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Sample records for effector ssei mediates

  1. Type III effector-mediated processes in Salmonella infection.

    PubMed

    van der Heijden, Joris; Finlay, B Brett

    2012-06-01

    Salmonella is one of the most successful bacterial pathogens that infect humans in both developed and developing countries. In order to cause infection, Salmonella uses type III secretion systems to inject bacterial effector proteins into host cells. In the age of antibiotic resistance, researchers have been looking for new strategies to reduce Salmonella infection. To understand infection and to analyze type III secretion as a potential therapeutic target, research has focused on identification of effectors, characterization of effector functions and how they contribute to disease. Many effector-mediated processes have been identified that contribute to infection but thus far no specific treatment has been found. In this perspective we discuss our current understanding of effector-mediated processes and discuss new techniques and approaches that may help us to find a solution to this worldwide problem.

  2. Armet is an effector protein mediating aphid-plant interactions.

    PubMed

    Wang, Wei; Dai, Huaien; Zhang, Yi; Chandrasekar, Raman; Luo, Lan; Hiromasa, Yasuaki; Sheng, Changzhong; Peng, Gongxin; Chen, Shaoliang; Tomich, John M; Reese, John; Edwards, Owain; Kang, Le; Reeck, Gerald; Cui, Feng

    2015-05-01

    Aphid saliva is predicted to contain proteins that modulate plant defenses and facilitate feeding. Armet is a well-characterized bifunctional protein in mammalian systems. Here we report a new role of Armet, namely as an effector protein in the pea aphid, Acyrthosiphon pisum. Pea aphid Armet's physical and chemical properties and its intracellular role are comparable to those reported for mammalian Armets. Uniquely, we detected Armet in aphid watery saliva and in the phloem sap of fava beans fed on by aphids. Armet's transcript level is several times higher in the salivary gland when aphids feed on bean plants than when they feed on an artificial diet. Knockdown of the Armet transcript by RNA interference disturbs aphid feeding behavior on fava beans measured by the electrical penetration graph technique and leads to a shortened life span. Inoculation of pea aphid Armet protein into tobacco leaves induced a transcriptional response that included pathogen-responsive genes. The data suggest that Armet is an effector protein mediating aphid-plant interactions. © FASEB.

  3. Cellular Signaling Pathways and Posttranslational Modifications Mediated by Nematode Effector Proteins1

    PubMed Central

    Hewezi, Tarek

    2015-01-01

    Plant-parasitic cyst and root-knot nematodes synthesize and secrete a suite of effector proteins into infected host cells and tissues. These effectors are the major virulence determinants mediating the transformation of normal root cells into specialized feeding structures. Compelling evidence indicates that these effectors directly hijack or manipulate refined host physiological processes to promote the successful parasitism of host plants. Here, we provide an update on recent progress in elucidating the molecular functions of nematode effectors. In particular, we emphasize how nematode effectors modify plant cell wall structure, mimic the activity of host proteins, alter auxin signaling, and subvert defense signaling and immune responses. In addition, we discuss the emerging evidence suggesting that nematode effectors target and recruit various components of host posttranslational machinery in order to perturb the host signaling networks required for immunity and to regulate their own activity and subcellular localization. PMID:26315856

  4. TAL effector-mediated susceptibility to bacterial blight of cotton

    USDA-ARS?s Scientific Manuscript database

    Bacterial blight of cotton (BBC) caused by Xanthomonas campestris pv. malvacearum (Xcm) is a destructive disease that has recently re-emerged in the U.S. Xcm injects transcription activator-like (TAL) effectors that directly induce the expression of host susceptibility (S) or resistance (R) genes. ...

  5. Structure of the catalytic domain of the Salmonella virulence factor SseI.

    PubMed

    Bhaskaran, Shyam S; Stebbins, C Erec

    2012-12-01

    SseI is secreted into host cells by Salmonella and contributes to the establishment of systemic infections. The crystal structure of the C-terminal domain of SseI has been solved to 1.70 Å resolution, revealing it to be a member of the cysteine protease superfamily with a catalytic triad consisting of Cys178, His216 and Asp231 that is critical to its virulence activities. Structure-based analysis revealed that SseI is likely to possess either acyl hydrolase or acyltransferase activity, placing this virulence factor in the rapidly growing class of enzymes of this family utilized by bacterial pathogens inside eukaryotic cells.

  6. External lipid PI3P mediates entry of eukaryotic pathogen effectors into plant and animal host cells.

    PubMed

    Kale, Shiv D; Gu, Biao; Capelluto, Daniel G S; Dou, Daolong; Feldman, Emily; Rumore, Amanda; Arredondo, Felipe D; Hanlon, Regina; Fudal, Isabelle; Rouxel, Thierry; Lawrence, Christopher B; Shan, Weixing; Tyler, Brett M

    2010-07-23

    Pathogens of plants and animals produce effector proteins that are transferred into the cytoplasm of host cells to suppress host defenses. One type of plant pathogens, oomycetes, produces effector proteins with N-terminal RXLR and dEER motifs that enable entry into host cells. We show here that effectors of another pathogen type, fungi, contain functional variants of the RXLR motif, and that the oomycete and fungal RXLR motifs enable binding to the phospholipid, phosphatidylinositol-3-phosphate (PI3P). We find that PI3P is abundant on the outer surface of plant cell plasma membranes and, furthermore, on some animal cells. All effectors could also enter human cells, suggesting that PI3P-mediated effector entry may be very widespread in plant, animal and human pathogenesis. Entry into both plant and animal cells involves lipid raft-mediated endocytosis. Blocking PI3P binding inhibited effector entry, suggesting new therapeutic avenues.

  7. Host FIH-Mediated Asparaginyl Hydroxylation of Translocated Legionella pneumophila Effectors

    PubMed Central

    Price, Christopher; Merchant, Michael; Jones, Snake; Best, Ashley; Von Dwingelo, Juanita; Lawrenz, Matthew B.; Alam, Nawsad; Schueler-Furman, Ora; Kwaik, Yousef A.

    2017-01-01

    FIH-mediated post-translational modification through asparaginyl hydroxylation of eukaryotic proteins impacts regulation of protein-protein interaction. We have identified the FIH recognition motif in 11 Legionella pneumophila translocated effectors, YopM of Yersinia, IpaH4.5 of Shigella and an ankyrin protein of Rickettsia. Mass spectrometry analyses of the AnkB and AnkH effectors of L. pneumophila confirm their asparaginyl hydroxylation. Consistent with localization of the AnkB effector to the Legionella-containing vacuole (LCV) membrane and its modification by FIH, our data show that FIH and its two interacting proteins, Mint3 and MT1-MMP are acquired by the LCV in a Dot/Icm type IV secretion-dependent manner. Chemical inhibition or RNAi-mediated knockdown of FIH promotes LCV-lysosomes fusion, diminishes decoration of the LCV with polyubiquitinated proteins, and abolishes intra-vacuolar replication of L. pneumophila. These data show acquisition of the host FIH by a pathogen-containing vacuole and that asparaginyl-hydroxylation of translocated effectors is indispensable for their function. PMID:28321389

  8. Cell-mediated cytotoxicity. Characterization of the effector cells.

    PubMed Central

    De Bracco, M M; Isturiz, M A; Manni, J A

    1976-01-01

    Isolated human mononuclear cells were fractionated according to their membrane characteristics or physical properties. Adherent cells were depleted by filtration through glass columns; phagocytic cells were removed by iron treatment and cell subpopulations capable of forming rosettes with sheep erythrocytes (E), erythrocyte-antibody-complement (EAC) and chicken erythrocyte-antibody complexes (CEA) were separated by centrifugation of Ficoll-Hypaque gradients. The functional activity of the cell subpopulations obtained was assayed by testing PHA-induced cytoxicity (PIC), antibody-dependent cytoxicity (ADCC) and blast transformation by PHA. The results of this study demonstrate that: (1) cells reacting in PIC and ADCC assays are different, adherent and phagocytic cells being necessary for full expression of PIC and not for ADCC; (2) PHA induces direct blast transformation of purified E-RFC in the absence of PIC cytotoxic cells; (3) cell populations specifically enriched in E or EAC rosette-forming cells are not cytotoxic neither in the PHA nor in antibody mediated cytotoxic assays; (4) cells participating in ADCC can be selectively purified by centrifugation of CEA rosettes. PMID:1254320

  9. The 3 major types of innate and adaptive cell-mediated effector immunity.

    PubMed

    Annunziato, Francesco; Romagnani, Chiara; Romagnani, Sergio

    2015-03-01

    The immune system has tailored its effector functions to optimally respond to distinct species of microbes. Based on emerging knowledge on the different effector T-cell and innate lymphoid cell (ILC) lineages, it is clear that the innate and adaptive immune systems converge into 3 major kinds of cell-mediated effector immunity, which we propose to categorize as type 1, type 2, and type 3. Type 1 immunity consists of T-bet(+) IFN-γ-producing group 1 ILCs (ILC1 and natural killer cells), CD8(+) cytotoxic T cells (TC1), and CD4(+) TH1 cells, which protect against intracellular microbes through activation of mononuclear phagocytes. Type 2 immunity consists of GATA-3(+) ILC2s, TC2 cells, and TH2 cells producing IL-4, IL-5, and IL-13, which induce mast cell, basophil, and eosinophil activation, as well as IgE antibody production, thus protecting against helminthes and venoms. Type 3 immunity is mediated by retinoic acid-related orphan receptor γt(+) ILC3s, TC17 cells, and TH17 cells producing IL-17, IL-22, or both, which activate mononuclear phagocytes but also recruit neutrophils and induce epithelial antimicrobial responses, thus protecting against extracellular bacteria and fungi. On the other hand, type 1 and 3 immunity mediate autoimmune diseases, whereas type 2 responses can cause allergic diseases.

  10. Effector-repressor interactions, binding of a single effector molecule to the operator-bound TtgR homodimer mediates derepression.

    PubMed

    Terán, Wilson; Krell, Tino; Ramos, Juan Luis; Gallegos, María-Trinidad

    2006-03-17

    The RND family transporter TtgABC and its cognate repressor TtgR from Pseudomonas putida DOT-T1E were both shown to possess multidrug recognition properties. Structurally unrelated molecules such as chloramphenicol, butyl paraben, 1,3-dihydroxynaphthalene, and several flavonoids are substrates of TtgABC and activate pump expression by binding to the TtgR-operator complex. Isothermal titration calorimetry was employed to determine the thermodynamic parameters for the binding of these molecules to TtgR. Dissociation constants were in the range from 1 to 150 microm, the binding stoichiometry was one effector molecule per dimer of TtgR, and the process was driven by favorable enthalpy changes. Although TtgR exhibits a large multidrug binding profile, the plant-derived compounds phloretin and quercetin were shown to bind with the highest affinity (K(D) of around 1 microm), in contrast to other effectors (chloramphenicol and aromatic solvents) for which exhibited a more reduced affinity. Structure-function studies of effectors indicate that the presence of aromatic rings as well as hydroxyl groups are determinants for TtgR binding. The binding of TtgR to its operator DNA does not alter the protein effector profile nor the effector binding stoichiometry. Moreover, we demonstrate here for the first time that the binding of a single effector molecule to the DNA-bound TtgR homodimer induces the dissociation of the repressor-operator complex. This provides important insight into the molecular mechanism of effector-mediated derepression.

  11. Structure of the catalytic domain of the Salmonella virulence factor SseI

    PubMed Central

    Bhaskaran, Shyam S.; Stebbins, C. Erec

    2012-01-01

    SseI is secreted into host cells by Salmonella and contributes to the establishment of systemic infections. The crystal structure of the C-terminal domain of SseI has been solved to 1.70 Å resolution, revealing it to be a member of the cysteine protease superfamily with a catalytic triad consisting of Cys178, His216 and Asp231 that is critical to its virulence activities. Structure-based analysis revealed that SseI is likely to possess either acyl hydrolase or acyltransferase activity, placing this virulence factor in the rapidly growing class of enzymes of this family utilized by bacterial pathogens inside eukaryotic cells. PMID:23151626

  12. Rafts can trigger contact-mediated secretion of bacterial effectors via a lipid-based mechanism.

    PubMed

    van der Goot, Françoise G; Tran van Nhieu, Guy; Allaoui, Abdelmounaaïm; Sansonetti, Phillipe; Lafont, Frank

    2004-11-12

    Infection by the Gram-negative bacterial pathogen Shigella flexneri depends on its ability to invade host cells. Bacterial engulfment requires a functional type III secretion system (TTSS) allowing the translocation into host cells of bacterial effectors that activate cell-signaling cascades. We demonstrated previously that specialized lipid membrane domains enriched in cholesterol and sphingolipids (rafts) are involved during early steps of invasion, namely in binding and host cell entry. In this study, we addressed the issue of contact-mediated secretion by the TTSS. We show that contact-mediated and TTSS-induced hemolysis depend on the presence of cholesterol on the host cell surface. We found that purified detergent resistant membranes were able to activate TTSS. Finally, we found that artificial liposomes, devoid of proteins, were able to activate the TTSS but only when their composition mimicked that of lipid rafts. Altogether, these data indicate that specific lipid packing can trigger contact-mediated secretion by S. flexneri.

  13. Epitope target structures of Fc-mediated effector function during HIV-1 acquisition.

    PubMed

    Lewis, George K; Guan, Yongjun; Kamin-Lewis, Roberta; Sajadi, Mohammad; Pazgier, Marzena; Devico, Anthony L

    2014-05-01

    This review analyzes recent studies suggesting that highly conserved epitopes in the HIV-1 Env trimer are targets of potentially protective nonneutralizing antibodies that mediate antibody-dependent cellular cytotoxicity. Recent studies in both non-human primates and humans suggest that nonneutralizing antibodies play a role in blocking infection with hybrid simian HIV (SHIV)/simian immunodeficiency virus (SIV) or HIV-1 by Fc-mediated effector function, in particular antibody-dependent cellular cytotoxicity. Further, several studies implicate highly conserved epitopes in the C1 region of gp120 as targets of these antibodies. However, these suggestions are controversial, as passive immunization studies do not indicate that such antibodies can block acquisition in non-human primates. Potential reasons for this discrepancy are discussed in the structural context of potent antibody-dependent cellular cytotoxicity epitopes on target cells during the narrow window of opportunity when antibodies can block HIV-1 acquisition. Cumulative evidence suggests that, in addition to virus neutralization, Fc-mediated effector responses to highly conserved epitopes in the HIV-1 trimer play distinct as well as overlapping roles in blocking HIV-1 acquisition. Evidence will be discussed as to whether nonneutralizing antibodies specific for epitopes on the HIV-1 Env trimer that become exposed during viral entry contribute significantly to blocking HIV-1 acquisition.

  14. An Effector Peptide Family Required for Drosophila Toll-Mediated Immunity

    PubMed Central

    Wasserman, Steven A.

    2015-01-01

    In Drosophila melanogaster, recognition of an invading pathogen activates the Toll or Imd signaling pathway, triggering robust upregulation of innate immune effectors. Although the mechanisms of pathogen recognition and signaling are now well understood, the functions of the immune-induced transcriptome and proteome remain much less well characterized. Through bioinformatic analysis of effector gene sequences, we have defined a family of twelve genes – the Bomanins (Boms) – that are specifically induced by Toll and that encode small, secreted peptides of unknown biochemical activity. Using targeted genome engineering, we have deleted ten of the twelve Bom genes. Remarkably, inactivating these ten genes decreases survival upon microbial infection to the same extent, and with the same specificity, as does eliminating Toll pathway function. Toll signaling, however, appears unaffected. Assaying bacterial load post-infection in wild-type and mutant flies, we provide evidence that the Boms are required for resistance to, rather than tolerance of, infection. In addition, by generating and assaying a deletion of a smaller subset of the Bom genes, we find that there is overlap in Bom activity toward particular pathogens. Together, these studies deepen our understanding of Toll-mediated immunity and provide a new in vivo model for exploration of the innate immune effector repertoire. PMID:25915418

  15. Vaccination with a single CD4 T cell peptide epitope from a Salmonella type III-secreted effector protein provides protection against lethal infection.

    PubMed

    Kurtz, Jonathan R; Petersen, Hailey E; Frederick, Daniel R; Morici, Lisa A; McLachlan, James B

    2014-06-01

    Salmonella infections affect millions worldwide and remain a significant cause of morbidity and mortality. It is known from mouse studies that CD4 T cells are essential mediators of immunity against Salmonella infection, yet it is not clear whether targeting CD4 T cell responses directly with peptide vaccines against Salmonella can be effective in combating infection. Additionally, it is not known whether T cell responses elicited against Salmonella secreted effector proteins can provide protective immunity against infection. In this study, we investigated both of these possibilities using prime-boost immunization of susceptible mice with a single CD4 T cell peptide epitope from Salmonella secreted effector protein I (SseI), a component of the Salmonella type III secretion system. This immunization conferred significant protection against lethal oral infection, equivalent to that conferred by whole heat-killed Salmonella bacteria. Surprisingly, a well-characterized T cell epitope from the flagellar protein FliC afforded no protection compared to immunization with an irrelevant control peptide. The protective response appeared to be most associated with polyfunctional CD4 T cells raised against the SseI peptide, since no antibodies were produced against any of the peptides and very little CD8 T cell response was observed. Overall, this study demonstrates that eliciting CD4 T cell responses against components of the Salmonella type III secretion system can contribute to protection against infection and should be considered in the design of future Salmonella subunit vaccines.

  16. The Fusarium oxysporum effector Six6 contributes to virulence and suppresses I-2-mediated cell death.

    PubMed

    Gawehns, F; Houterman, P M; Ichou, F Ait; Michielse, C B; Hijdra, M; Cornelissen, B J C; Rep, M; Takken, F L W

    2014-04-01

    Plant pathogens secrete effectors to manipulate their host and facilitate colonization. Fusarium oxysporum f. sp. lycopersici is the causal agent of Fusarium wilt disease in tomato. Upon infection, F. oxysporum f. sp. lycopersici secretes numerous small proteins into the xylem sap (Six proteins). Most Six proteins are unique to F. oxysporum, but Six6 is an exception; a homolog is also present in two Colletotrichum spp. SIX6 expression was found to require living host cells and a knockout of SIX6 in F. oxysporum f. sp. lycopersici compromised virulence, classifying it as a genuine effector. Heterologous expression of SIX6 did not affect growth of Agrobacterium tumefaciens in Nicotiana benthamiana leaves or susceptibility of Arabidopsis thaliana toward Verticillium dahliae, Pseudomonas syringae, or F. oxysporum, suggesting a specific function for F. oxysporum f. sp. lycopersici Six6 in the F. oxysporum f. sp. lycopersici- tomato pathosystem. Remarkably, Six6 was found to specifically suppress I-2-mediated cell death (I2CD) upon transient expression in N. benthamiana, whereas it did not compromise the activity of other cell-death-inducing genes. Still, this I2CD suppressing activity of Six6 does not allow the fungus to overcome I-2 resistance in tomato, suggesting that I-2-mediated resistance is independent from cell death.

  17. The rise of the undead:Pseudokinases as mediators of effector-triggered immunity

    USDA-ARS?s Scientific Manuscript database

    Pathogens use effector proteins to suppress host immunity and promote infection. However, plants can recognize specific effectors and mount an effector-triggered immune response that suppresses pathogen growth. The YopJ/HopZ family of type III secreted effector proteins is broadly distributed in bac...

  18. The rise of the undead: pseudokinases as mediators of effector-triggered immunity.

    PubMed

    Lewis, Jennifer D; Lo, Timothy; Bastedo, Patrick; Guttman, David S; Desveaux, Darrell

    2014-01-01

    Pathogens use effector proteins to suppress host immunity and promote infection. However, plants can recognize specific effectors and mount an effector-triggered immune response that suppresses pathogen growth. The YopJ/HopZ family of type III secreted effector proteins is broadly distributed in bacterial pathogens of both animals and plants. These effectors can either suppress host immunity or elicit defense responses depending on the host genotype. In a recent report, we identified an Arabidopsis thaliana pseudokinase ZED1 that is required for the recognition of the Pseudomonas syringae HopZ1a effector. Here we discuss the role of ZED1 in HopZ1a recognition, and present models of effector recognition in plants. We draw parallels between HopZ1a and YopJ effector proteins, and between ZED1 and other immunity-related kinases that can be targeted by pathogen effectors.

  19. Adiponectin-mediated changes in effector cells involved in the pathophysiology of rheumatoid arthritis.

    PubMed

    Frommer, Klaus W; Zimmermann, Birgit; Meier, Florian M P; Schröder, Dirk; Heil, Matthias; Schäffler, Andreas; Büchler, Christa; Steinmeyer, Jürgen; Brentano, Fabia; Gay, Steffen; Müller-Ladner, Ulf; Neumann, Elena

    2010-10-01

    Rheumatoid arthritis (RA) is associated with increased production of adipokines, which are cytokine-like mediators that are produced mainly in adipose tissue but also in synovial cells. Since RA synovial fibroblasts (RASFs), lymphocytes, endothelial cells, and chondrocytes are key players in the pathophysiology of RA, this study was undertaken to analyze the effects of the key adipokine adiponectin on proinflammatory and prodestructive synovial effector cells. Lymphocytes were activated in part prior to stimulation. All cells were stimulated with adiponectin, and changes in gene and protein expression were determined by Affymetrix and protein arrays. Messenger RNA and protein levels were confirmed using semiquantitative reverse transcription-polymerase chain reaction (PCR), real-time PCR, and immunoassays. Intracellular signal transduction was evaluated using chemical signaling inhibitors. Adiponectin stimulation of human RASFs predominantly induced the secretion of chemokines, as well as proinflammatory cytokines, prostaglandin synthases, growth factors, and factors of bone metabolism and matrix remodeling. Lymphocytes, endothelial cells, and chondrocytes responded to adiponectin stimulation with enhanced synthesis of cytokines and various chemokines. Additionally, chondrocytes released increased amounts of matrix metalloproteinases. In RASFs, adiponectin-mediated effects were p38 MAPK and protein kinase C dependent. Our previous findings indicated that adiponectin was present in inflamed synovium, at sites of cartilage invasion, in lymphocyte infiltrates, and in perivascular areas. The findings of the present study indicate that adiponectin induces gene expression and protein synthesis in human RASFs, lymphocytes, endothelial cells, and chondrocytes, supporting the concept of adiponectin being involved in the pathophysiologic modulation of RA effector cells. Adiponectin promotes inflammation through cytokine synthesis, attraction of inflammatory cells to the

  20. Characterization of in vitro antibody-dependent cell-mediated cytotoxicity activity of therapeutic antibodies - impact of effector cells.

    PubMed

    Chung, Shan; Lin, Yuwen L; Reed, Chae; Ng, Carl; Cheng, Zhijie Jey; Malavasi, Fabio; Yang, Jihong; Quarmby, Valerie; Song, An

    2014-05-01

    Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action implicated in the clinical efficacy of several therapeutic antibodies. In vitro ADCC assays employing effector cells capable of inducing lysis of target cells bound by antibodies are routinely performed to support the research and development of therapeutic antibodies. ADCC assays are commonly performed using peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells or engineered cell lines as effector cells. In this study we evaluated the impact of different effector cell types including primary PBMCs, primary NK cells, engineered NK cell lines, and an engineered reporter cell line, on the in vitro ADCC activity of two glycoforms of a humanized IgG1 antibody. The results of this study show the differential effects on both the efficacy and potency of the antibodies by different effector cells and the finding that both the allotype and the expression level of CD16a affect the potency of effector cells in ADCC assays. Our results also show that engineered NK or reporter cell lines provide reduced variability compared to primary effector cells for in vitro ADCC assays.

  1. A Downy Mildew Effector Attenuates Salicylic Acid–Triggered Immunity in Arabidopsis by Interacting with the Host Mediator Complex

    PubMed Central

    Caillaud, Marie-Cécile; Asai, Shuta; Rallapalli, Ghanasyam; Piquerez, Sophie; Fabro, Georgina; Jones, Jonathan D. G.

    2013-01-01

    Plants are continually exposed to pathogen attack but usually remain healthy because they can activate defences upon perception of microbes. However, pathogens have evolved to overcome plant immunity by delivering effectors into the plant cell to attenuate defence, resulting in disease. Recent studies suggest that some effectors may manipulate host transcription, but the specific mechanisms by which such effectors promote susceptibility remain unclear. We study the oomycete downy mildew pathogen of Arabidopsis, Hyaloperonospora arabidopsidis (Hpa), and show here that the nuclear-localized effector HaRxL44 interacts with Mediator subunit 19a (MED19a), resulting in the degradation of MED19a in a proteasome-dependent manner. The Mediator complex of ∼25 proteins is broadly conserved in eukaryotes and mediates the interaction between transcriptional regulators and RNA polymerase II. We found MED19a to be a positive regulator of immunity against Hpa. Expression profiling experiments reveal transcriptional changes resembling jasmonic acid/ethylene (JA/ET) signalling in the presence of HaRxL44, and also 3 d after infection with Hpa. Elevated JA/ET signalling is associated with a decrease in salicylic acid (SA)–triggered immunity (SATI) in Arabidopsis plants expressing HaRxL44 and in med19a loss-of-function mutants, whereas SATI is elevated in plants overexpressing MED19a. Using a PR1::GUS reporter, we discovered that Hpa suppresses PR1 expression specifically in cells containing haustoria, into which RxLR effectors are delivered, but not in nonhaustoriated adjacent cells, which show high PR1::GUS expression levels. Thus, HaRxL44 interferes with Mediator function by degrading MED19, shifting the balance of defence transcription from SA-responsive defence to JA/ET-signalling, and enhancing susceptibility to biotrophs by attenuating SA-dependent gene expression. PMID:24339748

  2. Transcription Activator-Like Effector Nucleases (TALEN)-Mediated Targeted DNA Insertion in Potato Plants

    PubMed Central

    Forsyth, Adrienne; Weeks, Troy; Richael, Craig; Duan, Hui

    2016-01-01

    Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. Specifically integrated transgenes are guaranteed to co-segregate, and expression level is more predictable, which makes downstream characterization and line selection more manageable. Because the site of DNA integration is known, the steps to deregulation of transgenic crops may be simplified. Here we describe a method that combines transcription activator-like effector nuclease (TALEN)-mediated induction of double strand breaks (DSBs) and non-autonomous marker selection to insert a transgene into a pre-selected, transcriptionally active region in the potato genome. In our experiment, TALEN was designed to create a DSB in the genome sequence following an endogenous constitutive promoter. A cytokinin vector was utilized for TALENs expression and prevention of stable integration of the nucleases. The donor vector contained a gene of interest cassette and a promoter-less plant-derived herbicide resistant gene positioned near the T-DNA left border which was used to select desired transgenic events. Our results indicated that TALEN induced T-DNA integration occurred with high frequency and resulting events have consistent expression of the gene of interest. Interestingly, it was found that, in most lines integration took place through one sided homology directed repair despite the minimal homologous sequence at the right border. An efficient transient assay for TALEN activity verification is also described. PMID:27826306

  3. Transcription Activator-Like Effector Nucleases (TALEN)-Mediated Targeted DNA Insertion in Potato Plants.

    PubMed

    Forsyth, Adrienne; Weeks, Troy; Richael, Craig; Duan, Hui

    2016-01-01

    Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. Specifically integrated transgenes are guaranteed to co-segregate, and expression level is more predictable, which makes downstream characterization and line selection more manageable. Because the site of DNA integration is known, the steps to deregulation of transgenic crops may be simplified. Here we describe a method that combines transcription activator-like effector nuclease (TALEN)-mediated induction of double strand breaks (DSBs) and non-autonomous marker selection to insert a transgene into a pre-selected, transcriptionally active region in the potato genome. In our experiment, TALEN was designed to create a DSB in the genome sequence following an endogenous constitutive promoter. A cytokinin vector was utilized for TALENs expression and prevention of stable integration of the nucleases. The donor vector contained a gene of interest cassette and a promoter-less plant-derived herbicide resistant gene positioned near the T-DNA left border which was used to select desired transgenic events. Our results indicated that TALEN induced T-DNA integration occurred with high frequency and resulting events have consistent expression of the gene of interest. Interestingly, it was found that, in most lines integration took place through one sided homology directed repair despite the minimal homologous sequence at the right border. An efficient transient assay for TALEN activity verification is also described.

  4. Fibroblast-like synoviocytes-dependent effector molecules as a critical mediator for rheumatoid arthritis: Current status and future directions.

    PubMed

    Ganesan, Ramamoorthi; Rasool, Mahaboobkhan

    2017-01-02

    Rheumatoid arthritis (RA) is a systemic-autoimmune-mediated disease characterized by synovial hyperplasia and progressive destruction of joint. Currently available biological agents and inhibitor therapy that specifically target tumor necrosis factor-α, interleukin 1β (IL-1β), IL-6, T cells, B cells, and subcellular molecules (p38 mitogen-activated protein kinase and janus kinase) cannot facilitate complete remission in all patients and are unable to cure the disease. Therefore, further potent therapeutic targets need to be identified for effective treatment and successful clinical outcomes in patients with RA. Scientific breakthroughs have brought new insights regarding fibroblast-like synoviocytes (FLS), a major constituent of the synovial hyperplasia. These play a pivotal role in RA invading cartilage and bone tissue. Currently there are no effective therapies available that specifically target these aggressive cells. Recent evidences indicate that FLS-dependent effector molecules (toll-like receptors, nodal effector molecules, hypoxia-inducible factor, and IL-17) have emerged as important mediators of RA. In this review, we discuss the pathological features and recent advances in understanding the role of FLS-dependent effector molecules in the disease onset of RA. Pharmacological inhibition of FLS-dependent effector molecules might be a promising option for FLS-targeted therapy in RA.

  5. HELZ2 Is an IFN Effector Mediating Suppression of Dengue Virus.

    PubMed

    Fusco, Dahlene N; Pratt, Henry; Kandilas, Stephen; Cheon, Scarlett Se Yun; Lin, Wenyu; Cronkite, D Alex; Basavappa, Megha; Jeffrey, Kate L; Anselmo, Anthony; Sadreyev, Ruslan; Yapp, Clarence; Shi, Xu; O'Sullivan, John F; Gerszten, Robert E; Tomaru, Takuya; Yoshino, Satoshi; Satoh, Tetsurou; Chung, Raymond T

    2017-01-01

    Flaviviral infections including dengue virus are an increasing clinical problem worldwide. Dengue infection triggers host production of the type 1 IFN, IFN alpha, one of the strongest and broadest acting antivirals known. However, dengue virus subverts host IFN signaling at early steps of IFN signal transduction. This subversion allows unbridled viral replication which subsequently triggers ongoing production of IFN which, again, is subverted. Identification of downstream IFN antiviral effectors will provide targets which could be activated to restore broad acting antiviral activity, stopping the signal to produce endogenous IFN at toxic levels. To this end, we performed a targeted functional genomic screen for IFN antiviral effector genes (IEGs), identifying 56 IEGs required for antiviral effects of IFN against fully infectious dengue virus. Dengue IEGs were enriched for genes encoding nuclear receptor interacting proteins, including HELZ2, MAP2K4, SLC27A2, HSP90AA1, and HSP90AB1. We focused on HELZ2 (Helicase With Zinc Finger 2), an IFN stimulated gene and IEG which encodes a promiscuous nuclear factor coactivator that exists in two isoforms. The two unique HELZ2 isoforms are both IFN responsive, contain ISRE elements, and gene products increase in the nucleus upon IFN stimulation. Chromatin immunoprecipitation-sequencing revealed that the HELZ2 complex interacts with triglyceride-regulator LMF1. Mass spectrometry revealed that HELZ2 knockdown cells are depleted of triglyceride subsets. We thus sought to determine whether HELZ2 interacts with a nuclear receptor known to regulate immune response and lipid metabolism, AHR, and identified HELZ2:AHR interactions via co-immunoprecipitation, found that AHR is a dengue IEG, and that an AHR ligand, FICZ, exhibits anti-dengue activity. Primary bone marrow derived macrophages from HELZ2 knockout mice, compared to wild type controls, exhibit enhanced dengue infectivity. Overall, these findings reveal that IFN antiviral

  6. HELZ2 Is an IFN Effector Mediating Suppression of Dengue Virus

    PubMed Central

    Fusco, Dahlene N.; Pratt, Henry; Kandilas, Stephen; Cheon, Scarlett Se Yun; Lin, Wenyu; Cronkite, D. Alex; Basavappa, Megha; Jeffrey, Kate L.; Anselmo, Anthony; Sadreyev, Ruslan; Yapp, Clarence; Shi, Xu; O'Sullivan, John F.; Gerszten, Robert E.; Tomaru, Takuya; Yoshino, Satoshi; Satoh, Tetsurou; Chung, Raymond T.

    2017-01-01

    Flaviviral infections including dengue virus are an increasing clinical problem worldwide. Dengue infection triggers host production of the type 1 IFN, IFN alpha, one of the strongest and broadest acting antivirals known. However, dengue virus subverts host IFN signaling at early steps of IFN signal transduction. This subversion allows unbridled viral replication which subsequently triggers ongoing production of IFN which, again, is subverted. Identification of downstream IFN antiviral effectors will provide targets which could be activated to restore broad acting antiviral activity, stopping the signal to produce endogenous IFN at toxic levels. To this end, we performed a targeted functional genomic screen for IFN antiviral effector genes (IEGs), identifying 56 IEGs required for antiviral effects of IFN against fully infectious dengue virus. Dengue IEGs were enriched for genes encoding nuclear receptor interacting proteins, including HELZ2, MAP2K4, SLC27A2, HSP90AA1, and HSP90AB1. We focused on HELZ2 (Helicase With Zinc Finger 2), an IFN stimulated gene and IEG which encodes a promiscuous nuclear factor coactivator that exists in two isoforms. The two unique HELZ2 isoforms are both IFN responsive, contain ISRE elements, and gene products increase in the nucleus upon IFN stimulation. Chromatin immunoprecipitation-sequencing revealed that the HELZ2 complex interacts with triglyceride-regulator LMF1. Mass spectrometry revealed that HELZ2 knockdown cells are depleted of triglyceride subsets. We thus sought to determine whether HELZ2 interacts with a nuclear receptor known to regulate immune response and lipid metabolism, AHR, and identified HELZ2:AHR interactions via co-immunoprecipitation, found that AHR is a dengue IEG, and that an AHR ligand, FICZ, exhibits anti-dengue activity. Primary bone marrow derived macrophages from HELZ2 knockout mice, compared to wild type controls, exhibit enhanced dengue infectivity. Overall, these findings reveal that IFN antiviral

  7. The effector SPRYSEC-19 of Globodera rostochiensis suppresses CC-NB-LRR-mediated disease resistance in plants.

    PubMed

    Postma, Wiebe J; Slootweg, Erik J; Rehman, Sajid; Finkers-Tomczak, Anna; Tytgat, Tom O G; van Gelderen, Kasper; Lozano-Torres, Jose L; Roosien, Jan; Pomp, Rikus; van Schaik, Casper; Bakker, Jaap; Goverse, Aska; Smant, Geert

    2012-10-01

    The potato cyst nematode Globodera rostochiensis invades roots of host plants where it transforms cells near the vascular cylinder into a permanent feeding site. The host cell modifications are most likely induced by a complex mixture of proteins in the stylet secretions of the nematodes. Resistance to nematodes conferred by nucleotide-binding-leucine-rich repeat (NB-LRR) proteins usually results in a programmed cell death in and around the feeding site, and is most likely triggered by the recognition of effectors in stylet secretions. However, the actual role of these secretions in the activation and suppression of effector-triggered immunity is largely unknown. Here we demonstrate that the effector SPRYSEC-19 of G. rostochiensis physically associates in planta with the LRR domain of a member of the SW5 resistance gene cluster in tomato (Lycopersicon esculentum). Unexpectedly, this interaction did not trigger defense-related programmed cell death and resistance to G. rostochiensis. By contrast, agroinfiltration assays showed that the coexpression of SPRYSEC-19 in leaves of Nicotiana benthamiana suppresses programmed cell death mediated by several coiled-coil (CC)-NB-LRR immune receptors. Furthermore, SPRYSEC-19 abrogated resistance to Potato virus X mediated by the CC-NB-LRR resistance protein Rx1, and resistance to Verticillium dahliae mediated by an unidentified resistance in potato (Solanum tuberosum). The suppression of cell death and disease resistance did not require a physical association of SPRYSEC-19 and the LRR domains of the CC-NB-LRR resistance proteins. Altogether, our data demonstrated that potato cyst nematodes secrete effectors that enable the suppression of programmed cell death and disease resistance mediated by several CC-NB-LRR proteins in plants.

  8. A Bacterial Parasite Effector Mediates Insect Vector Attraction in Host Plants Independently of Developmental Changes

    PubMed Central

    Orlovskis, Zigmunds; Hogenhout, Saskia A.

    2016-01-01

    Parasites can take over their hosts and trigger dramatic changes in host appearance and behavior that are typically interpreted as extended phenotypes that promote parasite survival and fitness. For example, Toxoplasma gondii is thought to manipulate the behaviors of infected rodents to aid transmission to cats and parasitic trematodes of the genus Ribeiroia alter limb development in their amphibian hosts to facilitate predation of the latter by birds. Plant parasites and pathogens also reprogram host development and morphology. However, whereas some parasite-induced morphological alterations may have a direct benefit to the fitness of the parasite and may therefore be adaptive, other host alterations may be side effects of parasite infections having no adaptive effects on parasite fitness. Phytoplasma parasites of plants often induce the development of leaf-like flowers (phyllody) in their host plants, and we previously found that the phytoplasma effector SAP54 generates these leaf-like flowers via the degradation of plant MADS-box transcription factors (MTFs), which regulate all major aspects of development in plants. Leafhoppers prefer to reproduce on phytoplasma-infected and SAP54-trangenic plants leading to the hypothesis that leafhopper vectors are attracted to plants with leaf-like flowers. Surprisingly, here we show that leafhopper attraction occurs independently of the presence of leaf-like flowers. First, the leafhoppers were also attracted to SAP54 transgenic plants without leaf-like flowers and to single leaves of these plants. Moreover, leafhoppers were not attracted to leaf-like flowers of MTF-mutant plants without the presence of SAP54. Thus, the primary role of SAP54 is to attract leafhopper vectors, which spread the phytoplasmas, and the generation of leaf-like flowers may be secondary or a side effect of the SAP54-mediated degradation of MTFs. PMID:27446117

  9. A Bacterial Parasite Effector Mediates Insect Vector Attraction in Host Plants Independently of Developmental Changes.

    PubMed

    Orlovskis, Zigmunds; Hogenhout, Saskia A

    2016-01-01

    Parasites can take over their hosts and trigger dramatic changes in host appearance and behavior that are typically interpreted as extended phenotypes that promote parasite survival and fitness. For example, Toxoplasma gondii is thought to manipulate the behaviors of infected rodents to aid transmission to cats and parasitic trematodes of the genus Ribeiroia alter limb development in their amphibian hosts to facilitate predation of the latter by birds. Plant parasites and pathogens also reprogram host development and morphology. However, whereas some parasite-induced morphological alterations may have a direct benefit to the fitness of the parasite and may therefore be adaptive, other host alterations may be side effects of parasite infections having no adaptive effects on parasite fitness. Phytoplasma parasites of plants often induce the development of leaf-like flowers (phyllody) in their host plants, and we previously found that the phytoplasma effector SAP54 generates these leaf-like flowers via the degradation of plant MADS-box transcription factors (MTFs), which regulate all major aspects of development in plants. Leafhoppers prefer to reproduce on phytoplasma-infected and SAP54-trangenic plants leading to the hypothesis that leafhopper vectors are attracted to plants with leaf-like flowers. Surprisingly, here we show that leafhopper attraction occurs independently of the presence of leaf-like flowers. First, the leafhoppers were also attracted to SAP54 transgenic plants without leaf-like flowers and to single leaves of these plants. Moreover, leafhoppers were not attracted to leaf-like flowers of MTF-mutant plants without the presence of SAP54. Thus, the primary role of SAP54 is to attract leafhopper vectors, which spread the phytoplasmas, and the generation of leaf-like flowers may be secondary or a side effect of the SAP54-mediated degradation of MTFs.

  10. Host Protein BSL1 Associates with Phytophthora infestans RXLR Effector AVR2 and the Solanum demissum Immune Receptor R2 to Mediate Disease Resistance[C][W

    PubMed Central

    Saunders, Diane G.O.; Breen, Susan; Win, Joe; Schornack, Sebastian; Hein, Ingo; Bozkurt, Tolga O.; Champouret, Nicolas; Vleeshouwers, Vivianne G.A.A.; Birch, Paul R.J.; Gilroy, Eleanor M.; Kamoun, Sophien

    2012-01-01

    Plant pathogens secrete effector proteins to modulate plant immunity and promote host colonization. Plant nucleotide binding leucine-rich repeat (NB-LRR) immunoreceptors recognize specific pathogen effectors directly or indirectly. Little is known about how NB-LRR proteins recognize effectors of filamentous plant pathogens, such as Phytophthora infestans. AVR2 belongs to a family of 13 sequence-divergent P. infestans RXLR effectors that are differentially recognized by members of the R2 NB-LRR family in Solanum demissum. We report that the putative plant phosphatase BSU-LIKE PROTEIN1 (BSL1) is required for R2-mediated perception of AVR2 and resistance to P. infestans. AVR2 associates with BSL1 and mediates the interaction of BSL1 with R2 in planta, possibly through the formation of a ternary complex. Strains of P. infestans that are virulent on R2 potatoes express an unrecognized form, Avr2-like (referred to as A2l). A2L can still interact with BSL1 but does not promote the association of BSL1 with R2. Our findings show that recognition of the P. infestans AVR2 effector by the NB-LRR protein R2 requires the putative phosphatase BSL1. This reveals that, similar to effectors of phytopathogenic bacteria, recognition of filamentous pathogen effectors can be mediated via a host protein that interacts with both the effector and the NB-LRR immunoreceptor. PMID:22885736

  11. Host protein BSL1 associates with Phytophthora infestans RXLR effector AVR2 and the Solanum demissum Immune receptor R2 to mediate disease resistance.

    PubMed

    Saunders, Diane G O; Breen, Susan; Win, Joe; Schornack, Sebastian; Hein, Ingo; Bozkurt, Tolga O; Champouret, Nicolas; Vleeshouwers, Vivianne G A A; Birch, Paul R J; Gilroy, Eleanor M; Kamoun, Sophien

    2012-08-01

    Plant pathogens secrete effector proteins to modulate plant immunity and promote host colonization. Plant nucleotide binding leucine-rich repeat (NB-LRR) immunoreceptors recognize specific pathogen effectors directly or indirectly. Little is known about how NB-LRR proteins recognize effectors of filamentous plant pathogens, such as Phytophthora infestans. AVR2 belongs to a family of 13 sequence-divergent P. infestans RXLR effectors that are differentially recognized by members of the R2 NB-LRR family in Solanum demissum. We report that the putative plant phosphatase BSU-LIKE PROTEIN1 (BSL1) is required for R2-mediated perception of AVR2 and resistance to P. infestans. AVR2 associates with BSL1 and mediates the interaction of BSL1 with R2 in planta, possibly through the formation of a ternary complex. Strains of P. infestans that are virulent on R2 potatoes express an unrecognized form, Avr2-like (referred to as A2l). A2L can still interact with BSL1 but does not promote the association of BSL1 with R2. Our findings show that recognition of the P. infestans AVR2 effector by the NB-LRR protein R2 requires the putative phosphatase BSL1. This reveals that, similar to effectors of phytopathogenic bacteria, recognition of filamentous pathogen effectors can be mediated via a host protein that interacts with both the effector and the NB-LRR immunoreceptor.

  12. A Phytophthora sojae cytoplasmic effector mediates disease resistance and abiotic stress tolerance in Nicotiana benthamiana

    PubMed Central

    Zhang, Meixiang; Ahmed Rajput, Nasir; Shen, Danyu; Sun, Peng; Zeng, Wentao; Liu, Tingli; Juma Mafurah, Joseph; Dou, Daolong

    2015-01-01

    Each oomycete pathogen encodes a large number of effectors. Some effectors can be used in crop disease resistance breeding, such as to accelerate R gene cloning and utilisation. Since cytoplasmic effectors may cause acute physiological changes in host cells at very low concentrations, we assume that some of these effectors can serve as functional genes for transgenic plants. Here, we generated transgenic Nicotiana benthamiana plants that express a Phytophthora sojae CRN (crinkling and necrosis) effector, PsCRN115. We showed that its expression did not significantly affect the growth and development of N. benthamiana, but significantly improved disease resistance and tolerance to salt and drought stresses. Furthermore, we found that expression of heat-shock-protein and cytochrome-P450 encoding genes were unregulated in PsCRN115-transgenic N. benthamiana based on digital gene expression profiling analyses, suggesting the increased plant defence may be achieved by upregulation of these stress-related genes in transgenic plants. Thus, PsCRN115 may be used to improve plant tolerance to biotic and abiotic stresses. PMID:26039925

  13. A Phytophthora sojae cytoplasmic effector mediates disease resistance and abiotic stress tolerance in Nicotiana benthamiana.

    PubMed

    Zhang, Meixiang; Ahmed Rajput, Nasir; Shen, Danyu; Sun, Peng; Zeng, Wentao; Liu, Tingli; Juma Mafurah, Joseph; Dou, Daolong

    2015-06-03

    Each oomycete pathogen encodes a large number of effectors. Some effectors can be used in crop disease resistance breeding, such as to accelerate R gene cloning and utilisation. Since cytoplasmic effectors may cause acute physiological changes in host cells at very low concentrations, we assume that some of these effectors can serve as functional genes for transgenic plants. Here, we generated transgenic Nicotiana benthamiana plants that express a Phytophthora sojae CRN (crinkling and necrosis) effector, PsCRN115. We showed that its expression did not significantly affect the growth and development of N. benthamiana, but significantly improved disease resistance and tolerance to salt and drought stresses. Furthermore, we found that expression of heat-shock-protein and cytochrome-P450 encoding genes were unregulated in PsCRN115-transgenic N. benthamiana based on digital gene expression profiling analyses, suggesting the increased plant defence may be achieved by upregulation of these stress-related genes in transgenic plants. Thus, PsCRN115 may be used to improve plant tolerance to biotic and abiotic stresses.

  14. Nicotinic acid adenine dinucleotide phosphate-mediated calcium signalling in effector T cells regulates autoimmunity of the central nervous system

    PubMed Central

    Cordiglieri, Chiara; Odoardi, Francesca; Zhang, Bo; Nebel, Merle; Kawakami, Naoto; Klinkert, Wolfgang E. F.; Lodygin, Dimtri; Lühder, Fred; Breunig, Esther; Schild, Detlev; Ulaganathan, Vijay Kumar; Dornmair, Klaus; Dammermann, Werner; Potter, Barry V. L.; Guse, Andreas H.

    2010-01-01

    Nicotinic acid adenine dinucleotide phosphate represents a newly identified second messenger in T cells involved in antigen receptor-mediated calcium signalling. Its function in vivo is, however, unknown due to the lack of biocompatible inhibitors. Using a recently developed inhibitor, we explored the role of nicotinic acid adenine dinucleotide phosphate in autoreactive effector T cells during experimental autoimmune encephalomyelitis, the animal model for multiple sclerosis. We provide in vitro and in vivo evidence that calcium signalling controlled by nicotinic acid adenine dinucleotide phosphate is relevant for the pathogenic potential of autoimmune effector T cells. Live two photon imaging and molecular analyses revealed that nicotinic acid adenine dinucleotide phosphate signalling regulates T cell motility and re-activation upon arrival in the nervous tissues. Treatment with the nicotinic acid adenine dinucleotide phosphate inhibitor significantly reduced both the number of stable arrests of effector T cells and their invasive capacity. The levels of pro-inflammatory cytokines interferon-gamma and interleukin-17 were strongly diminished. Consecutively, the clinical symptoms of experimental autoimmune encephalomyelitis were ameliorated. In vitro, antigen-triggered T cell proliferation and cytokine production were evenly suppressed. These inhibitory effects were reversible: after wash-out of the nicotinic acid adenine dinucleotide phosphate antagonist, the effector T cells fully regained their functions. The nicotinic acid derivative BZ194 induced this transient state of non-responsiveness specifically in post-activated effector T cells. Naïve and long-lived memory T cells, which express lower levels of the putative nicotinic acid adenine dinucleotide phosphate receptor, type 1 ryanodine receptor, were not targeted. T cell priming and recall responses in vivo were not reduced. These data indicate that the nicotinic acid adenine dinucleotide phosphate

  15. Johnston Atoll Chemical Agent Disposal System (JACADS) Final Second Supplemental Environmental Impact Statement (SSEIS)

    SciTech Connect

    Not Available

    1990-06-01

    This Final Second Supplemental Environmental Impact Statement (SSEIS) to the Johnston Atoll Chemical Agent Disposal System (JACADS) Environmental Impact Statement (EIS) assesses the effects of receiving, storing, and ultimately destructing the United States stockpile of lethal unitary chemical munitions currently stored in the Federal Republic of Germany (FRG) (European stockpile) at the Army's JACADS facility located on Johnston Atoll in the Pacific Ocean. This Final SSEIS addresses the effects of the following proposed European stockpile activities: the transport of the European stockpile from the territorial limit to Johnston Island, the unloading of munitions from transportation ships, the on-island munitions transport and handling, on-island munitions storage, the disposal of munitions in the JACADS facility, the disposal of incineration wastes, and alternatives to the proposed action. This document also updates information in the 1983 EIS and the 1988 SEIS, as appropriate. This volume contains reproduced letters from various agencies, reproduced written comments received from the public, and a transcript from the public meeting.

  16. Ehrlichia chaffeensis Exploits Host SUMOylation Pathways To Mediate Effector-Host Interactions and Promote Intracellular Survival

    PubMed Central

    Dunphy, Paige Selvy; Luo, Tian

    2014-01-01

    Ehrlichia chaffeensis is an obligately intracellular Gram-negative bacterium that selectively infects mononuclear phagocytes. We recently reported that E. chaffeensis utilizes a type 1 secretion (T1S) system to export tandem repeat protein (TRP) effectors and demonstrated that these effectors interact with a functionally diverse array of host proteins. By way of these interactions, TRP effectors modulate host cell functions; however, the molecular basis of these interactions and their roles in ehrlichial pathobiology are not well defined. In this study, we describe the first bacterial protein posttranslational modification (PTM) by the small ubiquitin-like modifier (SUMO). The E. chaffeensis T1S effector TRP120 is conjugated to SUMO at a carboxy-terminal canonical consensus SUMO conjugation motif in vitro and in human cells. In human cells, TRP120 was selectively conjugated with SUMO2/3 isoforms. Disruption of TRP120 SUMOylation perturbed interactions with known host proteins, through predicted SUMO interaction motif-dependent and -independent mechanisms. E. chaffeensis infection did not result in dramatic changes in the global host SUMOylated protein profile, but a robust colocalization of predominately SUMO1 with ehrlichial inclusions was observed. Inhibiting the SUMO pathway with a small-molecule inhibitor had a significant impact on E. chaffeensis replication and recruitment of the TRP120-interacting protein polycomb group ring finger protein 5 (PCGF5) to the inclusion, indicating that the SUMO pathway is critical for intracellular survival. This study reveals the novel exploitation of the SUMO pathway by Ehrlichia, which facilitates effector-eukaryote interactions necessary to usurp the host and create a permissive intracellular niche. PMID:25047847

  17. Interfering TAL effectors of Xanthomonas oryzae neutralize R-gene-mediated plant disease resistance

    PubMed Central

    Ji, Zhiyuan; Ji, Chonghui; Liu, Bo; Zou, Lifang; Chen, Gongyou; Yang, Bing

    2016-01-01

    Plant pathogenic bacteria of the genus Xanthomonas possess transcription activator-like effectors (TALEs) that activate transcription of disease susceptibility genes in the host, inducing a state of disease. Here we report that some isolates of the rice pathogen Xanthomonas oryzae use truncated versions of TALEs (which we term interfering TALEs, or iTALEs) to overcome disease resistance. In comparison with typical TALEs, iTALEs lack a transcription activation domain but retain nuclear localization motifs and are expressed from genes that were previously considered pseudogenes. We show that the rice gene Xa1, encoding a nucleotide-binding leucine-rich repeat protein, confers resistance against X. oryzae isolates by recognizing multiple TALEs. However, the iTALEs present in many isolates interfere with the otherwise broad-spectrum resistance conferred by Xa1. Our findings illustrate how bacterial effectors that trigger disease resistance in the host can evolve to interfere with the resistance process and, thus, promote disease. PMID:27811915

  18. The Effector Cig57 Hijacks FCHO-Mediated Vesicular Trafficking to Facilitate Intracellular Replication of Coxiella burnetii

    PubMed Central

    Latomanski, Eleanor A.; Newton, Patrice; Khoo, Chen Ai; Newton, Hayley J.

    2016-01-01

    Coxiella burnetii is an intracellular bacterial pathogen that infects alveolar macrophages and replicates within a unique lysosome-derived vacuole. When Coxiella is trafficked to a host cell lysosome the essential Dot/Icm type IV secretion system is activated allowing over 130 bacterial effector proteins to be translocated into the host cytosol. This cohort of effectors is believed to manipulate host cell functions to facilitate Coxiella-containing vacuole (CCV) biogenesis and bacterial replication. Transposon mutagenesis has demonstrated that the Dot/Icm effector Cig57 is required for CCV development and intracellular replication of Coxiella. Here, we demonstrate a role for Cig57 in subverting clathrin-mediated traffic through its interaction with FCHO2, an accessory protein of clathrin coated pits. A yeast two-hybrid screen identified FCHO2 as a binding partner of Cig57 and this interaction was confirmed during infection using immunoprecipitation experiments. The interaction between Cig57 and FCHO2 is dependent on one of three endocytic sorting motif encoded by Cig57. Importantly, complementation analysis demonstrated that this endocytic sorting motif is required for full function of Cig57. Consistent with the intracellular growth defect in cig57-disrupted Coxiella, siRNA gene silencing of FCHO2 or clathrin (CLTC) inhibits Coxiella growth and CCV biogenesis. Clathrin is recruited to the replicative CCV in a manner that is dependent on the interaction between Cig57 and FCHO2. Creation of an FCHO2 knockout cell line confirmed the importance of this protein for CCV expansion, intracellular replication of Coxiella and clathrin recruitment to the CCV. Collectively, these results reveal Cig57 to be a significant virulence factor that co-opts clathrin-mediated trafficking, via interaction with FCHO2, to facilitate the biogenesis of the fusogenic Coxiella replicative vacuole and enable intracellular success of this human pathogen. PMID:28002452

  19. Requirements for capsid-binding and an effector function in TRIMCyp-mediated restriction of HIV-1

    SciTech Connect

    Diaz-Griffero, Felipe; Vandegraaff, Nick; Li Yuan; McGee-Estrada, Kathleen; Stremlau, Matthew; Welikala, Sohanya; Si Zhihai; Engelman, Alan; Sodroski, Joseph . E-mail: joseph_sodroski@dfci.harvard.edu

    2006-08-01

    In owl monkeys, a retrotransposition event replaced the gene encoding the retroviral restriction factor TRIM5{alpha} with one encoding TRIMCyp, a fusion between the RING, B-box 2 and coiled-coil domains of TRIM5 and cyclophilin A. TRIMCyp restricts human immunodeficiency virus (HIV-1) infection by a mechanism dependent on the interaction of the cyclophilin A moiety and the HIV-1 capsid protein. Here, we show that infection by retroviruses other than HIV-1 can be restricted by TRIMCyp, providing an explanation for the evolutionary retention of the TRIMCyp gene in owl monkey lineages. The TRIMCyp-mediated block to HIV-1 infection occurs before the earliest step of reverse transcription. TRIMCyp-mediated restriction involves at least two functions: (1) capsid binding, which occurs most efficiently for trimeric TRIMCyp proteins that retain the coiled-coil and cyclophilin A domains, and (2) an effector function that depends upon the B-box 2 domain.

  20. Toxicity and SidJ-Mediated Suppression of Toxicity Require Distinct Regions in the SidE Family of Legionella pneumophila Effectors.

    PubMed

    Havey, James C; Roy, Craig R

    2015-09-01

    Intracellular bacteria use a variety of strategies to evade degradation and create a replicative niche. Legionella pneumophila is an intravacuolar pathogen that establishes a replicative niche through the secretion of more than 300 effector proteins. The function of most effectors remains to be determined. Toxicity in yeast has been used to identify functional domains and elucidate the biochemical function of effectors. A library of L. pneumophila effectors was screened using an expression plasmid that produces low levels of each protein. This screen identified the effector SdeA as a protein that confers a strong toxic phenotype that inhibits yeast replication. The toxicity of SdeA was suppressed in cells producing the effector SidJ. The effector SdeA is a member of the SidE family of L. pneumophila effector proteins. All SidE orthologs encoded by the Philadelphia isolate of Legionella pneumophila were toxic to yeast, and SidJ suppressed the toxicity of each. We identified a conserved central region in the SidE proteins that was sufficient to mediate yeast toxicity. Surprisingly, SidJ did not suppress toxicity when this central region was produced in yeast. We determined that the amino-terminal region of SidE was essential for SidJ-mediated suppression of toxicity. Thus, there is a genetic interaction that links the activity of SidJ and the amino-terminal region of SidE, which is required to modulate the toxic activity displayed by the central region of the SidE protein. This suggests a complex mechanism by which the L. pneumophila effector SidJ modulates the function of the SidE proteins after translocation into host cells.

  1. Integrin αvβ8-Mediated TGF-β Activation by Effector Regulatory T Cells Is Essential for Suppression of T-Cell-Mediated Inflammation

    PubMed Central

    Worthington, John J.; Kelly, Aoife; Smedley, Catherine; Bauché, David; Campbell, Simon; Marie, Julien C.; Travis, Mark A.

    2015-01-01

    Summary Regulatory T (Treg) cells play a pivotal role in suppressing self-harmful T cell responses, but how Treg cells mediate suppression to maintain immune homeostasis and limit responses during inflammation is unclear. Here we show that effector Treg cells express high amounts of the integrin αvβ8, which enables them to activate latent transforming growth factor-β (TGF-β). Treg-cell-specific deletion of integrin αvβ8 did not result in a spontaneous inflammatory phenotype, suggesting that this pathway is not important in Treg-cell-mediated maintenance of immune homeostasis. However, Treg cells lacking expression of integrin αvβ8 were unable to suppress pathogenic T cell responses during active inflammation. Thus, our results identify a mechanism by which Treg cells suppress exuberant immune responses, highlighting a key role for effector Treg-cell-mediated activation of latent TGF-β in suppression of self-harmful T cell responses during active inflammation. PMID:25979421

  2. Effector-Mediated Interaction of CbbRI and CbbRII Regulators with Target Sequences in Rhodobacter capsulatus

    PubMed Central

    Dubbs, Padungsri; Dubbs, James M.; Tabita, F. Robert

    2004-01-01

    In Rhodobacter capsulatus, genes encoding enzymes of the Calvin-Benson-Bassham reductive pentose phosphate pathway are located in the cbbI and cbbII operons. Each operon contains a divergently transcribed LysR-type transcriptional activator (CbbRI and CbbRII) that regulates the expression of its cognate cbb promoter in response to an as yet unidentified effector molecule(s). Both CbbRI and CbbRII were purified, and the ability of a variety of potential effector molecules to induce changes in their DNA binding properties at their target promoters was assessed. The responses of CbbRI and CbbRII to potential effectors were not identical. In gel mobility shift assays, the affinity of both CbbRI and CbbRII for their target promoters was enhanced in the presence of ribulose-1,5-bisphosphate (RuBP), phosphoenolpyruvate, 3-phosphoglycerate, 2-phosphoglycolate. ATP, 2-phosphoglycerate, and KH2PO4 were found to enhance only CbbRI binding, while fructose-1,6-bisphosphate enhanced the binding of only CbbRII. The DNase I footprint of CbbRI was reduced in the presence of RuBP, while reductions in the CbbRII DNase I footprint were induced by fructose-1,6-bisphosphate, 3-phosphoglycerate, and KH2PO4. The current in vitro results plus recent in vivo studies suggest that CbbR-mediated regulation of cbb transcription is controlled by multiple metabolic signals in R. capsulatus. This control reflects not only intracellular levels of Calvin-Benson-Bassham cycle metabolic intermediates but also the fixed (organic) carbon status and energy charge of the cell. PMID:15547275

  3. Effector-mediated interaction of CbbRI and CbbRII regulators with target sequences in Rhodobacter capsulatus.

    PubMed

    Dubbs, Padungsri; Dubbs, James M; Tabita, F Robert

    2004-12-01

    In Rhodobacter capsulatus, genes encoding enzymes of the Calvin-Benson-Bassham reductive pentose phosphate pathway are located in the cbb(I) and cbb(II) operons. Each operon contains a divergently transcribed LysR-type transcriptional activator (CbbR(I) and CbbR(II)) that regulates the expression of its cognate cbb promoter in response to an as yet unidentified effector molecule(s). Both CbbR(I) and CbbR(II) were purified, and the ability of a variety of potential effector molecules to induce changes in their DNA binding properties at their target promoters was assessed. The responses of CbbR(I) and CbbR(II) to potential effectors were not identical. In gel mobility shift assays, the affinity of both CbbR(I) and CbbR(II) for their target promoters was enhanced in the presence of ribulose-1,5-bisphosphate (RuBP), phosphoenolpyruvate, 3-phosphoglycerate, 2-phosphoglycolate. ATP, 2-phosphoglycerate, and KH(2)PO(4) were found to enhance only CbbR(I) binding, while fructose-1,6-bisphosphate enhanced the binding of only CbbR(II). The DNase I footprint of CbbR(I) was reduced in the presence of RuBP, while reductions in the CbbR(II) DNase I footprint were induced by fructose-1,6-bisphosphate, 3-phosphoglycerate, and KH(2)PO(4). The current in vitro results plus recent in vivo studies suggest that CbbR-mediated regulation of cbb transcription is controlled by multiple metabolic signals in R. capsulatus. This control reflects not only intracellular levels of Calvin-Benson-Bassham cycle metabolic intermediates but also the fixed (organic) carbon status and energy charge of the cell.

  4. Polyfunctional Fc-effector profiles mediated by IgG subclass selection distinguish RV144 and VAX003 vaccines.

    PubMed

    Chung, Amy W; Ghebremichael, Musie; Robinson, Hannah; Brown, Eric; Choi, Ickwon; Lane, Sophie; Dugast, Anne-Sophie; Schoen, Matthew K; Rolland, Morgane; Suscovich, Todd J; Mahan, Alison E; Liao, Larry; Streeck, Hendrik; Andrews, Charla; Rerks-Ngarm, Supachai; Nitayaphan, Sorachai; de Souza, Mark S; Kaewkungwal, Jaranit; Pitisuttithum, Punnee; Francis, Donald; Michael, Nelson L; Kim, Jerome H; Bailey-Kellogg, Chris; Ackerman, Margaret E; Alter, Galit

    2014-03-19

    The human phase 2B RV144 ALVAC-HIV vCP1521/AIDSVAX B/E vaccine trial, held in Thailand, resulted in an estimated 31.2% efficacy against HIV infection. By contrast, vaccination with VAX003 (consisting of only AIDSVAX B/E) was not protective. Because protection within RV144 was observed in the absence of neutralizing antibody activity or cytotoxic T cell responses, we speculated that the specificity or qualitative differences in Fc-effector profiles of nonneutralizing antibodies may have accounted for the efficacy differences observed between the two trials. We show that the RV144 regimen elicited nonneutralizing antibodies with highly coordinated Fc-mediated effector responses through the selective induction of highly functional immunoglobulin G3 (IgG3). By contrast, VAX003 elicited monofunctional antibody responses influenced by IgG4 selection, which was promoted by repeated AIDSVAX B/E protein boosts. Moreover, only RV144 induced IgG1 and IgG3 antibodies targeting the crown of the HIV envelope V2 loop, albeit with limited coverage of breakthrough viral sequences. These data suggest that subclass selection differences associated with coordinated humoral functional responses targeting strain-specific protective V2 loop epitopes may underlie differences in vaccine efficacy observed between these two vaccine trials.

  5. Neutrophils prime a long-lived effector macrophage phenotype that mediates accelerated helminth expulsion

    USDA-ARS?s Scientific Manuscript database

    The innate immune cell populations that mediate metazoan parasite expulsion remain largely undefined. We examined the role of innate cells in the immune response to the nematode parasite Nippostrongylus brasiliensis hypothesizing that they may mediate the markedly accelerated CD4+ T cell-independen...

  6. Novel mathematical models for cell-mediated cytotoxicity assays without applying enzyme kinetics but with combinations and probability: bystanders in bulk effector cells influence results of cell-mediated cytotoxicity assays.

    PubMed

    Takayanagi, Toshiaki

    2011-07-01

    Cell-mediated cytotoxicity assays are widely implemented to evaluate cell-mediated cytotoxic activity, and some assays are analyzed using the analogy of enzyme kinetics. In the analogy, the effector cell is regarded as the enzyme, the target cell as the substrate, the effector cell-target cell conjugate as the enzyme-substrate complex and the dead target cell as the product. However, the assumptions analogous to those of enzyme kinetics are not always true in cell-mediated cytotoxicity assays, and the parameter analogous to the Michaelis-Menten constant is not constant but is dependent on the number of effector cells. Therefore I present novel mathematical models for cell-mediated cytotoxicity assays without applying enzyme kinetics. I instead use combinations and probability, because analysis of cell-mediated cytotoxicity assays by applying enzyme kinetics seems controversial. With my original models, I demonstrate simulations of the data in previously published papers. The results are exhibited in the same forms as the corresponding data. Comparing the simulation results with the published data, the results seem to agree well with the data. From simulations of cytotoxic assays with bulk effector cells, it appears that bystanders in bulk effector cells increase both the cytotoxic activity and the motility of effector cells.

  7. A type III-B CRISPR-Cas effector complex mediating massive target DNA destruction

    PubMed Central

    Han, Wenyuan; Li, Yingjun; Deng, Ling; Feng, Mingxia; Peng, Wenfang; Hallstrøm, Søren; Zhang, Jing; Peng, Nan; Liang, Yun Xiang; White, Malcolm F.

    2017-01-01

    Abstract The CRISPR (clustered regularly interspaced short palindromic repeats) system protects archaea and bacteria by eliminating nucleic acid invaders in a crRNA-guided manner. The Sulfolobus islandicus type III-B Cmr–α system targets invading nucleic acid at both RNA and DNA levels and DNA targeting relies on the directional transcription of the protospacer in vivo. To gain further insight into the involved mechanism, we purified a native effector complex of III-B Cmr–α from S. islandicus and characterized it in vitro. Cmr–α cleaved RNAs complementary to crRNA present in the complex and its ssDNA destruction activity was activated by target RNA. The ssDNA cleavage required mismatches between the 5΄-tag of crRNA and the 3΄-flanking region of target RNA. An invader plasmid assay showed that mutation either in the histidine-aspartate acid (HD) domain (a quadruple mutation) or in the GGDD motif of the Cmr–2α protein resulted in attenuation of the DNA interference in vivo. However, double mutation of the HD motif only abolished the DNase activity in vitro. Furthermore, the activated Cmr–α binary complex functioned as a highly active DNase to destroy a large excess DNA substrate, which could provide a powerful means to rapidly degrade replicating viral DNA. PMID:27986854

  8. Type III chaperones & Co in bacterial plant pathogens: a set of specialized bodyguards mediating effector delivery.

    PubMed

    Lohou, David; Lonjon, Fabien; Genin, Stéphane; Vailleau, Fabienne

    2013-11-22

    Gram-negative plant pathogenic bacteria possess a type III secretion system (T3SS) to inject bacterial proteins, called type III effectors (T3Es), into host cells through a specialized syringe structure. T3Es are virulence factors that can suppress plant immunity but they can also conversely be recognized by the plant and trigger specific resistance mechanisms. The T3SS and injected T3Es play a central role in determining the outcome of a host-pathogen interaction. Still little is known in plant pathogens on the assembly of the T3SS and the regulatory mechanisms involved in the temporal control of its biosynthesis and T3E translocation. However, recent insights point out the role of several proteins as prime candidates in the role of regulators of the type III secretion (T3S) process. In this review we report on the most recent advances on the regulation of the T3S by focusing on protein players involved in secretion/translocation regulations, including type III chaperones (T3Cs), type III secretion substrate specificity switch (T3S4) proteins and other T3S orchestrators.

  9. DUOX2-derived reactive oxygen species are effectors of NOD2-mediated antibacterial responses.

    PubMed

    Lipinski, Simone; Till, Andreas; Sina, Christian; Arlt, Alexander; Grasberger, Helmut; Schreiber, Stefan; Rosenstiel, Philip

    2009-10-01

    Generation of microbicidal reactive oxygen species (ROS) is a pivotal protective component of the innate immune system in many eukaryotes. NOD (nucleotide oligomerisation domain containing protein)-like receptors (NLRs) have been implicated as phylogenetically ancient sensors of intracellular pathogens or endogenous danger signals. NOD2 recognizes the bacterial cell wall component muramyldipeptide leading to NFkappaB and MAPK activation via induced proximity signalling through the serine-threonine kinase RIP2. In addition to the subsequent induction of cytokines and antimicrobial peptides, NOD2 has been shown also to exert a direct antibacterial effect. Using a fluorescence-based ROS detection assay we demonstrate controlled ROS generation as an integral component of NOD2-induced signalling in epithelial cells. We demonstrate that the NAD(P)H oxidase family member DUOX2 is involved in NOD2-dependent ROS production. Coimmunoprecipitation and fluorescence microscopy were used to show that DUOX2 interacts and colocalizes with NOD2 at the plasma membrane. Moreover, simultaneous overexpression of NOD2 and DUOX2 was found to result in cooperative protection against bacterial cytoinvasion using the Listeria monocytogenes infection model. RNAi-based studies revealed that DUOX2 is required for the direct bactericidal properties of NOD2. Our results demonstrate a new role of ROS as effector molecules of protective cellular signalling in response to a defined danger signal carried out by a mammalian intracellular NLR system.

  10. Expression of membrane-bound burst-promoting activity is mediated by allogeneic effector cells.

    PubMed

    Guha, A; Tuck, D; Sorba, S; Dainiak, N

    1993-09-01

    To investigate whether "self" and "non-self" recognition processes are involved in murine erythropoiesis, the expression of membrane-bound burst-promoting activity (mBPA) was determined for B lymphocytes purified from spleens of CF-1, C57 BL/6J, B6021-7115, and CAF-1J mice using syngeneic and allogeneic bone marrow cultures. Addition of B lymphocyte conditioned medium (LCM), shed membrane-derived vesicles, or intact plasma membranes prepared from syngeneic murine cells stimulated erythroid burst-forming unit (BFU-E) proliferation by two- to three-fold above control levels. BFU-E proliferation was increased by six- to eight-fold, however, when LCM, shed membrane vesicles, or plasma membranes purified from allogenic B lymphocytes were used as sources of growth-stimulatory activity. Bioactivity was immunoprecipitated from detergent extracts of membranes purified from both allogeneic and syngeneic lymphocytes with a monoclonal antibody that specifically recognizes mBPA, suggesting that the factors expressed by these cells share antigenic determinants. The results indicate that allogeneic effector cells are a more potent source of mBPA-like molecules than are syngeneic cells, suggesting that immune mechanisms may be involved in inducing erythroid growth factor expression at the B cell surface.

  11. EDS1 mediates pathogen resistance and virulence function of a bacterial effector in soybean

    USDA-ARS?s Scientific Manuscript database

    Enhanced disease susceptibility 1 (EDS1) and phytoalexin deficient 4 (PAD4) are well known regulators of both basal and resistance (R) protein-mediated plant defense. We identified two EDS1- (GmEDS1a/b) and one PAD4-like (GmPAD4) protein that are required for resistance signaling in soybean. Consist...

  12. Cervical Remodeling/Ripening at Term and Preterm Delivery: The Same Mechanism Initiated by Different Mediators and Different Effector Cells

    PubMed Central

    Gonzalez, Juan M.; Dong, Zhong; Romero, Roberto; Girardi, Guillermina

    2011-01-01

    Background Premature cervical remodeling/ripening is believed to contribute to preterm delivery (PTD), the leading cause of perinatal morbidity and mortality. Despite considerable research, the causes of term and PTD remain unclear, and there is no effective treatment for PTD. We previously demonstrated that complement activation plays a causative role in cervical remodeling that leads to PTD in mice. Methodology/Principal Findings Here we found that complement activation is not required for the physiological process that leads to term delivery in mice. Neither increased C3 cervical deposition nor increased C3a and C5a serum levels were observed at term. In addition, macrophages infiltration was found in PTD in contrast to term delivery were no leukocytes were found. Despite the different role of complement and different cellular effector cells, PTD and term delivery share a common dowsntream pathway characterized by increased metalloproteinases (MMPs) release and increased collagen degradation. However, different sources of MMPs were identified. Macrophages are the source of MMPs in PTD while cervical fibroblasts and columnar epithelial cells synthesize MMPs at term delivery. A dramatic diminution in serum progesterone levels precedes parturition at term but not in PTD, suggesting that progesterone withdrawal initiates cervical remodeling at term. On the other hand, MMPs release in PTD is triggered by C5a. Conclusion and Significance In conclusion, preterm and term cervical remodeling occur through the same mechanism but they are initiated by different mediators and effector cells. That complement activation is required for PTD but not for the physiological process that leads to term delivery, suggests that complement is a potential specific biomarker and selective target to prevent PTD and thus avert neonatal mortality and morbidity. PMID:22073213

  13. Eosinophilia of dystrophin-deficient muscle is promoted by perforin-mediated cytotoxicity by T cell effectors

    NASA Technical Reports Server (NTRS)

    Cai, B.; Spencer, M. J.; Nakamura, G.; Tseng-Ong, L.; Tidball, J. G.

    2000-01-01

    Previous investigations have shown that cytotoxic T lymphocytes (CTLs) contribute to muscle pathology in the dystrophin-null mutant mouse (mdx) model of Duchenne muscular dystrophy through perforin-dependent and perforin-independent mechanisms. We have assessed whether the CTL-mediated pathology includes the promotion of eosinophilia in dystrophic muscle, and thereby provides a secondary mechanism through which CTLs contribute to muscular dystrophy. Quantitative immunohistochemistry confirmed that eosinophilia is a component of the mdx dystrophy. In addition, electron microscopic observations show that eosinophils traverse the basement membrane of mdx muscle fibers and display sites of close apposition of eosinophil and muscle membranes. The close membrane apposition is characterized by impingement of eosinophilic rods of major basic protein into the muscle cell membrane. Transfer of mdx splenocytes and mdx muscle extracts to irradiated C57 mice by intraperitoneal injection resulted in muscle eosinophilia in the recipient mice. Double-mutant mice lacking dystrophin and perforin showed less eosinophilia than was displayed by mdx mice that expressed perforin. Finally, administration of prednisolone, which has been shown previously to reduce the concentration of CTLs in dystrophic muscle, produced a significant reduction in eosinophilia. These findings indicate that eosinophilia is a component of the mdx pathology that is promoted by perforin-dependent cytotoxicity of effector T cells. However, some eosinophilia of mdx muscle is independent of perforin-mediated processes.

  14. TCF-1-mediated Wnt signaling regulates Paneth cell innate immune defense effectors HD-5 and -6: implications for Crohn's disease.

    PubMed

    Beisner, Julia; Teltschik, Zora; Ostaff, Maureen J; Tiemessen, Machteld M; Staal, Frank J T; Wang, Guoxing; Gersemann, Michael; Perminow, Gori; Vatn, Morten H; Schwab, Matthias; Stange, Eduard F; Wehkamp, Jan

    2014-09-01

    Wnt signaling regulates small intestinal stem cell maintenance and Paneth cell differentiation. In patients with ileal Crohn's disease (CD), a decrease of Paneth cell α-defensins has been observed that is partially caused by impaired TCF-4 and LRP6 function. Here we show reduced expression of the Wnt signaling effector TCF-1 (also known as TCF-7) in patients with ileal CD. Reporter gene assays and in vitro promoter binding analysis revealed that TCF-1 activates α-defensin HD-5 and HD-6 transcription in cooperation with β-catenin and that activation is mediated by three distinct TCF binding sites. EMSA analysis showed binding of TCF-1 to the respective motifs. In ileal CD patients, TCF-1 mRNA expression levels were significantly reduced. Moreover, we found specifically reduced expression of active TCF-1 mRNA isoforms. Tcf-1 knockout mice exhibited reduced cryptdin expression in the jejunum, which was not consistently seen at other small intestinal locations. Our data provide evidence that TCF-1-mediated Wnt signaling is disturbed in small intestinal CD, which might contribute to the observed barrier dysfunction in the disease.

  15. Eosinophilia of dystrophin-deficient muscle is promoted by perforin-mediated cytotoxicity by T cell effectors

    NASA Technical Reports Server (NTRS)

    Cai, B.; Spencer, M. J.; Nakamura, G.; Tseng-Ong, L.; Tidball, J. G.

