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Sample records for host cell-targeted effectors

  1. Comparative Genomics of the Zoonotic Pathogen Ehrlichia chaffeensis Reveals Candidate Type IV Effectors and Putative Host Cell Targets

    PubMed Central

    Noroy, Christophe; Meyer, Damien F.

    2017-01-01

    During infection, some intracellular pathogenic bacteria use a dedicated multiprotein complex known as the type IV secretion system to deliver type IV effector (T4E) proteins inside the host cell. These T4Es allow the bacteria to evade host defenses and to subvert host cell processes to their own advantage. Ehrlichia chaffeensis is a tick-transmitted obligate intracellular pathogenic bacterium, which causes human monocytic ehrlichiosis. Using comparative whole genome analysis, we identified the relationship between eight available E. chaffeensis genomes isolated from humans and show that these genomes are highly conserved. We identified the candidate core type IV effectome of E. chaffeensis and some conserved intracellular adaptive strategies. We assigned the West Paces strain to genetic group II and predicted the repertoires of T4Es encoded by E. chaffeensis genomes, as well as some putative host cell targets. We demonstrated that predicted T4Es are preferentially distributed in gene sparse regions of the genome. In addition to the identification of the two known type IV effectors of Anaplasmataceae, we identified two novel candidates T4Es, ECHLIB_RS02720 and ECHLIB_RS04640, which are not present in all E. chaffeensis strains and could explain some variations in inter-strain virulence. We also identified another novel candidate T4E, ECHLIB_RS02720, a hypothetical protein exhibiting EPIYA, and NLS domains as well as a classical type IV secretion signal, suggesting an important role inside the host cell. Overall, our results agree with current knowledge of Ehrlichia molecular pathogenesis, and reveal novel candidate T4Es that require experimental validation. This work demonstrates that comparative effectomics enables identification of important host pathways targeted by the bacterial pathogen. Our study, which focuses on the type IV effector repertoires among several strains of E. chaffeensis species, is an original approach and provides rational putative targets

  2. 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.

  3. 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

  4. 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

  5. An Aphid Effector Targets Trafficking Protein VPS52 in a Host-Specific Manner to Promote Virulence1[OPEN

    PubMed Central

    2017-01-01

    Plant- and animal-feeding insects secrete saliva inside their hosts, containing effectors, which may promote nutrient release and suppress immunity. Although for plant pathogenic microbes it is well established that effectors target host proteins to modulate host cell processes and promote disease, the host cell targets of herbivorous insects remain elusive. Here, we show that the existing plant pathogenic microbe effector paradigm can be extended to herbivorous insects in that effector-target interactions inside host cells modify critical host processes to promote plant susceptibility. We showed that the effector Mp1 from Myzus persicae associates with the host Vacuolar Protein Sorting Associated Protein52 (VPS52). Using natural variants, we provide a strong link between effector virulence activity and association with VPS52, and show that the association is highly specific to M. persicae-host interactions. Also, coexpression of Mp1, but not Mp1-like variants, specifically with host VPS52s resulted in effector relocalization to vesicle-like structures that associate with prevacuolar compartments. We show that high VPS52 levels negatively impact virulence, and that aphids are able to reduce VPS52 levels during infestation, indicating that VPS52 is an important virulence target. Our work is an important step forward in understanding, at the molecular level, how a major agricultural pest promotes susceptibility during infestation of crop plants. We give evidence that an herbivorous insect employs effectors that interact with host proteins as part of an effective virulence strategy, and that these effectors likely function in a species-specific manner. PMID:28100451

  6. 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.

  7. 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.

  8. 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

  9. 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

  10. 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.

  11. Identification of Novel Host Interactors of Effectors Secreted by Salmonella and Citrobacter

    PubMed Central

    Sontag, Ryan L.; Nakayasu, Ernesto S.; Brown, Roslyn N.; Niemann, George S.; Sydor, Michael A.; Sanchez, Octavio; Ansong, Charles; Lu, Shao-Yeh; Choi, Hyungwon; Valleau, Dylan; Weitz, Karl K.; Savchenko, Alexei; Cambronne, Eric D.

    2016-01-01

    ABSTRACT Many pathogenic bacteria of the family Enterobacteriaceae use type III secretion systems to inject virulence proteins, termed “effectors,” into the host cell cytosol. Although host-cellular activities of several effectors have been demonstrated, the function and host-targeted pathways of most of the effectors identified to date are largely undetermined. To gain insight into host proteins targeted by bacterial effectors, we performed coaffinity purification of host proteins from cell lysates using recombinant effectors from the Enterobacteriaceae intracellular pathogens Salmonella enterica serovar Typhimurium and Citrobacter rodentium. We identified 54 high-confidence host interactors for the Salmonella effectors GogA, GtgA, GtgE, SpvC, SrfH, SseL, SspH1, and SssB collectively and 21 interactors for the Citrobacter effectors EspT, NleA, NleG1, and NleK. We biochemically validated the interaction between the SrfH Salmonella protein and the extracellular signal-regulated kinase 2 (ERK2) host protein kinase, which revealed a role for this effector in regulating phosphorylation levels of this enzyme, which plays a central role in signal transduction. IMPORTANCE During infection, pathogenic bacteria face an adverse environment of factors driven by both cellular and humoral defense mechanisms. To help evade the immune response and ultimately proliferate inside the host, many bacteria evolved specialized secretion systems to deliver effector proteins directly into host cells. Translocated effector proteins function to subvert host defense mechanisms. Numerous pathogenic bacteria use a specialized secretion system called type III secretion to deliver effectors into the host cell cytosol. Here, we identified 75 new host targets of Salmonella and Citrobacter effectors, which will help elucidate their mechanisms of action. PMID:27822540

  12. 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.

  13. 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.

  14. At the Frontier; RXLR Effectors Crossing the Phytophthora–Host Interface

    PubMed Central

    Bouwmeester, Klaas; Meijer, Harold J. G.; Govers, Francine

    2011-01-01

    Plants are constantly beset by pathogenic organisms. To successfully infect their hosts, plant pathogens secrete effector proteins, many of which are translocated to the inside of the host cell where they manipulate normal physiological processes and undermine host defense. The way by which effectors cross the frontier to reach the inside of the host cell varies among different classes of pathogens. For oomycete plant pathogens – like the potato late blight pathogen Phytophthora infestans – it has been shown that effector translocation to the host cell cytoplasm is dependent on conserved amino acid motifs that are present in the N-terminal part of effector proteins. One of these motifs, known as the RXLR motif, has a strong resemblance with a host translocation motif found in effectors secreted by Plasmodium species. These malaria parasites, that reside inside specialized vacuoles in red blood cells, make use of a specific protein translocation complex to export effectors from the vacuole into the red blood cell. Whether or not also oomycete RXLR effectors require a translocation complex to cross the frontier is still under investigation. For one P. infestans RXLR effector named IPI-O we have found a potential host target that could play a role in establishing the first contact between this effector and the host cell. This membrane spanning lectin receptor kinase, LecRK-I.9, interacts with IPI-O via the tripeptide RGD that overlaps with the RXLR motif. In animals, RGD is a well-known cell adhesion motif; it binds to integrins, which are membrane receptors that regulate many cellular processes and which can be hijacked by pathogens for either effector translocation or pathogen entry into host cells. PMID:22645549

  15. Effectors of biotrophic fungi and oomycetes: pathogenicity factors and triggers of host resistance.

    PubMed

    Dodds, Peter N; Rafiqi, Maryam; Gan, Pamela H P; Hardham, Adrienne R; Jones, David A; Ellis, Jeffrey G

    2009-01-01

    Many biotrophic fungal and oomycete pathogens share a common infection process involving the formation of haustoria, which penetrate host cell walls and form a close association with plant membranes. Recent studies have identified a class of pathogenicity effector proteins from these pathogens that is transferred into host cells from haustoria during infection. This insight stemmed from the identification of avirulence (Avr) proteins from these pathogens that are recognized by intracellular host resistance (R) proteins. Oomycete effectors contain a conserved translocation motif that directs their uptake into host cells independently of the pathogen, and is shared with the human malaria pathogen. Genome sequence information indicates that oomycetes may express several hundred such host-translocated effectors. Elucidating the transport mechanism of fungal and oomycete effectors and their roles in disease offers new opportunities to understand how these pathogens are able to manipulate host cells to establish a parasitic relationship and to develop new disease-control measures.

  16. An effector of the Irish potato famine pathogen antagonizes a host autophagy cargo receptor

    PubMed Central

    Dagdas, Yasin F; Belhaj, Khaoula; Maqbool, Abbas; Chaparro-Garcia, Angela; Pandey, Pooja; Petre, Benjamin; Tabassum, Nadra; Cruz-Mireles, Neftaly; Hughes, Richard K; Sklenar, Jan; Win, Joe; Menke, Frank; Findlay, Kim; Banfield, Mark J; Kamoun, Sophien; Bozkurt, Tolga O

    2016-01-01

    Plants use autophagy to safeguard against infectious diseases. However, how plant pathogens interfere with autophagy-related processes is unknown. Here, we show that PexRD54, an effector from the Irish potato famine pathogen Phytophthora infestans, binds host autophagy protein ATG8CL to stimulate autophagosome formation. PexRD54 depletes the autophagy cargo receptor Joka2 out of ATG8CL complexes and interferes with Joka2's positive effect on pathogen defense. Thus, a plant pathogen effector has evolved to antagonize a host autophagy cargo receptor to counteract host defenses. DOI: http://dx.doi.org/10.7554/eLife.10856.001 PMID:26765567

  17. Legionella pneumophila type IV effectors hijack the transcription and translation machinery of the host cell.

    PubMed

    Rolando, Monica; Buchrieser, Carmen

    2014-12-01

    Intracellular bacterial pathogens modulate the host response to persist and replicate inside a eukaryotic cell and cause disease. Legionella pneumophila, the causative agent of Legionnaires' disease, is present in freshwater environments and represents one of these pathogens. During coevolution with protozoan cells, L. pneumophila has acquired highly sophisticated and diverse strategies to hijack host cell processes. It secretes hundreds of effectors into the host cell, and these manipulate host signaling pathways and key cellular processes. Recently it has been shown that L. pneumophila is also able to alter the transcription and translation machinery of the host and to exploit epigenetic mechanisms in the cells it resides in to counteract host responses.

  18. Structural Basis of Host Autophagy-related Protein 8 (ATG8) Binding by the Irish Potato Famine Pathogen Effector Protein PexRD54*♦

    PubMed Central

    Tregidgo, Nicholas; Zess, Erin; Belhaj, Khaoula

    2016-01-01

    Filamentous plant pathogens deliver effector proteins to host cells to promote infection. The Phytophthora infestans RXLR-type effector PexRD54 binds potato ATG8 via its ATG8 family-interacting motif (AIM) and perturbs host-selective autophagy. However, the structural basis of this interaction remains unknown. Here, we define the crystal structure of PexRD54, which includes a modular architecture, including five tandem repeat domains, with the AIM sequence presented at the disordered C terminus. To determine the interface between PexRD54 and ATG8, we solved the crystal structure of potato ATG8CL in complex with a peptide comprising the effector's AIM sequence, and we established a model of the full-length PexRD54-ATG8CL complex using small angle x-ray scattering. Structure-informed deletion of the PexRD54 tandem domains reveals retention of ATG8CL binding in vitro and in planta. This study offers new insights into structure/function relationships of oomycete RXLR effectors and how these proteins engage with host cell targets to promote disease. PMID:27458016

  19. Identification of Novel Host Interactors of Effectors Secreted by Salmonella and Citrobacter

    SciTech Connect

    Sontag, Ryan L.; Nakayasu, Ernesto S.; Brown, Roslyn N.; Niemann, George S.; Sydor, Michael A.; Sanchez, Octavio; Ansong, Charles; Lu, Shao-Yeh; Choi, Hyungwon; Valleau, Dylan; Weitz, Karl K.; Savchenko, Alexei; Cambronne, Eric D.; Adkins, Joshua N.; McFall-Ngai, Margaret J.

    2016-07-12

    Many pathogenic bacteria of the familyEnterobacteriaceaeuse type III secretion systems to inject virulence proteins, termed “effectors,” into the host cell cytosol. Although host-cellular activities of several effectors have been demonstrated, the function and host-targeted pathways of most of the effectors identified to date are largely undetermined. To gain insight into host proteins targeted by bacterial effectors, we performed coaffinity purification of host proteins from cell lysates using recombinant effectors from theEnterobacteriaceaeintracellular pathogensSalmonella entericaserovar Typhimurium andCitrobacter rodentium. We identified 54 high-confidence host interactors for theSalmonellaeffectors GogA, GtgA, GtgE, SpvC, SrfH, SseL, SspH1, and SssB collectively and 21 interactors for theCitrobactereffectors EspT, NleA, NleG1, and NleK. We biochemically validated the interaction between the SrfHSalmonellaprotein and the extracellular signal-regulated kinase 2 (ERK2) host protein kinase, which revealed a role for this effector in regulating phosphorylation levels of this enzyme, which plays a central role in signal transduction.

    IMPORTANCEDuring infection, pathogenic bacteria face an adverse environment of factors driven by both cellular and humoral defense mechanisms. To help evade the immune response and ultimately proliferate inside the host, many bacteria evolved specialized secretion systems to deliver effector proteins directly into host cells. Translocated effector proteins function to subvert host defense mechanisms. Numerous pathogenic bacteria use a specialized secretion system called type III secretion to deliver effectors into the host cell cytosol. Here, we identified 75 new host targets ofSalmonellaandCitrobactereffectors, which will help elucidate their mechanisms of

  20. 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.

  1. Host/pathogen interactions and immune effector mechanisms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An understanding of the host/pathogen interactions for mycobacterial infections underpins many of the outcomes required for improving disease control, including better diagnostic tests, vaccines and breeding for disease resistance. Upon infection these mycobacteria come in contact with cells of the ...

  2. Host-Induced gene silencing in barley powdery mildew reveals a class of ribonuclease-like effectors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Obligate biotrophic pathogens of plants require the ability to circumvent host defenses to enable colonization. To establish compatibility, pathogens secrete a variety of effectors, which regulate host immunity, and thus, facilitate the establishment of haustorial feeding structures. These structur...

  3. TRANSGENIC EXPRESSION OF THE ERWINIA AMYLOVORA (FIRE BLIGHT) EFFECTOR PROTEIN EOP1 SUPRESSES HOST BASAL DEFENSE MECHANISMS IN MALUS (APPLE)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Erwinia amylovora (Ea) is the causative agent of fire blight, a devastating disease of apple and pear. Like many other plant and animal bacterial pathogens Ea utilizes a type three secretion system (TTSS) to deliver effector proteins into plant host cells. Once inside the host cell, effector protei...

  4. 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.

