The presence of AvrRpt2 also was correlated with a further increase in free IAA levels during infection with P. syringae pv. tomato strain DC3000 (PstDC3000). These results indicate that AvrRpt2 alters A. thaliana auxin physiology. Application of the auxin analog 1-naphthaleneacetic acid promoted disease symptom development in PstDC3000-infected plants, suggesting that elevated auxin levels within host tissue promote PstDC3000 virulence. Thus, AvrRpt2 may be among the virulence factors of P. syringae that modulate host auxin physiology to promote disease. PMID:18056646

  9. The Type III Secretion System-Related CPn0809 from Chlamydia pneumoniae.

    PubMed

    Engel, Astrid C; Herbst, Frauke; Kerres, Anne; Galle, Jan N; Hegemann, Johannes H

    2016-01-01

    Chlamydia pneumoniae is an intracellular Gram-negative bacterium that possesses a type III secretion system (T3SS), which enables the pathogen to deliver, in a single step, effector proteins for modulation of host-cell functions into the human host cell cytosol to establish a unique intracellular niche for replication. The translocon proteins located at the top of the T3SS needle filament are essential for its function, as they form pores in the host-cell membrane. Interestingly, unlike other Gram-negative bacteria, C. pneumoniae has two putative translocon operons, named LcrH_1 and LcrH_2. However, little is known about chlamydial translocon proteins. In this study, we analyzed CPn0809, one of the putative hydrophobic translocators encoded by the LcrH_1 operon, and identified an 'SseC-like family' domain characteristic of T3S translocators. Using bright-field and confocal microscopy, we found that CPn0809 is associated with EBs during early and very late phases of a C. pneumoniae infection. Furthermore, CPn0809 forms oligomers, and interacts with the T3SS chaperone LcrH_1, via its N-terminal segment. Moreover, expression of full-length CPn0809 in the heterologous host Escherichia coli causes a grave cytotoxic effect that leads to cell death. Taken together, our data indicate that CPn0809 likely represents one of the translocon proteins of the C. pneumoniae T3SS, and possibly plays a role in the translocation of effector proteins in the early stages of infection. PMID:26895250

  10. The Type III Secretion System-Related CPn0809 from Chlamydia pneumoniae

    PubMed Central

    Engel, Astrid C.; Herbst, Frauke; Kerres, Anne; Galle, Jan N.; Hegemann, Johannes H.

    2016-01-01

    Chlamydia pneumoniae is an intracellular Gram-negative bacterium that possesses a type III secretion system (T3SS), which enables the pathogen to deliver, in a single step, effector proteins for modulation of host-cell functions into the human host cell cytosol to establish a unique intracellular niche for replication. The translocon proteins located at the top of the T3SS needle filament are essential for its function, as they form pores in the host-cell membrane. Interestingly, unlike other Gram-negative bacteria, C. pneumoniae has two putative translocon operons, named LcrH_1 and LcrH_2. However, little is known about chlamydial translocon proteins. In this study, we analyzed CPn0809, one of the putative hydrophobic translocators encoded by the LcrH_1 operon, and identified an ‘SseC-like family’ domain characteristic of T3S translocators. Using bright-field and confocal microscopy, we found that CPn0809 is associated with EBs during early and very late phases of a C. pneumoniae infection. Furthermore, CPn0809 forms oligomers, and interacts with the T3SS chaperone LcrH_1, via its N-terminal segment. Moreover, expression of full-length CPn0809 in the heterologous host Escherichia coli causes a grave cytotoxic effect that leads to cell death. Taken together, our data indicate that CPn0809 likely represents one of the translocon proteins of the C. pneumoniae T3SS, and possibly plays a role in the translocation of effector proteins in the early stages of infection. PMID:26895250

  11. ROLE OF TYPE III PROTEIN SECRETION SYSTEM IN SINORHIZOBIUM FREDII USDA257 AND SOYBEAN INTERACTIONS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant and animal pathogenic bacteria have evolved a specialized protein secretion system called type III to directly inject proteins into their host cells. The Type III secretion system (TTSS) plays an important role in plant-microbe interactions since mutation in TTSS causes a loss of bacterial pa...

  12. A Bacterial Pathogen uses Distinct Type III Secretion Systems to Alternate between Host Kingdom

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gram-negative bacterial pathogens of eukaryotes often secrete proteins directly into host cells via a needle-like protein channel called a ‘type III secretion system’ (T3SS). Bacteria that are adapted to either animal or plant hosts use phylogenetically distinct T3SSs for secreting proteins. Here, ...

  13. Decreased abundance of type III secretion system-inducing signals in Arabidopsis mkp1 enhances resistance against Pseudomonas syringae

    SciTech Connect

    Anderson, Jeffrey C.; Wan, Ying; Kim, Young-Mo; Pasa-Tolic, Ljiljana; Metz, Thomas O.; Peck, Scott C.

    2014-04-21

    Many phytopathogenic bacteria use a type III secretion system (T3SS) to inject defense-suppressing effector proteins into host cells. Genes encoding the T3SS are induced at the start of infection, yet host signals that initiate T3SS gene expression are poorly understood. Here we identify several plant-derived metabolites that induce the T3SS in the bacterial pathogen Pseudomonas syringae pv tomato DC3000. In addition, we report that mkp1 (mapk phosphatase 1), an Arabidopsis mutant that is more resistant to bacterial infection, produces decreased levels of these T3SS-inducing metabolites. Consistent with the observed decrease in these metabolites, T3SS effector delivery by DC3000 was impaired in mkp1. Addition of the bioactive metabolites to the mkp1-DC3000 interaction fully restored T3SS effector delivery and suppressed enhanced resistance in mkp1. Together, these results demonstrate that DC3000 perceives multiple signals derived from plants to initiate their virulence program, and reveal a new layer of molecular communication between plants and these pathogenic bacteria.

  14. Type III Secretion-Dependent Sensitivity of Escherichia coli O157 to Specific Ketolides

    PubMed Central

    Fernandez-Brando, Romina J.; Yamaguchi, Nao; Tahoun, Amin; McAteer, Sean P.; Gillespie, Trudi; Wang, Dai; Argyle, Sally A.; Palermo, Marina S.

    2015-01-01

    A subset of Gram-negative bacterial pathogens uses a type III secretion system (T3SS) to open up a conduit into eukaryotic cells in order to inject effector proteins. These modulate pathways to enhance bacterial colonization. In this study, we screened established bioactive compounds for any that could repress T3SS expression in enterohemorrhagic Escherichia coli (EHEC) O157. The ketolides telithromycin and, subsequently, solithromycin both demonstrated repressive effects on expression of the bacterial T3SS at sub-MICs, leading to significant reductions in bacterial binding and actin-rich pedestal formation on epithelial cells. Preincubation of epithelial cells with solithromycin resulted in significantly less attachment of E. coli O157. Moreover, bacteria expressing the T3SS were more susceptible to solithromycin, and there was significant preferential killing of E. coli O157 bacteria when they were added to epithelial cells that had been preexposed to the ketolide. This killing was dependent on expression of the T3SS. Taken together, this research indicates that the ketolide that has accumulated in epithelial cells may traffic back into the bacteria via the T3SS. Considering that neither ketolide induces the SOS response, nontoxic members of this class of antibiotics, such as solithromycin, should be considered for future testing and trials evaluating their use for treatment of EHEC infections. These antibiotics may also have broader significance for treating infections caused by other pathogenic bacteria, including intracellular bacteria, that express a T3SS. PMID:26525795

  15. HrcQ Provides a Docking Site for Early and Late Type III Secretion Substrates from Xanthomonas

    PubMed Central

    Lorenz, Christian; Hausner, Jens; Büttner, Daniela

    2012-01-01

    Pathogenicity of many Gram-negative bacteria depends on a type III secretion (T3S) system which translocates bacterial effector proteins into eukaryotic cells. The membrane-spanning secretion apparatus is associated with a cytoplasmic ATPase complex and a predicted cytoplasmic (C) ring structure which is proposed to provide a substrate docking platform for secreted proteins. In this study, we show that the putative C ring component HrcQ from the plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria is essential for bacterial pathogenicity and T3S. Fractionation studies revealed that HrcQ localizes to the cytoplasm and associates with the bacterial membranes under T3S-permissive conditions. HrcQ binds to the cytoplasmic T3S-ATPase HrcN, its predicted regulator HrcL and the cytoplasmic domains of the inner membrane proteins HrcV and HrcU. Furthermore, we observed an interaction between HrcQ and secreted proteins including early and late T3S substrates. HrcQ might therefore act as a general substrate acceptor site of the T3S system and is presumably part of a larger protein complex. Interestingly, the N-terminal export signal of the T3S substrate AvrBs3 is dispensable for the interaction with HrcQ, suggesting that binding of AvrBs3 to HrcQ occurs after its initial targeting to the T3S system. PMID:23226460

  16. Lysogeny with Shiga toxin 2-encoding bacteriophages represses type III secretion in enterohemorrhagic Escherichia coli.

    PubMed

    Xu, Xuefang; McAteer, Sean P; Tree, Jai J; Shaw, Darren J; Wolfson, Eliza B K; Beatson, Scott A; Roe, Andrew J; Allison, Lesley J; Chase-Topping, Margo E; Mahajan, Arvind; Tozzoli, Rosangela; Woolhouse, Mark E J; Morabito, Stefano; Gally, David L

    2012-01-01

    Lytic or lysogenic infections by bacteriophages drive the evolution of enteric bacteria. Enterohemorrhagic Escherichia coli (EHEC) have recently emerged as a significant zoonotic infection of humans with the main serotypes carried by ruminants. Typical EHEC strains are defined by the expression of a type III secretion (T3S) system, the production of Shiga toxins (Stx) and association with specific clinical symptoms. The genes for Stx are present on lambdoid bacteriophages integrated into the E. coli genome. Phage type (PT) 21/28 is the most prevalent strain type linked with human EHEC infections in the United Kingdom and is more likely to be associated with cattle shedding high levels of the organism than PT32 strains. In this study we have demonstrated that the majority (90%) of PT 21/28 strains contain both Stx2 and Stx2c phages, irrespective of source. This is in contrast to PT 32 strains for which only a minority of strains contain both Stx2 and 2c phages (28%). PT21/28 strains had a lower median level of T3S compared to PT32 strains and so the relationship between Stx phage lysogeny and T3S was investigated. Deletion of Stx2 phages from EHEC strains increased the level of T3S whereas lysogeny decreased T3S. This regulation was confirmed in an E. coli K12 background transduced with a marked Stx2 phage followed by measurement of a T3S reporter controlled by induced levels of the LEE-encoded regulator (Ler). The presence of an integrated Stx2 phage was shown to repress Ler induction of LEE1 and this regulation involved the CII phage regulator. This repression could be relieved by ectopic expression of a cognate CI regulator. A model is proposed in which Stx2-encoding bacteriophages regulate T3S to co-ordinate epithelial cell colonisation that is promoted by Stx and secreted effector proteins. PMID:22615557

  17. Lysogeny with Shiga Toxin 2-Encoding Bacteriophages Represses Type III Secretion in Enterohemorrhagic Escherichia coli

    PubMed Central

    Xu, Xuefang; McAteer, Sean P.; Tree, Jai J.; Shaw, Darren J.; Wolfson, Eliza B. K.; Beatson, Scott A.; Roe, Andrew J.; Allison, Lesley J.; Chase-Topping, Margo E.; Mahajan, Arvind; Tozzoli, Rosangela; Woolhouse, Mark E. J.; Morabito, Stefano; Gally, David L.

    2012-01-01

    Lytic or lysogenic infections by bacteriophages drive the evolution of enteric bacteria. Enterohemorrhagic Escherichia coli (EHEC) have recently emerged as a significant zoonotic infection of humans with the main serotypes carried by ruminants. Typical EHEC strains are defined by the expression of a type III secretion (T3S) system, the production of Shiga toxins (Stx) and association with specific clinical symptoms. The genes for Stx are present on lambdoid bacteriophages integrated into the E. coli genome. Phage type (PT) 21/28 is the most prevalent strain type linked with human EHEC infections in the United Kingdom and is more likely to be associated with cattle shedding high levels of the organism than PT32 strains. In this study we have demonstrated that the majority (90%) of PT 21/28 strains contain both Stx2 and Stx2c phages, irrespective of source. This is in contrast to PT 32 strains for which only a minority of strains contain both Stx2 and 2c phages (28%). PT21/28 strains had a lower median level of T3S compared to PT32 strains and so the relationship between Stx phage lysogeny and T3S was investigated. Deletion of Stx2 phages from EHEC strains increased the level of T3S whereas lysogeny decreased T3S. This regulation was confirmed in an E. coli K12 background transduced with a marked Stx2 phage followed by measurement of a T3S reporter controlled by induced levels of the LEE-encoded regulator (Ler). The presence of an integrated Stx2 phage was shown to repress Ler induction of LEE1 and this regulation involved the CII phage regulator. This repression could be relieved by ectopic expression of a cognate CI regulator. A model is proposed in which Stx2-encoding bacteriophages regulate T3S to co-ordinate epithelial cell colonisation that is promoted by Stx and secreted effector proteins. PMID:22615557

  18. Structure of the Yersinia pestis type III secretion chaperone SycH in complex with a stable fragment of YscM2

    SciTech Connect

    Phan, Jason; Tropea, Joseph E.; Waugh, David S.

    2010-11-16

    Pathogenic Yersinia species use a type III secretion system to inject cytotoxic effector proteins directly into the cytosol of mammalian cells, where they neutralize the innate immune response by interfering with the signal-transduction pathways that control phagocytosis and inflammation. To be exported efficiently, some effectors must transiently associate with cognate cytoplasmic secretion chaperones. SycH is the chaperone for YopH, a potent eukaryotic-like protein tyrosine phosphatase that is essential for virulence. SycH also binds two negative regulators of type III secretion, YscM1 and YscM2, both of which share significant sequence homology with the chaperone-binding domain of YopH. Here, the structure of a complex between SycH and a stable fragment of YscM2 that was designed on the basis of limited proteolysis experiments is presented. The overall fold of SycH is very similar to the structures of other homodimeric secretion chaperones that have been determined to date. YscM2 wraps around SycH in an extended fashion, with some secondary but no tertiary structure, assuming a conformation distinct from the globular fold that it is predicted to adopt in the absence of SycH.

  19. Identification of 17 HrpX-regulated proteins including two novel type III effectors, XOC_3956 and XOC_1550, in Xanthomonas oryzae pv. oryzicola.