    2000-01-01

    Previous investigations have shown that cytotoxic T lymphocytes (CTLs) contribute to muscle pathology in the dystrophin-null mutant mouse (mdx) model of Duchenne muscular dystrophy through perforin-dependent and perforin-independent mechanisms. We have assessed whether the CTL-mediated pathology includes the promotion of eosinophilia in dystrophic muscle, and thereby provides a secondary mechanism through which CTLs contribute to muscular dystrophy. Quantitative immunohistochemistry confirmed that eosinophilia is a component of the mdx dystrophy. In addition, electron microscopic observations show that eosinophils traverse the basement membrane of mdx muscle fibers and display sites of close apposition of eosinophil and muscle membranes. The close membrane apposition is characterized by impingement of eosinophilic rods of major basic protein into the muscle cell membrane. Transfer of mdx splenocytes and mdx muscle extracts to irradiated C57 mice by intraperitoneal injection resulted in muscle eosinophilia in the recipient mice. Double-mutant mice lacking dystrophin and perforin showed less eosinophilia than was displayed by mdx mice that expressed perforin. Finally, administration of prednisolone, which has been shown previously to reduce the concentration of CTLs in dystrophic muscle, produced a significant reduction in eosinophilia. These findings indicate that eosinophilia is a component of the mdx pathology that is promoted by perforin-dependent cytotoxicity of effector T cells. However, some eosinophilia of mdx muscle is independent of perforin-mediated processes.

  16. Neutrophils as effector cells of T-cell-mediated, acquired immunity in murine listeriosis.

    PubMed Central

    Appelberg, R; Castro, A G; Silva, M T

    1994-01-01

    The control of the infections caused by Listeria monocytogenes, considered an example of an intracellular parasite, is thought to involve co-operation between antigen-specific T cells and activated macrophages. Here we investigated the participation of polymorphonuclear leucocytes in the mechanisms of resistance during the immune phase of the antimicrobial response to L. monocytogenes infection. We found that BALB/c mice were unable to express T-cell-mediated (acquired) immunity to this pathogen in the absence of granulocytes. We propose that neutrophils should be included in the concept of cell-mediated immunity and that their antimicrobial role is not exclusively expressed during the early phases of a primary infection. PMID:7835951

  17. Osteopontin is a proximal effector of leptin-mediated non-alcoholic steatohepatitis (NASH) fibrosis

    PubMed Central

    Coombes, Jason D.; Choi, Steve S.; Swiderska-Syn, Marzena; Manka, Paul P.; Reid, Danielle; Palma, Elena; Briones-Orta, Marco A.; Xie, Guanhua; Younis, Rasha; Kitamura, Naoto; Peruta, Marco della; Bitencourt, Shanna; Dollé, Laurent; Oo, Ye Htun; Mi, Zhiyong; Kuo, Paul C.; Williams, Roger; Chokshi, Shilpa; Canbay, Ali; Claridge, Lee C.; Eksteen, Bertus; Diehl, Anna Mae; Syn, Wing-Kin

    2016-01-01

    Introduction Liver fibrosis develops when hepatic stellate cells (HSC) are activated into collagen-producing myofibroblasts. In non-alcoholic steatohepatitis (NASH), the adipokine leptin is upregulated, and promotes liver fibrosis by directly activating HSC via the hedgehog pathway. We reported that hedgehog-regulated osteopontin (OPN) plays a key role in promoting liver fibrosis. Herein, we evaluated if OPN mediates leptin-profibrogenic effects in NASH. Methods Leptin-deficient (ob/ob) and wild-type (WT) mice were fed control or methionine-choline deficient (MCD) diet. Liver tissues were assessed by Sirius-red, OPN and αSMA IHC, and qRT-PCR for fibrogenic genes. In vitro, HSC with stable OPN (or control) knockdown were treated with recombinant (r)leptin and OPN-neutralizing or sham-aptamers. HSC response to OPN loss was assessed by wound healing assay. OPN-aptamers were also added to precision-cut liver slices (PCLS), and administered to MCD-fed WT (leptin-intact) mice to determine if OPN neutralization abrogated fibrogenesis. Results MCD-fed WT mice developed NASH-fibrosis, upregulated OPN, and accumulated αSMA+ cells. Conversely, MCD-fed ob/ob mice developed less fibrosis and accumulated fewer αSMA+ and OPN+ cells. In vitro, leptin-treated HSC upregulated OPN, αSMA, collagen 1α1 and TGFβ mRNA by nearly 3-fold, but this effect was blunted by OPN loss. Inhibition of PI3K and transduction of dominant negative-Akt abrogated leptin-mediated OPN induction, while constitutive active-Akt upregulated OPN. Finally, OPN neutralization reduced leptin-mediated fibrogenesis in both PCLS and MCD-fed mice. Conclusion OPN overexpression in NASH enhances leptin-mediated fibrogenesis via PI3K/Akt. OPN neutralization significantly reduces NASH fibrosis, reinforcing the potential utility of targeting OPN in the treatment of patients with advanced NASH. PMID:26529285

  18. Effector CD4+ T cell expression signatures and immune-mediated disease associated genes.

    PubMed

    Zhang, Wei; Ferguson, John; Ng, Sok Meng; Hui, Ken; Goh, Gerald; Lin, Aiping; Esplugues, Enric; Flavell, Richard A; Abraham, Clara; Zhao, Hongyu; Cho, Judy H

    2012-01-01

    Genome-wide association studies (GWAS) in immune-mediated diseases have identified over 150 associated genomic loci. Many of these loci play a role in T cell responses, and regulation of T cell differentiation plays a critical role in immune-mediated diseases; however, the relationship between implicated disease loci and T cell differentiation is incompletely understood. To further address this relationship, we examined differential gene expression in naïve human CD4+ T cells, as well as in in vitro differentiated Th1, memory Th17-negative and Th17-enriched CD4+ T cells subsets using microarray and RNASeq. We observed a marked enrichment for increased expression in memory CD4+ compared to naïve CD4+ T cells of genes contained among immune-mediated disease loci. Within memory T cells, expression of disease-associated genes was typically increased in Th17-enriched compared to Th17-negative cells. Utilizing RNASeq and promoter methylation studies, we identified a differential regulation pattern for genes solely expressed in Th17 cells (IL17A and CCL20) compared to genes expressed in both Th17 and Th1 cells (IL23R and IL12RB2), where high levels of promoter methylation are correlated to near zero RNASeq levels for IL17A and CCL20. These findings have implications for human Th17 celI plasticity and for the regulation of Th17-Th1 expression signatures. Importantly, utilizing RNASeq we found an abundant isoform of IL23R terminating before the transmembrane domain that was enriched in Th17 cells. In addition to molecular resolution, we find that RNASeq provides significantly improved power to define differential gene expression and identify alternative gene variants relative to microarray analysis. The comprehensive integration of differential gene expression between cell subsets with disease-association signals, and functional pathways provides insight into disease pathogenesis.

  19. Ectopic expression of EFFECTOR OF TRANSCRIPTION perturbs gibberellin-mediated plant developmental processes.

    PubMed

    Ellerström, M; Reidt, W; Ivanov, R; Tiedemann, J; Melzer, M; Tewes, A; Moritz, T; Mock, H-P; Sitbon, F; Rask, L; Bäumlein, H

    2005-11-01

    The plant hormone gibberellin (GA) is known to modulate various aspects of plant cell differentiation and development. The current model of GA-mediated regulation is based on a de-repressible system and includes specific protein modification and degradation. HRT, a zinc finger protein from barley has been shown to have GA-dependent transcriptional repressing activity on the seed-specific alpha-amylase promoter [Raventos, D., Skriver, K., Schlein, M., Karnahl, K., Rogers, S.W., Rogers, J.C. and Mundy, J. 1998. J. Biol. Chem. 273: 23313-23320]. Here we report the characterization of a dicot homologue from Brassica napus (BnET) and provide evidence for its role in GA response modulation suggesting that this could be a conserved feature of this gene family. When BnET is ectopically expressed in either Arabidopsis or tobacco the phenotypes include dwarfism due to shorter internodes and late flowering, reduced germination rate, increased anthocyanin content and reduced xylem lignification as a marker for terminal cell differentiation. Transient expression in protoplasts supports the notion that this most likely is due to a transcriptional repression of GA controlled genes. Finally, histological analysis showed that in contrast to other GA deficient mutants the shorter internodes were due to fewer but not smaller cells, suggesting a function of BnET in GA-mediated cell division control.

  20. Amotl1 mediates sequestration of the Hippo effector Yap1 downstream of Fat4 to restrict heart growth

    PubMed Central

    Ragni, Chiara V.; Diguet, Nicolas; Le Garrec, Jean-François; Novotova, Marta; Resende, Tatiana P.; Pop, Sorin; Charon, Nicolas; Guillemot, Laurent; Kitasato, Lisa; Badouel, Caroline; Dufour, Alexandre; Olivo-Marin, Jean-Christophe; Trouvé, Alain; McNeill, Helen; Meilhac, Sigolène M

    2017-01-01

    Although in flies the atypical cadherin Fat is an upstream regulator of Hippo signalling, the closest mammalian homologue, Fat4, has been shown to regulate tissue polarity rather than growth. Here we show in the mouse heart that Fat4 modulates Hippo signalling to restrict growth. Fat4 mutant myocardium is thicker, with increased cardiomyocyte size and proliferation, and this is mediated by an upregulation of the transcriptional activity of Yap1, an effector of the Hippo pathway. Fat4 is not required for the canonical activation of Hippo kinases but it sequesters a partner of Yap1, Amotl1, out of the nucleus. The nuclear translocation of Amotl1 is accompanied by Yap1 to promote cardiomyocyte proliferation. We, therefore, identify Amotl1, which is not present in flies, as a mammalian intermediate for non-canonical Hippo signalling, downstream of Fat4. This work uncovers a mechanism for the restriction of heart growth at birth, a process which impedes the regenerative potential of the mammalian heart. PMID:28239148

  1. Nonsense-Mediated mRNA Decay Effectors Are Essential for Zebrafish Embryonic Development and Survival ▿ †

    PubMed Central

    Wittkopp, Nadine; Huntzinger, Eric; Weiler, Catrin; Saulière, Jérôme; Schmidt, Steffen; Sonawane, Mahendra; Izaurralde, Elisa

    2009-01-01

    The nonsense-mediated mRNA decay (NMD) pathway promotes rapid degradation of mRNAs containing premature translation termination codons (PTCs or nonsense codons), preventing accumulation of potentially detrimental truncated proteins. In metazoa, seven genes (upf1, upf2, upf3, smg1, smg5, smg6, and smg7) have been identified as essential for NMD; here we show that the zebrafish genome encodes orthologs of upf1, upf2, smg1, and smg5 to smg7 and two upf3 paralogs. We also show that Upf1 is required for degradation of PTC-containing mRNAs in zebrafish embryos. Moreover, its depletion has a severe impact on embryonic development, early patterning, and viability. Similar phenotypes are observed in Upf2-, Smg5-, or Smg6-depleted embryos, suggesting that zebrafish embryogenesis requires an active NMD pathway. Using cultured cells, we demonstrate that the ability of a PTC to trigger NMD is strongly stimulated by downstream exon-exon boundaries. Thus, as in mammals and plants but in contrast to invertebrates and fungi, NMD is coupled to splicing in zebrafish. Our results together with previous studies show that NMD effectors are essential for vertebrate embryogenesis and suggest that the coupling of splicing and NMD has been maintained in vertebrates but lost in fungi and invertebrates. PMID:19414594

  2. Akt Phosphorylates Wnt Coactivator and Chromatin Effector Pygo2 at Serine 48 to Antagonize Its Ubiquitin/Proteasome-mediated Degradation*

    PubMed Central

    Li, Qiuling; Li, Yuewei; Gu, Bingnan; Fang, Lei; Zhou, Pengbo; Bao, Shilai; Huang, Lan; Dai, Xing

    2015-01-01

    Pygopus 2 (Pygo2/PYGO2) is an evolutionarily conserved coactivator and chromatin effector in the Wnt/β-catenin signaling pathway that regulates cell growth and differentiation in various normal and malignant tissues. Although PYGO2 is highly overexpressed in a number of human cancers, the molecular mechanism underlying its deregulation is largely unknown. Here we report that Pygo2 protein is degraded through the ubiquitin/proteasome pathway and is posttranslationally stabilized through phosphorylation by activated phosphatidylinositol 3-kinase/Akt signaling. Specifically, Pygo2 is stabilized upon inhibition of the proteasome, and its intracellular level is regulated by Cullin 4 (Cul4) and DNA damage-binding protein 1 (DDB1), components of the Cul4-DDB1 E3 ubiquitin ligase complex. Furthermore, Pygo2 is phosphorylated at multiple residues, and Akt-mediated phosphorylation at serine 48 leads to its decreased ubiquitylation and increased stability. Finally, we provide evidence that Akt and its upstream growth factors act in parallel with Wnt to stabilize Pygo2. Taken together, our findings highlight chromatin regulator Pygo2 as a common node downstream of oncogenic Wnt and Akt signaling pathways and underscore posttranslational modification, particularly phosphorylation and ubiquitylation, as a significant mode of regulation of Pygo2 protein expression. PMID:26170450

  3. Versatile strategy for isolating transcription activator-like effector nuclease-mediated knockout mutants in Caenorhabditis elegans.

    PubMed

    Sugi, Takuma; Sakuma, Tetsushi; Ohtani, Yasuko; Yamamoto, Takashi

    2014-01-01

    Targeted genome editing using transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems has recently emerged as a potentially powerful method for creating locus-specific mutations in Caenorhabditis elegans. Due to the low mutation frequencies, one of the crucial steps in using these technologies is screening animals that harbor a targeted mutation. In previous studies, identifying targeted mutations in C. elegans usually depended on observations of fluorescent markers such as a green fluorescent protein or visible phenotypes such as dumpy and uncoordinated phenotypes. However, this strategy is limited in practice because the phenotypes caused by targeted mutations such as defects in sensory behaviors are often apparently invisible. Here, we describe a versatile strategy for isolating C. elegans knockout mutants by TALEN-mediated genome editing and a heteroduplex mobility assay. We applied TALENs to engineer the locus of the neural gene glr-1, which is a C. elegans AMPA-type receptor orthologue that is known to have crucial roles in various sensory behaviors. Knockout mutations in the glr-1 locus, which caused defective mechanosensory behaviors, were efficiently identified by the heteroduplex mobility assay. Thus, we demonstrated the utility of a TALEN-based knockout strategy for creating C. elegans with mutations that cause invisible phenotypes. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

  4. PTEX component HSP101 mediates export of diverse malaria effectors into host erythrocytes.

    PubMed

    Beck, Josh R; Muralidharan, Vasant; Oksman, Anna; Goldberg, Daniel E

    2014-07-31

    To mediate its survival and virulence, the malaria parasite Plasmodium falciparum exports hundreds of proteins into the host erythrocyte. To enter the host cell, exported proteins must cross the parasitophorous vacuolar membrane (PVM) within which the parasite resides, but the mechanism remains unclear. A putative Plasmodium translocon of exported proteins (PTEX) has been suggested to be involved for at least one class of exported proteins; however, direct functional evidence for this has been elusive. Here we show that export across the PVM requires heat shock protein 101 (HSP101), a ClpB-like AAA+ ATPase component of PTEX. Using a chaperone auto-inhibition strategy, we achieved rapid, reversible ablation of HSP101 function, resulting in a nearly complete block in export with substrates accumulating in the vacuole in both asexual and sexual parasites. Surprisingly, this block extended to all classes of exported proteins, revealing HSP101-dependent translocation across the PVM as a convergent step in the multi-pathway export process. Under export-blocked conditions, association between HSP101 and other components of the PTEX complex was lost, indicating that the integrity of the complex is required for efficient protein export. Our results demonstrate an essential and universal role for HSP101 in protein export and provide strong evidence for PTEX function in protein translocation into the host cell.

  5. Multiple Effector Functions Mediated by Human Immunodeficiency Virus-Specific CD4+ T-Cell Clones

    PubMed Central

    Norris, Philip J.; Sumaroka, Marina; Brander, Christian; Moffett, Howell F.; Boswell, Steven L.; Nguyen, Tam; Sykulev, Yuri; Walker, Bruce D.; Rosenberg, Eric S.

    2001-01-01

    Mounting evidence suggests that human immunodeficiency virus type 1 (HIV-1) Gag-specific T helper cells contribute to effective antiviral control, but their functional characteristics and the precise epitopes targeted by this response remain to be defined. In this study, we generated CD4+ T-cell clones specific for Gag from HIV-1-infected persons with vigorous Gag-specific responses detectable in peripheral blood mononuclear cells. Multiple peptides containing T helper epitopes were identified, including a minimal peptide, VHAGPIAG (amino acids 218 to 226), in the cyclophilin binding domain of Gag. Peptide recognition by all clones examined induced cell proliferation, gamma interferon (IFN-γ) secretion, and cytolytic activity. Cytolysis was abrogated by concanamycin A and EGTA but not brefeldin A or anti-Fas antibody, implying a perforin-mediated mechanism of cell lysis. Additionally, serine esterase release into the extracellular medium, a marker for cytolytic granules, was demonstrated in an antigen-specific, dose-dependent fashion. These data indicate that T helper cells can target multiple regions of the p24 Gag protein and suggest that cytolytic activity may be a component of the antiviral effect of these cells. PMID:11559810

  6. Ezrin Is an Effector of Hepatocyte Growth Factor–mediated Migration and Morphogenesis in Epithelial Cells

    PubMed Central

    Crepaldi, Tiziana; Gautreau, Alexis; Comoglio, Paolo M.; Louvard, Daniel; Arpin, Monique

    1997-01-01

    The dissociation, migration, and remodeling of epithelial monolayers induced by hepatocyte growth factor (HGF) entail modifications in cell adhesion and in the actin cytoskeleton through unknown mechanisms. Here we report that ezrin, a membrane–cytoskeleton linker, is crucial to HGF-mediated morphogenesis in a polarized kidney-derived epithelial cell line, LLC-PK1. Ezrin is a substrate for the tyrosine kinase HGF receptor both in vitro and in vivo. HGF stimulation causes enrichment of ezrin recovered in the detergent-insoluble cytoskeleton fraction. Overproduction of wild-type ezrin, by stable transfection in LLC-PK1 cells, enhances cell migration and tubulogenesis induced by HGF stimulation. Overproduction of a truncated variant of ezrin causes mislocalization of endogenous ezrin from microvilli into lateral surfaces. This is concomitant with altered cell shape, characterized by loss of microvilli and cell flattening. Moreover, the truncated variant of ezrin impairs the morphogenic and motogenic response to HGF, thus suggesting a dominant-negative mechanism of action. Site-directed mutagenesis of ezrin codons Y145 and Y353 to phenylalanine does not affect the localization of ezrin at microvilli, but perturbs the motogenic and morphogenic responses to HGF. These results provide evidence that ezrin displays activities that can control cell shape and signaling. PMID:9230083

  7. Differential requirement of lipid rafts for FcγRIIA mediated effector activities

    PubMed Central

    Vieth, Joshua A.; Kim, Moo-kyung; Pan, Xiao Qing; Schreiber, Alan D.

    2010-01-01

    Immunoglobulin G (IgG) dependent activities are important in host defense and autoimmune diseases. Various cell types including macrophages and neutrophils contribute to pathogen destruction and tissue damage through binding of IgG to Fcγ receptors (FcγR). One member of this family, FcγRIIA, is a transmembrane glycoprotein known to mediate binding and internalization of IgG-containing targets. FcγRIIA has been observed to translocate into lipids rafts upon binding IgG-containing targets. We hypothesize that lipid rafts participate to different extents in binding and internalizing targets of different sizes. We demonstrate that disruption of lipid rafts with 8mM methyl-β-cyclodextrin (MβCD) nearly abolishes binding (91% reduction) and phagocytosis (60% reduction) of large IgG-coated targets. Conversely, binding and internalization of small IgG-complexes is less dependent on lipid rafts (49% and 17% inhibition at 8mM MβCD respectively). These observations suggest that differences between phagocytosis and endocytosis may arise as early as the initial stages of ligand recognition. PMID:20728077

  8. Lactose inhibits regulatory T-cell-mediated suppression of effector T-cell interferon-γ and IL-17 production.

    PubMed

    Paasela, Monika; Kolho, Kaija-Leena; Vaarala, Outi; Honkanen, Jarno

    2014-12-14

    Our interest in lactose as an immunomodulatory molecule results from studies showing that lactose binds to galectin-9, which has been shown to have various regulatory functions in the immune system including regulation of T-cell responses. Impaired regulation of T helper (Th)1 and Th17 type immune responses and dysfunction of regulatory T cells (Treg) have been implicated in many human immune-mediated diseases. In the present study, we investigated the effects of lactose on immune regulation using co-cultures of human peripheral blood mononuclear cell (PBMC)-derived Treg and effector T cells (Teff) obtained from twenty healthy adults. Treg, i.e. CD4+CD25+CD127-, were isolated from PBMC by immunomagnetic separation. The fraction of CD4+CD127- cells that was depleted of CD25+ cells was used as Teff. Treg and Teff at a ratio 1:5 were activated and the effects of lactose on the secretion of interferon-γ (IFN-γ) and IL-17 were analysed using ELISA for protein and quantitative RT-PCR for mRNA. Treg down-regulated the secretion of both IFN-γ (8.8-3.9 ng/ml, n 20, P= 0.003) and IL-17 (0.83-0.64 ng/ml, n 15, P= 0.04) in co-cultures, while in the presence of lactose the levels of secreted IFN-γ and IL-17 remained high and no down-regulation was observed (16.4 v. 3.99 ng/ml, n 20, P< 0.0001, and 0.74 v. 0.64 ng/ml, n 15, P= 0.005, respectively). We showed that lactose inhibits human Treg-mediated suppression of Th1 and Th17 immune responses in vitro.

  9. The Effector SPRYSEC-19 of Globodera rostochiensis Suppresses CC-NB-LRR-Mediated Disease Resistance in Plants1[C][W][OA

    PubMed Central

    Postma, Wiebe J.; Slootweg, Erik J.; Rehman, Sajid; Finkers-Tomczak, Anna; Tytgat, Tom O.G.; van Gelderen, Kasper; Lozano-Torres, Jose L.; Roosien, Jan; Pomp, Rikus; van Schaik, Casper; Bakker, Jaap; Goverse, Aska; Smant, Geert

    2012-01-01

    The potato cyst nematode Globodera rostochiensis invades roots of host plants where it transforms cells near the vascular cylinder into a permanent feeding site. The host cell modifications are most likely induced by a complex mixture of proteins in the stylet secretions of the nematodes. Resistance to nematodes conferred by nucleotide-binding-leucine-rich repeat (NB-LRR) proteins usually results in a programmed cell death in and around the feeding site, and is most likely triggered by the recognition of effectors in stylet secretions. However, the actual role of these secretions in the activation and suppression of effector-triggered immunity is largely unknown. Here we demonstrate that the effector SPRYSEC-19 of G. rostochiensis physically associates in planta with the LRR domain of a member of the SW5 resistance gene cluster in tomato (Lycopersicon esculentum). Unexpectedly, this interaction did not trigger defense-related programmed cell death and resistance to G. rostochiensis. By contrast, agroinfiltration assays showed that the coexpression of SPRYSEC-19 in leaves of Nicotiana benthamiana suppresses programmed cell death mediated by several coiled-coil (CC)-NB-LRR immune receptors. Furthermore, SPRYSEC-19 abrogated resistance to Potato virus X mediated by the CC-NB-LRR resistance protein Rx1, and resistance to Verticillium dahliae mediated by an unidentified resistance in potato (Solanum tuberosum). The suppression of cell death and disease resistance did not require a physical association of SPRYSEC-19 and the LRR domains of the CC-NB-LRR resistance proteins. Altogether, our data demonstrated that potato cyst nematodes secrete effectors that enable the suppression of programmed cell death and disease resistance mediated by several CC-NB-LRR proteins in plants. PMID:22904163

  10. Ralstonia solanacearum Requires PopS, an Ancient AvrE-Family Effector, for Virulence and To Overcome Salicylic Acid-Mediated Defenses during Tomato Pathogenesis

    PubMed Central

    Jacobs, Jonathan M.; Milling, Annett; Mitra, Raka M.; Hogan, Clifford S.; Ailloud, Florent; Prior, Philippe; Allen, Caitilyn

    2013-01-01

    ABSTRACT During bacterial wilt of tomato, the plant pathogen Ralstonia solanacearum upregulates expression of popS, which encodes a type III-secreted effector in the AvrE family. PopS is a core effector present in all sequenced strains in the R. solanacearum species complex. The phylogeny of popS mirrors that of the species complex as a whole, suggesting that this is an ancient, vertically inherited effector needed for association with plants. A popS mutant of R. solanacearum UW551 had reduced virulence on agriculturally important Solanum spp., including potato and tomato plants. However, the popS mutant had wild-type virulence on a weed host, Solanum dulcamara, suggesting that some species can avoid the effects of PopS. The popS mutant was also significantly delayed in colonization of tomato stems compared to the wild type. Some AvrE-type effectors from gammaproteobacteria suppress salicylic acid (SA)-mediated plant defenses, suggesting that PopS, a betaproteobacterial ortholog, has a similar function. Indeed, the popS mutant induced significantly higher expression of tomato SA-triggered pathogenesis-related (PR) genes than the wild type. Further, pretreatment of roots with SA exacerbated the popS mutant virulence defect. Finally, the popS mutant had no colonization defect on SA-deficient NahG transgenic tomato plants. Together, these results indicate that this conserved effector suppresses SA-mediated defenses in tomato roots and stems, which are R. solanacearum’s natural infection sites. Interestingly, PopS did not trigger necrosis when heterologously expressed in Nicotiana leaf tissue, unlike the AvrE homolog DspEPcc from the necrotroph Pectobacterium carotovorum subsp. carotovorum. This is consistent with the differing pathogenesis modes of necrosis-causing gammaproteobacteria and biotrophic R. solanacearum. PMID:24281716

  11. Copper metabolism domain-containing 1 represses the mediators involved in the terminal effector pathways of human labour and delivery.

    PubMed

    Lappas, Martha

    2016-04-01

    Does Copper Metabolism MURR1 Domain 1 (COMMD1) play a role in regulating the mediators involved in the terminal processes of human labour and delivery? COMMD1 plays a critical role in the termination of nuclear factor-κB (NF-κB) activity and the control of pro-inflammatory and pro-labour mediators. Inflammation and infection are the biggest aetiological factors associated with preterm birth. NF-κB drives the transcription of pro-inflammatory mediators involved in the terminal effector pathways of human labour and delivery. In non-gestational tissues, COMMD1 is a negative regulator of NF-κB-induced inflammation. The mRNA and/or protein level of COMMD1 was assessed in myometrium (n = 8 per group) and fetal membranes (n = 8 per group) obtained from term non-labouring and labouring women at term, and fetal membranes (n = 8 per group) at preterm with and without histological chorioamnionitis. Primary human myometrial cells were used to determine the effect of pro-inflammatory mediators on COMMD1 level, and the effect of COMMD1 small interfering RNA (siRNA) on pro-labour mediators. Statistical significance was ascribed to a P < 0.05. COMMD1 expression was significantly decreased with spontaneous term labour in myometrium; in fetal membranes with histologically confirmed chorioamnionitis and in myometrial cells treated with pro-inflammatory cytokines interleukin (IL)-1β and tumour necrosis factor (TNF)-α, the bacterial product fibroblast-stimulating lipopeptide and the viral double stranded RNA analogue polyinosinic polycytidilic acid. Loss-of-function studies revealed an increase in inflammation- and infection-induced TNF-α, IL-1α, IL-1β, IL-6, IL-8 and/or monocyte chemoattractant protein-1 mRNA abundance and/or release; and cyclo-oxygenase-2 mRNA level, release of prostaglandin (PG) F2α and mRNA level of the PGF2α receptor FP. In addition, siRNA knockdown of COMMD1 was associated with significantly increased NF-κB activation as evidenced by increased IL-1

  12. A Phytophthora sojae effector suppresses endoplasmic reticulum stress-mediated immunity by stabilizing plant Binding immunoglobulin Proteins

    PubMed Central

    Jing, Maofeng; Guo, Baodian; Li, Haiyang; Yang, Bo; Wang, Haonan; Kong, Guanghui; Zhao, Yao; Xu, Huawei; Wang, Yan; Ye, Wenwu; Dong, Suomeng; Qiao, Yongli; Tyler, Brett M.; Ma, Wenbo; Wang, Yuanchao

    2016-01-01

    Phytophthora pathogens secrete an array of specific effector proteins to manipulate host innate immunity to promote pathogen colonization. However, little is known about the host targets of effectors and the specific mechanisms by which effectors increase susceptibility. Here we report that the soybean pathogen Phytophthora sojae uses an essential effector PsAvh262 to stabilize endoplasmic reticulum (ER)-luminal binding immunoglobulin proteins (BiPs), which act as negative regulators of plant resistance to Phytophthora. By stabilizing BiPs, PsAvh262 suppresses ER stress-triggered cell death and facilitates Phytophthora infection. The direct targeting of ER stress regulators may represent a common mechanism of host manipulation by microbes. PMID:27256489

  13. Five phylogenetically close rice SWEET genes confer TAL effector-mediated susceptibility to Xanthomonas oryzae pv. oryzae.

    PubMed

    Streubel, Jana; Pesce, Céline; Hutin, Mathilde; Koebnik, Ralf; Boch, Jens; Szurek, Boris

    2013-11-01

    Bacterial plant-pathogenic Xanthomonas strains translocate transcription activator-like (TAL) effectors into plant cells to function as specific transcription factors. Only a few plant target genes of TAL effectors have been identified, so far. Three plant SWEET genes encoding putative sugar transporters are known to be induced by TAL effectors from rice-pathogenic Xanthomonas oryzae pv. oryzae (Xoo). We predict and validate that expression of OsSWEET14 is induced by a novel TAL effector, Tal5, from an African Xoo strain. Artificial TAL effectors (ArtTALs) were constructed to individually target 20 SWEET orthologs in rice. They were used as designer virulence factors to study which rice SWEET genes can support Xoo virulence. The Tal5 target box differs from those of the already known TAL effectors TalC, AvrXa7 and PthXo3, which also induce expression of OsSWEET14, suggesting evolutionary convergence on key targets. ArtTALs efficiently complemented an Xoo talC mutant, demonstrating that specific induction of OsSWEET14 is the key target of TalC. ArtTALs that specifically target individual members of the rice SWEET family revealed three known and two novel SWEET genes to support bacterial virulence. Our results demonstrate that five phylogenetically close SWEET proteins, which presumably act as sucrose transporters, can support Xoo virulence.

  14. Suppression of Xo1-Mediated Disease Resistance in Rice by a Truncated, Non-DNA-Binding TAL Effector of Xanthomonas oryzae

    PubMed Central

    Read, Andrew C.; Rinaldi, Fabio C.; Hutin, Mathilde; He, Yong-Qiang; Triplett, Lindsay R.; Bogdanove, Adam J.

    2016-01-01

    Delivered into plant cells by type III secretion from pathogenic Xanthomonas species, TAL (transcription activator-like) effectors are nuclear-localized, DNA-binding proteins that directly activate specific host genes. Targets include genes important for disease, genes that confer resistance, and genes inconsequential to the host-pathogen interaction. TAL effector specificity is encoded by polymorphic repeats of 33–35 amino acids that interact one-to-one with nucleotides in the recognition site. Activity depends also on N-terminal sequences important for DNA binding and C-terminal nuclear localization signals (NLS) and an acidic activation domain (AD). Coding sequences missing much of the N- and C-terminal regions due to conserved, in-frame deletions are present and annotated as pseudogenes in sequenced strains of Xanthomonas oryzae pv. oryzicola (Xoc) and pv. oryzae (Xoo), which cause bacterial leaf streak and bacterial blight of rice, respectively. Here we provide evidence that these sequences encode proteins we call “truncTALEs,” for “truncated TAL effectors.” We show that truncTALE Tal2h of Xoc strain BLS256, and by correlation truncTALEs in other strains, specifically suppress resistance mediated by the Xo1 locus recently described in the heirloom rice variety Carolina Gold. Xo1-mediated resistance is triggered by different TAL effectors from diverse X. oryzae strains, irrespective of their DNA binding specificity, and does not require the AD. This implies a direct protein-protein rather than protein-DNA interaction. Similarly, truncTALEs exhibit diverse predicted DNA recognition specificities. And, in vitro, Tal2h did not bind any of several potential recognition sites. Further, a single candidate NLS sequence in Tal2h was dispensable for resistance suppression. Many truncTALEs have one 28 aa repeat, a length not observed previously. Tested in an engineered TAL effector, this repeat required a single base pair deletion in the DNA, suggesting that it

  15. Ralstonia solanacearum requires PopS, an ancient AvrE-family effector, for virulence and To overcome salicylic acid-mediated defenses during tomato pathogenesis.

    PubMed

    Jacobs, Jonathan M; Milling, Annett; Mitra, Raka M; Hogan, Clifford S; Ailloud, Florent; Prior, Philippe; Allen, Caitilyn

    2013-11-26

    During bacterial wilt of tomato, the plant pathogen Ralstonia solanacearum upregulates expression of popS, which encodes a type III-secreted effector in the AvrE family. PopS is a core effector present in all sequenced strains in the R. solanacearum species complex. The phylogeny of popS mirrors that of the species complex as a whole, suggesting that this is an ancient, vertically inherited effector needed for association with plants. A popS mutant of R. solanacearum UW551 had reduced virulence on agriculturally important Solanum spp., including potato and tomato plants. However, the popS mutant had wild-type virulence on a weed host, Solanum dulcamara, suggesting that some species can avoid the effects of PopS. The popS mutant was also significantly delayed in colonization of tomato stems compared to the wild type. Some AvrE-type effectors from gammaproteobacteria suppress salicylic acid (SA)-mediated plant defenses, suggesting that PopS, a betaproteobacterial ortholog, has a similar function. Indeed, the popS mutant induced significantly higher expression of tomato SA-triggered pathogenesis-related (PR) genes than the wild type. Further, pretreatment of roots with SA exacerbated the popS mutant virulence defect. Finally, the popS mutant had no colonization defect on SA-deficient NahG transgenic tomato plants. Together, these results indicate that this conserved effector suppresses SA-mediated defenses in tomato roots and stems, which are R. solanacearum's natural infection sites. Interestingly, PopS did not trigger necrosis when heterologously expressed in Nicotiana leaf tissue, unlike the AvrE homolog DspEPcc from the necrotroph Pectobacterium carotovorum subsp. carotovorum. This is consistent with the differing pathogenesis modes of necrosis-causing gammaproteobacteria and biotrophic R. solanacearum. The type III-secreted AvrE effector family is widely distributed in high-impact plant-pathogenic bacteria and is known to suppress plant defenses for virulence. We

  16. Effector-mediated discovery of a novel resistance gene against Bremia lactucae in a nonhost lettuce species.

    PubMed

    Giesbers, Anne K J; Pelgrom, Alexandra J E; Visser, Richard G F; Niks, Rients E; Van den Ackerveken, Guido; Jeuken, Marieke J W

    2017-08-21

    Candidate effectors from lettuce downy mildew (Bremia lactucae) enable high-throughput germplasm screening for the presence of resistance (R) genes. The nonhost species Lactuca saligna comprises a source of B. lactucae R genes that has hardly been exploited in lettuce breeding. Its cross-compatibility with the host species L. sativa enables the study of inheritance of nonhost resistance (NHR). We performed transient expression of candidate RXLR effector genes from B. lactucae in a diverse Lactuca germplasm set. Responses to two candidate effectors (BLR31 and BLN08) were genetically mapped and tested for co-segregation with disease resistance. BLN08 induced a hypersensitive response (HR) in 55% of the L. saligna accessions, but responsiveness did not co-segregate with resistance to Bl:24. BLR31 triggered an HR in 5% of the L. saligna accessions, and revealed a novel R gene providing complete B. lactucae race Bl:24 resistance. Resistant hybrid plants that were BLR31 nonresponsive indicated other unlinked R genes and/or nonhost QTLs. We have identified a candidate avirulence effector of B. lactucae (BLR31) and its cognate R gene in L. saligna. Concurrently, our results suggest that R genes are not required for NHR of L. saligna. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  17. Polycomb Repressive Complex 2-Mediated Chromatin Repression Guides Effector CD8(+) T Cell Terminal Differentiation and Loss of Multipotency.

    PubMed

    Gray, Simon M; Amezquita, Robert A; Guan, Tianxia; Kleinstein, Steven H; Kaech, Susan M

    2017-04-04

    Understanding immunological memory formation depends on elucidating how multipotent memory precursor (MP) cells maintain developmental plasticity and longevity to provide long-term immunity while other effector cells develop into terminally differentiated effector (TE) cells with limited survival. Profiling active (H3K27ac) and repressed (H3K27me3) chromatin in naive, MP, and TE CD8(+) T cells during viral infection revealed increased H3K27me3 deposition at numerous pro-memory and pro-survival genes in TE relative to MP cells, indicative of fate restriction, but permissive chromatin at both pro-memory and pro-effector genes in MP cells, indicative of multipotency. Polycomb repressive complex 2 deficiency impaired clonal expansion and TE cell differentiation, but minimally impacted CD8(+) memory T cell maturation. Abundant H3K27me3 deposition at pro-memory genes occurred late during TE cell development, probably from diminished transcription factor FOXO1 expression. These results outline a temporal model for loss of memory cell potential through selective epigenetic silencing of pro-memory genes in effector T cells.

  18. Antibody repertoire complexity and effector cell biology determined by assays for IgE-mediated basophil and T-cell activation.

    PubMed

    Lund, Gitte; Willumsen, Nicholas; Holm, Jens; Christensen, Lars Harder; Würtzen, Peter Adler; Lund, Kaare

    2012-09-28

    Effector cell activation and T-cell activation, the latter mediated by facilitated antigen presentation, are immunological mechanisms that play crucial roles in the manifestation and maintenance of allergic disease. In addition to their relevance for the pathogenesis of allergy in-vivo, in-vitro assays based on these immunological mechanisms have been established and used for diagnostics, for monitoring the progression of disease and for the effect of specific immunotherapy as well as for basic research purposes. Here we review different parameters that affect effector cell activation and facilitated antigen uptake and presentation, including assay designs, readout parameters and critical experimental conditions. Central to the two immunological mechanisms is complex formation between allergen-specific IgE, allergen, and cell surface-anchored immunoglobulin receptor; the high affinity IgE-receptor FcεRI on basophils and mast cells, and the low affinity IgE-receptor FcεRII (CD23) on B-cells. Accordingly, the effect of IgE repertoire complexity and allergen diversity on effector cell and facilitated antigen presentation is discussed in detail.

  19. Cdc42p regulation of the yeast formin Bni1p mediated by the effector Gic2p

    PubMed Central

    Chen, Hsin; Kuo, Chun-Chen; Kang, Hui; Howell, Audrey S.; Zyla, Trevin R.; Jin, Michelle; Lew, Daniel J.

    2012-01-01

    Actin filaments are dynamically reorganized to accommodate ever-changing cellular needs for intracellular transport, morphogenesis, and migration. Formins, a major family of actin nucleators, are believed to function as direct effectors of Rho GTPases, such as the polarity regulator Cdc42p. However, the presence of extensive redundancy has made it difficult to assess the in vivo significance of the low-affinity Rho GTPase–formin interaction and specifically whether Cdc42p polarizes the actin cytoskeleton via direct formin binding. Here we exploit a synthetically rewired budding yeast strain to eliminate the redundancy, making regulation of the formin Bni1p by Cdc42p essential for viability. Surprisingly, we find that direct Cdc42p–Bni1p interaction is dispensable for Bni1p regulation. Alternative paths linking Cdc42p and Bni1p via “polarisome” components Spa2p and Bud6p are also collectively dispensable. We identify a novel regulatory input to Bni1p acting through the Cdc42p effector, Gic2p. This pathway is sufficient to localize Bni1p to the sites of Cdc42p action and promotes a polarized actin organization in both rewired and wild-type contexts. We suggest that an indirect mechanism linking Rho GTPases and formins via Rho effectors may provide finer spatiotemporal control for the formin-nucleated actin cytoskeleton. PMID:22918946

  20. Chimeric Coupling Proteins Mediate Transfer of Heterologous Type IV Effectors through the Escherichia coli pKM101-Encoded Conjugation Machine

    PubMed Central

    Whitaker, Neal; Berry, Trista M.; Rosenthal, Nathan; Gordon, Jay E.; Gonzalez-Rivera, Christian; Sheehan, Kathy B.; Truchan, Hilary K.; VieBrock, Lauren; Newton, Irene L. G.; Carlyon, Jason A.

    2016-01-01

    ABSTRACT Bacterial type IV secretion systems (T4SSs) are composed of two major subfamilies, conjugation machines dedicated to DNA transfer and effector translocators for protein transfer. We show here that the Escherichia coli pKM101-encoded conjugation system, coupled with chimeric substrate receptors, can be repurposed for transfer of heterologous effector proteins. The chimeric receptors were composed of the N-terminal transmembrane domain of pKM101-encoded TraJ fused to soluble domains of VirD4 homologs functioning in Agrobacterium tumefaciens, Anaplasma phagocytophilum, or Wolbachia pipientis. A chimeric receptor assembled from A. tumefaciens VirD4 (VirD4At) mediated transfer of a MOBQ plasmid (pML122) and A. tumefaciens effector proteins (VirE2, VirE3, and VirF) through the pKM101 transfer channel. Equivalent chimeric receptors assembled from the rickettsial VirD4 homologs similarly supported the transfer of known or candidate effectors from rickettsial species. These findings establish a proof of principle for use of the dedicated pKM101 conjugation channel, coupled with chimeric substrate receptors, to screen for translocation competency of protein effectors from recalcitrant species. Many T4SS receptors carry sequence-variable C-terminal domains (CTDs) with unknown function. While VirD4At and the TraJ/VirD4At chimera with their CTDs deleted supported pML122 transfer at wild-type levels, ΔCTD variants supported transfer of protein substrates at strongly diminished or elevated levels. We were unable to detect binding of VirD4At's CTD to the VirE2 effector, although other VirD4At domains bound this substrate in vitro. We propose that CTDs evolved to govern the dynamics of substrate presentation to the T4SS either through transient substrate contacts or by controlling substrate access to other receptor domains. IMPORTANCE Bacterial type IV secretion systems (T4SSs) display striking versatility in their capacity to translocate DNA and protein substrates to

  1. Glycovariant anti-CD37 monospecific protein therapeutic exhibits enhanced effector cell-mediated cytotoxicity against chronic and acute B cell malignancies

    PubMed Central

    Rafiq, Sarwish; Siadak, Anthony; Butchar, Jonathan P.; Cheney, Carolyn; Lozanski, Gerard; Jacob, Naduparambil K.; Lapalombella, Rosa; McGourty, Jackie; Moledor, Meghan; Lowe, Richard; Setter, Ben; Jones, Jeffrey; Flynn, Joseph M.; Andritsos, Leslie; Devine, Steven; Mo, Xiaokui; Jarjoura, David; Tridandapani, Susheela; Algate, Paul; Byrd, John C.; Muthusamy, Natarajan

    2013-01-01

    TRU-016 is a SMIPTM (monospecific protein therapeutic) molecule against the tetraspanin transmembrane family protein CD37 that is currently in Phase 2 trials in Chronic Lymphocytic Leukemia (CLL) and Non-Hodgkin Lymphoma (NHL). In an attempt to enhance the ADCC function of SMIP-016, the chimeric version of TRU-016, SMIP-016GV was engineered with a modification in a glycosylation site in the Fc domain. The wild-type and glycovariant SMIP proteins mediate comparable Type I antibody-like direct cytotoxicity in the presence of anti-human Fc crosslinker and show a similar tyrosine phosphorylation pattern post-treatment. However, NK cells stimulated with the SMIP-016GV exhibit enhanced activation and release 3-fold more interferon-γ compared with SMIP-016. SMIP-016GV shows enhanced ADCC function against cells expressing CD37 with NK cell effectors derived from both normal and CLL-affected individuals. Enhanced ADCC is observed against CLL cells and is sustained at concentrations of SMIP-016GV as low at 5E−6 µg/mL on cells expressing minimal CD37 antigen. In support of the biological relevance of this, SMIP-016GV mediates effective ADCC against primary acute lymphoblastic leukemia (ALL) cells with low surface expression of CD37. Collectively, these data suggest potential use of the novel therapeutic agent SMIP-016GV with enhanced effector function for B cell malignancies, including CLL and ALL therapy. PMID:23883821

  2. Enhanced Disease Susceptibility1 Mediates Pathogen Resistance and Virulence Function of a Bacterial Effector in Soybean1[C][W][OPEN

    PubMed Central

    Wang, Jialin; Shine, M.B.; Gao, Qing-Ming; Navarre, Duroy; Jiang, Wei; Liu, Chunyan; Chen, Qingshan; Hu, Guohua; Kachroo, Aardra

    2014-01-01

    Enhanced disease susceptibility1 (EDS1) and phytoalexin deficient4 (PAD4) are well-known regulators of both basal and resistance (R) protein-mediated plant defense. We identified two EDS1-like (GmEDS1a/GmEDS1b) proteins and one PAD4-like (GmPAD4) protein that are required for resistance signaling in soybean (Glycine max). Consistent with their significant structural conservation to Arabidopsis (Arabidopsis thaliana) counterparts, constitutive expression of GmEDS1 or GmPAD4 complemented the pathogen resistance defects of Arabidopsis eds1 and pad4 mutants, respectively. Interestingly, however, the GmEDS1 and GmPAD4 did not complement pathogen-inducible salicylic acid accumulation in the eds1/pad4 mutants. Furthermore, the GmEDS1a/GmEDS1b proteins were unable to complement the turnip crinkle virus coat protein-mediated activation of the Arabidopsis R protein Hypersensitive reaction to Turnip crinkle virus (HRT), even though both interacted with HRT. Silencing GmEDS1a/GmEDS1b or GmPAD4 reduced basal and pathogen-inducible salicylic acid accumulation and enhanced soybean susceptibility to virulent pathogens. The GmEDS1a/GmEDS1b and GmPAD4 genes were also required for Resistance to Pseudomonas syringae pv glycinea2 (Rpg2)-mediated resistance to Pseudomonas syringae. Notably, the GmEDS1a/GmEDS1b proteins interacted with the cognate bacterial effector AvrA1 and were required for its virulence function in rpg2 plants. Together, these results show that despite significant structural similarities, conserved defense signaling components from diverse plants can differ in their functionalities. In addition, we demonstrate a role for GmEDS1 in regulating the virulence function of a bacterial effector. PMID:24872380

  3. Autoacetylation of the Ralstonia solanacearum Effector PopP2 Targets a Lysine Residue Essential for RRS1-R-Mediated Immunity in Arabidopsis

    PubMed Central

    Tasset, Céline; Bernoux, Maud; Jauneau, Alain; Pouzet, Cécile; Brière, Christian; Kieffer-Jacquinod, Sylvie; Rivas, Susana

    2010-01-01

    Type III effector proteins from bacterial pathogens manipulate components of host immunity to suppress defence responses and promote pathogen development. In plants, host proteins targeted by some effectors called avirulence proteins are surveyed by plant disease resistance proteins referred to as “guards”. The Ralstonia solanacearum effector protein PopP2 triggers immunity in Arabidopsis following its perception by the RRS1-R resistance protein. Here, we show that PopP2 interacts with RRS1-R in the nucleus of living plant cells. PopP2 belongs to the YopJ-like family of cysteine proteases, which share a conserved catalytic triad that includes a highly conserved cysteine residue. The catalytic cysteine mutant PopP2-C321A is impaired in its avirulence activity although it is still able to interact with RRS1-R. In addition, PopP2 prevents proteasomal degradation of RRS1-R, independent of the presence of an integral PopP2 catalytic core. A liquid chromatography/tandem mass spectrometry analysis showed that PopP2 displays acetyl-transferase activity leading to its autoacetylation on a particular lysine residue, which is well conserved among all members of the YopJ family. These data suggest that this lysine residue may correspond to a key binding site for acetyl-coenzyme A required for protein activity. Indeed, mutation of this lysine in PopP2 abolishes RRS1-R-mediated immunity. In agreement with the guard hypothesis, our results favour the idea that activation of the plant immune response by RRS1-R depends not only on the physical interaction between the two proteins but also on its perception of PopP2 enzymatic activity. PMID:21124938

  4. The cyclin dependent kinase inhibitor (R)-roscovitine mediates selective suppression of alloreactive human T cells but preserves pathogen-specific and leukemia-specific effectors

    PubMed Central

    Nellore, Anoma; Liu, Bianling; Patsoukis, Nikolaos; Boussiotis, Vassiliki A.; Li, Lequn

    2014-01-01

    Graft versus host disease (GvHD), mediated by donor T cells, remains the primary cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation and novel therapeutic approaches are required. Cdk2 is a critical node of signal integration and programming of T cell responses towards immunity versus anergy but is dispensable for hematopoiesis and thymocyte development. We examined the effects of pharmacologic Cdk2 inhibition on alloreactive human T cells. Inhibition of Cdk2 blocked expansion of alloreactive T cells upon culture with HLA-mismatched dendritic cells and prevented generation of IFN-γ-producing alloantigen-specific effectors. In contrast, Cdk2 inhibition preserved effectors specific for Wilms’ tumor 1 (WT1) leukemia antigen and for CMV as determined by WT1-specific and CMV-specific pentamers. Cdk2 inhibition preserved Treg cells, which have the ability to prevent GvHD while maintaining GvL. Thus, Cdk inhibitors may improve allogeneic HSCT by reducing alloreactivity and GvHD without loss of pathogen-specific and leukemia-specific immunity. PMID:24631965

  5. The Cyst Nematode Effector Protein 10A07 Targets and Recruits Host Posttranslational Machinery to Mediate Its Nuclear Trafficking and to Promote Parasitism in Arabidopsis

    PubMed Central

    Hewezi, Tarek; Juvale, Parijat S.; Piya, Sarbottam; Maier, Tom R.; Rambani, Aditi; Rice, J. Hollis; Mitchum, Melissa G.; Davis, Eric L.; Hussey, Richard S.; Baum, Thomas J.

    2015-01-01

    Plant-parasitic cyst nematodes synthesize and secrete effector proteins that are essential for parasitism. One such protein is the 10A07 effector from the sugar beet cyst nematode, Heterodera schachtii, which is exclusively expressed in the nematode dorsal gland cell during all nematode parasitic stages. Overexpression of H. schachtii 10A07 in Arabidopsis thaliana produced a hypersusceptible phenotype in response to H. schachtii infection along with developmental changes reminiscent of auxin effects. The 10A07 protein physically associates with a plant kinase and the IAA16 transcription factor in the cytoplasm and nucleus, respectively. The interacting plant kinase (IPK) phosphorylates 10A07 at Ser-144 and Ser-231 and mediates its trafficking from the cytoplasm to the nucleus. Translocation to the nucleus is phosphorylation dependent since substitution of Ser-144 and Ser-231 by alanine resulted in exclusive cytoplasmic accumulation of 10A07. IPK and IAA16 are highly upregulated in the nematode-induced syncytium (feeding cells), and deliberate manipulations of their expression significantly alter plant susceptibility to H. schachtii in an additive fashion. An inactive variant of IPK functioned antagonistically to the wild-type IPK and caused a dominant-negative phenotype of reduced plant susceptibility. Thus, exploitation of host processes to the advantage of the parasites is one mechanism by which cyst nematodes promote parasitism of host plants. PMID:25715285

  6. Obesity-Induced Metabolic Stress Leads to Biased Effector Memory CD4(+) T Cell Differentiation via PI3K p110δ-Akt-Mediated Signals.

    PubMed

    Mauro, Claudio; Smith, Joanne; Cucchi, Danilo; Coe, David; Fu, Hongmei; Bonacina, Fabrizia; Baragetti, Andrea; Cermenati, Gaia; Caruso, Donatella; Mitro, Nico; Catapano, Alberico L; Ammirati, Enrico; Longhi, Maria P; Okkenhaug, Klaus; Norata, Giuseppe D; Marelli-Berg, Federica M

    2017-03-07

    Low-grade systemic inflammation associated to obesity leads to cardiovascular complications, caused partly by infiltration of adipose and vascular tissue by effector T cells. The signals leading to T cell differentiation and tissue infiltration during obesity are poorly understood. We tested whether saturated fatty acid-induced metabolic stress affects differentiation and trafficking patterns of CD4(+) T cells. Memory CD4(+) T cells primed in high-fat diet-fed donors preferentially migrated to non-lymphoid, inflammatory sites, independent of the metabolic status of the hosts. This was due to biased CD4(+) T cell differentiation into CD44(hi)-CCR7(lo)-CD62L(lo)-CXCR3(+)-LFA1(+) effector memory-like T cells upon priming in high-fat diet-fed animals. Similar phenotype was observed in obese subjects in a cohort of free-living people. This developmental bias was independent of any crosstalk between CD4(+) T cells and dendritic cells and was mediated via direct exposure of CD4(+) T cells to palmitate, leading to increased activation of a PI3K p110δ-Akt-dependent pathway upon priming.

  7. ISG15 functions as an interferon-mediated antiviral effector early in the murine norovirus life cycle.

    PubMed

    Rodriguez, Marisela R; Monte, Kristen; Thackray, Larissa B; Lenschow, Deborah J

    2014-08-01

    Human noroviruses (HuNoV) are the leading cause of nonbacterial gastroenteritis worldwide. Similar to HuNoV, murine noroviruses (MNV) are enteric pathogens spread via the fecal-oral route and have been isolated from numerous mouse facilities worldwide. Type I and type II interferons (IFN) restrict MNV-1 replication; however, the antiviral effectors impacting MNV-1 downstream of IFN signaling are largely unknown. Studies using dendritic cells, macrophages, and mice deficient in free and conjugated forms of interferon-stimulated gene 15 (ISG15) revealed that ISG15 conjugation contributes to protection against MNV-1 both in vitro and in vivo. ISG15 inhibited a step early in the viral life cycle upstream of viral genome transcription. Directly transfecting MNV-1 RNA into IFN-stimulated mouse embryonic fibroblasts (MEFs) and bone marrow-derived dendritic cells (BMDC) lacking ISG15 conjugates bypassed the antiviral activity of ISG15, further suggesting that ISG15 conjugates restrict the MNV-1 life cycle at the viral entry/uncoating step. These results identify ISG15 as the first type I IFN effector regulating MNV-1 infection both in vitro and in vivo and for the first time implicate the ISG15 pathway in the regulation of early stages of MNV-1 replication. Type I IFNs are important in controlling murine norovirus 1 (MNV-1) infections; however, the proteins induced by IFNs that restrict viral growth are largely unknown. This report reveals that interferon-stimulated gene 15 (ISG15) mitigates MNV-1 replication both in vitro and in vivo. In addition, it shows that ISG15 inhibits MNV-1 replication by targeting an early step in the viral life cycle, MNV-1 entry and/or uncoating. These results identify ISG15 as the first type I IFN effector regulating MNV-1 infection both in vitro and in vivo and for the first time implicate the ISG15 pathway in the regulation of viral entry/uncoating. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  8. The type III effectors of Xanthomonas.

    PubMed

    White, Frank F; Potnis, Neha; Jones, Jeffrey B; Koebnik, Ralf

    2009-11-01

    A review of type III effectors (T3 effectors) from strains of Xanthomonas reveals a growing list of candidate and known effectors based on functional assays and sequence and structural similarity searches of genomic data. We propose that the effectors and suspected effectors should be distributed into 39 so-called Xop groups reflecting sequence similarity. Some groups have structural motifs for putative enzymatic functions, and recent studies have provided considerable insight into the interaction with host factors in their function as mediators of virulence and elicitors of resistance for a few specific T3 effectors. Many groups are related to T3 effectors of plant and animal pathogenic bacteria, and several groups appear to have been exploited primarily by Xanthomonas species based on available data. At the same time, a relatively large number of candidate effectors remain to be examined in more detail with regard to their function within host cells.

  9. The spontaneously adhesive leukocyte function-associated antigen-1 (LFA-1) integrin in effector T cells mediates rapid actin- and calmodulin-dependent adhesion strengthening to ligand under shear flow.

    PubMed

    Lek, Hwee San; Morrison, Vicky L; Conneely, Michael; Campbell, Paul A; McGloin, David; Kliche, Stefanie; Watts, Colin; Prescott, Alan; Fagerholm, Susanna C

    2013-05-24

    Integrins in effector T cells are highly expressed and important for trafficking of these cells and for their effector functions. However, how integrins are regulated in effector T cells remains poorly characterized. Here, we have investigated effector T cell leukocyte function-associated antigen-1 (LFA-1) regulation in primary murine effector T cells. These cells have high LFA-1 integrin expression and display high spontaneous binding to intercellular adhesion molecule-1 (ICAM-1) ligand under static conditions. In addition, these cells are able to migrate spontaneously on ICAM-1. Atomic force microscopy measurements showed that the force required for unbinding of integrin-ligand interactions increases over time (0.5-20-s contact time). The maximum unbinding force for this interaction was ∼140 piconewtons at 0.5-s contact time, increasing to 580 piconewtons at 20-s contact time. Also, the total work required to disrupt the interaction increased over the 20-s contact time, indicating LFA-1-mediated adhesion strengthening in primary effector T cells over a very quick time frame. Effector T cells adhered spontaneously to ICAM-1 under conditions of shear flow, in the absence of chemokine stimulation, and this binding was independent of protein kinase B/Akt and protein kinase C kinase activity, but dependent on calcium/calmodulin signaling and an intact actin cytoskeleton. These results indicate that effector T cell integrins are highly expressed and spontaneously adhesive in the absence of inside-out integrin signaling but that LFA-1-mediated firm adhesion under conditions of shear flow requires downstream integrin signaling, which is dependent on calcium/calmodulin and the actin cytoskeleton.

  10. Forced LIGHT expression in prostate tumors overcomes Treg mediated immunosuppression and synergizes with a prostate tumor therapeutic vaccine by recruiting effector T lymphocytes

    PubMed Central

    Yan, Lisa; Da Silva, Diane M.; Verma, Bhavna; Gray, Andrew; Brand, Heike E.; Skeate, Joseph G.; Porras, Tania B.; Kanodia, Shreya; Kast, W. Martin

    2014-01-01

    Background LIGHT, a ligand for lymphotoxin-β receptor (LTβR) and herpes virus entry mediator, is predominantly expressed on activated immune cells and LTβR signaling leads to the recruitment of lymphocytes. The interaction between LIGHT and LTβR has been previously shown in a virus induced tumor model to activate immune cells and result in tumor regression, but the role of LIGHT in tumor immunosuppression or in a prostate cancer setting, where self antigens exist, has not been explored. We hypothesized that forced expression of LIGHT in prostate tumors would shift the pattern of immune cell infiltration, would inhibit T regulatory cells (Tregs) and would induce prostate cancer tumor associated antigen (TAA) specific T cells that would eradicate tumors. Methods Real Time PCR was used to evaluate expression of forced LIGHT and various other genes in prostate tumors samples. Adenovirus encoding murine LIGHT was injected intratumorally into TRAMP C2 prostate cancer cell tumor bearing mice for in vivo studies. Chemokine and cytokine concentrations were determined by multiplex ELISA. Flow cytometry was used to phenotype tumor infiltrating lymphocytes and expression of LIGHT on the tumor cell surface. Tumor specific lymphocytes were quantified via an ELISpot assay. Treg induction and Treg suppression assays determined Treg functionality after LIGHT treatment. Results LIGHT expression peaked within 48 hours of infection, recruited effector T cells into the tumor microenvironment that recognized mouse prostate stem cell antigen (PSCA) and inhibited the infiltration of Tregs. Tregs isolated from tumor draining lymph nodes had impaired suppressive capability after LIGHT treatment. LIGHT in combination with a therapeutic vaccine, PSCA TriVax, reduced tumor burden. Conclusion Forced LIGHT treatment combined with PSCA TriVax therapeutic vaccination delays prostate cancer progression in mice by recruiting effector T lymphocytes to the tumor and inhibiting Treg mediated

  11. DNA Methylation Affects the Efficiency of Transcription Activator-Like Effector Nucleases-Mediated Genome Editing in Rice

    PubMed Central

    Kaya, Hidetaka; Numa, Hisataka; Nishizawa-Yokoi, Ayako; Toki, Seiichi; Habu, Yoshiki

    2017-01-01

    Genome editing in plants becomes popular since the advent of sequence-specific nucleases (SSNs) that are simple to set up and efficient in various plant species. Although transcription activator-like effector nucleases (TALENs) are one of the most prevalent SSNs and have a potential to provide higher target specificity by their dimeric property, TALENs are sensitive to methylated cytosines that are present not only in transposons but also in active genes in plants. In mammalian cells, the methylation sensitivity of TALENs could be overcome by using a base-recognition module (N∗) that has a higher affinity to methylated cytosine. In contrast to mammals, plants carry DNA methylation at all cytosine contexts (CG, CHG, and CHH, where H represents A, C, or T) with various degrees and effectiveness of N∗ module in genome editing in plants has not been explored. In this study, we designed sets of TALENs with or without N∗ modules and examined their efficiency in genome editing of methylated regions in rice. Although improvement in genome editing efficiency was observed with N∗-TALENs designed to a stably methylated target, another target carrying cytosines with various levels of methylation showed resistance to both normal and N∗-TALENs. The results suggest that variability of cytosine methylation in target regions is an additional factor affecting the genome editing efficiency of TALENs. PMID:28348570

  12. Transcription activator-like effector nucleases mediated metabolic engineering for enhanced fatty acids production in Saccharomyces cerevisiae.

    PubMed

    Aouida, Mustapha; Li, Lixin; Mahjoub, Ali; Alshareef, Sahar; Ali, Zahir; Piatek, Agnieszka; Mahfouz, Magdy M

    2015-10-01

    Targeted engineering of microbial genomes holds much promise for diverse biotechnological applications. Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/Cas9 systems are capable of efficiently editing microbial genomes, including that of Saccharomyces cerevisiae. Here, we demonstrate the use of TALENs to edit the genome of S. cerevisiae with the aim of inducing the overproduction of fatty acids. Heterodimeric TALENs were designed to simultaneously edit the FAA1 and FAA4 genes encoding acyl-CoA synthetases in S. cerevisiae. Functional yeast double knockouts generated using these TALENs over-produce large amounts of free fatty acids into the cell. This study demonstrates the use of TALENs for targeted engineering of yeast and demonstrates that this technology can be used to stimulate the enhanced production of free fatty acids, which are potential substrates for biofuel production. This proof-of-principle study extends the utility of TALENs as excellent genome editing tools and highlights their potential use for metabolic engineering of yeast and other organisms, such as microalgae and plants, for biofuel production.

  13. NKp46-mediated Dicer1 inactivation results in defective NK-cell differentiation and effector functions in mice.

    PubMed

    Degouve, Sophie; Tavares, Armelle; Viel, Sébastien; Walzer, Thierry; Marçais, Antoine

    2016-08-01

    MicroRNAs control developmental pathways and effector functions in immune cells. Previous studies have studied the role of microRNAs in natural killer (NK) cells. However, the mouse models of microRNA depletion used were nonNK-specific and only partially depleting, hampering the interpretation of the data obtained. To clarify the role of microRNAs in murine NK cells, we deleted the RNase III enzyme Dicer1 in NKp46-expressing cells. We observed a drastic decrease in several microRNAs specifically in NK cells. Furthermore, the overall size of the "NK-cell" pool was severely decreased, a phenotype associated with compromised survival. Moreover, performing a broad flow cytometry profiling, we show that Dicer1-deficient NK cells failed to complete their differentiation program. In particular, several integrins were inappropriately expressed in mature NK cells. These defects coincided with decreased response to IL-15, a cytokine responsible for "NK-cell" maturation and survival. In addition, Dicer1 deletion impaired key "NK-cell" functions: target cell killing and production of IFN-γ, leading to defective control of metastasis. Dicer1 deletion thus affects "NK-cell" biology in a cell intrinsic manner at several distinct stages.

  14. Induction of CD4(+) and CD8(+) anti-tumor effector T cell responses by bacteria mediated tumor therapy.

    PubMed

    Stern, Christian; Kasnitz, Nadine; Kocijancic, Dino; Trittel, Stephanie; Riese, Peggy; Guzman, Carlos A; Leschner, Sara; Weiss, Siegfried

    2015-10-15

    Facultative anaerobic bacteria like E. coli can colonize solid tumors often resulting in tumor growth retardation or even clearance. Little mechanistic knowledge is available for this phenomenon which is however crucial for optimization and further implementation in the clinic. Here, we show that intravenous injections with E. coli TOP10 can induce clearance of CT26 tumors in BALB/c mice. Importantly, re-challenging mice which had cleared tumors showed that clearance was due to a specific immune reaction. Accordingly, lymphopenic mice never showed tumor clearance after infection. Depletion experiments revealed that during induction phase, CD8(+) T cells are the sole effectors responsible for tumor clearance while in the memory phase CD8(+) and CD4(+) T cells were involved. This was confirmed by adoptive transfer. CD4(+) and CD8(+) T cells could reject newly set tumors while CD8(+) T cells could even reject established tumors. Detailed analysis of adoptively transferred CD4(+) T cells during tumor challenge revealed expression of granzyme B, FasL, TNF-α and IFN-γ in such T cells that might be involved in the anti-tumor activity. Our findings should pave the way for further optimization steps of this promising therapy.

  15. End-point effector stress mediators in neuroimmune interactions: their role in immune system homeostasis and autoimmune pathology.

    PubMed

    Dimitrijevic, Mirjana; Stanojevic, Stanislava; Kustrimovic, Natasa; Leposavic, Gordana

    2012-04-01

    Much evidence has identified a direct anatomical and functional link between the brain and the immune system, with glucocorticoids (GCs), catecholamines (CAs), and neuropeptide Y (NPY) as its end-point mediators. This suggests the important role of these mediators in immune system homeostasis and the pathogenesis of inflammatory autoimmune diseases. However, although it is clear that these mediators can modulate lymphocyte maturation and the activity of distinct immune cell types, their putative role in the pathogenesis of autoimmune disease is not yet completely understood. We have contributed to this field by discovering the influence of CAs and GCs on fine-tuning thymocyte negative selection and, in particular, by pointing to the putative CA-mediated mechanisms underlying this influence. Furthermore, we have shown that CAs are implicated in the regulation of regulatory T-cell development in the thymus. Moreover, our investigations related to macrophage biology emphasize the complex interaction between GCs, CAs and NPY in the modulation of macrophage functions and their putative significance for the pathogenesis of autoimmune inflammatory diseases.

  16. TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASE-Mediated Generation and Metabolic Analysis of Camalexin-Deficient cyp71a12 cyp71a13 Double Knockout Lines.

    PubMed

    Müller, Teresa M; Böttcher, Christoph; Morbitzer, Robert; Götz, Cornelia C; Lehmann, Johannes; Lahaye, Thomas; Glawischnig, Erich

    2015-07-01

    In Arabidopsis (Arabidopsis thaliana), a number of defense-related metabolites are synthesized via indole-3-acetonitrile (IAN), including camalexin and indole-3-carboxylic acid (ICOOH) derivatives. Cytochrome P450 71A13 (CYP71A13) is a key enzyme for camalexin biosynthesis and catalyzes the conversion of indole-3-acetaldoxime (IAOx) to IAN. The CYP71A13 gene is located in tandem with its close homolog CYP71A12, also encoding an IAOx dehydratase. However, for CYP71A12, indole-3-carbaldehyde and cyanide were identified as major reaction products. To clarify CYP71A12 function in vivo and to better understand IAN metabolism, we generated two cyp71a12 cyp71a13 double knockout mutant lines. CYP71A12-specific transcription activator-like effector nucleases were introduced into the cyp71a13 background, and very efficient somatic mutagenesis was achieved. We observed stable transmission of the cyp71a12 mutation to the following generations, which is a major challenge for targeted mutagenesis in Arabidopsis. In contrast to cyp71a13 plants, in which camalexin accumulation is partially reduced, double mutants synthesized only traces of camalexin, demonstrating that CYP71A12 contributes to camalexin biosynthesis in leaf tissue. A major role of CYP71A12 was identified for the inducible biosynthesis of ICOOH. Specifically, the ICOOH methyl ester was reduced to 12% of the wild-type level in AgNO3-challenged cyp71a12 leaves. In contrast, indole-3-carbaldehyde derivatives apparently are synthesized via alternative pathways, such as the degradation of indole glucosinolates. Based on these results, we present a model for this surprisingly complex metabolic network with multiple IAN sources and channeling of IAOx-derived IAN into camalexin biosynthesis. In conclusion, transcription activator-like effector nuclease-mediated mutation is a powerful tool for functional analysis of tandem genes in secondary metabolism.

  17. The Arabidopsis ZED1 pseudokinase is required for ZAR1-mediated immunity induced by the Pseudomonas syringae type III effector HopZ1a

    PubMed Central

    Lewis, Jennifer D.; Lee, Amy Huei-Yi; Hassan, Jana A.; Wan, Janet; Hurley, Brenden; Jhingree, Jacquelyn R.; Wang, Pauline W.; Lo, Timothy; Youn, Ji-Young; Guttman, David S.; Desveaux, Darrell

    2013-01-01

    Plant and animal pathogenic bacteria can suppress host immunity by injecting type III secreted effector (T3SE) proteins into host cells. However, T3SEs can also elicit host immunity if the host has evolved a means to recognize the presence or activity of specific T3SEs. The diverse YopJ/HopZ/AvrRxv T3SE superfamily, which is found in both animal and plant pathogens, provides examples of T3SEs playing this dual role. The T3SE HopZ1a is an acetyltransferase carried by the phytopathogen Pseudomonas syringae that elicits effector-triggered immunity (ETI) when recognized in Arabidopsis thaliana by the nucleotide-binding leucine-rich repeat (NB-LRR) protein ZAR1. However, recognition of HopZ1a does not require any known ETI-related genes. Using a forward genetics approach, we identify a unique ETI-associated gene that is essential for ZAR1-mediated immunity. The hopZ-ETI-deficient1 (zed1) mutant is specifically impaired in the recognition of HopZ1a, but not the recognition of other unrelated T3SEs or in pattern recognition receptor (PRR)-triggered immunity. ZED1 directly interacts with both HopZ1a and ZAR1 and is acetylated on threonines 125 and 177 by HopZ1a. ZED1 is a nonfunctional kinase that forms part of small genomic cluster of kinases in Arabidopsis. We hypothesize that ZED1 acts as a decoy to lure HopZ1a to the ZAR1–resistance complex, resulting in ETI activation. PMID:24170858

  18. Mycosphaerella graminicola LysM effector-mediated stealth pathogenesis subverts recognition through both CERK1 and CEBiP homologues in wheat.

    PubMed

    Lee, Wing-Sham; Rudd, Jason J; Hammond-Kosack, Kim E; Kanyuka, Kostya

    2014-03-01

    Fungal cell-wall chitin is a well-recognized pathogen-associated molecular pattern. Recognition of chitin in plants by pattern recognition receptors activates pathogen-triggered immunity (PTI). In Arabidopsis, this process is mediated by a plasma membrane receptor kinase, CERK1, whereas in rice, a receptor-like protein, CEBiP, in addition to CERK1 is required. Secreted chitin-binding lysin motif (LysM) containing fungal effector proteins, such as Ecp6 from the biotrophic fungus Cladosporium fulvum, have been reported to interfere with PTI. Here, we identified wheat homologues of CERK1 and CEBiP and investigated their role in the interaction with the nonbiotrophic pathogen of wheat Mycosphaerella graminicola (synonym Zymoseptoria tritici). We show that silencing of either CERK1 or CEBiP in wheat, using Barley stripe mosaic virus-mediated virus-induced gene silencing, is sufficient in allowing leaf colonization by the normally nonpathogenic M. graminicola Mg3LysM (homologue of Ecp6) deletion mutant, while the Mg1LysM deletion mutant was fully pathogenic toward both silenced and wild-type wheat leaves. These data indicate that Mg3LysM is important for fungal evasion of PTI in wheat leaf tissue and that both CERK1 and CEBiP are required for activation of chitin-induced defenses, a feature conserved between rice and wheat, and perhaps, also in other cereal species.

  19. Spontaneous and natural cytotoxicity receptor-mediated cytotoxicity are effector functions of distinct natural killer subsets in hepatitis C virus-infected chimpanzees.

    PubMed

    Verstrepen, B E; Nieuwenhuis, I G; Mooij, P; Bogers, W M; Boonstra, A; Koopman, G

    2016-07-01

    In humans, CD16 and CD56 are used to identify functionally distinct natural killer (NK) subsets. Due to ubiquitous CD56 expression, this marker cannot be used to distinguish between NK cell subsets in chimpanzees. Therefore, functional analysis of distinct NK subsets during hepatitis C virus (HCV) infection has never been performed in these animals. In the present study an alternative strategy was used to identify four distinct NK subsets on the basis of the expression of CD16 and CD94. The expression of activating and inhibiting surface receptors showed that these subsets resemble human NK subsets. CD107 expression was used to determine degranulation of the different subsets in naive and HCV-infected chimpanzees. In HCV-infected chimpanzees increased spontaneous cytotoxicity was observed in CD94(high/dim) CD16(pos) and CD94(low) CD16(pos) subsets. By contrast, increased natural cytotoxicity receptor (NCR)- mediated degranulation after NKp30 and NKp44 triggering was demonstrated in the CD94(dim) CD16(neg) subset. Our findings suggest that spontaneous and NCR-mediated cytotoxicity are effector functions of distinct NK subsets in HCV-infected chimpanzees.

  20. Transcription mediated by NFAT is highly inducible in effector CD4+ T helper 2 (Th2) cells but not in Th1 cells.

    PubMed Central

    Rincón, M; Flavell, R A

    1997-01-01

    Transcriptional factors of the NFAT family play an important role in regulating the expression of several cytokine genes during the immune response, such as the genes for interleukin 2 (IL-2) and IL-4, among others. Upon antigen stimulation, precursor CD4+ T helper (pTh) cells proliferate and differentiate into two populations of effector cells (eTh1 and eTh2), each one expressing a specific pattern of cytokines that distinguishes them from their precursors. eTh2 cells are the major source of IL-4, while gamma interferon is produced by eTh1 cells. Here we have used reporter transgenic mice to show that DNA binding and transcriptional activities of NFAT are transiently induced during the differentiation of pTh cells into either eTh1 or eTh2 cells to mediate the expression of IL-2 as a common growth factor in both pathways. However, although NFAT DNA binding is similarly induced in both eTh1 and eTh2 cells upon antigen stimulation, only the NFAT complexes present in eTh2 cells are able to mediate high-level transcription, and relatively little NFAT transcriptional activity was induced in eTh1 cells. In contrast to activated pTh cells, neither eTh1 nor eTh2 cells produced significant IL-2 upon stimulation, but the high levels of NFAT transcriptional activities directly correlate with the IL-4 production induced in response to antigen stimulation in eTh2 cells. These data suggest that activated NFAT is involved in the effector function of eTh2 cells and that the failure of eTh1 cells to produce IL-4 in response to an antigen is due, at least partially, to a failure to induce high-level transcription of the IL-4 gene by NFAT. Regulation of NFAT could be therefore a critical element in the polarization to eTh1 or eTh2. PMID:9032280

  1. Effects of cryopreservation on effector cells for antibody dependent cell-mediated cytotoxicity (ADCC) and natural killer (NK) cell activity in (51)Cr-release and CD107a assays.

    PubMed

    Mata, Mariana M; Mahmood, Fareeha; Sowell, Ryan T; Baum, Linda L

    2014-04-01

    Freshly isolated PBMC are broadly used as effector cells in functional assays that evaluate antibody-dependent cell mediated cytotoxicity (ADCC) and NK activity; however, they introduce natural-individual donor-to-donor variability. Cryopreserved PBMC provide a more consistent source of effectors than fresh cells in cytotoxicity assays. Our objective was to determine the effects of cryopreservation of effector PBMC on cell frequency, and on the magnitude and specificity of ADCC and NK activity. Fresh, frozen/overnight rested and frozen/not rested PBMC were used as effector cells in (51)Cr-release and CD107a degranulation assays. Frozen/overnight rested PBMC had higher ADCC and NK activity in both assays when compared to fresh PBMC; however, when using frozen/not rested PBMC, ADCC and NK activities were significantly lower than fresh PBMC. Background CD107a degranulation in the absence of target cell stimulation was greater in PBMC that were frozen/not rested when compared to fresh PBMC or PBMC that were frozen overnight and rested. The percentages of CD16(+)CD56(dim) NK cells and CD14(+) monocytes were lower in PBMC that were frozen and rested overnight than in fresh PBMC. CD16 expression on CD56(dim) NK cells was similar for all PBMC treatments. PBMC that were frozen and rested overnight were comparable to fresh PBMC effectors. PBMC that were frozen and used immediately when evaluating ADCC or NK activity using either a (51)Cr-release assay or a CD107a degranulation assay had the lowest activity. Clinical studies of antibodies that mediate ADCC would benefit from using effector cells that have been frozen, thawed and rested overnight prior to assay.

  2. Production of α1,3-galactosyltransferase targeted pigs using transcription activator-like effector nuclease-mediated genome editing technology.

    PubMed

    Kang, Jung-Taek; Kwon, Dae-Kee; Park, A-Rum; Lee, Eun-Jin; Yun, Yun-Jin; Ji, Dal-Young; Lee, Kiho; Park, Kwang-Wook

    2016-03-01

    Recent developments in genome editing technology using meganucleases demonstrate an efficient method of producing gene edited pigs. In this study, we examined the effectiveness of the transcription activator-like effector nuclease (TALEN) system in generating specific mutations on the pig genome. Specific TALEN was designed to induce a double-strand break on exon 9 of the porcine α1,3-galactosyltransferase (GGTA1) gene as it is the main cause of hyperacute rejection after xenotransplantation. Human decay-accelerating factor (hDAF) gene, which can produce a complement inhibitor to protect cells from complement attack after xenotransplantation, was also integrated into the genome simultaneously. Plasmids coding for the TALEN pair and hDAF gene were transfected into porcine cells by electroporation to disrupt the porcine GGTA1 gene and express hDAF. The transfected cells were then sorted using a biotin-labeled IB4 lectin attached to magnetic beads to obtain GGTA1 deficient cells. As a result, we established GGTA1 knockout (KO) cell lines with biallelic modification (35.0%) and GGTA1 KO cell lines expressing hDAF (13.0%). When these cells were used for somatic cell nuclear transfer, we successfully obtained live GGTA1 KO pigs expressing hDAF. Our results demonstrate that TALEN-mediated genome editing is efficient and can be successfully used to generate gene edited pigs.