  5. 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

  6. A translocation signal for delivery of oomycete effector proteins into host plant cells.

    PubMed

    Whisson, Stephen C; Boevink, Petra C; Moleleki, Lucy; Avrova, Anna O; Morales, Juan G; Gilroy, Eleanor M; Armstrong, Miles R; Grouffaud, Severine; van West, Pieter; Chapman, Sean; Hein, Ingo; Toth, Ian K; Pritchard, Leighton; Birch, Paul R J

    2007-11-01

    Bacterial, oomycete and fungal plant pathogens establish disease by translocation of effector proteins into host cells, where they may directly manipulate host innate immunity. In bacteria, translocation is through the type III secretion system, but analogous processes for effector delivery are uncharacterized in fungi and oomycetes. Here we report functional analyses of two motifs, RXLR and EER, present in translocated oomycete effectors. We use the Phytophthora infestans RXLR-EER-containing protein Avr3a as a reporter for translocation because it triggers RXLR-EER-independent hypersensitive cell death following recognition within plant cells that contain the R3a resistance protein. We show that Avr3a, with or without RXLR-EER motifs, is secreted from P. infestans biotrophic structures called haustoria, demonstrating that these motifs are not required for targeting to haustoria or for secretion. However, following replacement of Avr3a RXLR-EER motifs with alanine residues, singly or in combination, or with residues KMIK-DDK--representing a change that conserves physicochemical properties of the protein--P. infestans fails to deliver Avr3a or an Avr3a-GUS fusion protein into plant cells, demonstrating that these motifs are required for translocation. We show that RXLR-EER-encoding genes are transcriptionally upregulated during infection. Bioinformatic analysis identifies 425 potential genes encoding secreted RXLR-EER class proteins in the P. infestans genome. Identification of this class of proteins provides unparalleled opportunities to determine how oomycetes manipulate hosts to establish infection.

  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. Modulation of the host transcriptome by Coxiella burnetii nuclear effector Cbu1314.

    PubMed

    Weber, Mary M; Faris, Robert; McLachlan, Juanita; Tellez, Andres; Wright, William U; Galvan, Gloria; Luo, Zhao-Qing; Samuel, James E

    2016-05-01

    Coxiella burnetii is a Gram-negative, obligate intracellular pathogen that directs the formation of a parasitophorous vacuole derived from the host lysosomal network. Biogenesis and maintenance of this replicative compartment is dependent on bacterial protein synthesis and results in differential expression of specific host genes. However, the mechanisms by which the pathogen induces changes in the host transcriptome is poorly understood. In the current study we identified a Dot/Icm secreted effector, Cbu1314, which encodes two nuclear localization signals that are required for nuclear localization and association with host chromatin. Chromatin immunoprecipitation (ChIP) and deep sequencing revealed that Cbu1314 associated with host genes involved in transcription, cell signaling, and the immune response. RNA sequencing of cells overexpressing Cbu1314 demonstrated that Cbu1314 modulates the host transcriptome and these transcriptional changes required a functional nuclear localization signal. Of the differentially expressed genes, sixteen were also identified as Cbu1314 targets using ChIP sequencing. Collectively these results suggest that Cbu1314 associates with host chromatin and plays a role in modulating the host transcriptome.

  9. Multiple Candidate Effectors from the Oomycete Pathogen Hyaloperonospora arabidopsidis Suppress Host Plant Immunity

    PubMed Central

    Fabro, Georgina; Steinbrenner, Jens; Coates, Mary; Ishaque, Naveed; Baxter, Laura; Studholme, David J.; Körner, Evelyn; Allen, Rebecca L.; Piquerez, Sophie J. M.; Rougon-Cardoso, Alejandra; Greenshields, David; Lei, Rita; Badel, Jorge L.; Caillaud, Marie-Cecile; Sohn, Kee-Hoon; Van den Ackerveken, Guido; Parker, Jane E.; Beynon, Jim; Jones, Jonathan D. G.

    2011-01-01

    Oomycete pathogens cause diverse plant diseases. To successfully colonize their hosts, they deliver a suite of effector proteins that can attenuate plant defenses. In the oomycete downy mildews, effectors carry a signal peptide and an RxLR motif. Hyaloperonospora arabidopsidis (Hpa) causes downy mildew on the model plant Arabidopsis thaliana (Arabidopsis). We investigated if candidate effectors predicted in the genome sequence of Hpa isolate Emoy2 (HaRxLs) were able to manipulate host defenses in different Arabidopsis accessions. We developed a rapid and sensitive screening method to test HaRxLs by delivering them via the bacterial type-three secretion system (TTSS) of Pseudomonas syringae pv tomato DC3000-LUX (Pst-LUX) and assessing changes in Pst-LUX growth in planta on 12 Arabidopsis accessions. The majority (∼70%) of the 64 candidates tested positively contributed to Pst-LUX growth on more than one accession indicating that Hpa virulence likely involves multiple effectors with weak accession-specific effects. Further screening with a Pst mutant (ΔCEL) showed that HaRxLs that allow enhanced Pst-LUX growth usually suppress callose deposition, a hallmark of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). We found that HaRxLs are rarely strong avirulence determinants. Although some decreased Pst-LUX growth in particular accessions, none activated macroscopic cell death. Fewer HaRxLs conferred enhanced Pst growth on turnip, a non-host for Hpa, while several reduced it, consistent with the idea that turnip's non-host resistance against Hpa could involve a combination of recognized HaRxLs and ineffective HaRxLs. We verified our results by constitutively expressing in Arabidopsis a sub-set of HaRxLs. Several transgenic lines showed increased susceptibility to Hpa and attenuation of Arabidopsis PTI responses, confirming the HaRxLs' role in Hpa virulence. This study shows TTSS screening system provides a useful tool to test whether candidate

  10. Multiple candidate effectors from the oomycete pathogen Hyaloperonospora arabidopsidis suppress host plant immunity.

    PubMed

    Fabro, Georgina; Steinbrenner, Jens; Coates, Mary; Ishaque, Naveed; Baxter, Laura; Studholme, David J; Körner, Evelyn; Allen, Rebecca L; Piquerez, Sophie J M; Rougon-Cardoso, Alejandra; Greenshields, David; Lei, Rita; Badel, Jorge L; Caillaud, Marie-Cecile; Sohn, Kee-Hoon; Van den Ackerveken, Guido; Parker, Jane E; Beynon, Jim; Jones, Jonathan D G

    2011-11-01

    Oomycete pathogens cause diverse plant diseases. To successfully colonize their hosts, they deliver a suite of effector proteins that can attenuate plant defenses. In the oomycete downy mildews, effectors carry a signal peptide and an RxLR motif. Hyaloperonospora arabidopsidis (Hpa) causes downy mildew on the model plant Arabidopsis thaliana (Arabidopsis). We investigated if candidate effectors predicted in the genome sequence of Hpa isolate Emoy2 (HaRxLs) were able to manipulate host defenses in different Arabidopsis accessions. We developed a rapid and sensitive screening method to test HaRxLs by delivering them via the bacterial type-three secretion system (TTSS) of Pseudomonas syringae pv tomato DC3000-LUX (Pst-LUX) and assessing changes in Pst-LUX growth in planta on 12 Arabidopsis accessions. The majority (~70%) of the 64 candidates tested positively contributed to Pst-LUX growth on more than one accession indicating that Hpa virulence likely involves multiple effectors with weak accession-specific effects. Further screening with a Pst mutant (ΔCEL) showed that HaRxLs that allow enhanced Pst-LUX growth usually suppress callose deposition, a hallmark of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). We found that HaRxLs are rarely strong avirulence determinants. Although some decreased Pst-LUX growth in particular accessions, none activated macroscopic cell death. Fewer HaRxLs conferred enhanced Pst growth on turnip, a non-host for Hpa, while several reduced it, consistent with the idea that turnip's non-host resistance against Hpa could involve a combination of recognized HaRxLs and ineffective HaRxLs. We verified our results by constitutively expressing in Arabidopsis a sub-set of HaRxLs. Several transgenic lines showed increased susceptibility to Hpa and attenuation of Arabidopsis PTI responses, confirming the HaRxLs' role in Hpa virulence. This study shows TTSS screening system provides a useful tool to test whether candidate

  11. Mechanisms of nuclear suppression of host immunity by effectors from the Arabidopsis downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa).

    PubMed

    Caillaud, M-C; Wirthmueller, L; Fabro, G; Piquerez, S J M; Asai, S; Ishaque, N; Jones, J D G

    2012-01-01

    Filamentous phytopathogens form sophisticated intracellular feeding structures called haustoria in plant cells. Pathogen effectors are likely to play a role in the establishment and maintenance of haustoria additional to their more characterized role of suppressing plant defense. Recent studies suggest that effectors may manipulate host transcription or other nuclear regulatory components for the benefit of pathogen development. However, the specific mechanisms by which these effectors promote susceptibility remain unclear. Of two recent screenings, we identified 15 nuclear-localized Hpa effectors (HaRxLs) that interact directly or indirectly with host nuclear components. When stably expressed in planta, nuclear HaRxLs cause diverse developmental phenotypes highlighting that nuclear effectors might interfere with fundamental plant regulatory mechanisms. Here, we report recent advances in understanding how a pathogen can manipulate nuclear processes in order to cause disease.

  12. Identification of Two Legionella pneumophila Effectors that Manipulate Host Phospholipids Biosynthesis

    PubMed Central

    Viner, Ram; Chetrit, David; Ehrlich, Marcelo; Segal, Gil

    2012-01-01

    The intracellular pathogen Legionella pneumophila translocates a large number of effector proteins into host cells via the Icm/Dot type-IVB secretion system. Some of these effectors were shown to cause lethal effect on yeast growth. Here we characterized one such effector (LecE) and identified yeast suppressors that reduced its lethal effect. The LecE lethal effect was found to be suppressed by the over expression of the yeast protein Dgk1 a diacylglycerol (DAG) kinase enzyme and by a deletion of the gene encoding for Pah1 a phosphatidic acid (PA) phosphatase that counteracts the activity of Dgk1. Genetic analysis using yeast deletion mutants, strains expressing relevant yeast genes and point mutations constructed in the Dgk1 and Pah1 conserved domains indicated that LecE functions similarly to the Nem1-Spo7 phosphatase complex that activates Pah1 in yeast. In addition, by using relevant yeast genetic backgrounds we examined several L. pneumophila effectors expected to be involved in phospholipids biosynthesis and identified an effector (LpdA) that contains a phospholipase-D (PLD) domain which caused lethal effect only in a dgk1 deletion mutant of yeast. Additionally, LpdA was found to enhance the lethal effect of LecE in yeast cells, a phenomenon which was found to be dependent on its PLD activity. Furthermore, to determine whether LecE and LpdA affect the levels or distribution of DAG and PA in-vivo in mammalian cells, we utilized fluorescent DAG and PA biosensors and validated the notion that LecE and LpdA affect the in-vivo levels and distribution of DAG and PA, respectively. Finally, we examined the intracellular localization of both LecE and LpdA in human macrophages during L. pneumophila infection and found that both effectors are localized to the bacterial phagosome. Our results suggest that L. pneumophila utilize at least two effectors to manipulate important steps in phospholipids biosynthesis. PMID:23133385

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

    PubMed Central

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

    2015-01-01

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

  14. Convergent targeting of a common host protein-network by pathogen effectors from three kingdoms of life.

    PubMed

    Weßling, Ralf; Epple, Petra; Altmann, Stefan; He, Yijian; Yang, Li; Henz, Stefan R; McDonald, Nathan; Wiley, Kristin; Bader, Kai Christian; Gläßer, Christine; Mukhtar, M Shahid; Haigis, Sabine; Ghamsari, Lila; Stephens, Amber E; Ecker, Joseph R; Vidal, Marc; Jones, Jonathan D G; Mayer, Klaus F X; Ver Loren van Themaat, Emiel; Weigel, Detlef; Schulze-Lefert, Paul; Dangl, Jeffery L; Panstruga, Ralph; Braun, Pascal

    2014-09-10

    While conceptual principles governing plant immunity are becoming clear, its systems-level organization and the evolutionary dynamic of the host-pathogen interface are still obscure. We generated a systematic protein-protein interaction network of virulence effectors from the ascomycete pathogen Golovinomyces orontii and Arabidopsis thaliana host proteins. We combined this data set with corresponding data for the eubacterial pathogen Pseudomonas syringae and the oomycete pathogen Hyaloperonospora arabidopsidis. The resulting network identifies host proteins onto which intraspecies and interspecies pathogen effectors converge. Phenotyping of 124 Arabidopsis effector-interactor mutants revealed a correlation between intraspecies and interspecies convergence and several altered immune response phenotypes. Several effectors and the most heavily targeted host protein colocalized in subnuclear foci. Products of adaptively selected Arabidopsis genes are enriched for interactions with effector targets. Our data suggest the existence of a molecular host-pathogen interface that is conserved across Arabidopsis accessions, while evolutionary adaptation occurs in the immediate network neighborhood of effector targets.

  15. Convergent targeting of a common host protein-network by pathogen effectors from three kingdoms of life

    PubMed Central

    Weßling, Ralf; Epple, Petra; Altmann, Stefan; He, Yijian; Yang, Li; Henz, Stefan R.; McDonald, Nathan; Wiley, Kristin; Bader, Kai Christian; Gläßer, Christine; Mukhtar, M. Shahid; Haigis, Sabine; Ghamsari, Lila; Stephens, Amber E.; Ecker, Joseph R.; Vidal, Marc; Jones, Jonathan D. G.; Mayer, Klaus F. X.; van Themaat, Emiel Ver Loren; Weigel, Detlef; Schulze-Lefert, Paul; Dangl, Jeffery L.; Panstruga, Ralph; Braun, Pascal

    2014-01-01

    SUMMARY While conceptual principles governing plant immunity are becoming clear, its systems-level organization and the evolutionary dynamic of the host-pathogen interface are still obscure. We generated a systematic protein-protein interaction network of virulence effectors from the ascomycete pathogen Golovinomyces orontii and Arabidopsis thaliana host proteins. We combined this dataset with corresponding data for the eubacterial pathogen Pseudomonas syringae and the oomycete pathogen Hyaloperonospora arabidopsidis. The resulting network identifies host proteins onto which intraspecies and interspecies pathogen effectors converge. Phenotyping of 124 Arabidopsis effector-interactor mutants revealed a correlation between intra- and interspecies convergence and several altered immune response phenotypes. The effectors and most heavily targeted host protein co-localized in sub-nuclear foci. Products of adaptively selected Arabidopsis genes are enriched for interactions with effector targets. Our data suggest the existence of a molecular host-pathogen interface that is conserved across Arabidopsis accessions, while evolutionary adaptation occurs in the immediate network neighborhood of effector targets. PMID:25211078

  16. Copper Is a Host Effector Mobilized to Urine during Urinary Tract Infection To Impair Bacterial Colonization.

    PubMed

    Hyre, Amanda N; Kavanagh, Kylie; Kock, Nancy D; Donati, George L; Subashchandrabose, Sargurunathan

    2017-03-01

    Urinary tract infection (UTI) is a major global infectious disease affecting millions of people annually. Human urinary copper (Cu) content is elevated during UTI caused by uropathogenic Escherichia coli (UPEC). UPEC upregulates the expression of Cu efflux genes during clinical UTI in patients as an adaptive response to host-derived Cu. Whether Cu is mobilized to urine as a host response to UTI and its role in protection against UTI remain unresolved. To address these questions, we tested the hypothesis that Cu is a host effector mobilized to urine during UTI to limit bacterial growth. Our results reveal that Cu is mobilized to urine during UTI caused by the major uropathogens Proteus mirabilis and Klebsiella pneumoniae, in addition to UPEC, in humans. Ceruloplasmin, a Cu-containing ferroxidase, is found at higher levels in UTI urine than in healthy control urine and serves as the molecular source of urinary Cu during UTI. Our results demonstrate that ceruloplasmin decreases the bioavailability of iron in urine by a transferrin-dependent mechanism. Experimental UTI with UPEC in nonhuman primates recapitulates the increased urinary Cu content observed during clinical UTI. Furthermore, Cu-deficient mice are highly colonized by UPEC, indicating that Cu is involved in the limiting of bacterial growth within the urinary tract. Collectively, our results indicate that Cu is a host effector that is involved in protection against pathogen colonization of the urinary tract. Because urinary Cu levels are amenable to modulation, augmentation of the Cu-based host defense against UTI represents a novel approach to limiting bacterial colonization during UTI.