    PubMed

    Xue, Xiao-bo; Zou, Li-fang; Ma, Wen-xiu; Liu, Zhi-yang; Chen, Gong-you

    2014-01-01

    The function of some hypothetical proteins, possibly regulated by key hrp regulators, in the pathogenicity of phytopathogenic bacteria remains largely unknown. In the present study, in silicon microarray data demonstrated that the expression of 17 HrpX-regulated protein (Xrp) genes of X. oryzae pv. oryzicola (Xoc), which causes bacterial leaf streak in rice, were either positively or negatively regulated by HrpX or/and HrpG. Bioinformatics analysis demonstrated that five Xrps possess a putative type III secretion (T3S) signal in the first 50 N-terminal amino acids, six xrp genes contain a PIP-box-like sequence (TTCGB-NX-TTCGB, 9 ≤ X ≤ 25) in the promoter regions, and two Xrps have both motifs. Twelve Xrps are widely conserved in Xanthomonas spp., whereas four are specific for X. oryzae (Xrp6) or Xoc (Xrp8, Xrp14 and Xrp17). In addition to the regulation by HrpG/HrpX, some of the 17 genes were also modulated by another hrp regulator HrpD6. Mutagenesis of these 17 genes indicated that five Xrps (Xrp1, Xrp2, Xrp5, Xrp8 and Xrp14) were required for full virulence and bacterial growth in planta. Immunoblotting assays and fusion with N-terminally truncated AvrXa10 indicated that Xrp3 and Xrp5 were secreted and translocated into rice cells through the type-III secretion system (T3S), suggesting they are novel T3S effectors. Our results suggest that Xoc exploits an orchestra of proteins that are regulated by HrpG, HrpX and HrpD6, and these proteins facilitate both infection and metabolism. PMID:24675748

  20. Identification of 17 HrpX-Regulated Proteins Including Two Novel Type III Effectors, XOC_3956 and XOC_1550, in Xanthomonas oryzae pv. oryzicola

    PubMed Central

    Xue, Xiao-bo; Zou, Li-fang; Ma, Wen-xiu; Liu, Zhi-yang; Chen, Gong-you

    2014-01-01

    The function of some hypothetical proteins, possibly regulated by key hrp regulators, in the pathogenicity of phytopathogenic bacteria remains largely unknown. In the present study, in silicon microarray data demonstrated that the expression of 17 HrpX-regulated protein (Xrp) genes of X. oryzae pv. oryzicola (Xoc), which causes bacterial leaf streak in rice, were either positively or negatively regulated by HrpX or/and HrpG. Bioinformatics analysis demonstrated that five Xrps possess a putative type III secretion (T3S) signal in the first 50 N-terminal amino acids, six xrp genes contain a PIP-box-like sequence (TTCGB-NX-TTCGB, 9≤X≤25) in the promoter regions, and two Xrps have both motifs. Twelve Xrps are widely conserved in Xanthomonas spp., whereas four are specific for X. oryzae (Xrp6) or Xoc (Xrp8, Xrp14 and Xrp17). In addition to the regulation by HrpG/HrpX, some of the 17 genes were also modulated by another hrp regulator HrpD6. Mutagenesis of these 17 genes indicated that five Xrps (Xrp1, Xrp2, Xrp5, Xrp8 and Xrp14) were required for full virulence and bacterial growth in planta. Immunoblotting assays and fusion with N-terminally truncated AvrXa10 indicated that Xrp3 and Xrp5 were secreted and translocated into rice cells through the type-III secretion system (T3S), suggesting they are novel T3S effectors. Our results suggest that Xoc exploits an orchestra of proteins that are regulated by HrpG, HrpX and HrpD6, and these proteins facilitate both infection and metabolism. PMID:24675748

  1. Implications of Spatiotemporal Regulation of Shigella flexneri Type Three Secretion Activity on Effector Functions: Think Globally, Act Locally.

    PubMed

    Campbell-Valois, F-X; Pontier, Stéphanie M

    2016-01-01

    Shigella spp. are Gram-negative bacterial pathogens that infect human colonic epithelia and cause bacterial dysentery. These bacteria express multiple copies of a syringe-like protein complex, the Type Three Secretion apparatus (T3SA), which is instrumental in the etiology of the disease. The T3SA triggers the plasma membrane (PM) engulfment of the bacteria by host cells during the initial entry process. It then enables bacteria to escape the resulting phagocytic-like vacuole. Freed bacteria form actin comets to move in the cytoplasm, which provokes bacterial collision with the inner leaflet of the PM. This phenomenon culminates in T3SA-dependent secondary uptake and vacuolar rupture in neighboring cells in a process akin to what is observed during entry and named cell-to-cell spread. The activity of the T3SA of Shigella flexneri was recently demonstrated to display an on/off regulation during the infection. While the T3SA is active when bacteria are in contact with PM-derived compartments, it switches to an inactive state when bacteria are released within the cytosol. These observations indicate that effector proteins transiting through the T3SA are therefore translocated in a highly time and space constrained fashion, likely impacting on their cellular distribution. Herein, we present what is currently known about the composition, the assembly and the regulation of the T3SA activity and discuss the consequences of the on/off regulation of T3SA on Shigella effector properties and functions during the infection. Specific examples that will be developed include the role of effectors IcsB and VirA in the escape from LC3/ATG8-positive vacuoles formed during cell-to-cell spread and of IpaJ protease activity against N-miristoylated proteins. The conservation of a similar regulation of T3SA activity in other pathogens such as Salmonella or Enteropathogenic Escherichia coli will also be briefly discussed. PMID:27014638

  2. Implications of Spatiotemporal Regulation of Shigella flexneri Type Three Secretion Activity on Effector Functions: Think Globally, Act Locally

    PubMed Central

    Campbell-Valois, F.-X.; Pontier, Stéphanie M.

    2016-01-01

    Shigella spp. are Gram-negative bacterial pathogens that infect human colonic epithelia and cause bacterial dysentery. These bacteria express multiple copies of a syringe-like protein complex, the Type Three Secretion apparatus (T3SA), which is instrumental in the etiology of the disease. The T3SA triggers the plasma membrane (PM) engulfment of the bacteria by host cells during the initial entry process. It then enables bacteria to escape the resulting phagocytic-like vacuole. Freed bacteria form actin comets to move in the cytoplasm, which provokes bacterial collision with the inner leaflet of the PM. This phenomenon culminates in T3SA-dependent secondary uptake and vacuolar rupture in neighboring cells in a process akin to what is observed during entry and named cell-to-cell spread. The activity of the T3SA of Shigella flexneri was recently demonstrated to display an on/off regulation during the infection. While the T3SA is active when bacteria are in contact with PM-derived compartments, it switches to an inactive state when bacteria are released within the cytosol. These observations indicate that effector proteins transiting through the T3SA are therefore translocated in a highly time and space constrained fashion, likely impacting on their cellular distribution. Herein, we present what is currently known about the composition, the assembly and the regulation of the T3SA activity and discuss the consequences of the on/off regulation of T3SA on Shigella effector properties and functions during the infection. Specific examples that will be developed include the role of effectors IcsB and VirA in the escape from LC3/ATG8-positive vacuoles formed during cell-to-cell spread and of IpaJ protease activity against N-miristoylated proteins. The conservation of a similar regulation of T3SA activity in other pathogens such as Salmonella or Enteropathogenic Escherichia coli will also be briefly discussed. PMID:27014638

  3. Cell invasion of poultry-associated Salmonella enterica serovar Enteritidis isolates is associated with pathogenicity, motility and proteins secreted by the type III secretion system

    PubMed Central

    Zhou, Xiaohui; Addwebi, Tarek; Davis, Margaret A.; Orfe, Lisa; Call, Douglas R.; Guard, Jean; Besser, Thomas E.

    2011-01-01

    Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major cause of food-borne gastroenteritis in humans worldwide. Poultry and poultry products are considered the major vehicles of transmission to humans. Using cell invasiveness as a surrogate marker for pathogenicity, we tested the invasiveness of 53 poultry-associated isolates of S. Enteritidis in a well-differentiated intestinal epithelial cell model (Caco-2). The method allowed classification of the isolates into low (n = 7), medium (n = 18) and high (n = 30) invasiveness categories. Cell invasiveness of the isolates did not correlate with the presence of the virulence-associated gene spvB or the ability of the isolates to form biofilms. Testing of representative isolates with high and low invasiveness in a mouse model revealed that the former were more invasive in vivo and caused more and earlier mortalities, whereas the latter were significantly less invasive in vivo, causing few or no mortalities. Further characterization of representative isolates with low and high invasiveness showed that most of the isolates with low invasiveness had impaired motility and impaired secretion of either flagella-associated proteins (FlgK, FljB and FlgL) or type III secretion system (TTSS)-secreted proteins (SipA and SipD) encoded on Salmonella pathogenicity island-1. In addition, isolates with low invasiveness had impaired ability to invade and/or survive within chicken macrophages. These data suggest that not all isolates of S. Enteritidis recovered from poultry may be equally pathogenic, and that the pathogenicity of S. Enteritidis isolates is associated, in part, with both motility and secretion of TTSS effector proteins. PMID:21292746

  4. Functional diversification of the GALA type III effector family contributes to Ralstonia solanacearum adaptation on different plant hosts

    PubMed Central

    Remigi, Philippe; Anisimova, Maria; Guidot, Alice; Genin, Stéphane; Peeters, Nemo

    2011-01-01

    Type III effectors from phytopathogenic bacteria exhibit a high degree of functional redundancy, hampering the evaluation of their precise contribution to pathogenicity. This is illustrated by the GALA type III effectors from Ralstonia solanacearum, which have been shown to be collectively, but not individually, required for disease on Arabidopsis thaliana and tomato. We investigated evolution, redundancy and diversification of this family in order to understand the individual contribution of the GALA effectors to pathogenicity. From sequences available, we reconstructed GALA phylogeny and performed selection studies. We then focused on the GALAs from the reference strain GMI1000 to examine their ability to suppress plant defense responses and contribution to pathogenicity on three different host plants: A. thaliana, tomato (Lycopersicum esculentum) and eggplant (Solanum melongena). The GALA family is well conserved within R. solanacearum species. Patterns of selection detected on some GALA family members, together with experimental results, show that GALAs underwent functional diversification. We conclude that functional divergence of the GALA family likely accounts for its remarkable conservation during R. solanacearum evolution and could contribute to R. solanacearum’s adaptation on several host plants. PMID:21902695

  5. Type III secretion needle proteins induce cell signaling and cytokine secretion via Toll-like receptors.

    PubMed

    Jessen, Danielle L; Osei-Owusu, Patrick; Toosky, Melody; Roughead, William; Bradley, David S; Nilles, Matthew L

    2014-06-01

    Pathogens are recognized by hosts by use of various receptors, including the Toll-like receptor (TLR) and Nod-like receptor (NLR) families. Ligands for these varied receptors, including bacterial products, are identified by the immune system, resulting in development of innate immune responses. Only a couple of components from type III secretion (T3S) systems are known to be recognized by TLR or NLR family members. Known T3S components that are detected by pattern recognition receptors (PRRs) are (i) flagellin, detected by TLR5 and NLRC4 (Ipaf); and (ii) T3S rod proteins (PrgJ and homologs) and needle proteins (PrgI and homologs), detected by NAIP and the NLRC4 inflammasome. In this report, we characterize the induction of proinflammatory responses through TLRs by the Yersinia pestis T3S needle protein, YscF, the Salmonella enterica needle proteins PrgI and SsaG, and the Shigella needle protein, MxiH. More specifically, we determine that the proinflammatory responses occur through TLR2 and -4. These data support the hypothesis that T3S needles have an unrecognized role in bacterial pathogenesis by modulating immune responses. PMID:24643544

  6. Expression of the Bacterial Type III Effector DspA/E in Saccharomyces cerevisiae Down-regulates the Sphingolipid Biosynthetic Pathway Leading to Growth Arrest*

    PubMed Central

    Siamer, Sabrina; Guillas, Isabelle; Shimobayashi, Mitsugu; Kunz, Caroline; Hall, Michael N.; Barny, Marie-Anne

    2014-01-01

    Erwinia amylovora, the bacterium responsible for fire blight, relies on a type III secretion system and a single injected effector, DspA/E, to induce disease in host plants. DspA/E belongs to the widespread AvrE family of type III effectors that suppress plant defense responses and promote bacterial growth following infection. Ectopic expression of DspA/E in plant or in Saccharomyces cerevisiae is toxic, indicating that DspA/E likely targets a cellular process conserved between yeast and plant. To unravel the mode of action of DspA/E, we screened the Euroscarf S. cerevisiae library for mutants resistant to DspA/E-induced growth arrest. The most resistant mutants (Δsur4, Δfen1, Δipt1, Δskn1, Δcsg1, Δcsg2, Δorm1, and Δorm2) were impaired in the sphingolipid biosynthetic pathway. Exogenously supplied sphingolipid precursors such as the long chain bases (LCBs) phytosphingosine and dihydrosphingosine also suppressed the DspA/E-induced yeast growth defect. Expression of DspA/E in yeast down-regulated LCB biosynthesis and induced a rapid decrease in LCB levels, indicating that serine palmitoyltransferase (SPT), the first and rate-limiting enzyme of the sphingolipid biosynthetic pathway, was repressed. SPT down-regulation was mediated by dephosphorylation and activation of Orm proteins that negatively regulate SPT. A Δcdc55 mutation affecting Cdc55-PP2A protein phosphatase activity prevented Orm dephosphorylation and suppressed DspA/E-induced growth arrest. PMID:24828506

  7. Expression of the bacterial type III effector DspA/E in Saccharomyces cerevisiae down-regulates the sphingolipid biosynthetic pathway leading to growth arrest.

    PubMed

    Siamer, Sabrina; Guillas, Isabelle; Shimobayashi, Mitsugu; Kunz, Caroline; Hall, Michael N; Barny, Marie-Anne

    2014-06-27

    Erwinia amylovora, the bacterium responsible for fire blight, relies on a type III secretion system and a single injected effector, DspA/E, to induce disease in host plants. DspA/E belongs to the widespread AvrE family of type III effectors that suppress plant defense responses and promote bacterial growth following infection. Ectopic expression of DspA/E in plant or in Saccharomyces cerevisiae is toxic, indicating that DspA/E likely targets a cellular process conserved between yeast and plant. To unravel the mode of action of DspA/E, we screened the Euroscarf S. cerevisiae library for mutants resistant to DspA/E-induced growth arrest. The most resistant mutants (Δsur4, Δfen1, Δipt1, Δskn1, Δcsg1, Δcsg2, Δorm1, and Δorm2) were impaired in the sphingolipid biosynthetic pathway. Exogenously supplied sphingolipid precursors such as the long chain bases (LCBs) phytosphingosine and dihydrosphingosine also suppressed the DspA/E-induced yeast growth defect. Expression of DspA/E in yeast down-regulated LCB biosynthesis and induced a rapid decrease in LCB levels, indicating that serine palmitoyltransferase (SPT), the first and rate-limiting enzyme of the sphingolipid biosynthetic pathway, was repressed. SPT down-regulation was mediated by dephosphorylation and activation of Orm proteins that negatively regulate SPT. A Δcdc55 mutation affecting Cdc55-PP2A protein phosphatase activity prevented Orm dephosphorylation and suppressed DspA/E-induced growth arrest. PMID:24828506

  8. Direct and Indirect Targeting of PP2A by Conserved Bacterial Type-III Effector Proteins.

    PubMed

    Jin, Lin; Ham, Jong Hyun; Hage, Rosemary; Zhao, Wanying; Soto-Hernández, Jaricelis; Lee, Sang Yeol; Paek, Seung-Mann; Kim, Min Gab; Boone, Charles; Coplin, David L; Mackey, David

    2016-05-01

    Bacterial AvrE-family Type-III effector proteins (T3Es) contribute significantly to the virulence of plant-pathogenic species of Pseudomonas, Pantoea, Ralstonia, Erwinia, Dickeya and Pectobacterium, with hosts ranging from monocots to dicots. However, the mode of action of AvrE-family T3Es remains enigmatic, due in large part to their toxicity when expressed in plant or yeast cells. To search for targets of WtsE, an AvrE-family T3E from the maize pathogen Pantoea stewartii subsp. stewartii, we employed a yeast-two-hybrid screen with non-lethal fragments of WtsE and a synthetic genetic array with full-length WtsE. Together these screens indicate that WtsE targets maize protein phosphatase 2A (PP2A) heterotrimeric enzyme complexes via direct interaction with B' regulatory subunits. AvrE1, another AvrE-family T3E from Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000), associates with specific PP2A B' subunit proteins from its susceptible host Arabidopsis that are homologous to the maize B' subunits shown to interact with WtsE. Additionally, AvrE1 was observed to associate with the WtsE-interacting maize proteins, indicating that PP2A B' subunits are likely conserved targets of AvrE-family T3Es. Notably, the ability of AvrE1 to promote bacterial growth and/or suppress callose deposition was compromised in Arabidopsis plants with mutations of PP2A genes. Also, chemical inhibition of PP2A activity blocked the virulence activity of both WtsE and AvrE1 in planta. The function of HopM1, a Pto DC3000 T3E that is functionally redundant to AvrE1, was also impaired in specific PP2A mutant lines, although no direct interaction with B' subunits was observed. These results indicate that sub-component specific PP2A complexes are targeted by bacterial T3Es, including direct targeting by members of the widely conserved AvrE-family. PMID:27191168

  9. Direct and Indirect Targeting of PP2A by Conserved Bacterial Type-III Effector Proteins

    PubMed Central

    Jin, Lin; Ham, Jong Hyun; Hage, Rosemary; Zhao, Wanying; Soto-Hernández, Jaricelis; Lee, Sang Yeol; Paek, Seung-Mann; Kim, Min Gab; Boone, Charles; Coplin, David L.; Mackey, David