  3. Transcription activator-like effector-mediated regulation of gene expression based on the inducible packaging and delivery via designed extracellular vesicles.

    PubMed

    Lainšček, Duško; Lebar, Tina; Jerala, Roman

    2017-02-26

    Transcription activator-like effector (TALE) proteins present a powerful tool for genome editing and engineering, enabling introduction of site-specific mutations, gene knockouts or regulation of the transcription levels of selected genes. TALE nucleases or TALE-based transcription regulators are introduced into mammalian cells mainly via delivery of the coding genes. Here we report an extracellular vesicle-mediated delivery of TALE transcription regulators and their ability to upregulate the reporter gene in target cells. Designed transcriptional activator TALE-VP16 fused to the appropriate dimerization domain was enriched as a cargo protein within extracellular vesicles produced by mammalian HEK293 cells stimulated by Ca-ionophore and using blue light- or rapamycin-inducible dimerization systems. Blue light illumination or rapamycin increased the amount of the TALE-VP16 activator in extracellular vesicles and their addition to the target cells resulted in an increased expression of the reporter gene upon addition of extracellular vesicles to the target cells. This technology therefore represents an efficient delivery for the TALE-based transcriptional regulators.

  4. Inhibition of Cryptococcus neoformans replication by nitrogen oxides supports the role of these molecules as effectors of macrophage-mediated cytostasis

    SciTech Connect

    Alspaugh, J.A.; Granger, D.L. )

    1991-07-01

    Activated macrophages are able to inhibit the replication of intracellular microbes and tumor cells. In the murine system, this cytostatic effect is associated with the oxidation of L-arginine to L-citrulline, nitrite, and nitrate and is thought to be mediated by an intermediate of this reaction, possibly nitric oxide (NO.). By exposing replicating Cryptococcus neoformans cells to conditions under which NO. is chemically generated, we have observed a cytostatic effect similar to that caused by activated murine macrophages. Nitric oxide is formed as a decomposition product of nitrite salts in acidic, aqueous solutions. Although C. neoformans replicates well in the presence of high nitrite concentrations at physiologic pH, its growth in acidic media can be inhibited by the addition of low concentrations of sodium nitrite. The degree of cytostasis is dependent on both the pH and the nitrite concentration of the NO. generating solution. The cytostatic effector molecule appears to be a gas since, in addition to inhibiting C. neoformans replication in solution, it is able to exert its inhibitory effect across a gas-permeable but ion-impermeable membrane. At high nitrite concentrations, a fungicidal effect occurs. We propose that the growth inhibition of C. neoformans upon exposure to chemically generated NO. or some related oxide of nitrogen represents a cell-free system simulating the cytostatic effect of activated murine macrophages.

  5. Cancer-induced defective cytotoxic T lymphocyte effector function: another mechanism how antigenic tumors escape immune-mediated killing.

    PubMed Central

    Radoja, S.; Frey, A. B.

    2000-01-01

    BACKGROUND: The notion that a deficit in immune cell functions permits tumor growth has received experimental support with the discovery of several different biochemical defects in T lymphocytes that infiltrate cancers. Decreased levels of enzymes involved with T-cell signal transduction have been reported by several laboratories, suggesting that tumors or host cells recruited to the tumor site actively down-regulate antitumor T-cell immune response. This permits tumor escape from immune-mediated killing. The possibility that defects in T-cell signal transduction can be reversed, which would potentially permit successful vaccination or adoptive immunotherapy, motivates renewed interest in the field. Summarizing the literature concerning tumor-induced T-cell dysfunction, we focus on the end stage of immune response to human cancer, that of defective cytotoxic T lymphocyte killing function. Based on the data from several laboratories, we hypothesize a biochemical mechanism that accounts for the unusual phenotype of antitumor T-cell accumulation in tumors, but with defective killing function. PMID:10972084

  6. Nck adaptors, besides promoting N-WASP mediated actin-nucleation activity at pedestals, influence the cellular levels of enteropathogenic Escherichia coli Tir effector

    PubMed Central

    Nieto-Pelegrin, Elvira; Kenny, Brendan; Martinez-Quiles, Narcisa

    2014-01-01

    Enteropathogenic Escherichia coli (EPEC) binding to human intestinal cells triggers the formation of disease-associated actin rich structures called pedestals. The latter process requires the delivery, via a Type 3 secretion system, of the translocated Intimin receptor (Tir) protein into the host plasma membrane where binding of a host kinase-modified form to the bacterial surface protein Intimin triggers pedestal formation. Tir-Intimin interaction recruits the Nck adaptor to a Tir tyrosine phosphorylated residue where it activates neural Wiskott-Aldrich syndrome protein (N-WASP); initiating the major pathway to actin polymerization mediated by the actin-related protein (Arp) 2/3 complex. Previous studies with Nck-deficient mouse embryonic fibroblasts (MEFs) identified a key role for Nck in pedestal formation, presumed to reflect a lack of N-WASP activation. Here, we show the defect relates to reduced amounts of Tir within Nck-deficient cells. Indeed, Tir delivery and, thus, pedestal formation defects were much greater for MEFs than HeLa (human epithelial) cells. Crucially, the levels of two other effectors (EspB/EspF) within Nck-deficient MEFs were not reduced unlike that of Map (Mitochondrial associated protein) which, like Tir, requires CesT chaperone function for efficient delivery. Interestingly, drugs blocking various host protein degradation pathways failed to increase Tir cellular levels unlike an inhibitor of deacetylase activity (Trichostatin A; TSA). Treatments with TSA resulted in significant recovery of Tir levels, potentiation of actin polymerization and improvement in bacterial attachment to cells. Our findings have important implications for the current model of Tir-mediated actin polymerization and opens new lines of research in this area. PMID:25482634

  7. Effector-mediated eradication of precursor B acute lymphoblastic leukemia with a novel Fc engineered monoclonal antibody targeting the BAFF-R

    PubMed Central

    Parameswaran, Reshmi; Lim, Min; Fei, Fei; Abdel-Azim, Hisham; Arutyunyan, Anna; Schiffer, Isabelle; McLaughlin, Margaret E.; Gram, Hermann; Huet, Heather; Groffen, John; Heisterkamp, Nora

    2014-01-01

    B-cell activating factor receptor (BAFF-R) is expressed on precursor B acute lymphoblastic leukemia ALL (pre-B ALL) cells but not on their pre-B normal counterparts. Thus, selective killing of ALL cells is possible by targeting this receptor. Here we have further examined therapeutic targeting of pre-B ALL based on the presence of the BAFF-R. Mouse pre-B ALL cells lacking BAFF-R function had comparable viability and proliferation to wild type cells but were more sensitive to drug treatment. Viability of human pre-B ALL cells was further reduced when antibodies to the BAFF-R were combined with other drugs, even in the presence of stromal protection. This indicates that inhibition of BAFF-R function reduces fitness of stressed pre-B ALL cells. We tested a novel humanized anti-BAFF-R monoclonal antibody optimalized for FcRγIII mediated, antibody-dependent cell killing by effector cells. Antibody binding to human ALL cells was inhibitable, in a dose-dependent manner, by recombinant human BAFF. There was no evidence for internalization of the antibodies. The antibodies significantly stimulated NK cell-mediated killing of different human patient-derived ALL cells. Moreover, incubation of such ALL cells with these antibodies stimulated phagocytosis by macrophages. When this was tested in an immunodeficient transplant model, mice that were treated with the antibody had a significantly decreased leukemia burden in bone marrow and spleen. In view of the restricted expression of the BAFF-R on normal cells and the multiple anti-pre-B ALL activities stimulated by this antibody, a further examination of its use for treatment of pre-B ALL is warranted. PMID:24825858

  8. Nck adaptors, besides promoting N-WASP mediated actin-nucleation activity at pedestals, influence the cellular levels of enteropathogenic Escherichia coli Tir effector.

    PubMed

    Nieto-Pelegrin, Elvira; Kenny, Brendan; Martinez-Quiles, Narcisa

    2014-01-01

    Enteropathogenic Escherichia coli (EPEC) binding to human intestinal cells triggers the formation of disease-associated actin rich structures called pedestals. The latter process requires the delivery, via a Type 3 secretion system, of the translocated Intimin receptor (Tir) protein into the host plasma membrane where binding of a host kinase-modified form to the bacterial surface protein Intimin triggers pedestal formation. Tir-Intimin interaction recruits the Nck adaptor to a Tir tyrosine phosphorylated residue where it activates neural Wiskott-Aldrich syndrome protein (N-WASP); initiating the major pathway to actin polymerization mediated by the actin-related protein (Arp) 2/3 complex. Previous studies with Nck-deficient mouse embryonic fibroblasts (MEFs) identified a key role for Nck in pedestal formation, presumed to reflect a lack of N-WASP activation. Here, we show the defect relates to reduced amounts of Tir within Nck-deficient cells. Indeed, Tir delivery and, thus, pedestal formation defects were much greater for MEFs than HeLa (human epithelial) cells. Crucially, the levels of two other effectors (EspB/EspF) within Nck-deficient MEFs were not reduced unlike that of Map (Mitochondrial associated protein) which, like Tir, requires CesT chaperone function for efficient delivery. Interestingly, drugs blocking various host protein degradation pathways failed to increase Tir cellular levels unlike an inhibitor of deacetylase activity (Trichostatin A; TSA). Treatments with TSA resulted in significant recovery of Tir levels, potentiation of actin polymerization and improvement in bacterial attachment to cells. Our findings have important implications for the current model of Tir-mediated actin polymerization and opens new lines of research in this area.

  9. The Arthroderma benhamiae Hydrophobin HypA Mediates Hydrophobicity and Influences Recognition by Human Immune Effector Cells

    PubMed Central

    Heddergott, Christoph; Bruns, Sandra; Nietzsche, Sandor; Leonhardt, Ines; Kurzai, Oliver; Kniemeyer, Olaf

    2012-01-01

    Dermatophytes are the most common cause of superficial mycoses in humans and animals. They can coexist with their hosts for many years without causing significant symptoms but also cause highly inflammatory diseases. To identify mechanisms involved in the modulation of the host response during infection caused by the zoophilic dermatophyte Arthroderma benhamiae, cell wall-associated surface proteins were studied. By two-dimensional gel electrophoresis, we found that a hydrophobin protein designated HypA was the dominant cell surface protein. HypA was also detected in the supernatant during the growth and conidiation of the fungus. The A. benhamiae genome harbors only a single hydrophobin gene, designated hypA. A hypA deletion mutant was generated, as was a complemented hypA mutant strain (hypAC). In contrast to the wild type and the complemented strain, the hypA deletion mutant exhibited “easily wettable” mycelia and conidia, indicating the loss of surface hydrophobicity of both morphotypes. Compared with the wild type, the hypA deletion mutant triggered an increased activation of human neutrophil granulocytes and dendritic cells, characterized by an increased release of the immune mediators interleukin-6 (IL-6), IL-8, IL-10, and tumor necrosis factor alpha (TNF-α). For the first time, we observed the formation of neutrophil extracellular traps against dermatophytes, whose level of formation was increased by the ΔhypA mutant compared with the wild type. Furthermore, conidia of the ΔhypA strain were killed more effectively by neutrophils. Our data suggest that the recognition of A. benhamiae by the cellular immune defense system is notably influenced by the presence of the surface rodlet layer formed by the hydrophobin HypA. PMID:22408226

  10. The Arthroderma benhamiae hydrophobin HypA mediates hydrophobicity and influences recognition by human immune effector cells.

    PubMed

    Heddergott, Christoph; Bruns, Sandra; Nietzsche, Sandor; Leonhardt, Ines; Kurzai, Oliver; Kniemeyer, Olaf; Brakhage, Axel A

    2012-05-01

    Dermatophytes are the most common cause of superficial mycoses in humans and animals. They can coexist with their hosts for many years without causing significant symptoms but also cause highly inflammatory diseases. To identify mechanisms involved in the modulation of the host response during infection caused by the zoophilic dermatophyte Arthroderma benhamiae, cell wall-associated surface proteins were studied. By two-dimensional gel electrophoresis, we found that a hydrophobin protein designated HypA was the dominant cell surface protein. HypA was also detected in the supernatant during the growth and conidiation of the fungus. The A. benhamiae genome harbors only a single hydrophobin gene, designated hypA. A hypA deletion mutant was generated, as was a complemented hypA mutant strain (hypA(C)). In contrast to the wild type and the complemented strain, the hypA deletion mutant exhibited "easily wettable" mycelia and conidia, indicating the loss of surface hydrophobicity of both morphotypes. Compared with the wild type, the hypA deletion mutant triggered an increased activation of human neutrophil granulocytes and dendritic cells, characterized by an increased release of the immune mediators interleukin-6 (IL-6), IL-8, IL-10, and tumor necrosis factor alpha (TNF-α). For the first time, we observed the formation of neutrophil extracellular traps against dermatophytes, whose level of formation was increased by the ΔhypA mutant compared with the wild type. Furthermore, conidia of the ΔhypA strain were killed more effectively by neutrophils. Our data suggest that the recognition of A. benhamiae by the cellular immune defense system is notably influenced by the presence of the surface rodlet layer formed by the hydrophobin HypA.

  11. The Xanthomonas campestris type III effector XopJ targets the host cell proteasome to suppress salicylic-acid mediated plant defence.

    PubMed

    Üstün, Suayib; Bartetzko, Verena; Börnke, Frederik

    2013-01-01

    The phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) requires type III effector proteins (T3Es) for virulence. After translocation into the host cell, T3Es are thought to interact with components of host immunity to suppress defence responses. XopJ is a T3E protein from Xcv that interferes with plant immune responses; however, its host cellular target is unknown. Here we show that XopJ interacts with the proteasomal subunit RPT6 in yeast and in planta to inhibit proteasome activity. A C235A mutation within the catalytic triad of XopJ as well as a G2A exchange within the N-terminal myristoylation motif abolishes the ability of XopJ to inhibit the proteasome. Xcv ΔxopJ mutants are impaired in growth and display accelerated symptom development including tissue necrosis on susceptible pepper leaves. Application of the proteasome inhibitor MG132 restored the ability of the Xcv ΔxopJ to attenuate the development of leaf necrosis. The XopJ dependent delay of tissue degeneration correlates with reduced levels of salicylic acid (SA) and changes in defence- and senescence-associated gene expression. Necrosis upon infection with Xcv ΔxopJ was greatly reduced in pepper plants with reduced expression of NPR1, a central regulator of SA responses, demonstrating the involvement of SA-signalling in the development of XopJ dependent phenotypes. Our results suggest that XopJ-mediated inhibition of the proteasome interferes with SA-dependent defence response to attenuate onset of necrosis and to alter host transcription. A central role of the proteasome in plant defence is discussed.

  12. The Xanthomonas campestris Type III Effector XopJ Targets the Host Cell Proteasome to Suppress Salicylic-Acid Mediated Plant Defence

    PubMed Central

    Börnke, Frederik

    2013-01-01

    The phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) requires type III effector proteins (T3Es) for virulence. After translocation into the host cell, T3Es are thought to interact with components of host immunity to suppress defence responses. XopJ is a T3E protein from Xcv that interferes with plant immune responses; however, its host cellular target is unknown. Here we show that XopJ interacts with the proteasomal subunit RPT6 in yeast and in planta to inhibit proteasome activity. A C235A mutation within the catalytic triad of XopJ as well as a G2A exchange within the N-terminal myristoylation motif abolishes the ability of XopJ to inhibit the proteasome. Xcv ΔxopJ mutants are impaired in growth and display accelerated symptom development including tissue necrosis on susceptible pepper leaves. Application of the proteasome inhibitor MG132 restored the ability of the Xcv ΔxopJ to attenuate the development of leaf necrosis. The XopJ dependent delay of tissue degeneration correlates with reduced levels of salicylic acid (SA) and changes in defence- and senescence-associated gene expression. Necrosis upon infection with Xcv ΔxopJ was greatly reduced in pepper plants with reduced expression of NPR1, a central regulator of SA responses, demonstrating the involvement of SA-signalling in the development of XopJ dependent phenotypes. Our results suggest that XopJ-mediated inhibition of the proteasome interferes with SA-dependent defence response to attenuate onset of necrosis and to alter host transcription. A central role of the proteasome in plant defence is discussed. PMID:23785289

  13. Extracellular histones are essential effectors of C5aR- and C5L2-mediated tissue damage and inflammation in acute lung injury

    PubMed Central

    Bosmann, Markus; Grailer, Jamison J.; Ruemmler, Robert; Russkamp, Norman F.; Zetoune, Firas S.; Sarma, J. Vidya; Standiford, Theodore J.; Ward, Peter A.

    2013-01-01

    We investigated how complement activation promotes tissue injury and organ dysfunction during acute inflammation. Three models of acute lung injury (ALI) induced by LPS, IgG immune complexes, or C5a were used in C57BL/6 mice, all models requiring availability of both C5a receptors (C5aR and C5L2) for full development of ALI. Ligation of C5aR and C5L2 with C5a triggered the appearance of histones (H3 and H4) in bronchoalveolar lavage fluid (BALF). BALF from humans with ALI contained H4 histone. Histones were absent in control BALF from healthy volunteers. In mice with ALI, in vivo neutralization of H4 with IgG antibody reduced the intensity of ALI. Neutrophil depletion in mice with ALI markedly reduced H4 presence in BALF and was highly protective. The direct lung damaging effects of extracellular histones were demonstrated by airway administration of histones into mice and rats (Sprague-Dawley), which resulted in ALI that was C5a receptor-independent, and associated with intense inflammation, PMN accumulation, damage/destruction of alveolar epithelial cells, together with release into lung of cytokines/chemokines. High-resolution magnetic resonance imaging demonstrated lung damage, edema and consolidation in histone-injured lungs. These studies confirm the destructive C5a-dependent effects in lung linked to appearance of extracellular histones.—Bosmann, M., Grailer, J. J., Ruemmler, R., Russkamp, N. F., Zetoune, F. S., Sarma, J. V., Standiford, T. J., Ward, P. A. Extracellular histones are essential effectors of C5aR- and C5L2-mediated tissue damage and inflammation in acute lung injury. PMID:23982144

  14. The Anoikis Effector Bit1 Inhibits EMT through Attenuation of TLE1-Mediated Repression of E-Cadherin in Lung Cancer Cells

    PubMed Central

    Yao, Xin; Pham, Tri; Temple, Brandi; Gray, Selena; Cannon, Cornita; Chen, Renwei; Abdel-Mageed, Asim B.; Biliran, Hector

    2016-01-01

    The mitochondrial Bcl-2 inhibitor of transcription 1 (Bit1) protein is part of an anoikis-regulating pathway that is selectively dependent on integrins. We previously demonstrated that the caspase-independent apoptotic effector Bit1 exerts tumor suppressive function in lung cancer in part by inhibiting anoikis resistance and anchorage-independent growth in vitro and tumorigenicity in vivo. Herein we show a novel function of Bit1 as an inhibitor cell migration and epithelial–mesenchymal transition (EMT) in the human lung adenocarcinoma A549 cell line. Suppression of endogenous Bit1 expression via siRNA and shRNA strategies promoted mesenchymal phenotypes, including enhanced fibroblastoid morphology and cell migratory potential with concomitant downregulation of the epithelial marker E-cadherin expression. Conversely, ectopic Bit1 expression in A549 cells promoted epithelial transition characterized by cuboidal-like epithelial cell phenotype, reduced cell motility, and upregulated E-cadherin expression. Specific downregulation of E-cadherin in Bit1-transfected cells was sufficient to block Bit1-mediated inhibition of cell motility while forced expression of E-cadherin alone attenuated the enhanced migration of Bit1 knockdown cells, indicating that E-cadherin is a downstream target of Bit1 in regulating cell motility. Furthermore, quantitative real-time PCR and reporter analyses revealed that Bit1 upregulates E-cadherin expression at the transcriptional level through the transcriptional regulator Amino-terminal Enhancer of Split (AES) protein. Importantly, the Bit1/AES pathway induction of E-cadherin expression involves inhibition of the TLE1-mediated repression of E-cadherin, by decreasing TLE1 corepressor occupancy at the E-cadherin promoter as revealed by chromatin immunoprecipitation assays. Consistent with its EMT inhibitory function, exogenous Bit1 expression significantly suppressed the formation of lung metastases of A549 cells in an in vivo experimental

  15. The Anoikis Effector Bit1 Inhibits EMT through Attenuation of TLE1-Mediated Repression of E-Cadherin in Lung Cancer Cells.

    PubMed

    Yao, Xin; Pham, Tri; Temple, Brandi; Gray, Selena; Cannon, Cornita; Chen, Renwei; Abdel-Mageed, Asim B; Biliran, Hector

    The mitochondrial Bcl-2 inhibitor of transcription 1 (Bit1) protein is part of an anoikis-regulating pathway that is selectively dependent on integrins. We previously demonstrated that the caspase-independent apoptotic effector Bit1 exerts tumor suppressive function in lung cancer in part by inhibiting anoikis resistance and anchorage-independent growth in vitro and tumorigenicity in vivo. Herein we show a novel function of Bit1 as an inhibitor cell migration and epithelial-mesenchymal transition (EMT) in the human lung adenocarcinoma A549 cell line. Suppression of endogenous Bit1 expression via siRNA and shRNA strategies promoted mesenchymal phenotypes, including enhanced fibroblastoid morphology and cell migratory potential with concomitant downregulation of the epithelial marker E-cadherin expression. Conversely, ectopic Bit1 expression in A549 cells promoted epithelial transition characterized by cuboidal-like epithelial cell phenotype, reduced cell motility, and upregulated E-cadherin expression. Specific downregulation of E-cadherin in Bit1-transfected cells was sufficient to block Bit1-mediated inhibition of cell motility while forced expression of E-cadherin alone attenuated the enhanced migration of Bit1 knockdown cells, indicating that E-cadherin is a downstream target of Bit1 in regulating cell motility. Furthermore, quantitative real-time PCR and reporter analyses revealed that Bit1 upregulates E-cadherin expression at the transcriptional level through the transcriptional regulator Amino-terminal Enhancer of Split (AES) protein. Importantly, the Bit1/AES pathway induction of E-cadherin expression involves inhibition of the TLE1-mediated repression of E-cadherin, by decreasing TLE1 corepressor occupancy at the E-cadherin promoter as revealed by chromatin immunoprecipitation assays. Consistent with its EMT inhibitory function, exogenous Bit1 expression significantly suppressed the formation of lung metastases of A549 cells in an in vivo experimental

  16. Transcriptional Dynamics Driving MAMP-Triggered Immunity and Pathogen Effector-Mediated Immunosuppression in Arabidopsis Leaves Following Infection with Pseudomonas syringae pv tomato DC3000.

    PubMed

    Lewis, Laura A; Polanski, Krzysztof; de Torres-Zabala, Marta; Jayaraman, Siddharth; Bowden, Laura; Moore, Jonathan; Penfold, Christopher A; Jenkins, Dafyd J; Hill, Claire; Baxter, Laura; Kulasekaran, Satish; Truman, William; Littlejohn, George; Prusinska, Justyna; Mead, Andrew; Steinbrenner, Jens; Hickman, Richard; Rand, David; Wild, David L; Ott, Sascha; Buchanan-Wollaston, Vicky; Smirnoff, Nick; Beynon, Jim; Denby, Katherine; Grant, Murray

    2015-11-01

    Transcriptional reprogramming is integral to effective plant defense. Pathogen effectors act transcriptionally and posttranscriptionally to suppress defense responses. A major challenge to understanding disease and defense responses is discriminating between transcriptional reprogramming associated with microbial-associated molecular pattern (MAMP)-triggered immunity (MTI) and that orchestrated by effectors. A high-resolution time course of genome-wide expression changes following challenge with Pseudomonas syringae pv tomato DC3000 and the nonpathogenic mutant strain DC3000hrpA- allowed us to establish causal links between the activities of pathogen effectors and suppression of MTI and infer with high confidence a range of processes specifically targeted by effectors. Analysis of this information-rich data set with a range of computational tools provided insights into the earliest transcriptional events triggered by effector delivery, regulatory mechanisms recruited, and biological processes targeted. We show that the majority of genes contributing to disease or defense are induced within 6 h postinfection, significantly before pathogen multiplication. Suppression of chloroplast-associated genes is a rapid MAMP-triggered defense response, and suppression of genes involved in chromatin assembly and induction of ubiquitin-related genes coincide with pathogen-induced abscisic acid accumulation. Specific combinations of promoter motifs are engaged in fine-tuning the MTI response and active transcriptional suppression at specific promoter configurations by P. syringae. © 2015 American Society of Plant Biologists. All rights reserved.

  17. Transcriptional Dynamics Driving MAMP-Triggered Immunity and Pathogen Effector-Mediated Immunosuppression in Arabidopsis Leaves Following Infection with Pseudomonas syringae pv tomato DC3000[OPEN

    PubMed Central

    Lewis, Laura A.; Polanski, Krzysztof; de Torres-Zabala, Marta; Bowden, Laura; Jenkins, Dafyd J.; Hill, Claire; Baxter, Laura; Truman, William; Prusinska, Justyna; Hickman, Richard; Wild, David L.; Ott, Sascha; Buchanan-Wollaston, Vicky; Beynon, Jim

    2015-01-01

    Transcriptional reprogramming is integral to effective plant defense. Pathogen effectors act transcriptionally and posttranscriptionally to suppress defense responses. A major challenge to understanding disease and defense responses is discriminating between transcriptional reprogramming associated with microbial-associated molecular pattern (MAMP)-triggered immunity (MTI) and that orchestrated by effectors. A high-resolution time course of genome-wide expression changes following challenge with Pseudomonas syringae pv tomato DC3000 and the nonpathogenic mutant strain DC3000hrpA- allowed us to establish causal links between the activities of pathogen effectors and suppression of MTI and infer with high confidence a range of processes specifically targeted by effectors. Analysis of this information-rich data set with a range of computational tools provided insights into the earliest transcriptional events triggered by effector delivery, regulatory mechanisms recruited, and biological processes targeted. We show that the majority of genes contributing to disease or defense are induced within 6 h postinfection, significantly before pathogen multiplication. Suppression of chloroplast-associated genes is a rapid MAMP-triggered defense response, and suppression of genes involved in chromatin assembly and induction of ubiquitin-related genes coincide with pathogen-induced abscisic acid accumulation. Specific combinations of promoter motifs are engaged in fine-tuning the MTI response and active transcriptional suppression at specific promoter configurations by P. syringae. PMID:26566919

  18. New players in the same old game: a system level in silico study to predict type III secretion system and effector proteins in bacterial genomes reveals common themes in T3SS mediated pathogenesis.

    PubMed

    Sadarangani, Vineet; Datta, Sunando; Arunachalam, Manonmani

    2013-07-26

    Type III secretion system (T3SS) plays an important role in virulence or symbiosis of many pathogenic or symbiotic bacteria [CHM 2:291-294, 2007; Physiology (Bethesda) 20:326-339, 2005]. T3SS acts like a tunnel between a bacterium and its host through which the bacterium injects 'effector' proteins into the latter [Nature 444:567-573, 2006; COSB 18:258-266, 2008]. The effectors spatially and temporally modify the host signalling pathways [FEMS Microbiol Rev 35:1100-1125, 2011; Cell Host Microbe5:571-579, 2009]. In spite its crucial role in host-pathogen interaction, the study of T3SS and the associated effectors has been limited to a few bacteria [Cell Microbiol 13:1858-1869, 2011; Nat Rev Microbiol 6:11-16, 2008; Mol Microbiol 80:1420-1438, 2011]. Before one set out to perform systematic experimental studies on an unknown set of bacteria it would be beneficial to identify the potential candidates by developing an in silico screening algorithm. A system level study would also be advantageous over traditional laboratory methods to extract an overriding theme for host-pathogen interaction, if any, from the vast resources of data generated by sequencing multiple bacterial genomes. We have developed an in silico protocol in which the most conserved set of T3SS proteins was used as the query against the entire bacterial database with increasingly stringent search parameters. It enabled us to identify several uncharacterized T3SS positive bacteria. We adopted a similar strategy to predict the presence of the already known effectors in the newly identified T3SS positive bacteria. The huge resources of biochemical data [FEMS Microbiol Rev 35:1100-1125, 2011; Cell Host Microbe 5:571-579, 2009; BMC Bioinformatics 7(11):S4, 2010] on the T3SS effectors enabled us to search for the common theme in T3SS mediated pathogenesis. We identified few cellular signalling networks in the host, which are manipulated by most of the T3SS containing pathogens. We went on to look for

  19. The Arabidopsis ZED1 pseudokinase is required for ZAR1-mediated immunity induced by the Pseudomonas syringae type III effector HopZ1a

    USDA-ARS?s Scientific Manuscript database

    Plant and animal pathogenic bacteria can suppress host immunity by injecting type III secreted effector (T3SE) proteins into host cells (1-5). However, T3SEs can also elicit host immunity if the host has evolved a means to recognize the presence or activity of specific T3SEs (6). The diverse YopJ/Ho...

  20. Host-mediated gene silencing of a single effector gene from the potato pathogen Phytophthora infestans imparts partial resistance to late blight disease.

    PubMed

    Sanju, Suman; Siddappa, Sundaresha; Thakur, Aditi; Shukla, Pradeep K; Srivastava, Nidhi; Pattanayak, Debasis; Sharma, Sanjeev; Singh, B P

    2015-11-01

    RNA interference (RNAi) has proved a powerful genetic tool for silencing genes in plants. Host-induced gene silencing of pathogen genes has provided a gene knockout strategy for a wide range of biotechnological applications. The RXLR effector Avr3a gene is largely responsible for virulence of oomycete plant pathogen Phytophthora infestans. In this study, we attempted to silence the Avr3a gene of P. infestans through RNAi technology. The P. infestans inoculation resulted in lower disease progression and a reduction in pathogen load, as demonstrated by disease scoring and quantification of pathogen biomass in terms of Pi08 repetitive elements, respectively. Transgenic plants induced moderate silencing of Avr3a, and the presence and/or expression of small interfering RNAs, as determined through Northern hybridization, indicated siRNA targeted against Avr3a conferred moderate resistance to P. infestans. The single effector gene did not provide complete resistance against P. infestans. Although the Avr3a effector gene could confer moderate resistance, for complete resistance, the cumulative effect of effector genes in addition to Avr3a needs to be considered. In this study, we demonstrated that host-induced RNAi is an effective strategy for functional genomics in oomycetes.

  1. Xanthomonas axonopodis virulence is promoted by a transcription activator-like effector-mediated induction of a SWEET sugar transporter in cassava.

    PubMed

    Cohn, Megan; Bart, Rebecca S; Shybut, Mikel; Dahlbeck, Douglas; Gomez, Michael; Morbitzer, Robert; Hou, Bi-Huei; Frommer, Wolf B; Lahaye, Thomas; Staskawicz, Brian J

    2014-11-01

    The gene-for-gene concept has historically been applied to describe a specific resistance interaction wherein single genes from the host and the pathogen dictate the outcome. These interactions have been observed across the plant kingdom and all known plant microbial pathogens. In recent years, this concept has been extended to susceptibility phenotypes in the context of transcription activator-like (TAL) effectors that target SWEET sugar transporters. However, because this interaction has only been observed in rice, it was not clear whether the gene-for-gene susceptibility was unique to that system. Here, we show, through a combined systematic analysis of the TAL effector complement of Xanthomonas axonopodis pv. manihotis and RNA sequencing to identify targets in cassava, that TAL20Xam668 specifically induces the sugar transporter MeSWEET10a to promote virulence. Designer TAL effectors (dTALE) complement TAL20Xam668 mutant phenotypes, demonstrating that MeSWEET10a is a susceptibility gene in cassava. Sucrose uptake-deficient X. axonopodis pv. manihotis bacteria do not lose virulence, indicating that sucrose may be cleaved extracellularly and taken up as hexoses into X. axonopodis pv. manihotis. Together, our data suggest that pathogen hijacking of plant nutrients is not unique to rice blight but also plays a role in bacterial blight of the dicot cassava.

  2. Translocated effectors of Yersinia

    PubMed Central

    Matsumoto, Hiroyuki; Young, Glenn M.

    2009-01-01

    Summary Currently, all known translocated effectors of Yersinia are delivered into host cells by type III secretion systems (T3SSs). Pathogenic Yersinia maintain the plasmid-encoded Ysc T3SS for the specific delivery of the well-studied Yop effectors. New horizons for effector biology have opened with the discovery of the Ysps of Y. enterocolitica Biovar 1B, which are translocated into host cells by the chromosome-endoded Ysa T3SS. The reported arsenal of effectors is likely to expand since genomic analysis has revealed gene-clusters in some Yersinia that code for other T3SSs. These efforts also revealed possible type VI secretion (T6S) systems, which may indicate translocation of effectors occurs by multiple mechanisms. PMID:19185531

  3. The C-terminal half of Phytophthora infestans RXLR effector AVR3a is sufficient to trigger R3a-mediated hypersensitivity and suppress INF1-induced cell death in Nicotiana benthamiana.

    PubMed

    Bos, Jorunn I B; Kanneganti, Thirumala-Devi; Young, Carolyn; Cakir, Cahid; Huitema, Edgar; Win, Joe; Armstrong, Miles R; Birch, Paul R J; Kamoun, Sophien

    2006-10-01

    The RXLR cytoplasmic effector AVR3a of Phytophthora infestans confers avirulence on potato plants carrying the R3a gene. Two alleles of Avr3a encode secreted proteins that differ in only three amino acid residues, two of which are in the mature protein. Avirulent isolates carry the Avr3a allele, which encodes AVR3aKI (containing amino acids C19, K80 and I103), whereas virulent isolates express only the virulence allele avr3a, encoding AVR3aEM (S19, E80 and M103). Only the AVR3aKI protein is recognized inside the plant cytoplasm where it triggers R3a-mediated hypersensitivity. Similar to other oomycete avirulence proteins, AVR3aKI carries a signal peptide followed by a conserved motif centered on the consensus RXLR sequence that is functionally similar to a host cell-targeting signal of malaria parasites. The interaction between Avr3a and R3a can be reconstructed by their transient co-expression in Nicotiana benthamiana. We exploited the N. benthamiana experimental system to further characterize the Avr3a-R3a interaction. R3a activation by AVR3aKI is dependent on the ubiquitin ligase-associated protein SGT1 and heat-shock protein HSP90. The AVR3aKI and AVR3aEM proteins are equally stable in planta, suggesting that the difference in R3a-mediated death cannot be attributed to AVR3aEM protein instability. AVR3aKI is able to suppress cell death induced by the elicitin INF1 of P. infestans, suggesting a possible virulence function for this protein. Structure-function experiments indicated that the 75-amino acid C-terminal half of AVR3aKI, which excludes the RXLR region, is sufficient for avirulence and suppression functions, consistent with the view that the N-terminal region of AVR3aKI and other RXLR effectors is involved in secretion and targeting but is not required for effector activity. We also found that both polymorphic amino acids, K80 and I103, of mature AVR3a contribute to the effector functions.

  4. Differential Desensitization Observed at Multiple Effectors of Somatic μ-Opioid Receptors Underlies Sustained Agonist-Mediated Inhibition of Proopiomelanocortin Neuron Activity.

    PubMed

    Fox, Philip D; Hentges, Shane T

    2017-09-06

    Activation of somatic μ-opioid receptors (MORs) in hypothalamic proopiomelanocortin (POMC) neurons leads to the activation of G-protein-coupled inward rectifier potassium (GIRK) channels and hyperpolarization, but in response to continued signaling MORs undergo acute desensitization resulting in robust reduction in the peak GIRK current after minutes of agonist exposure. We hypothesized that the attenuation of the GIRK current would lead to a recovery of neuronal excitability whereby desensitization of the receptor would lead to a new steady state of POMC neuron activity reflecting the sustained GIRK current observed after the initial decline from peak with continued agonist exposure. However, electrophysiologic recordings and GCaMP6f Ca(2+) imaging in POMC neurons in mouse brain slices indicate that maximal inhibition of cellular activity by these measures can be maintained after the GIRK current declines. Blockade of the GIRK current by Ba(2+) or Tertiapin-Q did not disrupt the sustained inhibition of Ca(2+) transients in the continued presence of agonist, indicating the activation of an effector other than GIRK channels. Use of an irreversible MOR antagonist and Furchgott analysis revealed a low receptor reserve for the activation of GIRK channels but a >90% receptor reserve for the inhibition of Ca(2+) events. Altogether, the data show that somatodendritic MORs in POMC neurons inhibit neuronal activity through at least two effectors with distinct levels of receptor reserve and that differentially reflect receptor desensitization. Thus, in POMC cells, the decline in the GIRK current during prolonged MOR agonist exposure does not reflect an increase in cellular activity as expected.SIGNIFICANCE STATEMENT Desensitization of the μ-opioid receptor (MOR) is thought to underlie the development of cellular tolerance to opiate therapy. The present studies focused on MOR desensitization in hypothalamic proopiomelanocortin (POMC) neurons as these neurons produce the

  5. The Salmonella Typhimurium effector SteC inhibits Cdc42-mediated signaling through binding to the exchange factor Cdc24 in Saccharomyces cerevisiae

    PubMed Central

    Fernandez-Piñar, Pablo; Alemán, Ainel; Sondek, John; Dohlman, Henrik G.; Molina, María; Martín, Humberto

    2012-01-01

    Intracellular survival of Salmonella relies on the activity of proteins translocated into the host cell by type III secretion systems (T3SS). The protein kinase activity of the T3SS effector SteC is required for F-actin remodeling in host cells, although no SteC target has been identified so far. Here we show that expression of the N-terminal non-kinase domain of SteC down-regulates the mating and HOG pathways in Saccharomyces cerevisiae. Epistasis analyses using constitutively active components of these pathways indicate that SteC inhibits signaling at the level of the GTPase Cdc42. We demonstrate that SteC interacts through its N-terminal domain with the catalytic domain of Cdc24, the sole S. cerevisiae Cdc42 guanine nucleotide exchange factor (GEF). SteC also binds to the human Cdc24-like GEF protein Vav1. Moreover, expression of human Cdc42 suppresses growth inhibition caused by SteC. Of interest, the N-terminal SteC domain alters Cdc24 cellular localization, preventing its nuclear accumulation. These data reveal a novel functional domain within SteC, raising the possibility that this effector could also target GTPase function in mammalian cells. Our results also highlight the key role of the Cdc42 switch in yeast mating and HOG pathways and provide a new tool to study the functional consequences of Cdc24 localization. PMID:23015760

  6. End-effector microprocessor

    NASA Technical Reports Server (NTRS)

    Doggett, William R.

    1992-01-01

    The topics are presented in viewgraph form and include: automated structures assembly facility current control hierarchy; automated structures assembly facility purposed control hierarchy; end-effector software state transition diagram; block diagram for ideal install composite; and conclusions.

  7. Advanced Aerodynamic Control Effectors

    NASA Technical Reports Server (NTRS)

    Wood, Richard M.; Bauer, Steven X. S.

    1999-01-01

    A 1990 research program that focused on the development of advanced aerodynamic control effectors (AACE) for military aircraft has been reviewed and summarized. Data are presented for advanced planform, flow control, and surface contouring technologies. The data show significant increases in lift, reductions in drag, and increased control power, compared to typical aerodynamic designs. The results presented also highlighted the importance of planform selection in the design of a control effector suite. Planform data showed that dramatic increases in lift (greater than 25%) can be achieved with multiple wings and a sawtooth forebody. Passive porosity and micro drag generator control effector data showed control power levels exceeding that available from typical effectors (moving surfaces). Application of an advanced planform to a tailless concept showed benefits of similar magnitude as those observed in the generic studies.

  8. Type I interferons regulate effector and regulatory T cell accumulation and anti-inflammatory cytokine production during T cell-mediated colitis1

    PubMed Central

    Kole, Abhisake; He, JianPing; Rivollier, Aymeric; Silveira, Danielle D.; Kitamura, Kazuya; Maloy, Kevin J.; Kelsall, Brian L.

    2013-01-01

    We explored the function of endogenous type I interferons (IFN-1) in the colon using the T cell adoptive transfer model of colitis. Colon mononuclear phagocytes (MP) constitutively produced IFN-1 in a TRIF-dependent manner. Transfer of CD4+CD45RBhi T cells from wild type (WT) or interferon α/β receptor subunit 1 knockout (IFNAR1−/−) mice into RAG−/− hosts resulted in similar onset and severity of colitis. In contrast, RAG−/− x IFNAR1−/− double knockout (DKO) mice developed accelerated severe colitis compared to RAG−/− hosts when transferred WT CD4+CD45RBhi T cells. IFNAR signaling on host hematopoietic cells was required to delay colitis development. MPs isolated from the colon lamina propria of IFNAR1−/− mice produced less IL-10, IL-1 receptor antagonist (IL-1RA) and IL-27 compared to WT MPs. Accelerated colitis development in DKO mice was characterized by early T cell proliferation and accumulation of CD11b+CD103− dendritic cells in the mesenteric lymph nodes, both of which could be reversed by systemic administration of IL-1RA (anakinra). Co-transfer of CD4+CD25+ regulatory T cells (Tregs) from WT or IFNAR1−/− mice prevented disease caused by CD4+CD45RBhi T cells. However, WT CD4+CD25+Foxp3GFP+ Tregs co-transferred with CD4+CD45RBhi T cells into DKO hosts failed to expand or maintain Foxp3 expression and gained effector functions in the colon. These data are the first to demonstrate an essential role for IFN-1 in the production of anti-inflammatory cytokines by gut MPs and the indirect maintenance of intestinal T cell homeostasis by both limiting effector T cell expansion and promoting Treg stability. PMID:23913971

  9. Type I IFNs regulate effector and regulatory T cell accumulation and anti-inflammatory cytokine production during T cell-mediated colitis.

    PubMed

    Kole, Abhisake; He, JianPing; Rivollier, Aymeric; Silveira, Danielle D; Kitamura, Kazuya; Maloy, Kevin J; Kelsall, Brian L

    2013-09-01

    We explored the function of endogenous type I IFNs (IFN-1) in the colon using the T cell adoptive transfer model of colitis. Colon mononuclear phagocytes (MPs) constitutively produced IFN-1 in a Toll/IL-1R domain-containing adapter-inducing IFN-β-dependent manner. Transfer of CD4(+)CD45RB(hi) T cells from wild-type (WT) or IFN-α/β receptor subunit 1 knockout (IFNAR1(-/-)) mice into RAG(-/-) hosts resulted in similar onset and severity of colitis. In contrast, RAG(-/-) × IFNAR1(-/-) double knockout (DKO) mice developed accelerated severe colitis compared with RAG(-/-) hosts when transferred with WT CD4(+)CD45RB(hi) T cells. IFNAR signaling on host hematopoietic cells was required to delay colitis development. MPs isolated from the colon lamina propria of IFNAR1(-/-) mice produced less IL-10, IL-1R antagonist, and IL-27 compared with WT MPs. Accelerated colitis development in DKO mice was characterized by early T cell proliferation and accumulation of CD11b(+)CD103(-) dendritic cells in the mesenteric lymph nodes, both of which could be reversed by systemic administration of IL-1R antagonist (anakinra). Cotransfer of CD4(+)CD25(+) regulatory T cells (Tregs) from WT or IFNAR1(-/-) mice prevented disease caused by CD4(+)CD45RB(hi) T cells. However, WT CD4(+)CD25(+)Foxp3(GFP+) Tregs cotransferred with CD4(+)CD45RB(hi) T cells into DKO hosts failed to expand or maintain Foxp3 expression and gained effector functions in the colon. To our knowledge, these data are the first to demonstrate an essential role for IFN-1 in the production of anti-inflammatory cytokines by gut MPs and the indirect maintenance of intestinal T cell homeostasis by both limiting effector T cell expansion and promoting Treg stability.

  10. Modification of Bacterial Effector Proteins Inside Eukaryotic Host Cells.

    PubMed

    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.

  11. 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

  12. Effector-Mediated Suppression of Chitin-Triggered Immunity by Magnaporthe oryzae Is Necessary for Rice Blast Disease[C][W

    PubMed Central

    Mentlak, Thomas A.; Kombrink, Anja; Shinya, Tomonori; Ryder, Lauren S.; Otomo, Ippei; Saitoh, Hiromasa; Terauchi, Ryohei; Nishizawa, Yoko; Shibuya, Naoto; Thomma, Bart P.H.J.; Talbot, Nicholas J.

    2012-01-01

    Plants use pattern recognition receptors to defend themselves from microbial pathogens. These receptors recognize pathogen-associated molecular patterns (PAMPs) and activate signaling pathways that lead to immunity. In rice (Oryza sativa), the chitin elicitor binding protein (CEBiP) recognizes chitin oligosaccharides released from the cell walls of fungal pathogens. Here, we show that the rice blast fungus Magnaporthe oryzae overcomes this first line of plant defense by secreting an effector protein, Secreted LysM Protein1 (Slp1), during invasion of new rice cells. We demonstrate that Slp1 accumulates at the interface between the fungal cell wall and the rice plasma membrane, can bind to chitin, and is able to suppress chitin-induced plant immune responses, including generation of reactive oxygen species and plant defense gene expression. Furthermore, we show that Slp1 competes with CEBiP for binding of chitin oligosaccharides. Slp1 is required by M. oryzae for full virulence and exerts a significant effect on tissue invasion and disease lesion expansion. By contrast, gene silencing of CEBiP in rice allows M. oryzae to cause rice blast disease in the absence of Slp1. We propose that Slp1 sequesters chitin oligosaccharides to prevent PAMP-triggered immunity in rice, thereby facilitating rapid spread of the fungus within host tissue. PMID:22267486

  13. The type III effector HsvG of the gall-forming Pantoea agglomerans mediates expression of the host gene HSVGT.

    PubMed

    Nissan, Gal; Manulis-Sasson, Shulamit; Chalupowicz, Laura; Teper, Doron; Yeheskel, Adva; Pasmanik-Chor, Metsada; Sessa, Guido; Barash, Isaac

    2012-02-01

    The type III effector HsvG of the gall-forming Pantoea agglomerans pv. gypsophilae is a DNA-binding protein that is imported to the host nucleus and involved in host specificity. The DNA-binding region of HsvG was delineated to 266 amino acids located within a secondary structure region near the N-terminus of the protein but did not display any homology to canonical DNA-binding motifs. A binding site selection procedure was used to isolate a target gene of HsvG, named HSVGT, in Gypsophila paniculata. HSVGT is a predicted acidic protein of the DnaJ family with 244 amino acids. It harbors characteristic conserved motifs of a eukaryotic transcription factor, including a bipartite nuclear localization signal, zinc finger, and leucine zipper DNA-binding motifs. Quantitative real-time polymerase chain reaction analysis demonstrated that HSVGT transcription is specifically induced in planta within 2 h after inoculation with the wild-type P. agglomerans pv. gypsophilae compared with the hsvG mutant. Induction of HSVGT reached a peak of sixfold at 4 h after inoculation and progressively declined thereafter. Gel-shift assay demonstrated that HsvG binds to the HSVGT promoter, indicating that HSVGT is a direct target of HsvG. Our results support the hypothesis that HsvG functions as a transcription factor in gypsophila.

  14. A Shigella effector dampens inflammation by regulating epithelial release of danger signal ATP through production of the lipid mediator PtdIns5P.

    PubMed

    Puhar, Andrea; Tronchère, Hélène; Payrastre, Bernard; Nhieu, Guy Tran Van; Sansonetti, Philippe J

    2013-12-12

    Upon infection with Shigella flexneri, epithelial cells release ATP through connexin hemichannels. However, the pathophysiological consequence and the regulation of this process are unclear. Here we showed that in intestinal epithelial cell ATP release was an early alert response to infection with enteric pathogens that eventually promoted inflammation of the gut. Shigella evolved to escape this inflammatory reaction by its type III secretion effector IpgD, which blocked hemichannels via the production of the lipid PtdIns5P. Infection with an ipgD mutant resulted in rapid hemichannel-dependent accumulation of extracellular ATP in vitro and in vivo, which preceded the onset of inflammation. At later stages of infection, ipgD-deficient Shigella caused strong intestinal inflammation owing to extracellular ATP. We therefore describe a new paradigm of host-pathogen interaction based on endogenous danger signaling and identify extracellular ATP as key regulator of mucosal inflammation during infection. Our data provide new angles of attack for the development of anti-inflammatory molecules.

  15. NK cells require antigen-specific memory CD4+ T cells to mediate superior effector functions during HSV-2 recall responses in vitro.

    PubMed

    Chen, Branson; Lee, Amanda J; Chew, Marianne V; Ashkar, Ali A

    2016-12-14

    Natural killer (NK) cells have an important role in mounting protective innate responses against genital herpes simplex virus type 2 (HSV-2) infections. However their role as effectors in adaptive immune responses against HSV-2 is unclear. Here, we demonstrate that NK cells from C57BL/6 mice in an ex vivo splenocyte culture produce significantly more interferon γ (IFN-γ) upon re-exposure to HSV-2 antigens in a mouse model of genital HSV-2 immunization. We find that naïve NK cells do not require any prior stimulation or priming to be activated to produce IFN-γ. Our results demonstrate that HSV-2-experienced CD4(+) T cells have a crucial role in coordinating NK cell activation and that their presence during HSV-2 antigen presentation is required to activate NK cells in this model of secondary immune response. We also examined the requirement of cell-to-cell contacts for both CD4(+) T cells and NK cells. NK cells are dependent on direct interactions with other HSV-2-experienced splenocytes, and CD4(+) T cells need to be in close proximity to NK cells to activate them. This study revealed that NK cells do not exhibit any memory toward HSV-2 antigens and, in fact, require specific interactions with HSV-2-experienced CD4(+) T cells to produce IFN-γ.

  16. Exposure to double-stranded RNA mediated by tobacco rattle virus leads to transcription up-regulation of effector gene Mi-vap-2 from Meloidogyne incognita and promotion of pathogenicity in progeny.

    PubMed

    Chi, Yuankai; Wang, Xuan; Le, Xiuhu; Ju, Yuliang; Guan, Tinglong; Li, Hongmei

    2016-02-01

    Meloidogyne spp. are economically important plant parasites and cause enormous damage to agriculture world-wide. These nematodes use secreted effectors which modify host cells, allowing them to obtain the nutrients required for growth and development. A better understanding of the roles of effectors in nematode parasitism is critical for understanding the mechanisms of nematode-host interactions. In this study, Mi-vap-2 of Meloidogyne incognita, a gene encoding a venom allergen-like protein, was targeted by RNA interference mediated by the tobacco rattle virus. Unexpectedly, compared with a wild type line, a substantial up-regulation of Mi-vap-2 transcript was observed in juveniles collected at 7 days p.i. from Nicotiana benthamiana agroinfiltrated with TRV::vap-2. This up-regulation of the targeted transcript did not impact development of females or the production of galls, nor the number of females on the TRV::vap-2 line. In a positive control line, the transcript of Mi16D10 was knocked down in juveniles from the TRV::16D10 line at 7 days p.i., resulting in a significant inhibition of nematode development. The up-regulation of Mi-vap-2 triggered by TRV-RNAi was inherited by the progeny of the nematodes exposed to double-stranded RNA. Meanwhile, a substantial increase in Mi-VAP-2 expression in those juvenile progeny was revealed by ELISA. This caused an increase in the number of galls (71.2%) and females (84.6%) produced on seedlings of N. benthamiana compared with the numbers produced by control nematodes. Up-regulation of Mi-vap-2 and its encoded protein therefore enhanced pathogenicity of the nematodes, suggesting that Mi-vap-2 may be required for successful parasitism during the early parasitic stage of M. incognita.

  17. Transcription activator-like effector nuclease (TALEN)-mediated CLYBL targeting enables enhanced transgene expression and one-step generation of dual reporter human induced pluripotent stem cell (iPSC) and neural stem cell (NSC) lines.

    PubMed

    Cerbini, Trevor; Funahashi, Ray; Luo, Yongquan; Liu, Chengyu; Park, Kyeyoon; Rao, Mahendra; Malik, Nasir; Zou, Jizhong

    2015-01-01

    Targeted genome engineering to robustly express transgenes is an essential methodology for stem cell-based research and therapy. Although designer nucleases have been used to drastically enhance gene editing efficiency, targeted addition and stable expression of transgenes to date is limited at single gene/locus and mostly PPP1R12C/AAVS1 in human stem cells. Here we constructed transcription activator-like effector nucleases (TALENs) targeting the safe-harbor like gene CLYBL to mediate reporter gene integration at 38%-58% efficiency, and used both AAVS1-TALENs and CLYBL-TALENs to simultaneously knock-in multiple reporter genes at dual safe-harbor loci in human induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs). The CLYBL-TALEN engineered cell lines maintained robust reporter expression during self-renewal and differentiation, and revealed that CLYBL targeting resulted in stronger transgene expression and less perturbation on local gene expression than PPP1R12C/AAVS1. TALEN-mediated CLYBL engineering provides improved transgene expression and options for multiple genetic modification in human stem cells.

  18. Pneumatic inflatable end effector

    NASA Technical Reports Server (NTRS)

    Clark, K. H.; Johnston, J. D. (Inventor)

    1981-01-01

    The invention relates to an end effector device for robot or teleoperated type space vehicle which includes an inflatable balloon member carried on the end of tubular member which has a hollow center or conduit through which a suitable pressurized fluid is supplied. The device may be inserted into a variety of shaped openings or truss-type structures for handling in space.

  19. The Smac Mimetic BV6 Improves NK Cell-Mediated Killing of Rhabdomyosarcoma Cells by Simultaneously Targeting Tumor and Effector Cells.

    PubMed

    Fischer, Kyra; Tognarelli, Sara; Roesler, Stefanie; Boedicker, Cathinka; Schubert, Ralf; Steinle, Alexander; Klingebiel, Thomas; Bader, Peter; Fulda, Simone; Ullrich, Evelyn

    2017-01-01

    Rhabdomyosarcoma (RMS), the most common cancer of connective tissues in pediatrics, is often resistant to conventional therapies. One underlying mechanism of this resistance is the overexpression of Inhibitor of Apoptosis (IAP) proteins, leading to a dysfunctional cell death program within tumor cells. Smac mimetics (SM) are small molecules that can reactivate the cell death program by antagonizing IAP proteins and thereby compensating their overexpression. Here, we report that SM sensitize two RMS cell lines (RD and RH30) toward natural killer (NK) cell-mediated killing on the one hand, and increase the cytotoxic potential of NK cells on the other. The SM-induced sensitization of RH30 cells toward NK cell-mediated killing is significantly reduced through blocking tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on NK cells prior to coculture. In addition, the presence of zVAD.fmk, a pancaspase inhibitor, rescues tumor cells from the increase in killing, indicating an apoptosis-dependent cell death. On the NK cell side, the presence of SM in addition to IL-2 during the ex vivo expansion leads to an increase in their cytotoxic activity against RH30 cells. This effect is mainly TNFα-dependent and partially mediated by NK cell activation, which is associated with transcriptional upregulation of NF-κB target genes such as IκBα and RelB. Taken together, our findings implicate that SM represent a novel double-hit strategy, sensitizing tumor and activating NK cells with one single drug.

  20. The Smac Mimetic BV6 Improves NK Cell-Mediated Killing of Rhabdomyosarcoma Cells by Simultaneously Targeting Tumor and Effector Cells

    PubMed Central

    Fischer, Kyra; Tognarelli, Sara; Roesler, Stefanie; Boedicker, Cathinka; Schubert, Ralf; Steinle, Alexander; Klingebiel, Thomas; Bader, Peter; Fulda, Simone; Ullrich, Evelyn

    2017-01-01

    Rhabdomyosarcoma (RMS), the most common cancer of connective tissues in pediatrics, is often resistant to conventional therapies. One underlying mechanism of this resistance is the overexpression of Inhibitor of Apoptosis (IAP) proteins, leading to a dysfunctional cell death program within tumor cells. Smac mimetics (SM) are small molecules that can reactivate the cell death program by antagonizing IAP proteins and thereby compensating their overexpression. Here, we report that SM sensitize two RMS cell lines (RD and RH30) toward natural killer (NK) cell-mediated killing on the one hand, and increase the cytotoxic potential of NK cells on the other. The SM-induced sensitization of RH30 cells toward NK cell-mediated killing is significantly reduced through blocking tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on NK cells prior to coculture. In addition, the presence of zVAD.fmk, a pancaspase inhibitor, rescues tumor cells from the increase in killing, indicating an apoptosis-dependent cell death. On the NK cell side, the presence of SM in addition to IL-2 during the ex vivo expansion leads to an increase in their cytotoxic activity against RH30 cells. This effect is mainly TNFα-dependent and partially mediated by NK cell activation, which is associated with transcriptional upregulation of NF-κB target genes such as IκBα and RelB. Taken together, our findings implicate that SM represent a novel double-hit strategy, sensitizing tumor and activating NK cells with one single drug. PMID:28326081

  1. A STING-dependent innate sensing pathway mediates resistance to corneal HSV-1 infection via upregulation of the antiviral effector tetherin

    PubMed Central

    Royer, D.J.; Carr, D.J.J.

    2015-01-01

    Type 1 interferons (IFNα/β) mediate immunologic host resistance to numerous viral infections including herpes simplex virus type 1 (HSV-1). The pathways responsible for IFNα/β signaling during the innate immune response to acute HSV-1 infection in the cornea are incompletely understood. Using a murine ocular infection model, we hypothesized that the stimulator of IFN genes (STING) mediates resistance to HSV-1 infection at the ocular surface and preserves the structural integrity of this mucosal site. Viral pathogenesis, tissue pathology, and host immune responses during ocular HSV-1 infection were characterized by plaque assay, esthesiometry, pachymetry, immunohistochemistry, flow cytometry, and siRNA transfection in wildtype C57BL/6 (WT), STING-deficient (STING−/−), and IFNα/β receptor-deficient (CD118−/−) mice at days 3–5 post infection. The presence of STING was critical for sustained control of HSV-1 replication in the corneal epithelium and neuroinvasion, but loss of STING had a negligible impact with respect to gross tissue pathology. Auxiliary STING-independent IFNα/β signaling pathways were responsible for maintenance of the corneal integrity. Lymphatic vessels, mast cells, and sensory innervation were compromised in CD118−/− mice concurrent with increased tissue edema. STING-dependent signaling led to the upregulation of tetherin, a viral restriction factor we identify is important in containing the spread of HSV-1 in vivo. PMID:26627457

  2. Vinculin Interacts with the Chlamydia Effector TarP Via a Tripartite Vinculin Binding Domain to Mediate Actin Recruitment and Assembly at the Plasma Membrane.

    PubMed

    Thwaites, Tristan R; Pedrosa, Antonio T; Peacock, Thomas P; Carabeo, Rey A

    2015-01-01

    The mammalian protein vinculin is often a target of bacterial pathogens to subvert locally host cell actin dynamics. In Chlamydia infection, vinculin has been implicated in RNA interference screens, but the molecular basis for vinculin requirement has not been characterized. In this report, we show that vinculin was involved in the actin recruitment and F-actin assembly at the plasma membrane to facilitate invasion. Vinculin was recruited to the plasma membrane via its interaction with a specific tripartite motif within TarP that resembles the vinculin-binding domain (VBD) found in the Shigella invasion factor IpaA. The TarP-mediated plasma membrane recruitment of vinculin resulted in the localized recruitment of actin. In vitro pulldown assays for protein-protein interaction and imaging-based evaluation of recruitment to the plasma membrane demonstrated the essential role of the vinculin-binding site 1 (VBS1), and the dispensability of VBS2 and VBS3. As further support for the functionality of VBD-vinculin interaction, VBD-mediated actin recruitment required vinculin. Interestingly, while both vinculin and the focal adhesion kinase (FAK) colocalized at the sites of adhesion, the recruitment of one was independent of the other; and the actin recruitment function of the VBD/vinculin signaling axis was independent of the LD/FAK pathway.

  3. Robotic end effector

    DOEpatents

    Minichan, Richard L.

    1993-01-01

    An end effector for use in probing a surface with a robotic arm. The end effector has a first portion that carries a gimbal with a probe, the gimbal holding the probe normal to the surface, and a second portion with a set of three shafts within a housing for urging the gimbal and probe against the surface. The second portion contains a potentiometer connected by another shaft to the first portion to measure the position of the first portion with respect to the second so that the second portion can be moved to place and maintain the shafts at the midpoint of their travel. Then, as irregularities in the surface are encountered, the first portion can respond by moving closer to or farther from the second portion.

  4. Robotic end effector

    DOEpatents

    Minichan, R.L.

    1993-10-05

    An end effector is described for use in probing a surface with a robotic arm. The end effector has a first portion that carries a gimbal with a probe, the gimbal holding the probe normal to the surface, and a second portion with a set of three shafts within a housing for urging the gimbal and probe against the surface. The second portion contains a potentiometer connected by another shaft to the first portion to measure the position of the first portion with respect to the second so that the second portion can be moved to place and maintain the shafts at the midpoint of their travel. Then, as irregularities in the surface are encountered, the first portion can respond by moving closer to or farther from the second portion. 7 figures.

  5. Robotic end effector

    SciTech Connect

    Minichan, R.L.

    1991-12-31

    This invention is comprised of an end effector for use in probing a surface with a robotic arm. The end effector has a first portion that carries a gamble with a probe, the gamble holding the probe normal to the surface, and a second portion with a set of three shafts within a housing for urging the gamble and probe against the surface. The second portion contains a potentiometer connected by another shaft to the first portion to measure the position of the first portion with respect to the second so that the second portion can be moved to place and maintain the shafts at the midpoint of their travel. Then, as irregularities in the surface are encountered, the first portion can respond by moving closer to or farther from the second portion.

  6. TCRVα usage by effector CD4+Vβ11+ T cells mediating graft-versus-host disease directed to minor histocompatibility antigens

    PubMed Central

    DiRienzo, Christine G.; Murphy, George F.; Friedman, Thea M.; Korngold, Robert

    2007-01-01

    T cell receptor (TCR) Vα (TRAV) and Vβ (TRBV) chains provide the T cell specificity for recognition of major histocompatibility complex (MHC)-bound antigens. However, there is limited information on the diversity of TRAV usage within an antigen response. Previous investigation of CD4+ T cell-mediated graft-versus-host disease (GVHD) in the minor histocompatibility antigen (miHA)-mismatched C57BL/6 (B6) -> BALB.B irradiated murine model determined that Vβ11+ T cells were involved in the severity of disease. Polymerase chain reaction (PCR)-based complementarity determining region-3 (CDR3)-size spectratype analysis of B6 Vβ11+ T cells from the spleens of recipient BALB.B mice undergoing GVHD indicated biased usage within the Vα6, 9, 13, 14, 18, and 22 families. In order to probe deeper into this limited Vα response, the current study was undertaken to further define TRAV-Jα (TRAJ) nucleotide sequences found in host-presensitized B6 Vβ11+ T cells proliferating in response to in vitro stimulation with BALB.B splenocytes. Using the nonpalindromic adaptor-PCR method, we found dominant usage of the TRAV13-TRAJ16 transcript combination. Then, using laser capture microdissection (LMD), we found use of the identical TRAV-TRAJ nucleotide sequence in areas dominated by infiltrating Vβ11+ CD4+ T cells during development of GVHD in both the rete-like prominences of the dorsal lingual epithelium and the ileal crypts of the small intestine. PMID:17317580

  7. T-cell receptor V(alpha) usage by effector CD4+Vbeta11+ T cells mediating graft-versus-host disease directed to minor histocompatibility antigens.

    PubMed

    DiRienzo, Christine G; Murphy, George F; Friedman, Thea M; Korngold, Robert

    2007-03-01

    T-cell receptor (TCR) Valpha (TRAV) and Vbeta (TRBV) chains provide the T-cell specificity for recognition of major histocompatibility complex (MHC)-bound antigens. However, there is limited information on the diversity of TRAV use within an antigen response. Previous investigation of CD4(+) T-cell-mediated graft-versus-host disease (GVHD) in the minor histocompatibility antigen-mismatched C57BL/6 (B6)-->BALB.B irradiated murine model determined that Vbeta11(+) T cells were associated with disease severity. Polymerase chain reaction (PCR)-based complementarity-determining region 3 (CDR3)-sized spectratype analysis of B6 Vbeta11(+) T cells from the spleens of recipient BALB.B mice undergoing GVHD indicated biased use within the V(alpha)6, 9, 13, 14, 18, and 22 families. To probe deeper into this limited V(alpha) response, the current study was undertaken to further define TRAV-Jalpha (TRAJ) nucleotide sequences found in host-presensitized B6 Vbeta11(+) T cells proliferating in response to in vitro stimulation with BALB.B splenocytes. Using the nonpalindromic adaptor PCR method, we found dominant use of the TRAV13-TRAJ16 transcript combination. Then, using laser capture microdissection, we found use of the identical TRAV-TRAJ nucleotide sequence in areas dominated by infiltrating Vbeta11(+) CD4(+) T cells during the development of GVHD in both the rete-like prominences of the dorsal lingual epithelium and the ileal crypts of the small intestine.

  8. The aspartyl protease TgASP5 mediates the export of the Toxoplasma GRA16 and GRA24 effectors into host cells.

    PubMed

    Curt-Varesano, Aurélie; Braun, Laurence; Ranquet, Caroline; Hakimi, Mohamed-Ali; Bougdour, Alexandre

    2016-02-01

    Toxoplasma gondii and Plasmodium species are obligatory intracellular parasites that export proteins into the infected cells in order to interfere with host-signalling pathways, acquire nutrients or evade host defense mechanisms. With regard to export mechanism, a wealth of information in Plasmodium spp. is available, while the mechanisms operating in T. gondii remain uncertain. The recent discovery of exported proteins in T. gondii, mainly represented by dense granule resident proteins, might explain this discrepancy and offers a unique opportunity to study the export mechanism in T. gondii. Here, we report that GRA16 export is mediated by two protein elements present in its N-terminal region. Because the first element contains a putative Plasmodium export element linear motif (RRLAE), we hypothesized that GRA16 export depended on a maturation process involving protein cleavage. Using both N- and C-terminal epitope tags, we provide evidence for protein proteolysis occurring in the N-terminus of GRA16. We show that TgASP5, the T. gondii homolog of Plasmodium plasmepsin V, is essential for GRA16 export and is directly responsible for its maturation in a Plasmodium export element-dependent manner. Interestingly, TgASP5 is also involved in GRA24 export, although the GRA24 maturation mechanism is TgASP5-independent. Our data reveal different modus operandi for protein export, in which TgASP5 should play multiple functions.

  9. V-ATPase: a master effector of E2F1-mediated lysosomal trafficking, mTORC1 activation and autophagy.

    PubMed

    Meo-Evoli, Nathalie; Almacellas, Eugènia; Massucci, Francesco Alessandro; Gentilella, Antonio; Ambrosio, Santiago; Kozma, Sara C; Thomas, George; Tauler, Albert

    2015-09-29

    In addition to being a master regulator of cell cycle progression, E2F1 regulates other associated biological processes, including growth and malignancy. Here, we uncover a regulatory network linking E2F1 to lysosomal trafficking and mTORC1 signaling that involves v-ATPase regulation. By immunofluorescence and time-lapse microscopy we found that E2F1 induces the movement of lysosomes to the cell periphery, and that this process is essential for E2F1-induced mTORC1 activation and repression of autophagy. Gain- and loss-of-function experiments reveal that E2F1 regulates v-ATPase activity and inhibition of v-ATPase activity repressed E2F1-induced lysosomal trafficking and mTORC1 activation. Immunoprecipitation experiments demonstrate that E2F1 induces the recruitment of v-ATPase to lysosomal RagB GTPase, suggesting that E2F1 regulates v-ATPase activity by enhancing the association of V0 and V1 v-ATPase complex. Analysis of v-ATPase subunit expression identified B subunit of V0 complex, ATP6V0B, as a transcriptional target of E2F1. Importantly, ATP6V0B ectopic-expression increased v-ATPase and mTORC1 activity, consistent with ATP6V0B being responsible for mediating the effects of E2F1 on both responses. Our findings on lysosomal trafficking, mTORC1 activation and autophagy suppression suggest that pharmacological intervention at the level of v-ATPase may be an efficacious avenue for the treatment of metastatic processes in tumors overexpressing E2F1.

  10. V-ATPase: a master effector of E2F1-mediated lysosomal trafficking, mTORC1 activation and autophagy

    PubMed Central

    Meo-Evoli, Nathalie; Almacellas, Eugènia; Massucci, Francesco Alessandro; Gentilella, Antonio; Ambrosio, Santiago; Kozma, Sara C.; Thomas, George; Tauler, Albert

    2015-01-01

    In addition to being a master regulator of cell cycle progression, E2F1 regulates other associated biological processes, including growth and malignancy. Here, we uncover a regulatory network linking E2F1 to lysosomal trafficking and mTORC1 signaling that involves v-ATPase regulation. By immunofluorescence and time-lapse microscopy we found that E2F1 induces the movement of lysosomes to the cell periphery, and that this process is essential for E2F1-induced mTORC1 activation and repression of autophagy. Gain- and loss-of-function experiments reveal that E2F1 regulates v-ATPase activity and inhibition of v-ATPase activity repressed E2F1-induced lysosomal trafficking and mTORC1 activation. Immunoprecipitation experiments demonstrate that E2F1 induces the recruitment of v-ATPase to lysosomal RagB GTPase, suggesting that E2F1 regulates v-ATPase activity by enhancing the association of V0 and V1 v-ATPase complex. Analysis of v-ATPase subunit expression identified B subunit of V0 complex, ATP6V0B, as a transcriptional target of E2F1. Importantly, ATP6V0B ectopic-expression increased v-ATPase and mTORC1 activity, consistent with ATP6V0B being responsible for mediating the effects of E2F1 on both responses. Our findings on lysosomal trafficking, mTORC1 activation and autophagy suppression suggest that pharmacological intervention at the level of v-ATPase may be an efficacious avenue for the treatment of metastatic processes in tumors overexpressing E2F1. PMID:26356814

  11. Two-axis angular effector

    DOEpatents

    Vaughn, Mark R.; Robinett, III, Rush D.; Phelan, John R.; Van Zuiden, Don M.

    1997-01-21

    A new class of coplanar two-axis angular effectors. These effectors combine a two-axis rotational joint analogous to a Cardan joint with linear actuators in a manner to produce a wider range of rotational motion about both axes defined by the joint. This new class of effectors also allows design of robotic manipulators having very high strength and efficiency. These effectors are particularly suited for remote operation in unknown surroundings, because of their extraordinary versatility. An immediate application is to the problems which arise in nuclear waste remediation.

  12. Cell-mediated lympholysis of trinitrophenyl-modified autologous lymphocytes. Effector cell specificity to modified cell surface components controlled by H-2K and H-2D serological regions of the murine major histocompatibility complex

    PubMed Central

    1975-01-01

    Splenic lymphocytes from four C57BL/10 congenic resistant mouse strains were sensitized in vitro with trinitrophenyl (TNP)-modified autologous spleen cellsmthe effector cells generated were incubated with 51-Cr- labeled unmodified or TNP-modified spleen or tumor target cells, and the percentage of specific lympholysis determined. The results obtained using syngeneic-, congenic-, recombinante, and allogeneic-modified target cells indicated that TNP modification of the target cells was a necessary but insufficient requirement for lympholysis. Intra-H-2 homology either between modified stimulating cells and modified target cells or between responding lymphocytes and modified target cells was also important in the specificity for lysis. Homology at the K serological region or at K plus I-A in the B10.A and B10BR strains, and at either the D serological region or at some other region (possibly K) in the B10.D2 and C57BL/10 strains were shown to be necessary in order to detect lympholysis. Experiments using (B10itimes C57BL/10)F1 responding lymphocytes sensitized and assayed with TNP-modified parental cells indicated that the homology required for lympholysis was between modified stimulating and modified target cellsmthe possibility is raised that histocompatibility antigens may serve in the autologous system as cell surface components which are modified by viruses or autoimmune complexes to form cell-bound modified-self antigens, which are particularly suited for cell-mediated immune reactions. Evidence is presented suggesting that H-2-linked Ir genes are expressed in the TNP- modified autologous cytotoxic system. These findings imply that the major histocompatibility complex can be functionally involved both in the response potential to and in the formation of new antigenic determinants involving modified-self components. PMID:47900

  13. Effector Mechanisms of Rejection

    PubMed Central

    Moreau, Aurélie; Varey, Emilie; Anegon, Ignacio; Cuturi, Maria-Cristina

    2013-01-01

    Organ transplantation appears today to be the best alternative to replace the loss of vital organs induced by various diseases. Transplants can, however, also be rejected by the recipient. In this review, we provide an overview of the mechanisms and the cells/molecules involved in acute and chronic rejections. T cells and B cells mainly control the antigen-specific rejection and act either as effector, regulatory, or memory cells. On the other hand, nonspecific cells such as endothelial cells, NK cells, macrophages, or polymorphonuclear cells are also crucial actors of transplant rejection. Last, beyond cells, the high contribution of antibodies, chemokines, and complement molecules in graft rejection is discussed in this article. The understanding of the different components involved in graft rejection is essential as some of them are used in the clinic as biomarkers to detect and quantify the level of rejection. PMID:24186491

  14. Metal ions as enzyme effectors

    NASA Astrophysics Data System (ADS)

    Medyantseva, El'vina P.; Vertlib, Margarita G.; Budnikov, German K.

    1998-03-01

    The role of metal ions as enzyme effectors is considered. Data on inhibitory and activating effects of metal ions are summarised. The dual character of action of the effectors depending on their concentration and the nature of the enzyme is highlighted. The analytical applications of these effects are discussed. The bibliography includes 66 references.

  15. Avian Interferons and Their Antiviral Effectors

    PubMed Central

    Santhakumar, Diwakar; Rubbenstroth, Dennis; Martinez-Sobrido, Luis; Munir, Muhammad

    2017-01-01

    Interferon (IFN) responses, mediated by a myriad of IFN-stimulated genes (ISGs), are the most profound innate immune responses against viruses. Cumulatively, these IFN effectors establish a multilayered antiviral state to safeguard the host against invading viral pathogens. Considerable genetic and functional characterizations of mammalian IFNs and their effectors have been made, and our understanding on the avian IFNs has started to expand. Similar to mammalian counterparts, three types of IFNs have been genetically characterized in most avian species with available annotated genomes. Intriguingly, chickens are capable of mounting potent innate immune responses upon various stimuli in the absence of essential components of IFN pathways including retinoic acid-inducible gene I, IFN regulatory factor 3 (IRF3), and possibility IRF9. Understanding these unique properties of the chicken IFN system would propose valuable targets for the development of potential therapeutics for a broader range of viruses of both veterinary and zoonotic importance. This review outlines recent developments in the roles of avian IFNs and ISGs against viruses and highlights important areas of research toward our understanding of the antiviral functions of IFN effectors against viral infections in birds. PMID:28197148

  16. Avian Interferons and Their Antiviral Effectors.

    PubMed

    Santhakumar, Diwakar; Rubbenstroth, Dennis; Martinez-Sobrido, Luis; Munir, Muhammad

    2017-01-01

    Interferon (IFN) responses, mediated by a myriad of IFN-stimulated genes (ISGs), are the most profound innate immune responses against viruses. Cumulatively, these IFN effectors establish a multilayered antiviral state to safeguard the host against invading viral pathogens. Considerable genetic and functional characterizations of mammalian IFNs and their effectors have been made, and our understanding on the avian IFNs has started to expand. Similar to mammalian counterparts, three types of IFNs have been genetically characterized in most avian species with available annotated genomes. Intriguingly, chickens are capable of mounting potent innate immune responses upon various stimuli in the absence of essential components of IFN pathways including retinoic acid-inducible gene I, IFN regulatory factor 3 (IRF3), and possibility IRF9. Understanding these unique properties of the chicken IFN system would propose valuable targets for the development of potential therapeutics for a broader range of viruses of both veterinary and zoonotic importance. This review outlines recent developments in the roles of avian IFNs and ISGs against viruses and highlights important areas of research toward our understanding of the antiviral functions of IFN effectors against viral infections in birds.

  17. SNARC Effect in Different Effectors.

    PubMed

    Hesse, Philipp N; Fiehler, Katja; Bremmer, Frank

    2016-01-01

    The SNARC (spatial numerical association of response codes) effect, indicating that subjects react faster to the left for small numbers and to the right for large numbers, is used as evidence for the idea that humans use space to organize number representations. Previous studies compared the SNARC effect across sensory modalities within participants and concluded modality independence. So far, it is unknown what sensory-to-motor mappings are involved in generating the SNARC effect and whether these mappings are identical for different effectors within subjects. Hence, we tested whether the SNARC effect is effector specific. Participants performed an auditory parity judgment task and responded with three different effectors: finger (button release), eyes (saccades), and arm (pointing). The SNARC effect occurred in each effector but varied in strength across the effectors. Across subjects, we found a significant correlation of SNARC strength for finger and arm responses suggesting the use of a shared sensory-to-motor mapping. SNARC strength did not correlate, however, between finger and eyes or arm and eyes. An additional statistical analysis based on conditional probabilities provided further evidence for SNARC-effector specificity. Taken together, our results suggest that the sensory-to-motor mapping is not as tight as it would be expected if the SNARC effect was effector independent.