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

    SciTech Connect

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

    2014-07-11

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

  18. 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

  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. Genome plasticity in filamentous plant pathogens contributes to the emergence of novel effectors and their cellular processes in the host.

    PubMed

    Dong, Yanhan; Li, Ying; Qi, Zhongqiang; Zheng, Xiaobo; Zhang, Zhengguang

    2016-02-01

    Plant diseases cause extensive yield loss of crops worldwide, and secretory 'warfare' occurs between plants and pathogenic organisms all the time. Filamentous plant pathogens have evolved the ability to manipulate host processes and facilitate colonization through secreting effectors inside plant cells. The stresses from hosts and environment can drive the genome dynamics of plant pathogens. Remarkable advances in plant pathology have been made owing to these adaptable genome regions of several lineages of filamentous phytopathogens. Characterization new effectors and interaction analyses between pathogens and plants have provided molecular insights into the plant pathways perturbed during the infection process. In this mini-review, we highlight promising approaches of identifying novel effectors based on the genome plasticity. We also discuss the interaction mechanisms between plants and their filamentous pathogens and outline the possibilities of effector gene expression under epigenetic control that will be future directions for research.

  1. 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

  2. The inflammasomes: molecular effectors of host resistance against bacterial, viral, parasitic, and fungal infections.

    PubMed

    Skeldon, Alexander; Saleh, Maya

    2011-01-01

    The inflammasomes are large multi-protein complexes scaffolded by cytosolic pattern recognition receptors (PRRs) that form an important part of the innate immune system. They are activated following the recognition of microbial-associated molecular patterns or host-derived danger signals (danger-associated molecular patterns) by PRRs. This recognition results in the recruitment and activation of the pro-inflammatory protease caspase-1, which cleaves its preferred substrates pro-interleukin-1β (IL-1β) and pro-IL-18 into their mature biologically active cytokine forms. Through processing of a number of other cellular substrates, caspase-1 is also required for the release of "alarmins" and the induction and execution of an inflammatory form of cell death termed pyroptosis. A growing spectrum of inflammasomes have been identified in the host defense against a variety of pathogens. Reciprocally, pathogens have evolved effector strategies to antagonize the inflammasome pathway. In this review we discuss recent developments in the understanding of inflammasome-mediated recognition of bacterial, viral, parasitic, and fungal infections and the beneficial or detrimental effects of inflammasome signaling in host resistance.

  3. Phylogenomics of Xanthomonas field strains infecting pepper and tomato reveals diversity in effector repertoires and identifies determinants of host specificity

    PubMed Central

    Schwartz, Allison R.; Potnis, Neha; Timilsina, Sujan; Wilson, Mark; Patané, José; Martins, Joaquim; Minsavage, Gerald V.; Dahlbeck, Douglas; Akhunova, Alina; Almeida, Nalvo; Vallad, Gary E.; Barak, Jeri D.; White, Frank F.; Miller, Sally A.; Ritchie, David; Goss, Erica; Bart, Rebecca S.; Setubal, João C.; Jones, Jeffrey B.; Staskawicz, Brian J.

    2015-01-01

    Bacterial spot disease of pepper and tomato is caused by four distinct Xanthomonas species and is a severely limiting factor on fruit yield in these crops. The genetic diversity and the type III effector repertoires of a large sampling of field strains for this disease have yet to be explored on a genomic scale, limiting our understanding of pathogen evolution in an agricultural setting. Genomes of 67 Xanthomonas euvesicatoria (Xe), Xanthomonas perforans (Xp), and Xanthomonas gardneri (Xg) strains isolated from diseased pepper and tomato fields in the southeastern and midwestern United States were sequenced in order to determine the genetic diversity in field strains. Type III effector repertoires were computationally predicted for each strain, and multiple methods of constructing phylogenies were employed to understand better the genetic relationship of strains in the collection. A division in the Xp population was detected based on core genome phylogeny, supporting a model whereby the host-range expansion of Xp field strains on pepper is due, in part, to a loss of the effector AvrBsT. Xp-host compatibility was further studied with the observation that a double deletion of AvrBsT and XopQ allows a host range expansion for Nicotiana benthamiana. Extensive sampling of field strains and an improved understanding of effector content will aid in efforts to design disease resistance strategies targeted against highly conserved core effectors. PMID:26089818

  4. 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

  5. Quantification of disease expression conferred by three host gene-necrotrophic effector interactions in the wheat-Parastagonospora nodorum pathosystem

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The disease Septoria nodorum blotch (SNB) is caused by the necrotrophic fungal pathogen Parastagonospora nodorum, which induces cell death in wheat through the production of necrotrophic effectors (NEs). The objective of this project is to determine the relative importance of three host gene-NE int...

  6. Sterile Immunity to Malaria after DNA Prime/Adenovirus Boost Immunization Is Associated with Effector Memory CD8+T Cells Targeting AMA1 Class I Epitopes

    PubMed Central

    Sedegah, Martha; Hollingdale, Michael R.; Farooq, Fouzia; Ganeshan, Harini; Belmonte, Maria; Kim, Yohan; Peters, Bjoern; Sette, Alessandro; Huang, Jun; McGrath, Shannon; Abot, Esteban; Limbach, Keith; Shi, Meng; Soisson, Lorraine; Diggs, Carter; Chuang, Ilin; Tamminga, Cindy; Epstein, Judith E.; Villasante, Eileen; Richie, Thomas L.

    2014-01-01

    Background Fifteen volunteers were immunized with three doses of plasmid DNA encoding P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1) and boosted with human adenovirus-5 (Ad) expressing the same antigens (DNA/Ad). Four volunteers (27%) demonstrated sterile immunity to controlled human malaria infection and, overall, protection was statistically significantly associated with ELISpot and CD8+ T cell IFN-γ activities to AMA1 but not CSP. DNA priming was required for protection, as 18 additional subjects immunized with Ad alone (AdCA) did not develop sterile protection. Methodology/Principal Findings We sought to identify correlates of protection, recognizing that DNA-priming may induce different responses than AdCA alone. Among protected volunteers, two and three had higher ELISpot and CD8+ T cell IFN-γ responses to CSP and AMA1, respectively, than non-protected volunteers. Unexpectedly, non-protected volunteers in the AdCA trial showed ELISpot and CD8+ T cell IFN-γ responses to AMA1 equal to or higher than the protected volunteers. T cell functionality assessed by intracellular cytokine staining for IFN-γ, TNF-α and IL-2 likewise did not distinguish protected from non-protected volunteers across both trials. However, three of the four protected volunteers showed higher effector to central memory CD8+ T cell ratios to AMA1, and one of these to CSP, than non-protected volunteers for both antigens. These responses were focused on discrete regions of CSP and AMA1. Class I epitopes restricted by A*03 or B*58 supertypes within these regions of AMA1 strongly recalled responses in three of four protected volunteers. We hypothesize that vaccine-induced effector memory CD8+ T cells recognizing a single class I epitope can confer sterile immunity to P. falciparum in humans. Conclusions/Significance We suggest that better understanding of which epitopes within malaria antigens can confer sterile immunity and design of vaccine approaches

  7. Pseudomonas syringae Type III Effector HopBB1 Promotes Host Transcriptional Repressor Degradation to Regulate Phytohormone Responses and Virulence.

    PubMed

    Yang, Li; Teixeira, Paulo José Pereira Lima; Biswas, Surojit; Finkel, Omri M; He, Yijian; Salas-Gonzalez, Isai; English, Marie E; Epple, Petra; Mieczkowski, Piotr; Dangl, Jeffery L

    2017-02-08

    Independently evolved pathogen effectors from three branches of life (ascomycete, eubacteria, and oomycete) converge onto the Arabidopsis TCP14 transcription factor to manipulate host defense. However, the mechanistic basis for defense control via TCP14 regulation is unknown. We demonstrate that TCP14 regulates the plant immune system by transcriptionally repressing a subset of the jasmonic acid (JA) hormone signaling outputs. A previously unstudied Pseudomonas syringae (Psy) type III effector, HopBB1, interacts with TCP14 and targets it to the SCF(COI1) degradation complex by connecting it to the JA signaling repressor JAZ3. Consequently, HopBB1 de-represses the TCP14-regulated subset of JA response genes and promotes pathogen virulence. Thus, HopBB1 fine-tunes host phytohormone crosstalk by precisely manipulating part of the JA regulon to avoid pleiotropic host responses while promoting pathogen proliferation.

  8. Phytophthora infestans RXLR Effector PexRD2 Interacts with Host MAPKKKε to Suppress Plant Immune Signaling[W][OPEN

    PubMed Central

    King, Stuart R.F.; McLellan, Hazel; Boevink, Petra C.; Armstrong, Miles R.; Bukharova, Tatyana; Sukarta, Octavina; Win, Joe; Kamoun, Sophien; Birch, Paul R.J.; Banfield, Mark J.

    2014-01-01

    Mitogen-activated protein kinase cascades are key players in plant immune signaling pathways, transducing the perception of invading pathogens into effective defense responses. Plant pathogenic oomycetes, such as the Irish potato famine pathogen Phytophthora infestans, deliver RXLR effector proteins to plant cells to modulate host immune signaling and promote colonization. Our understanding of the molecular mechanisms by which these effectors act in plant cells is limited. Here, we report that the P. infestans RXLR effector PexRD2 interacts with the kinase domain of MAPKKKε, a positive regulator of cell death associated with plant immunity. Expression of PexRD2 or silencing MAPKKKε in Nicotiana benthamiana enhances susceptibility to P. infestans. We show that PexRD2 perturbs signaling pathways triggered by or dependent on MAPKKKε. By contrast, homologs of PexRD2 from P. infestans had reduced or no interaction with MAPKKKε and did not promote disease susceptibility. Structure-led mutagenesis identified PexRD2 variants that do not interact with MAPKKKε and fail to support enhanced pathogen growth or perturb MAPKKKε signaling pathways. Our findings provide evidence that P. infestans RXLR effector PexRD2 has evolved to interact with a specific host MAPKKK to perturb plant immunity–related signaling. PMID:24632534

  9. Phytophthora infestans RXLR effector PexRD2 interacts with host MAPKKK ε to suppress plant immune signaling.

    PubMed

    King, Stuart R F; McLellan, Hazel; Boevink, Petra C; Armstrong, Miles R; Bukharova, Tatyana; Sukarta, Octavina; Win, Joe; Kamoun, Sophien; Birch, Paul R J; Banfield, Mark J

    2014-03-01

    Mitogen-activated protein kinase cascades are key players in plant immune signaling pathways, transducing the perception of invading pathogens into effective defense responses. Plant pathogenic oomycetes, such as the Irish potato famine pathogen Phytophthora infestans, deliver RXLR effector proteins to plant cells to modulate host immune signaling and promote colonization. Our understanding of the molecular mechanisms by which these effectors act in plant cells is limited. Here, we report that the P. infestans RXLR effector PexRD2 interacts with the kinase domain of MAPKKKε, a positive regulator of cell death associated with plant immunity. Expression of PexRD2 or silencing MAPKKKε in Nicotiana benthamiana enhances susceptibility to P. infestans. We show that PexRD2 perturbs signaling pathways triggered by or dependent on MAPKKKε. By contrast, homologs of PexRD2 from P. infestans had reduced or no interaction with MAPKKKε and did not promote disease susceptibility. Structure-led mutagenesis identified PexRD2 variants that do not interact with MAPKKKε and fail to support enhanced pathogen growth or perturb MAPKKKε signaling pathways. Our findings provide evidence that P. infestans RXLR effector PexRD2 has evolved to interact with a specific host MAPKKK to perturb plant immunity-related signaling.

  10. A translocated effector required for Bartonella dissemination from derma to blood safeguards migratory host cells from damage by co-translocated effectors.

    PubMed

    Okujava, Rusudan; Guye, Patrick; Lu, Yun-Yueh; Mistl, Claudia; Polus, Florine; Vayssier-Taussat, Muriel; Halin, Cornelia; Rolink, Antonius G; Dehio, Christoph

    2014-06-01

    Numerous bacterial pathogens secrete multiple effectors to modulate host cellular functions. These effectors may interfere with each other to efficiently control the infection process. Bartonellae are Gram-negative, facultative intracellular bacteria using a VirB type IV secretion system to translocate a cocktail of Bartonella effector proteins (Beps) into host cells. Based on in vitro infection models we demonstrate here that BepE protects infected migratory cells from injurious effects triggered by BepC and is required for in vivo dissemination of bacteria from the dermal site of inoculation to blood. Human endothelial cells (HUVECs) infected with a ΔbepE mutant of B. henselae (Bhe) displayed a cell fragmentation phenotype resulting from Bep-dependent disturbance of rear edge detachment during migration. A ΔbepCE mutant did not show cell fragmentation, indicating that BepC is critical for triggering this deleterious phenotype. Complementation of ΔbepE with BepEBhe or its homologues from other Bartonella species abolished cell fragmentation. This cyto-protective activity is confined to the C-terminal Bartonella intracellular delivery (BID) domain of BepEBhe (BID2.EBhe). Ectopic expression of BID2.EBhe impeded the disruption of actin stress fibers by Rho Inhibitor 1, indicating that BepE restores normal cell migration via the RhoA signaling pathway, a major regulator of rear edge retraction. An intradermal (i.d.) model for B. tribocorum (Btr) infection in the rat reservoir host mimicking the natural route of infection by blood sucking arthropods allowed demonstrating a vital role for BepE in bacterial dissemination from derma to blood. While the Btr mutant ΔbepDE was abacteremic following i.d. inoculation, complementation with BepEBtr, BepEBhe or BIDs.EBhe restored bacteremia. Given that we observed a similar protective effect of BepEBhe on infected bone marrow-derived dendritic cells migrating through a monolayer of lymphatic endothelial cells we propose that

  11. A Translocated Effector Required for Bartonella Dissemination from Derma to Blood Safeguards Migratory Host Cells from Damage by Co-translocated Effectors

    PubMed Central

    Okujava, Rusudan; Guye, Patrick; Lu, Yun-Yueh; Mistl, Claudia; Polus, Florine; Vayssier-Taussat, Muriel; Halin, Cornelia; Rolink, Antonius G.; Dehio, Christoph

    2014-01-01

    Numerous bacterial pathogens secrete multiple effectors to modulate host cellular functions. These effectors may interfere with each other to efficiently control the infection process. Bartonellae are Gram-negative, facultative intracellular bacteria using a VirB type IV secretion system to translocate a cocktail of Bartonella effector proteins (Beps) into host cells. Based on in vitro infection models we demonstrate here that BepE protects infected migratory cells from injurious effects triggered by BepC and is required for in vivo dissemination of bacteria from the dermal site of inoculation to blood. Human endothelial cells (HUVECs) infected with a ΔbepE mutant of B. henselae (Bhe) displayed a cell fragmentation phenotype resulting from Bep-dependent disturbance of rear edge detachment during migration. A ΔbepCE mutant did not show cell fragmentation, indicating that BepC is critical for triggering this deleterious phenotype. Complementation of ΔbepE with BepEBhe or its homologues from other Bartonella species abolished cell fragmentation. This cyto-protective activity is confined to the C-terminal Bartonella intracellular delivery (BID) domain of BepEBhe (BID2.EBhe). Ectopic expression of BID2.EBhe impeded the disruption of actin stress fibers by Rho Inhibitor 1, indicating that BepE restores normal cell migration via the RhoA signaling pathway, a major regulator of rear edge retraction. An intradermal (i.d.) model for B. tribocorum (Btr) infection in the rat reservoir host mimicking the natural route of infection by blood sucking arthropods allowed demonstrating a vital role for BepE in bacterial dissemination from derma to blood. While the Btr mutant ΔbepDE was abacteremic following i.d. inoculation, complementation with BepEBtr, BepEBhe or BIDs.EBhe restored bacteremia. Given that we observed a similar protective effect of BepEBhe on infected bone marrow-derived dendritic cells migrating through a monolayer of lymphatic endothelial cells we propose that

  12. TAL effectors and activation of predicted host targets distinguish Asian from African strains of the rice pathogen Xanthomonas oryzae pv. oryzicola while strict conservation suggests universal importance of five TAL effectors.