    2016-01-01

    Bacterial AvrE-family Type-III effector proteins (T3Es) contribute significantly to the virulence of plant-pathogenic species of Pseudomonas, Pantoea, Ralstonia, Erwinia, Dickeya and Pectobacterium, with hosts ranging from monocots to dicots. However, the mode of action of AvrE-family T3Es remains enigmatic, due in large part to their toxicity when expressed in plant or yeast cells. To search for targets of WtsE, an AvrE-family T3E from the maize pathogen Pantoea stewartii subsp. stewartii, we employed a yeast-two-hybrid screen with non-lethal fragments of WtsE and a synthetic genetic array with full-length WtsE. Together these screens indicate that WtsE targets maize protein phosphatase 2A (PP2A) heterotrimeric enzyme complexes via direct interaction with B’ regulatory subunits. AvrE1, another AvrE-family T3E from Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000), associates with specific PP2A B’ subunit proteins from its susceptible host Arabidopsis that are homologous to the maize B’ subunits shown to interact with WtsE. Additionally, AvrE1 was observed to associate with the WtsE-interacting maize proteins, indicating that PP2A B’ subunits are likely conserved targets of AvrE-family T3Es. Notably, the ability of AvrE1 to promote bacterial growth and/or suppress callose deposition was compromised in Arabidopsis plants with mutations of PP2A genes. Also, chemical inhibition of PP2A activity blocked the virulence activity of both WtsE and AvrE1 in planta. The function of HopM1, a Pto DC3000 T3E that is functionally redundant to AvrE1, was also impaired in specific PP2A mutant lines, although no direct interaction with B’ subunits was observed. These results indicate that sub-component specific PP2A complexes are targeted by bacterial T3Es, including direct targeting by members of the widely conserved AvrE-family. PMID:27191168

  10. Structural Basis of Cytotoxicity Mediated by the Type III Secretion Toxin ExoU from Pseudomonas aeruginosa

    PubMed Central

    Gendrin, Claire; Contreras-Martel, Carlos; Bouillot, Stéphanie; Elsen, Sylvie; Lemaire, David; Skoufias, Dimitrios A.; Huber, Philippe; Attree, Ina; Dessen, Andréa

    2012-01-01

    The type III secretion system (T3SS) is a complex macromolecular machinery employed by a number of Gram-negative pathogens to inject effectors directly into the cytoplasm of eukaryotic cells. ExoU from the opportunistic pathogen Pseudomonas aeruginosa is one of the most aggressive toxins injected by a T3SS, leading to rapid cell necrosis. Here we report the crystal structure of ExoU in complex with its chaperone, SpcU. ExoU folds into membrane-binding, bridging, and phospholipase domains. SpcU maintains the N-terminus of ExoU in an unfolded state, required for secretion. The phospholipase domain carries an embedded catalytic site whose position within ExoU does not permit direct interaction with the bilayer, which suggests that ExoU must undergo a conformational rearrangement in order to access lipids within the target membrane. The bridging domain connects catalytic domain and membrane-binding domains, the latter of which displays specificity to PI(4,5)P2. Both transfection experiments and infection of eukaryotic cells with ExoU-secreting bacteria show that ExoU ubiquitination results in its co-localization with endosomal markers. This could reflect an attempt of the infected cell to target ExoU for degradation in order to protect itself from its aggressive cytotoxic action. PMID:22496657

  11. Evidence for alternative quaternary structure in a bacterial Type III secretion system chaperone

    SciTech Connect

    Barta, Michael L.; Zhang, Lingling; Picking, Wendy L.; Geisbrecht, Brian V.

    2010-10-05

    Type III secretion systems are a common virulence mechanism in many Gram-negative bacterial pathogens. These systems use a nanomachine resembling a molecular needle and syringe to provide an energized conduit for the translocation of effector proteins from the bacterial cytoplasm to the host cell cytoplasm for the benefit of the pathogen. Prior to translocation specialized chaperones maintain proper effector protein conformation. The class II chaperone, Invasion plasmid gene (Ipg) C, stabilizes two pore forming translocator proteins. IpgC exists as a functional dimer to facilitate the mutually exclusive binding of both translocators. In this study, we present the 3.3 {angstrom} crystal structure of an amino-terminally truncated form (residues 10-155, denoted IpgC10-155) of the class II chaperone IpgC from Shigella flexneri. Our structure demonstrates an alternative quaternary arrangement to that previously described for a carboxy-terminally truncated variant of IpgC (IpgC{sup 1-151}). Specifically, we observe a rotationally-symmetric 'head-to-head' dimerization interface that is far more similar to that previously described for SycD from Yersinia enterocolitica than to IpgC1-151. The IpgC structure presented here displays major differences in the amino terminal region, where extended coil-like structures are seen, as opposed to the short, ordered alpha helices and asymmetric dimerization interface seen within IpgC{sup 1-151}. Despite these differences, however, both modes of dimerization support chaperone activity, as judged by a copurification assay with a recombinant form of the translocator protein, IpaB. Conclusions: From primary to quaternary structure, these results presented here suggest that a symmetric dimerization interface is conserved across bacterial class II chaperones. In light of previous data which have described the structure and function of asymmetric dimerization, our results raise the possibility that class II chaperones may transition between

  12. Architecture of the major component of the type III secretion system export apparatus

    PubMed Central

    Abrusci, Patrizia; Vergara–Irigaray, Marta; Johnson, Steven; Beeby, Morgan D; Hendrixson, David; Roversi, Pietro; Friede, Miriam E; Deane, Janet E; Jensen, Grant J; Tang, Christoph M; Lea, Susan M

    2012-01-01

    Type III secretion systems (T3SSs) are bacterial membrane-embedded secretion nanomachines designed to export specifically targeted sets of proteins from the bacterial cytoplasm. Secretion through T3SS is governed by a subset of inner membrane proteins termed the ‘export apparatus’. We show that a key member of the Shigella flexneri export apparatus, MxiA, assembles into a ring essential for secretion in vivo. The ring forming interfaces are well conserved in both non-flagellar and flagellar homologues, implying that the ring is an evolutionary conserved feature in these systems. Electron cryo-tomography reveals a T3SS-associated cytoplasmic torus of size and shape corresponding to the MxiA ring aligned to the secretion channel located between the secretion pore and the ATPase complex. This defines the molecular architecture of the dominant component of the export apparatus and allows us to propose a model for the molecular mechanisms controlling secretion. PMID:23222644

  13. Intrinsic and Extrinsic Regulation of Type III Secretion Gene Expression in Pseudomonas Aeruginosa

    PubMed Central

    Diaz, Manisha R.; King, Jessica M.; Yahr, Timothy L.

    2011-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen that is particularly problematic in the healthcare setting where it is a frequent cause of pneumonia, bloodstream, and urinary tract infections. An important determinant of P. aeruginosa virulence is a type III secretion system (T3SS). T3SS-dependent intoxication is a complex process that minimally requires binding of P. aeruginosa to host cells, injection of the cytotoxic effector proteins through the host cell plasma membrane, and induction of T3SS gene expression. The latter process, referred to as contact-dependent expression, involves a well-characterized regulatory cascade that activates T3SS gene expression in response to host cell contact. Although host cell contact is a primary activating signal for T3SS gene expression, the involvement of multiple membrane-bound regulatory systems indicates that additional environmental signals also play a role in controlling expression of the T3SS. These regulatory systems coordinate T3SS gene expression with many other cellular activities including motility, mucoidy, polysaccharide production, and biofilm formation. The signals to which the organism responds are poorly understood but many seem to be coupled to the metabolic state of the cell and integrated within a master circuit that assimilates informational signals from endogenous and exogenous sources. Herein we review progress toward unraveling this complex circuitry, provide analysis of the current knowledge gaps, and highlight potential areas for future studies. Complete understanding of the regulatory networks that control T3SS gene expression will maximize opportunities for the development of strategies to treat P. aeruginosa infections. PMID:21833328

  14. A phospholipase A1 antibacterial Type VI secretion effector interacts directly with the C-terminal domain of the VgrG spike protein for delivery.

    PubMed

    Flaugnatti, Nicolas; Le, Thi Thu Hang; Canaan, Stéphane; Aschtgen, Marie-Stéphanie; Nguyen, Van Son; Blangy, Stéphanie; Kellenberger, Christine; Roussel, Alain; Cambillau, Christian; Cascales, Eric; Journet, Laure

    2016-03-01

    The Type VI secretion system (T6SS) is a multiprotein machine that delivers protein effectors in both prokaryotic and eukaryotic cells, allowing interbacterial competition and virulence. The mechanism of action of the T6SS requires the contraction of a sheath-like structure that propels a needle towards target cells, allowing the delivery of protein effectors. Here, we provide evidence that the entero-aggregative Escherichia coli Sci-1 T6SS is required to eliminate competitor bacteria. We further identify Tle1, a toxin effector encoded by this cluster and showed that Tle1 possesses phospholipase A1 and A2 activities required for the interbacterial competition. Self-protection of the attacker cell is secured by an outer membrane lipoprotein, Tli1, which binds Tle1 in a 1:1 stoichiometric ratio with nanomolar affinity, and inhibits its phospholipase activity. Tle1 is delivered into the periplasm of the prey cells using the VgrG1 needle spike protein as carrier. Further analyses demonstrate that the C-terminal extension domain of VgrG1, including a transthyretin-like domain, is responsible for the interaction with Tle1 and its subsequent delivery into target cells. Based on these results, we propose an additional mechanism of transport of T6SS effectors in which cognate effectors are selected by specific motifs located at the C-terminus of VgrG proteins. PMID:26714038

  15. Ketoglutarate Transport Protein KgtP Is Secreted through the Type III Secretion System and Contributes to Virulence in Xanthomonas oryzae pv. oryzae

    PubMed Central

    Guo, Wei; Cai, Lu-Lu; Zou, Hua-Song; Ma, Wen-Xiu; Liu, Xi-Ling; Zou, Li-Fang; Li, Yu-Rong

    2012-01-01

    The phytopathogenic prokaryote Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight (BB) of rice and utilizes a type III secretion system (T3SS) to deliver T3SS effectors into rice cells. In this report, we show that the ketoglutarate transport protein (KgtP) is secreted in an HpaB-independent manner through the T3SS of X. oryzae pv. oryzae PXO99A and localizes to the host cell membrane for α-ketoglutaric acid export. kgtP contained an imperfect PIP box (plant-inducible promoter) in the promoter region and was positively regulated by HrpX and HrpG. A kgtP deletion mutant was impaired in bacterial virulence and growth in planta; furthermore, the mutant showed reduced growth in minimal media containing α-ketoglutaric acid or sodium succinate as the sole carbon source. The reduced virulence and the deficiency in α-ketoglutaric acid utilization by the kgtP mutant were restored to wild-type levels by the presence of kgtP in trans. The expression of OsIDH, which is responsible for the synthesis of α-ketoglutaric acid in rice, was enhanced when KgtP was present in the pathogen. To our knowledge, this is the first report demonstrating that KgtP, which is regulated by HrpG and HrpX and secreted by the T3SS in Xanthomonas oryzae pv. oryzae, transports α-ketoglutaric acid when the pathogen infects rice. PMID:22685129

  16. Crystal structure of the Yersinia type III secretion protein YscE

    SciTech Connect

    Phan, Jason; Austin, Brian P.; Waugh, David S.

    2010-12-06

    The plague-causing bacterium Yersinia pestis utilizes a contact-dependent (type III) secretion system (T3SS) to transport virulence factors from the bacterial cytosol directly into the interior of mammalian cells where they interfere with signal transduction pathways that mediate phagocytosis and the inflammatory response. The type III secretion apparatus is composed of 20-25 different Yersinia secretion (Ysc) proteins. We report here the structure of YscE, the smallest Ysc protein, which is a dimer in solution. The probable mode of oligomerization is discussed.

  17. YopN and TyeA Hydrophobic Contacts Required for Regulating Ysc-Yop Type III Secretion Activity by Yersinia pseudotuberculosis

    PubMed Central

    Amer, Ayad A. A.; Gurung, Jyoti M.; Costa, Tiago R. D.; Ruuth, Kristina; Zavialov, Anton V.; Forsberg, Åke; Francis, Matthew S.

    2016-01-01

    Yersinia bacteria target Yop effector toxins to the interior of host immune cells by the Ysc-Yop type III secretion system. A YopN-TyeA heterodimer is central to controlling Ysc-Yop targeting activity. A + 1 frameshift event in the 3-prime end of yopN can also produce a singular secreted YopN-TyeA polypeptide that retains some regulatory function even though the C-terminal coding sequence of this YopN differs greatly from wild type. Thus, this YopN C-terminal segment was analyzed for its role in type III secretion control. Bacteria producing YopN truncated after residue 278, or with altered sequence between residues 279 and 287, had lost type III secretion control and function. In contrast, YopN variants with manipulated sequence beyond residue 287 maintained full control and function. Scrutiny of the YopN-TyeA complex structure revealed that residue W279 functioned as a likely hydrophobic contact site with TyeA. Indeed, a YopNW279G mutant lost all ability to bind TyeA. The TyeA residue F8 was also critical for reciprocal YopN binding. Thus, we conclude that specific hydrophobic contacts between opposing YopN and TyeA termini establishes a complex needed for regulating Ysc-Yop activity. PMID:27446813

  18. The Structures of Coiled-Coil Domains from Type III Secretion System Translocators Reveal Homology to Pore-Forming Toxins

    SciTech Connect

    Barta, Michael L.; Dickenson, Nicholas E.; Patil, Mrinalini; Keightley, Andrew; Wyckoff, Gerald J.; Picking, William D.; Picking, Wendy L.; Geisbrecht, Brian V.

    2012-03-26

    Many pathogenic Gram-negative bacteria utilize type III secretion systems (T3SSs) to alter the normal functions of target cells. Shigella flexneri uses its T3SS to invade human intestinal cells to cause bacillary dysentery (shigellosis) that is responsible for over one million deaths per year. The Shigella type III secretion apparatus is composed of a basal body spanning both bacterial membranes and an exposed oligomeric needle. Host altering effectors are secreted through this energized unidirectional conduit to promote bacterial invasion. The active needle tip complex of S. flexneri is composed of a tip protein, IpaD, and two pore-forming translocators, IpaB and IpaC. While the atomic structure of IpaD has been elucidated and studied, structural data on the hydrophobic translocators from the T3SS family remain elusive. We present here the crystal structures of a protease-stable fragment identified within the N-terminal regions of IpaB from S. flexneri and SipB from Salmonella enterica serovar Typhimurium determined at 2.1 {angstrom} and 2.8 {angstrom} limiting resolution, respectively. These newly identified domains are composed of extended-length (114 {angstrom} in IpaB and 71 {angstrom} in SipB) coiled-coil motifs that display a high degree of structural homology to one another despite the fact that they share only 21% sequence identity. Further structural comparisons also reveal substantial similarity to the coiled-coil regions of pore-forming proteins from other Gram-negative pathogens, notably, colicin Ia. This suggests that these mechanistically separate and functionally distinct membrane-targeting proteins may have diverged from a common ancestor during the course of pathogen-specific evolutionary events.

  19. Type III Secretion System Genes of Dickeya dadantii 3937 Are Induced by Plant Phenolic Acids

    PubMed Central

    Yang, Shihui; Peng, Quan; San Francisco, Michael; Wang, Yongjun; Zeng, Quan; Yang, Ching-Hong

    2008-01-01

    Background Dickeya dadantii is a broad-host range phytopathogen. D. dadantii 3937 (Ech3937) possesses a type III secretion system (T3SS), a major virulence factor secretion system in many Gram-negative pathogens of plants and animals. In Ech3937, the T3SS is regulated by two major regulatory pathways, HrpX/HrpY-HrpS-HrpL and GacS/GacA-rsmB-RsmA pathways. Although the plant apoplast environment, low pH, low temperature, and absence of complex nitrogen sources in media have been associated with the induction of T3SS genes of phytobacteria, no specific inducer has yet been identified. Methodology/Principal Findings In this work, we identified two novel plant phenolic compounds, o-coumaric acid (OCA) and t-cinnamic acid (TCA), that induced the expression of T3SS genes dspE (a T3SS effector), hrpA (a structural protein of the T3SS pilus), and hrpN (a T3SS harpin) in vitro. Assays by qRT-PCR showed higher amounts of mRNA of hrpL (a T3SS alternative sigma factor) and rsmB (an untranslated regulatory RNA), but not hrpS (a σ54-enhancer binding protein) of Ech3937 when these two plant compounds were supplemented into minimal medium (MM). However, promoter activity assays using flow cytometry showed similar promoter activities of hrpN in rsmB mutant Ech148 grown in MM and MM supplemented with these phenolic compounds. Compared with MM alone, only slightly higher promoter activities of hrpL were observed in bacterial cells grown in MM supplemented with OCA/TCA. Conclusion/Significance The induction of T3SS expression by OCA and TCA is moderated through the rsmB-RsmA pathway. This is the first report of plant phenolic compounds that induce the expression T3SS genes of plant pathogenic bacteria. PMID:18698421

  20. The Type III Secretion System and Cytotoxic Enterotoxin Alter the Virulence of Aeromonas hydrophila

    PubMed Central

    Sha, Jian; Pillai, Lakshmi; Fadl, Amin A.; Galindo, Cristi L.; Erova, Tatiana E.; Chopra, Ashok K.