  18. Orbital maneuvering end effectors

    NASA Technical Reports Server (NTRS)

    Myers, W. Neill (Inventor); Forbes, John C. (Inventor); Barnes, Wayne L. (Inventor)

    1986-01-01

    This invention relates to an end effector device for grasping and maneuvering objects such as berthing handles of a space telescope. The device includes a V-shaped capture window defined as inclined surfaces in parallel face plates which converge toward a retainer recess in which the handle is retained. A pivotal finger (30) meshes with a pair of pivoted fingers which rotate in counterrotation. The fingers rotate to pull a handle within the capture window into recess where latches lock handle in the recess. To align the capture window, plates may be cocked plus or minus five degrees on base. Drive means is included in the form of a motor coupled with a harmonic drive speed reducer, which provides for slow movement of the fingers at a high torque so that large articles may be handled. Novelty of the invention is believed to reside in the combined intermeshing finger structure, drive means and the harmonic drive speed reducer, which features provide the required maneuverability and strength.

  19. Effector-triggered defence against apoplastic fungal pathogens

    PubMed Central

    Stotz, Henrik U.; Mitrousia, Georgia K.; de Wit, Pierre J.G.M.; Fitt, Bruce D.L.

    2014-01-01

    R gene-mediated host resistance against apoplastic fungal pathogens is not adequately explained by the terms pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) or effector-triggered immunity (ETI). Therefore, it is proposed that this type of resistance is termed ‘effector-triggered defence’ (ETD). Unlike PTI and ETI, ETD is mediated by R genes encoding cell surface-localised receptor-like proteins (RLPs) that engage the receptor-like kinase SOBIR1. In contrast to this extracellular recognition, ETI is initiated by intracellular detection of pathogen effectors. ETI is usually associated with fast, hypersensitive host cell death, whereas ETD often triggers host cell death only after an elapsed period of endophytic pathogen growth. In this opinion, we focus on ETD responses against foliar fungal pathogens of crops. PMID:24856287

  20. Sequential Delivery of Host-Induced Virulence Effectors by Appressoria and Intracellular Hyphae of the Phytopathogen Colletotrichum higginsianum

    PubMed Central

    Kleemann, Jochen; Neumann, Ulla; van Themaat, Emiel Ver Loren; van der Does, H. Charlotte; Hacquard, Stéphane; Stüber, Kurt; Will, Isa; Schmalenbach, Wolfgang; Schmelzer, Elmon; O'Connell, Richard J.

    2012-01-01

    Phytopathogens secrete effector proteins to manipulate their hosts for effective colonization. Hemibiotrophic fungi must maintain host viability during initial biotrophic growth and elicit host death for subsequent necrotrophic growth. To identify effectors mediating these opposing processes, we deeply sequenced the transcriptome of Colletotrichum higginsianum infecting Arabidopsis. Most effector genes are host-induced and expressed in consecutive waves associated with pathogenic transitions, indicating distinct effector suites are deployed at each stage. Using fluorescent protein tagging and transmission electron microscopy-immunogold labelling, we found effectors localised to stage-specific compartments at the host-pathogen interface. In particular, we show effectors are focally secreted from appressorial penetration pores before host invasion, revealing new levels of functional complexity for this fungal organ. Furthermore, we demonstrate that antagonistic effectors either induce or suppress plant cell death. Based on these results we conclude that hemibiotrophy in Colletotrichum is orchestrated through the coordinated expression of antagonistic effectors supporting either cell viability or cell death. PMID:22496661

  1. Filamentous pathogen effectors interfering with small RNA silencing in plant hosts.

    PubMed

    Ye, Wenwu; Ma, Wenbo

    2016-08-01

    Filamentous eukaryotic pathogens including fungi and oomycetes are major threats of plant health. During the co-evolutionary arms race with the hosts, these pathogens have evolved a large repertoire of secreted virulence proteins, called effectors, to facilitate colonization and infection. Many effectors are believed to directly manipulate targeted processes inside the host cells; and a fundamental function of the effectors is to dampen immunity. Recent evidence suggests that the destructive oomycete pathogens in the genus Phytophthora encode RNA silencing suppressors. These effectors play an important virulence role during infection, likely through their inhibitory effect on host small RNA-mediated defense.

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

    PubMed

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

    2013-01-01

    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.

  3. 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

  4. MIX and match: mobile T6SS MIX-effectors enhance bacterial fitness.

    PubMed

    Salomon, Dor

    2016-01-01

    Protein secretion systems that mediate interbacterial competition secret a wide repertoire of antibacterial toxins. A major player in these competitions is the newly discovered bacterial type VI secretion system (T6SS). We recently found that a subset of polymorphic MIX-effectors, which are a widespread class of effectors secreted by T6SSs, are horizontally shared between marine bacteria and are used to diversify their T6SS effector repertoires, thus enhancing their environmental fitness. In this commentary, I expand on the ideas that were introduced in the previous report, and further speculate on the possible mobility of other MIX-effectors. In addition, I discuss the possible role of horizontal gene transfer in the dissemination of MIX-effectors through bacterial genomes, as well as its possible role in diversifying the T6SS effector repertoire.

  5. Lifestyles of the effector-rich: genome-enabled characterization of bacterial plant pathogens

    USDA-ARS?s Scientific Manuscript database

    Genome sequencing of bacterial plant pathogens is providing transformative insights into the complex network of molecular plant-microbe interactions mediated by extracellular effectors during pathogenesis. Bacterial pathogens sequenced to completion are phylogenetically diverse and vary significant...

  6. Effector and suppressor T cells in celiac disease.

    PubMed

    Mazzarella, Giuseppe

    2015-06-28

    Celiac disease (CD) is a T-cell mediated immune disease in which gliadin-derived peptides activate lamina propria effector CD4+ T cells. This activation leads to the release of cytokines, compatible with a Th1-like pattern, which play a crucial role in the pathogenesis of CD, controlling many aspects of the inflammatory immune response. Recent studies have shown that a novel subset of effector T cells, characterized by expression of high levels of IL-17A, termed Th17 cells, plays a pathogenic role in CD. While these effector T cell subsets produce proinflammatory cytokines, which cause substantial tissue injury in vivo in CD, recent studies have suggested the existence of additional CD4(+) T cell subsets with suppressor functions. These subsets include type 1 regulatory T cells and CD25(+)CD4(+) regulatory T cells, expressing the master transcription factor Foxp3, which have important implications for disease progression.

  7. Entry of oomycete and fungal effectors into plant and animal host cells.

    PubMed

    Kale, Shiv D; Tyler, Brett M

    2011-12-01

    Fungal and oomycete pathogens cause many destructive diseases of plants and important diseases of humans and other animals. Fungal and oomycete plant pathogens secrete numerous effector proteins that can enter inside host cells to condition susceptibility. Until recently it has been unknown if these effectors enter via pathogen-encoded translocons or via pathogen-independent mechanisms. Here we review recent evidence that many fungal and oomycete effectors enter via receptor-mediated endocytosis, and can do so in the absence of the pathogen. Surprisingly, a large number of these effectors utilize cell surface phosphatidyinositol-3-phosphate (PI-3-P) as a receptor, a molecule previously known only inside cells. Binding of effectors to PI-3-P appears to be mediated by the cell entry motif RXLR in oomycetes, and by diverse RXLR-like variants in fungi. PI-3-P appears to be present on the surface of animal cells also, suggesting that it may mediate entry of effectors of fungal and oomycete animal pathogens, for example, RXLR effectors found in the oomycete fish pathogen, Saprolegnia parasitica. Reagents that can block PI-3-P-mediated entry have been identified, suggesting new therapeutic strategies.

  8. Structural Analysis of Iac Repressor Bound to Allosteric Effectors

    SciTech Connect

    Daber,R.; Stayrook, S.; Rosenberg, A.; Lewis, M.

    2007-01-01

    The lac operon is a model system for understanding how effector molecules regulate transcription and are necessary for allosteric transitions. The crystal structures of the lac repressor bound to inducer and anti-inducer molecules provide a model for how these small molecules can modulate repressor function. The structures of the apo repressor and the repressor bound to effector molecules are compared in atomic detail. All effectors examined here bind to the repressor in the same location and are anchored to the repressor through hydrogen bonds to several hydroxyl groups of the sugar ring. Inducer molecules form a more extensive hydrogen-bonding network compared to anti-inducers and neutral effector molecules. The structures of these effector molecules suggest that the O6 hydroxyl on the galactoside is essential for establishing a water-mediated hydrogen bonding network that bridges the N-terminal and C-terminal sub-domains. The altered hydrogen bonding can account in part for the different structural conformations of the repressor, and is vital for the allosteric transition.

  9. Oomycetes, effectors, and all that jazz.

    PubMed

    Bozkurt, Tolga O; Schornack, Sebastian; Banfield, Mark J; Kamoun, Sophien

    2012-08-01

    Plant pathogenic oomycetes secrete a diverse repertoire of effector proteins that modulate host innate immunity and enable parasitic infection. Understanding how effectors evolve, translocate and traffic inside host cells, and perturb host processes are major themes in the study of oomycete-plant interactions. The last year has seen important progress in the study of oomycete effectors with, notably, the elucidation of the 3D structures of five RXLR effectors, and novel insights into how cytoplasmic effectors subvert host cells. In this review, we discuss these and other recent advances and highlight the most important open questions in oomycete effector biology.

  10. Effector proteins of rust fungi

    PubMed Central

    Petre, Benjamin; Joly, David L.; Duplessis, Sébastien

    2014-01-01

    Rust fungi include many species that are devastating crop pathogens. To develop resistant plants, a better understanding of rust virulence factors, or effector proteins, is needed. Thus far, only six rust effector proteins have been described: AvrP123, AvrP4, AvrL567, AvrM, RTP1, and PGTAUSPE-10-1. Although some are well established model proteins used to investigate mechanisms of immune receptor activation (avirulence activities) or entry into plant cells, how they work inside host tissues to promote fungal growth remains unknown. The genome sequences of four rust fungi (two Melampsoraceae and two Pucciniaceae) have been analyzed so far. Genome-wide analyses of these species, as well as transcriptomics performed on a broader range of rust fungi, revealed hundreds of small secreted proteins considered as rust candidate secreted effector proteins (CSEPs). The rust community now needs high-throughput approaches (effectoromics) to accelerate effector discovery/characterization and to better understand how they function in planta. However, this task is challenging due to the non-amenability of rust pathosystems (obligate biotrophs infecting crop plants) to traditional molecular genetic approaches mainly due to difficulties in culturing these species in vitro. The use of heterologous approaches should be promoted in the future. PMID:25191335

  11. Improving a Gripper End Effector

    SciTech Connect

    Mullen, O Dennis; Smith, Christopher M.; Gervais, Kevin L.

    2001-01-31

    This paper discusses the improvement made to an existing four-bar linkage gripping end effector to adapt it for use in a current project. The actuating linkage was modified to yield higher jaw force overall and particularly in the critical range of jaw displacement

  12. A Plethora of Virulence Strategies Hidden Behind Nuclear Targeting of Microbial Effectors

    PubMed Central

    Rivas, Susana; Genin, Stéphane

    2011-01-01

    Plant immune responses depend on the ability to couple rapid recognition of the invading microbe to an efficient response. During evolution, plant pathogens have acquired the ability to deliver effector molecules inside host cells in order to manipulate cellular and molecular processes and establish pathogenicity. Following translocation into plant cells, microbial effectors may be addressed to different subcellular compartments. Intriguingly, a significant number of effector proteins from different pathogenic microorganisms, including viruses, oomycetes, fungi, nematodes, and bacteria, is targeted to the nucleus of host cells. In agreement with this observation, increasing evidence highlights the crucial role played by nuclear dynamics, and nucleocytoplasmic protein trafficking during a great variety of analyzed plant–pathogen interactions. Once in the nucleus, effector proteins are able to manipulate host transcription or directly subvert essential host components to promote virulence. Along these lines, it has been suggested that some effectors may affect histone packing and, thereby, chromatin configuration. In addition, microbial effectors may either directly activate transcription or target host transcription factors to alter their regular molecular functions. Alternatively, nuclear translocation of effectors may affect subcellular localization of their cognate resistance proteins in a process that is essential for resistance protein-mediated plant immunity. Here, we review recent progress in our field on the identification of microbial effectors that are targeted to the nucleus of host plant cells. In addition, we discuss different virulence strategies deployed by microbes, which have been uncovered through examination of the mechanisms that guide nuclear localization of effector proteins. PMID:22639625

  13. Computational Predictions Provide Insights into the Biology of TAL Effector Target Sites

    PubMed Central

    Grau, Jan; Wolf, Annett; Reschke, Maik; Bonas, Ulla; Posch, Stefan; Boch, Jens

    2013-01-01

    Transcription activator-like (TAL) effectors are injected into host plant cells by Xanthomonas bacteria to function as transcriptional activators for the benefit of the pathogen. The DNA binding domain of TAL effectors is composed of conserved amino acid repeat structures containing repeat-variable diresidues (RVDs) that determine DNA binding specificity. In this paper, we present TALgetter, a new approach for predicting TAL effector target sites based on a statistical model. In contrast to previous approaches, the parameters of TALgetter are estimated from training data computationally. We demonstrate that TALgetter successfully predicts known TAL effector target sites and often yields a greater number of predictions that are consistent with up-regulation in gene expression microarrays than an existing approach, Target Finder of the TALE-NT suite. We study the binding specificities estimated by TALgetter and approve that different RVDs are differently important for transcriptional activation. In subsequent studies, the predictions of TALgetter indicate a previously unreported positional preference of TAL effector target sites relative to the transcription start site. In addition, several TAL effectors are predicted to bind to the TATA-box, which might constitute one general mode of transcriptional activation by TAL effectors. Scrutinizing the predicted target sites of TALgetter, we propose several novel TAL effector virulence targets in rice and sweet orange. TAL-mediated induction of the candidates is supported by gene expression microarrays. Validity of these targets is also supported by functional analogy to known TAL effector targets, by an over-representation of TAL effector targets with similar function, or by a biological function related to pathogen infection. Hence, these predicted TAL effector virulence targets are promising candidates for studying the virulence function of TAL effectors. TALgetter is implemented as part of the open-source Java library

  14. Computational predictions provide insights into the biology of TAL effector target sites.

    PubMed

    Grau, Jan; Wolf, Annett; Reschke, Maik; Bonas, Ulla; Posch, Stefan; Boch, Jens

    2013-01-01

    Transcription activator-like (TAL) effectors are injected into host plant cells by Xanthomonas bacteria to function as transcriptional activators for the benefit of the pathogen. The DNA binding domain of TAL effectors is composed of conserved amino acid repeat structures containing repeat-variable diresidues (RVDs) that determine DNA binding specificity. In this paper, we present TALgetter, a new approach for predicting TAL effector target sites based on a statistical model. In contrast to previous approaches, the parameters of TALgetter are estimated from training data computationally. We demonstrate that TALgetter successfully predicts known TAL effector target sites and often yields a greater number of predictions that are consistent with up-regulation in gene expression microarrays than an existing approach, Target Finder of the TALE-NT suite. We study the binding specificities estimated by TALgetter and approve that different RVDs are differently important for transcriptional activation. In subsequent studies, the predictions of TALgetter indicate a previously unreported positional preference of TAL effector target sites relative to the transcription start site. In addition, several TAL effectors are predicted to bind to the TATA-box, which might constitute one general mode of transcriptional activation by TAL effectors. Scrutinizing the predicted target sites of TALgetter, we propose several novel TAL effector virulence targets in rice and sweet orange. TAL-mediated induction of the candidates is supported by gene expression microarrays. Validity of these targets is also supported by functional analogy to known TAL effector targets, by an over-representation of TAL effector targets with similar function, or by a biological function related to pathogen infection. Hence, these predicted TAL effector virulence targets are promising candidates for studying the virulence function of TAL effectors. TALgetter is implemented as part of the open-source Java library

  15. EffectorP: predicting fungal effector proteins from secretomes using machine learning.

    PubMed

    Sperschneider, Jana; Gardiner, Donald M; Dodds, Peter N; Tini, Francesco; Covarelli, Lorenzo; Singh, Karam B; Manners, John M; Taylor, Jennifer M

    2016-04-01

    Eukaryotic filamentous plant pathogens secrete effector proteins that modulate the host cell to facilitate infection. Computational effector candidate identification and subsequent functional characterization delivers valuable insights into plant-pathogen interactions. However, effector prediction in fungi has been challenging due to a lack of unifying sequence features such as conserved N-terminal sequence motifs. Fungal effectors are commonly predicted from secretomes based on criteria such as small size and cysteine-rich, which suffers from poor accuracy. We present EffectorP which pioneers the application of machine learning to fungal effector prediction. EffectorP improves fungal effector prediction from secretomes based on a robust signal of sequence-derived properties, achieving sensitivity and specificity of over 80%. Features that discriminate fungal effectors from secreted noneffectors are predominantly sequence length, molecular weight and protein net charge, as well as cysteine, serine and tryptophan content. We demonstrate that EffectorP is powerful when combined with in planta expression data for predicting high-priority effector candidates. EffectorP is the first prediction program for fungal effectors based on machine learning. Our findings will facilitate functional fungal effector studies and improve our understanding of effectors in plant-pathogen interactions. EffectorP is available at http://effectorp.csiro.au.

  16. Protein-Protein Interaction Assays with Effector-GFP Fusions in Nicotiana benthamiana.

    PubMed

    Petre, Benjamin; Win, Joe; Menke, Frank L H; Kamoun, Sophien

    2017-01-01

    Plant parasites secrete proteins known as effectors into host tissues to manipulate host cell structures and functions. One of the major goals in effector biology is to determine the host cell compartments and the protein complexes in which effectors accumulate. Here, we describe a five-step pipeline that we routinely use in our lab to achieve this goal, which consists of (1) Golden Gate assembly of pathogen effector-green fluorescent protein (GFP) fusions into binary vectors, (2) Agrobacterium-mediated heterologous protein expression in Nicotiana benthamiana leaf cells, (3) laser-scanning confocal microscopy assay, (4) anti-GFP coimmunoprecipitation-liquid chromatography-tandem mass spectrometry (coIP/MS) assay, and (5) anti-GFP western blotting. This pipeline is suitable for rapid, cost-effective, and medium-throughput screening of pathogen effectors in planta.

  17. Dexterous end effector flight demonstration

    NASA Astrophysics Data System (ADS)

    Carter, Edward L.; Monford, Leo G.

    1994-01-01

    The Dexterous End Effector Flight Experiment is a flight demonstration of newly developed equipment and methods which make for more dexterous manipulation of robotic arms. The following concepts are to be demonstrated: The Force Torque Sensor is a six axis load cell located at the end of the RMS which displays load data to the operator on the orbiter CCTV monitor. TRAC is a target system which provides six axis positional information to the operator. It has the characteristic of having high sensitivity to attitude misalignment while being flat. AUTO-TRAC is a variation of TRAC in which a computer analyzes a target, displays translational and attitude misalignment information, and provides cues to the operator for corrective inputs. The Magnetic End Effector is a fault tolerant end effector which grapples payloads using magnetic attraction. The Carrier Latch Assembly is a fault tolerant payload carrier, which uses mechanical latches and/or magnetic attraction to hold small payloads during launch/landing and to release payloads as desired. The flight experiment goals and objectives are explained. The experiment equipment is described, and the tasks to be performed during the demonstration are discussed.

  18. Cyclic-di-GMP binding induces structural rearrangements in the PlzA and PlzC proteins of the Lyme disease and relapsing fever spirochetes: a possible switch mechanism for c-di-GMP-mediated effector functions.

    PubMed

    Mallory, Katherine L; Miller, Daniel P; Oliver, Lee D; Freedman, John C; Kostick-Dunn, Jessica L; Carlyon, Jason A; Marion, James D; Bell, Jessica K; Marconi, Richard T

    2016-11-01

    The c-di-GMP network of Borrelia burgdorferi, a causative agent of Lyme disease, consists of Rrp1, a diguanylate cyclase/response regulator; Hpk1, a histidine kinase; PdeA and PdeB, c-di-GMP phosphodiesterases; and PlzA, a PilZ domain c-di-GMP receptor. Borrelia hermsii, a causative agent of tick-borne relapsing fever, possesses a putative c-di-GMP regulatory network that is uncharacterized. While B. burgdorferi requires c-di-GMP to survive within ticks, the associated effector mechanisms are poorly defined. Using site-directed mutagenesis, size exclusion chromatography, isothermal titration calorimetry and fluorescence resonance energy transfer, we investigate the interaction of c-di-GMP with the Borrelia PilZ domain-containing Plz proteins: B. burgdorferi PlzA and B. hermsii PlzC. The Plz proteins were determined to be monomeric in their apo and holo forms and to bind c-di-GMP with high affinity with a 1:1 stoichiometry. C-di-GMP binding induced structural rearrangements in PlzA and PlzC. C-di-GMP binding proved to be dependent on positive charge at R145 of the PilZ domain motif, R145xxxR. Comparative sequence analyses led to the identification of Borrelia consensus sequences for the PilZ domain signature motifs. This study provides insight into c-di-GMP:Plz receptor interaction and identifies a possible switch mechanism that may regulate Plz protein effector functions. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  19. Systemic immunodominant CD8 responses with an effector-like phenotype are induced by intravaginal immunization with attenuated HSV vectors expressing HIV Tat and mediate protection against HSV infection.

    PubMed

    Nicoli, Francesco; Gallerani, Eleonora; Skarlis, Charalampos; Sicurella, Mariaconcetta; Cafaro, Aurelio; Ensoli, Barbara; Caputo, Antonella; Marconi, Peggy C; Gavioli, Riccardo

    2016-04-27

    Mucosal HSV infection remains a public health issue in developing and developed world. However, an effective vaccine is still missing, partly because of the incomplete knowledge of correlates of protection. In this study we have investigated the kinetics and quality of immunity elicited by an attenuated HSV1 vector expressing the immunomodulatory Tat protein of HIV-1 (HSV1-Tat). Animals were immunized by intravaginal (IVag) or intradermal (ID) route with HSV1-Tat or with a control HSV1 vector expressing the LacZ gene (HSV1-LacZ) and immune responses were characterized in different anatomical districts. IVag immunization with HSV1-Tat enhanced both expansion and memory phases of HSV-specific immunodominant CD8 responses at systemic, but not local, level and induced short- and long-term protection against mucosal challenge. Conversely, ID immunization with HSV1-Tat favored HSV-subdominant CD8 responses, which protected mice only at early time points after immunization. IVag immunization, in particular with HSV1-Tat, compared to ID immunization, induced the differentiation of CD8(+) T lymphocytes into short-lived effector (SLEC) and effector memory (Tem) cells, generating more robust recall responses associated with increased control of virus replication. Notably, systemic SLEC and Tem contributed to generate protective local secondary responses, demonstrating their importance for mucosal control of HSV. Finally, IgG responses were observed mostly in IVag HSV1-Tat immunized animals, although seemed dispensable for protection, which occurred even in few IgG negative mice. Thus, HSV1 vectors expressing Tat induce protective anti-HSV1 immune responses.

  20. A widespread bacterial type VI secretion effector superfamily identified using a heuristic approach.

    PubMed

    Russell, Alistair B; Singh, Pragya; Brittnacher, Mitchell; Bui, Nhat Khai; Hood, Rachel D; Carl, Mike A; Agnello, Danielle M; Schwarz, Sandra; Goodlett, David R; Vollmer, Waldemar; Mougous, Joseph D

    2012-05-17

    Sophisticated mechanisms are employed to facilitate information exchange between interfacing bacteria. A type VI secretion system (T6SS) of Pseudomonas aeruginosa was shown to deliver cell wall-targeting effectors to neighboring cells. However, the generality of bacteriolytic effectors and, moreover, of antibacterial T6S remained unknown. Using parameters derived from experimentally validated bacterial T6SS effectors we identified a phylogenetically disperse superfamily of T6SS-associated peptidoglycan-degrading effectors. The effectors separate into four families composed of peptidoglycan amidase enzymes of differing specificities. Effectors strictly co-occur with cognate immunity proteins, indicating that self-intoxication is a general property of antibacterial T6SSs and effector delivery by the system exerts a strong selective pressure in nature. The presence of antibacterial effectors in a plethora of organisms, including many that inhabit or infect polymicrobial niches in the human body, suggests that the system could mediate interbacterial interactions of both environmental and clinical significance.

  1. Manipulation of plant cells by cyst and root-knot nematode effectors.

    PubMed

    Hewezi, Tarek; Baum, Thomas J

    2013-01-01

    A key feature of sedentary plant-parasitic nematodes is the release of effector proteins from their esophageal gland cells through their stylets into host roots. These proteinaceous stylet secretions have been shown to be crucial for successful parasitism by mediating the transition of normal root cells into specialized feeding sites and by negating plant defenses. Recent technical advances of purifying mRNA from esophageal gland cells of plant-parasitic nematodes coupled with emerging sequencing technologies is steadily expanding our knowledge of nematode effector repertoires. Host targets and biological activities of a number of nematode effectors are continuously being reported and, by now, a first picture of the complexity of sedentary nematode parasitism at the molecular level is starting to take shape. In this review, we highlight effector mechanisms that recently have been uncovered by studying the host-pathogen interaction. These mechanisms range from mediating susceptibility of host plants to the actual triggering of defense responses. In particular, we portray and discuss the mechanisms by which nematode effectors modify plant cell walls, negate host defense responses, alter auxin and polyamine signaling, mimic plant molecules, regulate stress signaling, and activate hypersensitive responses. Continuous molecular characterization of newly discovered nematode effectors will be needed to determine how these effectors orchestrate host signaling pathways and biological processes leading to successful parasitism.

  2. Plasmodium cellular effector mechanisms and the hepatic microenvironment

    PubMed Central

    Frevert, Ute; Krzych, Urszula

    2015-01-01

    Plasmodium falciparum malaria remains one of the most serious health problems globally. Immunization with attenuated parasites elicits multiple cellular effector mechanisms capable of eliminating Plasmodium liver stages. However, malaria liver stage (LS) immunity is complex and the mechanisms effector T cells use to locate the few infected hepatocytes in the large liver in order to kill the intracellular LS parasites remain a mystery to date. Here, we review our current knowledge on the behavior of CD8 effector T cells in the hepatic microvasculature, in malaria and other hepatic infections. Taking into account the unique immunological and lymphogenic properties of the liver, we discuss whether classical granule-mediated cytotoxicity might eliminate infected hepatocytes via direct cell contact or whether cytokines might operate without cell–cell contact and kill Plasmodium LSs at a distance. A thorough understanding of the cellular effector mechanisms that lead to parasite death hence sterile protection is a prerequisite for the development of a successful malaria vaccine to protect the 40% of the world’s population currently at risk of Plasmodium infection. PMID:26074888

  3. 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

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

    PubMed

    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.

  5. Marker for type VI secretion system effectors

    PubMed Central

    Salomon, Dor; Kinch, Lisa N.; Trudgian, David C.; Guo, Xiaofeng; Klimko, John A.; Grishin, Nick V.; Mirzaei, Hamid; Orth, Kim

    2014-01-01

    Bacteria use diverse mechanisms to kill, manipulate, and compete with other cells. The recently discovered type VI secretion system (T6SS) is widespread in bacterial pathogens and used to deliver virulence effector proteins into target cells. Using comparative proteomics, we identified two previously unidentified T6SS effectors that contained a conserved motif. Bioinformatic analyses revealed that this N-terminal motif, named MIX (marker for type six effectors), is found in numerous polymorphic bacterial proteins that are primarily located in the T6SS genome neighborhood. We demonstrate that several MIX-containing proteins are T6SS effectors and that they are not required for T6SS activity. Thus, we propose that MIX-containing proteins are T6SS effectors. Our findings allow for the identification of numerous uncharacterized T6SS effectors that will undoubtedly lead to the discovery of new biological mechanisms. PMID:24927539

  6. Type VI Secretion Effectors: Methodologies and Biology

    PubMed Central

    Lien, Yun-Wei; Lai, Erh-Min

    2017-01-01

    The type VI secretion system (T6SS) is a nanomachine deployed by many Gram-negative bacteria as a weapon against eukaryotic hosts or prokaryotic competitors. It assembles into a bacteriophage tail-like structure that can transport effector proteins into the environment or target cells for competitive survival or pathogenesis. T6SS effectors have been identified by a variety of approaches, including knowledge/hypothesis-dependent and discovery-driven approaches. Here, we review and discuss the methods that have been used to identify T6SS effectors and the biological and biochemical functions of known effectors. On the basis of the nature and transport mechanisms of T6SS effectors, we further propose potential strategies that may be applicable to identify new T6SS effectors. PMID:28664151

  7. A Legionella Effector Disrupts Host Cytoskeletal Structure by Cleaving Actin

    PubMed Central

    Liu, Yao; Zhu, Wenhan; Tan, Yunhao; Nakayasu, Ernesto S.; Staiger, Christopher J.

    2017-01-01

    Legionella pneumophila, the etiological agent of Legionnaires’ disease, replicates intracellularly in protozoan and human hosts. Successful colonization and replication of this pathogen in host cells requires the Dot/Icm type IVB secretion system, which translocates approximately 300 effector proteins into the host cell to modulate various cellular processes. In this study, we identified RavK as a Dot/Icm substrate that targets the host cytoskeleton and reduces actin filament abundance in mammalian cells upon ectopic expression. RavK harbors an H95EXXH99 motif associated with diverse metalloproteases, which is essential for the inhibition of yeast growth and for the induction of cell rounding in HEK293T cells. We demonstrate that the actin protein itself is the cellular target of RavK and that this effector cleaves actin at a site between residues Thr351 and Phe352. Importantly, RavK-mediated actin cleavage also occurs during L. pneumophila infection. Cleavage by RavK abolishes the ability of actin to form polymers. Furthermore, an F352A mutation renders actin resistant to RavK-mediated cleavage; expression of the mutant in mammalian cells suppresses the cell rounding phenotype caused by RavK, further establishing that actin is the physiological substrate of RavK. Thus, L. pneumophila exploits components of the host cytoskeleton by multiple effectors with distinct mechanisms, highlighting the importance of modulating cellular processes governed by the actin cytoskeleton in the intracellular life cycle of this pathogen. PMID:28129393

  8. Space Station end effector strategy study

    NASA Technical Reports Server (NTRS)

    Katzberg, Stephen J.; Jensen, Robert L.; Willshire, Kelli F.; Satterthwaite, Robert E.

    1987-01-01

    The results of a study are presented for terminology definition, identification of functional requirements, technolgy assessment, and proposed end effector development strategies for the Space Station Program. The study is composed of a survey of available or under-developed end effector technology, identification of requirements from baselined Space Station documents, a comparative assessment of the match between technology and requirements, and recommended strategies for end effector development for the Space Station Program.

  9. Orbital maneuvering vehicle end effectors

    NASA Technical Reports Server (NTRS)

    Myers, W. Neill (Inventor); Forbes, John C. (Inventor); Barnes, Wayne L. (Inventor)

    1988-01-01

    An end effector device (A) for grasping and holding an article such as a handle (18) of a space telescope is disclosed. The device includes a V-shaped capture window (74) defined as inclined surfaces (76, 78) in parallel face plates (22, 24) which converge toward a retainer recess (54) in which the handle is retained. A pivotal finger (30) meshes with a pair of pivoted fingers (26, 28) which rotate in counterrotation. The fingers rotate to pull a handle within the capture window into recess (54) where latches (50) lock handle (18) in the recess. To align the capture window, plates (22, 24) may be cocked plus or minus five degrees on base (64).

  10. Blockade of TNF-α signaling benefits cancer therapy by suppressing effector regulatory T cell expansion.

    PubMed

    Chang, Li-Yuan; Lin, Yung-Chang; Chiang, Jy-Ming; Mahalingam, Jayashri; Su, Shih-Huan; Huang, Ching-Tai; Chen, Wei-Ting; Huang, Chien-Hao; Jeng, Wen-Juei; Chen, Yi-Cheng; Lin, Shi-Ming; Sheen, I-Shyan; Lin, Chun-Yen

    2015-10-01

    Effector but not naive regulatory T cells (Treg cells) can accumulate in the peripheral blood as well as the tumor microenvironment, expand during tumor progression and be one of the main suppressors for antitumor immunity. However, the underlying mechanisms for effector Treg cell expansion in tumor are still unknown. We demonstrate that effector Treg cell-mediated suppression of antitumor CD8(+) T cells is tumor-nonspecific. Furthermore, TNFR2 expression is increased in these Treg cells by Affymetrix chip analysis which was confirmed by monoclonal antibody staining in both hepatocellular carcinoma (HCC) and colorectal cancer (CRC) patients and murine models. Correspondingly, increased levels of TNF-α in both tissue and serum were also demonstrated. Interestingly, TNF-α could not only expand effector Treg cells through TNFR2 signaling, but also enhanced their suppressive activity against antitumor immunity of CD8(+) T cells. Furthermore, targeting TNFR2 signaling with a TNF-α inhibitor could selectively reduce rapid resurgence of effector Treg cells after cyclophosphamide-induced lymphodepletion and markedly inhibit the growth of established tumors. Herein, we propose a novel mechanism in which TNF-α could promote tumor-associated effector Treg cell expansion and suggest a new cancer immunotherapy strategy using TNF-α inhibitors to reduce effector Treg cells expansion after cyclophosphamide-induced lymphodepletion.

  11. Chimeric adaptor proteins translocate diverse type VI secretion system effectors in Vibrio cholerae.

    PubMed

    Unterweger, Daniel; Kostiuk, Benjamin; Ötjengerdes, Rina; Wilton, Ashley; Diaz-Satizabal, Laura; Pukatzki, Stefan

    2015-08-13

    Vibrio cholerae is a diverse species of Gram-negative bacteria, commonly found in the aquatic environment and the causative agent of the potentially deadly disease cholera. These bacteria employ a type VI secretion system (T6SS) when they encounter prokaryotic and eukaryotic competitors. This contractile puncturing device translocates a set of effector proteins into neighboring cells. Translocated effectors are toxic unless the targeted cell produces immunity proteins that bind and deactivate incoming effectors. Comparison of multiple V. cholerae strains indicates that effectors are encoded in T6SS effector modules on mobile genetic elements. We identified a diverse group of chimeric T6SS adaptor proteins required for the translocation of diverse effectors encoded in modules. An example for a T6SS effector that requires T6SS adaptor protein 1 (Tap-1) is TseL found in pandemic V. cholerae O1 serogroup strains and other clinical isolates. We propose a model in which Tap-1 is required for loading TseL onto the secretion apparatus. After T6SS-mediated TseL export is completed, Tap-1 is retained in the bacterial cell to load other T6SS machines. © 2015 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

  12. ER-Dependent Ca++-mediated Cytosolic ROS as an Effector for Induction of Mitochondrial Apoptotic and ATM-JNK Signal Pathways in Gallic Acid-treated Human Oral Cancer Cells.

    PubMed

    Lu, Yao-Cheng; Lin, Meng-Liang; Su, Hong-Lin; Chen, Shih-Shun

    2016-02-01

    Release of calcium (Ca(++)) from the endoplasmic reticulum (ER) has been proposed to be involved in induction of apoptosis by oxidative stress. Using inhibitor of ER Ca(++) release dantrolene and inhibitor of mitochondrial Ca(++) uptake Ru-360, we demonstrated that Ca(++) release from the ER was associated with generation of reactive oxygen species (ROS), loss of mitochondrial membrane potential, and apoptosis of human oral cancer (OC) cells induced by gallic acid (GA). Small interfering RNA-mediated suppression of protein kinase RNA-like endoplasmic reticulum kinase inhibited tunicamycin-induced induction of 78 kDa glucose-regulated protein, C/EBP homologous protein, pro-caspase-12 cleavage, cytosolic Ca(++) increase and apoptosis, but did not attenuate the increase in cytosolic Ca(++) level and apoptosis induced by GA. Ataxia telangiectasia mutated (ATM)-mediated c-Jun N-terminal kinase (JNK) phosphorylation and apoptosis by GA was blocked by dantrolene. The specificity of ROS-mediated ATM-JNK activation was confirmed by treatment with N-acetylcysteine, a ROS scavenger. Blockade of ATM activation by specific inhibitor KU55933, short hairpin RNA, or kinase-dead ATM overexpression suppressed JNK phosphorylation but did not completely inhibit cytosolic ROS production, mitochondrial cytochrome c release, pro-caspase-3 cleavage, and apoptosis induced by GA. Taken together, these results indicate that GA induces OC cell apoptosis by inducing the activation of mitochondrial apoptotic and ATM-JNK signal pathways, likely through ER Ca(++)-mediated ROS production. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  13. Live cell imaging reveals novel functions of Salmonella enterica SPI2-T3SS effector proteins in remodeling of the host cell endosomal system.

    PubMed

    Rajashekar, Roopa; Liebl, David; Chikkaballi, Deepak; Liss, Viktoria; Hensel, Michael

    2014-01-01

    Intracellular Salmonella enterica induce a massive remodeling of the endosomal system in infected host cells. One dramatic consequence of this interference is the induction of various extensive tubular aggregations of membrane vesicles, and tubules positive for late endosomal/lysosomal markers are referred to as Salmonella-induced filaments or SIF. SIF are highly dynamic in nature with extension and collapse velocities of 0.4-0.5 µm x sec-1. The induction of SIF depends on the function of the Salmonella Pathogenicity Island 2 (SPI2) encoded type III secretion system (T3SS) and a subset of effector proteins. In this study, we applied live cell imaging and electron microscopy to analyze the role of individual effector proteins in SIF morphology and dynamic properties of SIF. SIF in cells infected with sifB, sseJ, sseK1, sseK2, sseI, sseL, sspH1, sspH2, slrP, steC, gogB or pipB mutant strains showed a morphology and dynamics comparable to SIF induced by WT Salmonella. SIF were absent in cells infected with the sifA-deficient strain and live cell analyses allowed tracking of the loss of the SCV membrane of intracellular sifA Salmonella. In contrast to analyses in fixed cells, in living host cells SIF induced by sseF- or sseG-deficient strains were not discontinuous, but rather continuous and thinner in diameter. A very dramatic phenotype was observed for the pipB2-deficient strain that induced very bulky, non-dynamic aggregations of membrane vesicles. Our study underlines the requirement of the study of Salmonella-host interaction in living systems and reveals new phenotypes due to the intracellular activities of Salmonella.

  14. Human memory CD8 T cell effector potential is epigenetically preserved during in vivo homeostasis.

    PubMed

    Abdelsamed, Hossam A; Moustaki, Ardiana; Fan, Yiping; Dogra, Pranay; Ghoneim, Hazem E; Zebley, Caitlin C; Triplett, Brandon M; Sekaly, Rafick-Pierre; Youngblood, Ben

    2017-06-05

    Antigen-independent homeostasis of memory CD8 T cells is vital for sustaining long-lived T cell-mediated immunity. In this study, we report that maintenance of human memory CD8 T cell effector potential during in vitro and in vivo homeostatic proliferation is coupled to preservation of acquired DNA methylation programs. Whole-genome bisulfite sequencing of primary human naive, short-lived effector memory (TEM), and longer-lived central memory (TCM) and stem cell memory (TSCM) CD8 T cells identified effector molecules with demethylated promoters and poised for expression. Effector-loci demethylation was heritably preserved during IL-7- and IL-15-mediated in vitro cell proliferation. Conversely, cytokine-driven proliferation of TCM and TSCM memory cells resulted in phenotypic conversion into TEM cells and was coupled to increased methylation of the CCR7 and Tcf7 loci. Furthermore, haploidentical donor memory CD8 T cells undergoing in vivo proliferation in lymphodepleted recipients also maintained their effector-associated demethylated status but acquired TEM-associated programs. These data demonstrate that effector-associated epigenetic programs are preserved during cytokine-driven subset interconversion of human memory CD8 T cells. © 2017 Abdelsamed et al.

  15. Characterization of effectors from Fusarium graminearum

    USDA-ARS?s Scientific Manuscript database

    Fusarium graminearum is the causal agent of Fusarium head blight (FHB), which reduces crop yield and quality by producing various mycotoxins. Effectors play an important role in the pathogenesis of many bacterial and fungal pathogens. In this study, 26 effector candidates were selected for investiga...

  16. Comparative reactivity of human IgE to cynomolgus monkey and human effector cells and effects on IgE effector cell potency

    PubMed Central

    Saul, Louise; Saul, Louise; Josephs, Debra H; Josephs, Debra H; Cutler, Keith; Cutler, Keith; Bradwell, Andrew; Bradwell, Andrew; Karagiannis, Panagiotis; Karagiannis, Panagiotis; Selkirk, Chris; Selkirk, Chris; Gould, Hannah J; Gould, Hannah J; Jones, Paul; Jones, Paul; Spicer, James F; Spicer, James F; Karagiannis, Sophia N; Karagiannis, Sophia N

    2014-01-01

    Background: Due to genetic similarities with humans, primates of the macaque genus such as the cynomolgus monkey are often chosen as models for toxicology studies of antibody therapies. IgE therapeutics in development depend upon engagement with the FcεRI and FcεRII receptors on immune effector cells for their function. Only limited knowledge of the primate IgE immune system is available to inform the choice of models for mechanistic and safety evaluations.   Methods: The recognition of human IgE by peripheral blood lymphocytes from cynomolgus monkey and man was compared. We used effector cells from each species in ex vivo affinity, dose-response, antibody-receptor dissociation and potency assays. Results: We report cross-reactivity of human IgE Fc with cynomolgus monkey cells, and comparable binding kinetics to peripheral blood lymphocytes from both species. In competition and dissociation assays, however, human IgE dissociated faster from cynomolgus monkey compared with human effector cells. Differences in association and dissociation kinetics were reflected in effector cell potency assays of IgE-mediated target cell killing, with higher concentrations of human IgE needed to elicit effector response in the cynomolgus monkey system. Additionally, human IgE binding on immune effector cells yielded significantly different cytokine release profiles in each species. Conclusion: These data suggest that human IgE binds with different characteristics to human and cynomolgus monkey IgE effector cells. This is likely to affect the potency of IgE effector functions in these two species, and so has relevance for the selection of biologically-relevant model systems when designing pre-clinical toxicology and functional studies. PMID:24492303

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

    PubMed

    Hallstrom, Kelly N; McCormick, Beth A

    2016-01-01

    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.

  18. Enhancement of Immune Effector Functions by Modulating IgG’s Intrinsic Affinity for Target Antigen

    PubMed Central

    Mazor, Yariv; Yang, Chunning; Borrok, M. Jack; Ayriss, Joanne; Aherne, Karen; Wu, Herren; Dall’Acqua, William F.

    2016-01-01

    Antibody-mediated immune effector functions play an essential role in the anti-tumor efficacy of many therapeutic mAbs. While much of the effort to improve effector potency has focused on augmenting the interaction between the antibody-Fc and activating Fc-receptors expressed on immune cells, the role of antibody binding interactions with the target antigen remains poorly understood. We show that antibody intrinsic affinity to the target antigen clearly influences the extent and efficiency of Fc-mediated effector mechanisms, and report the pivotal role of antibody binding valence on the ability to regulate effector functions. More particularly, we used an array of affinity modulated variants of three different mAbs, anti-CD4, anti-EGFR and anti-HER2 against a panel of target cell lines expressing disparate levels of the target antigen. We found that at saturating antibody concentrations, IgG variants with moderate intrinsic affinities, similar to those generated by the natural humoral immune response, promoted superior effector functions compared to higher affinity antibodies. We hypothesize that at saturating concentrations, effector function correlates most directly with the amount of Fc bound to the cell surface. Thus, high affinity antibodies exhibiting slow off-rates are more likely to interact bivalently with the target cell, occupying two antigen sites with a single Fc. In contrast, antibodies with faster off-rates are likely to dissociate each binding arm more rapidly, resulting in a higher likelihood of monovalent binding. Monovalent binding may in turn increase target cell opsonization and lead to improved recruitment of effector cells. This unpredicted relationship between target affinity and effector function potency suggests a careful examination of antibody design and engineering for the development of next-generation immunotherapeutics. PMID:27322177

  19. A functional genomics approach identifies candidate effectors from the aphid species Myzus persicae (green peach aphid).

    PubMed

    Bos, Jorunn I B; Prince, David; Pitino, Marco; Maffei, Massimo E; Win, Joe; Hogenhout, Saskia A

    2010-11-18

    Aphids are amongst the most devastating sap-feeding insects of plants. Like most plant parasites, aphids require intimate associations with their host plants to gain access to nutrients. Aphid feeding induces responses such as clogging of phloem sieve elements and callose formation, which are suppressed by unknown molecules, probably proteins, in aphid saliva. Therefore, it is likely that aphids, like plant pathogens, deliver proteins (effectors) inside their hosts to modulate host cell processes, suppress plant defenses, and promote infestation. We exploited publicly available aphid salivary gland expressed sequence tags (ESTs) to apply a functional genomics approach for identification of candidate effectors from Myzus persicae (green peach aphid), based on common features of plant pathogen effectors. A total of 48 effector candidates were identified, cloned, and subjected to transient overexpression in Nicotiana benthamiana to assay for elicitation of a phenotype, suppression of the Pathogen-Associated Molecular Pattern (PAMP)-mediated oxidative burst, and effects on aphid reproductive performance. We identified one candidate effector, Mp10, which specifically induced chlorosis and local cell death in N. benthamiana and conferred avirulence to recombinant Potato virus X (PVX) expressing Mp10, PVX-Mp10, in N. tabacum, indicating that this protein may trigger plant defenses. The ubiquitin-ligase associated protein SGT1 was required for the Mp10-mediated chlorosis response in N. benthamiana. Mp10 also suppressed the oxidative burst induced by flg22, but not by chitin. Aphid fecundity assays revealed that in planta overexpression of Mp10 and Mp42 reduced aphid fecundity, whereas another effector candidate, MpC002, enhanced aphid fecundity. Thus, these results suggest that, although Mp10 suppresses flg22-triggered immunity, it triggers a defense response, resulting in an overall decrease in aphid performance in the fecundity assays. Overall, we identified aphid

  20. ROBOTIC TANK INSPECTION END EFFECTOR

    SciTech Connect

    Rachel Landry

    1999-10-01

    The objective of this contract between Oceaneering Space Systems (OSS) and the Department of Energy (DOE) was to provide a tool for the DOE to inspect the inside tank walls of underground radioactive waste storage tanks in their tank farms. Some of these tanks are suspected to have leaks, but the harsh nature of the environment within the tanks precludes human inspection of tank walls. As a result of these conditions only a few inspection methods can fulfill this task. Of the methods available, OSS chose to pursue Alternating Current Field Measurement (ACFM), because it does not require clean surfaces for inspection, nor any contact with the Surface being inspected, and introduces no extra by-products in the inspection process (no coupling fluids or residues are left behind). The tool produced by OSS is the Robotic Tank Inspection End Effector (RTIEE), which is initially deployed on the tip of the Light Duty Utility Arm (LDUA). The RTEE combines ACFM with a color video camera for both electromagnetic and visual inspection The complete package consists of an end effector, its corresponding electronics and software, and a user's manual to guide the operator through an inspection. The system has both coarse and fine inspection modes and allows the user to catalog defects and suspected areas of leakage in a database for further examination, which may lead to emptying the tank for repair, decommissioning, etc.. The following is an updated report to OSS document OSS-21100-7002, which was submitted in 1995. During the course of the contract, two related subtasks arose, the Wall and Coating Thickness Sensor and the Vacuum Scarifying and Sampling Tool Assembly. The first of these subtasks was intended to evaluate the corrosion and wall thinning of 55-gallon steel drums. The second was retrieved and characterized the waste material trapped inside the annulus region of the underground tanks on the DOE's tank farms. While these subtasks were derived from the original intent of

  1. A secreted fungal effector of Glomus intraradices promotes symbiotic biotrophy.

    PubMed

    Kloppholz, Silke; Kuhn, Hannah; Requena, Natalia

    2011-07-26

    Biotrophic fungi interacting with plants establish long-term relationships with their hosts to fulfill their life cycles. In contrast to necrotrophs, they need to contend with the defense mechanisms of the plant to develop within the host and feed on living cells. It is generally accepted that microbial pathogens produce and deliver a myriad of effector proteins to hijack the cellular program of their hosts. Arbuscular mycorrhizal (AM) fungi are the most widespread biotrophs of plant roots. We investigated whether AM fungi use effector proteins to short-circuit the plant defense program. Here we show that Glomus intraradices secretes a protein, SP7, that interacts with the pathogenesis-related transcription factor ERF19 in the plant nucleus. ERF19 is highly induced in roots by the fungal pathogen Colletotrichum trifolii as well as by several fungal extracts, but only transiently during mycorrhiza colonization. When constitutively expressed in roots, SP7 leads to higher mycorrhization while reducing the levels of C. trifolii-mediated defense responses. Furthermore, expression of SP7 in the rice blast fungus Magnaporthe oryzae attenuates root decay symptoms. Taken together, these results suggest that SP7 is an effector that contributes to develop the biotrophic status of AM fungi in roots by counteracting the plant immune program.

  2. The Functions of Effector Proteins in Yersinia Virulence.

    PubMed

    Zhang, Linglin; Mei, Meng; Yu, Chan; Shen, Wenwen; Ma, Lixin; He, Jiewang; Yi, Li

    2016-01-01

    Yersinia species are bacterial pathogens that can cause plague and intestinal diseases after invading into human cells through the Three Secretion System (TTSS). The effect of pathogenesis is mediated by Yersinia outer proteins (Yop) and manifested as down-regulation of the cytokine genes expression by inhibiting nuclear factor-κ-gene binding (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. In addition, its pathogenesis can also manipulate the disorder of host innate immune system and cell death such as apoptosis, pyroptosis, and autophagy. Among the Yersinia effector proteins, YopB and YopD assist the injection of other virulence effectors into the host cytoplasm, while YopE, YopH, YopJ, YopO, and YopT target on disrupting host cell signaling pathways in the host cytosols. Many efforts have been applied to reveal that intracellular proteins such as Rho-GTPase, and transmembrane receptors such as Toll-like receptors (TLRs) both play critical roles in Yersinia pathogenesis, establishing a connection between the pathogenic process and the signaling response. This review will mainly focus on how the effector proteins of Yersinia modulate the intrinsic signals in host cells and disturb the innate immunity of hosts through TTSS.

  3. B cells as effectors and regulators of autoimmunity.

    PubMed

    Mariño, Eliana; Grey, Shane T

    2012-08-01

    A classic understanding of the interplay between B and T cell components of the immune system that drive autoimmunity, where B cells provide an effector function, is represented by systemic lupus erythematosus (SLE), an autoimmune condition characterised by the production of auto-antibodies. In SLE, CD4+T cells provide cognate help to self-reactive B cells, which in turn produce pathogenic auto-antibodies (1). Thus, B cells act as effectors by producing auto-antibody aided by T cell help such that B and T cell interactions are unidirectional. However, this paradigm of B and T cell interactions is challenged by new clinical data demonstrating that B cell depletion is effective for T cell mediated autoimmune diseases including type I diabetes mellitus (T1D) (2), rheumatoid arthritis (3), and multiple sclerosis (4). These clinical data indicate a model whereby B cells can influence the developing autoimmune T cell response, and therefore act as effectors, in ways that extend beyond the production of autoantibody (5). In this review by largely focusing on type I diabetes we will develop a hypothesis that bi-directional B and T interactions control the course of autoimmunity.

  4. Interferon-Inducible Oligoadenylate Synthetase-Like Protein Acts as an Antiviral Effector against Classical Swine Fever Virus via the MDA5-Mediated Type I Interferon-Signaling Pathway.

    PubMed

    Li, Lian-Feng; Yu, Jiahui; Zhang, Yuexiu; Yang, Qian; Li, Yongfeng; Zhang, Lingkai; Wang, Jinghan; Li, Su; Luo, Yuzi; Sun, Yuan; Qiu, Hua-Ji

    2017-06-01

    Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), which poses a serious threat to the global pig industry. Interferons (IFNs) and IFN-stimulated genes (ISGs) play a key role in host antiviral defense. We have previously screened the porcine 2'-5'-oligoadenylate synthetase-like protein (pOASL) as a potential anti-CSFV ISG using a reporter CSFV. This study aimed to clarify the underlying antiviral mechanism of pOASL against CSFV. We confirmed that CSFV replication was significantly suppressed in lentivirus-delivered, pOASL-overexpressing PK-15 cells, whereas silencing the expression of endogenous pOASL by small interfering RNAs markedly enhanced CSFV growth. In addition, the transcriptional level of pOASL was upregulated both in vitro and in vivo upon CSFV infection. Interestingly, the anti-CSFV effects of pOASL are independent of the canonical RNase L pathway but depend on the activation of the type I IFN response. Glutathione S-transferase pulldown and coimmunoprecipitation assays revealed that pOASL interacts with MDA5, a double-stranded RNA sensor, and further enhances MDA5-mediated type I IFN signaling. Moreover, we showed that pOASL exerts anti-CSFV effects in an MDA5-dependent manner. In conclusion, pOASL suppresses CSFV replication via the MDA5-mediated type I IFN-signaling pathway.IMPORTANCE The host innate immune response plays an important role in mounting the initial resistance to viral infection. Here, we identify the porcine 2'-5'-oligoadenylate synthetase-like protein (pOASL) as an interferon (IFN)-stimulated gene (ISG) against classical swine fever virus (CSFV). We demonstrate that the anti-CSFV effects of pOASL depend on the activation of type I IFN response. In addition, we show that pOASL, as an MDA5-interacting protein, is a coactivator of MDA5-mediated IFN induction to exert anti-CSFV actions. This work will be beneficial to the development of novel anti-CSFV strategies by targeting pOASL. Copyright © 2017

  5. Interferon-Inducible Oligoadenylate Synthetase-Like Protein Acts as an Antiviral Effector against Classical Swine Fever Virus via the MDA5-Mediated Type I Interferon-Signaling Pathway

    PubMed Central

    Li, Lian-Feng; Yu, Jiahui; Zhang, Yuexiu; Yang, Qian; Li, Yongfeng; Zhang, Lingkai; Wang, Jinghan; Li, Su; Luo, Yuzi

    2017-01-01

    ABSTRACT Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), which poses a serious threat to the global pig industry. Interferons (IFNs) and IFN-stimulated genes (ISGs) play a key role in host antiviral defense. We have previously screened the porcine 2′-5′-oligoadenylate synthetase-like protein (pOASL) as a potential anti-CSFV ISG using a reporter CSFV. This study aimed to clarify the underlying antiviral mechanism of pOASL against CSFV. We confirmed that CSFV replication was significantly suppressed in lentivirus-delivered, pOASL-overexpressing PK-15 cells, whereas silencing the expression of endogenous pOASL by small interfering RNAs markedly enhanced CSFV growth. In addition, the transcriptional level of pOASL was upregulated both in vitro and in vivo upon CSFV infection. Interestingly, the anti-CSFV effects of pOASL are independent of the canonical RNase L pathway but depend on the activation of the type I IFN response. Glutathione S-transferase pulldown and coimmunoprecipitation assays revealed that pOASL interacts with MDA5, a double-stranded RNA sensor, and further enhances MDA5-mediated type I IFN signaling. Moreover, we showed that pOASL exerts anti-CSFV effects in an MDA5-dependent manner. In conclusion, pOASL suppresses CSFV replication via the MDA5-mediated type I IFN-signaling pathway. IMPORTANCE The host innate immune response plays an important role in mounting the initial resistance to viral infection. Here, we identify the porcine 2′-5′-oligoadenylate synthetase-like protein (pOASL) as an interferon (IFN)-stimulated gene (ISG) against classical swine fever virus (CSFV). We demonstrate that the anti-CSFV effects of pOASL depend on the activation of type I IFN response. In addition, we show that pOASL, as an MDA5-interacting protein, is a coactivator of MDA5-mediated IFN induction to exert anti-CSFV actions. This work will be beneficial to the development of novel anti-CSFV strategies by targeting p

  6. Oxysterols and Their Cellular Effectors

    PubMed Central

    Olkkonen, Vesa M.; Béaslas, Olivier; Nissilä, Eija

    2012-01-01

    Oxysterols are oxidized 27-carbon cholesterol derivatives or by-products of cholesterol biosynthesis, with a spectrum of biologic activities. Several oxysterols have cytotoxic and pro-apoptotic activities, the ability to interfere with the lateral domain organization, and packing of membrane lipids. These properties may account for their suggested roles in the pathology of diseases such as atherosclerosis, age-onset macular degeneration and Alzheimer’s disease. Oxysterols also have the capacity to induce inflammatory responses and play roles in cell differentiation processes. The functions of oxysterols as intermediates in the synthesis of bile acids and steroid hormones, and as readily transportable forms of sterol, are well established. Furthermore, their actions as endogenous regulators of gene expression in lipid metabolism via liver X receptors and the Insig (insulin-induced gene) proteins have been investigated in detail. The cytoplasmic oxysterol-binding protein (OSBP) homologues form a group of oxysterol/cholesterol sensors that has recently attracted a lot of attention. However, their mode of action is, as yet, poorly understood. Retinoic acid receptor-related orphan receptors (ROR) α and γ, and Epstein-Barr virus induced gene 2 (EBI2) have been identified as novel oxysterol receptors, revealing new physiologic oxysterol effector mechanisms in development, metabolism, and immunity, and evoking enhanced interest in these compounds in the field of biomedicine. PMID:24970128

  7. The effector AvrRxo1 phosphorylates NAD in planta.

    PubMed

    Shidore, Teja; Broeckling, Corey D; Kirkwood, Jay S; Long, John J; Miao, Jiamin; Zhao, Bingyu; Leach, Jan E; Triplett, Lindsay R

    2017-06-01

    Gram-negative bacterial pathogens of plants and animals employ type III secreted effectors to suppress innate immunity. Most characterized effectors work through modification of host proteins or transcriptional regulators, although a few are known to modify small molecule targets. The Xanthomonas type III secreted avirulence factor AvrRxo1 is a structural homolog of the zeta toxin family of sugar-nucleotide kinases that suppresses bacterial growth. AvrRxo1 was recently reported to phosphorylate the central metabolite and signaling molecule NAD in vitro, suggesting that the effector might enhance bacterial virulence on plants through manipulation of primary metabolic pathways. In this study, we determine that AvrRxo1 phosphorylates NAD in planta, and that its kinase catalytic sites are necessary for its toxic and resistance-triggering phenotypes. A global metabolomics approach was used to independently identify 3'-NADP as the sole detectable product of AvrRxo1 expression in yeast and bacteria, and NAD kinase activity was confirmed in vitro. 3'-NADP accumulated upon transient expression of AvrRxo1 in Nicotiana benthamiana and in rice leaves infected with avrRxo1-expressing strains of X. oryzae. Mutation of the catalytic aspartic acid residue D193 abolished AvrRxo1 kinase activity and several phenotypes of AvrRxo1, including toxicity in yeast, bacteria, and plants, suppression of the flg22-triggered ROS burst, and ability to trigger an R gene-mediated hypersensitive response. A mutation in the Walker A ATP-binding motif abolished the toxicity of AvrRxo1, but did not abolish the 3'-NADP production, virulence enhancement, ROS suppression, or HR-triggering phenotypes of AvrRxo1. These results demonstrate that a type III effector targets the central metabolite and redox carrier NAD in planta, and that this catalytic activity is required for toxicity and suppression of the ROS burst.

  8. The effector AvrRxo1 phosphorylates NAD in planta

    PubMed Central

    Long, John J.; Zhao, Bingyu; Leach, Jan E.

    2017-01-01

    Gram-negative bacterial pathogens of plants and animals employ type III secreted effectors to suppress innate immunity. Most characterized effectors work through modification of host proteins or transcriptional regulators, although a few are known to modify small molecule targets. The Xanthomonas type III secreted avirulence factor AvrRxo1 is a structural homolog of the zeta toxin family of sugar-nucleotide kinases that suppresses bacterial growth. AvrRxo1 was recently reported to phosphorylate the central metabolite and signaling molecule NAD in vitro, suggesting that the effector might enhance bacterial virulence on plants through manipulation of primary metabolic pathways. In this study, we determine that AvrRxo1 phosphorylates NAD in planta, and that its kinase catalytic sites are necessary for its toxic and resistance-triggering phenotypes. A global metabolomics approach was used to independently identify 3’-NADP as the sole detectable product of AvrRxo1 expression in yeast and bacteria, and NAD kinase activity was confirmed in vitro. 3’-NADP accumulated upon transient expression of AvrRxo1 in Nicotiana benthamiana and in rice leaves infected with avrRxo1-expressing strains of X. oryzae. Mutation of the catalytic aspartic acid residue D193 abolished AvrRxo1 kinase activity and several phenotypes of AvrRxo1, including toxicity in yeast, bacteria, and plants, suppression of the flg22-triggered ROS burst, and ability to trigger an R gene-mediated hypersensitive response. A mutation in the Walker A ATP-binding motif abolished the toxicity of AvrRxo1, but did not abolish the 3’-NADP production, virulence enhancement, ROS suppression, or HR-triggering phenotypes of AvrRxo1. These results demonstrate that a type III effector targets the central metabolite and redox carrier NAD in planta, and that this catalytic activity is required for toxicity and suppression of the ROS burst. PMID:28628666

  9. Mycobacterium tuberculosis effectors interfering host apoptosis signaling.

    PubMed

    Liu, Minqiang; Li, Wu; Xiang, Xiaohong; Xie, Jianping

    2015-07-01

    Tuberculosis remains a serious human public health concern. The coevolution between its pathogen Mycobacterium tuberculosis and human host complicated the way to prevent and cure TB. Apoptosis plays subtle role in this interaction. The pathogen endeavors to manipulate the apoptosis via diverse effectors targeting key signaling nodes. In this paper, we summarized the effectors pathogen used to subvert the apoptosis, such as LpqH, ESAT-6/CFP-10, LAMs. The interplay between different forms of cell deaths, such as apoptosis, autophagy, necrosis, is also discussed with a focus on the modes of action of effectors, and implications for better TB control.

  10. Epigenetic control of effectors in plant pathogens.

    PubMed

    Gijzen, Mark; Ishmael, Chelsea; Shrestha, Sirjana D

    2014-01-01

    Plant pathogens display impressive versatility in adapting to host immune systems. Pathogen effector proteins facilitate disease but can become avirulence (Avr) factors when the host acquires discrete recognition capabilities that trigger immunity. The mechanisms that lead to changes to pathogen Avr factors that enable escape from host immunity are diverse, and include epigenetic switches that allow for reuse or recycling of effectors. This perspective outlines possibilities of how epigenetic control of Avr effector gene expression may have arisen and persisted in filamentous plant pathogens, and how it presents special problems for diagnosis and detection of specific pathogen strains or pathotypes.

  11. Novel type III effectors in Pseudomonas aeruginosa.

    PubMed

    Burstein, David; Satanower, Shirley; Simovitch, Michal; Belnik, Yana; Zehavi, Meital; Yerushalmi, Gal; Ben-Aroya, Shay; Pupko, Tal; Banin, Ehud

    2015-03-17

    Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that causes chronic and acute infections in immunocompromised patients. Most P. aeruginosa strains encode an active type III secretion system (T3SS), utilized by the bacteria to deliver effector proteins from the bacterial cell directly into the cytoplasm of the host cell. Four T3SS effectors have been discovered and extensively studied in P. aeruginosa: ExoT, ExoS, ExoU, and ExoY. This is especially intriguing in light of P. aeruginosa's ability to infect a wide range of hosts. We therefore hypothesized that additional T3SS effectors that have not yet been discovered are encoded in the genome of P. aeruginosa. Here, we applied a machine learning classification algorithm to identify novel P. aeruginosa effectors. In this approach, various types of data are integrated to differentiate effectors from the rest of the open reading frames of the bacterial genome. Due to the lack of a sufficient learning set of positive effectors, our machine learning algorithm integrated genomic information from another Pseudomonas species and utilized dozens of features accounting for various aspects of the effector coding genes and their products. Twelve top-ranking predictions were experimentally tested for T3SS-specific translocation, leading to the discovery of two novel T3SS effectors. We demonstrate that these effectors are not part of the injection structural complex and report initial efforts toward their characterization. Pseudomonas aeruginosa uses a type III secretion system (T3SS) to secrete toxic proteins, termed effectors, directly into the cytoplasm of the host cell. The activation of this secretion system is correlated with disease severity and patient death. Compared with many other T3SS-utilizing pathogenic bacteria, P. aeruginosa has a fairly limited arsenal of effectors that have been identified. This is in sharp contrast with the wide range of hosts that this bacterium can infect. The discovery of

  12. CD152 (CTLA-4) regulates effector functions of CD8+ T lymphocytes by repressing Eomesodermin.

    PubMed

    Hegel, Johannes K; Knieke, Karin; Kolar, Paula; Reiner, Steven L; Brunner-Weinzierl, Monika C

    2009-03-01

    CD8(+) T lymphocytes are required for effective host defense against pathogens and also for mediating effector responses against uncontrolled proliferating self-tissues. In this study, we determine that individual CD8(+) T cells are tightly controlled in their effector functions by CD152 (CTLA-4). We demonstrate that signals induced by CD152 reduce the frequency of IFN-gamma and granzyme B expressing CD8(+) T cells independently of the transcription factors T-bet or cKrox by selectively inhibiting accumulation of Eomesodermin mRNA and protein. Ectopic expression of Eomesodermin reversed the CD152-mediated inhibition of effector molecule production. Additionally, enhanced cytotoxicity of individual CD8(+) T cells differentiated in the absence of CD152 signaling was determined in vivo. These novel insights extend our understanding of how immune responses of CD8(+) T cells are selectively modulated.

  13. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein-induced lysosomal translocation of proapoptotic effectors is mediated by phosphofurin acidic cluster sorting protein-2 (PACS-2).

    PubMed

    Werneburg, Nathan W; Bronk, Steve F; Guicciardi, Maria Eugenia; Thomas, Laurel; Dikeakos, Jimmy D; Thomas, Gary; Gores, Gregory J

    2012-07-13

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis of liver cancer cell lines requires death receptor-5 (DR5)-dependent permeabilization of lysosomal membranes. Ligated DR5 triggers recruitment of the proapoptotic proteins Bim and Bax to lysosomes, releasing cathepsin B into the cytosol where it mediates mitochondria membrane permeabilization and activation of executioner caspases. Despite the requirement for lysosome membrane permeabilization during TRAIL-induced apoptosis, little is known about the mechanism that controls recruitment of Bim and Bax to lysosomal membranes. Here we report that TRAIL induces recruitment of the multifunctional sorting protein phosphofurin acidic cluster sorting protein-2 (PACS-2) to DR5-positive endosomes in Huh-7 cells where it forms an immunoprecipitatable complex with Bim and Bax on lysosomal membranes. shRNA-targeted knockdown of PACS-2 prevents recruitment of Bim or Bax to lysosomes, blunting the TRAIL-induced lysosome membrane permeabilization. Consistent with the reduced lysosome membrane permeabilization, shRNA knockdown of PACS-2 in Huh-7 cells reduced TRAIL-induced apoptosis and increased clonogenic cell survival. The determination that recombinant PACS-2 bound Bim but not Bax in vitro and that shRNA knockdown of Bim blocked Bax recruitment to lysosomes suggests that TRAIL/DR5 triggers endosomal PACS-2 to recruit Bim and Bax to lysosomes to release cathepsin B and induce apoptosis. Together, these findings provide insight into the lysosomal pathway of apoptosis.

  14. Interactions between effectors linked to serotonin receptors.

    PubMed

    Berg, K A; Maayani, S; Clarke, W P

    1998-12-15

    In general, there are two types of interactions between effector signaling pathways. "Homologous" interactions are those that occur within a receptor system to alter its own responsiveness, for example the loss of responsiveness (desensitization) that can occur upon agonist occupancy of a receptor. "Heterologous" interactions are those that occur between different receptor systems where the responsiveness of one receptor system is regulated (positively or negatively) by activation of another receptor system (i.e., "cross-talk"). Many, if not all receptors, couple to multiple cellular effector pathways and alterations in the responsiveness of a receptor system can be effector pathway-dependent which underscores the importance of studying each effector coupled to a receptor. Regulation of receptor system responsiveness, and consequently the efficacy of drugs, is a highly dynamic process. Perhaps by exploiting these interactions, new targets for pharmacotherapy may be uncovered which will provide for increased efficacy and specificity of drug action.

  15. Functional dissection of SseF, a membrane-integral effector protein of intracellular Salmonella enterica.

    PubMed

    Müller, Petra; Chikkaballi, Deepak; Hensel, Michael

    2012-01-01

    During intracellular life, the bacterial pathogen Salmonella enterica translocates a complex cocktail of effector proteins by means of the SPI2-encoded type III secretions system. The effectors jointly modify the endosomal system and vesicular transport in host cells. SseF and SseG are two effectors encoded by genes within Salmonella Pathogenicity Island 2 and both effector associate with endosomal membranes and microtubules and are involved in the formation of Salmonella-induced filaments. Our previous deletional analyses identified protein domains of SseF required for the effector function. Here we present a detailed mutational analysis that identifies a short hydrophobic motif as functionally essential. We demonstrate that SseF and SseG are still functional if translocated as a single fusion protein, but also mediate effector function if translocated in cells co-infected with sseF and sseG strains. SseF has characteristics of an integral membrane protein after translocation into host cells.

  16. SPRYSEC Effectors: A Versatile Protein-Binding Platform to Disrupt Plant Innate Immunity

    PubMed Central

    Diaz-Granados, Amalia; Petrescu, Andrei-José; Goverse, Aska; Smant, Geert

    2016-01-01

    Persistent infections by sedentary plant-parasitic nematodes are a major threat to important food crops all over the world. These roundworms manipulate host plant cell morphology and physiology to establish sophisticated feeding structures. Key modifications to plant cells during their transition into feeding structures are largely attributed to the activity of effectors secreted by the nematodes. The SPRYSEC effectors were initially identified in the potato cyst nematodes Globodera rostochiensis and G. pallida, and are characterized by a single SPRY domain, a non-catalytic domain present in modular proteins with different functions. The SPRY domain is wide-spread among eukaryotes and thought to be involved in mediating protein–protein interactions. Thus far, the SPRY domain is only reported as a functional domain in effectors of plant-parasitic nematodes, but not of other plant pathogens. SPRYSEC effectors have been implicated in both suppression and activation of plant immunity, but other possible roles in nematode virulence remain undefined. Here, we review the latest reports on the structure, function, and sequence diversity of SPRYSEC effectors, which provide support for a model featuring these effectors as a versatile protein-binding platform for the nematodes to target a wide range of host proteins during parasitism. PMID:27812363

  17. Jet Engine Exhaust Nozzle Flow Effector

    NASA Technical Reports Server (NTRS)

    Turner, Travis L. (Inventor); Cano, Roberto J. (Inventor); Silcox, Richard J. (Inventor); Buehrle, Ralph D. (Inventor); Cagle, Christopher M. (Inventor); Cabell, Randolph H. (Inventor); Hilton, George C. (Inventor)

    2011-01-01

    A jet engine exhaust nozzle flow effector is a chevron formed with a radius of curvature with surfaces of the flow effector being defined and opposing one another. At least one shape memory alloy (SMA) member is embedded in the chevron closer to one of the chevron's opposing surfaces and substantially spanning from at least a portion of the chevron's root to the chevron's tip.

  18. Jet Engine Exhaust Nozzle Flow Effector

    NASA Technical Reports Server (NTRS)

    Turner, Travis L. (Inventor); Cano, Roberto J. (Inventor); Silox, Richard J. (Inventor); Buehrle, Ralph D. (Inventor); Cagle, Christopher M. (Inventor); Cabell, Randolph H. (Inventor); Hilton, George C. (Inventor)

    2014-01-01

    A jet engine exhaust nozzle flow effector is a chevron formed with a radius of curvature with surfaces of the flow effector being defined and opposing one another. At least one shape memory alloy (SMA) member is embedded in the chevron closer to one of the chevron's opposing surfaces and substantially spanning from at least a portion of the chevron's root to the chevron's tip.

  19. A Legionella Effector Disrupts Host Cytoskeletal Structure by Cleaving Actin

    DOE PAGES

    Liu, Yao; Zhu, Wenhan; Tan, Yunhao; ...

    2017-01-27

    Legionella pneumophila, the etiological agent of Legionnaires' disease, replicates intracellularly in protozoan and human hosts. Successful colonization and replication of this pathogen in host cells requires the Dot/Icm type IVB secretion system, which translocates approximately 300 effector proteins into the host cell to modulate various cellular processes. In this study, we identified RavK as a Dot/Icm substrate that targets the host cytoskeleton and reduces actin filament abundance in mammalian cells upon ectopic expression. RavK harbors an H95EXXH99 motif associated with diverse metalloproteases, which is essential for the inhibition of yeast growth and for the induction of cell rounding in HEK293Tmore » cells. We demonstrate that the actin protein itself is the cellular target of RavK and that this effector cleaves actin at a site between residues Thr351 and Phe352. Importantly, RavK-mediated actin cleavage also occurs during L. pneumophila infection. Cleavage by RavK abolishes the ability of actin to form polymers. Furthermore, an F352A mutation renders actin resistant to RavK-mediated cleavage; expression of the mutant in mammalian cells suppresses the cell rounding phenotype caused by RavK, further establishing that actin is the physiological substrate of RavK. Furthermore, L. pneumophila exploits components of the host cytoskeleton by multiple effectors with distinct mechanisms, highlighting the importance of modulating cellular processes governed by the actin cytoskeleton in the intracellular life cycle of this pathogen.« less

  20. The Cell Death Triggered by the Nuclear Localized RxLR Effector PITG_22798 from Phytophthora infestans Is Suppressed by the Effector AVR3b.

    PubMed

    Wang, Hongyang; Ren, Yajuan; Zhou, Jing; Du, Juan; Hou, Juan; Jiang, Rui; Wang, Haixia; Tian, Zhendong; Xie, Conghua

    2017-02-14

    Phytopathogenic oomycetes, such as Phytophthora infestans, potentially secrete many RxLR effector proteins into plant cells to modulate plant immune responses and promote colonization. However, the molecular mechanisms by which these RxLR effectors suppress plant immune responses are largely unknown. Here we describe an RxLR effector PITG_22798 (Gene accession: XM_002998349) that was upregulated during early infection of potato by P. infestans. By employment of agroinfiltration, we observed that PITG_22798 triggers cell death in Nicotiana benthamiana. Confocal microscopic examination showed that PITG_22798-GFP (Green Fluorescent Protein) located in the host nucleus when expressed transiently in N. benthamiana leaves. A nuclear localization signal (NLS) domain of PITG_22798 is important for nuclear localization and cell death-inducing activity. Sequence alignment and transient expression showed that PITG_22798 from diverse P. infestans isolates are conserved, and transient expression of PITG_22798 enhances P. infestans colonization of N. benthamiana leaves, which suggests that PITG_22798 contributes to P. infestans infection. PITG_22798-triggered cell death is dependent on SGT1-mediated signaling and is suppressed by the P. infestans avirulence effector 3b (AVR3b). The present research provides a clue for further investigation of how P. infestans effector PITG_22798 associates with and modulates host immunity.

  1. The Cell Death Triggered by the Nuclear Localized RxLR Effector PITG_22798 from Phytophthora infestans Is Suppressed by the Effector AVR3b

    PubMed Central

    Wang, Hongyang; Ren, Yajuan; Zhou, Jing; Du, Juan; Hou, Juan; Jiang, Rui; Wang, Haixia; Tian, Zhendong; Xie, Conghua

    2017-01-01

    Phytopathogenic oomycetes, such as Phytophthora infestans, potentially secrete many RxLR effector proteins into plant cells to modulate plant immune responses and promote colonization. However, the molecular mechanisms by which these RxLR effectors suppress plant immune responses are largely unknown. Here we describe an RxLR effector PITG_22798 (Gene accession: XM_002998349) that was upregulated during early infection of potato by P. infestans. By employment of agroinfiltration, we observed that PITG_22798 triggers cell death in Nicotiana benthamiana. Confocal microscopic examination showed that PITG_22798-GFP (Green Fluorescent Protein) located in the host nucleus when expressed transiently in N. benthamiana leaves. A nuclear localization signal (NLS) domain of PITG_22798 is important for nuclear localization and cell death-inducing activity. Sequence alignment and transient expression showed that PITG_22798 from diverse P. infestans isolates are conserved, and transient expression of PITG_22798 enhances P. infestans colonization of N. benthamiana leaves, which suggests that PITG_22798 contributes to P. infestans infection. PITG_22798-triggered cell death is dependent on SGT1-mediated signaling and is suppressed by the P. infestans avirulence effector 3b (AVR3b). The present research provides a clue for further investigation of how P. infestans effector PITG_22798 associates with and modulates host immunity. PMID:28216607

  2. In planta expression or delivery of potato aphid Macrosiphum euphorbiae effectors Me10 and Me23 enhances aphid fecundity.

    PubMed

    Atamian, Hagop S; Chaudhary, Ritu; Cin, Valeriano Dal; Bao, Ergude; Girke, Thomas; Kaloshian, Isgouhi

    2013-01-01

    The interactions between aphids and their host plants seem to be analogous to those of plant-microbial pathogens. Unlike microbial pathogen effectors, little is known about aphid effectors and their ability to interfere with host immunity. To date, only three functional aphid effectors have been reported. To identify potato aphid (Macrosiphum euphorbiae) effectors, we developed a salivary gland transcriptome using Illumina technology. We generated 85 million Illumina reads from salivary glands and assembled them into 646 contigs. Ab initio sequence analysis predicted secretion signal peptides in 24% of these sequences, suggesting that they might be secreted into the plant during aphid feeding. Eight of these candidate effectors with secretion signal peptides were functionally characterized using Agrobacterium tumefaciens-mediated transient overexpression in Nicotiana benthamiana. Two candidate effectors, Me10 and Me23, increased aphid fecundity, suggesting their ability to suppress N. benthamiana defenses. Five of these candidate effectors, including Me10 and Me23, were also analyzed in tomato by delivering them through the Pseudomonas syringae type three secretion system. In tomato, only Me10 increased aphid fecundity. This work identified two additional aphid effectors with ability to manipulate the host for their advantage.