    PubMed

    Wilkins, Katherine E; Booher, Nicholas J; Wang, Li; Bogdanove, Adam J

    2015-01-01

    Xanthomonas oryzae pv. oryzicola (Xoc) causes the increasingly important disease bacterial leaf streak of rice (BLS) in part by type III delivery of repeat-rich transcription activator-like (TAL) effectors to upregulate host susceptibility genes. By pathogen whole genome, single molecule, real-time sequencing and host RNA sequencing, we compared TAL effector content and rice transcriptional responses across 10 geographically diverse Xoc strains. TAL effector content is surprisingly conserved overall, yet distinguishes Asian from African isolates. Five TAL effectors are conserved across all strains. In a prior laboratory assay in rice cv. Nipponbare, only two contributed to virulence in strain BLS256 but the strict conservation indicates all five may be important, in different rice genotypes or in the field. Concatenated and aligned, TAL effector content across strains largely reflects relationships based on housekeeping genes, suggesting predominantly vertical transmission. Rice transcriptional responses did not reflect these relationships, and on average, only 28% of genes upregulated and 22% of genes downregulated by a strain are up- and down- regulated (respectively) by all strains. However, when only known TAL effector targets were considered, the relationships resembled those of the TAL effectors. Toward identifying new targets, we used the TAL effector-DNA recognition code to predict effector binding elements in promoters of genes upregulated by each strain, but found that for every strain, all upregulated genes had at least one. Filtering with a classifier we developed previously decreases the number of predicted binding elements across the genome, suggesting that it may reduce false positives among upregulated genes. Applying this filter and eliminating genes for which upregulation did not strictly correlate with presence of the corresponding TAL effector, we generated testable numbers of candidate targets for four of the five strictly conserved TAL

  13. TAL effectors and activation of predicted host targets distinguish Asian from African strains of the rice pathogen Xanthomonas oryzae pv. oryzicola while strict conservation suggests universal importance of five TAL effectors

    PubMed Central

    Wilkins, Katherine E.; Booher, Nicholas J.; Wang, Li; Bogdanove, Adam J.

    2015-01-01

    Xanthomonas oryzae pv. oryzicola (Xoc) causes the increasingly important disease bacterial leaf streak of rice (BLS) in part by type III delivery of repeat-rich transcription activator-like (TAL) effectors to upregulate host susceptibility genes. By pathogen whole genome, single molecule, real-time sequencing and host RNA sequencing, we compared TAL effector content and rice transcriptional responses across 10 geographically diverse Xoc strains. TAL effector content is surprisingly conserved overall, yet distinguishes Asian from African isolates. Five TAL effectors are conserved across all strains. In a prior laboratory assay in rice cv. Nipponbare, only two contributed to virulence in strain BLS256 but the strict conservation indicates all five may be important, in different rice genotypes or in the field. Concatenated and aligned, TAL effector content across strains largely reflects relationships based on housekeeping genes, suggesting predominantly vertical transmission. Rice transcriptional responses did not reflect these relationships, and on average, only 28% of genes upregulated and 22% of genes downregulated by a strain are up- and down- regulated (respectively) by all strains. However, when only known TAL effector targets were considered, the relationships resembled those of the TAL effectors. Toward identifying new targets, we used the TAL effector-DNA recognition code to predict effector binding elements in promoters of genes upregulated by each strain, but found that for every strain, all upregulated genes had at least one. Filtering with a classifier we developed previously decreases the number of predicted binding elements across the genome, suggesting that it may reduce false positives among upregulated genes. Applying this filter and eliminating genes for which upregulation did not strictly correlate with presence of the corresponding TAL effector, we generated testable numbers of candidate targets for four of the five strictly conserved TAL

  14. A Virulence Essential CRN Effector of Phytophthora capsici Suppresses Host Defense and Induces Cell Death in Plant Nucleus

    PubMed Central

    Mafurah, Joseph Juma; Ma, Huifei; Zhang, Meixiang; Xu, Jing; He, Feng; Ye, Tingyue; Shen, Danyu; Chen, Yanyu; Rajput, Nasir Ahmed; Dou, Daolong

    2015-01-01

    Phytophthora capsici is a soil-borne plant pathogen with a wide range of hosts. The pathogen secretes a large array of effectors during infection of host plants, including Crinkler (CRN) effectors. However, it remains largely unknown on the roles of these effectors in virulence especially in P. capsici. In this study, we identified a cell death-inducing CRN effector PcCRN4 using agroinfiltration approach. Transient expression of PcCRN4 gene induced cell death in N. benthamiana, N. tabacum and Solanum lycopersicum. Overexpression of the gene in N. benthamiana enhanced susceptibility to P. capsici. Subcellular localization results showed that PcCRN4 localized to the plant nucleus, and the localization was required for both of its cell death-inducing activity and virulent function. Silencing PcCRN4 gene in P. capsici significantly reduced pathogen virulence. The expression of the pathogenesis-related gene PR1b in N. benthamiana was significantly induced when plants were inoculated with PcCRN4-silenced P. capsici transformant compared to the wilt-type. Callose deposits were also abundant at sites inoculated with PcCRN4-silenced transformant, indicating that silencing of PcCRN4 in P. capsici reduced the ability of the pathogen to suppress plant defenses. Transcriptions of cell death-related genes were affected when PcCRN4-silenced line were inoculated on Arabidopsis thaliana, suggesting that PcCRN4 may induce cell death by manipulating cell death-related genes. Overall, our results demonstrate that PcCRN4 is a virulence essential effector and it needs target to the plant nucleus to suppress plant immune responses. PMID:26011314

  15. Effector CD8+ T cell engraftment and anti-tumor immunity in lymphodepleted hosts is IL-7Rα dependent

    PubMed Central

    Johnson, C. Bryce; Riesenberg, Brian P.; May, Bennett R.; Gilreath, Stuart C.; Li, Guangfu; Staveley-O’Carroll, Kevin F.; Garrett-Mayer, Elizabeth; Mehrotra, Shikhar; Cole, David J.; Rubinstein, Mark P.

    2016-01-01

    Adoptive cellular therapy, in which activated tumor-reactive T cells are transferred into murine lymphodepleted hosts, is a promising cancer treatment option. Activation of T cells decreases IL-7 responsiveness; therefore, IL-15 is generally considered the main driver of effector T cell responses in this setting. However, we found in lymphodepleted hosts that CD8+ T cells activated with IL-12 showed enhanced engraftment that was initially dependent on host IL-7, but not IL-15. Mechanistically, enhanced IL-7 responsiveness was conferred by elevated IL-7Rα expression, which was critical for anti-tumor immunity. Elevated IL-7Rα expression was achievable without IL-12, as polyclonal CD8+ T cells activated with high TCR stimulation depended on T cell IL-7Rα expression and host IL-7 for maximal engraftment. Finally, IL-12 conditioning during the activation of human CD8+ T cells, including TCR-modified T cells generated using a clinically relevant protocol, led to enhanced IL-7Rα expression. Our results demonstrate the importance of the donor IL-7Rα/host IL-7 axis for effector CD8+ T cell engraftment and suggest novel strategies to improve adoptive cellular therapy as a cancer treatment. PMID:26297711

  16. Analysis of three Xanthomonas axonopodis pv. citri effector proteins in pathogenicity and their interactions with host plant proteins.

    PubMed

    Dunger, Germán; Garofalo, Cecilia G; Gottig, Natalia; Garavaglia, Betiana S; Rosa, María C Pereda; Farah, Chuck S; Orellano, Elena G; Ottado, Jorgelina

    2012-10-01

    Xanthomonas axonopodis pv. citri, the bacterium responsible for citrus canker, uses effector proteins secreted by a type III protein secretion system to colonize its hosts. Among the putative effector proteins identified for this bacterium, we focused on the analysis of the roles of AvrXacE1, AvrXacE2 and Xac3090 in pathogenicity and their interactions with host plant proteins. Bacterial deletion mutants in avrXacE1, avrXacE2 and xac3090 were constructed and evaluated in pathogenicity assays. The avrXacE1 and avrXacE2 mutants presented lesions with larger necrotic areas relative to the wild-type strain when infiltrated in citrus leaves. Yeast two-hybrid studies were used to identify several plant proteins likely to interact with AvrXacE1, AvrXacE2 and Xac3090. We also assessed the localization of these effector proteins fused to green fluorescent protein in the plant cell, and observed that they co-localized to the subcellular spaces in which the plant proteins with which they interacted were predicted to be confined. Our results suggest that, although AvrXacE1 localizes to the plant cell nucleus, where it interacts with transcription factors and DNA-binding proteins, AvrXacE2 appears to be involved in lesion-stimulating disease 1-mediated cell death, and Xac3090 is directed to the chloroplast where its function remains to be clarified.

  17. 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

  18. Bordetella evades the host immune system by inducing IL-10 through a type III effector, BopN

    PubMed Central

    Nagamatsu, Kanna; Kuwae, Asaomi; Konaka, Tadashi; Nagai, Shigenori; Yoshida, Sei; Eguchi, Masahiro; Watanabe, Mineo; Mimuro, Hitomi; Koyasu, Shigeo

    2009-01-01

    The inflammatory response is one of several host alert mechanisms that recruit neutrophils from the circulation to the area of infection. We demonstrate that Bordetella, a bacterial pathogen, exploits an antiinflammatory cytokine, interleukin-10 (IL-10), to evade the host immune system. We identified a Bordetella effector, BopN, that is translocated into the host cell via the type III secretion system, where it induces enhanced production of IL-10. Interestingly, the BopN effector translocates itself into the nucleus and is involved in the down-regulation of mitogen-activated protein kinases. Using pharmacological blockade, we demonstrated that BopN-induced IL-10 production is mediated, at least in part, by its ability to block the extracellular signal-regulated kinase pathway. We also showed that BopN blocks nuclear translocation of nuclear factor κB p65 (NF-κBp65) but, in contrast, promotes nuclear translocation of NF-κBp50. A BopN-deficient strain was unable to induce IL-10 production in mice, resulting in the elimination of bacteria via neutrophil infiltration into the pulmonary alveoli. Furthermore, IL-10–deficient mice effectively eliminated wild-type as well as BopN mutant bacteria. Thus, Bordetella exploits BopN as a stealth strategy to shut off the host inflammatory reaction. These results explain the ability of Bordetella species to avoid induction of the inflammatory response. PMID:20008527

  19. [Mechanisms of in vivo suppressive effect of togaviridae and bunyaviridae on the activity of effectors of graft vs host reaction].

    PubMed

    Khozinskiĭ, V V; Semenov, B F

    1982-02-01

    Experiments on mice demonstrated the ability of 3 flaviviruses and 1 bunyavirus to suppress the activity of the effectors of the graft-versus-host (GVH) reaction. The conditions of the suppression of the primary immunological recognition were shown to differ in infections caused by different viruses. In experimental flavivirus infections caused by Langat, dengue 2 or yellow fever (strain 17D) viruses T-suppressor cells were activated, and their activity was realized only in respect to syngeneic or semisyngeneic target cells. In mice infected with Tahyna virus (a bunyavirus) no suppressor cells capable of suppressing the activity of the effectors of the GVH reaction were detected. The suppression of this reaction, not linked with the activity of the detected T-suppressor cells, was observed in the Langat virus infection under conditions of bilateral incompatibility when both the donor and the recipient were infected.

  20. 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

  1. Variations in type III effector repertoires, pathological phenotypes and host range of Xanthomonas citri pv. citri pathotypes.

    PubMed

    Escalon, Aline; Javegny, Stéphanie; Vernière, Christian; Noël, Laurent D; Vital, Karine; Poussier, Stéphane; Hajri, Ahmed; Boureau, Tristan; Pruvost, Olivier; Arlat, Matthieu; Gagnevin, Lionel

    2013-06-01

    The mechanisms determining the host range of Xanthomonas are still undeciphered, despite much interest in their potential roles in the evolution and emergence of plant pathogenic bacteria. Xanthomonas citri pv. citri (Xci) is an interesting model of host specialization because of its pathogenic variants: pathotype A strains infect a wide range of Rutaceous species, whereas pathotype A*/A(W) strains have a host range restricted to Mexican lime (Citrus aurantifolia) and alemow (Citrus macrophylla). Based on a collection of 55 strains representative of Xci worldwide diversity assessed by amplified fragment length polymorphism (AFLP), we investigated the distribution of type III effectors (T3Es) in relation to host range. We examined the presence of 66 T3Es from xanthomonads in Xci and identified a repertoire of 28 effectors, 26 of which were shared by all Xci strains, whereas two (xopAG and xopC1) were present only in some A*/A(W) strains. We found that xopAG (=avrGf1) was present in all A(W) strains, but also in three A* strains genetically distant from A(W) , and that all xopAG-containing strains induced the hypersensitive response (HR) on grapefruit and sweet orange. The analysis of xopAD and xopAG suggested horizontal transfer between X. citri pv. bilvae, another citrus pathogen, and some Xci strains. A strains were genetically less diverse, induced identical phenotypic responses and possessed indistinguishable T3E repertoires. Conversely, A*/A(W) strains exhibited a wider genetic diversity in which clades correlated with geographical origin and T3E repertoire, but not with pathogenicity, according to T3E deletion experiments. Our data outline the importance of taking into account the heterogeneity of Xci A*/A(W) strains when analysing the mechanisms of host specialization.

  2. 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

  3. 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.

  4. Pseudomonas type III effector AvrPtoB induces plant disease susceptibility by inhibition of host programmed cell death

    PubMed Central

    Abramovitch, Robert B.; Kim, Young-Jin; Chen, Shaorong; Dickman, Martin B.; Martin, Gregory B.

    2003-01-01

    The AvrPtoB type III effector protein is conserved among diverse genera of plant pathogens suggesting it plays an important role in pathogenesis. Here we report that Pseudomonas AvrPtoB acts inside the plant cell to inhibit programmed cell death (PCD) initiated by the Pto and Cf9 disease resistance proteins and, remarkably, the pro-apoptotic mouse protein Bax. AvrPtoB also suppressed PCD in yeast, demonstrating that AvrPtoB functions as a cell death inhibitor across kingdoms. Using truncated AvrPtoB proteins, we identified distinct N- and C-terminal domains of AvrPtoB that are sufficient for host recognition and PCD inhibition, respectively. We also identified a novel resistance phenotype, Rsb, that is triggered by an AvrPtoB truncation disrupted in the anti-PCD domain. A Pseudomonas syringae pv. tomato DC3000 strain with a chromosomal mutation in the AvrPtoB C-terminus elicited Rsb-mediated immunity in previously susceptible tomato plants and disease was restored when full-length AvrPtoB was expressed in trans. Thus, our results indicate that a type III effector can induce plant susceptibility to bacterial infection by inhibiting host PCD. PMID:12505984

  5. A T4SS Effector Targets Host Cell Alpha-Enolase Contributing to Brucella abortus Intracellular Lifestyle

    PubMed Central

    Marchesini, María I.; Morrone Seijo, Susana M.; Guaimas, Francisco F.; Comerci, Diego J.