    2005-01-01

    Many gram-negative bacteria use a type III secretion system (TTSS) to deliver effector proteins into host cells. Here we report the characterization of a TTSS chromosomal operon from the diarrheal isolate SSU of Aeromonas hydrophila. We deleted the gene encoding Aeromonas outer membrane protein B (AopB), which is predicted to be involved in the formation of the TTSS translocon, from wild-type (WT) A. hydrophila as well as from a previously characterized cytotoxic enterotoxin gene (act)-minus strain of A. hydrophila, thus generating aopB and act/aopB isogenic mutants. The act gene encodes a type II-secreted cytotoxic enterotoxin (Act) that has hemolytic, cytotoxic, and enterotoxic activities and induces lethality in a mouse model. These isogenic mutants (aopB, act, and act/aopB) were highly attenuated in their ability to induce cytotoxicity in RAW 264.7 murine macrophages and HT-29 human colonic epithelial cells. The act/aopB mutant demonstrated the greatest reduction in cytotoxicity to cultured cells after 4 h of infection, as measured by the release of lactate dehydrogenase enzyme, and was avirulent in mice, with a 90% survival rate compared to that of animals infected with Act and AopB mutants, which caused 50 to 60% of the animals to die at a dose of three 50% lethal doses. In contrast, WT A. hydrophila killed 100% of the mice within 48 h. The effects of these mutations on cytotoxicity could be complemented with the native genes. Our studies further revealed that the production of lactones, which are involved in quorum sensing (QS), was decreased in the act (32%) and aopB (64%) mutants and was minimal (only 8%) in the act/aopB mutant, compared to that of WT A. hydrophila SSU. The effects of act and aopB gene deletions on lactone production could also be complemented with the native genes, indicating specific effects of Act and the TTSS on lactone production. Although recent studies with other bacteria have indicated TTSS regulation by QS, this is the first

  1. Effectors of animal and plant pathogens use a common domain to bind host phosphoinositides

    PubMed Central

    Salomon, Dor; Guo, Yirui; Kinch, Lisa N.; Grishin, Nick V.; Gardner, Kevin H.; Orth, Kim

    2016-01-01

    Bacterial Type III Secretion Systems deliver effectors into host cells to manipulate cellular processes to the advantage of the pathogen. Many host targets of these effectors are found on membranes. Therefore, to identify their targets, effectors often use specialized membrane-localization domains to localize to appropriate host membranes. However, the molecular mechanisms used by many domains are unknown. Here we identify a conserved bacterial phosphoinositide-binding domain (BPD) that is found in functionally diverse Type III effectors of both plant and animal pathogens. We show that members of the BPD family functionally bind phosphoinositides and mediate localization to host membranes. Moreover, NMR studies reveal that the BPD of the newly identified Vibrio parahaemolyticus Type III effector VopR is unfolded in solution, but folds into a specific structure upon binding its ligand phosphatidylinositol-(4,5)-bisphosphate. Thus, our findings suggest a possible mechanism for promoting refolding of Type III effectors after delivery into host cells. PMID:24346350

  2. Pseudomonas syringae pv. tomato DC3000 HopPtoM (CEL ORF3) is important for lesion formation but not growth in tomato and is secreted and translocated by the Hrp type III secretion system in a chaperone-dependent manner.

    PubMed

    Badel, Jorge L; Nomura, Kinya; Bandyopadhyay, Sruti; Shimizu, Rena; Collmer, Alan; He, Sheng Yang

    2003-09-01

    Pseudomonas syringae pv. tomato DC3000 is a pathogen of tomato and Arabidopsis that injects virulence effector proteins into host cells via a type III secretion system (TTSS). TTSS-deficient mutants have a Hrp- phenotype, that is, they cannot elicit the hypersensitive response (HR) in non-host plants or pathogenesis in host plants. Mutations in effector genes typically have weak virulence phenotypes (apparently due to redundancy), but deletion of six open reading frames (ORF) in the DC3000 conserved effector locus (CEL) reduces parasitic growth and abolishes disease symptoms without affecting function of the TTSS. The inability of the DeltaCEL mutant to cause disease symptoms in tomato was restored by a clone expressing two of the six ORF that had been deleted: CEL ORF3 (HopPtoM) and ORF4 (ShcM). A DeltahopPtoM::nptII mutant was constructed and found to grow like the wild type in tomato but to be strongly reduced in its production of necrotic lesion symptoms. HopPtoM expression in DC3000 was activated by the HrpL alternative sigma factor, and the protein was secreted by the Hrp TTSS in culture and translocated into Arabidopsis cells by the Hrp TTSS during infection. Secretion and translocation were dependent on ShcM, which was neither secreted nor translocated but, like typical TTSS chaperones, could be shown to interact with HopPtoM, its cognate effector, in yeast two-hybrid experiments. Thus, HopPtoM is a type III effector that, among known plant pathogen effectors, is unusual in making a major contribution to the elicitation of lesion symptoms but not growth in host tomato leaves. PMID:12940984

  3. Edwardsiella tarda EsaE (Orf19 protein) is required for the secretion of type III substrates, and pathogenesis in fish.

    PubMed

    Zhou, Ying; Liu, Lu Yi; He, Tian Tian; Laghari, Zubair Ahmed; Nie, Pin; Gao, Qian; Xie, Hai Xia

    2016-07-15

    Type III secretion system (T3SS) is a large macromolecular assembly found on the surface of many pathogenic Gram-negative bacteria. Edwardsiella tarda is an important Gram-negative pathogen that employs T3SS to deliver effectors into host cells to facilitate its survival and replication. EseB, EseC, and EseD, when secreted, form a translocon complex EseBCD on host membranes through which effectors are translocated. The orf19 gene (esaE) of E. tarda is located upstream of esaK, and downstream of esaJ, esaI, esaH and esaG in the T3SS gene cluster. When its domains were searched using Delta-Blast, the EsaE protein was found to belong to the T3SS YscJ/PrgK family. In the present study, it is found that EsaE is not secreted into culture supernatant, and the deletion of esaE abolished the secretion of T3SS translocon proteins EseBCD and T3SS effector EseG. Increased steady-state protein level of EseC and EseD was detected in bacterial pellet of ΔesaE strain although a reduced level was observed for the eseC and eseD transcription. EsaE was found to localize on membrane but not in the cytoplasm of E. tarda by fractionation. In blue gourami fish infection model, 87.88% of blue gourami infected with ΔesaE strain survived whereas only 3.03% survived when infected with wild-type strain. Taken together, our study demonstrated that EsaE is probably an apparatus protein of T3SS, which contributes to the pathogenesis of E. tarda in fish. PMID:27283851

  4. Differential regulation of type III secretion and virulence genes in Bordetella pertussis and Bordetella bronchiseptica by a secreted anti-σ factor

    PubMed Central

    Ahuja, Umesh; Shokeen, Bhumika; Cheng, Ning; Cho, Yeonjoo; Blum, Charles; Coppola, Giovanni; Miller, Jeff F.

    2016-01-01

    The BvgAS phosphorelay regulates ∼10% of the annotated genomes of Bordetella pertussis and Bordetella bronchiseptica and controls their infectious cycles. The hierarchical organization of the regulatory network allows the integration of contextual signals to control all or specific subsets of BvgAS-regulated genes. Here, we characterize a regulatory node involving a type III secretion system (T3SS)-exported protein, BtrA, and demonstrate its role in determining fundamental differences in T3SS phenotypes among Bordetella species. We show that BtrA binds and antagonizes BtrS, a BvgAS-regulated extracytoplasmic function (ECF) sigma factor, to couple the secretory activity of the T3SS apparatus to gene expression. In B. bronchiseptica, a remarkable spectrum of expression states can be resolved by manipulating btrA, encompassing over 80 BtrA-activated loci that include genes encoding toxins, adhesins, and other cell surface proteins, and over 200 BtrA-repressed genes that encode T3SS apparatus components, secretion substrates, the BteA effector, and numerous additional factors. In B. pertussis, BtrA retains activity as a BtrS antagonist and exerts tight negative control over T3SS genes. Most importantly, deletion of btrA in B. pertussis revealed T3SS-mediated, BteA-dependent cytotoxicity, which had previously eluded detection. This effect was observed in laboratory strains and in clinical isolates from a recent California pertussis epidemic. We propose that the BtrA-BtrS regulatory node determines subspecies-specific differences in T3SS expression among Bordetella species and that B. pertussis is capable of expressing a full range of T3SS-dependent phenotypes in the presence of appropriate contextual cues. PMID:26884180

  5. Modification of Bacterial Effector Proteins Inside Eukaryotic Host Cells

    PubMed Central

    Popa, Crina M.; Tabuchi, Mitsuaki; Valls, Marc

    2016-01-01

    Pathogenic bacteria manipulate their hosts by delivering a number of virulence proteins -called effectors- directly into the plant or animal cells. Recent findings have shown that such effectors can suffer covalent modifications inside the eukaryotic cells. Here, we summarize the recent reports where effector modifications by the eukaryotic machinery have been described. We restrict our focus on proteins secreted by the type III or type IV systems, excluding other bacterial toxins. We describe the known examples of effectors whose enzymatic activity is triggered by interaction with plant and animal cell factors, including GTPases, E2-Ubiquitin conjugates, cyclophilin and thioredoxins. We focus on the structural interactions with these factors and their influence on effector function. We also review the described examples of host-mediated post-translational effector modifications which are required for proper subcellular location and function. These host-specific covalent modifications include phosphorylation, ubiquitination, SUMOylation, and lipidations such as prenylation, fatty acylation and phospholipid binding. PMID:27489796

  6. The Chlamydial Type III Secretion Mechanism: Revealing Cracks in a Tough Nut

    PubMed Central

    Betts-Hampikian, Helen Jennifer; Fields, Kenneth A.

    2010-01-01

    Present-day members of the Chlamydiaceae contain parasitic bacteria that have been co-evolving with their eukaryotic hosts over hundreds of millions of years. Likewise, a type III secretion system encoded within all genomes has been refined to complement the unique obligate intracellular niche colonized so successfully by Chlamydia spp. All this adaptation has occurred in the apparent absence of the horizontal gene transfer responsible for creating the wide range of diversity in other Gram-negative, type III-expressing bacteria. The result is a system that is, in many ways, uniquely chlamydial. A critical mass of information has been amassed that sheds significant light on how the chlamydial secretion system functions and contributes to an obligate intracellular lifestyle. Although the overall mechanism is certainly similar to homologous systems, an image has emerged where the chlamydial secretion system is essential for both survival and virulence. Numerous apparent differences, some subtle and some profound, differentiate chlamydial type III secretion from others. Herein, we provide a comprehensive review of the current state of knowledge regarding the Chlamydia type III secretion mechanism. We focus on the aspects that are distinctly chlamydial and comment on how this important system influences chlamydial pathogenesis. Gaining a grasp on this fascinating system has been challenging in the absence of a tractable genetic system. However, the surface of this tough nut has been scored and the future promises to be fruitful and revealing. PMID:21738522

  7. Genome-Wide Analysis of Small Secreted Cysteine-Rich Proteins Identifies Candidate Effector Proteins Potentially Involved in Fusarium graminearum-Wheat Interactions.

    PubMed

    Lu, Shunwen; Edwards, Michael C

    2016-02-01

    Pathogen-derived, small secreted cysteine-rich proteins (SSCPs) are known to be a common source of fungal effectors that trigger resistance or susceptibility in specific host plants. This group of proteins has not been well studied in Fusarium graminearum, the primary cause of Fusarium head blight (FHB), a devastating disease of wheat. We report here a comprehensive analysis of SSCPs encoded in the genome of this fungus and selection of candidate effector proteins through proteomics and sequence/transcriptional analyses. A total of 190 SSCPs were identified in the genome of F. graminearum (isolate PH-1) based on the presence of N-terminal signal peptide sequences, size (≤200 amino acids), and cysteine content (≥2%) of the mature proteins. Twenty-five (approximately 13%) SSCPs were confirmed to be true extracellular proteins by nanoscale liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) analysis of a minimal medium-based in vitro secretome. Sequence analysis suggested that 17 SSCPs harbor conserved functional domains, including two homologous to Ecp2, a known effector produced by the tomato pathogen Cladosporium fulvum. Transcriptional analysis revealed that at least 34 SSCPs (including 23 detected in the in vitro secretome) are expressed in infected wheat heads; about half are up-regulated with expression patterns correlating with the development of FHB. This work provides a solid candidate list for SSCP-derived effectors that may play roles in mediating F. graminearum-wheat interactions. The in vitro secretome-based method presented here also may be applicable for identifying candidate effectors in other ascomycete pathogens of crop plants. PMID:26524547

  8. Type III secretion system expression in oxygen-limited Pseudomonas aeruginosa cultures is stimulated by isocitrate lyase activity

    PubMed Central

    Chung, Jade C. S.; Rzhepishevska, Olena; Ramstedt, Madeleine; Welch, Martin

    2013-01-01

    Pseudomonas aeruginosa is an opportunistic human pathogen and a common cause of chronic infections in individuals with cystic fibrosis (CF). Oxygen limitation was recently reported to regulate the expression of a major virulence determinant in P. aeruginosa, the type III secretion system (T3SS). Here, we show that expression of the T3SS in oxygen-limited growth conditions is strongly dependent on the glyoxylate shunt enzyme, isocitrate lyase (ICL; encoded by aceA), which was previously shown to be highly expressed in CF isolates. ICL-dependent regulation of the T3SS did not alter the expression level of the master transcriptional regulator, ExsA, but did affect expression of the T3 structural proteins, effectors and regulators (ExsC, ExsD and ExsE). An aceA mutant displayed enhanced biofilm formation during anaerobic growth, which suggested that AceA-dependent modulation of type III secretion might impinge upon the RetS/LadS signalling pathways. Indeed, our data suggest that RetS is able to mediate some of its effects through AceA, as expression of aceA in trans partially restored T3SS expression in a retS mutant. Our findings indicate that AceA is a key player in the metabolic regulation of T3SS expression during oxygen-limited growth of P. aeruginosa. To the best of our knowledge, this is the first demonstration that the T3SS can be regulated by factors that do not affect ExsA expression levels. PMID:23363478

  9. Multiple activities of the plant pathogen type III effector proteins WtsE and AvrE require WxxxE motifs.

    PubMed

    Ham, Jong Hyun; Majerczak, Doris R; Nomura, Kinya; Mecey, Christy; Uribe, Francisco; He, Sheng-Yang; Mackey, David; Coplin, David L

    2009-06-01

    The broadly conserved AvrE-family of type III effectors from gram-negative plant-pathogenic bacteria includes important virulence factors, yet little is known about the mechanisms by which these effectors function inside plant cells to promote disease. We have identified two conserved motifs in AvrE-family effectors: a WxxxE motif and a putative C-terminal endoplasmic reticulum membrane retention/retrieval signal (ERMRS). The WxxxE and ERMRS motifs are both required for the virulence activities of WtsE and AvrE, which are major virulence factors of the corn pathogen Pantoea stewartii subsp. stewartii and the tomato or Arabidopsis pathogen Pseudomonas syringae pv. tomato, respectively. The WxxxE and the predicted ERMRS motifs are also required for other biological activities of WtsE, including elicitation of the hypersensitive response in nonhost plants and suppression of defense responses in Arabidopsis. A family of type III effectors from mammalian bacterial pathogens requires WxxxE and subcellular targeting motifs for virulence functions that involve their ability to mimic activated G-proteins. The conservation of related motifs and their necessity for the function of type III effectors from plant pathogens indicates that disturbing host pathways by mimicking activated host G-proteins may be a virulence mechanism employed by plant pathogens as well. PMID:19445595