  3. Endocannabinoids: Effectors of glucocorticoid signaling.

    PubMed

    Balsevich, Georgia; Petrie, Gavin N; Hill, Matthew N

    2017-10-01

    For decades, there has been speculation regarding the interaction of cannabinoids with glucocorticoid systems. Given the functional redundancy between many of the physiological effects of glucocorticoids and cannabinoids, it was originally speculated that the biological mechanisms of cannabinoids were mediated by direct interactions with glucocorticoid systems. With the discovery of the endocannabinoid system, additional research demonstrated that it was actually the opposite; glucocorticoids recruit endocannabinoid signaling, and that the engagement of endocannabinoid signaling mediated many of the neurobiological and physiological effects of glucocorticoids. With the development of advances in pharmacology and genetics, significant advances in this area have been made, and it is now clear that functional interactions between these systems are critical for a wide array of physiological processes. The current review acts a comprehensive summary of the contemporary state of knowledge regarding the biological interactions between glucocorticoids and endocannabinoids, and their potential role in health and disease. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Assembly of Customized TAL Effectors Through Advanced ULtiMATE System.

    PubMed

    Yang, Junjiao; Guo, Shengjie; Yuan, Pengfei; Wei, Wensheng

    2016-01-01

    Transcription activator-like effectors (TALEs) have been widely applied in gene targeting. Here we describe an advanced ULtiMATE (USER-based Ligation-Mediated Assembly of TAL Effector) system that utilizes USER fusion technique and archive of 512 tetramer templates to achieve highly efficient construction of TALEs, which takes only half a day to accomplish the assembly of any given TALE construct. This system is also suitable for large-scale assembly of TALENs and any other TALE-based constructions.

  5. End effector with astronaut foot restraint

    NASA Technical Reports Server (NTRS)

    Monford, Leo G., Jr. (Inventor)

    1991-01-01

    The combination of a foot restraint platform designed primarily for use by an astronaut being rigidly and permanently attached to an end effector which is suitable for attachment to the manipulator arm of a remote manipulating system is described. The foot restraint platform is attached by a brace to the end effector at a location away from the grappling interface of the end effector. The platform comprises a support plate provided with a pair of stirrups for receiving the toe portion of an astronaut's boots when standing on the platform and a pair of heel retainers in the form of raised members which are fixed to the surface of the platform and located to provide abutment surfaces for abutting engagement with the heels of the astronaut's boots when his toes are in the stirrups. The heel retainers preclude a backward sliding movement of the feet on the platform and instead require a lifting of the heels in order to extract the feet. The brace for attaching the foot restraint platform to the end effector may include a pivot or swivel joint to permit various orientations of the platform with respect to the end effector.

  6. Bacterial Effector Nanoparticles as Breast Cancer Therapeutics.

    PubMed

    Herrera Estrada, Lina; Padmore, Trudy J; Champion, Julie A

    2016-03-07

    Bacterial pathogens trigger cell death by a variety of mechanisms, including injection of effector proteins. Effector proteins have great potential as anticancer agents because they efficiently subvert a variety of eukaryotic signaling pathways involved in cancer development, drug resistance, and metastasis. In breast cancer, MAPK and NFκB pathways are known to be dysregulated. YopJ, an effector from Yersinia pestis, downregulates MAPK and NFκB pathways to induce cell death in specific cell types. We expressed YopJ in Escherichia coli as a fusion protein with glutathione S-transferase (GST), forming self-assembled protein nanoparticles with diameters of 100 nm. YopJ-GST nanoparticles efficiently delivered protein to cells, replacing the need for the pathogen secretion mechanism for effector delivery to cells. These nanoparticles induced dose and time dependent death in SKBR-3 breast cancer cells. After 72 h, 97% of cells died, significantly more than with the same molar dose of doxorubicin. Treatment with sublethal doses of nanoparticles decreased cell migration in vitro and downregulated the MAPK ERK 1/2 pathway, which has been correlated to metastasis. Exposure to a panel of breast cancer cell lines showed that YopJ-GST nanoparticles are cytotoxic to different subtypes, including doxorubicin resistant cells. However, they were not cytotoxic to NIH/3T3 fibroblasts or HeLa cells. Thus, YopJ-GST nanoparticles demonstrate the potential of effector proteins as breast cancer therapeutics with selective cytotoxicity and the capacity to decrease metastatic predictive behaviors.

  7. Cellular senescence and its effector programs

    PubMed Central

    Salama, Rafik; Sadaie, Mahito; Hoare, Matthew; Narita, Masashi

    2014-01-01

    Cellular senescence is a stress response that accompanies stable exit from the cell cycle. Classically, senescence, particularly in human cells, involves the p53 and p16/Rb pathways, and often both of these tumor suppressor pathways need to be abrogated to bypass senescence. In parallel, a number of effector mechanisms of senescence have been identified and characterized. These studies suggest that senescence is a collective phenotype of these multiple effectors, and their intensity and combination can be different depending on triggers and cell types, conferring a complex and diverse nature to senescence. Series of studies on senescence-associated secretory phenotype (SASP) in particular have revealed various layers of functionality of senescent cells in vivo. Here we discuss some key features of senescence effectors and attempt to functionally link them when it is possible. PMID:24449267

  8. Murine interstitial nephritis. III. The selection of phenotypic (Lyt and L3T4) and idiotypic (RE-Id) T cell preferences by genes in Igh-1 and H-2K characterizes the cell-mediated potential for disease expression: susceptible mice provide a unique effector T cell repertoire in response to tubular antigen.

    PubMed

    Neilson, E G; McCafferty, E; Mann, R; Michaud, L; Clayman, M

    1985-04-01

    The effector T cell repertoire in experimental interstitial nephritis was examined in a variety of susceptible and nonsusceptible mice. We observed that L3T4+ effector T cells in disease-susceptible mice disappear soon after immunization in preference to the emergence of Lyt-2+ effector cells. These latter cells respond with delayed-type hypersensitivity to tubular antigen in the context of H-2K. Such cells also express idiotypes (RE-Id) shared with kidney-bound alpha TBM-Ab that are regulated by an interactional effect of genes in Igh-1 and H-2K. These Lyt-2+ effector cells can be removed from renal infiltrates, and the transfer of similar cells under the renal capsule of naive mice results, within 5 days, in local interstitial nephritis. Nonsusceptible mice, however, not having these immune response genes, produce either L3T4+, Lyt-1+, RE-Id- effector T cells, which only respond to tubular antigen in the context of I-A, or Lyt-2+, RE-Id- T cells, which may lack very fine specificity. These findings suggest that susceptible mice carry a unique set of immune response genes that promote a T cell selection process that operates after induction, during the differentiation and development of disease-producing effector T cells.

  9. Transcription Factor Networks Directing the Development, Function, and Evolution of Innate Lymphoid Effectors

    PubMed Central

    Kang, Joonsoo; Malhotra, Nidhi

    2015-01-01

    Mammalian lymphoid immunity is mediated by fast and slow responders to pathogens. Fast innate lymphocytes are active within hours after infections in mucosal tissues. Slow adaptive lymphocytes are conventional T and B cells with clonal antigen receptors that function days after pathogen exposure. A transcription factor (TF) regulatory network guiding early T cell development is at the core of effector function diversification in all innate lymphocytes, and the kinetics of immune responses is set by developmental programming. Operational units within the innate lymphoid system are not classified by the types of pathogen-sensing machineries but rather by discrete effector functions programmed by regulatory TF networks. Based on the evolutionary history of TFs of the regulatory networks, fast effectors likely arose earlier in the evolution of animals to fortify body barriers, and in mammals they often develop in fetal ontogeny prior to the establishment of fully competent adaptive immunity. PMID:25650177

  10. A systems biology perspective on plant-microbe interactions: biochemical and structural targets of pathogen effectors.

    PubMed

    Pritchard, Leighton; Birch, Paul

    2011-04-01

    Plants have biochemical defences against stresses from predators, parasites and pathogens. In this review we discuss the interaction of plant defences with microbial pathogens such as bacteria, fungi and oomycetes, and viruses. We examine principles of complex dynamic networks that allow identification of network components that are differentially and predictably sensitive to perturbation, thus making them likely effector targets. We relate these principles to recent developments in our understanding of known effector targets in plant-pathogen systems, and propose a systems-level framework for the interpretation and modelling of host-microbe interactions mediated by effectors. We describe this framework briefly, and conclude by discussing useful experimental approaches for populating this framework.

  11. Transcription factor networks directing the development, function, and evolution of innate lymphoid effectors.

    PubMed

    Kang, Joonsoo; Malhotra, Nidhi

    2015-01-01

    Mammalian lymphoid immunity is mediated by fast and slow responders to pathogens. Fast innate lymphocytes are active within hours after infections in mucosal tissues. Slow adaptive lymphocytes are conventional T and B cells with clonal antigen receptors that function days after pathogen exposure. A transcription factor (TF) regulatory network guiding early T cell development is at the core of effector function diversification in all innate lymphocytes, and the kinetics of immune responses is set by developmental programming. Operational units within the innate lymphoid system are not classified by the types of pathogen-sensing machineries but rather by discrete effector functions programmed by regulatory TF networks. Based on the evolutionary history of TFs of the regulatory networks, fast effectors likely arose earlier in the evolution of animals to fortify body barriers, and in mammals they often develop in fetal ontogeny prior to the establishment of fully competent adaptive immunity.

  12. Conserved fungal LysM effector Ecp6 prevents chitin-triggered immunity in plants.

    PubMed

    de Jonge, Ronnie; van Esse, H Peter; Kombrink, Anja; Shinya, Tomonori; Desaki, Yoshitake; Bours, Ralph; van der Krol, Sander; Shibuya, Naoto; Joosten, Matthieu H A J; Thomma, Bart P H J

    2010-08-20

    Multicellular organisms activate immunity upon recognition of pathogen-associated molecular patterns (PAMPs). Chitin is the major component of fungal cell walls, and chitin oligosaccharides act as PAMPs in plant and mammalian cells. Microbial pathogens deliver effector proteins to suppress PAMP-triggered host immunity and to establish infection. Here, we show that the LysM domain-containing effector protein Ecp6 of the fungal plant pathogen Cladosporium fulvum mediates virulence through perturbation of chitin-triggered host immunity. During infection, Ecp6 sequesters chitin oligosaccharides that are released from the cell walls of invading hyphae to prevent elicitation of host immunity. This may represent a common strategy of host immune suppression by fungal pathogens, because LysM effectors are widely conserved in the fungal kingdom.

  13. Effector functions of pathogenic Yersinia species.

    PubMed

    Aepfelbacher, Martin; Trasak, Claudia; Ruckdeschel, Klaus

    2007-09-01

    Pathogenic species of the genus Yersinia suppress and reorient the immune system to infect lymphatic tissues, inner organs and at times also the vasculature. For this purpose yersiniae employ a type III secretion system to translocate effector proteins (Yersinia outer proteins; Yops) into immune cells. Yops often exert unique biochemical activities for modulating the activity of Rho GTP-binding proteins, focal adhesion proteins, inflammatory pathways and cell survival/apoptosis. In this review we will put emphasis on the biochemistry, cell- and infection biology of Yersinia effector Yops.

  14. RXLR effectors of plant pathogenic oomycetes.

    PubMed

    Morgan, William; Kamoun, Sophien

    2007-08-01

    Oomycetes are a phylogenetically distinct group of organisms that include some of the most devastating plant pathogens. Recent characterization of four oomycete Avr genes revealed that they encode effector proteins with a common modular structure, including a N-terminal conserved RXLR motif. Several lines of evidence initially indicated, with support from more recent works, that these Avr proteins are secreted by the pathogen and then translocated into the host cell during infection. In addition to elucidating the machinery required for host-cell transport, future works remain to determine the myriad virulence functions of oomycete RXLR effector proteins.

  15. Cell-mediated lympholysis of N-(3-nitro-4-hydroxy-5-iodophenylacetyl)- beta-anaylglycylglycyl-modified autologous lymphocytes. Effector cell specificity to modified cell surface components controlled by the H-2K and H-2D serological regions of the murine major histocompatibility complex

    PubMed Central

    1976-01-01

    Splenic lymphocytes from four C57BL/10 congenic mouse strains were sensitized in vitro to N(-3-nitro-4-hydroxy-5-iodophenylacetyl)-beta- alanylglycylglycyl-(N) modified autologous lymphoctyes. The effector cells generated after 5 days of culture were assayed on a series of either N-modified phytohemagglutinin-stimulated spleen cells or N- modified tumor cells. The results indicated inall cases that both N modification of the targets and H-2 homology between the modified stimulating and target cells are required for lysis to occur. In each case the effector cells were found to lyse N-modified target cells only when there was homology at either or both ends of the major histocompatibility complex (MHC) between the stimulator and target cells. B10.BR lysed targets sharing alleles at K (or K plus I-A) and/or at D. B10.A effector cell specificity was mapped to K (or K plus I-A) and/or the D half of the MHC (D or D plus I-C and/or S). The two regions of specificity determined for B10.D2 effector cells were D (or D plus S plus I-C) and a region not including D of the MHC. C57BL/10 effector cells lysed N-modified targets only if there was target cell H- 2 homology at K, I-A, and I-B or at the D serological region. As in the trinitrophenyl (TNP) system (6) B10.BR and B10.A effector cells lysed targets sharing K end H-2 serological regions greater than target cells sharing D-end serological regions. The C57BL/10 effector cells were shown to react to the K end greater than the D end, which differed from the equal reactivity seen in the TNP system for this strain. The data are consistent with the hypothesis that the antigen recognized by the effector cell includes an altered H-2 serological cell surface product. That the reaction is not "hapten specific" and the H-2 homology is required only for effector:target cell interaction was excluded by the use of two F1 combinations in which lysis of only N-modified target cells sharing the H-2 haplotype with the stimulating parental

  16. Translocation of surface-localized effectors in type III secretion

    PubMed Central

    Edgren, Tomas; Wang-Edgren, Helen; Rosqvist, Roland; Fahlgren, Anna; Wolf-Watz, Hans; Fallman, Maria

    2011-01-01

    Pathogenic Yersinia species suppress the host immune response by using a plasmid-encoded type III secretion system (T3SS) to translocate virulence proteins into the cytosol of the target cells. T3SS-dependent protein translocation is believed to occur in one step from the bacterial cytosol to the target-cell cytoplasm through a conduit created by the T3SS upon target cell contact. Here, we report that T3SS substrates on the surface of Yersinia pseudotuberculosis are translocated into target cells. Upon host cell contact, purified YopH coated on Y. pseudotuberculosis was specifically and rapidly translocated across the target-cell membrane, which led to a physiological response in the infected cell. In addition, translocation of externally added YopH required a functional T3SS and a specific translocation domain in the effector protein. Efficient, T3SS-dependent translocation of purified YopH added in vitro was also observed when using coated Salmonella typhimurium strains, which implies that T3SS-mediated translocation of extracellular effector proteins is conserved among T3SS-dependent pathogens. Our results demonstrate that polarized T3SS-dependent translocation of proteins can be achieved through an intermediate extracellular step that can be reconstituted in vitro. These results indicate that translocation can occur by a different mechanism from the assumed single-step conduit model. PMID:21220342

  17. Intermediate filaments enable pathogen docking to trigger type 3 effector translocation

    PubMed Central

    Russo, Brian C.; Stamm, Luisa M.; Raaben, Matthijs; Kim, Caleb M.; Kahoud, Emily; Robinson, Lindsey R.; Bose, Sayantan; Queiroz, Ana L.; Herrera, Bobby Brooke; Baxt, Leigh A.; Mor-Vaknin, Nirit; Fu, Yang; Molina, Gabriel; Markovitz, David M.; Whelan, Sean P.; Goldberg, Marcia B.

    2016-01-01

    Type 3 secretion systems (T3SSs) of bacterial pathogens translocate bacterial effector proteins that mediate disease into the eukaryotic cytosol. Effectors traverse the plasma membrane through a translocon pore formed by T3SS proteins. In a genome-wide selection, we identified the intermediate filament vimentin as required for infection by the T3SS-dependent pathogen Shigella flexneri. We found that vimentin is required for efficient T3SS translocation of effectors by S. flexneri and other pathogens that use T3SS, Salmonella Typhimurium and Yersinia pseudotuberculosis. Vimentin and the intestinal epithelial intermediate filament keratin 18 interact with the C-terminus of the Shigella translocon pore protein IpaC. Vimentin and its interaction with IpaC are dispensable for pore formation, but are required for stable docking of S. flexneri to cells; moreover, stable docking triggers effector secretion. These findings establish that stable docking of the bacterium specifically requires intermediate filaments, is a process distinct from pore formation, and is a prerequisite for effector secretion. PMID:27572444

  18. Allergic pulmonary inflammation in mice is dependent on eosinophil-induced recruitment of effector T cells

    PubMed Central

    Jacobsen, Elizabeth A.; Ochkur, Sergei I.; Pero, Ralph S.; Taranova, Anna G.; Protheroe, Cheryl A.; Colbert, Dana C.; Lee, Nancy A.; Lee, James J.

    2008-01-01

    The current paradigm surrounding allergen-mediated T helper type 2 (Th2) immune responses in the lung suggests an almost hegemonic role for T cells. Our studies propose an alternative hypothesis implicating eosinophils in the regulation of pulmonary T cell responses. In particular, ovalbumin (OVA)-sensitized/challenged mice devoid of eosinophils (the transgenic line PHIL) have reduced airway levels of Th2 cytokines relative to the OVA-treated wild type that correlated with a reduced ability to recruit effector T cells to the lung. Adoptive transfer of Th2-polarized OVA-specific transgenic T cells (OT-II) alone into OVA-challenged PHIL recipient mice failed to restore Th2 cytokines, airway histopathologies, and, most importantly, the recruitment of pulmonary effector T cells. In contrast, the combined transfer of OT-II cells and eosinophils into PHIL mice resulted in the accumulation of effector T cells and a concomitant increase in both airway Th2 immune responses and histopathologies. Moreover, we show that eosinophils elicit the expression of the Th2 chemokines thymus- and activation-regulated chemokine/CCL17 and macrophage-derived chemokine/CCL22 in the lung after allergen challenge, and blockade of these chemokines inhibited the recruitment of effector T cells. In summary, the data suggest that pulmonary eosinophils are required for the localized recruitment of effector T cells. PMID:18316417

  19. Effector prediction in host-pathogen interaction based on a Markov model of a ubiquitous EPIYA motif

    PubMed Central

    2010-01-01

    Background Effector secretion is a common strategy of pathogen in mediating host-pathogen interaction. Eight EPIYA-motif containing effectors have recently been discovered in six pathogens. Once these effectors enter host cells through type III/IV secretion systems (T3SS/T4SS), tyrosine in the EPIYA motif is phosphorylated, which triggers effectors binding other proteins to manipulate host-cell functions. The objectives of this study are to evaluate the distribution pattern of EPIYA motif in broad biological species, to predict potential effectors with EPIYA motif, and to suggest roles and biological functions of potential effectors in host-pathogen interactions. Results A hidden Markov model (HMM) of five amino acids was built for the EPIYA-motif based on the eight known effectors. Using this HMM to search the non-redundant protein database containing 9,216,047 sequences, we obtained 107,231 sequences with at least one EPIYA motif occurrence and 3115 sequences with multiple repeats of the EPIYA motif. Although the EPIYA motif exists among broad species, it is significantly over-represented in some particular groups of species. For those proteins containing at least four copies of EPIYA motif, most of them are from intracellular bacteria, extracellular bacteria with T3SS or T4SS or intracellular protozoan parasites. By combining the EPIYA motif and the adjacent SH2 binding motifs (KK, R4, Tarp and Tir), we built HMMs of nine amino acids and predicted many potential effectors in bacteria and protista by the HMMs. Some potential effectors for pathogens (such as Lawsonia intracellularis, Plasmodium falciparum and Leishmania major) are suggested. Conclusions Our study indicates that the EPIYA motif may be a ubiquitous functional site for effectors that play an important pathogenicity role in mediating host-pathogen interactions. We suggest that some intracellular protozoan parasites could secrete EPIYA-motif containing effectors through secretion systems similar to the

  20. Kinematic evaluation of end effector design

    NASA Astrophysics Data System (ADS)

    Edwards, Gary W.

    1992-09-01

    The complex, many degree-of-freedom end effectors at the leading edge of technology would be unusable in the sea bottom research environment. Simpler designs are required to provide adequate reliability for subsea use. This work examines selection of end effector designs to achieve optimum grasping ability with minimal mechanical complexity. A new method of calculating grasp stability is developed, incorporating elements of previous works in the field. Programs are developed which evaluate the ability of different end effector configurations to grasp representative objects (a cube, sphere, and infinite cylinder). End effector designs considered had circular palms with fingers located at the periphery, oriented so that each pointed to the center of the palm. The program tested configurations of from 1 to 4 fingers and from 1 to 3 links per finger. Three sets of finger proportions were considered: equal length links, half length links, and anthropomorphic proportions. The 2 finger, 2 link per finger configuration was determined to be the optimum design, and the half length proportions were selected as the best set of proportions.

  1. Minimal Mimicry: Mere Effector Matching Induces Preference

    ERIC Educational Resources Information Center

    Sparenberg, Peggy; Topolinski, Sascha; Springer, Anne; Prinz, Wolfgang

    2012-01-01

    Both mimicking and being mimicked induces preference for a target. The present experiments investigate the minimal sufficient conditions for this mimicry-preference link to occur. We argue that mere effector matching between one's own and the other person's movement is sufficient to induce preference, independent of which movement is actually…

  2. Cellular effector mechanisms against Plasmodium liver stages.

    PubMed

    Frevert, Ute; Nardin, Elizabeth

    2008-10-01

    Advances in our understanding of the molecular and cell biology of the malaria parasite have led to new vaccine development efforts resulting in a pipeline of over 40 candidates undergoing clinical phase I-III trials. Vaccine-induced CD4+ and CD8+ T cells specific for pre-erythrocytic stage antigens have been found to express cytolytic and multi-cytokine effector functions that support a key role for these T cells within the hepatic environment. However, little is known of the cellular interactions that occur during the effector phase in which the intracellular hepatic stage of the parasite is targeted and destroyed. This review focuses on cell biological aspects of the interaction between malaria-specific effector cells and the various antigen-presenting cells that are known to exist within the liver, including hepatocytes, dendritic cells, Kupffer cells, stellate cells and sinusoidal endothelia. Considering the unique immune properties of the liver, it is conceivable that these different hepatic antigen-presenting cells fulfil distinct but complementary roles during the effector phase against Plasmodium liver stages.

  3. Minimal mimicry: mere effector matching induces preference.

    PubMed

    Sparenberg, Peggy; Topolinski, Sascha; Springer, Anne; Prinz, Wolfgang

    2012-12-01

    Both mimicking and being mimicked induces preference for a target. The present experiments investigate the minimal sufficient conditions for this mimicry-preference link to occur. We argue that mere effector matching between one's own and the other person's movement is sufficient to induce preference, independent of which movement is actually performed. In Experiments 1 and 2, participants moved either their arms or legs, and watched avatars that moved either their arms or legs, respectively, without any instructions to mimic. The executed movements themselves and their pace were completely different between participants (fast circular movements) and targets (slow linear movements). Participants preferred avatars that moved the same body part as they did over avatars that moved a different body part. In Experiment 3, using human targets and differently paced movements, movement similarity was manipulated in addition to effector overlap (moving forward-backward or sideways with arms or legs, respectively). Only effector matching, but not movement matching, influenced preference ratings. These findings suggest that mere effector overlap is sufficient to trigger preference by mimicry.

  4. Minimal Mimicry: Mere Effector Matching Induces Preference

    ERIC Educational Resources Information Center

    Sparenberg, Peggy; Topolinski, Sascha; Springer, Anne; Prinz, Wolfgang

    2012-01-01

    Both mimicking and being mimicked induces preference for a target. The present experiments investigate the minimal sufficient conditions for this mimicry-preference link to occur. We argue that mere effector matching between one's own and the other person's movement is sufficient to induce preference, independent of which movement is actually…

  5. MARTX toxins as effector delivery platforms.

    PubMed

    Gavin, Hannah E; Satchell, Karla J F

    2015-12-01

    Bacteria frequently manipulate their host environment via delivery of microbial 'effector' proteins to the cytosol of eukaryotic cells. In the case of the multifunctional autoprocessing repeats-in-toxins (MARTX) toxin, this phenomenon is accomplished by a single, >3500 amino acid polypeptide that carries information for secretion, translocation, autoprocessing and effector activity. MARTX toxins are secreted from bacteria by dedicated Type I secretion systems. The released MARTX toxins form pores in target eukaryotic cell membranes for the delivery of up to five cytopathic effectors, each of which disrupts a key cellular process. Targeted cellular processes include modulation or modification of small GTPases, manipulation of host cell signaling and disruption of cytoskeletal integrity. More recently, MARTX toxins have been shown to be capable of heterologous protein translocation. Found across multiple bacterial species and genera--frequently in pathogens lacking Type 3 or Type 4 secretion systems--MARTX toxins in multiple cases function as virulence factors. Innovative research at the intersection of toxin biology and bacterial genetics continues to elucidate the intricacies of the toxin as well as the cytotoxic mechanisms of its diverse effector collection. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  6. Evaluating the cytotoxicity of innate immune effector cells using the GrB ELISPOT assay

    PubMed Central

    Shafer-Weaver, Kimberly A; Sayers, Thomas; Kuhns, Douglas B; Strobl, Susan L; Burkett, Mark W; Baseler, Michael; Malyguine, Anatoli

    2004-01-01

    Background This study assessed the Granzyme B (GrB) ELISPOT as a viable alternative to the 51Cr-release assay for measuring cytotoxic activity of innate immune effector cells. We strategically selected the GrB ELISPOT assay because GrB is a hallmark effector molecule of cell-mediated destruction of target cells. Methods We optimized the GrB ELISPOT assay using the human-derived TALL-104 cytotoxic cell line as effectors against K562 target cells. Titration studies were performed to assess whether the ELISPOT assay could accurately enumerate the number of GrB-secreting effector cells. TALL-104 were treated with various secretion inhibitors and utilized in the GrB ELISPOT to determine if GrB measured in the ELISPOT was due to degranulation of effector cells. Additionally, CD107a expression on effector cells after effector-target interaction was utilized to further confirm the mechanism of GrB release by TALL-104 and lymphokine-activated killer (LAK) cells. Direct comparisons between the GrB ELISPOT, the IFN-γ ELISPOT and the standard 51Cr-release assays were made using human LAK cells. Results Titration studies demonstrated a strong correlation between the number of TALL-104 and LAK effector cells and the number of GrB spots per well. GrB secretion was detectable within 10 min of effector-target contact with optimal secretion observed at 3–4 h; in contrast, optimal IFN-γ secretion was not observed until 24 h. The protein secretion inhibitor, brefeldin A, did not inhibit the release of GrB but did abrogate IFN-γ production by TALL-104 cells. GrB secretion was abrogated by BAPTA-AM (1,2-bis-(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid tetra(acetoxymethyl) ester), which sequesters intracellular Ca2+, thereby preventing degranulation. The number of effector cells expressing the degranulation associated glycoprotein CD107a increased after interaction with target cells and correlated with the stimulated release of GrB measured in the ELISPOT assay. Conclusions

  7. Modulation of innate immunity by Toxoplasma gondii virulence effectors

    PubMed Central

    Hunter, Christopher A.; Sibley, L. David

    2013-01-01

    Preface Toxoplasma gondii is a common parasite of animals and humans that can cause serious opportunistic infections. However, the majority of infections are asymptomatic possibly because the organism has co-evolved with its many vertebrate hosts and has developed multiple strategies to persist asymptomatically for the lifetime of the host. Over the past two decades, infection studies in the mouse, combined with forward genetic approaches aimed at unraveling the molecular basis of infection, have revealed that T. gondii virulence is mediated, in part, by secretion of effector proteins into the host cell during invasion. Here, we review recent advances that illustrate how these virulence factors disarm innate immunity and promote survival of the parasite. PMID:23070557

  8. Direct targeting of Rab-GTPase-effector interactions.

    PubMed

    Spiegel, Jochen; Cromm, Philipp M; Itzen, Aymelt; Goody, Roger S; Grossmann, Tom N; Waldmann, Herbert

    2014-02-24

    Small GTPases are molecular switches using GDP/GTP alternation to control numerous vital cellular processes. Although aberrant function and regulation of GTPases are implicated in various human diseases, direct targeting of this class of proteins has proven difficult, as GTPase signaling and regulation is mediated by extensive and shallow protein interfaces. Here we report the development of inhibitors of protein-protein interactions involving Rab proteins, a subfamily of GTPases, which are key regulators of vesicular transport. Hydrocarbon-stapled peptides were designed based on crystal structures of Rab proteins bound to their interaction partners. These modified peptides exhibit significantly increased affinities and include a stapled peptide (StRIP3) that selectively binds to activated Rab8a and inhibits a Rab8a-effector interaction in vitro.

  9. 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

  10. Adoptive cell therapy: genetic modification to redirect effector cell specificity.

    PubMed

    Morgan, Richard A; Dudley, Mark E; Rosenberg, Steven A

    2010-01-01

    Building on the principals that the adoptive transfer of T cells can lead to the regression of established tumors in humans, investigators are now further manipulating these cells using genetic engineering. Two decades of human gene transfer experiments have resulted in the translation of laboratory technology into robust clinical applications. The purpose of this review is to give the reader an introduction to the 2 major approaches being developed to redirect effector T-cell specificity. Primary human T cells can be engineered to express exogenous T-cell receptors or chimeric antigen receptors directed against multiple human tumor antigens. Initial clinical trial results have demonstrated that both T-cell receptor- and chimeric antigen receptor-engineered T cells can be administered to cancer patients and mediate tumor regression.

  11. TAL effector driven induction of a SWEET gene confers susceptibility to bacterial blight of cotton

    PubMed Central

    Cox, Kevin L.; Meng, Fanhong; Wilkins, Katherine E.; Li, Fangjun; Wang, Ping; Booher, Nicholas J.; Carpenter, Sara C. D.; Chen, Li-Qing; Zheng, Hui; Gao, Xiquan; Zheng, Yi; Fei, Zhangjun; Yu, John Z.; Isakeit, Thomas; Wheeler, Terry; Frommer, Wolf B.; He, Ping; Bogdanove, Adam J.; Shan, Libo

    2017-01-01

    Transcription activator-like (TAL) effectors from Xanthomonas citri subsp. malvacearum (Xcm) are essential for bacterial blight of cotton (BBC). Here, by combining transcriptome profiling with TAL effector-binding element (EBE) prediction, we show that GhSWEET10, encoding a functional sucrose transporter, is induced by Avrb6, a TAL effector determining Xcm pathogenicity. Activation of GhSWEET10 by designer TAL effectors (dTALEs) restores virulence of Xcm avrb6 deletion strains, whereas silencing of GhSWEET10 compromises cotton susceptibility to infections. A BBC-resistant line carrying an unknown recessive b6 gene bears the same EBE as the susceptible line, but Avrb6-mediated induction of GhSWEET10 is reduced, suggesting a unique mechanism underlying b6-mediated resistance. We show via an extensive survey of GhSWEET transcriptional responsiveness to different Xcm field isolates that additional GhSWEETs may also be involved in BBC. These findings advance our understanding of the disease and resistance in cotton and may facilitate the development cotton with improved resistance to BBC. PMID:28537271

  12. Optimal effector functions in human natural killer cells rely upon autocrine bone morphogenetic protein signaling.

    PubMed

    Robson, Neil C; Hidalgo, Laura; McAlpine, Tristan; Wei, Heng; Martínez, Víctor G; Entrena, Ana; Melen, Gustavo J; MacDonald, Andrew S; Phythian-Adams, Alexander; Sacedón, Rosa; Maraskovsky, Eugene; Cebon, Jonathan; Ramírez, Manuel; Vicente, Angeles; Varas, Alberto

    2014-09-15

    Natural killer (NK) cells are critical for innate tumor immunity due to their specialized ability to recognize and kill neoplastically transformed cells. However, NK cells require a specific set of cytokine-mediated signals to achieve optimal effector function. Th1-associated cytokines promote effector functions that are inhibited by the prototypic Th2 cytokine IL4 and the TGFβ superfamily members TGFβ1 and activin-A. Interestingly, the largest subgroup of the TGFβ superfamily are the bone morphogenetic proteins (BMP), but the effects of BMP signaling on NK cell effector functions have not been evaluated. Here, we demonstrate that blood-circulating NK cells express type I and II BMP receptors, BMP-2 and BMP-6 ligands, and phosphorylated isoforms of Smad-1/-5/-8, which mediate BMP family member signaling. In opposition to the inhibitory effects of TGFβ1 or activin-A, autocrine BMP signaling was supportive to NK cell function. Mechanistic investigations in cytokine and TLR-L-activated NK cells revealed that BMP signaling optimized IFNγ and global cytokine and chemokine production, phenotypic activation and proliferation, and autologous dendritic cell activation and target cytotoxicity. Collectively, our findings identify a novel auto-activatory pathway that is essential for optimal NK cell effector function, one that might be therapeutically manipulated to help eradicate tumors. Cancer Res; 74(18); 5019-31. ©2014 AACR.

  13. Ionic immune suppression within the tumour microenvironment limits T cell effector function

    PubMed Central

    Eil, Robert; Vodnala, Suman K; Clever, David; Klebanoff, Christopher A; Sukumar, Madhusudhanan; Pan, Jenny H; Palmer, Douglas C; Gros, Alena; Yamamoto, Tori N; Patel, Shashank J; Guittard, Geoffrey C; Yu, Zhiya; Carbonaro, Valentina; Okkenhaug, Klaus; Schrump, David S; Linehan, W Marston; Roychoudhuri, Rahul; Restifo, Nicholas P

    2016-01-01

    Tumours progress despite being infiltrated by tumour-specific effector T cells1. Tumours contain areas of cellular necrosis, which is associated with poor survival in a variety of cancers2. Here, we show that necrosis releases an intracellular ion, potassium, into the extracellular fluid of mouse and human tumours causing profound suppression of T cell effector function. We find that elevations in the extracellular potassium concentration [K+]e act to impair T cell receptor (TCR)-driven Akt-mTOR phosphorylation and effector programmes, this potassium-mediated suppression of Akt-mTOR signalling and T cell function is dependent upon the activity of the serine/threonine phosphatase PP2A3,4. While the suppressive effect mediated by elevated [K+]e is independent of changes in plasma membrane potential (Vm), it does require an increase in intracellular potassium ([K+]i). Concordantly, ionic reprogramming of tumour-specific T cells through overexpression of the potassium channel Kv1.3 lowers [K+]i and improves effector functions in vitro and in vivo. Consequently, Kv1.3 T cell overexpression enhances tumour clearance and survival of melanoma-bearing mice. These results uncover a previously undescribed ionic checkpoint blocking T cell function within tumours and identify new strategies for cancer immunotherapy. PMID:27626381

  14. Optimal effector functions in human natural killer cells rely upon autocrine bone morphogenetic protein signaling

    PubMed Central

    Mc Alpine, Tristan; Wei, Heng; Martínez, Víctor G.; Entrena, Ana; Melen, Gustavo J; MacDonald, Andrew S.; Phythian-Adams, Alexander; Sacedón, Rosa; Maraskovsky, Eugene; Cebon, Jonathan; Ramírez, Manuel

    2014-01-01

    Natural killer (NK) cells are critical for innate tumor immunity due to their specialized ability to recognize and kill neoplastically transformed cells. However, NK cells require a specific set of cytokine-mediated signals to achieve optimal effector function. Th1-associated cytokines promote effector functions which are inhibited by the prototypic Th-2 cytokine IL-4 and the TGF-β superfamily members TGF-β1 and activin-A. Interestingly, the largest subgroup of the TGF-β superfamily are the bone morphogenetic proteins (BMP), but the effects of BMP signaling to NK cell effector functions have not been evaluated. Here we demonstrate that blood-circulating NK cells express type I and II BMP receptors, BMP-2 and BMP-6 ligands, and phosphorylated isoforms of Smad-1/-5/-8 which mediate BMP family member signaling. In opposition to the inhibitory effects of TGF-β1 or activin-A, autocrine BMP signaling was supportive to NK cell function. Mechanistic investigations in cytokine and TLR-L activated NK cells revealed that BMP signaling optimized IFN-γ and global cytokine and chemokine production; phenotypic activation and proliferation; autologous DC activation and target cytotoxicity. Collectively, our findings identify a novel auto-activatory pathway that is essential for optimal NK cell effector function, one which might be therapeutically manipulated to help eradicate tumors. PMID:25038228

  15. Role of chemokines and chemokine receptors in shaping the effector phase of the antitumor immune response.

    PubMed

    Franciszkiewicz, Katarzyna; Boissonnas, Alexandre; Boutet, Marie; Combadière, Christophe; Mami-Chouaib, Fathia

    2012-12-15

    Immune system-mediated eradication of neoplastic cells requires induction of a strong long-lasting antitumor T-cell response. However, generation of tumor-specific effector T cells does not necessarily result in tumor clearance. CTL must first be able to migrate to the tumor site, infiltrate the tumor tissue, and interact with the target to finally trigger effector functions indispensable for tumor destruction. Chemokines are involved in circulation, homing, retention, and activation of immunocompetent cells. Although some of them are known to contribute to tumor growth and metastasis, others are responsible for changes in the tumor microenvironment that lead to extensive infiltration of lymphocytes, resulting in tumor eradication. Given their chemoattractive and activating properties, a role for chemokines in the development of the effector phase of the antitumor immune response has been suggested. Here, we emphasize the role of the chemokine-chemokine receptor network at multiple levels of the T-cell-mediated antitumor immune response. The identification of chemokine-dependent molecular mechanisms implicated in tumor-specific CTL trafficking, retention, and regulation of their in situ effector functions may offer new perspectives for development of innovative immunotherapeutic approaches to cancer treatment.

  16. A catalogue of the effector secretome of plant pathogenic oomycetes.

    PubMed

    Kamoun, Sophien

    2006-01-01

    The oomycetes form a phylogenetically distinct group of eukaryotic microorganisms that includes some of the most notorious pathogens of plants. Oomycetes accomplish parasitic colonization of plants by modulating host cell defenses through an array of disease effector proteins. The biology of effectors is poorly understood but tremendous progress has been made in recent years. This review classifies and catalogues the effector secretome of oomycetes. Two classes of effectors target distinct sites in the host plant: Apoplastic effectors are secreted into the plant extracellular space, and cytoplasmic effectors are translocated inside the plant cell, where they target different subcellular compartments. Considering that five species are undergoing genome sequencing and annotation, we are rapidly moving toward genome-wide catalogues of oomycete effectors. Already, it is evident that the effector secretome of pathogenic oomycetes is more complex than expected, with perhaps several hundred proteins dedicated to manipulating host cell structure and function.

  17. The RAS-Effector Interface: Isoform-Specific Differences in the Effector Binding Regions

    PubMed Central

    Nakhaeizadeh, Hossein; Amin, Ehsan; Nakhaei-Rad, Saeideh; Dvorsky, Radovan; Ahmadian, Mohammad Reza

    2016-01-01

    RAS effectors specifically interact with the GTP-bound form of RAS in response to extracellular signals and link them to downstream signaling pathways. The molecular nature of effector interaction by RAS is well-studied but yet still incompletely understood in a comprehensive and systematic way. Here, structure-function relationships in the interaction between different RAS proteins and various effectors were investigated in detail by combining our in vitro data with in silico data. Equilibrium dissociation constants were determined for the binding of HRAS, KRAS, NRAS, RRAS1 and RRAS2 to both the RAS binding (RB) domain of CRAF and PI3Kα, and the RAS association (RA) domain of RASSF5, RALGDS and PLCε, respectively, using fluorescence polarization. An interaction matrix, constructed on the basis of available crystal structures, allowed identification of hotspots as critical determinants for RAS-effector interaction. New insights provided by this study are the dissection of the identified hotspots in five distinct regions (R1 to R5) in spite of high sequence variability not only between, but also within, RB/RA domain-containing effectors proteins. Finally, we propose that intermolecular β-sheet interaction in R1 is a central recognition region while R3 may determine specific contacts of RAS versus RRAS isoforms with effectors. PMID:27936046

  18. Roadmap for future research on plant pathogen effectors.

    PubMed

    Alfano, James R

    2009-11-01

    Bacterial and eukaryotic plant pathogens deliver effector proteins into plant cells to promote pathogenesis. Bacterial pathogens containing type III protein secretion systems are known to inject many of these effectors into plant cells. More recently, oomycete pathogens have been shown to possess a large family of effectors containing the RXLR motif, and many effectors are also being discovered in fungal pathogens. Although effector activities are largely unknown, at least a subset suppress plant immunity. A plethora of new plant pathogen genomes that will soon be available thanks to next-generation sequencing technologies will allow the identification of many more effectors. This article summarizes the key approaches used to identify plant pathogen effectors, many of which will continue to be useful for future effector discovery. Thus, it can be viewed as a 'roadmap' for effector and effector target identification. Because effectors can be used as tools to elucidate components of innate immunity, advances in our understanding of effectors and their targets should lead to improvements in agriculture.

  19. Structures of the flax-rust effector AvrM reveal insights into the molecular basis of plant-cell entry and effector-triggered immunity.

    PubMed

    Ve, Thomas; Williams, Simon J; Catanzariti, Ann-Maree; Rafiqi, Maryam; Rahman, Motiur; Ellis, Jeffrey G; Hardham, Adrienne R; Jones, David A; Anderson, Peter A; Dodds, Peter N; Kobe, Bostjan

    2013-10-22

    Fungal and oomycete pathogens cause some of the most devastating diseases in crop plants, and facilitate infection by delivering a large number of effector molecules into the plant cell. AvrM is a secreted effector protein from flax rust (Melampsora lini) that can internalize into plant cells in the absence of the pathogen, binds to phosphoinositides (PIPs), and is recognized directly by the resistance protein M in flax (Linum usitatissimum), resulting in effector-triggered immunity. We determined the crystal structures of two naturally occurring variants of AvrM, AvrM-A and avrM, and both reveal an L-shaped fold consisting of a tandem duplicated four-helix motif, which displays similarity to the WY domain core in oomycete effectors. In the crystals, both AvrM variants form a dimer with an unusual nonglobular shape. Our functional analysis of AvrM reveals that a hydrophobic surface patch conserved between both variants is required for internalization into plant cells, whereas the C-terminal coiled-coil domain mediates interaction with M. AvrM binding to PIPs is dependent on positive surface charges, and mutations that abrogate PIP binding have no significant effect on internalization, suggesting that AvrM binding to PIPs is not essential for transport of AvrM across the plant membrane. The structure of AvrM and the identification of functionally important surface regions advance our understanding of the molecular mechanisms underlying how effectors enter plant cells and how they are detected by the plant immune system.

  20. The Biological Functions of T Helper 17 Cell Effector Cytokines in Inflammation

    PubMed Central

    Ouyang, Wenjun; Kolls, Jay K.; Zheng, Yan

    2012-01-01

    T helper 17 (Th17) cells belong to a recently identified T helper subset, in addition to the traditional Th1 and Th2 subsets. These cells are characterized as preferential producers of interleukin-17A (IL-17A), IL-17F, IL-21, and IL-22. Th17 cells and their effector cytokines mediate host defensive mechanisms to various infections, especially extracellular bacteria infections, and are involved in the pathogenesis of many autoimmune diseases. The receptors for IL-17 and IL-22 are broadly expressed on various epithelial tissues. The effector cytokines of Th17 cells, therefore, mediate the crucial crosstalk between immune system and tissues, and play indispensable roles in tissue immunity. PMID:18400188

  1. Natural effector T lymphocytes in normal mice.

    PubMed Central

    Pereira, P; Larsson, E L; Forni, L; Bandeira, A; Coutinho, A

    1985-01-01

    The "natural" T-cell activity in normal unimmunized mice was studied. By double-parameter fluorescence-activated cell sorter analysis, it was found that 5-10% of all splenic Lyt-2+ and L3T4+ lymphocytes are large, of which more than half are in mitotic cycle. In contrast with small resting cells of the same phenotype, activated (large) T cells isolated from normal mice are functional effector cells: L3T4+ large cells induce normal B lymphocytes into proliferation and antibody secretion, while large Lyt-2+ cells efficiently suppress B-lymphocyte responses. No effector cell cytolytic activity could be detected among naturally activated T cells. The significance of these findings for the internal activity in the normal immune system is discussed. PMID:2933744

  2. Wheat PR-1 proteins are targeted by necrotrophic pathogen effector proteins.

    PubMed

    Breen, Susan; Williams, Simon J; Winterberg, Britta; Kobe, Bostjan; Solomon, Peter S

    2016-10-01

    Recent studies have identified that proteinaceous effectors secreted by Parastagonospora nodorum are required to cause disease on wheat. These effectors interact in a gene-for-gene manner with host-dominant susceptibilty loci, resulting in disease. However, whilst the requirement of these effectors for infection is clear, their mechanisms of action remain poorly understood. A yeast-two-hybrid library approach was used to search for wheat proteins that interacted with the necrotrophic effector SnTox3. Using this strategy we indentified an interaction between SnTox3 and the wheat pathogenicity-related protein TaPR-1-1, and confirmed it by in-planta co-immunprecipitation. PR-1 proteins represent a large family (23 in wheat) of proteins that are upregulated early in the defence response; however, their function remains ellusive. Interestingly, the P. nodorum effector SnToxA has recently been shown to interact specifically with TaPR-1-5. Our analysis of the SnTox3-TaPR-1 interaction demonstrated that SnTox3 can interact with a broader range of TaPR-1 proteins. Based on these data we utilised homology modeling to predict, and validate, regions on TaPR-1 proteins that are likely to be involved in the SnTox3 interaction. Precipitating from this work, we identified that a PR-1-derived defence signalling peptide from the C-terminus of TaPR-1-1, known as CAPE1, enhanced the infection of wheat by P. nodorum in an SnTox3-dependent manner, but played no role in ToxA-mediated disease. Collectively, our data suggest that P. nodorum has evolved unique effectors that target a common host-protein involved in host defence, albeit with different mechanisms and potentially outcomes.

  3. Novel Control Effectors for Truss Braced Wing

    NASA Technical Reports Server (NTRS)

    White, Edward V.; Kapania, Rakesh K.; Joshi, Shiv

    2015-01-01

    At cruise flight conditions very high aspect ratio/low sweep truss braced wings (TBW) may be subject to design requirements that distinguish them from more highly swept cantilevered wings. High aspect ratio, short chord length and relative thinness of the airfoil sections all contribute to relatively low wing torsional stiffness. This may lead to aeroelastic issues such as aileron reversal and low flutter margins. In order to counteract these issues, high aspect ratio/low sweep wings may need to carry additional high speed control effectors to operate when outboard ailerons are in reversal and/or must carry additional structural weight to enhance torsional stiffness. The novel control effector evaluated in this study is a variable sweep raked wing tip with an aileron control surface. Forward sweep of the tip allows the aileron to align closely with the torsional axis of the wing and operate in a conventional fashion. Aft sweep of the tip creates a large moment arm from the aileron to the wing torsional axis greatly enhancing aileron reversal. The novelty comes from using this enhanced and controllable aileron reversal effect to provide roll control authority by acting as a servo tab and providing roll control through intentional twist of the wing. In this case the reduced torsional stiffness of the wing becomes an advantage to be exploited. The study results show that the novel control effector concept does provide roll control as described, but only for a restricted class of TBW aircraft configurations. For the configuration studied (long range, dual aisle, Mach 0.85 cruise) the novel control effector provides significant benefits including up to 12% reduction in fuel burn.

  4. PCNASUMO and Srs2: a model SUMO substrate-effector pair.

    PubMed

    Ulrich, H D

    2007-12-01

    Attachment of the SUMO (small ubiquitin-related modifier) to the replication factor PCNA (proliferating-cell nuclear antigen) in the budding yeast has been shown to recruit a helicase, Srs2, to active replication forks, which in turn prevents unscheduled recombination events. In the present review, I will discuss how the interaction between SUMOylated PCNA and Srs2 serves as an example for a mechanism by which SUMO modulates the properties of its targets and mediates the activation of downstream effector proteins.

  5. Impact of end effector technology on telemanipulation performance

    NASA Technical Reports Server (NTRS)

    Bejczy, A. K.; Szakaly, Z.; Ohm, T.

    1990-01-01

    Generic requirements for end effector design are briefly summarized as derived from generic functional and operational requirements. Included is a brief summary of terms and definitions related to end effector technology. The second part contains a brief overview of end effector technology work as JPL during the past ten years, with emphasis on the evolution of new mechanical, sensing and control capabilities of end effectors. The third and major part is devoted to the description of current end effector technology. The ongoing work addresses mechanical, sensing and control details with emphasis on mechanical ruggedness, increased resolution in sensing, and close electronic and control integration with overall telemanipulator control system.

  6. Nematode effector proteins: an emerging paradigm of parasitism.

    PubMed

    Mitchum, Melissa G; Hussey, Richard S; Baum, Thomas J; Wang, Xiaohong; Elling, Axel A; Wubben, Martin; Davis, Eric L

    2013-09-01

    Phytonematodes use a stylet and secreted effectors to modify host cells and ingest nutrients to support their growth and development. The molecular function of nematode effectors is currently the subject of intense investigation. In this review, we summarize our current understanding of nematode effectors, with a particular focus on proteinaceous stylet-secreted effectors of sedentary endoparasitic phytonematodes, for which a wealth of information has surfaced in the past 10 yr. We provide an update on the effector repertoires of several of the most economically important genera of phytonematodes and discuss current approaches to dissecting their function. Lastly, we highlight the latest breakthroughs in effector discovery that promise to shed new light on effector diversity and function across the phylum Nematoda. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

  7. Legionella Metaeffector Exploits Host Proteasome to Temporally Regulate Cognate Effector

    PubMed Central

    Kubori, Tomoko; Shinzawa, Naoaki; Kanuka, Hirotaka; Nagai, Hiroki

    2010-01-01

    Pathogen-associated secretion systems translocate numerous effector proteins into eukaryotic host cells to coordinate cellular processes important for infection. Spatiotemporal regulation is therefore important for modulating distinct activities of effectors at different stages of infection. Here we provide the first evidence of “metaeffector,” a designation for an effector protein that regulates the function of another effector within the host cell. Legionella LubX protein functions as an E3 ubiquitin ligase that hijacks the host proteasome to specifically target the bacterial effector protein SidH for degradation. Delayed delivery of LubX to the host cytoplasm leads to the shutdown of SidH within the host cells at later stages of infection. This demonstrates a sophisticated level of coevolution between eukaryotic cells and L. pneumophila involving an effector that functions as a key regulator to temporally coordinate the function of a cognate effector protein. PMID:21151961

  8. PRC2 Epigenetically Silences Th1-Type Chemokines to Suppress Effector T-Cell Trafficking in Colon Cancer.

    PubMed

    Nagarsheth, Nisha; Peng, Dongjun; Kryczek, Ilona; Wu, Ke; Li, Wei; Zhao, Ende; Zhao, Lili; Wei, Shuang; Frankel, Timothy; Vatan, Linda; Szeliga, Wojciech; Dou, Yali; Owens, Scott; Marquez, Victor; Tao, Kaixiong; Huang, Emina; Wang, Guobin; Zou, Weiping

    2016-01-15

    Infiltration of tumors with effector T cells is positively associated with therapeutic efficacy and patient survival. However, the mechanisms underlying effector T-cell trafficking to the tumor microenvironment remain poorly understood in patients with colon cancer. The polycomb repressive complex 2 (PRC2) is involved in cancer progression, but the regulation of tumor immunity by epigenetic mechanisms has yet to be investigated. In this study, we examined the relationship between the repressive PRC2 machinery and effector T-cell trafficking. We found that PRC2 components and demethylase JMJD3-mediated histone H3 lysine 27 trimethylation (H3K27me3) repress the expression and subsequent production of Th1-type chemokines CXCL9 and CXCL10, mediators of effector T-cell trafficking. Moreover, the expression levels of PRC2 components, including EZH2, SUZ12, and EED, were inversely associated with those of CD4, CD8, and Th1-type chemokines in human colon cancer tissue, and this expression pattern was significantly associated with patient survival. Collectively, our findings reveal that PRC2-mediated epigenetic silencing is not only a crucial oncogenic mechanism, but also a key circuit controlling tumor immunosuppression. Therefore, targeting epigenetic programs may have significant implications for improving the efficacy of current cancer immunotherapies relying on effective T-cell-mediated immunity at the tumor site.

  9. Uptake of the Fusarium Effector Avr2 by Tomato Is Not a Cell Autonomous Event

    PubMed Central

    Di, Xiaotang; Gomila, Jo; Ma, Lisong; van den Burg, Harrold A.; Takken, Frank L. W.

    2016-01-01

    Pathogens secrete effector proteins to manipulate the host for their own proliferation. Currently it is unclear whether the uptake of effector proteins from extracellular spaces is a host autonomous process. We study this process using the Avr2 effector protein from Fusarium oxysporum f. sp. lycopersici (Fol). Avr2 is an important virulence factor that is secreted into the xylem sap of tomato following infection. Besides that, it is also an avirulence factor triggering immune responses in plants carrying the I-2 resistance gene. Recognition of Avr2 by I-2 occurs inside the plant nucleus. Here, we show that pathogenicity of an Avr2 knockout Fusarium (FolΔAvr2) strain is fully complemented on transgenic tomato lines that express either a secreted (Avr2) or cytosolic Avr2 (ΔspAvr2) protein, indicating that Avr2 exerts its virulence functions inside the host cells. Furthermore, our data imply that secreted Avr2 is taken up from the extracellular spaces in the presence of the fungus. Grafting studies were performed in which scions of I-2 tomato plants were grafted onto either a ΔspAvr2 or on an Avr2 rootstock. Although the Avr2 protein could readily be detected in the xylem sap of the grafted plant tissues, no I-2-mediated immune responses were induced suggesting that I-2-expressing tomato cells cannot autonomously take up the effector protein from the xylem sap. Additionally, ΔspAvr2 and Avr2 plants were crossed with I-2 plants. Whereas ΔspAvr2/I-2 F1 plants showed a constitutive immune response, immunity was not triggered in the Avr2/I-2 plants confirming that Avr2 is not autonomously taken up from the extracellular spaces to trigger I-2. Intriguingly, infiltration of Agrobacterium tumefaciens in leaves of Avr2/I-2 plants triggered I-2 mediated cell death, which indicates that Agrobacterium triggers effector uptake. To test whether, besides Fol, effector uptake could also be induced by other fungal pathogens the ΔspAvr2 and Avr2 transgenic lines were inoculated

  10. The Machinery at Endoplasmic Reticulum-Plasma Membrane Contact Sites Contributes to Spatial Regulation of Multiple Legionella Effector Proteins

    PubMed Central

    Hubber, Andree; Arasaki, Kohei; Nakatsu, Fubito; Hardiman, Camille; Lambright, David; De Camilli, Pietro; Nagai, Hiroki; Roy, Craig R.

    2014-01-01

    The Dot/Icm system of the intracellular pathogen Legionella pneumophila has the capacity to deliver over 270 effector proteins into host cells during infection. Important questions remain as to spatial and temporal mechanisms used to regulate such a large array of virulence determinants after they have been delivered into host cells. Here we investigated several L. pneumophila effector proteins that contain a conserved phosphatidylinositol-4-phosphate (PI4P)-binding domain first described in the effector DrrA (SidM). This PI4P binding domain was essential for the localization of effectors to the early L. pneumophila-containing vacuole (LCV), and DrrA-mediated recruitment of Rab1 to the LCV required PI4P-binding activity. It was found that the host cell machinery that regulates sites of contact between the plasma membrane (PM) and the endoplasmic reticulum (ER) modulates PI4P dynamics on the LCV to control localization of these effectors. Specifically, phosphatidylinositol-4-kinase IIIα (PI4KIIIα) was important for generating a PI4P signature that enabled L. pneumophila effectors to localize to the PM-derived vacuole, and the ER-associated phosphatase Sac1 was involved in metabolizing the PI4P on the vacuole to promote the dissociation of effectors. A defect in L. pneumophila replication in macrophages deficient in PI4KIIIα was observed, highlighting that a PM-derived PI4P signature is critical for biogenesis of a vacuole that supports intracellular multiplication of L. pneumophila. These data indicate that PI4P metabolism by enzymes controlling PM-ER contact sites regulate the association of L. pneumophila effectors to coordinate early stages of vacuole biogenesis. PMID:24992562

  11. Macroautophagy regulates energy metabolism during effector T cell activation.

    PubMed

    Hubbard, Vanessa M; Valdor, Rut; Patel, Bindi; Singh, Rajat; Cuervo, Ana Maria; Macian, Fernando

    2010-12-15

    Macroautophagy is a highly conserved mechanism of lysosomal-mediated protein degradation that plays a key role in maintaining cellular homeostasis by recycling amino acids, reducing the amount of damaged proteins, and regulating protein levels in response to extracellular signals. We have found that macroautophagy is induced after effector T cell activation. Engagement of the TCR and CD28 results in enhanced microtubule-associated protein 1 light chain 3 (LC3) processing, increased numbers of LC3-containing vesicles, and increased LC3 flux, indicating active autophagosome formation and clearance. The autophagosomes formed in stimulated T cells actively fuse with lysosomes to degrade their cargo. Using a conditional KO mouse model where Atg7, a critical gene for macroautophagy, is specifically deleted in T cells, we have found that macroautophagy-deficient effector Th cells have defective IL-2 and IFN-γ production and reduced proliferation after stimulation, with no significant increase in apoptosis. We have found that ATP generation is decreased when autophagy is blocked, and defects in activation-induced cytokine production are restored when an exogenous energy source is added to macroautophagy-deficient T cells. Furthermore, we present evidence showing that the nature of the cargo inside autophagic vesicles found in resting T cells differs from the cargo of autophagosomes in activated T cells, where mitochondria and other organelles are selectively excluded. These results suggest that macroautophagy is an actively regulated process in T cells that can be induced in response to TCR engagement to accommodate the bioenergetic requirements of activated T cells.

  12. TGF-β1 Attenuates the Acquisition and Expression of Effector Function by Tumor Antigen-Specific Human Memory CD8 T Cells

    PubMed Central

    Ahmadzadeh, Mojgan; Rosenberg, Steven A.

    2008-01-01

    TGF-β1 is a potent immunoregulatory cytokine. However, its impact on the generation and effector function of Ag-specific human effector memory CD8 T cells had not been evaluated. Using Ag-specific CD8 T cells derived from melanoma patients immunized with the gp100 melanoma Ag, we demonstrate that the addition of TGF-β1 to the initial Ag activation cultures attenuated the gain of effector function by Ag-specific memory CD8 T cells while the phenotypic changes associated with activation and differentiation into effector memory were comparable to control cultures. These activated memory CD8 T cells consistently expressed lower mRNA levels for T-bet, suggesting a mechanism for TGF-β1-mediated suppression of gain of effector function in memory T cells. Moreover, TGF-β1 induced a modest expression of CCR7 on Ag-activated memory CD8 T cells. TGF-β1 also suppressed cytokine secretion by Ag-specific effector memory CD8 T cells, as well as melanoma-reactive tumor-infiltrating lymphocytes and CD8 T cell clones. These results demonstrate that TGF-β1 suppresses not only the acquisition but also expression of effector function on human memory CD8 T cells and tumor-infiltrating lymphocytes reactive against melanoma, suggesting that TGF-β1-mediated suppression can hinder the therapeutic benefits of vaccination, as well as immunotherapy in cancer patients. PMID:15843517

  13. Suppression of HIV-1 replication by microRNA effectors

    PubMed Central

    Chable-Bessia, Christine; Meziane, Oussama; Latreille, Daniel; Triboulet, Robinson; Zamborlini, Alessia; Wagschal, Alexandre; Jacquet, Jean-Marc; Reynes, Jacques; Levy, Yves; Saib, Ali; Bennasser, Yamina; Benkirane, Monsef

    2009-01-01

    The rate of HIV-1 gene expression is a key step that determines the kinetics of virus spread and AIDS progression. Viral entry and gene expression were described to be the key determinants for cell permissiveness to HIV. Recent reports highlighted the involvement of miRNA in regulating HIV-1 replication post-transcriptionally. In this study we explored the role of cellular factors required for miRNA-mediated mRNA translational inhibition in regulating HIV-1 gene expression. Here we show that HIV-1 mRNAs associate and co-localize with components of the RNA Induced Silencing Complex (RISC), and we characterize some of the proteins required for miRNA-mediated silencing (miRNA effectors). RCK/p54, GW182, LSm-1 and XRN1 negatively regulate HIV-1 gene expression by preventing viral mRNA association with polysomes. Interestingly, knockdown of RCK/p54 or DGCR8 resulted in virus reactivation in PBMCs isolated from HIV infected patients treated with suppressive HAART. PMID:19272132

  14. The relationship of stress and blood pressure effectors.

    PubMed

    Ayada, C; Toru, Ü; Korkut, Y

    2015-01-01

    Exaggerated cardiovascular response to acute and chronic stresses increases the risk for hypertension and cardiovascular disease. Stress also can be broadly defined as a disruption of homeostasis. The re-establishment and maintenance of homeostasis entail the coordinated activation and control of neuroendocrine and autonomic stress systems. Stressor-related information from all major sensory systems is conveyed to the brain.  Brain activates neural and neuroendocrine systems to minimize the harmful effects of stress. Stress is generally thought to contribute to the development of hypertension. On the other hand, the evidence is still inconclusive. It is generally accepted that stress-induced hypertension occurs because of increases in sympathoadrenal activity, which enhances vascular tone, but complete α-adrenoreceptor blockade cannot prevent the long-lasting vasoconstriction induced by sympathetic nerve stimulation. That is why it is suggested that sympathetic nerve-mediated vasoconstriction may also be mediated by factors other than catecholamines. In this review, we aim to present the relationship between blood pressure effectors and stress altogether, along with evaluating the relationship between stress and blood pressure. In this respect, we have identified topics to explain the relationship between stress and the renin angiotensin aldosterone system, glucocorticoids, endothelial nitric oxide, endothelin-1 and L-type Ca2+ channels. Hippokratia 2015; 19 (2): 99-108.

  15. A Phytophthora infestans RXLR effector targets plant PP1c isoforms that promote late blight disease.

    PubMed

    Boevink, Petra C; Wang, Xiaodan; McLellan, Hazel; He, Qin; Naqvi, Shaista; Armstrong, Miles R; Zhang, Wei; Hein, Ingo; Gilroy, Eleanor M; Tian, Zhendong; Birch, Paul R J

    2016-01-29

    Plant pathogens deliver effectors to alter host processes. Knowledge of how effectors target and manipulate host proteins is critical to understand crop disease. Here, we show that in planta expression of the RXLR effector Pi04314 enhances leaf colonization by Phytophthora infestans via activity in the host nucleus and attenuates induction of jasmonic and salicylic acid-responsive genes. Pi04314 interacts with three host protein phosphatase 1 catalytic (PP1c) isoforms, causing their re-localization from the nucleolus to the nucleoplasm. Re-localization of PP1c-1 also occurs during infection and is dependent on an R/KVxF motif in the effector. Silencing the PP1c isoforms or overexpression of a phosphatase-dead PP1c-1 mutant attenuates infection, demonstrating that host PP1c activity is required for disease. Moreover, expression of PP1c-1mut abolishes enhanced leaf colonization mediated by in planta Pi04314 expression. We argue that PP1c isoforms are susceptibility factors forming holoenzymes with Pi04314 to promote late blight disease.

  16. 24-hour control of body temperature in rats. I. Integration of behavioral and autonomic effectors.

    PubMed

    Gordon, C J

    1994-07-01

    Some studies suggest that the nocturnal elevation in core temperature (Tc) of the rat is mediated by an elevation in the set point. The role of set point can be assessed if behavioral effectors are measured simultaneously with other thermoregulatory effectors and Tc over a 24-h period. Selected ambient temperature (STa) and motor activity (MA) were measured in rats housed in a temperature gradient system with a 12:12-h photoperiod (lights on 0600 h). Tc and heart rate (HR) were monitored by telemetry. During the light phase, STa, Tc, HR, and MA were relatively stable with values 29.0 degrees C, 37.1 degrees C, 310 beats/min, and 1-2 m/h, respectively. During the light-to-dark transition there were abrupt elevations in Tc, HR, and MA but no change in STa. STa decreased during the dark phase and reached a nadir of 23 degrees C at 0500 h. All variables recovered to basal levels within 3-4 h after the onset of the light phase. Overall, autonomic effectors control the elevation in Tc during the onset of the dark phase while behavioral effectors have little if any role. Behavioral thermoregulation is important in two ways: 1) the selection of cooler Ta values at night to prevent an excess elevation in Tc and 2) a preference for cooler Ta values before the light phase to facilitate the recovery of Tc.

  17. A Phytophthora infestans RXLR effector targets plant PP1c isoforms that promote late blight disease

    PubMed Central

    Boevink, Petra C.; Wang, Xiaodan; McLellan, Hazel; He, Qin; Naqvi, Shaista; Armstrong, Miles R.; Zhang, Wei; Hein, Ingo; Gilroy, Eleanor M.; Tian, Zhendong; Birch, Paul R. J.

    2016-01-01

    Plant pathogens deliver effectors to alter host processes. Knowledge of how effectors target and manipulate host proteins is critical to understand crop disease. Here, we show that in planta expression of the RXLR effector Pi04314 enhances leaf colonization by Phytophthora infestans via activity in the host nucleus and attenuates induction of jasmonic and salicylic acid-responsive genes. Pi04314 interacts with three host protein phosphatase 1 catalytic (PP1c) isoforms, causing their re-localization from the nucleolus to the nucleoplasm. Re-localization of PP1c-1 also occurs during infection and is dependent on an R/KVxF motif in the effector. Silencing the PP1c isoforms or overexpression of a phosphatase-dead PP1c-1 mutant attenuates infection, demonstrating that host PP1c activity is required for disease. Moreover, expression of PP1c–1mut abolishes enhanced leaf colonization mediated by in planta Pi04314 expression. We argue that PP1c isoforms are susceptibility factors forming holoenzymes with Pi04314 to promote late blight disease. PMID:26822079

  18. Radiation-induced CXCL16 release by breast cancer cells attracts effector T cells1

    PubMed Central

    Matsumura, Satoko; Wang, Baomei; Kawashima, Noriko; Braunstein, Steve; Badura, Michelle; Cameron, Thomas O.; Babb, James S.; Schneider, Robert J.; Formenti, Silvia C.; Dustin, Michael L.; Demaria, Sandra

    2008-01-01

    Recruitment of effector T cells to inflamed peripheral tissues is regulated by chemokines and their receptors, but the factors regulating recruitment to tumors remain largely undefined. Ionizing radiation (IR) therapy is a common treatment modality for breast and other cancers. Used as a cytocidal agent for proliferating cancer cells, IR in combination with immunotherapy has been shown to promote immune-mediated tumor destruction in pre-clinical studies. Here we demonstrate that IR markedly enhanced the secretion by mouse and human breast cancer cells of CXCL16, a chemokine that binds to CXCR6 on Th1 and activated CD8 effector T cells, and plays an important role in their recruitment to sites of inflammation. Employing a poorly immunogenic mouse model of breast cancer, we found that irradiation increased the migration of CD8+CXCR6+ activated T cells to tumors in vitro and in vivo. CXCR6-deficient mice showed reduced infiltration of tumors by activated CD8 T cells and impaired tumor regression following treatment with local IR to the tumor and antibodies blocking the negative regulator of T cell activation CTLA-4. These results provide the first evidence that IR can induce the secretion by cancer cells of pro-inflammatory chemotactic factors that recruit anti-tumor effector T cells. The ability of IR to convert tumors into “inflamed” peripheral tissues could be exploited to overcome obstacles at the effector phase of the anti-tumor immune response and improve the therapeutic efficacy of immunotherapy. PMID:18713980

  19. Secreted Effectors Encoded within and outside of the Francisella Pathogenicity Island Promote Intramacrophage Growth.

    PubMed

    Eshraghi, Aria; Kim, Jungyun; Walls, Alexandra C; Ledvina, Hannah E; Miller, Cheryl N; Ramsey, Kathryn M; Whitney, John C; Radey, Matthew C; Peterson, S Brook; Ruhland, Brittany R; Tran, Bao Q; Goo, Young Ah; Goodlett, David R; Dove, Simon L; Celli, Jean; Veesler, David; Mougous, Joseph D

    2016-11-09

    The intracellular bacterial pathogen Francisella tularensis causes tularemia, a zoonosis that can be fatal. The type VI secretion system (T6SS) encoded by the Francisella pathogenicity island (FPI) is critical for the virulence of this organism. Existing studies suggest that the complete repertoire of T6SS effectors delivered to host cells is encoded by the FPI. Using a proteome-wide approach, we discovered that the FPI-encoded T6SS exports at least three effectors encoded outside of the island. These proteins share features with virulence determinants of other pathogens, and we provide evidence that they can contribute to intramacrophage growth. The remaining proteins that we identified are encoded within the FPI. Two of these FPI-encoded proteins constitute effectors, whereas the others form a unique complex required for core function of the T6SS apparatus. The discovery of secreted effectors mediating interactions between Francisella and its host significantly advances our understanding of the pathogenesis of this organism. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Inhibition of murine nephritogenic effector T cells by a clone-specific suppressor factor.

    PubMed Central

    Meyers, C M; Kelly, C J

    1994-01-01

    We have used a murine model of organ-specific autoimmunity to characterize therapeutic modalities capable of down-regulating the cellular limb of the autoimmune response. Murine interstitial nephritis is an autoimmune disease mediated by tubular antigen-specific CD8+ nephritogenic effector T cells which are delayed-type hypersensitivity (DTH) reactive and cytotoxic to renal epithelial cells. Previous studies have demonstrated that disease can be suppressed with experimentally induced populations of T cells (Ts1 and Ts2 cells) obtained after injection of tubular antigen-coupled splenocytes into syngeneic mice. As the target of Ts2 is the CD8+ effector T cell, we have evaluated its effects on nephritogenic effector T cell clones isolated from diseased animals. Our studies demonstrate that soluble proteins expressed by Ts2 cells (TsF2) specifically abrogate the DTH, cytotoxic, and nephritogenic potential of M52 cells, although T cell receptor and IL-2 receptor expression are unchanged in these unresponsive M52 clones. TsF2-induced inhibition is dependent on new mRNA and protein synthesis. In a cytotoxic clone, M52.26, exposure to TsF2 induces expression of TGF-beta 1 which is, in turn, required for inhibition of cytotoxicity and nephritogenicity. Our studies are consistent with TGF-beta 1 behaving, at least in some T cells, as a nonspecific final effector of clone-specific suppression. Images PMID:7962556

  1. Effector discovery in the fungal wheat pathogen Zymoseptoria tritici.

    PubMed

    Mirzadi Gohari, Amir; Ware, Sarah B; Wittenberg, Alexander H J; Mehrabi, Rahim; Ben M'Barek, Sarrah; Verstappen, Els C P; van der Lee, Theo A J; Robert, Olivier; Schouten, Henk J; de Wit, Pierre P J G M; Kema, Gert H J

    2015-12-01

    Fungal plant pathogens, such as Zymoseptoria tritici (formerly known as Mycosphaerella graminicola), secrete repertoires of effectors to facilitate infection or trigger host defence mechanisms. The discovery and functional characterization of effectors provides valuable knowledge that can contribute to the design of new and effective disease management strategies. Here, we combined bioinformatics approaches with expression profiling during pathogenesis to identify candidate effectors of Z. tritici. In addition, a genetic approach was conducted to map quantitative trait loci (QTLs) carrying putative effectors, enabling the validation of both complementary strategies for effector discovery. In planta expression profiling revealed that candidate effectors were up-regulated in successive waves corresponding to consecutive stages of pathogenesis, contrary to candidates identified by QTL mapping that were, overall, expressed at low levels. Functional analyses of two top candidate effectors (SSP15 and SSP18) showed their dispensability for Z. tritici pathogenesis. These analyses reveal that generally adopted criteria, such as protein size, cysteine residues and expression during pathogenesis, may preclude an unbiased effector discovery. Indeed, genetic mapping of genomic regions involved in specificity render alternative effector candidates that do not match the aforementioned criteria, but should nevertheless be considered as promising new leads for effectors that are crucial for the Z. tritici-wheat pathosystem. © 2015 BSPP AND JOHN WILEY & SONS LTD.

  2. Repeat-containing protein effectors of plant-associated organisms

    PubMed Central

    Mesarich, Carl H.; Bowen, Joanna K.; Hamiaux, Cyril; Templeton, Matthew D.

    2015-01-01

    Many plant-associated organisms, including microbes, nematodes, and insects, deliver effector proteins into the apoplast, vascular tissue, or cell cytoplasm of their prospective hosts. These effectors function to promote colonization, typically by altering host physiology or by modulating host immune responses. The same effectors however, can also trigger host immunity in the presence of cognate host immune receptor proteins, and thus prevent colonization. To circumvent effector-triggered immunity, or to further enhance host colonization, plant-associated organisms often rely on adaptive effector evolution. In recent years, it has become increasingly apparent that several effectors of plant-associated organisms are repeat-containing proteins (RCPs) that carry tandem or non-tandem arrays of an amino acid sequence or structural motif. In this review, we highlight the diverse roles that these repeat domains play in RCP effector function. We also draw attention to the potential role of these repeat domains in adaptive evolution with regards to RCP effector function and the evasion of effector-triggered immunity. The aim of this review is to increase the profile of RCP effectors from plant-associated organisms. PMID:26557126

  3. Design and fabrication of an end effector

    NASA Technical Reports Server (NTRS)

    Crossley, F. R. E.; Umholtz, F. G.

    1975-01-01

    The construction is described of a prototype mechanical hand or 'end effector' for use on a remotely controlled robot, but with possible application as a prosthetic device. An analysis of hand motions is reported, from which it is concluded that the two most important manipulations (apart from grasps) are to be able to pick up a tool and draw it into a nested grip against the palm, and to be able to hold a pistol-grip tool such as an electric drill and pull the trigger. A model was tested and found capable of both these operations.

  4. A rheumatoid factor paradox: inhibition of rituximab effector function

    PubMed Central

    2013-01-01

    Introduction Rituximab (RTX) therapy of rheumatoid arthritis (RA) exhibits enhanced effectiveness in seropositive patients. Using patient sera, we tested if this improved efficacy was associated with enhanced RTX mediated complement-dependent cytotoxicity (RTX-CDC). Methods We developed an in vitro assay for RTX-CDC using patient sera and the Daudi human B cell line. Using propidium iodide uptake and flow cytometry, we compared RTX-CDC with rheumatoid factor (RF)+ sera relative to normal volunteer, non-RA and RF- sera. Additional studies examined mixing studies of RF+ and RF- sera, as well as the effect of monoclonal IgA or IgM RF. Finally, the effect of RF on RTX mediated trogocytosis of normal B cells was evaluated. Results Using human sera, addition of RTX resulted in rapid and profound (> 50%) Daudi cell death that was complement dependent. Surprisingly, RF+ patient sera exhibited reduced RTX-CDC relative to RF- sera, with an inverse relationship of RTX-CDC and RF titer. Mixing studies indicated the presence of an inhibitor of RTX-CDC in RF+ sera. The addition of monoclonal IgM or IgA RF to RF- sera markedly inhibited RTX-CDC. This effect was specific for RF binding to the Fc portion of RTX as it was not apparent with the F(ab)' domains of RTX engineered onto IgG3 heavy chain. RF also modestly inhibited RTX mediated trogocytosis. Conclusions Contrary to expectations, RF+ sera exhibits reduced RTX-CDC due to the presence of RF. The enhanced efficacy of RTX in seropositive RA patients cannot be attributed to improved B cell depletion through CDC. This result indicates that high RF levels may potentially modulate the efficacy of any therapeutic monoclonal antibody dependent on Fc effector function. PMID:23351360

  5. Metabolic priming by a secreted fungal effector.

    PubMed

    Djamei, Armin; Schipper, Kerstin; Rabe, Franziska; Ghosh, Anupama; Vincon, Volker; Kahnt, Jörg; Osorio, Sonia; Tohge, Takayuki; Fernie, Alisdair R; Feussner, Ivo; Feussner, Kirstin; Meinicke, Peter; Stierhof, York-Dieter; Schwarz, Heinz; Macek, Boris; Mann, Matthias; Kahmann, Regine

    2011-10-05

    Maize smut caused by the fungus Ustilago maydis is a widespread disease characterized by the development of large plant tumours. U. maydis is a biotrophic pathogen that requires living plant tissue for its development and establishes an intimate interaction zone between fungal hyphae and the plant plasma membrane. U. maydis actively suppresses plant defence responses by secreted protein effectors. Its effector repertoire comprises at least 386 genes mostly encoding proteins of unknown function and expressed exclusively during the biotrophic stage. The U. maydis secretome also contains about 150 proteins with probable roles in fungal nutrition, fungal cell wall modification and host penetration as well as proteins unlikely to act in the fungal-host interface like a chorismate mutase. Chorismate mutases are key enzymes of the shikimate pathway and catalyse the conversion of chorismate to prephenate, the precursor for tyrosine and phenylalanine synthesis. Root-knot nematodes inject a secreted chorismate mutase into plant cells likely to affect development. Here we show that the chorismate mutase Cmu1 secreted by U. maydis is a virulence factor. The enzyme is taken up by plant cells, can spread to neighbouring cells and changes the metabolic status of these cells through metabolic priming. Secreted chorismate mutases are found in many plant-associated microbes and might serve as general tools for host manipulation.