    2016-01-01

    Brucella abortus, the causative agent of bovine brucellosis, invades and replicates within cells inside a membrane-bound compartment known as the Brucella containing vacuole (BCV). After trafficking along the endocytic and secretory pathways, BCVs mature into endoplasmic reticulum-derived compartments permissive for bacterial replication. Brucella Type IV Secretion System (VirB) is a major virulence factor essential for the biogenesis of the replicative organelle. Upon infection, Brucella uses the VirB system to translocate effector proteins from the BCV into the host cell cytoplasm. Although the functions of many translocated proteins remain unknown, some of them have been demonstrated to modulate host cell signaling pathways to favor intracellular survival and replication. BPE123 (BAB2_0123) is a B. abortus VirB-translocated effector protein recently identified by our group whose function is yet unknown. In an attempt to identify host cell proteins interacting with BPE123, a pull-down assay was performed and human alpha-enolase (ENO-1) was identified by LC/MS-MS as a potential interaction partner of BPE123. These results were confirmed by immunoprecipitation assays. In bone-marrow derived macrophages infected with B. abortus, ENO-1 associates to BCVs in a BPE123-dependent manner, indicating that interaction with translocated BPE123 is also occurring during the intracellular phase of the bacterium. Furthermore, ENO-1 depletion by siRNA impaired B. abortus intracellular replication in HeLa cells, confirming a role for α-enolase during the infection process. Indeed, ENO-1 activity levels were enhanced upon B. abortus infection of THP-1 macrophagic cells, and this activation is highly dependent on BPE123. Taken together, these results suggest that interaction between BPE123 and host cell ENO-1 contributes to the intracellular lifestyle of B. abortus. PMID:27900285

  6. A T4SS Effector Targets Host Cell Alpha-Enolase Contributing to Brucella abortus Intracellular Lifestyle.

    PubMed

    Marchesini, María I; Morrone Seijo, Susana M; Guaimas, Francisco F; Comerci, Diego J

    2016-01-01

    Brucella abortus, the causative agent of bovine brucellosis, invades and replicates within cells inside a membrane-bound compartment known as the Brucella containing vacuole (BCV). After trafficking along the endocytic and secretory pathways, BCVs mature into endoplasmic reticulum-derived compartments permissive for bacterial replication. Brucella Type IV Secretion System (VirB) is a major virulence factor essential for the biogenesis of the replicative organelle. Upon infection, Brucella uses the VirB system to translocate effector proteins from the BCV into the host cell cytoplasm. Although the functions of many translocated proteins remain unknown, some of them have been demonstrated to modulate host cell signaling pathways to favor intracellular survival and replication. BPE123 (BAB2_0123) is a B. abortus VirB-translocated effector protein recently identified by our group whose function is yet unknown. In an attempt to identify host cell proteins interacting with BPE123, a pull-down assay was performed and human alpha-enolase (ENO-1) was identified by LC/MS-MS as a potential interaction partner of BPE123. These results were confirmed by immunoprecipitation assays. In bone-marrow derived macrophages infected with B. abortus, ENO-1 associates to BCVs in a BPE123-dependent manner, indicating that interaction with translocated BPE123 is also occurring during the intracellular phase of the bacterium. Furthermore, ENO-1 depletion by siRNA impaired B. abortus intracellular replication in HeLa cells, confirming a role for α-enolase during the infection process. Indeed, ENO-1 activity levels were enhanced upon B. abortus infection of THP-1 macrophagic cells, and this activation is highly dependent on BPE123. Taken together, these results suggest that interaction between BPE123 and host cell ENO-1 contributes to the intracellular lifestyle of B. abortus.

  7. Phenotypic analysis of apoplastic effectors from the phytopathogenic nematode, Globodera rostochiensis demonstrates that an expansin can induce and suppress host defenses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The potato cyst nematode Globodera rostochiensis (Woll.) is an important pest of potato. Like other biotrophic pathogens, plant parasitic nematodes are presumed to employ effector proteins, secreted into the apoplast as well as the host cytoplasm to successfully infect their hosts. We have identifie...

  8. Broadly conserved fungal effector BEC1019 suppresses host cell death and enhances pathogen virulence in powdery mildew of barley (Hordeum vulgare L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The interaction of barley, Hordeum vulgare L., with the biotrophic powdery mildew fungus, Blumeria graminis f. sp. hordei, is an ideal model to address fundamental questions in host resistance and susceptibility. Effector proteins secreted by B. graminis act to inhibit, induce, or accelerate host pr...

  9. The role of effectors and host immunity in plant-necrotrophic fungal interactions.

    PubMed

    Wang, Xuli; Jiang, Nan; Liu, Jinling; Liu, Wende; Wang, Guo-Liang

    2014-01-01

    Fungal diseases pose constant threats to the global economy and food safety. As the largest group of plant fungal pathogens, necrotrophic fungi cause heavy crop losses worldwide. The molecular mechanisms of the interaction between necrotrophic fungi and plants are complex and involve sophisticated recognition and signaling networks. Here, we review recent findings on the roles of phytotoxin and proteinaceous effectors, pathogen-associated molecular patterns (PAMPs), and small RNAs from necrotrophic fungi. We also consider the functions of damage-associated molecular patterns (DAMPs), the receptor-like protein kinase BIK1, and epigenetic regulation in plant immunity to necrotrophic fungi.

  10. A competitive index assay identifies several Ralstonia solanacearum type III effector mutant strains with reduced fitness in host plants.

    PubMed

    Macho, Alberto P; Guidot, Alice; Barberis, Patrick; Beuzón, Carmen R; Genin, Stéphane

    2010-09-01

    Ralstonia solanacearum, the causal agent of bacterial wilt, is a soil bacterium which can naturally infect a wide range of host plants through the root system. Pathogenicity relies on a type III secretion system which delivers a large set of approximately 75 type III effectors (T3E) into plant cells. On several plants, pathogenicity assays based on quantification of wilting symptoms failed to detect a significant contribution of R. solanacearum T3E in this process, thus revealing the collective effect of T3E in pathogenesis. We developed a mixed infection-based method with R. solanacearum to monitor bacterial fitness in plant leaf tissues as a virulence assay. This accurate and sensitive assay provides evidence that growth defects can be detected for T3E mutants: we identified 12 genes contributing to bacterial fitness in eggplant leaves and 3 of them were also implicated in bacterial fitness on two other hosts, tomato and bean. Contribution to fitness of several T3E appears to be host specific, and we show that some known avirulence determinants such as popP2 or avrA do provide competitive advantages on some susceptible host plants. In addition, this assay revealed that the efe gene, which directs the production of ethylene by bacteria in plant tissues, and hdfB, involved in the biosynthesis of the secondary metabolite 3-hydroxy-oxindole, are also required for optimal growth in plant leaf tissues.

  11. Role of Hcp, a type 6 secretion system effector, of Aeromonas hydrophila in modulating activation of host immune cells.

    PubMed

    Suarez, Giovanni; Sierra, Johanna C; Kirtley, Michelle L; Chopra, Ashok K

    2010-12-01

    Recently, we reported that the type 6 secretion system (T6SS) of Aeromonas hydrophila SSU plays an important role in bacterial virulence in a mouse model, and immunization of animals with the T6SS effector haemolysin co-regulated protein (Hcp) protected them against lethal infections with wild-type bacteria. Additionally, we showed that the mutant bacteria deleted for the vasH gene within the T6SS gene cluster did not express the hcp gene, while the vasK mutant could express and translocate Hcp, but was unable to secrete it into the extracellular milieu. Both of these A. hydrophila SSU mutants were readily phagocytosed by murine macrophages, pointing to the possible role of the secreted form of Hcp in the evasion of the host innate immunity. By using the ΔvasH mutant of A. hydrophila, our in vitro data showed that the addition of exogenous recombinant Hcp (rHcp) reduced bacterial uptake by macrophages. These results were substantiated by increased bacterial virulence when rHcp was added along with the ΔvasH mutant in a septicaemic mouse model of infection. Analysis of the cytokine profiling in the intraperitoneal lavage as well as activation of host cells after 4 h of infection with the ΔvasH mutant supplemented with rHcp indicated that this T6SS effector inhibited production of pro-inflammatory cytokines and induced immunosuppressive cytokines, such as interleukin-10 and transforming growth factor-β, which could circumvent macrophage activation and maturation. This mechanism of innate immune evasion by Hcp possibly inhibited the recruitment of cellular immune components, which allowed bacterial multiplication and dissemination in animals, thereby leading to their mortality.

  12. 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.

  13. Analysis of Putative Apoplastic Effectors from the Nematode, Globodera rostochiensis, and Identification of an Expansin-Like Protein That Can Induce and Suppress Host Defenses

    PubMed Central

    Ali, Shawkat; Magne, Maxime; Chen, Shiyan; Côté, Olivier; Stare, Barbara Gerič; Obradovic, Natasa; Jamshaid, Lubna; Wang, Xiaohong; Bélair, Guy; Moffett, Peter

    2015-01-01

    The potato cyst nematode, Globodera rostochiensis, is an important pest of potato. Like other pathogens, plant parasitic nematodes are presumed to employ effector proteins, secreted into the apoplast as well as the host cytoplasm, to alter plant cellular functions and successfully infect their hosts. We have generated a library of ORFs encoding putative G. rostochiensis putative apoplastic effectors in vectors for expression in planta. These clones were assessed for morphological and developmental effects on plants as well as their ability to induce or suppress plant defenses. Several CLAVATA3/ESR-like proteins induced developmental phenotypes, whereas predicted cell wall-modifying proteins induced necrosis and chlorosis, consistent with roles in cell fate alteration and tissue invasion, respectively. When directed to the apoplast with a signal peptide, two effectors, an ubiquitin extension protein (GrUBCEP12) and an expansin-like protein (GrEXPB2), suppressed defense responses including NB-LRR signaling induced in the cytoplasm. GrEXPB2 also elicited defense response in species- and sequence-specific manner. Our results are consistent with the scenario whereby potato cyst nematodes secrete effectors that modulate host cell fate and metabolism as well as modifying host cell walls. Furthermore, we show a novel role for an apoplastic expansin-like protein in suppressing intra-cellular defense responses. PMID:25606855

  14. Bacterial effector binds host cell adenylyl cyclase to potentiate Gαs-dependent cAMP production.

    PubMed

    Pulliainen, Arto T; Pieles, Kathrin; Brand, Cameron S; Hauert, Barbara; Böhm, Alex; Quebatte, Maxime; Wepf, Alexander; Gstaiger, Matthias; Aebersold, Ruedi; Dessauer, Carmen W; Dehio, Christoph

    2012-06-12

    Subversion of host organism cAMP signaling is an efficient and widespread mechanism of microbial pathogenesis. Bartonella effector protein A (BepA) of vasculotumorigenic Bartonella henselae protects the infected human endothelial cells against apoptotic stimuli by elevation of cellular cAMP levels by an as yet unknown mechanism. Here, adenylyl cyclase (AC) and the α-subunit of the AC-stimulating G protein (Gαs) were identified as potential cellular target proteins for BepA by gel-free proteomics. Results of the proteomics screen were evaluated for physical and functional interaction by: (i) a heterologous in vivo coexpression system, where human AC activity was reconstituted under the regulation of Gαs and BepA in Escherichia coli; (ii) in vitro AC assays with membrane-anchored full-length human AC and recombinant BepA and Gαs; (iii) surface plasmon resonance experiments; and (iv) an in vivo fluorescence bimolecular complementation-analysis. The data demonstrate that BepA directly binds host cell AC to potentiate the Gαs-dependent cAMP production. As opposed to the known microbial mechanisms, such as ADP ribosylation of G protein α-subunits by cholera and pertussis toxins, the fundamentally different BepA-mediated elevation of host cell cAMP concentration appears subtle and is dependent on the stimulus of a G protein-coupled receptor-released Gαs. We propose that this mechanism contributes to the persistence of Bartonella henselae in the chronically infected vascular endothelium.

  15. Bacterial effector binds host cell adenylyl cyclase to potentiate Gαs-dependent cAMP production

    PubMed Central

    Pulliainen, Arto T.; Pieles, Kathrin; Brand, Cameron S.; Hauert, Barbara; Böhm, Alex; Quebatte, Maxime; Wepf, Alexander; Gstaiger, Matthias; Aebersold, Ruedi; Dessauer, Carmen W.; Dehio, Christoph

    2012-01-01

    Subversion of host organism cAMP signaling is an efficient and widespread mechanism of microbial pathogenesis. Bartonella effector protein A (BepA) of vasculotumorigenic Bartonella henselae protects the infected human endothelial cells against apoptotic stimuli by elevation of cellular cAMP levels by an as yet unknown mechanism. Here, adenylyl cyclase (AC) and the α-subunit of the AC-stimulating G protein (Gαs) were identified as potential cellular target proteins for BepA by gel-free proteomics. Results of the proteomics screen were evaluated for physical and functional interaction by: (i) a heterologous in vivo coexpression system, where human AC activity was reconstituted under the regulation of Gαs and BepA in Escherichia coli; (ii) in vitro AC assays with membrane-anchored full-length human AC and recombinant BepA and Gαs; (iii) surface plasmon resonance experiments; and (iv) an in vivo fluorescence bimolecular complementation-analysis. The data demonstrate that BepA directly binds host cell AC to potentiate the Gαs-dependent cAMP production. As opposed to the known microbial mechanisms, such as ADP ribosylation of G protein α-subunits by cholera and pertussis toxins, the fundamentally different BepA-mediated elevation of host cell cAMP concentration appears subtle and is dependent on the stimulus of a G protein-coupled receptor-released Gαs. We propose that this mechanism contributes to the persistence of Bartonella henselae in the chronically infected vascular endothelium. PMID:22635269

  16. 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

  17. Broadly Conserved Fungal Effector BEC1019 Suppresses Host Cell Death and Enhances Pathogen Virulence in Powdery Mildew of Barley (Hordeum vulgare L.).