  10. Inheritance of Pantoea type III secretion systems through both vertical and horizontal transfer.

    PubMed

    Kirzinger, Morgan W B; Butz, Cory J; Stavrinides, John

    2015-12-01

    The type III secretion system (T3SS) is an extracellular apparatus used by many Gram-negative bacteria to deliver effector proteins directly into plant and animal cells, thereby facilitating host-specific association. Strains of the enterobacterial genus, Pantoea, have been isolated from a wide variety of hosts, including plants, insects, and humans, yet it is unclear whether the T3SS may be involved in these associations. In this study, we use comparative genomics and phylogenetic methods to examine the origin and distribution of T3SSs in 35 sequenced environmental and clinical strains of Pantoea. We began our analysis by examining the distribution of the previously characterized plant cell-specific PSI-1 and animal cell-specific PSI-2 of the plant pathogenic Pantoea stewartii subsp. stewartii DC283 (PstDC283), and showed that both had a somewhat limited distribution. Our analysis, however, identified two variants of a unique plant cell-specific T3SS (PSI-1a and PSI-1b) in six Pantoea strains, including a clinical isolate. Our genome analysis of PstDC283 also identified a third T3SS that we named PSI-3, which has a similar genetic content and organization to the Salmonella, animal cell-specific SPI-2 system. Phylogenetic analysis of all three systems suggests that the PSI-1 system has been inherited vertically, whereas the newly identified PSI-1a and PSI-1b systems have been acquired independently from other genera within the Enterobacteriaceae. PSI-2 appears to have been acquired horizontally as far back as the Erwinia/Pantoea common ancestor, with evidence of more recent horizontal acquisition of the PSI-3 system. Our results suggest that Pantoea is a relatively old plant pathogen that has lost and subsequently regained different plant-associated T3SSs. This work has broad implications for understanding the host-associating capacity of Pantoea strains, and reveals the propensity for Pantoea isolates to exchange pathogenicity determinants with human

  11. The Aeromonas salmonicida subsp. salmonicida exoproteome: determination of the complete repertoire of Type-Three Secretion System effectors and identification of other virulence factors

    PubMed Central

    2013-01-01

    Background Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen of fisheries worldwide. Several virulence factors have been described, but the type-three secretion system (T3SS) is recognized as having a major effect on virulence by injecting effectors directly into fish cells. In this study we used high-throughput proteomics to display the differences between in vitro secretome of A. salmonicida wild-type (wt, hypervirulent, JF2267) and T3SS-deficient (isogenic ΔascV, extremely low-virulent, JF2747) strains in exponential and stationary phases of growth. Results Results confirmed the secretion of effectors AopH, AexT, AopP and AopO via T3SS, and for the first time demonstrated the impact of T3SS in secretion of Ati2, AopN and ExsE that are known as effectors in other pathogens. Translocators, needle subunits, Ati1, and AscX were also secreted in supernatants (SNs) dependent on T3SS. AopH, Ati2, AexT, AopB and AopD were in the top seven most abundant excreted proteins. EF-G, EF-Tu, DnaK, HtpG, PNPase, PepN and MdeA were moderately secreted in wt SNs and predicted to be putative T3 effectors by bioinformatics. Pta and ASA_P5G088 were increased in wt SNs and T3-associated in other bacteria. Ten conserved cytoplasmic proteins were more abundant in wt SNs than in the ΔascV mutant, but without any clear association to a secretion system. T1-secreted proteins were predominantly found in wt SNs: OmpAI, OmpK40, DegQ, insulinase ASA_0716, hypothetical ASA_0852 and ASA_3619. Presence of T3SS components in pellets was clearly decreased by ascV deletion, while no impact was observed on T1- and T2SS. Our results demonstrated that the ΔascV mutant strain excreted well-described (VapA, AerA, AerB, GCAT, Pla1, PlaC, TagA, Ahe2, GbpA and enolase) and yet uncharacterized potential toxins, adhesins and enzymes as much as or even more than the wt strain. Other putative important virulence factors were not detected. Conclusions We

  12. Functional Characterization of SsaE, a Novel Chaperone Protein of the Type III Secretion System Encoded by Salmonella Pathogenicity Island 2▿

    PubMed Central

    Miki, Tsuyoshi; Shibagaki, Yoshio; Danbara, Hirofumi; Okada, Nobuhiko

    2009-01-01

    The type III secretion system (T3SS) encoded by Salmonella pathogenicity island 2 (SPI-2) is involved in systemic infection and intracellular replication of Salmonella enterica serovar Typhimurium. In this study, we investigated the function of SsaE, a small cytoplasmic protein encoded within the SPI-2 locus, which shows structural similarity to the T3SS class V chaperones. An S. enterica serovar Typhimurium ssaE mutant failed to secrete SPI-2 translocator SseB and SPI-2-dependent effector PipB proteins. Coimmunoprecipitation and mass spectrometry analyses using an SsaE-FLAG fusion protein indicated that SsaE interacts with SseB and a putative T3SS-associated ATPase, SsaN. A series of deleted and point-mutated SsaE-FLAG fusion proteins revealed that the C-terminal coiled-coil domain of SsaE is critical for protein-protein interactions. Although SseA was reported to be a chaperone for SseB and to be required for its secretion and stability in the bacterial cytoplasm, an sseA deletion mutant was able to secrete the SseB in vitro when plasmid-derived SseB was overexpressed. In contrast, ssaE mutant strains could not transport SseB extracellularly under the same assay conditions. In addition, an ssaE(I55G) point-mutated strain that expresses the SsaE derivative lacking the ability to form a C-terminal coiled-coil structure showed attenuated virulence comparable to that of an SPI-2 T3SS null mutant, suggesting that the coiled-coil interaction of SsaE is absolutely essential for the functional SPI-2 T3SS and for Salmonella virulence. Based on these findings, we propose that SsaE recognizes translocator SseB and controls its secretion via SPI-2 type III secretion machinery. PMID:19767440

  13. Effect of the O-antigen length of lipopolysaccharide on the functions of Type III secretion systems in Salmonella enterica.

    PubMed

    Hölzer, Stefanie U; Schlumberger, Markus C; Jäckel, Daniela; Hensel, Michael

    2009-12-01

    The virulence of Salmonella enterica critically depends on the functions of two type III secretion systems (T3SS), with the Salmonella pathogenicity island 1 (SPI1)-encoded T3SS required for host cell invasion and the SPI2-T3SS enabling Salmonella to proliferate within host cells. A further T3SS is required for the assembly of the flagella. Most serovars of Salmonella also possess a lipopolysaccharide with a complex O-antigen (OAg) structure. The number of OAg units attached to the core polysaccharide varies between 16 and more than 100 repeats, with a trimodal distribution. This work investigated the correlation of the OAg length with the functions of the SPI1-T3SS and the SPI2-T3SS. We observed that the number of repeats of OAg units had no effect on bacterial motility. The interaction of Salmonella with epithelial cells was altered if the OAg structure was changed by mutations in regulators of OAg. Strains defective in synthesis of very long or long and very long OAg species showed increased translocation of a SPI1-T3SS effector protein and increased invasion. Invasion of a strain entirely lacking OAg was increased, but this mutant strain also showed increased adhesion. In contrast, translocation of a SPI2-T3SS effector protein and intracellular replication were not affected by modification of the OAg length. Mutant strains lacking the entire OAg or long and very long OAg were highly susceptible to complement killing. These observations indicate that the architecture of the outer membrane of Salmonella is balanced to permit sufficient T3SS function but also to confer optimal protection against antimicrobial defense mechanisms. PMID:19797066

  14. A common assembly module in injectisome and flagellar type III secretion sorting platforms

    NASA Astrophysics Data System (ADS)

    Notti, Ryan Q.; Bhattacharya, Shibani; Lilic, Mirjana; Stebbins, C. Erec

    2015-05-01

    Translocating proteins across the double membrane of Gram-negative bacteria, type III secretion systems (T3SS) occur in two evolutionarily related forms: injectisomes, delivering virulence factors into host cells, and the flagellar system, secreting the polymeric filament used for motility. While both systems share related elements of a cytoplasmic sorting platform that facilitates the hierarchical secretion of protein substrates, its assembly and regulation remain unclear. Here we describe a module mediating the assembly of the sorting platform in both secretion systems, and elucidate the structural basis for segregation of homologous components among these divergent T3SS subtypes sharing a common cytoplasmic milieu. These results provide a foundation for the subtype-specific assembly of T3SS sorting platforms and will support further mechanistic analysis and anti-virulence drug design.

  15. Live attenuated Salmonella vaccines against Mycobacterium tuberculosis with antigen delivery via the type III secretion system.

    PubMed

    Juárez-Rodríguez, María Dolores; Arteaga-Cortés, Lourdes T; Kader, Rebin; Curtiss, Roy; Clark-Curtiss, Josephine E

    2012-02-01

    Tuberculosis remains a global health threat, and there is dire need to develop a vaccine that is safe and efficacious and confers long-lasting protection. In this study, we constructed recombinant attenuated Salmonella vaccine (RASV) strains with plasmids expressing fusion proteins consisting of the 80 amino-terminal amino acids of the type 3 secretion system effector SopE of Salmonella and the Mycobacterium tuberculosis antigens early secreted antigenic target 6-kDa (ESAT-6) protein and culture filtrate protein 10 (CFP-10). We demonstrated that the SopE-mycobacterial antigen fusion proteins were translocated into the cytoplasm of INT-407 cells in cell culture assays. Oral immunization of mice with RASV strains synthesizing SopE-ESAT-6-CFP-10 fusion proteins resulted in significant protection of the mice against aerosol challenge with M. tuberculosis H37Rv that was similar to the protection afforded by immunization with Mycobacterium bovis bacillus Calmette-Guérin (BCG) administered subcutaneously. In addition, oral immunization with the RASV strains specifying these mycobacterial antigens elicited production of significant antibody titers to ESAT-6 and production of ESAT-6- or CFP-10-specific gamma interferon (IFN-γ)-secreting and tumor necrosis factor alpha (TNF-α)-secreting splenocytes. PMID:22144486

  16. Perturbation of maize phenylpropanoid metabolism by an AvrE family type III effector from Pantoea stewartii.

    PubMed

    Asselin, Jo Ann E; Lin, Jinshan; Perez-Quintero, Alvaro L; Gentzel, Irene; Majerczak, Doris; Opiyo, Stephen O; Zhao, Wanying; Paek, Seung-Mann; Kim, Min Gab; Coplin, David L; Blakeslee, Joshua J; Mackey, David

    2015-03-01

    AvrE family type III effector proteins share the ability to suppress host defenses, induce disease-associated cell death, and promote bacterial growth. However, despite widespread contributions to numerous bacterial diseases in agriculturally important plants, the mode of action of these effectors remains largely unknown. WtsE is an AvrE family member required for the ability of Pantoea stewartii ssp. stewartii (Pnss) to proliferate efficiently and cause wilt and leaf blight symptoms in maize (Zea mays) plants. Notably, when WtsE is delivered by a heterologous system into the leaf cells of susceptible maize seedlings, it alone produces water-soaked disease symptoms reminiscent of those produced by Pnss. Thus, WtsE is a pathogenicity and virulence factor in maize, and an Escherichia coli heterologous delivery system can be used to study the activity of WtsE in isolation from other factors produced by Pnss. Transcriptional profiling of maize revealed the effects of WtsE, including induction of genes involved in secondary metabolism and suppression of genes involved in photosynthesis. Targeted metabolite quantification revealed that WtsE perturbs maize metabolism, including the induction of coumaroyl tyramine. The ability of mutant WtsE derivatives to elicit transcriptional and metabolic changes in susceptible maize seedlings correlated with their ability to promote disease. Furthermore, chemical inhibitors that block metabolic flux into the phenylpropanoid pathways targeted by WtsE also disrupted the pathogenicity and virulence activity of WtsE. While numerous metabolites produced downstream of the shikimate pathway are known to promote plant defense, our results indicate that misregulated induction of phenylpropanoid metabolism also can be used to promote pathogen virulence. PMID:25635112

  17. Perturbation of Maize Phenylpropanoid Metabolism by an AvrE Family Type III Effector from Pantoea stewartii1[OPEN

    PubMed Central

    Asselin, Jo Ann E.; Lin, Jinshan; Perez-Quintero, Alvaro L.; Gentzel, Irene; Majerczak, Doris; Opiyo, Stephen O.; Zhao, Wanying; Paek, Seung-Mann; Kim, Min Gab; Coplin, David L.; Blakeslee, Joshua J.; Mackey, David

    2015-01-01

    AvrE family type III effector proteins share the ability to suppress host defenses, induce disease-associated cell death, and promote bacterial growth. However, despite widespread contributions to numerous bacterial diseases in agriculturally important plants, the mode of action of these effectors remains largely unknown. WtsE is an AvrE family member required for the ability of Pantoea stewartii ssp. stewartii (Pnss) to proliferate efficiently and cause wilt and leaf blight symptoms in maize (Zea mays) plants. Notably, when WtsE is delivered by a heterologous system into the leaf cells of susceptible maize seedlings, it alone produces water-soaked disease symptoms reminiscent of those produced by Pnss. Thus, WtsE is a pathogenicity and virulence factor in maize, and an Escherichia coli heterologous delivery system can be used to study the activity of WtsE in isolation from other factors produced by Pnss. Transcriptional profiling of maize revealed the effects of WtsE, including induction of genes involved in secondary metabolism and suppression of genes involved in photosynthesis. Targeted metabolite quantification revealed that WtsE perturbs maize metabolism, including the induction of coumaroyl tyramine. The ability of mutant WtsE derivatives to elicit transcriptional and metabolic changes in susceptible maize seedlings correlated with their ability to promote disease. Furthermore, chemical inhibitors that block metabolic flux into the phenylpropanoid pathways targeted by WtsE also disrupted the pathogenicity and virulence activity of WtsE. While numerous metabolites produced downstream of the shikimate pathway are known to promote plant defense, our results indicate that misregulated induction of phenylpropanoid metabolism also can be used to promote pathogen virulence. PMID:25635112

  18. Correlating levels of type III secretion and secreted proteins with fecal shedding of Escherichia coli O157:H7 in cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The locus of enterocyte effacement (LEE) encodes a type III secretion system (T3SS) for secreting factors that enable Escherichia coli O157:H7 to produce attaching and effacing lesions (A/E) on epithelial cells. The importance of LEE-encoded proteins in intestinal colonization of cattle is well-stud...

  19. Escherichia coli Type III Secretion System 2 ATPase EivC Is Involved in the Motility and Virulence of Avian Pathogenic Escherichia coli

    PubMed Central

    Wang, Shaohui; Liu, Xin; Xu, Xuan; Yang, Denghui; Wang, Dong; Han, Xiangan; Shi, Yonghong; Tian, Mingxing; Ding, Chan; Peng, Daxin; Yu, Shengqing

    2016-01-01

    Type III secretion systems (T3SSs) are crucial for bacterial infections because they deliver effector proteins into host cells. The Escherichia coli type III secretion system 2 (ETT2) is present in the majority of E. coli strains, and although it is degenerate, ETT2 regulates bacterial virulence. An ATPase is essential for T3SS secretion, but the function of the ETT2 ATPase has not been demonstrated. Here, we show that EivC is homologous to the β subunit of F0F1 ATPases and it possesses ATPase activity. To investigate the effects of ETT2 ATPase EivC on the phenotype and virulence of avian pathogenic Escherichia coli (APEC), eivC mutant and complemented strains were constructed and characterized. Inactivation of eivC led to impaired flagella production and augmented fimbriae on the bacterial surface, and, consequently, reduced bacterial motility. In addition, the eivC mutant strain exhibited attenuated virulence in ducks, diminished serum resistance, reduced survival in macrophage cells and in ducks, upregulated fimbrial gene expression, and downregulated flagellar and virulence gene expression. The expression of the inflammatory cytokines interleukin (IL)-1β and IL-8 were increased in HD-11 macrophages infected with the eivC mutant strain, compared with the wild-type strain. These virulence-related phenotypes were restored by genetic complementation. These findings demonstrate that ETT2 ATPase EivC is involved in the motility and pathogenicity of APEC.