  6. The Rab7 effector PLEKHM1 binds Arl8b to promote cargo traffic to lysosomes

    PubMed Central

    Marwaha, Rituraj; Arya, Subhash B.; Jagga, Divya; Kaur, Harmeet

    2017-01-01

    Endocytic, autophagic, and phagocytic vesicles move on microtubule tracks to fuse with lysosomes. Small GTPases, such as Rab7 and Arl8b, recruit their downstream effectors to mediate this transport and fusion. However, the potential cross talk between these two GTPases is unclear. Here, we show that the Rab7 effector PLEKHM1 simultaneously binds Rab7 and Arl8b, bringing about clustering and fusion of late endosomes and lysosomes. We show that the N-terminal RUN domain of PLEKHM1 is necessary and sufficient for interaction with Arl8b and its subsequent localization to lysosomes. Notably, we also demonstrate that Arl8b mediates recruitment of HOPS complex to PLEKHM1-positive vesicle contact sites. Consequently, Arl8b binding to PLEKHM1 is required for its function in delivery and, therefore, degradation of endocytic and autophagic cargo in lysosomes. Finally, we also show that PLEKHM1 competes with SKIP for Arl8b binding, which dictates lysosome positioning. These findings suggest that Arl8b, along with its effectors, orchestrates lysosomal transport and fusion. PMID:28325809

  7. Timing of CD8 T cell effector responses in viral infections

    PubMed Central

    Stipp, Shaun R.; Iniguez, Abdon; Wan, Frederic; Wodarz, Dominik

    2016-01-01

    CD8 T cell or cytotoxic T lymphocyte (CTL) responses are an important branch of the immune system in the fight against viral infections. The dynamics of anti-viral CTL responses have been characterized in some detail, both experimentally and with mathematical models. An interesting experimental observation concerns the timing of CTL responses. A recent study reported that in pneumonia virus of mice the effector CTL tended to arrive in the lung only after maximal virus loads had been achieved, an observation that seems at first counterintuitive because prevention of pathology would require earlier CTL-mediated activity. A delay in CTL-mediated effector activity has also been quoted as a possible explanation for the difficulties associated with CTL-based vaccines. This paper uses mathematical models to show that in specific parameter regimes, delayed CTL effector activity can be advantageous for the host in the sense that it can increase the chances of virus clearance. The increased ability of the CTL to clear the infection, however, is predicted to come at the cost of acute pathology, giving rise to a trade-off, which is discussed in the light of evolutionary processes. This work provides a theoretical basis for understanding the described experimental observations. PMID:26998338

  8. Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions.

    PubMed

    Wolters, Manuel; Zobiak, Bernd; Nauth, Theresa; Aepfelbacher, Martin

    2015-10-13

    Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

  9. Allelic barley MLA immune receptors recognize sequence-unrelated avirulence effectors of the powdery mildew pathogen

    PubMed Central

    Lu, Xunli; Kracher, Barbara; Saur, Isabel M. L.; Bauer, Saskia; Ellwood, Simon R.; Wise, Roger; Yaeno, Takashi; Maekawa, Takaki; Schulze-Lefert, Paul

    2016-01-01

    Disease-resistance genes encoding intracellular nucleotide-binding domain and leucine-rich repeat proteins (NLRs) are key components of the plant innate immune system and typically detect the presence of isolate-specific avirulence (AVR) effectors from pathogens. NLR genes define the fastest-evolving gene family of flowering plants and are often arranged in gene clusters containing multiple paralogs, contributing to copy number and allele-specific NLR variation within a host species. Barley mildew resistance locus a (Mla) has been subject to extensive functional diversification, resulting in allelic resistance specificities each recognizing a cognate, but largely unidentified, AVRa gene of the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). We applied a transcriptome-wide association study among 17 Bgh isolates containing different AVRa genes and identified AVRa1 and AVRa13, encoding candidate-secreted effectors recognized by Mla1 and Mla13 alleles, respectively. Transient expression of the effector genes in barley leaves or protoplasts was sufficient to trigger Mla1 or Mla13 allele-specific cell death, a hallmark of NLR receptor-mediated immunity. AVRa1 and AVRa13 are phylogenetically unrelated, demonstrating that certain allelic MLA receptors evolved to recognize sequence-unrelated effectors. They are ancient effectors because corresponding loci are present in wheat powdery mildew. AVRA1 recognition by barley MLA1 is retained in transgenic Arabidopsis, indicating that AVRA1 directly binds MLA1 or that its recognition involves an evolutionarily conserved host target of AVRA1. Furthermore, analysis of transcriptome-wide sequence variation among the Bgh isolates provides evidence for Bgh population structure that is partially linked to geographic isolation. PMID:27702901

  10. Allelic barley MLA immune receptors recognize sequence-unrelated avirulence effectors of the powdery mildew pathogen.

    PubMed

    Lu, Xunli; Kracher, Barbara; Saur, Isabel M L; Bauer, Saskia; Ellwood, Simon R; Wise, Roger; Yaeno, Takashi; Maekawa, Takaki; Schulze-Lefert, Paul

    2016-10-18

    Disease-resistance genes encoding intracellular nucleotide-binding domain and leucine-rich repeat proteins (NLRs) are key components of the plant innate immune system and typically detect the presence of isolate-specific avirulence (AVR) effectors from pathogens. NLR genes define the fastest-evolving gene family of flowering plants and are often arranged in gene clusters containing multiple paralogs, contributing to copy number and allele-specific NLR variation within a host species. Barley mildew resistance locus a (Mla) has been subject to extensive functional diversification, resulting in allelic resistance specificities each recognizing a cognate, but largely unidentified, AVRa gene of the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). We applied a transcriptome-wide association study among 17 Bgh isolates containing different AVRa genes and identified AVRa1 and AVRa13, encoding candidate-secreted effectors recognized by Mla1 and Mla13 alleles, respectively. Transient expression of the effector genes in barley leaves or protoplasts was sufficient to trigger Mla1 or Mla13 allele-specific cell death, a hallmark of NLR receptor-mediated immunity. AVRa1 and AVRa13 are phylogenetically unrelated, demonstrating that certain allelic MLA receptors evolved to recognize sequence-unrelated effectors. They are ancient effectors because corresponding loci are present in wheat powdery mildew. AVRA1 recognition by barley MLA1 is retained in transgenic Arabidopsis, indicating that AVRA1 directly binds MLA1 or that its recognition involves an evolutionarily conserved host target of AVRA1 Furthermore, analysis of transcriptome-wide sequence variation among the Bgh isolates provides evidence for Bgh population structure that is partially linked to geographic isolation.

  11. Role of effector cells (CCR7(-)CD27(-)) and effector-memory cells (CCR7(-)CD27(+)) in drug-induced maculopapular exanthema.

    PubMed

    Fernandez, T D; Torres, M J; Lopez, S; Antunez, C; Gomez, E; Del Prado, M F; Canto, G; Blanca, M; Mayorga, C

    2010-01-01

    Maculopapular exanthema (MPE) induced by drugs is a T-cell mediated reaction and effector cells may play an important role in its development. We assessed the effector and cutaneous homing phenotype in peripheral blood cells from allergic patients after drug stimulation. This study included 10 patients and 10 controls. The effector phenotype (CCR7(-)CD27(+/-)), chemokine receptors (CCR4 and CCR10), and activation (CD25(low)) and regulatory markers (CD25(high)) were measured by flow cytometry in both peripheral blood mononuclear cells (PBMCs) and CD4-T-lymphocytes. Proliferation was determined by 5-(-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) assay and the migratory capacity by a chemotaxis assay using CCL17 and CCL27. Compared to controls, CCR7(-)CD27(-) cells were increased in patients without (p=0.003) and with drug stimulation (p less than 0.001) and had significantly higher proliferation (p=0.010). CCR10 expression was increased in patients after drug stimulation in total and memory CD27(+) T-cells. Lymphocyte migration with CCL27 was higher in patients with drug stimulation (p=0.048), with a decrease in CCR7(-)CD27(-) (p less than 0.0001) and an increase in CCR7(-)CD27(+) (p=0.017). In patients, CD4-T-lymphocytes were significantly activated after drug stimulation (p less than 0.001). In conclusion, we show that effector memory CD4(+) T-cells (CCR7(-)CD27(+)) respond specifically to the drug responsible for MPE and confirm previous data about the involvement of CCR10 in cell trafficking to the skin.

  12. Fibre optic sensor on robot end effector for flexible assembly

    SciTech Connect

    Yung, K.L.; Lau, W.S.; Choi, C.K.; Shan, Y.Y.

    1995-12-31

    A fibre optic sensor system was constructed for use on robot end effectors for flexible assembly. The sensor detected the deviations between robot end effector and the workpiece. The signal was fed back to robot controller to shift the end effector until the centre of end effector and the centre of workpiece were aligned at the correct orientation. Then workpiece can be grasped symmetrically. Sensor fusion concept was used to guard against sensor system failure. Fuzzy linguistic variable and control rule concept were introduced in the sensor integration. The experimental setup for the sensor integrated system was shown. The accuracy was also discussed.

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

    PubMed Central

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

    2016-01-01

    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

  14. Potential effector and immunoregulatory functions of mast cells in mucosal immunity

    PubMed Central

    Reber, Laurent L; Sibilano, Riccardo; Mukai, Kaori; Galli, Stephen J

    2016-01-01

    Mast cells (MCs) are cells of hematopoietic origin that normally reside in mucosal tissues, often near epithelial cells, glands, smooth muscle cells, and nerves. Best known for their contributions to pathology during IgE-associated disorders such as food allergy, asthma, and anaphylaxis, MCs are also thought to mediate IgE-associated effector functions during certain parasite infections. However, various MC populations also can be activated to express functional programs – such as secreting pre-formed and/or newly synthesized biologically active products – in response to encounters with products derived from diverse pathogens, other host cells (including leukocytes and structural cells), damaged tissue, or the activation of the complement or coagulation systems, as well as by signals derived from the external environment (including animal toxins, plant products, and physical agents). In this review, we will discuss evidence suggesting that MCs can perform diverse effector and immunoregulatory roles that contribute to homeostasis or pathology in mucosal tissues. PMID:25669149

  15. Inhibition of effector antigen-specific T cells by intradermal administration of heme oxygenase-1 inducers.

    PubMed

    Simon, Thomas; Pogu, Julien; Rémy, Séverine; Brau, Frédéric; Pogu, Sylvie; Maquigneau, Maud; Fonteneau, Jean-François; Poirier, Nicolas; Vanhove, Bernard; Blancho, Gilles; Piaggio, Eliane; Anegon, Ignacio; Blancou, Philippe

    2017-03-22

    Developing protocols aimed at inhibiting effector T cells would be key for the treatment of T cell-dependent autoimmune diseases including type 1 autoimmune diabetes (T1D) and multiple sclerosis (MS). While heme oxygenase-1 (HO-1) inducers are clinically approved drugs for non-immune-related diseases, they do have immunosuppressive properties when administered systemically in rodents. Here we show that HO-1 inducers inhibit antigen-specific effector T cells when injected intradermally together with the T cell cognate antigens in mice. This phenomenon was observed in both a CD8(+) T cell-mediated model of T1D and in a CD4(+) T cell-dependent MS model. Intradermal injection of HO-1 inducers induced the recruitment of HO-1(+) monocyte-derived dendritic cell (MoDCs) exclusively to the lymph nodes (LN) draining the site of intradermal injection. After encountering HO-1(+)MoDCs, effector T-cells exhibited a lower velocity and a reduced ability to migrate towards chemokine gradients resulting in impaired accumulation to the inflamed organ. Intradermal co-injection of a clinically approved HO-1 inducer and a specific antigen to non-human primates also induced HO-1(+) MoDCs to accumulate in dermal draining LN and to suppress delayed-type hypersensitivity. Therefore, in both mice and non-human primates, HO-1 inducers delivered locally inhibited effector T-cells in an antigen-specific manner, paving the way for repositioning these drugs for the treatment of immune-mediated diseases.

  16. The Enteropathogenic E. coli (EPEC) Tir Effector Inhibits NF-κB Activity by Targeting TNFα Receptor-Associated Factors

    PubMed Central

    Ruchaud-Sparagano, Marie-Hélène; Mühlen, Sabrina; Dean, Paul; Kenny, Brendan

    2011-01-01

    Enteropathogenic Escherichia coli (EPEC) disease depends on the transfer of effector proteins into epithelia lining the human small intestine. EPEC E2348/69 has at least 20 effector genes of which six are located with the effector-delivery system genes on the Locus of Enterocyte Effacement (LEE) Pathogenicity Island. Our previous work implied that non-LEE-encoded (Nle) effectors possess functions that inhibit epithelial anti-microbial and inflammation-inducing responses by blocking NF-κB transcription factor activity. Indeed, screens by us and others have identified novel inhibitory mechanisms for NleC and NleH, with key co-operative functions for NleB1 and NleE1. Here, we demonstrate that the LEE-encoded Translocated-intimin receptor (Tir) effector has a potent and specific ability to inhibit NF-κB activation. Indeed, biochemical, imaging and immunoprecipitation studies reveal a novel inhibitory mechanism whereby Tir interaction with cytoplasm-located TNFα receptor-associated factor (TRAF) adaptor proteins induces their proteasomal-independent degradation. Infection studies support this Tir-TRAF relationship but reveal that Tir, like NleC and NleH, has a non-essential contribution in EPEC's NF-κB inhibitory capacity linked to Tir's activity being suppressed by undefined EPEC factors. Infections in a disease-relevant intestinal model confirm key NF-κB inhibitory roles for the NleB1/NleE1 effectors, with other studies providing insights on host targets. The work not only reveals a second Intimin-independent property for Tir and a novel EPEC effector-mediated NF-κB inhibitory mechanism but also lends itself to speculations on the evolution of EPEC's capacity to inhibit NF-κB function. PMID:22144899

  17. The enteropathogenic E. coli (EPEC) Tir effector inhibits NF-κB activity by targeting TNFα receptor-associated factors.

    PubMed

    Ruchaud-Sparagano, Marie-Hélène; Mühlen, Sabrina; Dean, Paul; Kenny, Brendan

    2011-12-01

    Enteropathogenic Escherichia coli (EPEC) disease depends on the transfer of effector proteins into epithelia lining the human small intestine. EPEC E2348/69 has at least 20 effector genes of which six are located with the effector-delivery system genes on the Locus of Enterocyte Effacement (LEE) Pathogenicity Island. Our previous work implied that non-LEE-encoded (Nle) effectors possess functions that inhibit epithelial anti-microbial and inflammation-inducing responses by blocking NF-κB transcription factor activity. Indeed, screens by us and others have identified novel inhibitory mechanisms for NleC and NleH, with key co-operative functions for NleB1 and NleE1. Here, we demonstrate that the LEE-encoded Translocated-intimin receptor (Tir) effector has a potent and specific ability to inhibit NF-κB activation. Indeed, biochemical, imaging and immunoprecipitation studies reveal a novel inhibitory mechanism whereby Tir interaction with cytoplasm-located TNFα receptor-associated factor (TRAF) adaptor proteins induces their proteasomal-independent degradation. Infection studies support this Tir-TRAF relationship but reveal that Tir, like NleC and NleH, has a non-essential contribution in EPEC's NF-κB inhibitory capacity linked to Tir's activity being suppressed by undefined EPEC factors. Infections in a disease-relevant intestinal model confirm key NF-κB inhibitory roles for the NleB1/NleE1 effectors, with other studies providing insights on host targets. The work not only reveals a second Intimin-independent property for Tir and a novel EPEC effector-mediated NF-κB inhibitory mechanism but also lends itself to speculations on the evolution of EPEC's capacity to inhibit NF-κB function.

  18. Early Decision: Effector and Effector Memory T Cell Differentiation in Chronic Infection

    PubMed Central

    Opata, Michael M.; Stephens, Robin

    2013-01-01

    As effector memory T cells (Tem) are the predominant population elicited by chronic parasitic infections, increasing our knowledge of their function, survival and derivation, as phenotypically and functionally distinct from central memory and effector T cells will be critical to vaccine development for these diseases. In some infections, memory T cells maintain increased effector functions, however; this may require the presence of continued antigen, which can also lead to T cell exhaustion. Alternatively, in the absence of antigen, only the increase in the number of memory cells remains, without enhanced functionality as central memory. In order to understand the requirement for antigen and the potential for longevity or protection, the derivation of each type of memory must be understood. A thorough review of the data establishes the existence of both memory (Tmem) precursors and effector T cells (Teff) from the first hours of an immune response. This suggests a new paradigm of Tmem differentiation distinct from the proposition that Tmem only appear after the contraction of Teff. Several signals have been shown to be important in the generation of memory T cells, such as the integrated strength of “signals 1-3” of antigen presentation (antigen receptor, co-stimulation, cytokines) as perceived by each T cell clone. Given that these signals integrated at antigen presentation cells have been shown to determine the outcome of Teff and Tmem phenotypes and numbers, this decision must be made at a very early stage. It would appear that the overwhelming expansion of effector T cells and the inability to phenotypically distinguish memory T cells at early time points has masked this important decision point. This does not rule out an effect of repeated stimulation or chronic inflammatory milieu on populations generated in these early stages. Recent studies suggest that Tmem are derived from early Teff, and we suggest that this includes Tem as well as Tcm. Therefore, we

  19. Miniature Trailing Edge Effector for Aerodynamic Control

    NASA Technical Reports Server (NTRS)

    Lee, Hak-Tae (Inventor); Bieniawski, Stefan R. (Inventor); Kroo, Ilan M. (Inventor)

    2008-01-01

    Improved miniature trailing edge effectors for aerodynamic control are provided. Three types of devices having aerodynamic housings integrated to the trailing edge of an aerodynamic shape are presented, which vary in details of how the control surface can move. A bucket type device has a control surface which is the back part of a C-shaped member having two arms connected by the back section. The C-shaped section is attached to a housing at the ends of the arms, and is rotatable about an axis parallel to the wing trailing edge to provide up, down and neutral states. A flip-up type device has a control surface which rotates about an axis parallel to the wing trailing edge to provide up, down, neutral and brake states. A rotating type device has a control surface which rotates about an axis parallel to the chord line to provide up, down and neutral states.

  20. The mast cell: a multifunctional effector cell.

    PubMed

    Crivellato, Enrico; Ribatti, Domenico; Mallardi, Franco; Beltrami, Carlo Alberto

    2003-01-01

    Mast cells (MC) are recognized key cells of type I hypersensitivity reactions. Several lines of evidence, however, indicate that MC not only express critical effector functions in classic IgE-associated allergic disorders, but also play important roles in host defence against parasites, bacteria and perhaps even viruses. Indeed, it is now clear that MC can contribute to host defence in the context of either acquired or innate immune responses through the release of a myriad of pro-inflammatory and immunoregulatory molecules and the expression of a wide spectrum of surface receptors for cytokines and chemokines. Moreover, there is growing evidence that MC exert distinct nonimmunological functions, playing a relevant role in tissue homeostasis, remodeling and fibrosis as well as in the processes of tissue angiogenesis. In this review, we provide a small insight into the biology of mast cells and their potential implications in human pathology.

  1. Active membrane cholesterol as a physiological effector.

    PubMed

    Lange, Yvonne; Steck, Theodore L

    2016-09-01

    Sterols associate preferentially with plasma membrane sphingolipids and saturated phospholipids to form stoichiometric complexes. Cholesterol in molar excess of the capacity of these polar bilayer lipids has a high accessibility and fugacity; we call this fraction active cholesterol. This review first considers how active cholesterol serves as an upstream regulator of cellular sterol homeostasis. The mechanism appears to utilize the redistribution of active cholesterol down its diffusional gradient to the endoplasmic reticulum and mitochondria, where it binds multiple effectors and directs their feedback activity. We have also reviewed a broad literature in search of a role for active cholesterol (as opposed to bulk cholesterol or lipid domains such as rafts) in the activity of diverse membrane proteins. Several systems provide such evidence, implicating, in particular, caveolin-1, various kinds of ABC-type cholesterol transporters, solute transporters, receptors and ion channels. We suggest that this larger role for active cholesterol warrants close attention and can be tested easily.

  2. Rack Insertion End Effector (RIEE) automation

    NASA Technical Reports Server (NTRS)

    Malladi, Narasimha

    1993-01-01

    NASA is developing a mechanism to manipulate and insert Racks into the Space Station Logistic modules. The mechanism consists of the following: a base with three motorized degrees of freedom, a 3 section motorized boom that goes from 15 to 44 feet in length, and a Rack Insertion End Effector (RIEE) with 5 hand wheels for precise alignment. The robotics section was tasked with the automation of the RIEE unit. In this report, for the automation of the RIEE unit, application of the Perceptics Vision System was conceptually developed to determine the position and orientation of the RIEE relative to the logistic module, and a MathCad program is written to display the needed displacements for precise alignment and final insertion of the Rack. The uniqueness of this report is that the whole report is in fact a MathCad program including text, derivations, and executable equations with example inputs and outputs.

  3. Specific DNA-RNA hybrid recognition by TAL effectors.

    PubMed

    Yin, Ping; Deng, Dong; Yan, Chuangye; Pan, Xiaojing; Xi, Jianzhong Jeff; Yan, Nieng; Shi, Yigong

    2012-10-25

    The transcription activator-like (TAL) effector targets specific host promoter through its central DNA-binding domain, which comprises multiple tandem repeats (TALE repeats). Recent structural analyses revealed that the TALE repeats form a superhelical structure that tracks along the forward strand of the DNA duplex. Here, we demonstrate that TALE repeats specifically recognize a DNA-RNA hybrid where the DNA strand determines the binding specificity. The crystal structure of a designed TALE in complex with the DNA-RNA hybrid was determined at a resolution of 2.5 Å. Although TALE repeats are in direct contact with only the DNA strand, the phosphodiester backbone of the RNA strand is inaccessible by macromolecules such as RNases. Consistent with this observation, sequence-specific recognition of an HIV-derived DNA-RNA hybrid by an engineered TALE efficiently blocked RNase H-mediated degradation of the RNA strand. Our study broadens the utility of TALE repeats and suggests potential applications in processes involving DNA replication and retroviral infections.

  4. Natural resistance of lethally irradiated F1 hybrid mice to parental marrow grafts is a function of H-2/Hh-restricted effectors

    SciTech Connect

    Daley, J.P.; Nakamura, I.

    1984-04-01

    The natural resistance of F1 hybrid mice against parental bone marrow grafts is thought to be mediated by natural killer (NK)-like effector cells. However, unlike the NK cell activity against a wide range of tumors and normal cells, hybrid resistance is characterized by the immunogenetic specificity controlled by a set of unique noncodominant genes denoted as Hh. Two alternative hypotheses can account for the specificity. Thus, the specificity may reflect either the Hh restriction of effectors or the Hh gene control of mechanisms regulating non-Hh-restricted effector activity. In this study, therefore, we tested the recognition specificity of putative effectors mediating hybrid resistance in lethally irradiated H-2b/d and H-2b/k F1 hybrid mice to the engraftment of parental H-2b bone marrow. As a direct means of defining the effector specificity, rejection of parental bone marrow grafts was subjected to competitive inhibition in situ by irradiated tumor cells. Of the 16 independent lines of lymphoma and other hemopoietic tumor cells tested, the ability to inhibit hybrid resistance was the exclusive property of all tumors derived from mice homozygous for the H-2Db region, regardless of whether the tumor cells were susceptible or resistant to NK cell-mediated cytotoxicity in vitro. Four cell lines heterozygous for the H-2Db were noninhibitory, including one that is susceptible to natural killing. Pretreatment of the F1 hosts with an interferon inducer augmented the resistance with no alteration in the recognition specificity of effector cells. Therefore, natural resistance to parental H-2b bone marrow grafts was mediated by effectors restricted by the H-2Db/Hh-1b gene(s), and not by the nonrestricted NK cells detectable in conventional in vitro assays.

  5. The Pseudomonas syringae effector protein HopZ1a suppresses effector-triggered immunity.

    PubMed

    Macho, Alberto P; Guevara, Carlos M; Tornero, Pablo; Ruiz-Albert, Javier; Beuzón, Carmen R

    2010-09-01

    *The Pseudomonas syringae pv syringae type III effector HopZ1a is a member of the HopZ effector family of cysteine-proteases that triggers immunity in Arabidopsis. This immunity is dependent on HopZ1a cysteine-protease activity, and independent of known resistance genes. We have previously shown that HopZ1a-triggered immunity is partially additive to that triggered by AvrRpt2. These partially additive effects could be caused by at least two mechanisms: their signalling pathways share a common element(s), or one effector interferes with the response triggered by the other. *Here, we investigate the molecular basis for the partially additive effect displayed by AvrRpt2- and HopZ1a-triggered immunities, by analysing competitive indices, hypersensitive response and symptom induction, PR-1 accumulation, expression of PR genes, and systemic acquired resistance (SAR) induction. *Partially additive effects between these defence responses require HopZ1a cysteine-protease activity, and also take place between HopZ1a and AvrRps4 or AvrRpm1-triggered responses. We establish that HopZ1a-triggered immunity is independent of salicylic acid (SA), EDS1, jasmonic acid (JA) and ethylene (ET)-dependent pathways, and show that HopZ1a suppresses the induction of PR-1 and PR-5 associated with P. syringae pv tomato (Pto)-triggered effector-triggered immunity (ETI)-like defences, AvrRpt2-triggered immunity, and Pto or Pto (avrRpt2) activation of SAR, and that suppression requires HopZ1a cysteine-protease activity. *Our results indicate that HopZ1a triggers an unusual resistance independent of known pathways and suppresses SA and EDS1-dependent resistance.

  6. Nematode effector proteins: an emerging paradigm of parasitism

    USDA-ARS?s Scientific Manuscript database

    Phytonematodes use a stylet and secreted effectors to invade host tissues and extract nutrients to support their growth and development. The molecular function of nematode effectors is currently the subject of intense investigation. In this review, we summarize our current understanding of nematode ...

  7. Computational Prediction of Effector Proteins in Fungi: Opportunities and Challenges

    PubMed Central

    Sonah, Humira; Deshmukh, Rupesh K.; Bélanger, Richard R.

    2016-01-01

    Effector proteins are mostly secretory proteins that stimulate plant infection by manipulating the host response. Identifying fungal effector proteins and understanding their function is of great importance in efforts to curb losses to plant diseases. Recent advances in high-throughput sequencing technologies have facilitated the availability of several fungal genomes and 1000s of transcriptomes. As a result, the growing amount of genomic information has provided great opportunities to identify putative effector proteins in different fungal species. There is little consensus over the annotation and functionality of effector proteins, and mostly small secretory proteins are considered as effector proteins, a concept that tends to overestimate the number of proteins involved in a plant–pathogen interaction. With the characterization of Avr genes, criteria for computational prediction of effector proteins are becoming more efficient. There are 100s of tools available for the identification of conserved motifs, signature sequences and structural features in the proteins. Many pipelines and online servers, which combine several tools, are made available to perform genome-wide identification of effector proteins. In this review, available tools and pipelines, their strength and limitations for effective identification of fungal effector proteins are discussed. We also present an exhaustive list of classically secreted proteins along with their key conserved motifs found in 12 common plant pathogens (11 fungi and one oomycete) through an analytical pipeline. PMID:26904083

  8. Prostaglandin D2-loaded microspheres effectively activate macrophage effector functions.

    PubMed

    Pereira, Priscilla Aparecida Tartari; Bitencourt, Claudia da Silva; dos Santos, Daiane Fernanda; Nicolete, Roberto; Gelfuso, Guilherme Martins; Faccioli, Lúcia Helena

    2015-10-12

    Biodegradable lactic-co-glycolic acid (PLGA) microspheres (MS) improve the stability of biomolecules stability and allow enable their sustained release. Lipid mediators represent a strategy for improving host defense; however, most of these mediators, such as prostaglandin D2 (PGD2), have low water solubility and are unstable. The present study aimed to develop and characterize MS loaded with PGD2 (PGD2-MS) to obtain an innovative tool to activate macrophages. PGD2-MS were prepared using an oil-in-water emulsion solvent extraction-evaporation process, and the size, zeta potential, surface morphology and encapsulation efficiency were determined. It was also evaluated in vitro the phagocytic index, NF-κB activation, as well as nitric oxide and cytokine production by alveolar macrophages (AMs) in response to PGD2-MS. PGD2-MS were spherical with a diameter of 5.0±3.3 μm and regular surface, zeta potential of -13.4±5.6 mV, and 36% of encapsulation efficiency, with 16-26% release of entrapped PGD2 at 4 and 48 h, respectively. PGD2-MS were more efficiently internalized by AMs than unloaded-MS, and activated NF-κB more than free PGD2. Moreover, PGD2-MS stimulated the production of nitric oxide, TNF-α, IL-1β, and TGF-β, more than free PGD2, indicating that microencapsulation increased the activating effect of PGD2 on cells. In LPS-pre-treated AMs, PGD2-MS decreased the release of IL-6 but increased the production of nitric oxide and IL-1β. These results show that the morphological characteristics of PGD2-MS facilitated interaction with, and activation of phagocytic cells; moreover, PGD2-MS retained the biological activities of PGD2 to trigger effector mechanisms in AMs. It is suggested that PGD2-MS represent a strategy for therapeutic intervention in the lungs of immunocompromised subjects.

  9. Secretion, delivery and function of oomycete effector proteins.

    PubMed

    Wawra, Stephan; Belmonte, Rodrigo; Löbach, Lars; Saraiva, Marcia; Willems, Ariane; van West, Pieter

    2012-12-01

    Oomycetes are responsible for multi-billion dollar damages in aquaculture, agriculture and forestry. One common strategy they share with most cellular disease agents is the secretion of effector proteins. Effectors are molecules that change host physiology by initiating and allowing an infection to develop. Oomycetes secrete both extracellular and intracellular effectors. Studying secretion, delivery and function of effectors will hopefully lead to alternative control measures, which is much needed as several chemicals to control plant and animal pathogenic oomycetes cannot be used anymore; due to resistance in the host, or because the control measures have been prohibited as a result of toxicity to the environment and/or consumers. Here the latest findings on oomycete effector secretion, delivery and function are discussed.

  10. Double lead spiral platen parallel jaw end effector

    NASA Technical Reports Server (NTRS)

    Beals, David C.

    1989-01-01

    The double lead spiral platen parallel jaw end effector is an extremely powerful, compact, and highly controllable end effector that represents a significant improvement in gripping force and efficiency over the LaRC Puma (LP) end effector. The spiral end effector is very simple in its design and has relatively few parts. The jaw openings are highly predictable and linear, making it an ideal candidate for remote control. The finger speed is within acceptable working limits and can be modified to meet the user needs; for instance, greater finger speed could be obtained by increasing the pitch of the spiral. The force relaxation is comparable to the other tested units. Optimization of the end effector design would involve a compromise of force and speed for a given application.

  11. Advanced Aerodynamic Design of Passive Porosity Control Effectors

    NASA Technical Reports Server (NTRS)

    Hunter, Craig A.; Viken, Sally A.; Wood, Richard M.; Bauer, Steven X. S.

    2001-01-01

    This paper describes aerodynamic design work aimed at developing a passive porosity control effector system for a generic tailless fighter aircraft. As part of this work, a computational design tool was developed and used to layout passive porosity effector systems for longitudinal and lateral-directional control at a low-speed, high angle of attack condition. Aerodynamic analysis was conducted using the NASA Langley computational fluid dynamics code USM3D, in conjunction with a newly formulated surface boundary condition for passive porosity. Results indicate that passive porosity effectors can provide maneuver control increments that equal and exceed those of conventional aerodynamic effectors for low-speed, high-alpha flight, with control levels that are a linear function of porous area. This work demonstrates the tremendous potential of passive porosity to yield simple control effector systems that have no external moving parts and will preserve an aircraft's fixed outer mold line.

  12. Fungal effector Ecp6 outcompetes host immune receptor for chitin binding through intrachain LysM dimerization

    PubMed Central

    Kombrink, Anja; Hansen, Guido; Valkenburg, Dirk-Jan

    2013-01-01

    While host immune receptors detect pathogen-associated molecular patterns to activate immunity, pathogens attempt to deregulate host immunity through secreted effectors. Fungi employ LysM effectors to prevent recognition of cell wall-derived chitin by host immune receptors, although the mechanism to compete for chitin binding remained unclear. Structural analysis of the LysM effector Ecp6 of the fungal tomato pathogen Cladosporium fulvum reveals a novel mechanism for chitin binding, mediated by intrachain LysM dimerization, leading to a chitin-binding groove that is deeply buried in the effector protein. This composite binding site involves two of the three LysMs of Ecp6 and mediates chitin binding with ultra-high (pM) affinity. Intriguingly, the remaining singular LysM domain of Ecp6 binds chitin with low micromolar affinity but can nevertheless still perturb chitin-triggered immunity. Conceivably, the perturbation by this LysM domain is not established through chitin sequestration but possibly through interference with the host immune receptor complex. DOI: http://dx.doi.org/10.7554/eLife.00790.001 PMID:23840930

  13. Fungal effector Ecp6 outcompetes host immune receptor for chitin binding through intrachain LysM dimerization.

    PubMed

    Sánchez-Vallet, Andrea; Saleem-Batcha, Raspudin; Kombrink, Anja; Hansen, Guido; Valkenburg, Dirk-Jan; Thomma, Bart P H J; Mesters, Jeroen R

    2013-07-02

    While host immune receptors detect pathogen-associated molecular patterns to activate immunity, pathogens attempt to deregulate host immunity through secreted effectors. Fungi employ LysM effectors to prevent recognition of cell wall-derived chitin by host immune receptors, although the mechanism to compete for chitin binding remained unclear. Structural analysis of the LysM effector Ecp6 of the fungal tomato pathogen Cladosporium fulvum reveals a novel mechanism for chitin binding, mediated by intrachain LysM dimerization, leading to a chitin-binding groove that is deeply buried in the effector protein. This composite binding site involves two of the three LysMs of Ecp6 and mediates chitin binding with ultra-high (pM) affinity. Intriguingly, the remaining singular LysM domain of Ecp6 binds chitin with low micromolar affinity but can nevertheless still perturb chitin-triggered immunity. Conceivably, the perturbation by this LysM domain is not established through chitin sequestration but possibly through interference with the host immune receptor complex. DOI:http://dx.doi.org/10.7554/eLife.00790.001.

  14. Expression of Pseudomonas syringae type III effectors in yeast under stress conditions reveals that HopX1 attenuates activation of the high osmolarity glycerol MAP kinase pathway.

    PubMed

    Salomon, Dor; Bosis, Eran; Dar, Daniel; Nachman, Iftach; Sessa, Guido

    2012-11-01

    The Gram-negative bacterium Pseudomonas syringae pv. tomato (Pst) is the causal agent of speck disease in tomato. Pst pathogenicity depends on a type III secretion system that delivers effector proteins into host cells, where they promote disease by manipulating processes to the advantage of the pathogen. Previous studies identified seven Pst effectors that inhibit growth when expressed in yeast under normal growth conditions, suggesting that they interfere with cellular processes conserved in yeast and plants. We hypothesized that effectors also target conserved cellular processes that are required for yeast growth only under stress conditions. We therefore examined phenotypes induced by expression of Pst effectors in yeast grown in the presence of various stressors. Out of 29 effectors tested, five (HopX1, HopG1, HopT1-1, HopH1 and AvrPtoB) displayed growth inhibition phenotypes only in combination with stress conditions. Viability assays revealed that the HopX1 effector caused loss of cell viability under prolonged osmotic stress. Using transcription reporters, we found that HopX1 attenuated the activation of the high osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway, which is responsible for yeast survival under osmotic stress, while other MAPK pathways were mildly affected by HopX1. Interestingly, HopX1-mediated phenotypes in yeast were dependent on the putative transglutaminase catalytic triad of the effector. This study enlarges the pool of phenotypes available for the functional analysis of Pst type III effectors in yeast, and exemplifies how analysis of phenotypes detected in yeast under stress conditions can lead to the identification of eukaryotic cellular processes affected by bacterial effectors.

  15. Action selection in multi-effector decision making

    PubMed Central

    Madlon-Kay, Seth; Pesaran, Bijan; Daw, Nathaniel D.

    2012-01-01

    Decision making and reinforcement learning over movements suffer from the curse of dimensionality: the space of possible movements is too vast to search or even represent in its entirety. When actions involve only a single effector, this problem can be ameliorated by considering that effector separately; accordingly, the brain’s sensorimotor systems can subdivide choice by representing values and actions separately for each effector. However, for many actions, such as playing the piano, the value of an action by an effector (e.g., a hand) depends inseparably on the actions of other effectors. By definition, the values of such coordinated multi-effector actions cannot be represented by effector-specific action values, such as those that have been most extensively investigated in parietal and premotor regions. For such actions, one possible solution is to choose according to more abstract valuations over different goods or options, which can then be mapped onto the necessary motor actions. Such an approach separates the learning and decision problem, which will often be lower-dimensional than the space of possible movements, from the multi-effector movement planning problem. The ventromedial prefrontal cortex (vmPFC) is thought to contain goods-based value signals, so we hypothesized that this region might preferentially drive multi-effector action selection. To examine how the brain solves this problem, we used fMRI to compare patterns of BOLD activity in humans during reward learning tasks in which options were selected through either unimanual or bimanual actions, and in which the response requirements in the latter condition inseparably coupled valuation across both hands. We found value signals in the bilateral medial motor cortex and vmPFC, and consistent with previous studies, the medial motor value signals contained contra-lateral biases indicating effector-specificity, while the vmPFC value signals did not exhibit detectable effector specificity. Although

  16. Phospholipase C-delta1 and oxytocin receptor signalling: evidence of its role as an effector.

    PubMed

    Park, E S; Won, J H; Han, K J; Suh, P G; Ryu, S H; Lee, H S; Yun, H Y; Kwon, N S; Baek, K J

    1998-04-01

    Although the oxytocin receptor modulates intracellular Ca2+ ion levels in myometrium, the identities of signal molecules have not been clearly clarified. Our previous studies on oxytocin receptor signalling demonstrated that 80 kDa Ghalpha is a signal mediator [Baek, Kwon, Lee, Kim, Muralidhar and Im (1996) Biochem. J. 315, 739-744]. To elucidate the effector in the oxytocin receptor signalling pathway, we evaluated the oxytocin-mediated activation of phospholipase C (PLC) by using solubilized membranes from human myometrium and a three-component preparation containing the oxytocin receptor-Ghalpha-PLC-delta1 complex. PLC-delta1 activity in the three-component preparation, as well as PLC activity in solubilized membranes, was increased by oxytocin in the presence of Ca2+ and activated Ghalpha (GTP-bound Ghalpha). Furthermore the stimulated PLC-delta1 activity resulting from activation of Ghalpha via the oxytocin receptor was significantly attenuated by the selective oxytocin antagonist desGly-NH2d(CH2)5[Tyr(Me)2,Thr4]ornithine vasotocin or GDP. Consistent with these observations, co-immunoprecipitation and co-immunoadsorption of PLC-delta1 in the three-component preparation by anti-Gh7alpha antibody resulted in the PLC-delta1 being tightly coupled to activated Ghalpha on stimulation of the oxytocin receptor. These results indicate that PLC-delta1 is the effector for Ghalpha-mediated oxytocin receptor signalling.

  17. A Pseudomonas aeruginosa type VI secretion phospholipase D effector targets both prokaryotic and eukaryotic cells.

    PubMed

    Jiang, Feng; Waterfield, Nicholas R; Yang, Jian; Yang, Guowei; Jin, Qi

    2014-05-14

    Widely found in animal and plant-associated proteobacteria, type VI secretion systems (T6SSs) are potentially capable of facilitating diverse interactions with eukaryotes and/or other bacteria. Pseudomonas aeruginosa encodes three distinct T6SS haemolysin coregulated protein (Hcp) secretion islands (H1, H2, and H3-T6SS), each involved in different aspects of the bacterium's interaction with other organisms. Here we describe the characterization of a P. aeruginosa H3-T6SS-dependent phospholipase D effector, PldB, and its three tightly linked cognate immunity proteins. PldB targets the periplasm of prokaryotic cells and exerts an antibacterial activity. Surprisingly, PldB also facilitates intracellular invasion of host eukaryotic cells by activation of the PI3K/Akt pathway, revealing it to be a trans-kingdom effector. Our findings imply a potentially widespread T6SS-mediated mechanism, which deploys a single phospholipase effector to influence both prokaryotic cells and eukaryotic hosts.

  18. Suppression of rice immunity by Xanthomonas oryzae type III effector Xoo2875.

    PubMed

    Yamaguchi, Koji; Nakamura, Yusuke; Ishikawa, Kazuya; Yoshimura, Yuya; Tsuge, Seiji; Kawasaki, Tsutomu

    2013-01-01

    Xanthomonas oryzae delivers effector proteins into host cells through a type III secretion system to inhibit host immune responses, but how these effectors suppress host immunity is largely unknown. Here we found that Xoo2875, one of the effectors of X. oryzae, strongly inhibited host resistance to X. oryzae. Transgenic rice plants expressing Xoo2875 exhibited semi-dwarfism and a reduction in Brassinolide-dependent laminar inclination, characteristics of brassinosteroid (BR)-insensitive mutants caused by mutations of the BR receptor. A yeast two-hybrid experiment indicated that Xoo2875 interacted with OsBAK1, an essential component of both microbe-associated molecular patterns (MAMPs) and BR receptors, suggesting that the virulent activity of Xoo2875 is mediated by inhibition of OsBAK1. Expression of Xoo2875 in Arabidopsis cells activated host immune responses, suggesting the presence of intracellular immune receptors that recognize Xoo2875. Because Xoo2875 homologs are highly conserved in Xanthomonas species, the development of Xoo2875-induced immunity is probably a useful strategy to avoid pathogen invasion.

  19. Unraveling Effector Functions of B Cells During Infection: The Hidden World Beyond Antibody Production

    PubMed Central

    León, Beatriz; Ballesteros-Tato, André; Misra, Ravi S.; Wojciechowski, Wojciech; Lund, Frances E.

    2015-01-01

    Antibodies made by B cells are critically important for immune protection to a variety of infectious agents. However, it is becoming increasingly clear that B cells do more than make antibodies and that B cells can both enhance and suppress immune responses. Furthermore, there is growing evidence that B cells modulate cellular immune responses by antibody dependent and independent mechanisms. Although we have a good understanding of the roles played by antibody-secreting effector B cells during immune responses, we know very little about the Ab independent “effector” functions of B cells in either health or disease. Given the recent data suggesting that B cells may contribute to autoimmune disease pathogenesis via an antibody independent mechanism and the increasing use of B cell depletion therapy in autoimmune patients, investigators are beginning to reassess the multiple roles for B cells during immune responses. In this article, we review data describing how B cells mediate protection to pathogens independently of antibody production. In particular, we will focus on the role that B cells play in facilitating dendritic cell and T cell interactions in lymph nodes, the importance of antigen-presenting B cells in sustaining effector T cell and T follicular helper responses to pathogens and the relevance of cytokine-producing effector and regulatory B cells in immune responses. PMID:22394173

  20. Trio's Rho-specific GEF domain is the missing Galpha q effector in C. elegans.

    PubMed

    Williams, Stacey L; Lutz, Susanne; Charlie, Nicole K; Vettel, Christiane; Ailion, Michael; Coco, Cassandra; Tesmer, John J G; Jorgensen, Erik M; Wieland, Thomas; Miller, Kenneth G

    2007-11-01

    The Galpha(q) pathway is essential for animal life and is a central pathway for driving locomotion, egg laying, and growth in Caenorhabditis elegans, where it exerts its effects through EGL-8 (phospholipase Cbeta [PLCbeta]) and at least one other effector. To find the missing effector, we performed forward genetic screens to suppress the slow growth and hyperactive behaviors of mutants with an overactive Galpha(q) pathway. Four suppressor mutations disrupted the Rho-specific guanine-nucleotide exchange factor (GEF) domain of UNC-73 (Trio). The mutations produce defects in neuronal function, but not neuronal development, that cause sluggish locomotion similar to animals lacking EGL-8 (PLCbeta). Strains containing null mutations in both EGL-8 (PLCbeta) and UNC-73 (Trio RhoGEF) have strong synthetic phenotypes that phenocopy the arrested growth and near-complete paralysis of Galpha(q)-null mutants. Using cell-based and biochemical assays, we show that activated C. elegans Galpha(q) synergizes with Trio RhoGEF to activate RhoA. Activated Galpha(q) and Trio RhoGEF appear to be part of a signaling complex, because they coimmunoprecipitate when expressed together in cells. Our results show that Trio's Rho-specific GEF domain is a major Galpha(q) effector that, together with PLCbeta, mediates the Galpha(q) signaling that drives the locomotion, egg laying, and growth of the animal.

  1. Complement Effectors of Inflammation in Cystic Fibrosis Lung Fluid Correlate with Clinical Measures of Disease

    PubMed Central

    Sass, Laura A.; Hair, Pamela S.; Perkins, Amy M.; Shah, Tushar A.; Krishna, Neel K.; Cunnion, Kenji M.

    2015-01-01

    In cystic fibrosis (CF), lung damage is mediated by a cycle of obstruction, infection, and inflammation. Here we explored complement inflammatory effectors in CF lung fluid. In this study soluble fractions (sols) from sputum samples of 15 CF patients were assayed for complement effectors and analyzed with clinical measurements. The pro-inflammatory peptide C5a was increased 4.8-fold (P = 0.04) in CF sols compared with controls. Incubation of CF sols with P. aeruginosa or S. aureus increased C5a concentration 2.3-fold (P = 0.02). A peptide inhibitor of complement C1 (PIC1) completely blocked the increase in C5a concentration from P. aeruginosa in CF sol in vitro (P = 0.001). C5a concentration in CF sol correlated inversely with body mass index (BMI) percentile in children (r = -0.77, P = 0.04). C3a, which has anti-inflammatory effects, correlated positively with FEV1% predicted (rs = 0.63, P = 0.02). These results suggest that complement effectors may significantly impact inflammation in CF lung fluid. PMID:26642048

  2. Complement Effectors of Inflammation in Cystic Fibrosis Lung Fluid Correlate with Clinical Measures of Disease.

    PubMed

    Sass, Laura A; Hair, Pamela S; Perkins, Amy M; Shah, Tushar A; Krishna, Neel K; Cunnion, Kenji M

    2015-01-01

    In cystic fibrosis (CF), lung damage is mediated by a cycle of obstruction, infection, and inflammation. Here we explored complement inflammatory effectors in CF lung fluid. In this study soluble fractions (sols) from sputum samples of 15 CF patients were assayed for complement effectors and analyzed with clinical measurements. The pro-inflammatory peptide C5a was increased 4.8-fold (P = 0.04) in CF sols compared with controls. Incubation of CF sols with P. aeruginosa or S. aureus increased C5a concentration 2.3-fold (P = 0.02). A peptide inhibitor of complement C1 (PIC1) completely blocked the increase in C5a concentration from P. aeruginosa in CF sol in vitro (P = 0.001). C5a concentration in CF sol correlated inversely with body mass index (BMI) percentile in children (r = -0.77, P = 0.04). C3a, which has anti-inflammatory effects, correlated positively with FEV1% predicted (rs = 0.63, P = 0.02). These results suggest that complement effectors may significantly impact inflammation in CF lung fluid.

  3. Signals for the execution of Th2 effector function.

    PubMed

    Fowell, Deborah J

    2009-04-01

    Appropriate control of infection depends on the generation of lymphocytes armed with a particular array of cytokine and chemokine effector molecules. The differentiation of naïve T cells into functionally distinct effector subsets is regulated by signals from the T cell receptor (TCR) and cytokine receptors. Using gene knock-out approaches, the initiation of discrete effector programs appears differentially sensitive to the loss of individual TCR signaling components; likely due to differences in the transcription factors needed to activate individual cytokine genes. Less well understood however, are the signal requirements for the execution of effector function. With a focus on Th2 cells and the kinase ITK, we review recent observations that point to differences between the signals needed for the initiation and implementation of cytokine programs in CD4+ T cells. Indeed, Th2 effector cells signal differently from both their naïve counterparts and from Th1 effectors suggesting they may transduce activation signals differently or may be selectively receptive to different activation signals. Potential regulation points for effector function lie at the level of transcription and translation of cytokine genes. We also discuss how provision of these execution signals may be spatially segregated in vivo occurring at tissue sites of inflammation and subject to modulation by the pathogen itself.

  4. Bioprospecting open reading frames for peptide effectors.

    PubMed

    Xiong, Ling; Scott, Charles

    2014-01-01

    Recent successes in the development of small-molecule antagonists of protein-protein interactions designed based on co-crystal structures of peptides bound to their biological targets confirm that short peptides derived from interacting proteins can be high-value ligands for pharmacologic validation of targets and for identification of druggable sites. Evolved sequence space is likely to be enriched for interacting peptides, but identifying minimal peptide effectors within genomic sequence can be labor intensive. Here we describe the use of incremental truncation to diversify genetic material on the scale of open reading frames into comprehensive libraries of constituent peptides. The approach is capable of generating peptides derived from both continuous and discontinuous sequence elements, and is compatible with the expression of free linear or backbone cyclic peptides, with peptides tethered to amino- or carboxyl-terminal fusion partners or with the expression of peptides displayed within protein scaffolds (peptide aptamers). Incremental truncation affords a valuable source of molecular diversity to interrogate the druggable genome or evaluate the therapeutic potential of candidate genes.

  5. Computational investigation of miniature trailing edge effectors

    NASA Astrophysics Data System (ADS)

    Lee, Hak-Tae

    Miniature trailing edge effectors (MiTEs) are small flaps (typically 1% to 5% chord) actuated with deflection angles of up to 90 degrees. The small size, combined with little required power and good control authority, enables the device to be used for high bandwidth control as well as conventional attitude control. However, some of the aerodynamic characteristics of these devices are complex and poorly understood. This research investigated the aerodynamics of MiTEs using incompressible Navier-Stokes flow solvers, INS2D and INS3D. To understand the flow structure and establish a parametric database, two dimensional steady-state computations were performed for MiTEs with various geometries and flow conditions. Time accurate computations were used to resolve the unsteady characteristics including transient response and vortex shedding phenomena. The frequency response was studied to fully identify the dynamics of MiTEs. Three dimensional computations show the change in control effectiveness with respect to the spanwise length of MiTEs as well as the spanwise lift distribution induced by these devices. Based on the CFD results, an approximate vortex panel model was developed for design purposes that reproduces the key characteristics of MiTEs. Two application areas for MiTEs were explored. Flutter suppression was demonstrated by combining a finite element structural model with the vortex panel model. The application of MiTEs to augment maximum lift and improve the post stall behavior of an airfoil was also investigated.

  6. Rack Insertion End Effector (RIEE) guidance

    NASA Technical Reports Server (NTRS)

    Malladi, Narasimha S.

    1994-01-01

    NASA-KSC has developed a mechanism to handle and insert Racks into the Space Station Logistic Modules. This mechanism consists of a Base with 3 motorized degrees of freedom, a 3 section motorized Boom that goes from 15 to 44 feet in length, and a Rack Insertion End Effector (RIEE) with 5 hand wheels for precise alignment. During the 1993 NASA-ASEE Summer Faculty Fellowship Program at KSC, I designed an Active Vision (Camera) Arrangement and developed an algorithm to determine (1) the displacements required by the Room for its initial positioning and (2) the rotations required at the five hand-wheels of the RIEE, for the insertion of the Rack, using the centroids fo the Camera Images of the Location Targets in the Logistic Module. Presently, during the summer of '94, I completed the preliminary design of an easily portable measuring instrument using encoders to obtain the 3-Dimensional Coordinates of Location Targets in the Logistics Module relative to the RIEE mechanism frame. The algorithm developed in '93 can use the output of this instrument also. Simplification of the '93 work and suggestions for the future work are discussed.

  7. Structure of the archaeal Cascade subunit Csa5: relating the small subunits of CRISPR effector complexes.

    PubMed

    Reeks, Judith; Graham, Shirley; Anderson, Linzi; Liu, Huanting; White, Malcolm F; Naismith, James H

    2013-05-01

    The Cascade complex for CRISPR-mediated antiviral immunity uses CRISPR RNA (crRNA) to target invading DNA species from mobile elements such as viruses, leading to their destruction. The core of the Cascade effector complex consists of the Cas5 and Cas7 subunits, which are widely conserved in prokaryotes. Cas7 binds crRNA and forms the helical backbone of Cascade. Many archaea encode a version of the Cascade complex (denoted Type I-A) that includes a Csa5 (or small) subunit, which interacts weakly with the core proteins. Here, we report the crystal structure of the Csa5 protein from Sulfolobus solfataricus. Csa5 comprises a conserved α-helical domain with a small insertion consisting of a weakly conserved β-strand domain. In the crystal, the Csa5 monomers have multimerized into infinite helical threads. At each interface is a strictly conserved intersubunit salt bridge, deletion of which disrupts multimerization. Structural analysis indicates a shared evolutionary history among the small subunits of the CRISPR effector complexes. The same α-helical domain is found in the C-terminal domain of Cse2 (from Type I-E Cascade), while the N-terminal domain of Cse2 is found in Cmr5 of the CMR (Type III-B) effector complex. As Cmr5 shares no match with Csa5, two possibilities present themselves: selective domain loss from an ancestral Cse2 to create two new subfamilies or domain fusion of two separate families to create a new Cse2 family. A definitive answer awaits structural studies of further small subunits from other CRISPR effector complexes.

  8. The Piriformospora indica effector PIIN_08944 promotes the mutualistic Sebacinalean symbiosis

    PubMed Central

    Akum, Fidele N.; Steinbrenner, Jens; Biedenkopf, Dagmar; Imani, Jafargholi; Kogel, Karl-Heinz

    2015-01-01

    Pathogenic and mutualistic microbes actively suppress plant defense by secreting effector proteins to manipulate the host responses for their own benefit. Current knowledge about fungal effectors has been mainly derived from biotrophic and hemibiotrophic plant pathogenic fungi and oomycetes with restricted host range. We studied colonization strategies of the root endophytic basidiomycete Piriformospora indica that colonizes a wide range of plant species thereby establishing long-term mutualistic relationships. The release of P. indica’s genome helped to identify hundreds of genes coding for candidate effectors and provides an opportunity to investigate the role of those proteins in a mutualistic symbiosis. We demonstrate that the candidate effector PIIN_08944 plays a crucial role during fungal colonization of Arabidopsis thaliana roots. PIIN_08944 expression was detected during chlamydospore germination, and fungal deletion mutants (PiΔ08944) showed delayed root colonization. Constitutive over-expression of PIIN_08944 in Arabidopsis rescued the delayed colonization phenotype of the deletion mutant. PIIN_08944-expressing Arabidopsis showed a reduced expression of flg22-induced marker genes of pattern-triggered immunity (PTI) and the salicylic acid (SA) defense pathway, and expression of PIIN_08944 in barley reduced the burst of reactive oxygen species (ROS) triggered by flg22 and chitin. These data suggest that PIIN_08944 contributes to root colonization by P. indica by interfering with SA-mediated basal immune responses of the host plant. Consistent with this, PIIN_08944-expressing Arabidopsis also supported the growth of the biotrophic oomycete Hyaloperonospora arabidopsidis while growth of the necrotrophic fungi Botrytis cinerea on Arabidopsis and Fusarium graminearum on barley was not affected. PMID:26579156

  9. System for exchanging tools and end effectors on a robot

    DOEpatents

    Burry, David B.; Williams, Paul M.

    1991-02-19

    A system and method for exchanging tools and end effectors on a robot permits exchange during a programmed task. The exchange mechanism is located off the robot, thus reducing the mass of the robot arm and permitting smaller robots to perform designated tasks. A simple spring/collet mechanism mounted on the robot is used which permits the engagement and disengagement of the tool or end effector without the need for a rotational orientation of the tool to the end effector/collet interface. As the tool changing system is not located on the robot arm no umbilical cords are located on robot.

  10. System for exchanging tools and end effectors on a robot

    DOEpatents

    Burry, D.B.; Williams, P.M.

    1991-02-19

    A system and method for exchanging tools and end effectors on a robot permits exchange during a programmed task. The exchange mechanism is located off the robot, thus reducing the mass of the robot arm and permitting smaller robots to perform designated tasks. A simple spring/collet mechanism mounted on the robot is used which permits the engagement and disengagement of the tool or end effector without the need for a rotational orientation of the tool to the end effector/collet interface. As the tool changing system is not located on the robot arm no umbilical cords are located on robot. 12 figures.

  11. TLR4 ligands lipopolysaccharide and monophosphoryl lipid a differentially regulate effector and memory CD8+ T Cell differentiation.

    PubMed

    Cui, Weiguo; Joshi, Nikhil S; Liu, Ying; Meng, Hailong; Kleinstein, Steven H; Kaech, Susan M

    2014-05-01

    Vaccines formulated with nonreplicating pathogens require adjuvants to help bolster immunogenicity. The role of adjuvants in Ab production has been well studied, but how they influence memory CD8(+) T cell differentiation remains poorly defined. In this study we implemented dendritic cell-mediated immunization to study the effects of commonly used adjuvants, TLR ligands, on effector and memory CD8(+) T cell differentiation in mice. Intriguingly, we found that the TLR4 ligand LPS was far more superior to other TLR ligands in generating memory CD8(+) T cells upon immunization. LPS boosted clonal expansion similar to the other adjuvants, but fewer of the activated CD8(+) T cells died during contraction, generating a larger pool of memory cells. Surprisingly, monophosphoryl lipid A (MPLA), another TLR4 ligand, enhanced clonal expansion of effector CD8(+) T cells, but it also promoted their terminal differentiation and contraction; thus, fewer memory CD8(+) T cells formed, and MPLA-primed animals were less protected against secondary infection compared with those primed with LPS. Furthermore, gene expression profiling revealed that LPS-primed effector cells displayed a stronger pro-memory gene expression signature, whereas the gene expression profile of MPLA-primed effector cells aligned closer with terminal effector CD8(+) T cells. Lastly, we demonstrated that the LPS-TLR4-derived "pro-memory" signals were MyD88, but not Toll/IL-1R domain-containing adapter inducing IFN-β, dependent. This study reveals the influential power of adjuvants on the quantity and quality of CD8(+) T cell memory, and that attention to adjuvant selection is crucial because boosting effector cell expansion may not always equate with more memory T cells or greater protection.

  12. Transition of late-stage effector T cells to CD27+ CD28+ tumor-reactive effector memory T cells in humans after adoptive cell transfer therapy

    PubMed Central

    Powell, Daniel J.; Dudley, Mark E.; Robbins, Paul F.; Rosenberg, Steven A.

    2007-01-01

    In humans, the pathways of memory T-cell differentiation remain poorly defined. Recently, adoptive cell transfer (ACT) of tumor-reactive T lymphocytes to metastatic melanoma patients after nonmyeloablative chemotherapy has resulted in persistence of functional, tumor-reactive lymphocytes, regression of disease, and induction of melanocyte-directed autoimmunity in some responding patients. In the current study, longitudinal phenotypic analysis was performed on melanoma antigen–specific CD8+ T cells during their transition from in vitro cultured effector cells to long-term persistent memory cells following ACT to 6 responding patients. Tumor-reactive T cells used for therapy were generally late-stage effector cells with a CD27Lo CD28Lo CD45RA− CD62 ligand− (CD62L−) CC chemokine receptor 7− (CCR7−) interleukin-7 receptor αLo (IL-7RαLo) phenotype. After transfer, rapid up-regulation and continued expression of IL-7Rα in vivo suggested an important role for IL-7R in immediate and long-term T-cell survival. Although the tumor antigen–specific T-cell population contracted between 1 and 4 weeks after transfer, stable numbers of CD27+ CD28+ tumor-reactive T cells were maintained, demonstrating their contribution to the development of long-term, melanoma-reactive memory CD8+ T cells in vivo. At 2 months after transfer, melanoma-reactive T cells persisted at high levels and displayed an effector memory phenotype, including a CD27+ CD28+ CD62L− CCR7− profile, which may explain in part their ability to mediate tumor destruction. PMID:15345595

  13. Actin dynamics shape microglia effector functions.

    PubMed

    Uhlemann, Ria; Gertz, Karen; Boehmerle, Wolfgang; Schwarz, Tobias; Nolte, Christiane; Freyer, Dorette; Kettenmann, Helmut; Endres, Matthias; Kronenberg, Golo

    2016-06-01

    Impaired actin filament dynamics have been associated with cellular senescence. Microglia, the resident immune cells of the brain, are emerging as a central pathophysiological player in neurodegeneration. Microglia activation, which ranges on a continuum between classical and alternative, may be of critical importance to brain disease. Using genetic and pharmacological manipulations, we studied the effects of alterations in actin dynamics on microglia effector functions. Disruption of actin dynamics did not affect transcription of genes involved in the LPS-triggered classical inflammatory response. By contrast, in consequence of impaired nuclear translocation of phospho-STAT6, genes involved in IL-4 induced alternative activation were strongly downregulated. Functionally, impaired actin dynamics resulted in reduced NO secretion and reduced release of TNFalpha and IL-6 from LPS-stimulated microglia and of IGF-1 from IL-4 stimulated microglia. However, pathological stabilization of the actin cytoskeleton increased LPS-induced release of IL-1beta and IL-18, which belong to an unconventional secretory pathway. Reduced NO release was associated with decreased cytoplasmic iNOS protein expression and decreased intracellular arginine uptake. Furthermore, disruption of actin dynamics resulted in reduced microglia migration, proliferation and phagocytosis. Finally, baseline and ATP-induced [Ca(2+)]int levels were significantly increased in microglia lacking gelsolin, a key actin-severing protein. Together, the dynamic state of the actin cytoskeleton profoundly and distinctly affects microglia behaviours. Disruption of actin dynamics attenuates M2 polarization by inhibiting transcription of alternative activation genes. In classical activation, the role of actin remodelling is complex, does not relate to gene transcription and shows a major divergence between cytokines following conventional and unconventional secretion.

  14. Changes in nucleoporin domain topology in response to chemical effectors.

    PubMed

    Paulillo, Sara M; Powers, Maureen A; Ullman, Katharine S; Fahrenkrog, Birthe

    2006-10-13

    Nucleoporins represent the molecular building blocks of nuclear pore complexes (NPCs), which mediate facilitated macromolecular trafficking between the cytoplasm and nucleus of eukaryotic cells. Phenylalanine-glycine (FG) repeat motifs are found in about one-third of the nucleoporins, and they provide major binding or docking sites for soluble transport receptors. We have shown recently that localization of the FG-repeat domains of vertebrate nucleoporins Nup153 and Nup214 within the NPC is influenced by its transport state. To test whether chemical effectors, such as calcium and ATP, influence the localization of the FG-repeat domains of Nup153 and Nup214 within the NPC, we performed immuno-electron microscopy of Xenopus oocyte nuclei using domain-specific antibodies against Nup153 and Nup214, respectively. Ca2+ and ATP are known to induce conformational changes in the NPC architecture, especially at the cytoplasmic face, but also at the nuclear basket of the NPC. We have found concentrations of calcium in the micromolar range or 1 mM ATP in the surrounding buffer leaves the spatial distribution of the FG-repeat of Nup153 and Nup214 largely unchanged. In contrast, ATP depletion, calcium store depletion by EGTA or thapsigargin, and high concentrations of divalent cation (i.e. 2 mM Ca2+ and 2 mM Mg2+) constrain the distribution of the FG-repeats of Nup153 and Nup214. Our data suggest that the location of the FG-repeat domains of Nup153 and Nup214 is sensitive to chemical changes within the near-field environment of the NPC.

  15. Genomic analysis of 38 Legionella species identifies large and diverse effector repertoires.

    PubMed

    Burstein, David; Amaro, Francisco; Zusman, Tal; Lifshitz, Ziv; Cohen, Ofir; Gilbert, Jack A; Pupko, Tal; Shuman, Howard A; Segal, Gil

    2016-02-01

    Infection by the human pathogen Legionella pneumophila relies on the translocation of ∼ 300 virulence proteins, termed effectors, which manipulate host cell processes. However, almost no information exists regarding effectors in other Legionella pathogens. Here we sequenced, assembled and characterized the genomes of 38 Legionella species and predicted their effector repertoires using a previously validated machine learning approach. This analysis identified 5,885 predicted effectors. The effector repertoires of different Legionella species were found to be largely non-overlapping, and only seven core effectors were shared by all species studied. Species-specific effectors had atypically low GC content, suggesting exogenous acquisition, possibly from the natural protozoan hosts of these species. Furthermore, we detected numerous new conserved effector domains and discovered new domain combinations, which allowed the inference of as yet undescribed effector functions. The effector collection and network of domain architectures described here can serve as a roadmap for future studies of effector function and evolution.

  16. An intelligent end-effector for a rehabilitation robot.

    PubMed

    Gosine, R G; Harwin, W S; Furby, L J; Jackson, R D

    1989-01-01

    A UMI RTX robot, modified with limited end-effector sensors and a restricted but effective vision system, is currently used in a developmental education setting for severely physically disabled children. The low physical and cognitive abilities of the children involved in the project require a semi-autonomous robot with environmental sensing capability to operate in a task oriented mode. A variety of low-cost sensors including proximity, distance, force and slip sensors, have been investigated for integration in end-effectors for the RTX robot. The sensors employed on a modified end-effector are detailed and experimental results are presented. A design for an end-effector with integrated sensors is discussed. The integration of the sensor information into a high-level, task-oriented programming language is detailed and examples of high-level control sequences using sensor inputs are presented. Finally, the development of intelligent gripping strategies based on sensor information is discussed.

  17. Effector-triggered immunity: from pathogen perception to robust defense.

    PubMed

    Cui, Haitao; Tsuda, Kenichi; Parker, Jane E

    2015-01-01

    In plant innate immunity, individual cells have the capacity to sense and respond to pathogen attack. Intracellular recognition mechanisms have evolved to intercept perturbations by pathogen virulence factors (effectors) early in host infection and convert it to rapid defense. One key to resistance success is a polymorphic family of intracellular nucleotide-binding/leucine-rich-repeat (NLR) receptors that detect effector interference in different parts of the cell. Effector-activated NLRs connect, in various ways, to a conserved basal resistance network in order to transcriptionally boost defense programs. Effector-triggered immunity displays remarkable robustness against pathogen disturbance, in part by employing compensatory mechanisms within the defense network. Also, the mobility of some NLRs and coordination of resistance pathways across cell compartments provides flexibility to fine-tune immune outputs. Furthermore, a number of NLRs function close to the nuclear chromatin by balancing actions of defense-repressing and defense-activating transcription factors to program cells dynamically for effective disease resistance.

  18. Gunite Scarifying End Effector. Innovative Technology Summary Report

    SciTech Connect

    2001-09-01

    The Gunite Scarifying End Effector (GSEE) is designed to remove a layer of the gunite tank walls, which are contaminated with radioactivity. Removing this radioactivity is necessary to close the tank.

  19. Interactions of legionella effector proteins with host phosphoinositide lipids.

    PubMed

    Weber, Stephen; Dolinsky, Stephanie; Hilbi, Hubert

    2013-01-01

    By means of the Icm/Dot type IV secretion system Legionella pneumophila translocates several effector proteins into host cells, where they anchor to the cytoplasmic face of the LCV membrane by binding to phosphoinositide (PI) lipids. Thus, phosphatidylinositol-4-phosphate anchors the effector proteins SidC and SidM, which promote the interaction of LCVs with the ER and the secretory vesicle trafficking -pathway. In this chapter, we describe protocols to (1) identify PI-binding proteins in Legionella lysates using PI-beads, (2) determine PI-binding specificities and affinities of recombinant Legionella effector proteins by protein-lipid overlays, and (3) use Legionella effectors to identify cellular PI lipids.

  20. Robotic end-effector for rewaterproofing shuttle tiles

    NASA Astrophysics Data System (ADS)

    Manouchehri, Davoud; Hansen, Joseph M.; Wu, Cheng M.; Yamamoto, Brian S.; Graham, Todd

    1992-11-01

    This paper summarizes work by Rockwell International's Space Systems Division's Robotics Group at Downey, California. The work is part of a NASA-led team effort to automate Space Shuttle rewaterproofing in the Orbiter Processing Facility at the Kennedy Space Center and the ferry facility at the Ames-Dryden Flight Research Facility. Rockwell's effort focuses on the rewaterproofing end-effector, whose function is to inject hazardous dimethylethyloxysilane into thousands of ceramic tiles on the underside of the orbiter after each flight. The paper has five sections. First, it presents background on the present manual process. Second, end-effector requirements are presented, including safety and interface control. Third, a design is presented for the five end-effector systems: positioning, delivery, containment, data management, and command and control. Fourth, end-effector testing and integrating to the total system are described. Lastly, future applications for this technology are discussed.

  1. Robotic end-effector for rewaterproofing shuttle tiles

    NASA Technical Reports Server (NTRS)

    Manouchehri, Davoud; Hansen, Joseph M.; Wu, Cheng M.; Yamamoto, Brian S.; Graham, Todd

    1992-01-01

    This paper summarizes work by Rockwell International's Space Systems Division's Robotics Group at Downey, California. The work is part of a NASA-led team effort to automate Space Shuttle rewaterproofing in the Orbiter Processing Facility at the Kennedy Space Center and the ferry facility at the Ames-Dryden Flight Research Facility. Rockwell's effort focuses on the rewaterproofing end-effector, whose function is to inject hazardous dimethylethyloxysilane into thousands of ceramic tiles on the underside of the orbiter after each flight. The paper has five sections. First, it presents background on the present manual process. Second, end-effector requirements are presented, including safety and interface control. Third, a design is presented for the five end-effector systems: positioning, delivery, containment, data management, and command and control. Fourth, end-effector testing and integrating to the total system are described. Lastly, future applications for this technology are discussed.

  2. Vision-based end-effector position error compensation

    NASA Technical Reports Server (NTRS)

    Bajracharya, Max; Backes, Paul; DiCicco, Matthew

    2006-01-01

    This paper describes a computationally efficient algorithm that provides the ability to accurately place an arm end-effector on a target designated in an image using low speed feed back from a fixed stero camera.

  3. Functional redundancy of necrotrophic effectors - consequences for exploitation for breeding.

    PubMed

    Tan, Kar-Chun; Phan, Huyen T T; Rybak, Kasia; John, Evan; Chooi, Yit H; Solomon, Peter S; Oliver, Richard P

    2015-01-01

    Necrotrophic diseases of wheat cause major losses in most wheat growing areas of world. Tan spot (caused by Pyrenophora tritici-repentis) and septoria nodorum blotch (SNB; Parastagonospora nodorum) have been shown to reduce yields by 10-20% across entire agri-ecological zones despite the application of fungicides and a heavy focus over the last 30 years on resistance breeding. Efforts by breeders to improve the resistance of cultivars has been compromised by the universal finding that resistance was quantitative and governed by multiple quantitative trait loci (QTL). Most QTL had a limited effect that was hard to measure precisely and varied significantly from site to site and season to season. The discovery of necrotrophic effectors has given breeding for disease resistance new methods and tools. In the case of tan spot in West Australia, a single effector, PtrToxA and its recogniser gene Tsn1, has a dominating impact in disease resistance. The delivery of ToxA to breeders has had a major impact on cultivar choice and breeding strategies. For P. nodorum, three effectors - SnToxA, SnTox1, and SnTox3 - have been well characterized. Unlike tan spot, no one effector has a dominating role. Genetic analysis of various mapping populations and pathogen isolates has shown that different effectors have varying impact and that epistatic interactions also occur. As a result of these factors the deployment of these effectors for SNB resistance breeding is more complex. We have deleted the three effectors in a strain of P. nodorum and measured effector activity and disease potential of the triple knockout mutant. The culture filtrate causes necrosis in several cultivars and the strain causes disease, albeit the overall levels are less than in the wild type. Modeling of the field disease resistance scores of cultivars from their reactions to the microbially expressed effectors SnToxA, SnTox1, and SnTox3 is significantly improved by including the response to the triple knockout

  4. Immune Effector Recovery in Chronic Myeloid Leukemia and Treatment-Free Remission.

    PubMed

    Hughes, Amy; Yong, Agnes S M

    2017-01-01

    Chronic myeloid leukemia (CML) is a hematological cancer, characterized by a reciprocal chromosomal translocation between chromosomes 9 and 22 [t(9;22)], producing the Bcr-Abl oncogene. Tyrosine kinase inhibitors (TKIs) represent the standard of care for CML patients and exert a dual mode of action: direct oncokinase inhibition and restoration of effector-mediated immune surveillance, which is rendered dysfunctional in CML patients at diagnosis, prior to TKI therapy. TKIs such as imatinib, and more potent second-generation nilotinib and dasatinib induce a high rate of deep molecular response (DMR, BCR-ABL1 ≤ 0.01%) in CML patients. As a result, the more recent goal of therapy in CML treatment is to induce a durable DMR as a prelude to successful treatment-free remission (TFR), which occurs in approximately half of all CML patients who cease TKI therapy. The lack of overt relapse in such patients has been attributed to immunological control of CML. In this review, we discuss an immunological timeline to successful TFR, focusing on the immunology of CML during TKI treatment; an initial period of immune suppression, limiting antitumor immune effector responses in newly diagnosed CML patients, linked to an expansion of immature myeloid-derived suppressor cells and regulatory T cells and aberrant expression of immune checkpoint signaling pathways, including programmed death-1/programmed death ligand-1. Commencement of TKI treatment is associated with immune system re-activation and restoration of effector-mediated [natural killer (NK) cell and T cell] immune surveillance in CML patients, albeit with differing frequencies in concert with differing levels of molecular response achieved on TKI. DMR is associated with maximal restoration of immune recovery in CML patients on TKI. Current data suggest a net balance between both the effector and suppressor arms of the immune system, at a minimum involving mature, cytotoxic CD56(dim) NK cells may be important in

  5. Specific in planta recognition of two GKLR proteins of the downy mildew Bremia lactucae revealed in a large effector screen in lettuce.

    PubMed

    Stassen, Joost H M; den Boer, Erik; Vergeer, Pim W J; Andel, Annemiek; Ellendorff, Ursula; Pelgrom, Koen; Pel, Mathieu; Schut, Johan; Zonneveld, Olaf; Jeuken, Marieke J W; Van den Ackerveken, Guido

    2013-11-01

    Breeding lettuce (Lactuca sativa) for resistance to the downy mildew pathogen Bremia lactucae is mainly achieved by introgression of dominant downy mildew resistance (Dm) genes. New Bremia races quickly render Dm genes ineffective, possibly by mutation of recognized host-translocated effectors or by suppression of effector-triggered immunity. We have previously identified 34 potential RXLR(-like) effector proteins of B. lactucae that were here tested for specific recognition within a collection of 129 B. lactucae-resistant Lactuca lines. Two effectors triggered a hypersensitive response: BLG01 in 52 lines, predominantly L. saligna, and BLG03 in two L. sativa lines containing Dm2 resistance. The N-terminal sequences of BLG01 and BLG03, containing the signal peptide and GKLR variant of the RXLR translocation motif, are not required for in planta recognition but function in effector delivery. The locus responsible for BLG01 recognition maps to the bottom of lettuce chromosome 9, whereas recognition of BLG03 maps in the RGC2 cluster on chromosome 2. Lactuca lines that recognize the BLG effectors are not resistant to Bremia isolate Bl:24 that expresses both BLG genes, suggesting that Bl:24 can suppress the triggered immune responses. In contrast, lettuce segregants displaying Dm2-mediated resistance to Bremia isolate Bl:5 are responsive to BLG03, suggesting that BLG03 is a candidate Avr2 protein.

  6. Design, testing and evaluation of latching end effector

    NASA Technical Reports Server (NTRS)

    Walker, B.; Vandersluis, R.

    1995-01-01

    The Latching End Effector (LEE) forms part of the Space Station Remote Manipulator System (SSRMS) for which Spar Aerospace Ltd, Space Systems Division is the prime contractor. The design, testing and performance evaluation of the Latching End Effector mechanisms is the subject of this paper focusing on: (1) ambient, thermal and vibration testing; (2) snare/rigidize performance testing and interaction during payload acquisition; and (3) latch/umbilical test results and performance.

  7. Views of the manipulator development facility end effector simulation

    NASA Technical Reports Server (NTRS)

    1985-01-01

    Closeup view of the manipulator development facility (MDF) during an end effector simulation showing the two specially made extensions deployed on April 16 by the STS 51-D crewmembers (30953); Medium closeup view of MDF. The white cylindar at right represents the Syncon IV (LEASAT) satellite. The robot device in the foreground is the forearm and end effector of a training version of the remote manipulator system (RMS). Attached to the arm's end are two flyswatter-like tools (30954-5).

  8. Views of the manipulator development facility end effector simulation

    NASA Technical Reports Server (NTRS)

    1985-01-01

    Closeup view of the manipulator development facility (MDF) during an end effector simulation showing the two specially made extensions deployed on April 16 by the STS 51-D crewmembers (30953); Medium closeup view of MDF. The white cylindar at right represents the Syncon IV (LEASAT) satellite. The robot device in the foreground is the forearm and end effector of a training version of the remote manipulator system (RMS). Attached to the arm's end are two flyswatter-like tools (30954-5).