    PubMed

    Whigham, Ehren; Qi, Shan; Mistry, Divya; Surana, Priyanka; Xu, Ruo; Fuerst, Gregory; Pliego, Clara; Bindschedler, Laurence V; Spanu, Pietro D; Dickerson, Julie A; Innes, Roger W; Nettleton, Dan; Bogdanove, Adam J; Wise, Roger P

    2015-09-01

    The interaction of barley, Hordeum vulgare L., with the powdery mildew fungus Blumeria graminis f. sp. hordei is a well-developed model to investigate resistance and susceptibility to obligate biotrophic pathogens. The 130-Mb Blumeria genome encodes approximately 540 predicted effectors that are hypothesized to suppress or induce host processes to promote colonization. Blumeria effector candidate (BEC)1019, a single-copy gene encoding a putative, secreted metalloprotease, is expressed in haustorial feeding structures, and host-induced gene silencing of BEC1019 restricts haustorial development in compatible interactions. Here, we show that Barley stripe mosaic virus-induced gene silencing of BEC1019 significantly reduces fungal colonization of barley epidermal cells, demonstrating that BEC1019 plays a central role in virulence. In addition, delivery of BEC1019 to the host cytoplasm via Xanthomonas type III secretion suppresses cultivar nonspecific hypersensitive reaction (HR) induced by Xanthomonas oryzae pv. oryzicola, as well as cultivar-specific HR induced by AvrPphB from Pseudomonas syringae pv. phaseolicola. BEC1019 homologs are present in 96 of 241 sequenced fungal genomes, including plant pathogens, human pathogens, and free-living nonpathogens. Comparative analysis revealed variation at several amino acid positions that correlate with fungal lifestyle and several highly conserved, noncorrelated motifs. Site-directed mutagenesis of one of these, ETVIC, compromises the HR-suppressing activity of BEC1019. We postulate that BEC1019 represents an ancient, broadly important fungal protein family, members of which have evolved to function as effectors in plant and animal hosts.

  18. Phytophthora infestans effector AVR3a is essential for virulence and manipulates plant immunity by stabilizing host E3 ligase CMPG1.

    PubMed

    Bos, Jorunn I B; Armstrong, Miles R; Gilroy, Eleanor M; Boevink, Petra C; Hein, Ingo; Taylor, Rosalind M; Zhendong, Tian; Engelhardt, Stefan; Vetukuri, Ramesh R; Harrower, Brian; Dixelius, Christina; Bryan, Glenn; Sadanandom, Ari; Whisson, Stephen C; Kamoun, Sophien; Birch, Paul R J

    2010-05-25

    Fungal and oomycete plant pathogens translocate effector proteins into host cells to establish infection. However, virulence targets and modes of action of their effectors are unknown. Effector AVR3a from potato blight pathogen Phytophthora infestans is translocated into host cells and occurs in two forms: AVR3a(KI), which is detected by potato resistance protein R3a, strongly suppresses infestin 1 (INF1)-triggered cell death (ICD), whereas AVR3a(EM), which evades recognition by R3a, weakly suppresses host ICD. Here we show that AVR3a interacts with and stabilizes host U-box E3 ligase CMPG1, which is required for ICD. In contrast, AVR3a(KI/Y147del), a mutant with a deleted C-terminal tyrosine residue that fails to suppress ICD, cannot interact with or stabilize CMPG1. CMPG1 is stabilized by the inhibitors MG132 and epoxomicin, indicating that it is degraded by the 26S proteasome. CMPG1 is degraded during ICD. However, it is stabilized by mutations in the U-box that prevent its E3 ligase activity. In stabilizing CMPG1, AVR3a thus modifies its normal activity. Remarkably, given the potential for hundreds of effector genes in the P. infestans genome, silencing Avr3a compromises P. infestans pathogenicity, suggesting that AVR3a is essential for virulence. Interestingly, Avr3a silencing can be complemented by in planta expression of Avr3a(KI) or Avr3a(EM) but not the Avr3a(KI/Y147del) mutant. Our data provide genetic evidence that AVR3a is an essential virulence factor that targets and stabilizes the plant E3 ligase CMPG1, potentially to prevent host cell death during the biotrophic phase of infection.

  19. 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.

  20. Computational prediction of secretion systems and secretomes of Brucella: identification of novel type IV effectors and their interaction with the host.

    PubMed

    Sankarasubramanian, Jagadesan; Vishnu, Udayakumar S; Dinakaran, Vasudevan; Sridhar, Jayavel; Gunasekaran, Paramasamy; Rajendhran, Jeyaprakash

    2016-01-01

    Brucella spp. are facultative intracellular pathogens that cause brucellosis in various mammals including humans. Brucella survive inside the host cells by forming vacuoles and subverting host defence systems. This study was aimed to predict the secretion systems and the secretomes of Brucella spp. from 39 complete genome sequences available in the databases. Furthermore, an attempt was made to identify the type IV secretion effectors and their interactions with host proteins. We predicted the secretion systems of Brucella by the KEGG pathway and SecReT4. Brucella secretomes and type IV effectors (T4SEs) were predicted through genome-wide screening using JVirGel and S4TE, respectively. Protein-protein interactions of Brucella T4SEs with their hosts were analyzed by HPIDB 2.0. Genes coding for Sec and Tat pathways of secretion and type I (T1SS), type IV (T4SS) and type V (T5SS) secretion systems were identified and they are conserved in all the species of Brucella. In addition to the well-known VirB operon coding for the type IV secretion system (T4SS), we have identified the presence of additional genes showing homology with T4SS of other organisms. On the whole, 10.26 to 14.94% of total proteomes were found to be either secreted (secretome) or membrane associated (membrane proteome). Approximately, 1.7 to 3.0% of total proteomes were identified as type IV secretion effectors (T4SEs). Prediction of protein-protein interactions showed 29 and 36 host-pathogen specific interactions between Bos taurus (cattle)-B. abortus and Ovis aries (sheep)-B. melitensis, respectively. Functional characterization of the predicted T4SEs and their interactions with their respective hosts may reveal the secrets of host specificity of Brucella.

  1. Effector Protein Cig2 Decreases Host Tolerance of Infection by Directing Constitutive Fusion of Autophagosomes with the Coxiella-Containing Vacuole

    PubMed Central

    Kohler, Lara J.; Reed, Shawna R.; Sarraf, Shireen A.; Arteaga, David D.

    2016-01-01

    ABSTRACT Coxiella burnetii replicates in an acidified lysosome-derived vacuole. Biogenesis of the Coxiella-containing vacuole (CCV) requires bacterial effector proteins delivered into host cells by the Dot/Icm secretion system. Genetic and cell biological analysis revealed that an effector protein called Cig2 promotes constitutive fusion of autophagosomes with the CCV to maintain this compartment in an autolysosomal stage of maturation. This distinguishes the CCV from other pathogen-containing vacuoles that are targeted by the host autophagy pathway, which typically confers host resistance to infection by delivering the pathogen to a toxic lysosomal environment. By maintaining the CCV in an autolysosomal stage of maturation, Cig2 enabled CCV homotypic fusion and enhanced bacterial virulence in the Galleria mellonella (wax moth) model of infection by a mechanism that decreases host tolerance. Thus, C. burnetii residence in an autolysosomal organelle alters host tolerance of infection, which indicates that Cig2-dependent manipulation of a lysosome-derived vacuole influences the host response to infection. PMID:27435465

  2. Lack of the programmed death-1 receptor renders host susceptible to enteric microbial infection through impairing the production of the mucosal natural killer cell effector molecules.

    PubMed

    Solaymani-Mohammadi, Shahram; Lakhdari, Omar; Minev, Ivelina; Shenouda, Steve; Frey, Blake F; Billeskov, Rolf; Singer, Steven M; Berzofsky, Jay A; Eckmann, Lars; Kagnoff, Martin F

    2016-03-01

    The programmed death-1 receptor is expressed on a wide range of immune effector cells, including T cells, natural killer T cells, dendritic cells, macrophages, and natural killer cells. In malignancies and chronic viral infections, increased expression of programmed death-1 by T cells is generally associated with a poor prognosis. However, its role in early host microbial defense at the intestinal mucosa is not well understood. We report that programmed death-1 expression is increased on conventional natural killer cells but not on CD4(+), CD8(+) or natural killer T cells, or CD11b(+) or CD11c(+) macrophages or dendritic cells after infection with the mouse pathogen Citrobacter rodentium. Mice genetically deficient in programmed death-1 or treated with anti-programmed death-1 antibody were more susceptible to acute enteric and systemic infection with Citrobacter rodentium. Wild-type but not programmed death-1-deficient mice infected with Citrobacter rodentium showed significantly increased expression of the conventional mucosal NK cell effector molecules granzyme B and perforin. In contrast, natural killer cells from programmed death-1-deficient mice had impaired expression of those mediators. Consistent with programmed death-1 being important for intracellular expression of natural killer cell effector molecules, mice depleted of natural killer cells and perforin-deficient mice manifested increased susceptibility to acute enteric infection with Citrobacter rodentium. Our findings suggest that increased programmed death-1 signaling pathway expression by conventional natural killer cells promotes host protection at the intestinal mucosa during acute infection with a bacterial gut pathogen by enhancing the expression and production of important effectors of natural killer cell function.

  3. 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

  4. Dual roles for the variable domain in protein trafficking and host-specific recognition of Heterodera glycines CLE effector proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean cyst nematodes (Heterodera glycines) produce secreted effector proteins that function as peptide mimics of plant CLAVATA3 / ESR (CLE)-like peptides probably involved in the developmental reprogramming of root cells to form specialized feeding cells called syncytia. The site of action and me...

  5. Quantification of disease expression conferred by three host gene-necrotrophic effector interactions in the wheat-Parastagonospora nodorum pathosystem

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Septoria nodorum blotch (SNB), which is a major foliar disease on wheat is caused by the necrotrophic fungus Parastagonospora nodorum. The wheat-P. nodorum pathosystem involves the recognition of necrotrophic effectors (NEs) secreted by P. nodorum by corresponding wheat NE sensitivity genes. Recogni...

  6. CRN13 candidate effectors from plant and animal eukaryotic pathogens are DNA-binding proteins which trigger host DNA damage response.

    PubMed

    Ramirez-Garcés, Diana; Camborde, Laurent; Pel, Michiel J C; Jauneau, Alain; Martinez, Yves; Néant, Isabelle; Leclerc, Catherine; Moreau, Marc; Dumas, Bernard; Gaulin, Elodie

    2016-04-01

    To successfully colonize their host, pathogens produce effectors that can interfere with host cellular processes. Here we investigated the function of CRN13 candidate effectors produced by plant pathogenic oomycetes and detected in the genome of the amphibian pathogenic chytrid fungus Batrachochytrium dendrobatidis (BdCRN13). When expressed in Nicotiana, AeCRN13, from the legume root pathogen Aphanomyces euteiches, increases the susceptibility of the leaves to the oomycete Phytophthora capsici. When transiently expressed in amphibians or plant cells, AeCRN13 and BdCRN13 localize to the cell nuclei, triggering aberrant cell development and eventually causing cell death. Using Förster resonance energy transfer experiments in plant cells, we showed that both CRN13s interact with nuclear DNA and trigger plant DNA damage response (DDR). Mutating key amino acid residues in a predicted HNH-like endonuclease motif abolished the interaction of AeCRN13 with DNA, the induction of DDR and the enhancement of Nicotiana susceptibility to P. capsici. Finally, H2AX phosphorylation, a marker of DNA damage, and enhanced expression of genes involved in the DDR were observed in A. euteiches-infected Medicago truncatula roots. These results show that CRN13 from plant and animal eukaryotic pathogens promotes host susceptibility by targeting nuclear DNA and inducing DDR.

  7. Human subtilase SKI-1/S1P is a master regulator of the HCV Lifecycle and a potential host cell target for developing indirect-acting antiviral agents.

    PubMed

    Olmstead, Andrea D; Knecht, Wolfgang; Lazarov, Ina; Dixit, Surjit B; Jean, François

    2012-01-01

    HCV infection is a major risk factor for liver cancer and liver transplantation worldwide. Overstimulation of host lipid metabolism in the liver by HCV-encoded proteins during viral infection creates a favorable environment for virus propagation and pathogenesis. In this study, we hypothesize that targeting cellular enzymes acting as master regulators of lipid homeostasis could represent a powerful approach to developing a novel class of broad-spectrum antivirals against infection associated with human Flaviviridae viruses such as hepatitis C virus (HCV), whose assembly and pathogenesis depend on interaction with lipid droplets (LDs). One such master regulator of cholesterol metabolic pathways is the host subtilisin/kexin-isozyme-1 (SKI-1)--or site-1 protease (S1P). SKI-1/S1P plays a critical role in the proteolytic activation of sterol regulatory element binding proteins (SREBPs), which control expression of the key enzymes of cholesterol and fatty-acid biosynthesis. Here we report the development of a SKI-1/S1P-specific protein-based inhibitor and its application to blocking the SREBP signaling cascade. We demonstrate that SKI-1/S1P inhibition effectively blocks HCV from establishing infection in hepatoma cells. The inhibitory mechanism is associated with a dramatic reduction in the abundance of neutral lipids, LDs, and the LD marker: adipose differentiation-related protein (ADRP)/perilipin 2. Reduction of LD formation inhibits virus assembly from infected cells. Importantly, we confirm that SKI-1/S1P is a key host factor for HCV infection by using a specific active, site-directed, small-molecule inhibitor of SKI-1/S1P: PF-429242. Our studies identify SKI-1/S1P as both a novel regulator of the HCV lifecycle and as a potential host-directed therapeutic target against HCV infection and liver steatosis. With identification of an increasing number of human viruses that use host LDs for infection, our results suggest that SKI-1/S1P inhibitors may allow development of

  8. 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.

  9. Insect-induced effects on plants and possible effectors used by galling and leaf-mining insects to manipulate their host-plant.

    PubMed

    Giron, David; Huguet, Elisabeth; Stone, Graham N; Body, Mélanie

    2016-01-01

    Gall-inducing insects are iconic examples in the manipulation and reprogramming of plant development, inducing spectacular morphological and physiological changes of host-plant tissues within which the insect feeds and grows. Despite decades of research, effectors involved in gall induction and basic mechanisms of gall formation remain unknown. Recent research suggests that some aspects of the plant manipulation shown by gall-inducers may be shared with other insect herbivorous life histories. Here, we illustrate similarities and contrasts by reviewing current knowledge of metabolic and morphological effects induced on plants by gall-inducing and leaf-mining insects, and ask whether leaf-miners can also be considered to be plant reprogrammers. We review key plant functions targeted by various plant reprogrammers, including plant-manipulating insects and nematodes, and functionally characterize insect herbivore-derived effectors to provide a broader understanding of possible mechanisms used in host-plant manipulation. Consequences of plant reprogramming in terms of ecology, coevolution and diversification of plant-manipulating insects are also discussed.

  10. 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

  11. Context-dependent protein folding of a virulence peptide in the bacterial and host environments: structure of an SycH–YopH chaperone–effector complex

    SciTech Connect

    Vujanac, Milos; Stebbins, C. Erec

    2013-04-01

    The structure of a SycH–YopH chaperone–effector complex from Yersinia reveals the bacterial state of a protein that adopts different folds in the host and pathogen environments. Yersinia pestis injects numerous bacterial proteins into host cells through an organic nanomachine called the type 3 secretion system. One such substrate is the tyrosine phosphatase YopH, which requires an interaction with a cognate chaperone in order to be effectively injected. Here, the first crystal structure of a SycH–YopH complex is reported, determined to 1.9 Å resolution. The structure reveals the presence of (i) a nonglobular polypeptide in YopH, (ii) a so-called β-motif in YopH and (iii) a conserved hydrophobic patch in SycH that recognizes the β-motif. Biochemical studies establish that the β-motif is critical to the stability of this complex. Finally, since previous work has shown that the N-terminal portion of YopH adopts a globular fold that is functional in the host cell, aspects of how this polypeptide adopts radically different folds in the host and in the bacterial environments are analysed.

  12. Characterization of effector mechanisms at the host:parasite interface during the immune response to tissue-dwelling intestinal nematode parasites

    PubMed Central

    Patel, Nirav; Kreider, Timothy; Urban, Joseph F.; Gause, William C.