  20. Oligomeric states of the Shigella translocator protein IpaB provide structural insights into formation of the type III secretion translocon

    PubMed Central

    Dickenson, Nicholas E; Choudhari, Shyamal P; Adam, Philip R; Kramer, Ryan M; Joshi, Sangeeta B; Middaugh, C Russell; Picking, Wendy L; Picking, William D

    2013-01-01

    The Shigella flexneri Type III secretion system (T3SS) senses contact with human intestinal cells and injects effector proteins that promote pathogen entry as the first step in causing life threatening bacillary dysentery (shigellosis). The Shigella Type III secretion apparatus (T3SA) consists of an anchoring basal body, an exposed needle, and a temporally assembled tip complex. Exposure to environmental small molecules recruits IpaB, the first hydrophobic translocator protein, to the maturing tip complex. IpaB then senses contact with a host cell membrane, forming the translocon pore through which effectors are delivered to the host cytoplasm. Within the bacterium, IpaB exists as a heterodimer with its chaperone IpgC; however, IpaB's structural state following secretion is unknown due to difficulties isolating stable protein. We have overcome this by coexpressing the IpaB/IpgC heterodimer and isolating IpaB by incubating the complex in mild detergents. Interestingly, preparation of IpaB with n-octyl-oligo-oxyethylene (OPOE) results in the assembly of discrete oligomers while purification in N,N-dimethyldodecylamine N-oxide (LDAO) maintains IpaB as a monomer. In this study, we demonstrate that IpaB tetramers penetrate phospholipid membranes to allow a size-dependent release of small molecules, suggesting the formation of discrete pores. Monomeric IpaB also interacts with liposomes but fails to disrupt them. From these and additional findings, we propose that IpaB can exist as a tetramer having inherent flexibility, which allows it to cooperatively interact with and insert into host cell membranes. This event may then lay the foundation for formation of the Shigella T3SS translocon pore. PMID:23456854

  1. Structural Characterization of the Yersinia pestis Type III Secretion System Needle Protein YscF in Complex with Its Heterodimeric Chaperone YscE/YscG

    SciTech Connect

    Sun, Ping; Tropea, Joseph E.; Austin, Brian P.; Cherry, Scott; Waugh, David S.

    2008-05-03

    The plague-causing bacterium Yersinia pestis utilizes a type III secretion system to deliver effector proteins into mammalian cells where they interfere with signal transduction pathways that mediate phagocytosis and the inflammatory response. Effector proteins are injected through a hollow needle structure composed of the protein YscF. YscG and YscE act as 'chaperones' to prevent premature polymerization of YscF in the cytosol of the bacterium prior to assembly of the needle. Here, we report the crystal structure of the YscEFG protein complex at 1.8 {angstrom} resolution. Overall, the structure is similar to that of the analogous PscEFG complex from the Pseudomonas aeruginosa type III secretion system, but there are noteworthy differences. The structure confirms that, like PscG, YscG is a member of the tetratricopeptide repeat family of proteins. YscG binds tightly to the C-terminal half of YscF, implying that it is this region of YscF that controls its polymerization into the needle structure. YscE interacts with the N-terminal tetratricopeptide repeat motif of YscG but makes very little direct contact with YscF. Its function may be to stabilize the structure of YscG and/or to participate in recruiting the complex to the secretion apparatus. No electron density could be observed for the 49 N-terminal residues of YscF. This and additional evidence suggest that the N-terminus of YscF is disordered in the complex with YscE and YscG. As expected, conserved residues in the C-terminal half of YscF mediate important intra- and intermolecular interactions in the complex. Moreover, the phenotypes of some previously characterized mutations in the C-terminal half of YscF can be rationalized in terms of the structure of the heterotrimeric YscEFG complex.

  2. Small-molecule inhibitors suppress the expression of both type III secretion and amylovoran biosynthesis genes in Erwinia amylovora.

    PubMed

    Yang, Fan; Korban, Schuyler S; Pusey, P Lawrence; Elofsson, Michael; Sundin, George W; Zhao, Youfu

    2014-01-01

    The type III secretion system (T3SS) and exopolysaccharide (EPS) amylovoran are two essential pathogenicity factors in Erwinia amylovora, the causal agent of the serious bacterial disease fire blight. In this study, small molecules that inhibit T3SS gene expression in E. amylovora under hrp (hypersensitive response and pathogenicity)-inducing conditions were identified and characterized using green fluorescent protein (GFP) as a reporter. These compounds belong to salicylidene acylhydrazides and also inhibit amylovoran production. Microarray analysis of E. amylovora treated with compounds 3 and 9 identified a total of 588 significantly differentially expressed genes. Among them, 95 and 78 genes were activated and suppressed by both compounds, respectively, when compared with the dimethylsulphoxide (DMSO) control. The expression of the majority of T3SS genes in E. amylovora, including hrpL and the avrRpt2 effector gene, was suppressed by both compounds. Compound 3 also suppressed the expression of amylovoran precursor and biosynthesis genes. However, both compounds induced significantly the expression of glycogen biosynthesis genes and siderophore biosynthesis, regulatory and transport genes. Furthermore, many membrane, lipoprotein and exported protein-encoding genes were also activated by both compounds. Similar expression patterns were observed for compounds 1, 2 and 4. Using crab apple flower as a model, compound 3 was capable of reducing disease development in pistils. These results suggest a common inhibition mechanism shared by salicylidene acylhydrazides and indicate that small-molecule inhibitors that disable T3SS function could be explored to control fire blight disease. PMID:23915008

  3. The NAIP-NLRC4 inflammasome in innate immune detection of bacterial flagellin and type III secretion apparatus.

    PubMed

    Zhao, Yue; Shao, Feng

    2015-05-01

    Bacterial flagella and type III secretion system (T3SS) are evolutionarily related molecular transport machineries. Flagella mediate bacterial motility; the T3SS delivers virulence effectors to block host defenses. The inflammasome is a cytosolic multi-protein complex that activates caspase-1. Active caspase-1 triggers interleukin-1β (IL-1β)/IL-18 maturation and macrophage pyroptotic death to mount an inflammatory response. Central to the inflammasome is a pattern recognition receptor that activates caspase-1 either directly or through an adapter protein. Studies in the past 10 years have established a NAIP-NLRC4 inflammasome, in which NAIPs are cytosolic receptors for bacterial flagellin and T3SS rod/needle proteins, while NLRC4 acts as an adapter for caspase-1 activation. Given the wide presence of flagella and the T3SS in bacteria, the NAIP-NLRC4 inflammasome plays a critical role in anti-bacteria defenses. Here, we review the discovery of the NAIP-NLRC4 inflammasome and further discuss recent advances related to its biochemical mechanism and biological function as well as its connection to human autoinflammatory disease. PMID:25879286

  4. Mathematical Model for Length Control by the Timing of Substrate Switching in the Type III Secretion System

    PubMed Central

    Nariya, Maulik K.; Israeli, Johnny; Shi, Jack J.; Deeds, Eric J.

    2016-01-01

    Type III Secretion Systems (T3SS) are complex bacterial structures that provide gram-negative pathogens with a unique virulence mechanism whereby they grow a needle-like structure in order to inject bacterial effector proteins into the cytoplasm of a host cell. Numerous experiments have been performed to understand the structural details of this nanomachine during the past decade. Despite the concerted efforts of molecular and structural biologists, several crucial aspects of the assembly of this structure, such as the regulation of the length of the needle itself, remain unclear. In this work, we used a combination of mathematical and computational techniques to better understand length control based on the timing of substrate switching, which is a possible mechanism for how bacteria ensure that the T3SS needles are neither too short nor too long. In particular, we predicted the form of the needle length distribution based on this mechanism, and found excellent agreement with available experimental data from Salmonella typhimurium with only a single free parameter. Although our findings provide preliminary evidence in support of the substrate switching model, they also make a set of quantitative predictions that, if tested experimentally, would assist in efforts to unambiguously characterize the regulatory mechanisms that control the growth of this crucial virulence factor. PMID:27078235

  5. Mathematical Model for Length Control by the Timing of Substrate Switching in the Type III Secretion System.

    PubMed

    Nariya, Maulik K; Israeli, Johnny; Shi, Jack J; Deeds, Eric J

    2016-04-01

    Type III Secretion Systems (T3SS) are complex bacterial structures that provide gram-negative pathogens with a unique virulence mechanism whereby they grow a needle-like structure in order to inject bacterial effector proteins into the cytoplasm of a host cell. Numerous experiments have been performed to understand the structural details of this nanomachine during the past decade. Despite the concerted efforts of molecular and structural biologists, several crucial aspects of the assembly of this structure, such as the regulation of the length of the needle itself, remain unclear. In this work, we used a combination of mathematical and computational techniques to better understand length control based on the timing of substrate switching, which is a possible mechanism for how bacteria ensure that the T3SS needles are neither too short nor too long. In particular, we predicted the form of the needle length distribution based on this mechanism, and found excellent agreement with available experimental data from Salmonella typhimurium with only a single free parameter. Although our findings provide preliminary evidence in support of the substrate switching model, they also make a set of quantitative predictions that, if tested experimentally, would assist in efforts to unambiguously characterize the regulatory mechanisms that control the growth of this crucial virulence factor. PMID:27078235

  6. Immunization with chlamydial type III secretion antigens reduces vaginal shedding and prevents fallopian tube pathology following live C. muridarum challenge.

    PubMed

    Bulir, David C; Liang, Steven; Lee, Amanda; Chong, Sylvia; Simms, Elizabeth; Stone, Christopher; Kaushic, Charu; Ashkar, Ali; Mahony, James B

    2016-07-25

    Chlamydia trachomatis infections in women are often asymptomatic and if left untreated can lead to significant late sequelae including pelvic inflammatory disease and tubal factor infertility. Vaccine development efforts over the past three decades have been unproductive and there is no vaccine approved for use in humans. The existence of serologically distinct strains or serovars of C. trachomatis mandates a vaccine that will provide protection against multiple serovars. Chlamydia spp. use a highly conserved type III secretion system (T3SS) composed of both structural and effector proteins which is an essential virulence factor for infection and intracellular replication. In this study we evaluated a novel fusion protein antigen (BD584) which consists of three T3SS proteins from C. trachomatis (CopB, CopD, and CT584) as a potential chlamydial vaccine candidate. Intranasal immunization with BD584 elicited serum neutralizing antibodies that inhibited C. trachomatis infection in vitro. Following intravaginal challenge with C. muridarum, immunized mice had a 95% reduction in chlamydial shedding from the vagina at the peak of infection and cleared the infection sooner than control mice. Immunization with BD584 also reduced the rate of hydrosalpinx by 87.5% compared to control mice. Together, these results suggest that highly conserved proteins of the chlamydial T3SS may represent good candidates for a Chlamydia vaccine. PMID:27325352

  7. Allele-Specific Virulence Attenuation of the Pseudomonas syringae HopZ1a Type III Effector via the Arabidopsis ZAR1 Resistance Protein

    PubMed Central

    Lewis, Jennifer D.; Wu, Ronald

    2010-01-01

    Plant resistance (R) proteins provide a robust surveillance system to defend against potential pathogens. Despite their importance in plant innate immunity, relatively few of the ∼170 R proteins in Arabidopsis have well-characterized resistance specificity. In order to identify the R protein responsible for recognition of the Pseudomonas syringae type III secreted effector (T3SE) HopZ1a, we assembled an Arabidopsis R gene T–DNA Insertion Collection (ARTIC) from publicly available Arabidopsis thaliana insertion lines and screened it for plants lacking HopZ1a-induced immunity. This reverse genetic screen revealed that the Arabidopsis R protein HOPZ-ACTIVATED RESISTANCE 1 (ZAR1; At3g50950) is required for recognition of HopZ1a in Arabidopsis. ZAR1 belongs to the coiled-coil (CC) class of nucleotide binding site and leucine-rich repeat (NBS–LRR) containing R proteins; however, the ZAR1 CC domain phylogenetically clusters in a clade distinct from other related Arabidopsis R proteins. ZAR1–mediated immunity is independent of several genes required by other R protein signaling pathways, including NDR1 and RAR1, suggesting that ZAR1 possesses distinct signaling requirements. The closely-related T3SE protein, HopZ1b, is still recognized by zar1 Arabidopsis plants indicating that Arabidopsis has evolved at least two independent R proteins to recognize the HopZ T3SE family. Also, in Arabidopsis zar1 plants HopZ1a promotes P. syringae growth indicative of an ancestral virulence function for this T3SE prior to the evolution of recognition by the host resistance protein ZAR1. Our results demonstrate that the Arabidopsis resistance protein ZAR1 confers allele-specific recognition and virulence attenuation of the Pseudomonas syringae T3SE protein HopZ1a. PMID:20368970

  8. pH sensing by intracellular Salmonella induces effector translocation.

    PubMed

    Yu, Xiu-Jun; McGourty, Kieran; Liu, Mei; Unsworth, Kate E; Holden, David W

    2010-05-21

    Salmonella enterica is an important intracellular bacterial pathogen of humans and animals. It replicates within host-cell vacuoles by delivering virulence (effector) proteins through a vacuolar membrane pore made by the Salmonella pathogenicity island 2 (SPI-2) type III secretion system (T3SS). T3SS assembly follows vacuole acidification, but when bacteria are grown at low pH, effector secretion is negligible. We found that effector secretion was activated at low pH from mutant strains lacking a complex of SPI-2-encoded proteins SsaM, SpiC, and SsaL. Exposure of wild-type bacteria to pH 7.2 after growth at pH 5.0 caused dissociation and degradation of SsaM/SpiC/SsaL complexes and effector secretion. In infected cells, loss of the pH 7.2 signal through acidification of host-cell cytosol prevented complex degradation and effector translocation. Thus, intravacuolar Salmonella senses host cytosolic pH, resulting in the degradation of regulatory complex proteins and effector translocation. PMID:20395475

  9. Membrane and chaperone recognition by the major translocator protein PopB of the type III secretion system of Pseudomonas aeruginosa.

    PubMed

    Discola, Karen F; Förster, Andreas; Boulay, François; Simorre, Jean-Pierre; Attree, Ina; Dessen, Andréa; Job, Viviana

    2014-02-01

    The type III secretion system is a widespread apparatus used by pathogenic bacteria to inject effectors directly into the cytoplasm of eukaryotic cells. A key component of this highly conserved system is the translocon, a pore formed in the host membrane that is essential for toxins to bypass this last physical barrier. In Pseudomonas aeruginosa the translocon is composed of PopB and PopD, both of which before secretion are stabilized within the bacterial cytoplasm by a common chaperone, PcrH. In this work we characterize PopB, the major translocator, in both membrane-associated and PcrH-bound forms. By combining sucrose gradient centrifugation experiments, limited proteolysis, one-dimensional NMR, and β-lactamase reporter assays on eukaryotic cells, we show that PopB is stably inserted into bilayers with its flexible N-terminal domain and C-terminal tail exposed to the outside. In addition, we also report the crystal structure of the complex between PcrH and an N-terminal region of PopB (residues 51-59), which reveals that PopB lies within the concave face of PcrH, employing mostly backbone residues for contact. PcrH is thus the first chaperone whose structure has been solved in complex with both type III secretion systems translocators, revealing that both molecules employ the same surface for binding and excluding the possibility of formation of a ternary complex. The characterization of the major type III secretion system translocon component in both membrane-bound and chaperone-bound forms is a key step for the eventual development of antibacterials that block translocon assembly. PMID:24297169

  10. Membrane and Chaperone Recognition by the Major Translocator Protein PopB of the Type III Secretion System of Pseudomonas aeruginosa*

    PubMed Central

    Discola, Karen F.; Förster, Andreas; Boulay, François; Simorre, Jean-Pierre; Attree, Ina; Dessen, Andréa; Job, Viviana

    2014-01-01

    The type III secretion system is a widespread apparatus used by pathogenic bacteria to inject effectors directly into the cytoplasm of eukaryotic cells. A key component of this highly conserved system is the translocon, a pore formed in the host membrane that is essential for toxins to bypass this last physical barrier. In Pseudomonas aeruginosa the translocon is composed of PopB and PopD, both of which before secretion are stabilized within the bacterial cytoplasm by a common chaperone, PcrH. In this work we characterize PopB, the major translocator, in both membrane-associated and PcrH-bound forms. By combining sucrose gradient centrifugation experiments, limited proteolysis, one-dimensional NMR, and β-lactamase reporter assays on eukaryotic cells, we show that PopB is stably inserted into bilayers with its flexible N-terminal domain and C-terminal tail exposed to the outside. In addition, we also report the crystal structure of the complex between PcrH and an N-terminal region of PopB (residues 51–59), which reveals that PopB lies within the concave face of PcrH, employing mostly backbone residues for contact. PcrH is thus the first chaperone whose structure has been solved in complex with both type III secretion systems translocators, revealing that both molecules employ the same surface for binding and excluding the possibility of formation of a ternary complex. The characterization of the major type III secretion system translocon component in both membrane-bound and chaperone-bound forms is a key step for the eventual development of antibacterials that block translocon assembly. PMID:24297169

  11. Pseudomonas syringae type III effector HopAF1 suppresses plant immunity by targeting methionine recycling to block ethylene induction

    PubMed Central

    Washington, Erica J.; Mukhtar, M. Shahid; Finkel, Omri M.; Wan, Li; Kieber, Joseph J.; Dangl, Jeffery L.