  9. Plant Photosynthetic Responses During Insect Effector-Triggered Plant Susceptibility and Immunity.

    PubMed

    Gramig, Greta G; Harris, Marion O

    2015-06-01

    Gall-inducing insects are known for altering source-sink relationships within plants. Changes in photosynthesis may contribute to this phenomenon. We investigated photosynthetic responses in wheat [Triticum aestivum L. (Poaceae: Triticeae)] seedlings attacked by the Hessian fly [Mayetiola destructor (Say) (Diptera: Cecidomyiidae], which uses a salivary effector-based strategy to induce a gall nutritive tissue in susceptible plants. Resistant plants have surveillance systems mediated by products of Resistance (R) genes. Detection of a specific salivary effector triggers downstream responses that result in a resistance that kills neonate larvae. A 2 × 2 factorial design was used to study maximum leaf photosynthetic assimilation and stomatal conductance rates. The plant treatments were-resistant or susceptible wheat lines expressing or not expressing the H13 resistance gene. The insect treatments were-no attack (control) or attack by larvae killed by H13 gene-mediated resistance. Photosynthesis was measured for the second and third leaves of the seedling, the latter being the only leaf directly attacked by larvae. We predicted effector-based attack would trigger increases in photosynthetic rates in susceptible but not resistant plants. For susceptible plants, attack was associated with increases (relative to controls) in photosynthesis for the third but not the second leaf. For resistant plants, attack was associated with increases in photosynthesis for both the second and third leaves. Mechanisms underlying the increases appeared to differ. Resistant plants exhibited responses suggesting altered source-sink relationships. Susceptible plants exhibited responses suggesting a mechanism other than altered source-sink relationships, possibly changes in water relations that contributed to increased stomatal conductance.

  10. Characterization of the Largest Effector Gene Cluster of Ustilago maydis

    PubMed Central

    Vincon, Volker; Kahmann, Regine

    2014-01-01

    In the genome of the biotrophic plant pathogen Ustilago maydis, many of the genes coding for secreted protein effectors modulating virulence are arranged in gene clusters. The vast majority of these genes encode novel proteins whose expression is coupled to plant colonization. The largest of these gene clusters, cluster 19A, encodes 24 secreted effectors. Deletion of the entire cluster results in severe attenuation of virulence. Here we present the functional analysis of this genomic region. We show that a 19A deletion mutant behaves like an endophyte, i.e. is still able to colonize plants and complete the infection cycle. However, tumors, the most conspicuous symptoms of maize smut disease, are only rarely formed and fungal biomass in infected tissue is significantly reduced. The generation and analysis of strains carrying sub-deletions identified several genes significantly contributing to tumor formation after seedling infection. Another of the effectors could be linked specifically to anthocyanin induction in the infected tissue. As the individual contributions of these genes to tumor formation were small, we studied the response of maize plants to the whole cluster mutant as well as to several individual mutants by array analysis. This revealed distinct plant responses, demonstrating that the respective effectors have discrete plant targets. We propose that the analysis of plant responses to effector mutant strains that lack a strong virulence phenotype may be a general way to visualize differences in effector function. PMID:24992561

  11. Characterization of the largest effector gene cluster of Ustilago maydis.

    PubMed

    Brefort, Thomas; Tanaka, Shigeyuki; Neidig, Nina; Doehlemann, Gunther; Vincon, Volker; Kahmann, Regine

    2014-07-01

    In the genome of the biotrophic plant pathogen Ustilago maydis, many of the genes coding for secreted protein effectors modulating virulence are arranged in gene clusters. The vast majority of these genes encode novel proteins whose expression is coupled to plant colonization. The largest of these gene clusters, cluster 19A, encodes 24 secreted effectors. Deletion of the entire cluster results in severe attenuation of virulence. Here we present the functional analysis of this genomic region. We show that a 19A deletion mutant behaves like an endophyte, i.e. is still able to colonize plants and complete the infection cycle. However, tumors, the most conspicuous symptoms of maize smut disease, are only rarely formed and fungal biomass in infected tissue is significantly reduced. The generation and analysis of strains carrying sub-deletions identified several genes significantly contributing to tumor formation after seedling infection. Another of the effectors could be linked specifically to anthocyanin induction in the infected tissue. As the individual contributions of these genes to tumor formation were small, we studied the response of maize plants to the whole cluster mutant as well as to several individual mutants by array analysis. This revealed distinct plant responses, demonstrating that the respective effectors have discrete plant targets. We propose that the analysis of plant responses to effector mutant strains that lack a strong virulence phenotype may be a general way to visualize differences in effector function.

  12. Functional roles of effectors of plant-parasitic nematodes.

    PubMed

    Haegeman, Annelies; Mantelin, Sophie; Jones, John T; Gheysen, Godelieve

    2012-01-15

    Plant pathogens have evolved a variety of different strategies that allow them to successfully infect their hosts. Plant-parasitic nematodes secrete numerous proteins into their hosts. These proteins, called effectors, have various functions in the plant cell. The most studied effectors to date are the plant cell wall degrading enzymes, which have an interesting evolutionary history since they are believed to have been acquired from bacteria or fungi by horizontal gene transfer. Extensive genome, transcriptome and proteome studies have shown that plant-parasitic nematodes secrete many additional effectors. The function of many of these is less clear although during the last decade, several research groups have determined the function of some of these effectors. Even though many effectors remain to be investigated, it has already become clear that they can have very diverse functions. Some are involved in suppression of plant defences, while others can specifically interact with plant signalling or hormone pathways to promote the formation of nematode feeding sites. In this review, the most recent progress in the understanding of the function of plant-parasitic nematode effectors is discussed. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. Functional heterogeneity of human effector CD8+ T cells.

    PubMed

    Takata, Hiroshi; Naruto, Takuya; Takiguchi, Masafumi

    2012-02-09

    Effector CD8(+) T cells are believed to be terminally differentiated cells having cytotoxic activity and the ability to produce effector cytokines such as INF-γ and TNF-α. We investigated the difference between CXCR1(+) and CXCR1(-) subsets of human effector CD27(-)CD28(-)CD8(+) T cells. The subsets expressed cytolytic molecules similarly and exerted substantial cytolytic activity, whereas only the CXCR1(-) subset had IL-2 productivity and self-proliferative activity and was more resistant to cell death than the CXCR1(+) subset. These differences were explained by the specific up-regulation of CAMK4, SPRY2, and IL-7R in the CXCR1(-) subset and that of pro-apoptotic death-associated protein kinase 1 (DAPK1) in the CXCR1(+) subset. The IL-2 producers were more frequently found in the IL-7R(+) subset of the CXCR1(-) effector CD8(+) T cells than in the IL-7R(-) subset. IL-7/IL-7R signaling promoted cell survival only in the CXCR1(-) subset. The present study has highlighted a novel subset of effector CD8(+) T cells producing IL-2 and suggests the importance of this subset in the homeostasis of effector CD8(+) T cells.

  14. Tc1 and Tc2 effector cell therapy elicit long-term tumor immunity by contrasting mechanisms that result in complementary endogenous type 1 antitumor responses.

    PubMed

    Dobrzanski, Mark J; Reome, Joyce B; Hollenbaugh, Joseph A; Dutton, Richard W

    2004-02-01

    Cytolytic CD8(+) effector cells fall into two subpopulations based on cytokine secretion. Type 1 CD8(+) T cells (Tc1) secrete IFN-gamma, whereas type 2 CD8(+) T cells (Tc2) secrete IL-4 and IL-5. Both effector cell subpopulations display predominantly perforin-dependent cytolysis in vitro. Using an OVA-transfected B16 lung metastases model, we show that adoptively transferred OVA-specific Tc1 and Tc2 cells induce considerable suppression, but not cure, of pulmonary metastases. However, long-term tumor immunity prolonged survival times indefinitely and was evident by resistance to lethal tumor rechallenge. At early stages after therapy, protection by Tc2 and Tc1 effector cells were dependent in part on effector cell-derived IL-4, IL-5, and IFN-gamma, respectively. Whereas effector cell-derived perforin was not necessary. Over time the numbers of both donor cells diminished to low, yet still detectable, levels. Concomitantly, Tc1 and Tc2 effector cell therapies potentiated endogenous recipient-derived antitumor responses by inducing 1) local T cell-derived chemokines associated with type 1-like immune responses; 2) elevated levels of recipient-derived OVA tetramer-positive CD8 memory T cells that were CD44(high), CD122(+), and Ly6C(high) that predominantly produced IFN-gamma and TNF-alpha; and 3) heightened numbers of activated recipient-derived Th1 and Tc1 T cell subpopulations expressing CD25(+), CD69(+), and CD95(+) cell surface activation markers. Moreover, both Tc2 and Tc1 effector cell therapies were dependent in part on recipient-derived IFN-gamma and TNF-alpha for long-term survival and protection. Collectively, Tc1 and Tc2 effector cell immunotherapy mediate long-term tumor immunity by different mechanisms that subsequently potentiate endogenous recipient-derived type 1 antitumor responses.

  15. TRAF3 regulates the effector function of regulatory T cells and humoral immune responses

    PubMed Central

    Chang, Jae-Hoon; Hu, Hongbo; Jin, Jin; Puebla-Osorio, Nahum; Xiao, Yichuan; Gilbert, Brian E.; Brink, Robert; Ullrich, Stephen E.

    2014-01-01

    Regulatory T cells (Treg cells) control different aspects of immune responses, but how the effector functions of Treg cells are regulated is incompletely understood. Here we identified TNF receptor–associated factor 3 (TRAF3) as a regulator of Treg cell function. Treg cell–specific ablation of TRAF3 impaired CD4 T cell homeostasis, characterized by an increase in the Th1 type of effector/memory T cells. Moreover, the ablation of TRAF3 in Treg cells resulted in increased antigen-stimulated activation of follicular T helper cells (TFH cells), coupled with heightened formation of germinal centers and production of high-affinity IgG antibodies. Although the loss of TRAF3 did not reduce the overall frequency of Treg cells, it attenuated the antigen-stimulated production of follicular Treg cells (TFR cells). TRAF3 signaling in Treg cells was required to maintain high level expression of inducible co-stimulator (ICOS), which in turn was required for TFR cell generation and inhibition of antibody responses. These findings establish TRAF3 as a mediator of Treg cell function in the regulation of antibody responses and suggest a role for TRAF3 in mediating ICOS expression in Treg cells. PMID:24378539

  16. Antigen-specific CD4{sup +} effector T cells: Analysis of factors regulating clonal expansion and cytokine production

    SciTech Connect

    Ohnuki, Kazunobu; Watanabe, Yuri; Takahashi, Yusuke; Kobayashi, Sakiko; Watanabe, Shiho; Ogawa, Shuhei; Kotani, Motoko; Kozono, Haruo; Tanabe, Kazunari; Abe, Ryo

    2009-03-20

    In order to fully understand T cell-mediated immunity, the mechanisms that regulate clonal expansion and cytokine production by CD4{sup +} antigen-specific effector T cells in response to a wide range of antigenic stimulation needs clarification. For this purpose, panels of antigen-specific CD4{sup +} T cell clones with different thresholds for antigen-induced proliferation were generated by repeated stimulation with high- or low-dose antigen. Differences in antigen sensitivities did not correlate with expression of TCR, CD4, adhesion or costimulatory molecules. There was no significant difference in antigen-dependent cytokine production by TG40 cells transfected with TCR obtained from either high- or low-dose-responding T cell clones, suggesting that the affinity of TCRs for their ligands is not primary determinant of T cell antigen reactivity. The proliferative responses of all T cell clones to both peptide stimulation and to TCR{beta} crosslinking revealed parallel dose-response curves. These results suggest that the TCR signal strength of effector T cells and threshold of antigen reactivity is determined by an intrinsic property, such as the TCR signalosome and/or intracellular signaling machinery. Finally, the antigen responses of high- and low-peptide-responding T cell clones reveal that clonal expansion and cytokine production of effector T cells occur independently of antigen concentration. Based on these results, the mechanisms underlying selection of high 'avidity' effector and memory T cells in response to pathogen are discussed.

  17. The novel Cladosporium fulvum lysin motif effector Ecp6 is a virulence factor with orthologues in other fungal species.

    PubMed

    Bolton, Melvin D; van Esse, H Peter; Vossen, Jack H; de Jonge, Ronnie; Stergiopoulos, Ioannis; Stulemeijer, Iris J E; van den Berg, Grardy C M; Borrás-Hidalgo, Orlando; Dekker, Henk L; de Koster, Chris G; de Wit, Pierre J G M; Joosten, Matthieu H A J; Thomma, Bart P H J

    2008-07-01

    During tomato leaf colonization, the biotrophic fungus Cladosporium fulvum secretes several effector proteins into the apoplast. Eight effectors have previously been characterized and show no significant homology to each other or to other fungal genes. To discover novel C. fulvum effectors that might play a role in virulence, we utilized two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to visualize proteins secreted during C. fulvum-tomato interactions. Three novel C. fulvum proteins were identified: CfPhiA, Ecp6 and Ecp7. CfPhiA shows homology to proteins found on fungal sporogenous cells called phialides. Ecp6 contains lysin motifs (LysM domains) that are recognized as carbohydrate-binding modules. Ecp7 encodes a small, cysteine-rich protein with no homology to known proteins. Heterologous expression of Ecp6 significantly increased the virulence of the vascular pathogen Fusarium oxysporum on tomato. Furthermore, by RNA interference (RNAi)-mediated gene silencing we demonstrate that Ecp6 is instrumental for C. fulvum virulence on tomato. Hardly any allelic variation was observed in the Ecp6 coding region of a worldwide collection of C. fulvum strains. Although none of the C. fulvum effectors identified so far have obvious orthologues in other organisms, conserved Ecp6 orthologues were identified in various fungal species. Homology-based modelling suggests that the LysM domains of C. fulvum Ecp6 may be involved in chitin binding.

  18. The Xanthomonas campestris effector protein XopDXcc8004 triggers plant disease tolerance by targeting DELLA proteins.

    PubMed

    Tan, Leitao; Rong, Wei; Luo, Hongli; Chen, Yinhua; He, Chaozu

    2014-11-01

    Plants protect themselves from the harmful effects of pathogens by resistance and tolerance. Disease resistance, which eliminates pathogens, can be modulated by bacterial type III effectors. Little is known about whether disease tolerance, which sustains host fitness with a given pathogen burden, is regulated by effectors. Here, we examined the effects of the Xanthomonas effector protein XopDXcc8004 on plant disease defenses by constructing knockout and complemented Xanthomonas strains, and performing inoculation studies in radish (Raphanus sativus L. var. radiculus XiaoJinZhong) and Arabidopsis plants. XopDXcc8004 suppresses disease symptoms without changing bacterial titers in infected leaves. In Arabidopsis, XopDXcc8004 delays the hormone gibberellin (GA)-mediated degradation of RGA (repressor of ga1-3), one of five DELLA proteins that repress GA signaling and promote plant tolerance under biotic and abiotic stresses. The ERF-associated amphiphilic repression (EAR) motif-containing region of XopDXcc8004 interacts with the DELLA domain of RGA and might interfere with the GA-induced binding of GID1, a GA receptor, to RGA. The EAR motif was found to be present in a number of plant transcriptional regulators. Thus, our data suggest that bacterial pathogens might have evolved effectors, which probably mimic host components, to initiate disease tolerance and enhance their survival.

  19. A common glycan structure on immunoglobulin G for enhancement of effector functions

    PubMed Central

    Lin, Chin-Wei; Tsai, Ming-Hung; Li, Shiou-Ting; Tsai, Tsung-I; Chu, Kuo-Ching; Liu, Ying-Chih; Lai, Meng-Yu; Wu, Chia-Yu; Tseng, Yung-Chieh; Shivatare, Sachin S.; Wang, Chia-Hung; Chao, Ping; Wang, Shi-Yun; Shih, Hao-Wei; Zeng, Yi-Fang; You, Tsai-Hong; Liao, Jung-Yu; Tu, Yu-Chen; Lin, Yih-Shyan; Chuang, Hong-Yang; Chen, Chia-Lin; Tsai, Charng-Sheng; Huang, Chiu-Chen; Lin, Nan-Horng; Ma, Che; Wu, Chung-Yi; Wong, Chi-Huey

    2015-01-01

    Antibodies have been developed as therapeutic agents for the treatment of cancer, infection, and inflammation. In addition to binding activity toward the target, antibodies also exhibit effector-mediated activities through the interaction of the Fc glycan and the Fc receptors on immune cells. To identify the optimal glycan structures for individual antibodies with desired activity, we have developed an effective method to modify the Fc-glycan structures to a homogeneous glycoform. In this study, it was found that the biantennary N-glycan structure with two terminal alpha-2,6-linked sialic acids is a common and optimized structure for the enhancement of antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and antiinflammatory activities. PMID:26253764

  20. Dual function of a bacterial protein as an adhesin and extracellular effector of host GTPase signaling

    PubMed Central

    Stones, Daniel Henry; Krachler, Anne Marie

    2015-01-01

    Bacterial pathogens often target conserved cellular mechanisms within their hosts to rewire signaling pathways and facilitate infection. Rho GTPases are important nodes within eukaryotic signaling networks and thus constitute a common target of pathogen-mediated manipulation. A diverse array of microbial mechanisms exists to interfere with Rho GTPase signaling. While targeting of GTPases by secreted bacterial effectors is a well-known strategy bacterial pathogens employ to interfere with the host, we have recently described pathogen adhesion as a novel extracellular stimulus that hijacks host GTPase signaling. The Multivalent Adhesion Molecule MAM7 from Vibrio parahaemolyticus directly binds host cell membrane lipids. The ensuing coalescence of phosphatidic acid ligands in the host membrane leads to downstream activation of RhoA and actin rearrangements. Herein, we discuss mechanistic models of lipid-mediated Rho activation and the implications from the infected host's and the pathogen's perspective. PMID:26156628

  1. The Type III Secretion System Effector SptP of Salmonella enterica Serovar Typhi

    PubMed Central

    Johnson, Rebecca; Byrne, Alexander; Berger, Cedric N.; Klemm, Elizabeth; Crepin, Valerie F.; Dougan, Gordon

    2016-01-01

    ABSTRACT Strains of the various Salmonella enterica serovars cause gastroenteritis or typhoid fever in humans, with virulence depending on the action of two type III secretion systems (Salmonella pathogenicity island 1 [SPI-1] and SPI-2). SptP is a Salmonella SPI-1 effector, involved in mediating recovery of the host cytoskeleton postinfection. SptP requires a chaperone, SicP, for stability and secretion. SptP has 94% identity between S. enterica serovar Typhimurium and S. Typhi; direct comparison of the protein sequences revealed that S. Typhi SptP has numerous amino acid changes within its chaperone-binding domain. Subsequent comparison of ΔsptP S. Typhi and S. Typhimurium strains demonstrated that, unlike SptP in S. Typhimurium, SptP in S. Typhi was not involved in invasion or cytoskeletal recovery postinfection. Investigation of whether the observed amino acid changes within SptP of S. Typhi affected its function revealed that S. Typhi SptP was unable to complement S. Typhimurium ΔsptP due to an absence of secretion. We further demonstrated that while S. Typhimurium SptP is stable intracellularly within S. Typhi, S. Typhi SptP is unstable, although stability could be recovered following replacement of the chaperone-binding domain with that of S. Typhimurium. Direct assessment of the strength of the interaction between SptP and SicP of both serovars via bacterial two-hybrid analysis demonstrated that S. Typhi SptP has a significantly weaker interaction with SicP than the equivalent proteins in S. Typhimurium. Taken together, our results suggest that changes within the chaperone-binding domain of SptP in S. Typhi hinder binding to its chaperone, resulting in instability, preventing translocation, and therefore restricting the intracellular activity of this effector. IMPORTANCE Studies investigating Salmonella pathogenesis typically rely on Salmonella Typhimurium, even though Salmonella Typhi causes the more severe disease in humans. As such, an understanding of

  2. The Yersinia pestis Effector YopM Inhibits Pyrin Inflammasome Activation

    PubMed Central

    Wang, Donghai; Gavrilin, Mikhail A.; Alnemri, Emad S.; Johnson, Peter F.; Lee, Bettina; Mecsas, Joan; Kayagaki, Nobuhiko; Goguen, Jon D.; Lien, Egil

    2016-01-01

    Type III secretion systems (T3SS) are central virulence factors for many pathogenic Gram-negative bacteria, and secreted T3SS effectors can block key aspects of host cell signaling. To counter this, innate immune responses can also sense some T3SS components to initiate anti-bacterial mechanisms. The Yersinia pestis T3SS is particularly effective and sophisticated in manipulating the production of pro-inflammatory cytokines IL-1β and IL-18, which are typically processed into their mature forms by active caspase-1 following inflammasome formation. Some effectors, like Y. pestis YopM, may block inflammasome activation. Here we show that YopM prevents Y. pestis induced activation of the Pyrin inflammasome induced by the RhoA-inhibiting effector YopE, which is a GTPase activating protein. YopM blocks YopE-induced Pyrin-mediated caspase-1 dependent IL-1β/IL-18 production and cell death. We also detected YopM in a complex with Pyrin and kinases RSK1 and PKN1, putative negative regulators of Pyrin. In contrast to wild-type mice, Pyrin deficient mice were also highly susceptible to an attenuated Y. pestis strain lacking YopM, emphasizing the importance of inhibition of Pyrin in vivo. A complex interplay between the Y. pestis T3SS and IL-1β/IL-18 production is evident, involving at least four inflammasome pathways. The secreted effector YopJ triggers caspase-8- dependent IL-1β activation, even when YopM is present. Additionally, the presence of the T3SS needle/translocon activates NLRP3 and NLRC4-dependent IL-1β generation, which is blocked by YopK, but not by YopM. Taken together, the data suggest YopM specificity for obstructing the Pyrin pathway, as the effector does not appear to block Y. pestis-induced NLRP3, NLRC4 or caspase-8 dependent caspase-1 processing. Thus, we identify Y. pestis YopM as a microbial inhibitor of the Pyrin inflammasome. The fact that so many of the Y. pestis T3SS components are participating in regulation of IL-1β/IL-18 release suggests

  3. Role of Fc-mediated antibody function in protective immunity against HIV-1.

    PubMed

    Lewis, George K

    2014-05-01

    The importance of Fc-mediated effector function in protective immunity to HIV-1 (hereafter referred to simply as HIV) is becoming increasingly apparent. A large of number of studies in natural infection cohorts, spanning the last 26 years, have associated Fc-mediated effector function, particularly antibody-dependent cellular cytotoxicity, with a favourable clinical course. These studies strongly suggest a role for Fc-mediated effector function in the post-infection control of viraemia. More recently, studies in both humans and non-human primates (NHPs) also implicate Fc-mediated effector function in blocking HIV acquisition. Accordingly, this review will discuss the results supporting a role of Fc-mediated effector function in both blocking acquisition and post-infection control of viraemia. Parallel studies in NHPs and humans will be compared for common themes. Context for this discussion will be provided by summarizing the temporal emergence of key host and virological events over the course of an untreated HIV infection framing where, when and how Fc-mediated effector function might be protective. A hypothesis that Fc-mediated effector function protects primarily in the early stages of both acquisition and post-infection control of viraemia will be developed.

  4. Unusual evolutionary mechanisms to escape effector-triggered immunity in the fungal phytopathogen Leptosphaeria maculans.

    PubMed

    Plissonneau, C; Blaise, F; Ollivier, B; Leflon, M; Carpezat, J; Rouxel, T; Balesdent, M-H

    2017-04-01

    Leptosphaeria maculans is the fungus responsible for the stem canker disease of oilseed rape (Brassica napus). AvrLm3 and AvrLm4-7, two avirulence effector genes of L. maculans, are involved in an unusual relationship: AvrLm4-7 suppresses the Rlm3-mediated resistance. Here, we assessed AvrLm3 polymorphism in a collection of 235 L. maculans isolates. No field isolates exhibited deletion or inactivating mutations in AvrLm3, as observed for other L. maculans avirulence genes. Eleven isoforms of the AvrLm3 protein were found. In isolates virulent towards both Rlm3 and Rlm7 (a3a7), the loss of the Rlm3-mediated resistance response was due to two distinct mechanisms. First, when AvrLm4-7 was inactivated (deletion or inactivating mutations), amino acid substitutions in AvrLm3 generated virulent isoforms of the protein. Second, when only point mutations were observed in AvrLm4-7, a3a7 isolates still contained an avirulent allele of AvrLm3. Directed mutagenesis confirmed that some point mutations in AvrLm4-7 were sufficient for the fungus to escape Rlm7-mediated resistance while maintaining the suppression of the AvrLm3 phenotype. Signatures of positive selection were also identified in AvrLm3. The complex evolutionary mechanisms enabling L. maculans to escape Rlm3-mediated resistance while preserving AvrLm3 integrity, along with observed reduced aggressiveness of isolates silenced for AvrLm3, serves to emphasize the importance of this effector in pathogenicity towards B. napus. While the common response to resistance gene pressure is local selection of isolates depleted in the cognate avirulence gene, this example contributes to complexify the gene-for-gene concept of plant-pathogen evolution with a 'camouflaged' model allowing retention of nondispensable avirulence effectors.

  5. Target selection biases from recent experience transfer across effectors.

    PubMed

    Moher, Jeff; Song, Joo-Hyun

    2016-02-01

    Target selection is often biased by an observer's recent experiences. However, not much is known about whether these selection biases influence behavior across different effectors. For example, does looking at a red object make it easier to subsequently reach towards another red object? In the current study, we asked observers to find the uniquely colored target object on each trial. Randomly intermixed pre-trial cues indicated the mode of action: either an eye movement or a visually guided reach movement to the target. In Experiment 1, we found that priming of popout, reflected in faster responses following repetition of the target color on consecutive trials, occurred regardless of whether the effector was repeated from the previous trial or not. In Experiment 2, we examined whether an inhibitory selection bias away from a feature could transfer across effectors. While priming of popout reflects both enhancement of the repeated target features and suppression of the repeated distractor features, the distractor previewing effect isolates a purely inhibitory component of target selection in which a previewed color is presented in a homogenous display and subsequently inhibited. Much like priming of popout, intertrial suppression biases in the distractor previewing effect transferred across effectors. Together, these results suggest that biases for target selection driven by recent trial history transfer across effectors. This indicates that representations in memory that bias attention towards or away from specific features are largely independent from their associated actions.

  6. C. elegans S6K Mutants Require a Creatine-Kinase-like Effector for Lifespan Extension.

    PubMed

    McQuary, Philip R; Liao, Chen-Yu; Chang, Jessica T; Kumsta, Caroline; She, Xingyu; Davis, Andrew; Chu, Chu-Chiao; Gelino, Sara; Gomez-Amaro, Rafael L; Petrascheck, Michael; Brill, Laurence M; Ladiges, Warren C; Kennedy, Brian K; Hansen, Malene

    2016-03-08

    Deficiency of S6 kinase (S6K) extends the lifespan of multiple species, but the underlying mechanisms are unclear. To discover potential effectors of S6K-mediated longevity, we performed a proteomics analysis of long-lived rsks-1/S6K C. elegans mutants compared to wild-type animals. We identified the arginine kinase ARGK-1 as the most significantly enriched protein in rsks-1/S6K mutants. ARGK-1 is an ortholog of mammalian creatine kinase, which maintains cellular ATP levels. We found that argk-1 is possibly a selective effector of rsks-1/S6K-mediated longevity and that overexpression of ARGK-1 extends C. elegans lifespan, in part by activating the energy sensor AAK-2/AMPK. argk-1 is also required for the reduced body size and increased stress resistance observed in rsks-1/S6K mutants. Finally, creatine kinase levels are increased in the brains of S6K1 knockout mice. Our study identifies ARGK-1 as a longevity effector in C. elegans with reduced RSKS-1/S6K levels. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Graft rejection by cytolytic T cells. Specificity of the effector mechanism in the rejection of allogeneic marrow

    SciTech Connect

    Nakamura, H.; Gress, R.E. )

    1990-02-01

    Cellular effector mechanisms of allograft rejection remain incompletely described. Characterizing the rejection of foreign-marrow allografts rather than solid-organ grafts has the advantage that the cellular composition of the marrow graft, as a single cell suspension, can be altered to include cellular components with differing antigen expression. Rejection of marrow grafts is sensitive to lethal doses of radiation in the mouse but resistant to sublethal levels of radiation. In an effort to identify cells mediating host resistance, lymphocytes were isolated and cloned from spleens of mice 7 days after sublethal TBI (650 cGy) and inoculation with allogeneic marrow. All clones isolated were cytolytic with specificity for MHC encoded gene products of the allogeneic marrow donor. When cloned cells were transferred in vivo into lethally irradiated (1025 cGy) recipients unable to reject allogeneic marrow, results utilizing splenic 125IUdR uptake indicated that these MHC-specific cytotoxic clones could suppress marrow proliferation. In order to characterize the effector mechanism and the ability of the clones to affect final engraftment, double donor chimeras were constructed so that 2 target cell populations differing at the MHC from each other and from the host were present in the same marrow allograft. Results directly demonstrated an ability of CTL of host MHC type to mediate graft rejection and characterized the effector mechanism as one with specificity for MHC gene products.

  8. Serine Protease Inhibitor-6 Differentially Affects the Survival of Effector and Memory Alloreactive CD8-T Cells

    PubMed Central

    Azzi, J.; Ohori, S.; Ting, C.; Uehara, M.; Abdoli, R.; Smith, B. D.; Safa, K.; Solhjou, Z.; Lukyanchykov, P.; Patel, J.; McGrath, M.; Abdi, R.

    2016-01-01

    The clonal expansion of effector T cells and subsequent generation of memory T cells are critical in determining the outcome of transplantation. While cytotoxic T lymphocytes induce direct cytolysis of target cells through secretion of Granzyme-B (GrB), they also express cytoplasmic serine protease inhibitor-6 (Spi6) to protect themselves from GrB that has leaked from granules. Here, we studied the role of GrB/Spi6 axis in determining clonal expansion of alloreactive CD8-T cells and subsequent generation of memory CD8-T cells in transplantation. CD8-T cells from Spi6−/− mice underwent more GrB mediated apoptosis upon alloantigen stimulation in vitro and in vivo following adoptive transfer into an allogeneic host. Interestingly, while OT1.Spi6−/− CD8 T cells showed significantly lower clonal expansion following skin transplants from OVA mice, there was no difference in the size of the effector memory CD8-T cells long after transplantation. Furthermore, lack of Spi6 resulted in a decrease of short-lived-effector-CD8-cells but did not impact the pool of memory-precursor-effector-CD8-cells. Similar results were found in heart transplant models. Our findings suggest that the final alloreactive CD8-memory-pool-size is independent from the initial clonal-proliferation as memory precursors express low levels of GrB and therefore are independent of Spi6 for survival. These data advance our understanding of memory T cells generation in transplantation and provide basis for Spi6 based strategies to target effector T cells. PMID:25534448

  9. Mast cells as effectors in atherosclerosis.

    PubMed

    Bot, Ilze; Shi, Guo-Ping; Kovanen, Petri T

    2015-02-01

    The mast cell is a potent immune cell known for its functions in host defense responses and diseases, such as asthma and allergies. In the past years, accumulating evidence established the contribution of the mast cell to cardiovascular diseases as well, in particular, by its effects on atherosclerotic plaque progression and destabilization. Through its release not only of mediators, such as the mast cell-specific proteases chymase and tryptase, but also of growth factors, histamine, and chemokines, activated mast cells can have detrimental effects on its immediate surroundings in the vessel wall. This results in matrix degradation, apoptosis, and enhanced recruitment of inflammatory cells, thereby actively contributing to cardiovascular diseases. In this review, we will discuss the current knowledge on mast cell function in cardiovascular diseases and speculate on potential novel therapeutic strategies to prevent acute cardiovascular syndromes via targeting of mast cells.

  10. Mast cells as effectors in atherosclerosis

    PubMed Central

    Bot, Ilze; Shi, Guo-Ping; Kovanen, Petri T.

    2014-01-01

    The mast cell is a potent immune cell known for its functions in host defense responses and diseases such as asthma and allergies. In the past years, accumulating evidence established the contribution of the mast cell to cardiovascular diseases as well, in particular by its effects on atherosclerotic plaque progression and destabilization. Through its release of mediators, such as the mast cell-specific proteases chymase and tryptase, but also of growth factors, histamine and chemokines, activated mast cells can have detrimental effects on its immediate surroundings in the vessel wall. This results in matrix degradation, apoptosis and enhanced recruitment of inflammatory cells, thereby actively contributing to cardiovascular diseases. In this review, we will discuss the current knowledge on mast cell function in cardiovascular diseases and speculate on potential novel therapeutic strategies to prevent acute cardiovascular syndromes via targeting of mast cells. PMID:25104798

  11. Signaling in Effector Lymphocytes: Insights toward Safer Immunotherapy

    PubMed Central

    Rajasekaran, Kamalakannan; Riese, Matthew J.; Rao, Sridhar; Wang, Li; Thakar, Monica S.; Sentman, Charles L.; Malarkannan, Subramaniam

    2016-01-01

    Receptors on T and NK cells systematically propagate highly complex signaling cascades that direct immune effector functions, leading to protective immunity. While extensive studies have delineated hundreds of signaling events that take place upon receptor engagement, the precise molecular mechanism that differentially regulates the induction or repression of a unique effector function is yet to be fully defined. Such knowledge can potentiate the tailoring of signal transductions and transform cancer immunotherapies. Targeted manipulations of signaling cascades can augment one effector function such as antitumor cytotoxicity while contain the overt generation of pro-inflammatory cytokines that contribute to treatment-related toxicity such as “cytokine storm” and “cytokine-release syndrome” or lead to autoimmune diseases. Here, we summarize how individual signaling molecules or nodes may be optimally targeted to permit selective ablation of toxic immune side effects. PMID:27242783

  12. Diacylglycerol Kinases in T Cell Tolerance and Effector Function

    PubMed Central

    Chen, Shelley S.; Hu, Zhiming; Zhong, Xiao-Ping

    2016-01-01

    Diacylglycerol kinases (DGKs) are a family of enzymes that regulate the relative levels of diacylglycerol (DAG) and phosphatidic acid (PA) in cells by phosphorylating DAG to produce PA. Both DAG and PA are important second messengers cascading T cell receptor (TCR) signal by recruiting multiple effector molecules, such as RasGRP1, PKCθ, and mTOR. Studies have revealed important physiological functions of DGKs in the regulation of receptor signaling and the development and activation of immune cells. In this review, we will focus on recent progresses in our understanding of two DGK isoforms, α and ζ, in CD8 T effector and memory cell differentiation, regulatory T cell development and function, and invariant NKT cell development and effector lineage differentiation. PMID:27891502

  13. Structural Insights into Rab27 Recruitment by its Effectors

    NASA Astrophysics Data System (ADS)

    M. G. Chavas, Leonard; Ihara, Kentaro; Kawasaki, Masato; Wakatsuki, Soichi

    An increasing number of Rab GTPases associated with partial dysfunction has been linked to several human diseases characterized by a diminution in vesicle transport. Due to its direct implication in human disorders, the Rab27 subfamily is considered as a standard for vesicle docking studies. By which mechanism Rab27 effectors distinguish among the pool of Rab GTPases? What is the underneath machinery rendering the interaction of eleven distinct effectors specific of Rab27 when compared to other Rabs of the secretory pathway? By solving the X-ray structures of Rab27, both in its inactive form and active form bound to the effector protein Slp2-a, attempts have been given to unravel the molecular basis of regulation of the delivering process of vesicles to fusion by the Rab27 subfamily.

  14. Effector proteins that modulate plant--insect interactions.

    PubMed

    Hogenhout, Saskia A; Bos, Jorunn I B

    2011-08-01

    Insect herbivores have highly diverse life cycles and feeding behaviors. They establish close interactions with their plant hosts and suppress plant defenses. Chewing herbivores evoke characteristic defense responses distinguishable from general mechanical damage. In addition, piercing-sucking hemipteran insects display typical feeding behavior that suggests active suppression of plant defense responses. Effectors that modulate plant defenses have been identified in the saliva of these insects. Tools for high-throughput effector identification and functional characterization have been developed. In addition, in some insect species it is possible to silence gene expression by RNAi. Together, this technological progress has enabled the identification of insect herbivore effectors and their targets that will lead to the development of novel strategies for pest resistances in plants.

  15. The sterol regulatory element binding proteins are essential for the metabolic programming of effector T cells and adaptive immunity

    PubMed Central

    Kidani, Yoko; Elsaesser, Heidi; Hock, M Benjamin; Vergnes, Laurent; Williams, Kevin J; Argus, Joseph P; Marbois, Beth N; Komisopoulou, Evangelia; Wilson, Elizabeth B; Osborne, Timothy F; Graeber, Thomas G; Reue, Karen; Brooks, David G; Bensinger, Steven J

    2013-01-01

    Newly activated CD8+ T cells reprogram their metabolism to meet the extraordinary biosynthetic demands of clonal expansion; however, the signals mediating metabolic reprogramming remain poorly defined. Herein, we demonstrate an essential role for sterol regulatory element binding proteins (SREBPs) in the acquisition of effector cell metabolism. Without SREBP signaling, CD8+ T cells are unable to blast, resulting in markedly attenuated clonal expansion during viral infection. Mechanistic studies indicate that SREBPs are essential to meet the heightened lipid requirements of membrane synthesis during blastogenesis. SREBPs are dispensable for homeostatic proliferation, indicating a context-specific requirement for SREBPs in effector responses. These studies provide insights into the molecular signals underlying metabolic reprogramming of CD8+ T cells during the transition from quiescence to activation. PMID:23563690

  16. The phytopathogenic virulent effector protein RipI induces apoptosis in budding yeast Saccharomyces cerevisiae.

    PubMed

    Deng, Meng-Ying; Sun, Yun-Hao; Li, Pai; Fu, Bei; Shen, Dong; Lu, Yong-Jun

    2016-10-01

    Virulent protein toxins secreted by the bacterial pathogens can cause cytotoxicity by various molecular mechanisms to combat host cell defense. On the other hand, these proteins can also be used as probes to investigate the defense pathway of host innate immunity. Ralstonia solanacearum, one of the most virulent bacterial phytopathogens, translocates more than 70 effector proteins via type III secretion system during infection. Here, we characterized the cytotoxicity of effector RipI in budding yeast Saccharomyce scerevisiae, an alternative host model. We found that over-expression of RipI resulted in severe growth defect and arginine (R) 117 within the predicted integrase motif was required for inhibition of yeast growth. The phenotype of death manifested the hallmarks of apoptosis. Our data also revealed that RipI-induced apoptosis was independent of Yca1 and mitochondria-mediated apoptotic pathways because Δyca1 and Δaif1 were both sensitive to RipI as compared with the wild type. We further demonstrated that RipI was localized in the yeast nucleus and the N-terminal 1-174aa was required for the localization. High-throughput RNA sequencing analysis showed that upon RipI over-expression, 101 unigenes of yeast ribosome presented lower expression level, and 42 GO classes related to the nucleus or recombination were enriched with differential expression levels. Taken together, our data showed that a nuclear-targeting effector RipI triggers yeast apoptosis, potentially dependent on its integrase function. Our results also provided an alternative strategy to dissect the signaling pathway of cytotoxicity induced by the protein toxins. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. The Xanthomonas campestris type III effector XopJ proteolytically degrades proteasome subunit RPT6.

    PubMed

    Üstün, Suayib; Börnke, Frederik

    2015-05-01

    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. © 2015 American Society of Plant Biologists. All Rights Reserved.

  18. 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

  19. CpG oligodeoxynucleotide induction of antiviral effector molecules in sheep.

    PubMed

    Nichani, Anil K; Kaushik, Radhey S; Mena, Angelo; Popowych, Yurij; Dent, Donna; Townsend, Hugh G G; Mutwiri, George; Hecker, Rolf; Babiuk, Lorne A; Griebel, Philip J

    2004-01-01

    Immunostimulatory CpG oligodeoxynucleotide (ODN) can protect mice against infection by many pathogens but the mechanisms mediating disease protection are not well defined. Furthermore, the mechanisms of CpG ODN induced disease protection in vivo have not been investigated in other species. We investigated the induction of antiviral effector molecules in sheep treated with a class B CpG ODN (2007). Subcutaneous injection of ODN 2007 induced a dose-dependent increase in serum levels of the antiviral effector molecule, 2'5'-A synthetase. Peak levels of enzyme were observed 4 days following ODN injection and enzyme levels remained elevated for the following 3-5 days. Repeated ODN injections induced a more sustained elevation of serum 2'5'-A synthetase activity. Finally, formulation of ODN 2007 in emulsigen increased the level of serum 2'5'-A synthetase activity and this response was CpG-specific. Elevated serum 2'5'-A synthetase activity suggested that CpG ODN acted through the induction of either interferon (IFN)-alpha or IFN-gamma. ODN 2007 did not induce detectable levels of IFN-alpha or IFN-gamma when incubated with peripheral blood mononuclear cells, but both IFN-alpha and IFN-gamma were detected following stimulation of lymph node cells with ODN 2007. CpG ODN induction of 2'5'-A synthetase in vitro correlated with the secretion of both IFN-alpha and IFN-gamma. Furthermore, immunohistochemical staining of skin revealed a marked cellular infiltration at the site of ODN 2007 injection. This cellular infiltration was CpG-specific and consisted of primarily CD172(+) myeloid cells. Many of the cells recruited to the site of ODN 2007 injection expressed IFN-alpha and some IFN-gamma. These observations support the conclusion that localized cell recruitment and activation contribute to CpG ODN induction of antiviral effector molecules, such as interferon and 2'5'-A synthetase.

  20. 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

  1. Identification and functional analysis of secreted effectors from phytoparasitic nematodes.

    PubMed

    Rehman, Sajid; Gupta, Vijai K; Goyal, Aakash K

    2016-03-21

    Plant parasitic nematodes develop an intimate and long-term feeding relationship with their host plants. They induce a multi-nucleate feeding site close to the vascular bundle in the roots of their host plant and remain sessile for the rest of their life. Nematode secretions, produced in the oesophageal glands and secreted through a hollow stylet into the host plant cytoplasm, are believed to play key role in pathogenesis. To combat these persistent pathogens, the identity and functional analysis of secreted effectors can serve as a key to devise durable control measures. In this review, we will recapitulate the knowledge over the identification and functional characterization of secreted nematode effector repertoire from phytoparasitic nematodes. Despite considerable efforts, the identity of genes encoding nematode secreted proteins has long been severely hampered because of their microscopic size, long generation time and obligate biotrophic nature. The methodologies such as bioinformatics, protein structure modeling, in situ hybridization microscopy, and protein-protein interaction have been used to identify and to attribute functions to the effectors. In addition, RNA interference (RNAi) has been instrumental to decipher the role of the genes encoding secreted effectors necessary for parasitism and genes attributed to normal development. Recent comparative and functional genomic approaches have accelerated the identification of effectors from phytoparasitic nematodes and offers opportunities to control these pathogens. Plant parasitic nematodes pose a serious threat to global food security of various economically important crops. There is a wealth of genomic and transcriptomic information available on plant parasitic nematodes and comparative genomics has identified many effectors. Bioengineering crops with dsRNA of phytonematode genes can disrupt the life cycle of parasitic nematodes and therefore holds great promise to develop resistant crops against plant

  2. Development and testing of the cooling coil cleaning end effector

    SciTech Connect

    Johnson, K.I.; Mullen, O.D.; Powell, M.R.; Daly, D.S.; Engel, D.W.

    1997-09-30

    The Retrieval Process Development and Enhancement (KPD{ampersand}E) program has developed and tested an end effector to support the waste retrieval mission at the Idaho National Engineering and Environmental Laboratory (INEEL). The end effector was developed specifically to remove a sticky waste material from the cooling coils in the High Level Liquid Waste (HLLW) tank, and to vacuum up a sediment layer that has settled beneath the cooling coils. An extensive testing program was conducted in the hydraulic test bed (HTB) at the Pacific Northwest National Laboratory (PNNL) to evaluate the performance of the end effector under simulated in-tank conditions. A mock up of the cooling coils was installed in the test bed tank, and simulated waste materials were included to represent the sticky waste on the tubes and the particulate waste settled beneath them. The testing program focused on assessing long-duration mining strategies for cleaning the cooling coils and removing the particulate waste forms. The report describes the results of the end effector testing program at PNNL. Section 2 describes the physical characteristics of the HLLW tanks, including the layout of the cooling coils, and it also describes what is known of the waste forms in the tanks. Section 3 describes the cleaning and retrieval strategy that was used in developing the end effector design. Section 4 describes the cooling coil mockup in the hydraulic test bed. Section 5 discusses the rationale used in selecting the simulants for the tarry waste and particulate waste forms. Section 6 describes the tests that were performed to evaluate cleaning of the cooling coils and retrieval of the particulate simulant. Section 7 summarizes the cleaning and retrieval tests, assesses the relative importance of cleaning the cooling coils and retrieving the particulate waste, and suggests modifications that would simplify the end effector design.

  3. Visual End-Effector Position Error Compensation for Planetary Robotics

    NASA Technical Reports Server (NTRS)

    Bajracharya, Max; DiCicco, Matthew; Backes, Paul; Nickels, Kevin

    2007-01-01

    This paper describes a vision-guided manipulation algorithm that improves arm end-effector positioning to subpixel accuracy and meets the highly restrictive imaging and computational constraints of a planetary robotic flight system. Analytical, simulation-based, and experimental analyses of the algorithm's effectiveness and sensitivity to camera and arm model error is presented along with results on several prototype research systems and 'ground-in-the-loop' technology experiments on the Mars Exploration Rover (MER) vehicles. A computationally efficient and robust subpixel end-effector fiducial detector that is instrumental to the algorithm's ability to achieve high accuracy is also described along with its validation results on MER data.

  4. Hemipteran and dipteran pests: Effectors and plant host immune regulators.

    PubMed

    Kaloshian, Isgouhi; Walling, Linda L

    2016-04-01

    Hemipteran and dipteran insects have behavioral, cellular and chemical strategies for evading or coping with the host plant defenses making these insects particularly destructive pests worldwide. A critical component of a host plant's defense to herbivory is innate immunity. Here we review the status of our understanding of the receptors that contribute to perception of hemipteran and dipteran pests and highlight the gaps in our knowledge in these early events in immune signaling. We also highlight recent advances in identification of the effectors that activate pattern-triggered immunity and those involved in effector-triggered immunity.

  5. Nanorobotic end-effectors: Design, fabrication, and in situ characterization

    NASA Astrophysics Data System (ADS)

    Fan, Zheng

    Nano-robotic end-effectors have promising applications for nano-fabrication, nano-manufacturing, nano-optics, nano-medical, and nano-sensing; however, low performances of the conventional end-effectors have prevented the widespread utilization of them in various fields. There are two major difficulties in developing the end-effectors: their nano-fabrication and their advanced characterization in the nanoscale. Here we introduce six types of end-effectors: the nanotube fountain pen (NFP), the super-fine nanoprobe, the metal-filled carbon nanotube (m CNT)-based sphere-on-pillar (SOP) nanoantennas, the tunneling nanosensor, and the nanowire-based memristor. The investigations on the NFP are focused on nano-fluidics and nano-fabrications. The NFP could direct write metallic "inks" and fabricating complex metal nanostructures from 0D to 3D with a position servo control, which is critically important to future large-scale, high-throughput nanodevice production. With the help of NFP, we could fabricate the end-effectors such as super-fine nanoprobe and m CNT-based SOP nanoantennas. Those end-effectors are able to detect local flaws or characterize the electrical/mechanical properties of the nanostructure. Moreover, using electron-energy-loss-spectroscopy (EELS) technique during the operation of the SOP optical antenna opens a new basis for the application of nano-robotic end-effectors. The technique allows advanced characterization of the physical changes, such as carrier diffusion, that are directly responsible for the device's properties. As the device was coupled with characterization techniques of scanning-trasmission-electron-microscopy (STEM), the development of tunneling nanosensor advances this field of science into quantum world. Furthermore, the combined STEM-EELS technique plays an important role in our understanding of the memristive switching performance in the nanowire-based memristor. The developments of those nano-robotic end-effectors expend the study

  6. Visual End-Effector Position Error Compensation for Planetary Robotics

    NASA Technical Reports Server (NTRS)

    Bajracharya, Max; DiCicco, Matthew; Backes, Paul; Nickels, Kevin

    2007-01-01

    This paper describes a vision-guided manipulation algorithm that improves arm end-effector positioning to subpixel accuracy and meets the highly restrictive imaging and computational constraints of a planetary robotic flight system. Analytical, simulation-based, and experimental analyses of the algorithm's effectiveness and sensitivity to camera and arm model error is presented along with results on several prototype research systems and 'ground-in-the-loop' technology experiments on the Mars Exploration Rover (MER) vehicles. A computationally efficient and robust subpixel end-effector fiducial detector that is instrumental to the algorithm's ability to achieve high accuracy is also described along with its validation results on MER data.

  7. How Do Filamentous Pathogens Deliver Effector Proteins into Plant Cells?

    PubMed Central

    Petre, Benjamin; Kamoun, Sophien

    2014-01-01

    Fungal and oomycete plant parasites are among the most devastating pathogens of food crops. These microbes secrete effector proteins inside plant cells to manipulate host processes and facilitate colonization. How these effectors reach the host cytoplasm remains an unclear and debated area of plant research. In this article, we examine recent conflicting findings that have generated discussion in the field. We also highlight promising approaches based on studies of both parasite and host during infection. Ultimately, this knowledge may inform future broad spectrum strategies for protecting crops from such pathogens. PMID:24586116

  8. Recombinant immunotoxins and retargeted killer cells: employing engineered antibody fragments for tumor-specific targeting of cytotoxic effectors.

    PubMed

    Wels, Winfried; Biburger, Markus; Müller, Tina; Dälken, Benjamin; Giesübel, Ulrike; Tonn, Torsten; Uherek, Christoph

    2004-03-01

    Over the past years, monoclonal antibodies have attracted enormous interest as targeted therapeutics, and a number of such reagents are in clinical use. However, responses could not be achieved in all patients with tumors expressing high levels of the respective target antigens, suggesting that other factors such as limited recruitment of endogenous immune effector mechanisms can also influence treatment outcome. This justifies the search for alternative, potentially more effective reagents. Antibody-toxins and cytolytic effector cells genetically modified to carry antibody-based receptors on the surface, represent such tailor-made targeting vehicles with the potential of improved tumor localization and enhanced efficacy. In this way, advances in recombinant antibody technology have made it possible to circumvent problems inherent in chemical coupling of antibodies and toxins, and have allowed construction via gene fusion of recombinant molecules which combine antibody-mediated recognition of tumor cells with specific delivery of potent protein toxins of bacterial or plant origin. Likewise, recombinant antibody fragments provide the basis for the construction of chimeric antigen receptors that, upon expression in cytotoxic T lymphocytes (CTLs) or natural killer (NK) cells, link antibody-mediated recognition of tumor antigens with these effector cells' potent cytolytic activities, thereby making them promising cellular therapeutics for adoptive cancer therapy. Here, general principles for the derivation of cytotoxic proteins and effector cells with antibody-dependent tumor specificity are summarized, and current strategies to employ these molecules and cells for directed cancer therapy are discussed, focusing mainly on the tumor-associated antigens epidermal growth factor receptor (EGFR) and the closely related ErbB2 (HER2) as targets.

  9. Effector and Naturally Occurring Regulatory T Cells Display No Abnormalities in Activation Induced Cell Death in NOD Mice

    PubMed Central

    Kaminitz, Ayelet; Yolcu, Esma S.; Askenasy, Enosh M.; Stein, Jerry; Yaniv, Isaac; Shirwan, Haval; Askenasy, Nadir

    2011-01-01

    Background Disturbed peripheral negative regulation might contribute to evolution of autoimmune insulitis in type 1 diabetes. This study evaluates the sensitivity of naïve/effector (Teff) and regulatory T cells (Treg) to activation-induced cell death mediated by Fas cross-linking in NOD and wild-type mice. Principal Findings Both effector (CD25−, FoxP3−) and suppressor (CD25+, FoxP3+) CD4+ T cells are negatively regulated by Fas cross-linking in mixed splenocyte populations of NOD, wild type mice and FoxP3-GFP tranegenes. Proliferation rates and sensitivity to Fas cross-linking are dissociated in Treg cells: fast cycling induced by IL-2 and CD3/CD28 stimulation improve Treg resistance to Fas-ligand (FasL) in both strains. The effector and suppressor CD4+ subsets display balanced sensitivity to negative regulation under baseline conditions, IL-2 and CD3/CD28 stimulation, indicating that stimulation does not perturb immune homeostasis in NOD mice. Effective autocrine apoptosis of diabetogenic cells was evident from delayed onset and reduced incidence of adoptive disease transfer into NOD.SCID by CD4+CD25− T cells decorated with FasL protein. Treg resistant to Fas-mediated apoptosis retain suppressive activity in vitro. The only detectable differential response was reduced Teff proliferation and upregulation of CD25 following CD3-activation in NOD mice. Conclusion These data document negative regulation of effector and suppressor cells by Fas cross-linking and dissociation between sensitivity to apoptosis and proliferation in stimulated Treg. There is no evidence that perturbed AICD in NOD mice initiates or promotes autoimmune insulitis. PMID:21738739

  10. LOCALIZER: subcellular localization prediction of both plant and effector proteins in the plant cell

    PubMed Central

    Sperschneider, Jana; Catanzariti, Ann-Maree; DeBoer, Kathleen; Petre, Benjamin; Gardiner, Donald M.; Singh, Karam B.; Dodds, Peter N.; Taylor, Jennifer M.

    2017-01-01

    Pathogens secrete effector proteins and many operate inside plant cells to enable infection. Some effectors have been found to enter subcellular compartments by mimicking host targeting sequences. Although many computational methods exist to predict plant protein subcellular localization, they perform poorly for effectors. We introduce LOCALIZER for predicting plant and effector protein localization to chloroplasts, mitochondria, and nuclei. LOCALIZER shows greater prediction accuracy for chloroplast and mitochondrial targeting compared to other methods for 652 plant proteins. For 107 eukaryotic effectors, LOCALIZER outperforms other methods and predicts a previously unrecognized chloroplast transit peptide for the ToxA effector, which we show translocates into tobacco chloroplasts. Secretome-wide predictions and confocal microscopy reveal that rust fungi might have evolved multiple effectors that target chloroplasts or nuclei. LOCALIZER is the first method for predicting effector localisation in plants and is a valuable tool for prioritizing effector candidates for functional investigations. LOCALIZER is available at http://localizer.csiro.au/. PMID:28300209

  11. The Effector Domain Region of the Vibrio vulnificus MARTX Toxin Confers Biphasic Epithelial Barrier Disruption and Is Essential for Systemic Spread from the Intestine.

    PubMed

    Gavin, Hannah E; Beubier, Nike T; Satchell, Karla J F

    2017-01-01

    Vibrio vulnificus causes highly lethal bacterial infections in which the Multifunctional Autoprocessing Repeats-in-Toxins (MARTX) toxin product of the rtxA1 gene is a key virulence factor. MARTX toxins are secreted proteins up to 5208 amino acids in size. Conserved MARTX N- and C-terminal repeat regions work in concert to form pores in eukaryotic cell membranes, through which the toxin's central region of modular effector domains is translocated. Upon inositol hexakisphosphate-induced activation of the of the MARTX cysteine protease domain (CPD) in the eukaryotic cytosol, effector domains are released from the holotoxin by autoproteolytic activity. We previously reported that the native MARTX toxin effector domain repertoire is dispensable for epithelial cellular necrosis in vitro, but essential for cell rounding and apoptosis prior to necrotic cell death. Here we use an intragastric mouse model to demonstrate that the effector domain region is required for bacterial virulence during intragastric infection. The MARTX effector domain region is essential for bacterial dissemination from the intestine, but dissemination occurs in the absence of overt intestinal tissue pathology. We employ an in vitro model of V. vulnificus interaction with polarized colonic epithelial cells to show that the MARTX effector domain region induces rapid intestinal barrier dysfunction and increased paracellular permeability prior to onset of cell lysis. Together, these results negate the inherent assumption that observations of necrosis in vitro directly predict bacterial virulence, and indicate a paradigm shift in our conceptual understanding of MARTX toxin function during intestinal infection. Results implicate the MARTX effector domain region in mediating early bacterial dissemination from the intestine to distal organs-a key step in V. vulnificus foodborne pathogenesis-even before onset of overt intestinal pathology.

  12. The Effector Domain Region of the Vibrio vulnificus MARTX Toxin Confers Biphasic Epithelial Barrier Disruption and Is Essential for Systemic Spread from the Intestine

    PubMed Central

    Gavin, Hannah E.; Beubier, Nike T.

    2017-01-01

    Vibrio vulnificus causes highly lethal bacterial infections in which the Multifunctional Autoprocessing Repeats-in-Toxins (MARTX) toxin product of the rtxA1 gene is a key virulence factor. MARTX toxins are secreted proteins up to 5208 amino acids in size. Conserved MARTX N- and C-terminal repeat regions work in concert to form pores in eukaryotic cell membranes, through which the toxin’s central region of modular effector domains is translocated. Upon inositol hexakisphosphate-induced activation of the of the MARTX cysteine protease domain (CPD) in the eukaryotic cytosol, effector domains are released from the holotoxin by autoproteolytic activity. We previously reported that the native MARTX toxin effector domain repertoire is dispensable for epithelial cellular necrosis in vitro, but essential for cell rounding and apoptosis prior to necrotic cell death. Here we use an intragastric mouse model to demonstrate that the effector domain region is required for bacterial virulence during intragastric infection. The MARTX effector domain region is essential for bacterial dissemination from the intestine, but dissemination occurs in the absence of overt intestinal tissue pathology. We employ an in vitro model of V. vulnificus interaction with polarized colonic epithelial cells to show that the MARTX effector domain region induces rapid intestinal barrier dysfunction and increased paracellular permeability prior to onset of cell lysis. Together, these results negate the inherent assumption that observations of necrosis in vitro directly predict bacterial virulence, and indicate a paradigm shift in our conceptual understanding of MARTX toxin function during intestinal infection. Results implicate the MARTX effector domain region in mediating early bacterial dissemination from the intestine to distal organs–a key step in V. vulnificus foodborne pathogenesis–even before onset of overt intestinal pathology. PMID:28060924

  13. Steady-State and Kinetics-Based Affinity Determination in Effector-Effector Target Interactions.

    PubMed

    Reinhard, André; Nürnberger, Thorsten

    2017-01-01

    Dissecting the functional basis of pathogenicity and resistance in the context of plant innate immunity benefits greatly from detailed knowledge about biomolecular interactions, as both resistance and virulence depend on specific interactions between pathogen and host biomolecules. While in vivo systems provide biological context to host-pathogen interactions, these experiments typically cannot provide quantitative biochemical characterization of biomolecular interactions. However, in many cases, the biological function does not only depend on whether an interaction occurs at all, but rather on the "intensity" of the interaction, as quantified by affinity. Specifically, microbial effector proteins may bind more than one host target to exert virulence functions, and looking at these interactions more closely than would be possible in a purely black-and-white qualitative assay (as classically based on plant or yeast systems) can reveal new insights into the evolutionary arms race between host and pathogen. Recent advances in biomolecular interaction assays that can be performed in vitro allow quantification of binding events with ever greater fidelity and application range. Here, we describe assays based on microscale thermophoresis (MST) and surface plasmon resonance (SPR). Using these technologies allows affinity determination both in steady-state and in kinetic configurations, providing two conceptually independent pathways to arrive at quantitative affinity data describing the interactions of pathogen and host biomolecules.

  14. Robotic End Effectors for Hard-Rock Climbing

    NASA Technical Reports Server (NTRS)

    Kennedy, Brett; Leger, Patrick

    2004-01-01

    Special-purpose robot hands (end effectors) now under development are intended to enable robots to traverse cliffs much as human climbers do. Potential applications for robots having this capability include scientific exploration (both on Earth and other rocky bodies in space), military reconnaissance, and outdoor search and rescue operations. Until now, enabling robots to traverse cliffs has been considered too difficult a task because of the perceived need of prohibitively sophisticated planning algorithms as well as end effectors as dexterous as human hands. The present end effectors are being designed to enable robots to attach themselves to typical rock-face features with less planning and simpler end effectors. This advance is based on the emulation of the equipment used by human climbers rather than the emulation of the human hand. Climbing-aid equipment, specifically cams, aid hooks, and cam hooks, are used by sport climbers when a quick ascent of a cliff is desired (see Figure 1). Currently two different end-effector designs have been created. The first, denoted the simple hook emulator, consists of three "fingers" arranged around a central "palm." Each finger emulates the function of a particular type of climbing hook (aid hook, wide cam hook, and a narrow cam hook). These fingers are connected to the palm via a mechanical linkage actuated with a leadscrew/nut. This mechanism allows the fingers to be extended or retracted. The second design, denoted the advanced hook emulator (see Figure 2), shares these features, but it incorporates an aid hook and a cam hook into each finger. The spring-loading of the aid hook allows the passive selection of the type of hook used. The end effectors can be used in several different modes. In the aid-hook mode, the aid hook on one of the fingers locks onto a horizontal ledge while the other two fingers act to stabilize the end effector against the cliff face. In the cam-hook mode, the broad, flat tip of the cam hook is

  15. Non-host Resistance Induced by the Xanthomonas Effector XopQ Is Widespread within the Genus Nicotiana and Functionally Depends on EDS1

    PubMed Central

    Adlung, Norman; Prochaska, Heike; Thieme, Sabine; Banik, Anne; Blüher, Doreen; John, Peter; Nagel, Oliver; Schulze, Sebastian; Gantner, Johannes; Delker, Carolin; Stuttmann, Johannes; Bonas, Ulla

    2016-01-01

    Most Gram-negative plant pathogenic bacteria translocate effector proteins (T3Es) directly into plant cells via a conserved type III secretion system, which is essential for pathogenicity in susceptible plants. In resistant plants, recognition of some T3Es is mediated by corresponding resistance (R) genes or R proteins and induces effector triggered immunity (ETI) that often results in programmed cell death reactions. The identification of R genes and understanding their evolution/distribution bears great potential for the generation of resistant crop plants. We focus on T3Es from Xanthomonas campestris pv. vesicatoria (Xcv), the causal agent of bacterial spot disease on pepper and tomato plants. Here, 86 Solanaceae lines mainly of the genus Nicotiana were screened for phenotypical reactions after Agrobacterium tumefaciens-mediated transient expression of 21 different Xcv effectors to (i) identify new plant lines for T3E characterization, (ii) analyze conservation/evolution of putative R genes and (iii) identify promising plant lines as repertoire for R gene isolation. The effectors provoked different reactions on closely related plant lines indicative of a high variability and evolution rate of potential R genes. In some cases, putative R genes were conserved within a plant species but not within superordinate phylogenetical units. Interestingly, the effector XopQ was recognized by several Nicotiana spp. lines, and Xcv infection assays revealed that XopQ is a host range determinant in many Nicotiana species. Non-host resistance against Xcv and XopQ recognition in N. benthamiana required EDS1, strongly suggesting the presence of a TIR domain-containing XopQ-specific R protein in these plant lines. XopQ is a conserved effector among most xanthomonads, pointing out the XopQ-recognizing RxopQ as candidate for targeted crop improvement. PMID:27965697

  16. Positive selection and intragenic recombination contribute to high allelic diversity in effector genes of Mycosphaerella fijiensis, causal agent of the black leaf streak disease of banana.

    PubMed

    Stergiopoulos, Ioannis; Cordovez, Viviane; Okmen, Bilal; Beenen, Henriek G; Kema, Gert H J; de Wit, Pierre J G M

    2014-06-01

    Previously, we have determined the nonhost-mediated recognition of the MfAvr4 and MfEcp2 effector proteins from the banana pathogen Mycosphaerella fijiensis in tomato, by the cognate Cf-4 and Cf-Ecp2 resistance proteins, respectively. These two resistance proteins could thus mediate resistance against M. fijiensis if genetically transformed into banana (Musa spp.). However, disease resistance controlled by single dominant genes can be overcome by mutated effector alleles, whose products are not recognized by the cognate resistance proteins. Here, we surveyed the allelic variation within the MfAvr4, MfEcp2, MfEcp2-2 and MfEcp2-3 effector genes of M. fijiensis in a global population of the pathogen, and assayed its impact on recognition by the tomato Cf-4 and Cf-Ecp2 resistance proteins, respectively. We identified a large number of polymorphisms that could reflect a co-evolutionary arms race between host and pathogen. The analysis of nucleotide substitution patterns suggests that both positive selection and intragenic recombination have shaped the evolution of M. fijiensis effectors. Clear differences in allelic diversity were observed between strains originating from South-East Asia relative to strains from other banana-producing continents, consistent with the hypothesis that M. fijiensis originated in the Asian-Pacific region. Furthermore, transient co-expression of the MfAvr4 effector alleles and the tomato Cf-4 resistance gene, as well as of MfEcp2, MfEcp2-2 and MfEcp2-3 and the putative Cf-Ecp2 resistance gene, indicated that effector alleles able to overcome these resistance genes are already present in natural populations of the pathogen, thus questioning the durability of resistance that can be provided by these genes in the field. © 2013 BSPP AND JOHN WILEY & SONS LTD.

  17. mTORC1-dependent metabolic reprogramming is a prerequisite for Natural Killer cell effector function

    PubMed Central

    Donnelly, Raymond P.; Loftus, Róisín M.; Keating, Sinéad E.; Liou, Kevin T.; Biron, Christine A.; Gardiner, Clair M.; Finlay, David K.

    2014-01-01

    The mammalian target of rapamcyin complex 1 (mTORC1) is a key regulator of cellular metabolism and also has fundamental roles in controlling immune responses. Emerging evidence suggests that these two functions of mTORC1 are integrally linked. However, little is known regarding mTORC1 function in controlling the metabolism and function of natural killer (NK) cells, lymphocytes that play key roles in anti-viral and anti-tumour immunity. This study investigated the hypothesis that mTORC1-controlled metabolism underpins normal NK cell pro-inflammatory function. We demonstrate that mTORC1 is robustly stimulated in NK cells activated in vivo and in vitro. This mTORC1 activity is required for the production of the key NK cell effector molecules IFNγ, important in delivering antimicrobial and immunoregulatory functions, and granzyme B, a critical component of NK cell cytotoxic granules. The data reveal that NK cells undergo dramatic metabolic reprogramming upon activation, up-regulating rates of glucose uptake and glycolysis, and that mTORC1 activity is essential for attaining this elevated glycolytic state. Directly limiting the rate of glycolysis is sufficient to inhibit IFNγ production and granzyme B expression. This study provides the highly novel insight that mTORC1-mediated metabolic reprogramming of NK cells is a prerequisite for the acquisition of normal effector functions. PMID:25261477

  18. The Escherichia coli effector EspJ blocks Src kinase activity via amidation and ADP ribosylation.

    PubMed

    Young, Joanna C; Clements, Abigail; Lang, Alexander E; Garnett, James A; Munera, Diana; Arbeloa, Ana; Pearson, Jaclyn; Hartland, Elizabeth L; Matthews, Stephen J; Mousnier, Aurelie; Barry, David J; Way, Michael; Schlosser, Andreas; Aktories, Klaus; Frankel, Gad

    2014-12-19

    The hallmark of enteropathogenic Escherichia coli (EPEC) infection is the formation of actin-rich pedestal-like structures, which are generated following phosphorylation of the bacterial effector Tir by cellular Src and Abl family tyrosine kinases. This leads to recruitment of the Nck-WIP-N-WASP complex that triggers Arp2/3-dependent actin polymerization in the host cell. The same phosphorylation-mediated signalling network is also assembled downstream of the Vaccinia virus protein A36 and the phagocytic Fc-gamma receptor FcγRIIa. Here we report that the EPEC type-III secretion system effector EspJ inhibits autophosphorylation of Src and phosphorylation of the Src substrates Tir and FcγRIIa. Consistent with this, EspJ inhibits actin polymerization downstream of EPEC, Vaccinia virus and opsonized red blood cells. We identify EspJ as a unique adenosine diphosphate (ADP) ribosyltransferase that directly inhibits Src kinase by simultaneous amidation and ADP ribosylation of the conserved kinase-domain residue, Src E310, resulting in glutamine-ADP ribose.

  19. ICAM-1-expressing neutrophils exhibit enhanced effector functions in murine models of endotoxemia.

    PubMed

    Woodfin, Abigail; Beyrau, Martina; Voisin, Mathieu-Benoit; Ma, Bin; Whiteford, James R; Hordijk, Peter L; Hogg, Nancy; Nourshargh, Sussan

    2016-02-18

    Intracellular adhesion molecule-1 (ICAM-1) is a transmembrane glycoprotein expressed on the cell surface of numerous cell types such as endothelial and epithelial cells, vascular smooth muscle cells, and certain leukocyte subsets. With respect to the latter, ICAM-1 has been detected on neutrophils in several clinical and experimental settings, but little is known about the regulation of expression or function of neutrophil ICAM-1. In this study, we report on the de novo induction of ICAM-1 on the cell surface of murine neutrophils by lipopolysaccharide (LPS), tumor necrosis factor, and zymosan particles in vitro. The induction of neutrophil ICAM-1 was associated with enhanced phagocytosis of zymosan particles and reactive oxygen species (ROS) generation. Conversely, neutrophils from ICAM-1-deficient mice were defective in these effector functions. Mechanistically, ICAM-1-mediated intracellular signaling appeared to support neutrophil ROS generation and phagocytosis. In vivo, LPS-induced inflammation in the mouse cremaster muscle and peritoneal cavity led to ICAM-1 expression on intravascular and locally transmigrated neutrophils. The use of chimeric mice deficient in ICAM-1 on myeloid cells demonstrated that neutrophil ICAM-1 was not required for local neutrophil transmigration, but supported optimal intravascular and extravascular phagocytosis of zymosan particles. Collectively, the present results shed light on regulation of expression and function of ICAM-1 on neutrophils and identify it as an additional regulator of neutrophil effector responses in host defense.

  20. Cervical cancer cells suppress effector functions of cytotoxic T cells through the adenosinergic pathway.

    PubMed

    Mora-García, M L; Ávila-Ibarra, L R; García-Rocha, R; Weiss-Steider, B; Hernández-Montes, J; Don-López, C A; Gutiérrez-Serrano, V; Titla-Vilchis, I J; Fuentes-Castañeda, M C; Monroy-Mora, A; Jave-Suárez, L F; Chacón-Salinas, R; Vallejo-Castillo, L; Pérez-Tapia, S M; Monroy-García, A

    2017-09-07

    The expression of CD73 in tumor cells plays a significant role in the production of adenosine (Ado) that suppresses antitumor effector cells. In this study we analyzed the capability of HPV-positive (HPV+) cervical cancer (CeCa) cell lines CaSki, SiHa, HeLa, and RoVa; and HPV-negative (HPV-) cell lines C33A and ViBo to produce Ado and inhibit effector functions of CD8+ T cells. HPV+ CeCa cells expressed significantly higher levels of CD73 in the membrane (p<0.01) than HPV- CeCa cells and this expression was associated with the production of larger amounts of Ado (>400μM) compared to HPV-CeCa cells (<200μM) in the presence of AMP, as well asa stronger inhibition of (>50%) proliferation, activation, and cytotoxic activity of CD8+ T cells via interaction with A2A adenosine receptor. We also provide evidence that silenced E6/E7 expression in CeCa cells, strongly reduced its CD73 expression level and its capability to generate Ado. This results suggest that HPV infection, which is associated with more than 99% of CeCa cases, may present an increased constitutive expression of CD73 in cervical neoplasia to contribute to the suppression of the immune response mediated by the production of large amounts of Ado. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Host Fate is Rapidly Determined by Innate Effector-Microbial Interactions During Acinetobacter baumannii Bacteremia

    PubMed Central

    Bruhn, Kevin W.; Pantapalangkoor, Paul; Nielsen, Travis; Tan, Brandon; Junus, Justin; Hujer, Kristine M.; Wright, Meredith S.; Bonomo, Robert A.; Adams, Mark D.; Chen, Wangxue; Spellberg, Brad

    2015-01-01

    Background. Acinetobacter baumannii is one of the most antibiotic-resistant pathogens. Defining mechanisms driving pathogenesis is critical to enable new therapeutic approaches. Methods. We studied virulence differences across a diverse panel of A. baumannii clinical isolates during murine bacteremia to elucidate host-microbe interactions that drive outcome. Results. We identified hypervirulent strains that were lethal at low intravenous inocula and achieved very high early, and persistent, blood bacterial densities. Virulent strains were nonlethal at low inocula but lethal at 2.5-fold higher inocula. Finally, relatively avirulent (hypovirulent) strains were nonlethal at 20-fold higher inocula and were efficiently cleared by early time points. In vivo virulence correlated with in vitro resistance to complement and macrophage uptake. Depletion of complement, macrophages, and neutrophils each independently increased bacterial density of the hypovirulent strain but insufficiently to change lethality. However, disruption of all 3 effector mechanisms enabled early bacterial densities similar to hypervirulent strains, rendering infection 100% fatal. Conclusions. The lethality of A. baumannii strains depends on distinct stages. Strains resistant to early innate effectors are able to establish very high early bacterial blood density, and subsequent sustained bacteremia leads to Toll-like receptor 4–mediated hyperinflammation and lethality. These results have important implications for translational efforts to develop therapies that modulate host-microbe interactions. PMID:25378635

  2. The bacterial effector Cif interferes with SCF ubiquitin ligase function by inhibiting deneddylation of Cullin1.

    PubMed

    Morikawa, Hanako; Kim, Minsoo; Mimuro, Hitomi; Punginelli, Claire; Koyama, Tomohiro; Nagai, Shinya; Miyawaki, Atsushi; Iwai, Kazuhiro; Sasakawa, Chihiro

    2010-10-15

    Cycle inhibiting factor (Cif) is one of the effectors delivered into epithelial cells by enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) via the type III secretion system (TTSS). Cif family proteins, which inhibit host cell-cycle progression via mechanisms not yet precisely understood, are highly conserved among EPEC, EHEC, Yersinia pseudotuberculosis, Photorhabdus luminescens and Burkholderia pseudomallei. Levels of several proteins relevant to cell-cycle progression are modulated by Cullin-RING ligases (CRLs), which in turn are activated by conjugation and deconjugation of NEDD8 to Cullins. Here we show that Cif interacts with NEDD8 and interferes with SCF (Skp1-Cullin1-F-box protein) complex ubiquitin ligase function. We found that neddylated Cullin family proteins accumulated and ubiquitination of p27 decreased in cells infected with EPEC. Consequently, Cif stabilized SCF substrates such as CyclinD1, Cdt1, and p27, and caused G1 cell-cycle arrest. Using time-lapse-imaging of fluorescent ubiquitination-based cell-cycle indicator (Fucci)-expressing cells, we were able to monitor cell-cycle progression during EPEC infection and confirmed the arrest of infected cells at G1. Our in vitro and in vivo data show that Cif-NEDD8 interaction inhibits deneddylation of Cullins, suppresses CRL activity and induces G1 arrest. We thus conclude that the bacterial effector Cif interferes with neddylation-mediated cell-cycle control. Copyright © 2010 Elsevier Inc. All rights reserved.

  3. IgE epitope proximity determines immune complex shape and effector cell activation capacity

    PubMed Central

    Gieras, Anna; Linhart, Birgit; Roux, Kenneth H.; Dutta, Moumita; Khodoun, Marat; Zafred, Domen; Cabauatan, Clarissa R.; Lupinek, Christian; Weber, Milena; Focke-Tejkl, Margarete; Keller, Walter; Finkelman, Fred D.; Valenta, Rudolf

    2016-01-01

    Background IgE-allergen complexes induce mast cell and basophil activation and thus immediate allergic inflammation. They are also important for IgE-facilitated allergen presentation to T cells by antigen-presenting cells. Objective To investigate whether the proximity of IgE binding sites on an allergen affects immune complex shape and subsequent effector cell activation in vitro and in vivo. Methods We constructed artificial allergens by grafting IgE epitopes in different numbers and proximity onto a scaffold protein. The shape of immune complexes formed between artificial allergens and the corresponding IgE was studied by negative-stain electron microscopy. Allergenic activity was determined using basophil activation assays. Mice were primed with IgE, followed by injection of artificial allergens to evaluate their in vivo allergenic activity. Severity of systemic anaphylaxis was measured by changes in body temperature. Results We could demonstrate simultaneous binding of 4 IgE antibodies in close vicinity to each other. The proximity of IgE binding sites on allergens influenced the shape of the resulting immune complexes and the magnitude of effector cell activation and in vivo inflammation. Conclusions Our results demonstrate that the proximity of IgE epitopes on an allergen affects its allergenic activity. We thus identified a novel mechanism by which IgE-allergen complexes regulate allergic inflammation. This mechanism should be important for allergy and other immune complex–mediated diseases. PMID:26684291

  4. IgG-effector functions: "the good, the bad and the ugly".

    PubMed

    Kapur, Rick; Einarsdottir, Helga K; Vidarsson, Gestur

    2014-08-01

    IgG-antibodies are potent and versatile mediators of host protection. They elicit their biological effects through specific interaction of the Fc-part with complement, specific cellular receptors, or both. Several factors should be taken into consideration when analyzing the nature and intensity of the immunological response elicited via IgG-effector functions, especially for the family of IgG-Fc receptors (FcγRs) exclusively expressed on immune cells. These include the various classes of leukocyte FcγR, expressed variably on different immune cells, each with distinct affinity for every IgG subclass, as well as genetic FcγR-polymorphisms affecting expression and affinity for IgG. Furthermore, various aspects of the IgG itself are also crucial for the outcome of the biological response. These include endogenously encoded IgG-polymorphisms, such as IgG3 polymorphisms, and post-transcriptional IgG-modifications, in particular IgG-Fc-glycosylation, affecting IgG effector functions through modified binding affinity to FcγR. These latter aspects concerning the variability in IgG3 on its half-life and placental transport and the clinical consequences of altered IgG-quality through glycosylation, will be the focus of this review.