    2010-01-01

    The protective immune response that develops following infection with many tissue-dwelling intestinal nematode parasites is characterized by elevations in IL-4 and IL-13 and increased numbers of CD4+ T cells, granulocytes and macrophages. These cells accumulate at the site of infection and in many cases can mediate resistance to these large multicellular pathogens. Recent studies suggest novel potential mechanisms mediated by these immune cell populations through their differential activation and ability to stimulate production of novel effector molecules. These newly discovered protective mechanisms may provide novel strategies to develop immunotherapies and vaccines against this group of pathogens. In this review, we will examine recent studies elucidating mechanisms of host protection against three widely-used experimental murine models of tissue-dwelling intestinal nematode parasites: Heligmosomoides polygyrus, Trichuris muris and Trichinella spiralis. PMID:18804113

  13. Host-selective toxins, Ptr ToxA and Ptr ToxB, as necrotrophic effectors in the Pyrenophora tritici-repentis-wheat interaction.

    PubMed

    Ciuffetti, Lynda M; Manning, Viola A; Pandelova, Iovanna; Betts, Melania Figueroa; Martinez, J Patrick

    2010-09-01

    Host-selective toxins (HSTs) are effectors produced by some necrotrophic pathogenic fungi that typically confer the ability to cause disease. Often, diseases caused by pathogens that produce HSTs follow an inverse gene-for-gene model where toxin production is required for the ability to cause disease and a single locus in the host is responsible for toxin sensitivity and disease susceptibility. Pyrenophora tritici-repentis represents an ideal pathogen for studying the biological significance of such inverse gene-for-gene interactions, because it displays a complex race structure based on its production of multiple HSTs. Ptr ToxA and Ptr ToxB are two proteinaceous HSTs produced by P. tritici-repentis that are structurally unrelated and appear to evoke different host responses, yet each toxin confers the ability to cause disease. This review will summarize the current knowledge of how these two dissimilar HSTs display distinct modes of action, yet each confers pathogenicity to P. tritici-repentis.

  14. 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

  15. Influence of NleH effector expression, host genetics, and inflammation on Citrobacter rodentium colonization of mice.

    PubMed

    Feuerbacher, Leigh Ann; Hardwidge, Philip R

    2014-05-01

    The Escherichia coli NleH1 and NleH2 virulence proteins differentially regulate host transcription of innate immunity genes. The mouse pathogen Citrobacter rodentium encodes one NleH protein, which functions equivalently to E. coli NleH1. We examined the impact of host genetics and intestinal inflammation on the contribution of NleH to C. rodentium colonization of mice differing in LPS responsiveness. NleH expression was detrimental to C. rodentium in C57BL/10ScNJ mice, which do not mount LPS-induced inflammatory responses. This phenotype was reversed if inflammation was induced by chemical means. C. rodentium that expressed both E. coli NleH1 and NleH2 was hypervirulent in C3H/HeJ mice.

  16. Host-Adaptation of Francisella tularensis Alters the Bacterium's Surface-Carbohydrates to Hinder Effectors of Innate and Adaptive Immunity

    PubMed Central

    Zarrella, Tiffany M.; Singh, Anju; Bitsaktsis, Constantine; Rahman, Tabassum; Sahay, Bikash; Feustel, Paul J.; Gosselin, Edmund J.; Sellati, Timothy J.; Hazlett, Karsten R. O.

    2011-01-01

    Background The gram-negative bacterium Francisella tularensis survives in arthropods, fresh water amoeba, and mammals with both intracellular and extracellular phases and could reasonably be expected to express distinct phenotypes in these environments. The presence of a capsule on this bacterium has been controversial with some groups finding such a structure while other groups report that no capsule could be identified. Previously we reported in vitro culture conditions for this bacterium which, in contrast to typical methods, yielded a bacterial phenotype that mimics that of the bacterium's mammalian, extracellular phase. Methods/Findings SDS-PAGE and carbohydrate analysis of differentially-cultivated F. tularensis LVS revealed that bacteria displaying the host-adapted phenotype produce both longer polymers of LPS O-antigen (OAg) and additional HMW carbohydrates/glycoproteins that are reduced/absent in non-host-adapted bacteria. Analysis of wildtype and OAg-mutant bacteria indicated that the induced changes in surface carbohydrates involved both OAg and non-OAg species. To assess the impact of these HMW carbohydrates on the access of outer membrane constituents to antibody we used differentially-cultivated bacteria in vitro to immunoprecipitate antibodies directed against outer membrane moieties. We observed that the surface-carbohydrates induced during host–adaptation shield many outer membrane antigens from binding by antibody. Similar assays with normal mouse serum indicate that the induced HMW carbohydrates also impede complement deposition. Using an in vitro macrophage infection assay, we find that the bacterial HMW carbohydrate impedes TLR2-dependent, pro-inflammatory cytokine production by macrophages. Lastly we show that upon host-adaptation, the human-virulent strain, F. tularensis SchuS4 also induces capsule production with the effect of reducing macrophage-activation and accelerating tularemia pathogenesis in mice. Conclusion F. tularensis undergoes

  17. Immunosuppressive Yersinia Effector YopM Binds DEAD Box Helicase DDX3 to Control Ribosomal S6 Kinase in the Nucleus of Host Cells

    PubMed Central

    Rumm, Andreas; Trasak, Claudia; Ruckdeschel, Klaus; Alawi, Malik; Grundhoff, Adam; Kikhney, Alexey G.; Koch-Nolte, Friedrich; Buck, Friedrich; Perbandt, Markus; Betzel, Christian; Svergun, Dmitri I.; Hentschke, Moritz; Aepfelbacher, Martin

    2016-01-01

    Yersinia outer protein M (YopM) is a crucial immunosuppressive effector of the plaque agent Yersinia pestis and other pathogenic Yersinia species. YopM enters the nucleus of host cells but neither the mechanisms governing its nucleocytoplasmic shuttling nor its intranuclear activities are known. Here we identify the DEAD-box helicase 3 (DDX3) as a novel interaction partner of Y. enterocolitica YopM and present the three-dimensional structure of a YopM:DDX3 complex. Knockdown of DDX3 or inhibition of the exportin chromosomal maintenance 1 (CRM1) increased the nuclear level of YopM suggesting that YopM exploits DDX3 to exit the nucleus via the CRM1 export pathway. Increased nuclear YopM levels caused enhanced phosphorylation of Ribosomal S6 Kinase 1 (RSK1) in the nucleus. In Y. enterocolitica infected primary human macrophages YopM increased the level of Interleukin-10 (IL-10) mRNA and this effect required interaction of YopM with RSK and was enhanced by blocking YopM's nuclear export. We propose that the DDX3/CRM1 mediated nucleocytoplasmic shuttling of YopM determines the extent of phosphorylation of RSK in the nucleus to control transcription of immunosuppressive cytokines. PMID:27300509

  18. 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.

  19. New Insights into the Roles of Host Gene-Necrotrophic Effector Interactions in Governing Susceptibility of Durum Wheat to Tan Spot and Septoria nodorum Blotch

    PubMed Central

    Virdi, Simerjot K.; Liu, Zhaohui; Overlander, Megan E.; Zhang, Zengcui; Xu, Steven S.; Friesen, Timothy L.; Faris, Justin D.

    2016-01-01

    Tan spot and Septoria nodorum blotch (SNB) are important diseases of wheat caused by the necrotrophic fungi Pyrenophora tritici-repentis and Parastagonospora nodorum, respectively. The P. tritici-repentis necrotrophic effector (NE) Ptr ToxB causes tan spot when recognized by the Tsc2 gene. The NE ToxA is produced by both pathogens and has been associated with the development of both tan spot and SNB when recognized by the wheat Tsn1 gene. Most work to study these interactions has been conducted in common wheat, but little has been done in durum wheat. Here, quantitative trait loci (QTL) analysis of a segregating biparental population indicated that the Tsc2-Ptr ToxB interaction plays a prominent role in the development of tan spot in durum. However, analysis of two biparental populations indicated that the Tsn1-ToxA interaction was not associated with the development of tan spot, but was strongly associated with the development of SNB. Pa. nodorum expressed ToxA at high levels in infected Tsn1 plants, whereas ToxA expression in P. tritici-repentis was barely detectable, suggesting that the differences in disease levels associated with the Tsn1-ToxA interaction were due to differences in pathogen expression of ToxA. These and previous results together indicate that: (1) the effects of Tsn1-ToxA on tan spot in common wheat can range from nonsignificant to highly significant depending on the host genetic background; (2) Tsn1-ToxA is not a significant factor for tan spot development in durum wheat; and (3) Tsn1-ToxA plays a major role in SNB development in both common and durum wheat. Durum and common wheat breeders alike should strive to remove both Tsc2 and Tsn1 from their materials to achieve disease resistance. PMID:27777262

  20. New Insights into the Roles of Host Gene-Necrotrophic Effector Interactions in Governing Susceptibility of Durum Wheat to Tan Spot and Septoria nodorum Blotch.

    PubMed

    Virdi, Simerjot K; Liu, Zhaohui; Overlander, Megan E; Zhang, Zengcui; Xu, Steven S; Friesen, Timothy L; Faris, Justin D

    2016-12-07

    Tan spot and Septoria nodorum blotch (SNB) are important diseases of wheat caused by the necrotrophic fungi Pyrenophora tritici-repentis and Parastagonospora nodorum, respectively. The P. tritici-repentis necrotrophic effector (NE) Ptr ToxB causes tan spot when recognized by the Tsc2 gene. The NE ToxA is produced by both pathogens and has been associated with the development of both tan spot and SNB when recognized by the wheat Tsn1 gene. Most work to study these interactions has been conducted in common wheat, but little has been done in durum wheat. Here, quantitative trait loci (QTL) analysis of a segregating biparental population indicated that the Tsc2-Ptr ToxB interaction plays a prominent role in the development of tan spot in durum. However, analysis of two biparental populations indicated that the Tsn1-ToxA interaction was not associated with the development of tan spot, but was strongly associated with the development of SNB. Pa. nodorum expressed ToxA at high levels in infected Tsn1 plants, whereas ToxA expression in P. tritici-repentis was barely detectable, suggesting that the differences in disease levels associated with the Tsn1-ToxA interaction were due to differences in pathogen expression of ToxA These and previous results together indicate that: (1) the effects of Tsn1-ToxA on tan spot in common wheat can range from nonsignificant to highly significant depending on the host genetic background; (2) Tsn1-ToxA is not a significant factor for tan spot development in durum wheat; and (3) Tsn1-ToxA plays a major role in SNB development in both common and durum wheat. Durum and common wheat breeders alike should strive to remove both Tsc2 and Tsn1 from their materials to achieve disease resistance.

  1. The lectin receptor kinase LecRK-I.9 is a novel Phytophthora resistance component and a potential host target for a RXLR effector.

    PubMed

    Bouwmeester, Klaas; de Sain, Mara; Weide, Rob; Gouget, Anne; Klamer, Sofieke; Canut, Herve; Govers, Francine

    2011-03-01

    In plants, an active defense against biotrophic pathogens is dependent on a functional continuum between the cell wall (CW) and the plasma membrane (PM). It is thus anticipated that proteins maintaining this continuum also function in defense. The legume-like lectin receptor kinase LecRK-I.9 is a putative mediator of CW-PM adhesions in Arabidopsis and is known to bind in vitro to the Phytophthora infestans RXLR-dEER effector IPI-O via a RGD cell attachment motif present in IPI-O. Here we show that LecRK-I.9 is associated with the plasma membrane, and that two T-DNA insertions lines deficient in LecRK-I.9 (lecrk-I.9) have a 'gain-of-susceptibility' phenotype specifically towards the oomycete Phytophthora brassicae. Accordingly, overexpression of LecRK-I.9 leads to enhanced resistance to P. brassicae. A similar 'gain-of-susceptibility' phenotype was observed in transgenic Arabidopsis lines expressing ipiO (35S-ipiO1). This phenocopy behavior was also observed with respect to other defense-related functions; lecrk-I.9 and 35S-ipiO1 were both disturbed in pathogen- and MAMP-triggered callose deposition. By site-directed mutagenesis, we demonstrated that the RGD cell attachment motif in IPI-O is not only essential for disrupting the CW-PM adhesions, but also for disease suppression. These results suggest that destabilizing the CW-PM continuum is one of the tactics used by Phytophthora to promote infection. As countermeasure the host may want to strengthen CW-PM adhesions and the novel Phytophthora resistance component LecRK-I.9 seems to function in this process.

  2. The Lectin Receptor Kinase LecRK-I.9 Is a Novel Phytophthora Resistance Component and a Potential Host Target for a RXLR Effector

    PubMed Central

    Bouwmeester, Klaas; de Sain, Mara; Weide, Rob; Gouget, Anne; Klamer, Sofieke; Canut, Herve; Govers, Francine

    2011-01-01

    In plants, an active defense against biotrophic pathogens is dependent on a functional continuum between the cell wall (CW) and the plasma membrane (PM). It is thus anticipated that proteins maintaining this continuum also function in defense. The legume-like lectin receptor kinase LecRK-I.9 is a putative mediator of CW-PM adhesions in Arabidopsis and is known to bind in vitro to the Phytophthora infestans RXLR-dEER effector IPI-O via a RGD cell attachment motif present in IPI-O. Here we show that LecRK-I.9 is associated with the plasma membrane, and that two T-DNA insertions lines deficient in LecRK-I.9 (lecrk-I.9) have a ‘gain-of-susceptibility’ phenotype specifically towards the oomycete Phytophthora brassicae. Accordingly, overexpression of LecRK-I.9 leads to enhanced resistance to P. brassicae. A similar ‘gain-of-susceptibility’ phenotype was observed in transgenic Arabidopsis lines expressing ipiO (35S-ipiO1). This phenocopy behavior was also observed with respect to other defense-related functions; lecrk-I.9 and 35S-ipiO1 were both disturbed in pathogen- and MAMP-triggered callose deposition. By site-directed mutagenesis, we demonstrated that the RGD cell attachment motif in IPI-O is not only essential for disrupting the CW-PM adhesions, but also for disease suppression. These results suggest that destabilizing the CW-PM continuum is one of the tactics used by Phytophthora to promote infection. As countermeasure the host may want to strengthen CW-PM adhesions and the novel Phytophthora resistance component LecRK-I.9 seems to function in this process. PMID:21483488

  3. Phosphorylation of HopQ1, a Type III Effector from Pseudomonas syringae, Creates a Binding Site for Host 14-3-3 Proteins1[C][W][OA

    PubMed Central

    Giska, Fabian; Lichocka, Małgorzata; Piechocki, Marcin; Dadlez, Michał; Schmelzer, Elmon; Hennig, Jacek; Krzymowska, Magdalena

    2013-01-01

    HopQ1 (for Hrp outer protein Q), a type III effector secreted by Pseudomonas syringae pv phaseolicola, is widely conserved among diverse genera of plant bacteria. It promotes the development of halo blight in common bean (Phaseolus vulgaris). However, when this same effector is injected into Nicotiana benthamiana cells, it is recognized by the immune system and prevents infection. Although the ability to synthesize HopQ1 determines host specificity, the role it plays inside plant cells remains unexplored. Following transient expression in planta, HopQ1 was shown to copurify with host 14-3-3 proteins. The physical interaction between HopQ1 and 14-3-3a was confirmed in planta using the fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy technique. Moreover, mass spectrometric analyses detected specific phosphorylation of the canonical 14-3-3 binding site (RSXpSXP, where pS denotes phosphoserine) located in the amino-terminal region of HopQ1. Amino acid substitution within this motif abrogated the association and led to altered subcellular localization of HopQ1. In addition, the mutated HopQ1 protein showed reduced stability in planta. These data suggest that the association between host 14-3-3 proteins and HopQ1 is important for modulating the properties of this bacterial effector. PMID:23396834

  4. 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.