    2016-01-01

    HopAF1 is a type III effector protein of unknown function encoded in the genomes of several strains of Pseudomonas syringae and other plant pathogens. Structural modeling predicted that HopAF1 is closely related to deamidase proteins. Deamidation is the irreversible substitution of an amide group with a carboxylate group. Several bacterial virulence factors are deamidases that manipulate the activity of specific host protein substrates. We identified Arabidopsis methylthioadenosine nucleosidase proteins MTN1 and MTN2 as putative targets of HopAF1 deamidation. MTNs are enzymes in the Yang cycle, which is essential for the high levels of ethylene biosynthesis in Arabidopsis. We hypothesized that HopAF1 inhibits the host defense response by manipulating MTN activity and consequently ethylene levels. We determined that bacterially delivered HopAF1 inhibits ethylene biosynthesis induced by pathogen-associated molecular patterns and that Arabidopsis mtn1 mtn2 mutant plants phenocopy the effect of HopAF1. Furthermore, we identified two conserved asparagines in MTN1 and MTN2 from Arabidopsis that confer loss of function phenotypes when deamidated via site-specific mutation. These residues are potential targets of HopAF1 deamidation. HopAF1-mediated manipulation of Yang cycle MTN proteins is likely an evolutionarily conserved mechanism whereby HopAF1 orthologs from multiple plant pathogens contribute to disease in a large variety of plant hosts. PMID:27274076

  12. Evaluation of the roles played by Hcp and VgrG type 6 secretion system effectors in Aeromonas hydrophila SSU pathogenesis

    PubMed Central

    Sha, Jian; Rosenzweig, Jason A.; Kozlova, Elena V.; Wang, Shaofei; Erova, Tatiana E.; Kirtley, Michelle L.; van Lier, Christina J.

    2013-01-01

    Aeromonas hydrophila, a Gram-negative bacterium, is an emerging human pathogen equipped with both a type 3 and a type 6 secretion system (T6SS). In this study, we evaluated the roles played by paralogous T6SS effector proteins, hemolysin co-regulated proteins (Hcp-1 and -2) and valine glycine repeat G (VgrG-1, -2 and -3) protein family members in A. hydrophila SSU pathogenesis by generating various combinations of deletion mutants of the their genes. In addition to their predicted roles as structural components and effector proteins of the T6SS, our data clearly demonstrated that paralogues of Hcp and VgrG also influenced bacterial motility, protease production and biofilm formation. Surprisingly, there was limited to no observed functional redundancy among and/or between the aforementioned T6SS effector paralogues in multiple assays. Our data indicated that Hcp and VgrG paralogues located within the T6SS cluster were more involved in forming T6SS structures, while the primary roles of Hcp-1 and VgrG-1, located outside of the T6SS cluster, were as T6SS effectors. In terms of influence on bacterial physiology, Hcp-1, but not Hcp-2, influenced bacterial motility and protease production, and in its absence, increases in both of the aforementioned activities were observed. Likewise, VgrG-1 played a major role in regulating bacterial protease production, while VgrG-2 and VgrG-3 were critical in regulating bacterial motility and biofilm formation. In an intraperitoneal murine model of infection, all Hcp and VgrG paralogues were required for optimal bacterial virulence and dissemination to mouse peripheral organs. Importantly, the observed phenotypic alterations of the T6SS mutants could be fully complemented. Taking these results together, we have further established the roles played by the two known T6SS effectors of A. hydrophila by defining their contributions to T6SS function and virulence in both in vitro and in vivo models of infection. PMID:23519162

  13. Expression and Quorum Sensing Regulation of Type III Secretion System Genes of Vibrio harveyi during Infection of Gnotobiotic Brine Shrimp

    PubMed Central

    Ruwandeepika, H. A. Darshanee; Karunasagar, Indrani; Bossier, Peter; Defoirdt, Tom

    2015-01-01

    Type III secretion systems enable pathogens to inject their virulence factors directly into the cytoplasm of the host cells. The type III secretion system of Vibrio harveyi, a major pathogen of aquatic organisms and a model species in quorum sensing studies, is repressed by the quorum sensing master regulator LuxR. In this study, we found that during infection of gnotobiotic brine shrimp larvae, the expression levels of three type III secretion operons in V. harveyi increased within the first 12h after challenge and decreased again thereafter. The in vivo expression levels were highest in a mutant with a quorum sensing system that is locked in low cell density configuration (minimal LuxR levels) and lowest in a mutant with a quorum sensing system that is locked in the high cell density configuration (maximal LuxR levels), which is consistent with repression of type III secretion by LuxR. Remarkably, in vivo expression levels of the type III secretion system genes were much (> 1000 fold) higher than the in vitro expression levels, indicating that (currently unknown) host factors significantly induce the type III secretion system. Given the fact that type III secretion is energy-consuming, repression by the quorum sensing master regulators might be a mechanism to save energy under conditions where it does not provide an advantage to the cells. PMID:26636765

  14. Expression and Quorum Sensing Regulation of Type III Secretion System Genes of Vibrio harveyi during Infection of Gnotobiotic Brine Shrimp.

    PubMed

    Ruwandeepika, H A Darshanee; Karunasagar, Indrani; Bossier, Peter; Defoirdt, Tom

    2015-01-01

    Type III secretion systems enable pathogens to inject their virulence factors directly into the cytoplasm of the host cells. The type III secretion system of Vibrio harveyi, a major pathogen of aquatic organisms and a model species in quorum sensing studies, is repressed by the quorum sensing master regulator LuxR. In this study, we found that during infection of gnotobiotic brine shrimp larvae, the expression levels of three type III secretion operons in V. harveyi increased within the first 12h after challenge and decreased again thereafter. The in vivo expression levels were highest in a mutant with a quorum sensing system that is locked in low cell density configuration (minimal LuxR levels) and lowest in a mutant with a quorum sensing system that is locked in the high cell density configuration (maximal LuxR levels), which is consistent with repression of type III secretion by LuxR. Remarkably, in vivo expression levels of the type III secretion system genes were much (> 1000 fold) higher than the in vitro expression levels, indicating that (currently unknown) host factors significantly induce the type III secretion system. Given the fact that type III secretion is energy-consuming, repression by the quorum sensing master regulators might be a mechanism to save energy under conditions where it does not provide an advantage to the cells. PMID:26636765

  15. Salmonella-secreted Virulence Factors

    SciTech Connect

    Heffron, Fred; Niemann, George; Yoon, Hyunjin; Kidwai, Afshan S.; Brown, Roslyn N.; McDermott, Jason E.; Smith, Richard D.; Adkins, Joshua N.

    2011-05-01

    In this short review we discuss secreted virulence factors of Salmonella, which directly affect Salmonella interaction with its host. Salmonella secretes protein to subvert host defenses but also, as discussed, to reduce virulence thereby permitting the bacteria to persist longer and more successfully disperse. The type III secretion system (TTSS) is the best known and well studied of the mechanisms that enable secretion from the bacterial cytoplasm to the host cell cytoplasm. Other secretion systems include outer membrane vesicles, which are present in all Gram-negative bacteria examined to date, two-partner secretion, and type VI secretion will also be addressed. Excellent reviews of Salmonella secreted effectors have focused on themes such as actin rearrangements, vesicular trafficking, ubiquitination, and the activities of the virulence factors themselves. This short review is based on S. Typhimurium infection of mice because it is a model of typhoid like disease in humans. We have organized effectors in terms of events that happen during the infection cycle and how secreted effectors may be involved.

  16. ESCRT-III drives the final stages of CUPS maturation for unconventional protein secretion.

    PubMed

    Curwin, Amy J; Brouwers, Nathalie; Alonso Y Adell, Manuel; Teis, David; Turacchio, Gabriele; Parashuraman, Seetharaman; Ronchi, Paolo; Malhotra, Vivek

    2016-01-01

    The unconventional secretory pathway exports proteins that bypass the endoplasmic reticulum. In Saccharomyces cerevisiae, conditions that trigger Acb1 secretion via this pathway generate a Grh1 containing compartment composed of vesicles and tubules surrounded by a cup-shaped membrane and collectively called CUPS. Here we report a quantitative assay for Acb1 secretion that reveals requirements for ESCRT-I, -II, and -III but, surprisingly, without the involvement of the Vps4 AAA-ATPase. The major ESCRT-III subunit Snf7 localizes transiently to CUPS and this was accelerated in vps4Δ cells, correlating with increased Acb1 secretion. Microscopic analysis suggests that, instead of forming intraluminal vesicles with the help of Vps4, ESCRT-III/Snf7 promotes direct engulfment of preexisting Grh1 containing vesicles and tubules into a saccule to generate a mature Acb1 containing compartment. This novel multivesicular / multilamellar compartment, we suggest represents the stable secretory form of CUPS that is competent for the release of Acb1 to cells exterior. PMID:27115345

  17. ESCRT-III drives the final stages of CUPS maturation for unconventional protein secretion

    PubMed Central

    Curwin, Amy J; Brouwers, Nathalie; Alonso Y Adell, Manuel; Teis, David; Turacchio, Gabriele; Parashuraman, Seetharaman; Ronchi, Paolo; Malhotra, Vivek

    2016-01-01

    The unconventional secretory pathway exports proteins that bypass the endoplasmic reticulum. In Saccharomyces cerevisiae, conditions that trigger Acb1 secretion via this pathway generate a Grh1 containing compartment composed of vesicles and tubules surrounded by a cup-shaped membrane and collectively called CUPS. Here we report a quantitative assay for Acb1 secretion that reveals requirements for ESCRT-I, -II, and -III but, surprisingly, without the involvement of the Vps4 AAA-ATPase. The major ESCRT-III subunit Snf7 localizes transiently to CUPS and this was accelerated in vps4Δ cells, correlating with increased Acb1 secretion. Microscopic analysis suggests that, instead of forming intraluminal vesicles with the help of Vps4, ESCRT-III/Snf7 promotes direct engulfment of preexisting Grh1 containing vesicles and tubules into a saccule to generate a mature Acb1 containing compartment. This novel multivesicular / multilamellar compartment, we suggest represents the stable secretory form of CUPS that is competent for the release of Acb1 to cells exterior. DOI: http://dx.doi.org/10.7554/eLife.16299.001 PMID:27115345

  18. Selective Purification of Recombinant Neuroactive Peptides Using the Flagellar Type III Secretion System

    PubMed Central

    Singer, Hanna M.; Erhardt, Marc; Steiner, Andrew M.; Zhang, Min-Min; Yoshikami, Doju; Bulaj, Grzegorz; Olivera, Baldomero M.; Hughes, Kelly T.

    2012-01-01

    ABSTRACT The structure, assembly, and function of the bacterial flagellum involves about 60 different proteins, many of which are selectively secreted via a specific type III secretion system (T3SS) (J. Frye et al., J. Bacteriol. 188:2233–2243, 2006). The T3SS is reported to secrete proteins at rates of up to 10,000 amino acid residues per second. In this work, we showed that the flagellar T3SS of Salmonella enterica serovar Typhimurium could be manipulated to export recombinant nonflagellar proteins through the flagellum and into the surrounding medium. We translationally fused various neuroactive peptides and proteins from snails, spiders, snakes, sea anemone, and bacteria to the flagellar secretion substrate FlgM. We found that all tested peptides of various sizes were secreted via the bacterial flagellar T3SS. We subsequently purified the recombinant μ-conotoxin SIIIA (rSIIIA) from Conus striatus by affinity chromatography and confirmed that T3SS-derived rSIIIA inhibited mammalian voltage-gated sodium channel NaV1.2 comparably to chemically synthesized SIIIA. PMID:22647788

  19. Requirement of the Lipopolysaccharide O-Chain Biosynthesis Gene wxocB for Type III Secretion and Virulence of Xanthomonas oryzae pv. Oryzicola

    PubMed Central

    Wang, Li; Vinogradov, Evgeny V.

    2013-01-01

    Xanthomonas oryzae pv. oryzicola causes bacterial leaf streak of rice. A mutant disrupted in wxocB, predicted to encode an enzyme for lipopolysaccharide (LPS) synthesis, was previously shown to suffer reduced virulence. Here, we confirm a role for wxocB in virulence and demonstrate its requirement for LPS O-chain assembly. Structure analysis indicated that wild-type LPS contains a polyrhamnose O chain with irregular, variant residues and a core oligosaccharide identical to that of other Xanthomonas spp. and that the wxocB mutant lacks the O chain. The mutant also showed moderate impairment in exopolysaccharide (EPS) production, but comparison with an EPS-deficient mutant demonstrated that this impairment could not account entirely for the reduced virulence. The wxocB mutant was not detectably different from the wild type in its induction of pathogenesis-related rice genes, type II secretion competence, flagellar motility, or resistance to two phytoalexins or resveratrol, and it was more, not less, resistant to oxidative stress and a third phytoalexin, indicating that none of these properties is involved. The mutant was more sensitive to SDS and to novobiocin, so increased sensitivity to some host-derived antimicrobials cannot be ruled out. However, the mutant showed a marked decrease in type III secretion into plant cells. This was not associated with any change in expression of genes for type III secretion or the ability to attach to plant cells in suspension. Thus, virulence of the wxocB mutant is likely reduced due primarily to a direct, possibly structural, effect of the loss of the O chain on type III delivery of effector proteins. PMID:23435979

  20. Derivatives of Plant Phenolic Compound Affect the Type III Secretion System of Pseudomonas aeruginosa via a GacS-GacA Two-Component Signal Transduction System

    PubMed Central

    Yamazaki, Akihiro; Li, Jin; Zeng, Quan; Khokhani, Devanshi; Hutchins, William C.; Yost, Angela C.; Biddle, Eulandria; Toone, Eric J.

    2012-01-01

    Antibiotic therapy is the most commonly used strategy to control pathogenic infections; however, it has contributed to the generation of antibiotic-resistant bacteria. To circumvent this emerging problem, we are searching for compounds that target bacterial virulence factors rather than their viability. Pseudomonas aeruginosa, an opportunistic human pathogen, possesses a type III secretion system (T3SS) as one of the major virulence factors by which it secretes and translocates T3 effector proteins into human host cells. The fact that this human pathogen also is able to infect several plant species led us to screen a library of phenolic compounds involved in plant defense signaling and their derivatives for novel T3 inhibitors. Promoter activity screening of exoS, which encodes a T3-secreted toxin, identified two T3 inhibitors and two T3 inducers of P. aeruginosa PAO1. These compounds alter exoS transcription by affecting the expression levels of the regulatory small RNAs RsmY and RsmZ. These two small RNAs are known to control the activity of carbon storage regulator RsmA, which is responsible for the regulation of the key T3SS regulator ExsA. As RsmY and RsmZ are the only targets directly regulated by GacA, our results suggest that these phenolic compounds affect the expression of exoS through the GacSA-RsmYZ-RsmA-ExsA regulatory pathway. PMID:21968370

  1. A Pseudomonas syringae pv. tomato DC3000 mutant lacking the type III effector HopQ1-1 is able to cause disease in the model plant Nicotiana benthamiana.