  5. Recognition of the Magnaporthe oryzae Effector AVR-Pia by the Decoy Domain of the Rice NLR Immune Receptor RGA5[OPEN

    PubMed Central

    Ortiz, Diana; de Guillen, Karine; Cesari, Stella; Chalvon, Véronique

    2017-01-01

    Nucleotide binding domain and leucine-rich repeat proteins (NLRs) are important receptors in plant immunity that allow recognition of pathogen effectors. The rice (Oryza sativa) NLR RGA5 recognizes the Magnaporthe oryzae effector AVR-Pia through direct interaction. Here, we gained detailed insights into the molecular and structural bases of AVR-Pia-RGA5 interaction and the role of the RATX1 decoy domain of RGA5. NMR titration combined with in vitro and in vivo protein-protein interaction analyses identified the AVR-Pia interaction surface that binds to the RATX1 domain. Structure-informed AVR-Pia mutants showed that, although AVR-Pia associates with additional sites in RGA5, binding to the RATX1 domain is necessary for pathogen recognition but can be of moderate affinity. Therefore, RGA5-mediated resistance is highly resilient to mutations in the effector. We propose a model that explains such robust effector recognition as a consequence, and an advantage, of the combination of integrated decoy domains with additional independent effector-NLR interactions. PMID:28087830

  6. Recognition of the Magnaporthe oryzae Effector AVR-Pia by the Decoy Domain of the Rice NLR Immune Receptor RGA5.

    PubMed

    Ortiz, Diana; de Guillen, Karine; Cesari, Stella; Chalvon, Véronique; Gracy, Jérome; Padilla, André; Kroj, Thomas

    2017-01-01

    Nucleotide binding domain and leucine-rich repeat proteins (NLRs) are important receptors in plant immunity that allow recognition of pathogen effectors. The rice (Oryza sativa) NLR RGA5 recognizes the Magnaporthe oryzae effector AVR-Pia through direct interaction. Here, we gained detailed insights into the molecular and structural bases of AVR-Pia-RGA5 interaction and the role of the RATX1 decoy domain of RGA5. NMR titration combined with in vitro and in vivo protein-protein interaction analyses identified the AVR-Pia interaction surface that binds to the RATX1 domain. Structure-informed AVR-Pia mutants showed that, although AVR-Pia associates with additional sites in RGA5, binding to the RATX1 domain is necessary for pathogen recognition but can be of moderate affinity. Therefore, RGA5-mediated resistance is highly resilient to mutations in the effector. We propose a model that explains such robust effector recognition as a consequence, and an advantage, of the combination of integrated decoy domains with additional independent effector-NLR interactions. © 2017 American Society of Plant Biologists. All rights reserved.

  7. Study on cellular events in postthymectomy autoimmune oophoritis in mice. I. Requirement of Lyt-1 effector cells for oocytes damage after adoptive transfer

    PubMed Central

    1982-01-01

    Neonatal thymectomy during the critical period, 2-4 d after birth, can induce various organ-specific autoimmune diseases including oophoritis in A/J mice. The oophoritis thus induced was passively transferred into neonatal mice by injection of spleen cells obtained from syngeneic donors with the disease. Recipient ovaries were rapidly damaged with remarkable mononuclear cell infiltration and destruction of follicular structures. The phenotype of effector cells responsible for successful adoptive transfer was found to be Thy-1+, Lyt-1+,23-, Ia-, Qa-1-, and was sensitive to antithymocyte serum treatment but resistant to cyclophosphamide treatment or in vitro X-ray irradiation. The compatibility between donor and recipient at the major histocompatibility complex was not required for the effector phase of transfer. The oophoritis induced in BALB/c (nu/+ or +/+) was also shown to be transferred into athymic BALB/c nude mice with resulting ovarian lesion and circulating autoantibodies against oocytes. In this transfer system, the effector cells were also demonstrated to be T cells with the Lyt-1+,23- phenotype. Adoptive transfer experiments in both systems revealed that the destruction of ovaries in postthymectomy autoimmune oophoritis was mediated by Lyt-1 T cells. Whether these T cells can be distinguished from other Lyt-1 cells, such as T helper cells and effector T cells in delayed-type hypersensitivity (DTH), is not clear at present, but the results suggest that the effector mechanisms may be closely related to a DTH reaction. PMID:6983557

  8. Combined Targeting of BRAF and CRAF or BRAF and PI3K Effector Pathways Is Required for Efficacy in NRAS Mutant Tumors

    PubMed Central

    Jaiswal, Bijay S.; Janakiraman, Vasantharajan; Kljavin, Noelyn M.; Eastham-Anderson, Jeffrey; Cupp, James E.; Liang, Yuxin; Davis, David P.; Hoeflich, Klaus P.; Seshagiri, Somasekar

    2009-01-01

    Background Oncogenic RAS is a highly validated cancer target. Attempts at targeting RAS directly have so far not succeeded in the clinic. Understanding downstream RAS-effectors that mediate oncogenesis in a RAS mutant setting will help tailor treatments that use RAS-effector inhibitors either alone or in combination to target RAS-driven tumors. Methodology/Principal Findings In this study, we have investigated the sufficiency of targeting RAS-effectors, RAF, MEK and PI3-Kinase either alone or in combination in RAS mutant lines, using an inducible shRNA in vivo mouse model system. We find that in colon cancer cells harboring a KRASG13D mutant allele, knocking down KRAS alone or the RAFs in combination or the RAF effectors, MEK1 and MEK2, together is effective in delaying tumor growth in vivo. In melanoma cells harboring an NRASQ61L or NRASQ61K mutant allele, we find that targeting NRAS alone or both BRAF and CRAF in combination or both BRAF and PIK3CA together showed efficacy. Conclusion/Significance Our data indicates that targeting oncogenic NRAS-driven melanomas require decrease in both pERK and pAKT downstream of RAS-effectors for efficacy. This can be achieved by either targeting both BRAF and CRAF or BRAF and PIK3CA simultaneously in NRAS mutant tumor cells. PMID:19492075

  9. T Cells: Soldiers and Spies--The Surveillance and Control of Effector T Cells by Regulatory T Cells.

    PubMed

    Hall, Bruce M

    2015-11-06

    Traditionally, T cells were CD4+ helper or CD8+ cytotoxic T cells, and with antibodies, they were the soldiers of immunity. Now, many functionally distinct subsets of activated CD4+ and CD8+ T cells have been described, each with distinct cytokine and transcription factor expression. For CD4+ T cells, these include Th1 cells expressing the transcription factor T-bet and cytokines IL-2, IFN-γ, and TNF-β; Th2 cells expressing GATA-3 and the cytokines IL-4, IL-5, and IL-13; and Th17 cells expressing RORγt and cytokines IL-17A, IL-17F, IL-21, and IL-22. The cytokines produced determine the immune inflammation that they mediate. T cells of the effector lineage can be naïve T cells, recently activated T cells, or memory T cells that can be distinguished by cell surface markers. T regulatory cells or spies were characterized as CD8+ T cells expressing I-J in the 1970s. In the 1980s, suppressor cells fell into disrepute when the gene for I-J was not present in the mouse MHC I region. At that time, a CD4+ T cell expressing CD25, the IL-2 receptor-α, was identified to transfer transplant tolerance. This was the same phenotype of activated CD4+ CD25+ T cells that mediated rejection. Thus, the cells that could induce tolerance and undermine rejection had similar badges and uniforms as the cells effecting rejection. Later, FOXP3, a transcription factor that confers suppressor function, was described and distinguishes T regulatory cells from effector T cells. Many subtypes of T regulatory cells can be characterized by different expressions of cytokines and receptors for cytokines or chemokines. In intense immune inflammation, T regulatory cells express cytokines characteristic of effector cells; for example, Th1-like T regulatory cells express T-bet, and IFN-γ-like Th1 cells and effector T cells can change sides by converting to T regulatory cells. Effector T cells and T regulatory cells use similar molecules to be activated and mediate their function, and thus, it can be

  10. Proteases in Fas-mediated apoptosis.

    PubMed

    Zhivotovsky, B; Burgess, D H; Schlegel, J; Pörn, M I; Vanags, D; Orrenius, S

    1997-01-01

    Involvement of a unique family of cysteine proteases in the multistep apoptotic process has been documented. Cloning of several mammalian genes identifies some components of this cellular response. However, it is currently unclear which protease plays a role as a signal and/or effector of apoptosis. We summarize contributions to the data concerning proteases in Fas-mediated apoptosis.

  11. Modulation of hemoglobin dynamics by an allosteric effector

    PubMed Central

    Maccarini, Marco; Fouquet, Peter; Ho, Nancy T.; Ho, Chien; Makowski, Lee

    2017-01-01

    Abstract Hemoglobin (Hb) is an extensively studied paradigm of proteins that alter their function in response to allosteric effectors. Models of its action have been used as prototypes for structure‐function relationships in many proteins, and models for the molecular basis of its function have been deeply studied and extensively argued. Recent reports suggest that dynamics may play an important role in its function. Relatively little is known about the slow, correlated motions of hemoglobin subunits in various structural states because experimental and computational strategies for their characterization are challenging. Allosteric effectors such as inositol hexaphosphate (IHP) bind to both deoxy‐Hb and HbCO, albeit at different sites, leading to a lowered oxygen affinity. The manner in which these effectors impact oxygen binding is unclear and may involve changes in structure, dynamics or both. Here we use neutron spin echo measurements accompanied by wide‐angle X‐ray scattering to show that binding of IHP to HbCO results in an increase in the rate of coordinated motions of Hb subunits relative to one another with little if any change in large scale structure. This increase of large‐scale dynamics seems to be coupled with a decrease in the average magnitude of higher frequency modes of individual residues. These observations indicate that enhanced dynamic motions contribute to the functional changes induced by IHP and suggest that they may be responsible for the lowered oxygen affinity triggered by these effectors. PMID:27977887

  12. The Coding and Effector Transfer of Movement Sequences

    ERIC Educational Resources Information Center

    Kovacs, Attila J.; Muhlbauer, Thomas; Shea, Charles H.

    2009-01-01

    Three experiments utilizing a 14-element arm movement sequence were designed to determine if reinstating the visual-spatial coordinates, which require movements to the same spatial locations utilized during acquisition, results in better effector transfer than reinstating the motor coordinates, which require the same pattern of homologous muscle…

  13. Effector proteins support the asymmetric apportioning of Salmonella during cytokinesis

    PubMed Central

    Zhao, Yaya; Gorvel, Jean-Pierre; Méresse, Stéphane

    2016-01-01

    ABSTRACT Salmonella-infected cells are characterized by the presence of intra-cellular membranous tubules that emerge from bacterial vacuoles and extend along microtubules. The formation of Salmonella-induced tubules depends on the Salmonella pathogenicity island 2-encoded type III secretion system (T3SS-2) that translocates bacterial effector proteins inside host cells. Effector proteins have enzymatic activities or allow for hijacking of cellular functions. The role of Salmonella-induced tubules in virulence remains unclear but their absence is correlated with virulence defects. This study describes the presence of inter-cellular tubules that arise between daughter cells during cytokinesis. Inter-cellular tubules connect bacterial vacuoles originally present in the parent cell and that have been apportioned between daughters. Their formation requires a functional T3SS-2 and effector proteins. Our data establish a correlation between the formation of inter-cellular tubules and the asymmetric distribution of bacterial vacuoles in daughters. Thus, by manipulating the distribution of bacteria in cytokinetic cells, Salmonella T3SS-2 effector proteins may increase bacterial spreading and the systemic character of the infection. PMID:27046257

  14. Modulation of hemoglobin dynamics by an allosteric effector

    DOE PAGES

    Lal, Jyotsana; Maccarini, Marco; Fouquet, Peter; ...

    2016-12-15

    Hemoglobin (Hb) is an extensively studied paradigm of proteins that alter their function in response to allosteric effectors. Models of its action have been used as prototypes for structure-function relationships in many proteins, and models for the molecular basis of its function have been deeply studied and extensively argued. Recent reports suggest that dynamics may play an important role in its function. Relatively little is known about the slow, correlated motions of hemoglobin subunits in various structural states because experimental and computational strategies for their characterization are challenging. Allosteric effectors such as inositol hexaphosphate (IHP) bind to both deoxy-Hb andmore » HbCO, albeit at different sites, leading to a lowered oxygen affinity. The manner in which these effectors impact oxygen binding is unclear and may involve changes in structure, dynamics or both. Here we use neutron spin echo (NSE) measurements accompanied by wideangle x-ray scattering (WAXS) to show that binding of IHP to HbCO results in an increase in the rate of coordinated motions of Hb subunits relative to one another with little if any change in large scale structure. This increase of large-scale dynamics seems to be coupled with a decrease in the average magnitude of higher frequency modes of individual residues. Furthermore, these observations indicate that enhanced dynamic motions contribute to the functional changes induced by IHP and suggest that they may be responsible for the lowered oxygen affinity triggered by these effectors.« less

  15. Concurrent simulation of a parallel jaw end effector

    NASA Technical Reports Server (NTRS)

    Bynum, Bill

    1985-01-01

    A system of programs developed to aid in the design and development of the command/response protocol between a parallel jaw end effector and the strategic planner program controlling it are presented. The system executes concurrently with the LISP controlling program to generate a graphical image of the end effector that moves in approximately real time in response to commands sent from the controlling program. Concurrent execution of the simulation program is useful for revealing flaws in the communication command structure arising from the asynchronous nature of the message traffic between the end effector and the strategic planner. Software simulation helps to minimize the number of hardware changes necessary to the microprocessor driving the end effector because of changes in the communication protocol. The simulation of other actuator devices can be easily incorporated into the system of programs by using the underlying support that was developed for the concurrent execution of the simulation process and the communication between it and the controlling program.

  16. Imitation of hand and tool actions is effector-independent.

    PubMed

    van Elk, M; van Schie, H T; Bekkering, H

    2011-10-01

    Following the theoretical notion that tools often extend one's body, in the present study, we investigated whether imitation of hand or tool actions is modulated by effector-specific information. Subjects performed grasping actions toward an object with either a handheld tool or their right hand. Actions were initiated in response to pictures representing a grip at an object that could be congruent or incongruent with the required action (grip-type congruency). Importantly, actions could be cued by means of a tool cue, a hand cue, and a symbolic cue (effector-type congruency). For both hand and tool actions, an action congruency effect was observed, reflected in faster reaction times if the observed grip type was congruent with the required movement. However, neither hand actions nor tool actions were differentially affected by the effector represented in the picture (i.e., when performing a tool action, the action congruency effect was similar for tool cues and hand cues). This finding suggests that imitation of hand and tool actions is effector-independent and thereby supports generalist rather than specialist theories of imitation.

  17. Targeting DNA double-strand breaks with TAL effector nucleases.

    PubMed

    Christian, Michelle; Cermak, Tomas; Doyle, Erin L; Schmidt, Clarice; Zhang, Feng; Hummel, Aaron; Bogdanove, Adam J; Voytas, Daniel F

    2010-10-01

    Engineered nucleases that cleave specific DNA sequences in vivo are valuable reagents for targeted mutagenesis. Here we report a new class of sequence-specific nucleases created by fusing transcription activator-like effectors (TALEs) to the catalytic domain of the FokI endonuclease. Both native and custom TALE-nuclease fusions direct DNA double-strand breaks to specific, targeted sites.

  18. Hand to Mouth: Automatic Imitation across Effector Systems

    ERIC Educational Resources Information Center

    Leighton, Jane; Heyes, Cecilia

    2010-01-01

    The effector dependence of automatic imitation was investigated using a stimulus-response compatibility (SRC) procedure during which participants were required to make an open or closed response with their hand or their mouth. The correct response for each trial was indicated by a pair of letters in Experiments 1 and 2 and by a colored square in…

  19. RMS end effector waiting for command and SPAS-01 nearby

    NASA Technical Reports Server (NTRS)

    1983-01-01

    The end effector of the remote manipulator system (RMS) appears to be waiting for its next command at the top of this frame and the Shuttle pallet satellite (SPAS-01), in its free flying mode, appears nearby. The three letters legible on the SPAS stand for Messerschmitt-Boelkow-Blohm Gmbit, a West German firm.

  20. Effector profiles distinguish formae speciales of Fusarium oxysporum.

    PubMed

    van Dam, Peter; Fokkens, Like; Schmidt, Sarah M; Linmans, Jasper H J; Kistler, H Corby; Ma, Li-Jun; Rep, Martijn

    2016-11-01

    Formae speciales (ff.spp.) of the fungus Fusarium oxysporum are often polyphyletic within the species complex, making it impossible to identify them on the basis of conserved genes. However, sequences that determine host-specific pathogenicity may be expected to be similar between strains within the same forma specialis. Whole genome sequencing was performed on strains from five different ff.spp. (cucumerinum, niveum, melonis, radicis-cucumerinum and lycopersici). In each genome, genes for putative effectors were identified based on small size, secretion signal, and vicinity to a "miniature impala" transposable element. The candidate effector genes of all genomes were collected and the presence/absence patterns in each individual genome were clustered. Members of the same forma specialis turned out to group together, with cucurbit-infecting strains forming a supercluster separate from other ff.spp. Moreover, strains from different clonal lineages within the same forma specialis harbour identical effector gene sequences, supporting horizontal transfer of genetic material. These data offer new insight into the genetic basis of host specificity in the F. oxysporum species complex and show that (putative) effectors can be used to predict host specificity in F. oxysporum. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

  1. Memory versus effector immune responses in oncolytic virotherapies.

    PubMed

    Macnamara, Cicely; Eftimie, Raluca

    2015-07-21

    The main priority when designing cancer immuno-therapies has been to seek viable biological mechanisms that lead to permanent cancer eradication or cancer control. Understanding the delicate balance between the role of effector and memory cells on eliminating cancer cells remains an elusive problem in immunology. Here we make an initial investigation into this problem with the help of a mathematical model for oncolytic virotherapy; although the model can in fact be made general enough to be applied also to other immunological problems. According to this model, we find that long-term cancer control is associated with a large number of persistent effector cells (irrespective of the initial peak in effector cell numbers). However, this large number of persistent effector cells is sustained by a relatively large number of memory cells. Moreover, the results of the mathematical model suggest that cancer control from a dormant state cannot be predicted by the size of the memory population. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Hand to Mouth: Automatic Imitation across Effector Systems

    ERIC Educational Resources Information Center

    Leighton, Jane; Heyes, Cecilia

    2010-01-01

    The effector dependence of automatic imitation was investigated using a stimulus-response compatibility (SRC) procedure during which participants were required to make an open or closed response with their hand or their mouth. The correct response for each trial was indicated by a pair of letters in Experiments 1 and 2 and by a colored square in…

  3. Electroporation of Functional Bacterial Effectors into Mammalian Cells

    DOE PAGES

    Sontag, Ryan L.; Mihai, Cosmin; Orr, Galya; ...

    2015-01-19

    Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

  4. Ehrlichia Moonlighting Effectors and Interkingdom Interactions with the Mononuclear Phagocyte

    PubMed Central

    Dunphy, Paige Selvy; Luo, Tian; McBride, Jere W.

    2013-01-01

    Ehrlichia chaffeensis is an obligately intracellular gram negative bacterium with a small genome that thrives in mammalian mononuclear phagoctyes by exploiting eukaryotic processes. Herein, we discuss the latest findings on moonlighting tandem repeat protein effectors and their secretion mechanisms, and novel molecular interkingdom interactions that provide insight into the intracellular pathobiology of ehrlichiae. PMID:24141087

  5. Structure Analysis Uncovers a Highly Diverse but Structurally Conserved Effector Family in Phytopathogenic Fungi

    PubMed Central

    Gracy, Jérome; Fournier, Elisabeth; Kroj, Thomas; Padilla, André

    2015-01-01

    Phytopathogenic ascomycete fungi possess huge effector repertoires that are dominated by hundreds of sequence-unrelated small secreted proteins. The molecular function of these effectors and the evolutionary mechanisms that generate this tremendous number of singleton genes are largely unknown. To get a deeper understanding of fungal effectors, we determined by NMR spectroscopy the 3-dimensional structures of the Magnaporthe oryzae effectors AVR1-CO39 and AVR-Pia. Despite a lack of sequence similarity, both proteins have very similar 6 β-sandwich structures that are stabilized in both cases by a disulfide bridge between 2 conserved cysteins located in similar positions of the proteins. Structural similarity searches revealed that AvrPiz-t, another effector from M. oryzae, and ToxB, an effector of the wheat tan spot pathogen Pyrenophora tritici-repentis have the same structures suggesting the existence of a family of sequence-unrelated but structurally conserved fungal effectors that we named MAX-effectors (Magnaporthe Avrs and ToxB like). Structure-informed pattern searches strengthened this hypothesis by identifying MAX-effector candidates in a broad range of ascomycete phytopathogens. Strong expansion of the MAX-effector family was detected in M. oryzae and M. grisea where they seem to be particularly important since they account for 5–10% of the effector repertoire and 50% of the cloned avirulence effectors. Expression analysis indicated that the majority of M. oryzae MAX-effectors are expressed specifically during early infection suggesting important functions during biotrophic host colonization. We hypothesize that the scenario observed for MAX-effectors can serve as a paradigm for ascomycete effector diversity and that the enormous number of sequence-unrelated ascomycete effectors may in fact belong to a restricted set of structurally conserved effector families. PMID:26506000

  6. Genome-Scale Identification of Legionella pneumophila Effectors Using a Machine Learning Approach

    PubMed Central

    Burstein, David; Zusman, Tal; Degtyar, Elena; Viner, Ram; Segal, Gil; Pupko, Tal

    2009-01-01

    A large number of highly pathogenic bacteria utilize secretion systems to translocate effector proteins into host cells. Using these effectors, the bacteria subvert host cell processes during infection. Legionella pneumophila translocates effectors via the Icm/Dot type-IV secretion system and to date, approximately 100 effectors have been identified by various experimental and computational techniques. Effector identification is a critical first step towards the understanding of the pathogenesis system in L. pneumophila as well as in other bacterial pathogens. Here, we formulate the task of effector identification as a classification problem: each L. pneumophila open reading frame (ORF) was classified as either effector or not. We computationally defined a set of features that best distinguish effectors from non-effectors. These features cover a wide range of characteristics including taxonomical dispersion, regulatory data, genomic organization, similarity to eukaryotic proteomes and more. Machine learning algorithms utilizing these features were then applied to classify all the ORFs within the L. pneumophila genome. Using this approach we were able to predict and experimentally validate 40 new effectors, reaching a success rate of above 90%. Increasing the number of validated effectors to around 140, we were able to gain novel insights into their characteristics. Effectors were found to have low G+C content, supporting the hypothesis that a large number of effectors originate via horizontal gene transfer, probably from their protozoan host. In addition, effectors were found to cluster in specific genomic regions. Finally, we were able to provide a novel description of the C-terminal translocation signal required for effector translocation by the Icm/Dot secretion system. To conclude, we have discovered 40 novel L. pneumophila effectors, predicted over a hundred additional highly probable effectors, and shown the applicability of machine learning algorithms for

  7. Active Flow Effectors for Noise and Separation Control

    NASA Technical Reports Server (NTRS)

    Turner, Travis L.

    2011-01-01

    New flow effector technology for separation control and enhanced mixing is based upon shape memory alloy hybrid composite (SMAHC) technology. The technology allows for variable shape control of aircraft structures through actively deformable surfaces. The flow effectors are made by embedding shape memory alloy actuator material in a composite structure. When thermally actuated, the flow effector def1ects into or out of the flow in a prescribed manner to enhance mixing or induce separation for a variety of applications, including aeroacoustic noise reduction, drag reduction, and f1ight control. The active flow effectors were developed for noise reduction as an alternative to fixed-configuration effectors, such as static chevrons, that cannot be optimized for airframe installation effects or variable operating conditions and cannot be retracted for off-design or fail-safe conditions. Benefits include: Increased vehicle control, overall efficiency, and reduced noise throughout all f1ight regimes, Reduced flow noise, Reduced drag, Simplicity of design and fabrication, Simplicity of control through direct current stimulation, autonomous re sponse to environmental heating, fast re sponse, and a high degree of geometric stability. The concept involves embedding prestrained SMA actuators on one side of the chevron neutral axis in order to generate a thermal moment and def1ect the structure out of plane when heated. The force developed in the host structure during def1ection and the aerodynamic load is used for returning the structure to the retracted position. The chevron design is highly scalable and versatile, and easily affords active and/or autonomous (environmental) control. The technology offers wide-ranging market applications, including aerospace, automotive, and any application that requires flow separation or noise control.

  8. [Nitric oxide as main effector in the interleukin-1 system in ovulation].

    PubMed

    Díaz Flores, M; Ortega-Camarillo, C; Rosales-Torres, A M; Baiza-Gutman, L A; Hicks, J J

    2001-01-01

    Ovulation is a complex process involving not only gonadotropins and steroid hormones, but also many local mediators common to inflammatory reactions, such as cytokines. Of particular interest is the ovarian interleukin-1 (IL-1) system, which may be an intermediary of gonadotropins in the ovulatory process. The preovulatory follicles have a complete and highly compartmentalized intraovarian IL-1 system including ligands, receptor, and receptor antagonist. IL-1 has been considered as the inductor of several ovulation-associated events such as prostaglandin and progesterone biosynthesis, plasminogen activator production, glycosaminoglycan generation, and enhancement of vascular permeability. The principal effector of the IL-1 system is nitric oxide. This paper analyzes the sites of synthesis and action of the IL-1 system in preovulatory follicle and its vascular dynamics as well as IL-1's mechanism of action in triggering follicular rupture.

  9. SAUR Proteins as Effectors of Hormonal and Environmental Signals in Plant Growth.

    PubMed

    Ren, Hong; Gray, William M

    2015-08-01

    The plant hormone auxin regulates numerous aspects of plant growth and development. Early auxin response genes mediate its genomic effects on plant growth and development. Discovered in 1987, small auxin up RNAs (SAURs) are the largest family of early auxin response genes. SAUR functions have remained elusive, however, presumably due to extensive genetic redundancy. However, recent molecular, genetic, biochemical, and genomic studies have implicated SAURs in the regulation of a wide range of cellular, physiological, and developmental processes. Recently, crucial mechanistic insight into SAUR function was provided by the demonstration that SAURs inhibit PP2C.D phosphatases to activate plasma membrane (PM) H(+)-ATPases and promote cell expansion. In addition to auxin, several other hormones and environmental factors also regulate SAUR gene expression. We propose that SAURs are key effector outputs of hormonal and environmental signals that regulate plant growth and development. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

  10. SAUR Proteins as Effectors of Hormonal and Environmental Signals in Plant Growth

    PubMed Central

    Ren, Hong; Gray, William M.

    2016-01-01

    The plant hormone auxin regulates numerous aspects of plant growth and development. Early auxin response genes mediate its genomic effects on plant growth and development. Discovered in 1987, SMALL AUXIN UP RNAs (SAURs) are the largest family of early auxin response genes. SAUR functions have remained elusive, however, presumably due to extensive genetic redundancy. However, recent molecular, genetic, biochemical, and genomic studies have implicated SAURs in the regulation of a wide range of cellular, physiological, and developmental processes. Recently, crucial mechanistic insight into SAUR function was provided by the demonstration that SAURs inhibit PP2C.D phosphatases to activate plasma membrane (PM) H+-ATPases and promote cell expansion. In addition to auxin, several other hormones and environmental factors also regulate SAUR gene expression. We propose that SAURs are key effector outputs of hormonal and environmental signals that regulate plant growth and development. PMID:25983207

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  12. The anoikis effector Bit1 displays tumor suppressive function in lung cancer cells.

    PubMed

    Yao, Xin; Jennings, Scott; Ireland, Shubha Kale; Pham, Tri; Temple, Brandi; Davis, Mya; Chen, Renwei; Davenport, Ian; Biliran, Hector

    2014-01-01

    The mitochondrial Bit1 (Bcl-2 inhibitor of transcription 1) protein is a part of an apoptotic pathway that is uniquely regulated by integrin-mediated attachment. As an anoikis effector, Bit1 is released into the cytoplasm following loss of cell attachment and induces a caspase-independent form of apoptosis. Considering that anoikis resistance is a critical determinant of transformation, we hypothesized that cancer cells may circumvent the Bit1 apoptotic pathway to attain anchorage-independence and tumorigenic potential. Here, we provide the first evidence of the tumor suppressive effect of Bit1 through a mechanism involving anoikis induction in human lung adenocarcinoma derived A549 cells. Restitution of Bit1 in anoikis resistant A549 cells is sufficient to induce detachment induced-apoptosis despite defect in caspase activation and impairs their anchorage-independent growth. Conversely, stable downregulation of Bit1 in these cells significantly enhances their anoikis resistance and anchorage-independent growth. The Bit1 knockdown cells exhibit significantly enhanced tumorigenecity in vivo. It has been previously shown that the nuclear TLE1 corepressor is a putative oncogene in lung cancer, and we show here that TLE1 blocks Bit1 mediated anoikis in part by sequestering the pro-apoptotic partner of Bit1, the Amino-terminal Enhancer of Split (AES) protein, in the nucleus. Taken together, these findings suggest a tumor suppressive role of the caspase-independent anoikis effector Bit1 in lung cancer. Consistent with its role as a tumor suppressor, we have found that Bit1 is downregulated in human non-small cell lung cancer (NSCLC) tissues.

  13. Highly individual patterns of virus-immune IgG effector responses in humans.

    PubMed

    Corrales-Aguilar, Eugenia; Trilling, Mirko; Reinhard, Henrike; Falcone, Valeria; Zimmermann, Albert; Adams, Ortwin; Santibanez, Sabine; Hengel, Hartmut

    2016-10-01

    IgG responses are fundamental to adaptive immunity and document immunological memory of previous pathogen encounter. While specific antigen recognition is mediated by the variable F(ab')2 domain of IgG, various effector functions become activated via the constant Fcγ part bridging IgG-opsonized targets to FcγR-expressing immune effector cells. Traditionally, neutralizing IgG is considered the most appropriate correlate of protective humoral immunity to viruses. However, evidence is increasing that antiviral IgG mediates protection to viruses via activation of FcγRs. Using a test system allowing quantitative detection of virus-immune IgG able to activate FcγRs, sera of healthy individuals and vaccinees were assessed with regard to two prototypical human pathogenic viruses: measles and human cytomegalovirus. Marked differences in the capacity of individuals to generate FcγRI-, FcγRII- and FcγRIII-activating responses were noted. Comparison of FcγR-activating IgG with neutralizing and ELISA IgG concentrations did not correlate for HCMV and only very poorly for MV. Since neither neutralizing IgG nor overall IgG responses faithfully predict the activation of FcγRs, only the simultaneous quantification of IgGs activating defined FcγRs will aid to delineate individual "immunograms" of virus IgG immunity. Such new multiparametric assessment of antiviral IgG qualities could be instrumental in defining correlates of protection and disease progression.

  14. The Secreted Effector Protein EspZ Is Essential for Virulence of Rabbit Enteropathogenic Escherichia coli

    PubMed Central

    Wilbur, John Scott; Byrd, Wyatt; Ramamurthy, Shylaja; Ledvina, Hannah E.; Khirfan, Khaldoon; Riggs, Michael W.; Boedeker, Edgar C.; Vedantam, Gayatri

    2015-01-01

    Attaching and effacing (A/E) pathogens adhere intimately to intestinal enterocytes and efface brush border microvilli. A key virulence strategy of A/E pathogens is the type III secretion system (T3SS)-mediated delivery of effector proteins into host cells. The secreted protein EspZ is postulated to promote enterocyte survival by regulating the T3SS and/or by modulating epithelial signaling pathways. To explore the role of EspZ in A/E pathogen virulence, we generated an isogenic espZ deletion strain (ΔespZ) and corresponding cis-complemented derivatives of rabbit enteropathogenic Escherichia coli and compared their abilities to regulate the T3SS and influence host cell survival in vitro. For virulence studies, rabbits infected with these strains were monitored for bacterial colonization, clinical signs, and intestinal tissue alterations. Consistent with data from previous reports, espZ-transfected epithelial cells were refractory to infection-dependent effector translocation. Also, the ΔespZ strain induced greater host cell death than did the parent and complemented strains. In rabbit infections, fecal ΔespZ strain levels were 10-fold lower than those of the parent strain at 1 day postinfection, while the complemented strain was recovered at intermediate levels. In contrast to the parent and complemented mutants, ΔespZ mutant fecal carriage progressively decreased on subsequent days. ΔespZ mutant-infected animals gained weight steadily over the infection period, failed to show characteristic disease symptoms, and displayed minimal infection-induced histological alterations. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining of intestinal sections revealed increased epithelial cell apoptosis on day 1 after infection with the ΔespZ strain compared to animals infected with the parent or complemented strains. Thus, EspZ-dependent host cell cytoprotection likely prevents epithelial cell death and sloughing and thereby

  15. Opening the Ralstonia solanacearum type III effector tool box: insights into host cell subversion mechanisms.

    PubMed

    Deslandes, Laurent; Genin, Stephane

    2014-08-01

    Effectors delivered to host cells by the Type III secretion system are essential to Ralstonia solanacearum pathogenicity, as in several other plant pathogenic bacteria. The establishment of exhaustive effector repertoires in multiple R. solanacearum strains drew a first picture of the evolutionary dynamics of the pathogen effector suites. Effector repertoires are diversified, with a core of 20-30 effectors present in most of the strains and the obtention of mutants lacking one or more effector genes revealed the functional overlap among this effector network. Recent functional studies have provided insights into the ability of single effectors to manipulate the host proteasome, elicit cell death, trigger the expression of plant genes, and/or display biochemical activities on plant protein targets. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Regulatory T Cells from Colon Cancer Patients Inhibit Effector T-cell Migration through an Adenosine-Dependent Mechanism.

    PubMed

    Sundström, Patrik; Stenstad, Hanna; Langenes, Veronica; Ahlmanner, Filip; Theander, Lisa; Ndah, Tapuka Gordon; Fredin, Kamilla; Börjesson, Lars; Gustavsson, Bengt; Bastid, Jérémy; Quiding-Järbrink, Marianne

    2016-03-01

    T cell-mediated immunity is a major component of antitumor immunity. In order to be efficient, effector T cells must leave the circulation and enter into the tumor tissue. Regulatory T cells (Treg) from gastric cancer patients, but not from healthy volunteers, potently inhibit migration of conventional T cells through activated endothelium. In this study, we compared T cells from colon cancer patients and healthy donors to determine the mechanisms used by Tregs from cancer patients to inhibit conventional T-cell migration. Our results showed that circulating Tregs from cancer patients expressed high levels of CD39, an ectoenzyme mediating hydrolysis of ATP to AMP, as a rate-determining first step in the generation of immunosuppressive adenosine. Tumor-associated Tregs expressed even more CD39, and we therefore examined the importance of adenosine in Treg-mediated inhibition of T-cell transendothelial migration in vitro. Exogenous adenosine significantly reduced migration of conventional T cells from healthy volunteers, and blocking either adenosine receptors or CD39 enzymatic activity during transmigration restored the ability of conventional T cells from cancer patients to migrate. Adenosine did not directly affect T cells or endothelial cells, but reduced the ability of monocytes to activate the endothelium. Taken together, our results indicate that Treg-derived adenosine acts on monocytes and contributes to reduced transendothelial migration of effector T cells into tumors. This effect of Tregs is specific for cancer patients, and our results indicate that Tregs may affect not only T-cell effector functions but also their migration into tumors.

  17. Early effector cells survive the contraction phase in malaria infection and generate both central and effector memory T cells.

    PubMed

    Opata, Michael M; Carpio, Victor H; Ibitokou, Samad A; Dillon, Brian E; Obiero, Joshua M; Stephens, Robin

    2015-06-01

    CD4 T cells orchestrate immunity against blood-stage malaria. However, a major challenge in designing vaccines to the disease is poor understanding of the requirements for the generation of protective memory T cells (Tmem) from responding effector T cells (Teff) in chronic parasite infection. In this study, we use a transgenic mouse model with T cells specific for the merozoite surface protein (MSP)-1 of Plasmodium chabaudi to show that activated T cells generate three distinct Teff subsets with progressive activation phenotypes. The earliest observed Teff subsets (CD127(-)CD62L(hi)CD27(+)) are less divided than CD62L(lo) Teff and express memory genes. Intermediate (CD62L(lo)CD27(+)) effector subsets include the most multicytokine-producing T cells, whereas fully activated (CD62L(lo)CD27(-)) late effector cells have a terminal Teff phenotype (PD-1(+), Fas(hi), AnnexinV(+)). We show that although IL-2 promotes expansion, it actually slows terminal effector differentiation. Using adoptive transfer, we show that only early Teff survive the contraction phase and generate the terminal late Teff subsets, whereas in uninfected recipients, they become both central and effector Tmem. Furthermore, we show that progression toward full Teff activation is promoted by increased duration of infection, which in the long-term promotes Tem differentiation. Therefore, we have defined markers of progressive activation of CD4 Teff at the peak of malaria infection, including a subset that survives the contraction phase to make Tmem, and show that Ag and cytokine levels during CD4 T cell expansion influence the proportion of activated cells that can survive contraction and generate memory in malaria infection.

  18. Planar cell polarity effector gene Intu regulates cell fate-specific differentiation of keratinocytes through the primary cilia.

    PubMed

    Dai, D; Li, L; Huebner, A; Zeng, H; Guevara, E; Claypool, D J; Liu, A; Chen, J

    2013-01-01

    Genes involved in the planar cell polarity (PCP) signaling pathway are essential for a number of developmental processes in mammals, such as convergent extension and ciliogenesis. Tissue-specific PCP effector genes of the PCP signaling pathway are believed to mediate PCP signals in a tissue- and cell type-specific manner. However, how PCP signaling controls the morphogenesis of mammalian tissues remains unclear. In this study, we investigated the role of inturned (Intu), a tissue-specific PCP effector gene, during hair follicle formation in mice. Tissue-specific disruption of Intu in embryonic epidermis resulted in hair follicle morphogenesis arrest because of the failure of follicular keratinocyte to differentiate. Targeting Intu in the epidermis resulted in almost complete loss of primary cilia in epidermal and follicular keratinocytes, and a suppressed hedgehog signaling pathway. Surprisingly, the epidermal stratification and differentiation programs and barrier function were not affected. These results demonstrate that tissue-specific PCP effector genes of the PCP signaling pathway control the differentiation of keratinocytes through the primary cilia in a cell fate- and context-dependent manner, which may be critical in orchestrating the propagation and interpretation of polarity signals established by the core PCP components.

  19. Generation of effector T cells in Hymenolepis nana-infected, FK-506-treated BALB/c mice.

    PubMed

    Asano, K; Matsuo, S; Okamoto, K

    1995-05-01

    FK-506 administered into mice daily at a dose of 10.0 mg/kg (but not 1.0 and 5.0 mg/kg) caused suppression of protective immunity to Hymenolepis nana, when the agent was injected intraperitoneally during the induction phase of protective immunity. Daily administration of 10.0 mg/kg FK-506, during the course of larval development from challenge, also suppressed protective immunity. Inhibition of protective immunity was only observed in mice that received FK-506 for 6 days at a daily dose of 10.0 mg/kg and were challenged 24 h after the final FK-506 injection. FK-506 did not inhibit formation of effector cells that mediate delayed-type hypersensitivity (DTH) to H. nana egg antigen when the agent was administered intraperitoneally at a dose of 10.0 mg/kg/day for 6 days before cell preparation. However, FK-506 did inhibit DTH effector cell activation when cells prepared from infected, saline-injected mice were transferred into 10.0 mg/kg FK-506-treated recipient mice. These results strongly indicate that FK-506 cannot inhibit the generation of effector cells but will suppress their function in vivo.

  20. Focusing and sustaining the antitumor CTL effector killer response by agonist anti-CD137 mAb

    PubMed Central

    Weigelin, Bettina; Bolaños, Elixabet; Teijeira, Alvaro; Martinez-Forero, Ivan; Labiano, Sara; Azpilikueta, Arantza; Morales-Kastresana, Aizea; Quetglas, José I.; Wagena, Esther; Sánchez-Paulete, Alfonso Rodríguez; Chen, Lieping; Friedl, Peter; Melero, Ignacio

    2015-01-01

    Cancer immunotherapy is undergoing significant progress due to recent clinical successes by refined adoptive T-cell transfer and immunostimulatory monoclonal Ab (mAbs). B16F10-derived OVA-expressing mouse melanomas resist curative immunotherapy with either adoptive transfer of activated anti-OVA OT1 CTLs or agonist anti-CD137 (4-1BB) mAb. However, when acting in synergistic combination, these treatments consistently achieve tumor eradication. Tumor-infiltrating lymphocytes that accomplish tumor rejection exhibit enhanced effector functions in both transferred OT-1 and endogenous cytotoxic T lymphocytes (CTLs). This is consistent with higher levels of expression of eomesodermin in transferred and endogenous CTLs and with intravital live-cell two-photon microscopy evidence for more efficacious CTL-mediated tumor cell killing. Anti-CD137 mAb treatment resulted in prolonged intratumor persistence of the OT1 CTL-effector cells and improved function with focused and confined interaction kinetics of OT-1 CTL with target cells and increased apoptosis induction lasting up to six days postadoptive transfer. The synergy of adoptive T-cell therapy and agonist anti-CD137 mAb thus results from in vivo enhancement and sustainment of effector functions. PMID:26034288

  1. CD161 DEFINES EFFECTOR T CELLS THAT EXPRESS LIGHT AND RESPOND TO TL1A-DR3 SIGNALING.

    PubMed

    Cohavy, O; Shih, D Q; Doherty, T M; Ware, C F; Targan, S R

    2011-03-01

    Expression of NK cell markers identifies pro-inflammatory T cell subsets in the liver and intestinal immune compartments. Specifically, CD161 is expressed on Th17 cells which play an important role in the regulation of mucosal inflammation. In this study, we characterized human peripheral blood CD161+ T cells as an effector population partially resembling a gut T cell phenotype. CD161+ CD4+ T cells express the gut-associated TNF family member, LIGHT, and respond to crosslinking of DR3, a receptor to another gut-associated cytokine, TL1A. Robust IFN-γ production in response to DR3 signaling correlated with enhanced expression of surface DR3 on CD161+ T cells and co-stimulation with IL12 and IL18. CD161+ T cell effector function was directly demonstrated by activation of responder monocytes in co-culture leading to CD40 upregulation and CD14 downregulation. CD161+ T cells reciprocally responded to activated monocytes, inducing expression of activation marker, CD69, and production of IL2 and IFN-γ, further demonstrating effective CD161+ T cell cross-talk with monocytes. Finally, CD161 defined a subset of T cells that co-express CD56, a second NK marker. Our findings implicate human CD161+ T cells in gut-associated signaling mechanisms, and suggest a monocyte mediated effector function in mucosal inflammation.

  2. CD161 defines effector T cells that express light and respond to TL1A-DR3 signaling

    PubMed Central

    Cohavy, O.; Shih, D. Q.; Doherty, T. M.; Ware, C. F.

    2011-01-01

    Expression of NK cell markers identifies pro-inflammatory T cell subsets in the liver and intestinal immune compartments. Specifically, CD161 is expressed on Th17 cells which play an important role in the regulation of mucosal inflammation. In this study, we characterized human peripheral blood CD161+ T cells as an effector population partially resembling a gut T cell phenotype. CD161+ CD4+ T cells express the gut-associated TNF family member, LIGHT, and respond to crosslinking of DR3, a receptor to another gut-associated cytokine, TL1A. Robust IFN-γ production in response to DR3 signaling correlated with enhanced expression of surface DR3 on CD161+ T cells and co-stimulation with IL12 and IL18. CD161+ T cell effector function was directly demonstrated by activation of responder monocytes in co-culture leading to CD40 upregulation and CD14 downregulation. CD161+ T cells reciprocally responded to activated monocytes, inducing expression of activation marker, CD69, and production of IL2 and IFN-γ, further demonstrating effective CD161+ T cell cross-talk with monocytes. Finally, CD161 defined a subset of T cells that co-express CD56, a second NK marker. Our findings implicate human CD161+ T cells in gut-associated signaling mechanisms, and suggest a monocyte mediated effector function in mucosal inflammation. PMID:22348196

  3. Distinct regions of the Phytophthora essential effector Avh238 determine its function in cell death activation and plant immunity suppression.

    PubMed

    Yang, Bo; Wang, Qunqing; Jing, Maofeng; Guo, Baodian; Wu, Jiawei; Wang, Haonan; Wang, Yang; Lin, Long; Wang, Yan; Ye, Wenwu; Dong, Suomeng; Wang, Yuanchao

    2017-04-01

    Phytophthora pathogens secrete effectors to manipulate host innate immunity, thus facilitating infection. Among the RXLR effectors highly induced during Phytophthora sojae infection, Avh238 not only contributes to pathogen virulence but also triggers plant cell death. However, the detailed molecular basis of Avh238 functions remains largely unknown. We mapped the regions responsible for Avh238 functions in pathogen virulence and plant cell death induction using a strategy that combines investigation of natural variation and large-scale mutagenesis assays. The correlation between cellular localization and Avh238 functions was also evaluated. We found that the 79(th) residue (histidine or leucine) of Avh238 determined its cell death-inducing activity, and that the 53 amino acids in its C-terminal region are responsible for promoting Phytophthora infection. Transient expression of Avh238 in Nicotiana benthamiana revealed that nuclear localization is essential for triggering cell death, while Avh238-mediated suppression of INF1-triggered cell death requires cytoplasmic localization. Our results demonstrate that a representative example of an essential Phytophthora RXLR effector can evolve to escape recognition by the host by mutating one nucleotide site, and can also retain plant immunosuppressive activity to enhance pathogen virulence in planta.

  4. The neuro-immunological interface in an evolutionary perspective: the dynamic relationship between effector and recognition systems.

    PubMed

    Ottaviani, E; Valensin, S; Franceschi, C

    1998-04-16

    The evolutionary perspective indicates that an immune-neuroendocrine effector system integrating innate immunity, stress and inflammation is present in invertebrates. This defense network, centered on the macrophage and exerting primitive and highly promiscuous recognition units, is very effective, ancestral and appears to have been conserved throughout evolution from invertebrates to higher vertebrates. It would seem that there was a "big bang" in the recognition system of lower vertebrates, and T and B cell repertoires, MHC and antibodies suddenly appeared. We argue that this phenomenon is the counterpart of the increasing complexity of the internal circuitry and recognition units in the effector system. The immediate consequences were a progressive enlargement of the pathogen repertoire and new problems regarding self/not-self discrimination. Probably not by chance, a new organ appeared, capable of purging cells able of excessive self recognition. This organ, the thymus, appears to be the result of a well known evolutionary strategy of re-using pre-existing material (neuroendocrine cells and mediators constituting the thymic microenvironment). This bricolage at an organ level is similar to the effect we have already described at the level of molecules and functions of the defense network, and has a general counterpart at genetic level. Thus, in vertebrates, the conserved immune-neuroendocrine effector system remains of fundamental importance in defense against pathogens, while its efficiency has increased through synergy with the new, clonotipical recognition repertoire.

  5. Old Mice Accumulate Activated Effector CD4 T Cells Refractory to Regulatory T Cell-Induced Immunosuppression

    PubMed Central

    Harpaz, Idan; Bhattacharya, Udayan; Elyahu, Yehezqel; Strominger, Itai; Monsonego, Alon

    2017-01-01

    Chronic low-grade inflammation and reduced lymphocyte potency are implicated in the pathogenesis of major illnesses associated with aging. Whether this immune phenotype results from a loss of cell-mediated regulation or intrinsic dysregulated function of effector T cells (Teffs) requires further research. Here, we report that, as compared with young C57BL6 mice, old mice show an increased frequency of CD4+CD62L− Teffs with a dysregulated activated phenotype and markedly increased effector functions. Analysis of the frequency and suppressive function of CD4+FoxP3+ regulatory T cells (Tregs) indicates an increase in the frequency of FoxP3+ T cells with aging which, however, occurs within the CD4+CD25− T cells. Furthermore, whereas Tregs from young and old mice similarly suppress Teffs from young mice, both have a compromised suppressive capacity of Teffs from old mice, a phenomenon which is partially recovered in the presence of IL-2-producing CD4+CD62L+ non-Teffs. Finally, we observed that Teff subsets from old mice are enriched with IL-17A-producing T cells and exhibit intrinsically dysregulated expression of genes encoding cell-surface molecules and transcription factors, which play a key role in T-cell activation and regulation. We, thus, demonstrate an age-related impairment in the regulation of effector CD4 T cells, which may underlie the higher risk for destructive inflammation associated with aging. PMID:28382033

  6. Developmentally determined reduction in CD31 during gestation is associated with CD8+ T cell effector differentiation in preterm infants.

    PubMed

    Scheible, Kristin M; Emo, Jason; Yang, Hongmei; Holden-Wiltse, Jeanne; Straw, Andrew; Huyck, Heidie; Misra, Sara; Topham, David J; Ryan, Rita M; Reynolds, Anne Marie; Mariani, Thomas J; Pryhuber, Gloria S

    2015-12-01

    Homeostatic T cell proliferation is more robust during human fetal development. In order to understand the relative effect of normal fetal homeostasis and perinatal exposures on CD8+ T cell behavior in PT infants, we characterized umbilical cord blood CD8+ T cells from infants born between 23-42weeks gestation. Subjects were recruited as part of the NHLBI-sponsored Prematurity and Respiratory Outcomes Program. Cord blood from PT infants had fewer naïve CD8+ T cells and lower regulatory CD31 expression on both naïve and effector, independent of prenatal exposures. CD8+ T cell in vitro effector function was greater at younger gestational ages, an effect that was exaggerated in infants with prior inflammatory exposures. These results suggest that CD8+ T cells earlier in gestation have loss of regulatory co-receptor CD31 and greater effector differentiation, which may place PT neonates at unique risk for CD8+ T cell-mediated inflammation and impaired T cell memory formation. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Developmentally determined reduction in CD31 during gestation is associated with CD8+ T cell effector differentiation in preterm infants

    PubMed Central

    Scheible, Kristin M.; Emo, Jason; Yang, Hongmei; Holden-Wiltse, Jeanne; Straw, Andrew; Huyck, Heidie; Misra, Sara; Topham, David J.; Ryan, Rita M.; Reynolds, Anne Marie; Mariani, Thomas J.; Pryhuber, Gloria S.

    2015-01-01

    Homeostatic T cell proliferation is more robust during human fetal development. In order to understand the relative effect of normal fetal homeostasis and perinatal exposures on CD8+ T cell behavior in PT infants, we characterized umbilical cord blood CD8+ T cells from infants born between 23–42 weeks gestation. Subjects were recruited as part of the NHLBI-sponsored Prematurity and Respiratory Outcomes Program. Cord blood from PT infants had fewer naïve CD8+ T cells and lower regulatory CD31 expression on both naïve and effector, independent of prenatal exposures. CD8+ T cell in vitro effector function was greater at younger gestational ages, an effect that was exaggerated in infants with prior inflammatory exposures. These results suggest that CD8+ T cells earlier in gestation have loss of regulatory co-receptor CD31 and greater effector differentiation, which may place PT neonates at unique risk for CD8+ T cell-mediated inflammation and impaired T cell memory formation. PMID:26232733

  8. Old Mice Accumulate Activated Effector CD4 T Cells Refractory to Regulatory T Cell-Induced Immunosuppression.

    PubMed

    Harpaz, Idan; Bhattacharya, Udayan; Elyahu, Yehezqel; Strominger, Itai; Monsonego, Alon

    2017-01-01

    Chronic low-grade inflammation and reduced lymphocyte potency are implicated in the pathogenesis of major illnesses associated with aging. Whether this immune phenotype results from a loss of cell-mediated regulation or intrinsic dysregulated function of effector T cells (Teffs) requires further research. Here, we report that, as compared with young C57BL6 mice, old mice show an increased frequency of CD4+CD62L- Teffs with a dysregulated activated phenotype and markedly increased effector functions. Analysis of the frequency and suppressive function of CD4+FoxP3+ regulatory T cells (Tregs) indicates an increase in the frequency of FoxP3+ T cells with aging which, however, occurs within the CD4+CD25- T cells. Furthermore, whereas Tregs from young and old mice similarly suppress Teffs from young mice, both have a compromised suppressive capacity of Teffs from old mice, a phenomenon which is partially recovered in the presence of IL-2-producing CD4+CD62L+ non-Teffs. Finally, we observed that Teff subsets from old mice are enriched with IL-17A-producing T cells and exhibit intrinsically dysregulated expression of genes encoding cell-surface molecules and transcription factors, which play a key role in T-cell activation and regulation. We, thus, demonstrate an age-related impairment in the regulation of effector CD4 T cells, which may underlie the higher risk for destructive inflammation associated with aging.

  9. Structural basis for the recognition and degradation of host TRIM proteins by Salmonella effector SopA

    PubMed Central

    Fiskin, Evgenij; Bhogaraju, Sagar; Herhaus, Lina; Kalayil, Sissy; Hahn, Marcel; Dikic, Ivan

    2017-01-01

    The hallmark of Salmonella Typhimurium infection is an acute intestinal inflammatory response, which is mediated through the action of secreted bacterial effector proteins. The pro-inflammatory Salmonella effector SopA is a HECT-like E3 ligase, which was previously proposed to activate host RING ligases TRIM56 and TRIM65. Here we elucidate an inhibitory mechanism of TRIM56 and TRIM65 targeting by SopA. We present the crystal structure of SopA in complex with the RING domain of human TRIM56, revealing the atomic details of their interaction and the basis for SopA selectivity towards TRIM56 and TRIM65. Structure-guided biochemical analysis shows that SopA inhibits TRIM56 E3 ligase activity by occluding the E2-interacting surface of TRIM56. We further demonstrate that SopA ubiquitinates TRIM56 and TRIM65, resulting in their proteasomal degradation during infection. Our results provide the basis for how a bacterial HECT ligase blocks host RING ligases and exemplifies the multivalent power of bacterial effectors during infection. PMID:28084320

  10. Metalloproteases regulate T-cell proliferation and effector function via LAG-3

    PubMed Central

    Li, Nianyu; Wang, Yao; Forbes, Karen; Vignali, Kate M; Heale, Bret S; Saftig, Paul; Hartmann, Dieter; Black, Roy A; Rossi, John J; Blobel, Carl P; Dempsey, Peter J; Workman, Creg J; Vignali, Dario A A

    2007-01-01

    Tight control of T-cell proliferation and effector function is essential to ensure an effective but appropriate immune response. Here, we reveal that this is controlled by the metalloprotease-mediated cleavage of LAG-3, a negative regulatory protein expressed by all activated T cells. We show that LAG-3 cleavage is mediated by two transmembrane metalloproteases, ADAM10 and ADAM17, with the activity of both modulated by two distinct T-cell receptor (TCR) signaling-dependent mechanisms. ADAM10 mediates constitutive LAG-3 cleavage but increases ∼12-fold following T-cell activation, whereas LAG-3 shedding by ADAM17 is induced by TCR signaling in a PKCθ-dependent manner. LAG-3 must be cleaved from the cell surface to allow for normal T-cell activation as noncleavable LAG-3 mutants prevented proliferation and cytokine production. Lastly, ADAM10 knockdown reduced wild-type but not LAG-3−/− T-cell proliferation. These data demonstrate that LAG-3 must be cleaved to allow efficient T-cell proliferation and cytokine production and establish a novel paradigm in which T-cell expansion and function are regulated by metalloprotease cleavage with LAG-3 as its sole molecular target. PMID:17245433

  11. Diabetes-associated cardiac fibrosis: cellular effectors, molecular mechanisms and therapeutic opportunities

    PubMed Central

    Russo, Ilaria; Frangogiannis, Nikolaos G

    2015-01-01

    Both type 1 and type 2 diabetes are associated with cardiac fibrosis that may reduce myocardial compliance, contribute to the pathogenesis of heart failure, and trigger arrhythmic events. Diabetes-associated fibrosis is mediated by activated cardiac fibroblasts, but may also involve fibrogenic actions of macrophages, cardiomyocytes and vascular cells. The molecular basis responsible for cardiac fibrosis in diabetes remains poorly understood. Hyperglycemia directly activates a fibrogenic program, leading to accumulation of advanced glycation end-products (AGEs) that crosslink extracellular matrix proteins, and transduce fibrogenic signals through reactive oxygen species generation, or through activation of Receptor for AGEs (RAGE)-mediated pathways. Pro-inflammatory cytokines and chemokines may recruit fibrogenic leukocyte subsets in the cardiac interstitium. Activation of transforming growth factor-β/Smad signaling may activate fibroblasts inducing deposition of structural extracellular matrix proteins and matricellular macromolecules. Adipokines, endothelin-1 and the renin-angiotensin-aldosterone system have also been implicated in the diabetic myocardium. This manuscript reviews our current understanding of the cellular effectors and molecular pathways that mediate fibrosis in diabetes. Based on the pathophysiologic mechanism, we propose therapeutic interventions that may attenuate the diabetes-associated fibrotic response and discuss the challenges that may hamper clinical translation. PMID:26705059

  12. Measurement of Effector Protein Translocation Using Phosphorylatable Epitope Tags and Phospho-Specific Antibodies.

    PubMed

    Bartra, Sara Schesser; Plano, Gregory V

    2017-01-01

    Numerous bacterial pathogens employ specialized protein secretion machineries to directly inject anti-host proteins, termed effector proteins, into eukaryotic cells. Effector proteins carrying small phosphorylatable tags can be used to detect and quantify effector protein injection. Here, we describe the use of the ELK- and GSK-tags to detect the translocation of the Y. pestis YopE effector protein into RAW 264.7 macrophage-like cells using immunoblot analysis with phospho-specific antibodies.

  13. End-Effector Development for the PIP Puck Handling Robot

    SciTech Connect

    Fowley, M.D.

    2001-01-31

    It has been decided that excess, weapons-grade plutonium shall be immobilized to prevent nuclear proliferation. The method of immobilization is to encapsulate the plutonium in a ceramic puck, roughly the size of a hockey puck, using a sintering process. This method has been officially identified as the Plutonium Immobilization Process (PIP). A Can-in-Canister storage method will be used to further immobilize the plutonium. The Can-in-Canister method uses the existing design of a Defense Waste Processing Facility (DWPF) canister to house the plutonium pucks. the process begins with several pucks being stacked in a stainless steel can. Several of the stainless steel cans are stacked in a cage-like magazine. Several of the magazines are then placed in a DWPF canister. The DWPF canister is then filled with molten glass containing high-level, radioactive waste from the DWPF vitrification process. The Can-in-Canister method makes reclamation of plutonium from the pucks technically difficult and highly undesirable. The mechanical requirements of the Can-in-Canister process, in conjunction with the amount of time required to immobilize the vast quantities of weapons-grade plutonium, will expose personnel to unnecessarily high levels of radiation if the processes were completed manually, in glove boxes. Therefore, automated equipment is designed into the process to reduce or eliminate personnel exposure. Robots are used whenever the automated handling operations become complicated. There are two such operations in the initial stages of the Can-in-Canister process, which required a six-axis robot. The first operation is a press unloading process. The second operation is a tray transfer process. To successfully accomplish the operational tasks described in the two operations, the end-effector of the robot must be versatile, lightweight, and rugged. As a result of these demands, an extensive development process was undertaken to design the optimum end-effector for these puck

  14. End-Effector Development for the PIP Puck Handling Robot

    SciTech Connect

    Fowley, M.D.

    2001-01-03

    It has been decided that excess, weapons-grade plutonium shall be immobilized to prevent nuclear proliferation. The method of immobilization is to encapsulate the plutonium in a ceramic puck, roughly the size of a hockey puck, using a sintering process. This method has been officially identified as the Plutonium Immobilization Process (PIP). A Can-in-Canister storage method will be used to further immobilize the plutonium. The Can-in-Canister method uses the existing design of a Defense Waste Processing Facility (DWPF) canister to house the plutonium pucks. the process begins with several pucks being stacked in a stainless steel can. Several of the stainless steel cans are stacked in a cage-like magazine. Several of the magazines are then placed in a DWPF canister. The DWPF canister is then filled with molten glass containing high-level, radioactive waste from the DWPF vitrification process. The Can-in-Canister method makes reclamation of plutonium from the pucks technically difficult and highly undesirable. The mechanical requirements of the Can-in-Canister process, in conjunction with the amount of time required to immobilize the vast quantities of weapons-grade plutonium, will expose personnel to unnecessarily high levels of radiation if the processes were completed manually, in glove boxes. Therefore, automated equipment is designed into the process to reduce or eliminate personnel exposure. Robots are used whenever the automated handling operations become complicated. There are two such operations in the initial stages of the Can-in-Canister process, which required a six-axis robot. The first operation is a press unloading process. The second operation is a tray transfer process. To successfully accomplish the operational tasks described in the two operations, the end-effector of the robot must be versatile, lightweight, and rugged. As a result of these demands, an extensive development process was undertaken to design the optimum end-effector for these puck

  15. The Salmonella effector SseJ disrupts microtubule dynamics when ectopically expressed in normal rat kidney cells

    PubMed Central

    Raines, Sally A.; Hodgkinson, Michael R.; Dowle, Adam A.

    2017-01-01

    Salmonella effector protein SseJ is secreted by Salmonella into the host cell cytoplasm where it can then modify host cell processes. Whilst host cell small GTPase RhoA has previously been shown to activate the acyl-transferase activity of SseJ we show here an un-described effect of SseJ protein production upon microtubule dynamism. SseJ prevents microtubule collapse and this is independent of SseJ’s acyl-transferase activity. We speculate that the effects of SseJ on microtubules would be mediated via its known interactions with the small GTPases of the Rho family. PMID:28235057

  16. Erwinia amylovora effector protein Eop1 suppresses PAMP-triggered immunity in Malus

    USDA-ARS?s Scientific Manuscript database

    Erwinia amylovora (Ea) utilizes a type three secretion system (T3SS) to deliver effector proteins into plant host cells. Several Ea effectors have been identified based on their sequence similarity to plant and animal bacterial pathogen effectors; however, the function of the majority of Ea effecto...

  17. Autophagy mediates the mitotic senescence transition.

    PubMed

    Young, Andrew R J; Narita, Masako; Ferreira, Manuela; Kirschner, Kristina; Sadaie, Mahito; Darot, Jeremy F J; Tavaré, Simon; Arakawa, Satoko; Shimizu, Shigeomi; Watt, Fiona M; Narita, Masashi

    2009-04-01

    As a stress response, senescence is a dynamic process involving multiple effector mechanisms whose combination determines the phenotypic quality. Here we identify autophagy as a new effector mechanism of senescence. Autophagy is activated during senescence and its activation is correlated with negative feedback in the PI3K-mammalian target of rapamycin (mTOR) pathway. A subset of autophagy-related genes are up-regulated during senescence: Overexpression of one of those genes, ULK3, induces autophagy and senescence. Furthermore, inhibition of autophagy delays the senescence phenotype, including senescence-associated secretion. Our data suggest that autophagy, and its consequent protein turnover, mediate the acquisition of the senescence phenotype.

  18. Phytoplasma protein effector SAP11 enhances insect vector reproduction by manipulating plant development and defense hormone biosynthesis.

    PubMed

    Sugio, Akiko; Kingdom, Heather N; MacLean, Allyson M; Grieve, Victoria M; Hogenhout, Saskia A

    2011-11-29

    Phytoplasmas are insect-transmitted phytopathogenic bacteria that can alter plant morphology and the longevity and reproduction rates and behavior of their insect vectors. There are various examples of animal and plant parasites that alter the host phenotype to attract insect vectors, but it is unclear how these parasites accomplish this. We hypothesized that phytoplasmas produce effectors that modulate specific targets in their hosts leading to the changes in plant development and insect performance. Previously, we sequenced and mined the genome of Aster Yellows phytoplasma strain Witches' Broom (AY-WB) and identified 56 candidate effectors. Here, we report that the secreted AY-WB protein 11 (SAP11) effector modulates plant defense responses to the advantage of the AY-WB insect vector Macrosteles quadrilineatus. SAP11 binds and destabilizes Arabidopsis CINCINNATA (CIN)-related TEOSINTE BRANCHED1, CYCLOIDEA, PROLIFERATING CELL FACTORS 1 and 2 (TCP) transcription factors, which control plant development and promote the expression of lipoxygenase (LOX) genes involved in jasmonate (JA) synthesis. Both the Arabidopsis SAP11 lines and AY-WB-infected plants produce less JA on wounding. Furthermore, the AY-WB insect vector produces more offspring on AY-WB-infected plants, SAP11 transgenic lines, and plants impaired in CIN-TCP and JA synthesis. Thus, SAP11-mediated destabilization of CIN-TCPs leads to the down-regulation of LOX2 expression and JA synthesis and an increase in M. quadrilineatus progeny. Phytoplasmas are obligate inhabitants of their plant host and insect vectors, in which the latter transmits AY-WB to a diverse range of plant species. This finding demonstrates that pathogen effectors can reach beyond the pathogen-host interface to modulate a third organism in the biological interaction.

  19. Inhibitory action of deoxyspergualin on effector/memory T cell generation during Hymenolepis nana infection in mice.

    PubMed

    Asano, K; Muramatsu, K; Ikeda, K; Okamoto, K

    1996-10-01

    The effects of deoxyspergualin (DSG), a newly developed immunosuppressive agent, on protective immunity to Hymenolepis nana reinfection were examined in BALB/c mice. Administration of DSG at daily doses of 10.0 mg/kg to 30.0 mg/kg (but not 5.0 mg/kg) caused suppression of protective immunity when the agent was injected intraperitoneally during the induction phase of the immunity. In contrast, daily administration of 30.0 mg/kg DSG, during effector phase, could not suppress protective immunity. DSG inhibited endogenous interferon-gamma production in mesenteric lymph nodes induced by H. nana challenge infection, when the agent was injected intraperitoneally at a daily dose of 10.0 mg/kg during the induction phase of immunity. Delayed type hypersensitivity (DTH) local transfer analysis revealed that administration of DSG at 10.0 mg/kg/day into donor mice during induction phase of immunity inhibited generation of effector/memory cells that mediate DTH to H. nana egg antigen. However, DSG could not inhibit DTH effector cell activation when cells prepared from H. nana-infected, saline-injected mice were transferred into recipient treated with 10.0 mg/kg DSG. Administration of DSG at a dose of 10.0 mg/kg daily for 5 days produced large DNA fragments in mesenteric lymph node (MLN) cells. These results strongly suggest that DSG suppresses generation of effector/memory cells by apoptotic cell death but cannot suppress lymphocyte activation in vivo.

  20. Helicases in antiviral immunity: dual properties as sensors and effectors

    PubMed Central

    Ahmad, Sadeem; Hur, Sun

    2015-01-01

    Many helicases have a unique ability to couple cognate RNA binding to ATP hydrolysis, which can induce a large conformational change that affects its interaction with RNA, position along RNA, or oligomeric state. A growing number of these helicases contribute to the innate immune system, either as sensors that detect foreign nucleic acids and/or as effectors that directly participate in the clearance of such foreign species. We here discuss a few examples, including retinoic acid-inducible gene-I (RIG-I), melanoma differentiation-associated gene 5 (MDA5) and Dicer, focusing on their dual functions as both sensors and effectors. We will also discuss the closely related, but less understood helicases, laboratory of genetics and physiology 2 (LGP2) and Dicer-related helicase-1 and -3 (DRH-1 and -3). PMID:26410598

  1. Interchangeable end effector tools utilized on the protoflight manipulator arm

    NASA Technical Reports Server (NTRS)

    1987-01-01

    A subset of teleoperator and effector tools was designed, fabricated, delivered and successfully demonstrated on the Marshall Space Flight Center (MSFC) protoflight manipulator arm (PFMA). The tools delivered included a rotary power tool with interchangeable collets and two fluid coupling mate/demate tools; one for a Fairchild coupling and the other for a Purolator coupling. An electrical interface connector was also provided for the rotary power tool. A tool set, from which the subset was selected, for performing on-orbit satellite maintenance was identified and conceptionally designed. Maintenance requirements were synthesized, evaluated and prioritized to develop design requirements for a set of end effector tools representative of those needed to provide on-orbit maintenance of satellites to be flown in the 1986 to 2000 timeframe.

  2. Interferon-inducible effector mechanisms in cell-autonomous immunity.

    PubMed

    MacMicking, John D

    2012-04-25

    Interferons (IFNs) induce the expression of hundreds of genes as part of an elaborate antimicrobial programme designed to combat infection in all nucleated cells - a process termed cell-autonomous immunity. As described in this Review, recent genomic and subgenomic analyses have begun to assign functional properties to novel IFN-inducible effector proteins that restrict bacteria, protozoa and viruses in different subcellular compartments and at different stages of the pathogen life cycle. Several newly described host defence factors also participate in canonical oxidative and autophagic pathways by spatially coordinating their activities to enhance microbial killing. Together, these IFN-induced effector networks help to confer vertebrate host resistance to a vast and complex microbial world.

  3. A robot end effector exchange mechanism for space applications

    NASA Technical Reports Server (NTRS)

    Gorin, Barney F.

    1990-01-01

    Efficient robot operation requires the use of specialized end effectors or tools for tasks. In spacecraft applications, the microgravity environment precludes the use of gravitational forces to retain the tools in holding fixture. As a result of this, a retention mechanism which forms a part of the tool storage container is required. A unique approach to this problem has resulted in the development of an end effector exchange mechanism that meets the requirements for spaceflight applications while avoiding the complexity usually involved. This mechanism uses multiple latching cams both on the manipulator and in the tool storage container, combined with a system of catch rings to provide retention in both locations and the required failure tolerance. Because of the cam configuration the mechanism operates passively, requiring no electrical commands except those needed to move the manipulator into position. Similarly, it inherently provides interlocks to prevent the release of one cam before its opposite number is engaged.

  4. Evidence for acquisition of virulence effectors in pathogenic chytrids.

    PubMed

    Sun, Guiling; Yang, Zefeng; Kosch, Tiffany; Summers, Kyle; Huang, Jinling

    2011-07-08

    The decline in amphibian populations across the world is frequently linked to the infection of the chytrid fungus Batrachochytrium dendrobatidis (Bd). This is particularly perplexing because Bd was only recently discovered in 1999 and no chytrid fungus had previously been identified as a vertebrate pathogen. In this study, we show that two large families of known virulence effector genes, crinkler (CRN) proteins and serine peptidases, were acquired by Bd from oomycete pathogens and bacteria, respectively. These two families have been duplicated after their acquisition by Bd. Additional selection analyses indicate that both families evolved under strong positive selection, suggesting that they are involved in the adaptation of Bd to its hosts. We propose that the acquisition of virulence effectors, in combination with habitat disruption and climate change, may have driven the Bd epidemics and the decline in amphibian populations. This finding provides a starting point for biochemical investigations of chytridiomycosis.

  5. TLR-exosomes exhibit distinct kinetics and effector function

    PubMed Central

    Srinivasan, Swetha; Su, Michelle; Ravishankar, Shashidhar; Moore, James; Head, PamelaSara; Dixon, J. Brandon; Vannberg, Fredrik

    2017-01-01

    The innate immune system is vital to rapidly responding to pathogens and Toll-like receptors (TLRs) are a critical component of this response. Nanovesicular exosomes play a role in immunity, but to date their exact contribution to the dissemination of the TLR response is unknown. Here we show that exosomes from TLR stimulated cells can largely recapitulate TLR activation in distal cells in vitro. We can abrogate the action-at-a-distance signaling of exosomes by UV irradiation, demonstrating that RNA is crucial for their effector function. We are the first to show that exosomes derived from poly(I:C) stimulated cells induce in vivo macrophage M1-like polarization within murine lymph nodes. These poly(I:C) exosomes demonstrate enhanced trafficking to the node and preferentially recruit neutrophils as compared to control exosomes. This work definitively establishes the differential effector function for exosomes in communicating the TLR activation state of the cell of origin. PMID:28290538

  6. Identification of novel type III effectors using latent Dirichlet allocation.

    PubMed

    Yang, Yang

    2012-01-01

    Among the six secretion systems identified in Gram-negative bacteria, the type III secretion system (T3SS) plays important roles in the disease development of pathogens. T3SS has attracted a great deal of research interests. However, the secretion mechanism has not been fully understood yet. Especially, the identification of effectors (secreted proteins) is an important and challenging task. This paper adopts machine learning methods to identify type III secreted effectors (T3SEs). We extract features from amino acid sequences and conduct feature reduction based on latent semantic information by using latent Dirichlet allocation model. The experimental results on Pseudomonas syringae data set demonstrate the good performance of the new methods.

  7. Design and Implementation of Multifunctional Automatic Drilling End Effector

    NASA Astrophysics Data System (ADS)

    Wang, Zhanxi; Qin, Xiansheng; Bai, Jing; Tan, Xiaoqun; Li, Jing

    2017-03-01

    In order to realize the automatic drilling in aircraft assembly, a drilling end effector is designed by integrating the pressure unit, drilling unit, measurement unit, control system and frame structure. In order to reduce the hole deviation, this paper proposes a vertical normal adjustment program based on 4 laser distance sensors. The actual normal direction of workpiece surface can be calculated through the sensors measurements, and then robot posture is adjusted to realize the hole deviation correction. A base detection method is proposed to detect and locate the hole automatically by using the camera and the reference hole. The experiment results show that the position accuracy of the system is less than 0.3mm, and the normal precision is less than 0.5°. The drilling end effector and robot can greatly improve the efficiency of the aircraft parts and assembly quality, and reduce the product development cycle.

  8. Functional metagenomic discovery of bacterial effectors in the human microbiome and isolation of commendamide, a GPCR G2A/132 agonist

    PubMed Central

    Cohen, Louis J.; Kang, Hahk-Soo; Chu, John; Huang, Yun-Han; Gordon, Emma A.; Reddy, Boojala Vijay B.; Ternei, Melinda A.; Craig, Jeffrey W.; Brady, Sean F.

    2015-01-01

    The trillions of bacteria that make up the human microbiome are believed to encode functions that are important to human health; however, little is known about the specific effectors that commensal bacteria use to interact with the human host. Functional metagenomics provides a systematic means of surveying commensal DNA for genes that encode effector functions. Here, we examine 3,000 Mb of metagenomic DNA cloned from three phenotypically distinct patients for effectors that activate NF-κB, a transcription factor known to play a central role in mediating responses to environmental stimuli. This screen led to the identification of 26 unique commensal bacteria effector genes (Cbegs) that are predicted to encode proteins with diverse catabolic, anabolic, and ligand-binding functions and most frequently interact with either glycans or lipids. Detailed analysis of one effector gene family (Cbeg12) recovered from all three patient libraries found that it encodes for the production of N-acyl-3-hydroxypalmitoyl-glycine (commendamide). This metabolite was also found in culture broth from the commensal bacterium Bacteroides vulgatus, which harbors a gene highly similar to Cbeg12. Commendamide resembles long-chain N-acyl-amides that function as mammalian signaling molecules through activation of G-protein–coupled receptors (GPCRs), which led us to the observation that commendamide activates the GPCR G2A/GPR132. G2A has been implicated in disease models of autoimmunity and atherosclerosis. This study shows the utility of functional metagenomics for identifying potential mechanisms used by commensal bacteria for host interactions and outlines a functional metagenomics-based pipeline for the systematic identification of diverse commensal bacteria effectors that impact host cellular functions. PMID:26283367

  9. Construction of a Plasmodium falciparum Rab-interactome identifies CK1 and PKA as Rab-effector kinases in malaria parasites

    PubMed Central

    Rached, Fathia Ben; Ndjembo-Ezougou, Carinne; Chandran, Syama; Talabani, Hana; Yera, Hélène; Dandavate, Vrushali; Bourdoncle, Pierre; Meissner, Markus; Tatu, Utpal; Langsley, Gordon

    2012-01-01

    Background information The pathology causing stages of the human malaria parasite Plasmodium falciparum reside within red blood cells that are devoid of any regulated transport system. The parasite, therefore, is entirely responsible for mediating vesicular transport within itself and in the infected erythrocyte cytoplasm, and it does so in part via its family of 11 Rab GTPases. Putative functions have been ascribed to Plasmodium Rabs due to their homology with Rabs of yeast, particularly with Saccharomyces that has an equivalent number of rab/ypt genes and where analyses of Ypt function is well characterized. Results Rabs are important regulators of vesicular traffic due to their capacity to recruit specific effectors. In order to identify P. falciparum Rab (PfRab) effectors, we first built a Ypt-interactome by exploiting genetic and physical binding data available at the Saccharomyces genome database (SGD). We then constructed a PfRab-interactome using putative parasite Rab-effectors identified by homology to Ypt-effectors. We demonstrate its potential by wet-bench testing three predictions; that casein kinase-1 (PfCK1) is a specific Rab5B interacting protein and that the catalytic subunit of cAMP-dependent protein kinase A (PfPKA-C) is a PfRab5A and PfRab7 effector. Conclusions The establishment of a shared set of physical Ypt/PfRab-effector proteins sheds light on a core set Plasmodium Rab-interactants shared with yeast. The PfRab-interactome should benefit vesicular trafficking studies in malaria parasites. The recruitment of PfCK1 to PfRab5B+ and PfPKA-C to PfRab5A+ and PfRab7+ vesicles, respectively, suggests that PfRab-recruited kinases potentially play a role in early and late endosome function in malaria parasites. PMID:22188458

  10. Xanthomonas and the TAL Effectors: Nature's Molecular Biologist.

    PubMed

    White, Frank

    2016-01-01

    Agrobacterium, due to the transfer of T-DNA to the host genome, is known as nature's genetic engineer. Once again, bacteria have led the way to newfound riches in biotechnology. Xanthomonas has emerged as nature's molecular biologist as the functional domains of the sequence-specific DNA transcription factors known as TAL effectors were characterized and associated with the cognate disease susceptibility and resistance genes of plants.