    PubMed

    Wei, Chia-Fong; Kvitko, Brian H; Shimizu, Rena; Crabill, Emerson; Alfano, James R; Lin, Nai-Chun; Martin, Gregory B; Huang, Hsiou-Chen; Collmer, Alan

    2007-07-01

    The model pathogen Pseudomonas syringae pv. tomato DC3000 causes bacterial speck in tomato and Arabidopsis, but Nicotiana benthamiana, an important model plant, is considered to be a non-host. Strain DC3000 injects approximately 28 effector proteins into plant cells via the type III secretion system (T3SS). These proteins were individually delivered into N. benthamiana leaf cells via T3SS-proficient Pseudomonas fluorescens, and eight, including HopQ1-1, showed some capacity to cause cell death in this test. Four gene clusters encoding 13 effectors were deleted from DC3000: cluster II (hopH1, hopC1), IV (hopD1, hopQ1-1, hopR1), IX (hopAA1-2, hopV1, hopAO1, hopG1), and native plasmid pDC3000A (hopAM1-2, hopX1, hopO1-1, hopT1-1). DC3000 mutants deleted for cluster IV or just hopQ1-1 acquired the ability to grow to high levels and produce bacterial speck lesions in N. benthamiana. HopQ1-1 showed other hallmarks of an avirulence determinant in N. benthamiana: expression in the tobacco wildfire pathogen P. syringae pv. tabaci 11528 rendered this strain avirulent in N. benthamiana, and elicitation of the hypersensitive response in N. benthamiana by HopQ1-1 was dependent on SGT1. DC3000 polymutants involving other effector gene clusters in a hopQ1-1-deficient background revealed that clusters II and IX contributed to the severity of lesion symptoms in N. benthamiana, as well as in Arabidopsis and tomato. The results support the hypothesis that the host ranges of P. syringae pathovars are limited by the complex interactions of effector repertoires with plant anti-effector surveillance systems, and they demonstrate that N. benthamiana can be a useful model host for DC3000. PMID:17559511

  2. EffectiveDB—updates and novel features for a better annotation of bacterial secreted proteins and Type III, IV, VI secretion systems

    PubMed Central

    Eichinger, Valerie; Nussbaumer, Thomas; Platzer, Alexander; Jehl, Marc-André; Arnold, Roland; Rattei, Thomas

    2016-01-01

    Protein secretion systems play a key role in the interaction of bacteria and hosts. EffectiveDB (http://effectivedb.org) contains pre-calculated predictions of bacterial secreted proteins and of intact secretion systems. Here we describe a major update of the database, which was previously featured in the NAR Database Issue. EffectiveDB bundles various tools to recognize Type III secretion signals, conserved binding sites of Type III chaperones, Type IV secretion peptides, eukaryotic-like domains and subcellular targeting signals in the host. Beyond the analysis of arbitrary protein sequence collections, the new release of EffectiveDB also provides a ‘genome-mode’, in which protein sequences from nearly complete genomes or metagenomic bins can be screened for the presence of three important secretion systems (Type III, IV, VI). EffectiveDB contains pre-calculated predictions for currently 1677 bacterial genomes from the EggNOG 4.0 database and for additional bacterial genomes from NCBI RefSeq. The new, user-friendly and informative web portal offers a submission tool for running the EffectiveDB prediction tools on user-provided data. PMID:26590402

  3. Salmonella Phage ST64B Encodes a Member of the SseK/NleB Effector Family

    PubMed Central

    Brown, Nat F.; Coombes, Brian K.; Bishop, Jenna L.; Wickham, Mark E.; Lowden, Michael J.; Gal-Mor, Ohad; Goode, David L.; Boyle, Erin C.; Sanderson, Kristy L.; Finlay, B. Brett

    2011-01-01

    Salmonella enterica is a species of bacteria that is a major cause of enteritis across the globe, while certain serovars cause typhoid, a more serious disease associated with a significant mortality rate. Type III secreted effectors are major contributors to the pathogenesis of Salmonella infections. Genes encoding effectors are acquired via horizontal gene transfer, and a subset are encoded within active phage lysogens. Because the acquisition of effectors is in flux, the complement of effectors possessed by various Salmonella strains frequently differs. By comparing the genome sequences of S. enterica serovar Typhimurium strain SL1344 with LT2, we identified a gene with significant similarity to SseK/NleB type III secreted effector proteins within a phage ST64B lysogen that is absent from LT2. We have named this gene sseK3. SseK3 was co-regulated with the SPI-2 type III secretion system in vitro and inside host cells, and was also injected into infected host cells. While no role for SseK3 in virulence could be identified, a role for the other family members in murine typhoid was found. SseK3 and other phage-encoded effectors were found to have a significant but sparse distribution in the available Salmonella genome sequences, indicating the potential for more uncharacterised effectors to be present in less studied serovars. These phage-encoded effectors may be principle subjects of contemporary selective processes shaping Salmonella-host interactions. PMID:21445262

  4. The Importance of the Pseudomonas aeruginosa Type III Secretion System in Epithelium Traversal Depends upon Conditions of Host Susceptibility

    PubMed Central

    Sullivan, Aaron B.; Tam, K. P. Connie; Metruccio, Matteo M. E.; Evans, David J.

    2015-01-01

    Pseudomonas aeruginosa is invasive or cytotoxic to host cells, depending on the type III secretion system (T3SS) effectors encoded. While the T3SS is known to be involved in disease in vivo, how it participates remains to be clarified. Here, mouse models of superficial epithelial injury (tissue paper blotting with EGTA treatment) and immunocompromise (MyD88 deficiency) were used to study the contribution of the T3SS transcriptional activator ExsA to epithelial traversal. Corneas of excised eyeballs were inoculated with green fluorescent protein (GFP)-expressing PAO1 or isogenic exsA mutants for 6 h ex vivo before bacterial traversal and epithelial thickness were quantified by using imaging. In the blotting-EGTA model, exsA mutants were defective in capacity for traversal. Accordingly, an ∼16-fold variability in exsA expression among PAO1 isolates from three sources correlated with epithelial loss. In contrast, MyD88−/− epithelia remained susceptible to P. aeruginosa traversal despite exsA mutation. Epithelial lysates from MyD88−/− mice had reduced antimicrobial activity compared to those from wild-type mice with and without prior antigen challenge, particularly 30- to 100-kDa fractions, for which mass spectrometry revealed multiple differences, including (i) lower baseline levels of histones, tubulin, and lumican and (ii) reduced glutathione S-transferase, annexin, and dermatopontin, after antigen challenge. Thus, the importance of ExsA in epithelial traversal by invasive P. aeruginosa depends on the compromise enabling susceptibility, suggesting that strategies for preventing infection will need to extend beyond targeting the T3SS. The data also highlight the importance of mimicking conditions allowing susceptibility in animal models and the need to monitor variability among bacterial isolates from different sources, even for the same strain. PMID:25667266

  5. The Burkholderia pseudomallei type III secretion system and BopA are required for evasion of LC3-associated phagocytosis.

    PubMed

    Gong, Lan; Cullinane, Meabh; Treerat, Puthayalai; Ramm, Georg; Prescott, Mark; Adler, Ben; Boyce, John D; Devenish, Rodney J

    2011-01-01

    Burkholderia pseudomallei is the causative agent of melioidosis, a fatal infectious disease endemic in tropical regions worldwide, and especially prevalent in southeast Asia and northern Australia. This intracellular pathogen can escape from phagosomes into the host cytoplasm, where it replicates and infects adjacent cells. We previously demonstrated that, in response to B. pseudomallei infection of macrophage cell line RAW 264.7, a subset of bacteria co-localized with the autophagy marker protein, microtubule-associated protein light chain 3 (LC3), implicating autophagy in host cell defence against infection. Recent reports have suggested that LC3 can be recruited to both phagosomes and autophagosomes, thereby raising questions regarding the identity of the LC3-positive compartments in which invading bacteria reside and the mechanism of the autophagic response to B. pseudomallei infection. Electron microscopy analysis of infected cells demonstrated that the invading bacteria were either free in the cytosol, or sequestered in single-membrane phagosomes rather than double-membrane autophagosomes, suggesting that LC3 is recruited to B. pseudomallei-containing phagosomes. Partial or complete loss of function of type III secretion system cluster 3 (TTSS3) in mutants lacking the BopA (effector) or BipD (translocator) proteins respectively, resulted in delayed or no escape from phagosomes. Consistent with these observations, bopA and bipD mutants both showed a higher level of co-localization with LC3 and the lysosomal marker LAMP1, and impaired survival in RAW264.7 cells, suggesting enhanced killing in phagolysosomes. We conclude that LC3 recruitment to phagosomes stimulates killing of B. pseudomallei trapped in phagosomes. Furthermore, BopA plays an important role in efficient escape of B. pseudomallei from phagosomes. PMID:21412437

  6. Type III Secretion System Translocon Component EseB Forms Filaments on and Mediates Autoaggregation of and Biofilm Formation by Edwardsiella tarda

    PubMed Central

    Gao, Zhi Peng; Nie, Pin; Lu, Jin Fang; Liu, Lu Yi; Xiao, Tiao Yi; Liu, Wei

    2015-01-01

    The type III secretion system (T3SS) of Edwardsiella tarda plays an important role in infection by translocating effector proteins into host cells. EseB, a component required for effector translocation, is reported to mediate autoaggregation of E. tarda. In this study, we demonstrate that EseB forms filamentous appendages on the surface of E. tarda and is required for biofilm formation by E. tarda in Dulbecco's modified Eagle's medium (DMEM). Biofilm formation by E. tarda in DMEM does not require FlhB, an essential component for assembling flagella. Dynamic analysis of EseB filament formation, autoaggregation, and biofilm formation shows that the formation of EseB filaments occurs prior to autoaggregation and biofilm formation. The addition of an EseB antibody to E. tarda cultures before bacterial autoaggregation prevents autoaggregation and biofilm formation in a dose-dependent manner, whereas the addition of the EseB antibody to E. tarda cultures in which biofilm is already formed does not destroy the biofilm. Therefore, EseB filament-mediated bacterial cell-cell interaction is a prerequisite for autoaggregation and biofilm formation. PMID:26116669

  7. YscU/FlhB of Yersinia pseudotuberculosis Harbors a C-terminal Type III Secretion Signal.

    PubMed

    Login, Frédéric H; Wolf-Watz, Hans

    2015-10-23

    All type III secretion systems (T3SS) harbor a member of the YscU/FlhB family of proteins that is characterized by an auto-proteolytic process that occurs at a conserved cytoplasmic NPTH motif. We have previously demonstrated that YscUCC, the C-terminal peptide generated by auto-proteolysis of Yersinia pseudotuberculosis YscU, is secreted by the T3SS when bacteria are grown in Ca(2+)-depleted medium at 37 °C. Here, we investigated the secretion of this early T3S-substrate and showed that YscUCC encompasses a specific C-terminal T3S signal within the 15 last residues (U15). U15 promoted C-terminal secretion of reporter proteins like GST and YopE lacking its native secretion signal. Similar to the "classical" N-terminal secretion signal, U15 interacted with the ATPase YscN. Although U15 is critical for YscUCC secretion, deletion of the C-terminal secretion signal of YscUCC did neither affect Yop secretion nor Yop translocation. However, these deletions resulted in increased secretion of YscF, the needle subunit. Thus, these results suggest that YscU via its C-terminal secretion signal is involved in regulation of the YscF secretion. PMID:26338709

  8. Solution structure of monomeric BsaL, the type III secretion needle protein of Burkholderia pseudomallei.

    PubMed

    Zhang, Lingling; Wang, Yu; Picking, Wendy L; Picking, William D; De Guzman, Roberto N

    2006-06-01

    Many gram-negative bacteria that are important human pathogens possess type III secretion systems as part of their required virulence factor repertoire. During the establishment of infection, these pathogens coordinately assemble greater than 20 different proteins into a macromolecular structure that spans the bacterial inner and outer membranes and, in many respects, resembles and functions like a syringe. This type III secretion apparatus (TTSA) is used to inject proteins into a host cell's membrane and cytoplasm to subvert normal cellular processes. The external portion of the TTSA is a needle that is composed of a single type of protein that is polymerized in a helical fashion to form an elongated tube with a central channel of 2-3 nm in diameter. TTSA needle proteins from a variety of bacterial pathogens share sequence conservation; however, no atomic structure for any TTSA needle protein is yet available. Here, we report the structure of a TTSA needle protein called BsaL from Burkholderia pseudomallei determined by nuclear magnetic resonance (NMR) spectroscopy. The central part of the protein assumes a helix-turn-helix core domain with two well-defined alpha-helices that are joined by an ordered, four-residue linker. This forms a two-helix bundle that is stabilized by interhelix hydrophobic contacts. Residues that flank this presumably exposed core region are not completely disordered, but adopt a partial helical conformation. The atomic structure of BsaL and its sequence homology with other TTSA needle proteins suggest potentially unique structural dynamics that could be linked with a universal mechanism for control of type III secretion in diverse gram-negative bacterial pathogens. PMID:16631790

  9. Ralstonia solanacearum Type III Effector RipAY Is a Glutathione-Degrading Enzyme That Is Activated by Plant Cytosolic Thioredoxins and Suppresses Plant Immunity

    PubMed Central

    Hatanaka, Tadashi; Nakano, Masahito; Oda, Kenji

    2016-01-01

    ABSTRACT The plant pathogen Ralstonia solanacearum uses a large repertoire of type III effector proteins to succeed in infection. To clarify the function of effector proteins in host eukaryote cells, we expressed effectors in yeast cells and identified seven effector proteins that interfere with yeast growth. One of the effector proteins, RipAY, was found to share homology with the ChaC family proteins that function as γ-glutamyl cyclotransferases, which degrade glutathione (GSH), a tripeptide that plays important roles in the plant immune system. RipAY significantly inhibited yeast growth and simultaneously induced rapid GSH depletion when expressed in yeast cells. The in vitro GSH degradation activity of RipAY is specifically activated by eukaryotic factors in the yeast and plant extracts. Biochemical purification of the yeast protein identified that RipAY is activated by thioredoxin TRX2. On the other hand, RipAY was not activated by bacterial thioredoxins. Interestingly, RipAY was activated by plant h-type thioredoxins that exist in large amounts in the plant cytosol, but not by chloroplastic m-, f-, x-, y- and z-type thioredoxins, in a thiol-independent manner. The transient expression of RipAY decreased the GSH level in plant cells and affected the flg22-triggered production of reactive oxygen species (ROS) and expression of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) marker genes in Nicotiana benthamiana leaves. These results indicate that RipAY is activated by host cytosolic thioredoxins and degrades GSH specifically in plant cells to suppress plant immunity. PMID:27073091

  10. An NF-κB-Based High-Throughput Screen Identifies Piericidins as Inhibitors of the Yersinia pseudotuberculosis Type III Secretion System

    PubMed Central

    Duncan, Miles C.; Wong, Weng Ruh; Dupzyk, Allison J.; Bray, Walter M.; Linington, Roger G.

    2014-01-01

    The type III secretion system (T3SS) is a bacterial appendage used by dozens of Gram-negative pathogens to subvert host defenses and cause disease, making it an ideal target for pathogen-specific antimicrobials. Here, we report the discovery and initial characterization of two related natural products with T3SS-inhibitory activity that were derived from a marine actinobacterium. Bacterial extracts containing piericidin A1 and the piericidin derivative Mer-A 2026B inhibited Yersinia pseudotuberculosis from triggering T3SS-dependent activation of the host transcription factor NF-κB in HEK293T cells but were not toxic to mammalian cells. As the Yersinia T3SS must be functional in order to trigger NF-κB activation, these data indicate that piericidin A1 and Mer-A 2026B block T3SS function. Consistent with this, purified piericidin A1 and Mer-A 2026B dose-dependently inhibited translocation of the Y. pseudotuberculosis T3SS effector protein YopM inside CHO cells. In contrast, neither compound perturbed bacterial growth in vitro, indicating that piericidin A1 and Mer-A 2026B do not function as general antibiotics in Yersinia. In addition, when Yersinia was incubated under T3SS-inducing culture conditions in the absence of host cells, Mer-A 2026B and piericidin A1 inhibited secretion of T3SS cargo as effectively as or better than several previously described T3SS inhibitors, such as MBX-1641 and aurodox. This suggests that Mer-A 2026B and piericidin A1 do not block type III secretion by blocking the bacterium-host cell interaction, but rather inhibit an earlier stage, such as T3SS needle assembly. In summary, the marine-derived natural products Mer-A 2026B and piericidin A1 possess previously uncharacterized activity against the bacterial T3SS. PMID:24295981