  5. New insights into the roles of host gene-necrotrophic effector interactions in governing susceptibility of durum wheat to tan spot and Septoria nodorum blotch

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tan spot and Septoria nodorum blotch (SNB) are important diseases of wheat caused by the necrotrophic fungi Pyrenophora tritici-repentis and Parastagonospora nodorum, respectively. The P. tritici-repentis necrotrophic effector (NE) Ptr ToxB causes tan spot when recognized by the Tsc2 gene. The NE To...

  6. Shigella flexneri T3SS effectors OspB and OspF target the nucleus to down-regulate the host inflammatory response via interactions with retinoblastoma protein

    PubMed Central

    Zurawski, Daniel V.; Mumy, Karen L.; Faherty, Christina S.; McCormick, Beth A.; Maurelli, Anthony T.

    2009-01-01

    Summary OspF, OspG, and IpaH9.8 are type III secretion system (T3SS) effectors of Shigella flexneri that down-regulate the host innate immune response. OspF modifies mitogen-activated protein kinase (MAPK) pathways and polymorphonuclear leukocyte (PMN) transepithelial migration associated with Shigella invasion. OspF also localizes in the nucleus to mediate chromatin remodeling resulting in reduced transcription of inflammatory cytokines. We now report that OspB can be added to the set of S. flexneri T3SS effectors required to modulate the innate immune response. T84 cells infected with a ΔospB mutant resulted in reduced PMN transepithelial migration and MAPK signaling. Tagged versions of OspB localized with endosomes and the nucleus. Further, T84 cells infected with the ΔospB mutant showed increased levels of secreted IL-8 compared to wild-type infected cells. Both GST-OspB and GST-OspF co-precipitated retinoblastoma protein (Rb) from host cell lysates. Because ΔospB and ΔospF mutants share similar phenotypes, and OspB and OspF share a host binding partner, we propose that OspB and OspF facilitate the remodeling of chromatin via interactions with Rb resulting in diminished inflammatory cytokine production. The requirement of multiple T3SS effectors to modulate the innate immune response correlates to the complexity of the human immune system. PMID:19017275

  7. 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.

  8. A functionally conserved Zn2 Cys6 binuclear cluster transcription factor class regulates necrotrophic effector gene expression and host-specific virulence of two major Pleosporales fungal pathogens of wheat.

    PubMed

    Rybak, Kasia; See, Pao Theen; Phan, Huyen T T; Syme, Robert A; Moffat, Caroline S; Oliver, Richard P; Tan, Kar-Chun

    2017-04-01

    The fungus Parastagonospora nodorum is the causal agent of Septoria nodorum blotch of wheat (Triticum aestivum). The interaction is mediated by multiple fungal necrotrophic effector-dominant host sensitivity gene interactions. The three best-characterized effector-sensitivity gene systems are SnToxA-Tsn1, SnTox1-Snn1 and SnTox3-Snn3. These effector genes are highly expressed during early infection, but expression decreases as the infection progresses to tissue necrosis and sporulation. However, the mechanism of regulation is unknown. We have identified and functionally characterized a gene, referred to as PnPf2, which encodes a putative zinc finger transcription factor. PnPf2 deletion resulted in the down-regulation of SnToxA and SnTox3 expression. Virulence on Tsn1 and Snn3 wheat cultivars was strongly reduced. The SnTox1-Snn1 interaction remained unaffected. Furthermore, we have also identified and deleted an orthologous PtrPf2 from the tan spot fungus Pyrenophora tritici-repentis which possesses a near-identical ToxA that was acquired from P. nodorum via horizontal gene transfer. PtrPf2 deletion also resulted in the down-regulation of PtrToxA expression and a near-complete loss of virulence on Tsn1 wheat. We have demonstrated, for the first time, evidence for a functionally conserved signalling component that plays a role in the regulation of a common/horizontally transferred effector found in two major fungal pathogens of wheat.

  9. Diversion at the ER: How Plasmodium falciparum exports proteins into host erythrocytes.

    PubMed

    Römisch, Karin

    2012-01-01

    Malaria is caused by parasites which live in host erythrocytes and remodel these cells to provide optimally for the parasites' needs by exporting effector proteins into the host cells. Eight years ago the discovery of a host cell targeting sequence present in both soluble and transmembrane  P. falciparum exported proteins generated a starting point for investigating the mechanism of parasite protein transport into infected erythrocytes. Since then many confusing facts about this targeting signal have emerged. In this paper, I try to make sense of them.

  10. 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.

  11. Depletion of tumor-induced regulatory T cells prior to reconstitution rescues enhanced priming of tumor-specific, therapeutic effector T cells in lymphopenic hosts

    PubMed Central

    Poehlein, Christian H.; Haley, Daniel; Walker, Edwin; Fox, Bernard A.

    2010-01-01

    We reported previously that vaccination of reconstituted-lymphopenic mice resulted in a higher frequency of tumor-specific effector T cells with therapeutic activity than vaccination of normal mice. Here we show that lymphopenic mice reconstituted with spleen cells from tumor-bearing mouse (TBM), a situation which resembles the clinical condition, failed to generate tumor-specific T cells with therapeutic efficacy. However, depletion of CD25+ Treg from the spleen cells of TBM restored tumor-specific priming and therapeutic efficacy. Adding back TBM CD25+ Treg to CD25- naïve and TBM donor T cells prior to reconstitution confirmed their suppressive role. CD25+ Treg from TBM prevented priming of tumor-specific T cells since subsequent depletion of CD4+ T cells did not restore therapeutic efficacy. This effect may not be antigen-specific as three histologically distinct tumors generated CD25+ Treg that could suppress the T cell immune response to a melanoma vaccine. Importantly, since ex vivo depletion of CD25+ Treg from TBM spleen cells prior to reconstitution and vaccination fully restored the generation of therapeutic effector T cells, even in animals with established tumor burden, we have initiated a translational clinical trial of this strategy in patients with metastatic melanoma. PMID:19839008

  12. Orientia tsutsugamushi Ank9 is a multifunctional effector that utilizes a novel GRIP-like Golgi localization domain for Golgi-to-endoplasmic reticulum trafficking and interacts with host COPB2.

    PubMed

    Beyer, Andrea R; Rodino, Kyle G; VieBrock, Lauren; Green, Ryan S; Tegels, Brittney K; Oliver, Lee D; Marconi, Richard T; Carlyon, Jason A

    2017-01-19

    Orientia tsutsugamushi causes scrub typhus, a potentially fatal infection that afflicts 1 million people annually. This obligate intracellular bacterium boasts one of the largest microbial arsenals of ankyrin repeat-containing protein (Ank) effectors, most of which target the endoplasmic reticulum (ER) by undefined mechanisms. Ank9 is the only one proven to function during infection. Here, we demonstrate that Ank9 bears a motif that mimics the GRIP domain of eukaryotic golgins and is necessary and sufficient for its Golgi localization. Ank9 reaches the ER exclusively by retrograde trafficking from the Golgi. Consistent with this observation, it binds COPB2, a host protein that mediates Golgi-to-ER transport. Ank9 destabilizes the Golgi and ER in a Golgi localization domain-dependent manner and induces the activating transcription factor 4-dependent unfolded protein response. The Golgi is also destabilized in cells infected with O. tsutsugamushi or treated with COPB2 small interfering RNA. COPB2 reduction and/or the cellular events that it invokes, such as Golgi destabilization, benefit Orientia replication. Thus, Ank9 or bacterial negative modulation of COPB2 might contribute to the bacterium's intracellular replication. This report identifies a novel microbial Golgi localization domain, links Ank9 to the ability of O. tsutsugamushi to perturb Golgi structure, and describes the first mechanism by which any Orientia effector targets the secretory pathway.

  13. 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

  14. Dendritic Cell-Targeted Vaccines

    PubMed Central

    Cohn, Lillian; Delamarre, Lélia

    2014-01-01

    Despite significant effort, the development of effective vaccines inducing strong and durable T-cell responses against intracellular pathogens and cancer cells has remained a challenge. The initiation of effector CD8+ T-cell responses requires the presentation of peptides derived from internalized antigen on class I major histocompatibility complex molecules by dendritic cells (DCs) in a process called cross-presentation. A current strategy to enhance the effectiveness of vaccination is to deliver antigens directly to DCs. This is done via selective targeting of antigen using monoclonal antibodies directed against endocytic receptors on the surface of the DCs. In this review, we will discuss considerations relevant to the design of such vaccines: the existence of DC subsets with specialized functions, the impact of the antigen intracellular trafficking on cross-presentation, and the influence of maturation signals received by DCs on the outcome of the immune response. PMID:24910635

  15. Magnaporthe oryzae Effector AVR-Pii Helps to Establish Compatibility by Inhibition of the Rice NADP-Malic Enzyme Resulting in Disruption of Oxidative Burst and Host Innate Immunity.

    PubMed

    Singh, Raksha; Dangol, Sarmina; Chen, Yafei; Choi, Jihyun; Cho, Yoon-Seong; Lee, Jea-Eun; Choi, Mi-Ok; Jwa, Nam-Soo

    2016-05-31

    Plant disease resistance occurs as a hypersensitive response (HR) at the site of attempted pathogen invasion. This specific event is initiated in response to recognition of pathogen-associated molecular pattern (PAMP) and subsequent PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). Both PTI and ETI mechanisms are tightly connected with reactive oxygen species (ROS) production and disease resistance that involves distinct biphasic ROS production as one of its pivotal plant immune responses. This unique oxidative burst is strongly dependent on the resistant cultivars because a monophasic ROS burst is a hallmark of the susceptible cultivars. However, the cause of the differential ROS burst remains unknown. In the study here, we revealed the plausible underlying mechanism of the differential ROS burst through functional understanding of the Magnaporthe oryzae (M. oryzae) AVR effector, AVR-Pii. We performed yeast two-hybrid (Y2H) screening using AVR-Pii as bait and isolated rice NADP-malic enzyme2 (Os-NADP-ME2) as the rice target protein. To our surprise, deletion of the rice Os-NADP-ME2 gene in a resistant rice cultivar disrupted innate immunity against the rice blast fungus. Malic enzyme activity and inhibition studies demonstrated that AVR-Pii proteins specifically inhibit in vitro NADP-ME activity. Overall, we demonstrate that rice blast fungus, M. oryzae attenuates the host ROS burst via AVR-Pii-mediated inhibition of Os-NADP-ME2, which is indispensable in ROS metabolism for the innate immunity of rice. This characterization of the regulation of the host oxidative burst will help to elucidate how the products of AVR genes function associated with virulence of the pathogen.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  17. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  19. 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.

  20. 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

  1. Pseudomonas syringae Effector Avirulence Protein E Localizes to the Host Plasma Membrane and Down-Regulates the Expression of the NONRACE-SPECIFIC DISEASE RESISTANCE1/HARPIN-INDUCED1-LIKE13 Gene Required for Antibacterial Immunity in Arabidopsis1[OPEN

    PubMed Central

    Xin, Xiu-Fang; Nomura, Kinya; Ding, Xinhua; Chen, Xujun; Wang, Kun; Aung, Kyaw; Uribe, Francisco; Rosa, Bruce; Yao, Jian; Chen, Jin; He, Sheng Yang

    2015-01-01

    Many bacterial pathogens of plants and animals deliver effector proteins into host cells to promote infection. Elucidation of how pathogen effector proteins function not only is critical for understanding bacterial pathogenesis but also provides a useful tool in discovering the functions of host genes. In this study, we characterized the Pseudomonas syringae pv tomato DC3000 effector protein Avirulence Protein E (AvrE), the founding member of a widely distributed, yet functionally enigmatic, bacterial effector family. We show that AvrE is localized in the plasma membrane (PM) and PM-associated vesicle-like structures in the plant cell. AvrE contains two physically interacting domains, and the amino-terminal portion contains a PM-localization signal. Genome-wide microarray analysis indicates that AvrE, as well as the functionally redundant effector Hypersensitive response and pathogenicity-dependent Outer Protein M1, down-regulates the expression of the NONRACE-SPECIFIC DISEASE RESISTANCE1/HARPIN-INDUCED1-LIKE13 (NHL13) gene in Arabidopsis (Arabidopsis thaliana). Mutational analysis shows that NHL13 is required for plant immunity, as the nhl13 mutant plant displayed enhanced disease susceptibility. Our results defined the action site of one of the most important bacterial virulence proteins in plants and the antibacterial immunity function of the NHL13 gene. PMID:26206852

  2. 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.

  3. Bacterial effector HopF2 interacts with AvrPto and suppresses Arabidopsis innate immunity at the plasma membrane

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant pathogenic bacteria inject a cocktail of effector proteins into host plant cells to modulate the host immune response, thereby promoting pathogenicity. How or whether these effectors work cooperatively is largely unknown. The Pseudomonas syringae DC3000 effector HopF2 suppresses the host plan...

  4. 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

  5. TAL effectors and the executor R genes

    PubMed Central

    Zhang, Junli; Yin, Zhongchao; White, Frank

    2015-01-01

    Transcription activator-like (TAL) effectors are bacterial type III secretion proteins that function as transcription factors in plants during Xanthomonas/plant interactions, conditioning either host susceptibility and/or host resistance. Three types of TAL effector associated resistance (R) genes have been characterized—recessive, dominant non-transcriptional, and dominant TAL effector-dependent transcriptional based resistance. Here, we discuss the last type of R genes, whose functions are dependent on direct TAL effector binding to discrete effector binding elements in the promoters. Only five of the so-called executor R genes have been cloned, and commonalities are not clear. We have placed the protein products in two groups for conceptual purposes. Group 1 consists solely of the protein from pepper, BS3, which is predicted to have catalytic function on the basis of homology to a large conserved protein family. Group 2 consists of BS4C-R, XA27, XA10, and XA23, all of which are relatively short proteins from pepper or rice with multiple potential transmembrane domains. Group 2 members have low sequence similarity to proteins of unknown function in closely related species. Firm predictions await further experimentation on these interesting new members to the R gene repertoire, which have potential broad application in new strategies for disease resistance. PMID:26347759

  6. 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.

  7. 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.

  8. 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.

  9. Erwinia amylovora effector protein Eop1 suppresses PAMP-triggered immunity in Malus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  10. Plant-Pathogen Effectors: Cellular Probes Interfering with Plant Defenses in Spatial and Temporal Manners

    PubMed Central

    Toruño, Tania Y.; Stergiopoulos, Ioannis; Coaker, Gitta

    2017-01-01

    Plants possess large arsenals of immune receptors capable of recognizing all pathogen classes. To cause disease, pathogenic organisms must be able to overcome physical barriers, suppress or evade immune perception, and derive nutrients from host tissues. Consequently, to facilitate some of these processes, pathogens secrete effector proteins that promote colonization. This review covers recent advances in the field of effector biology, focusing on conserved cellular processes targeted by effectors from diverse pathogens. The ability of effectors to facilitate pathogen entry into the host interior, suppress plant immune perception, and alter host physiology for pathogen benefit is discussed. Pathogens also deploy effectors in a spatial and temporal manner, depending on infection stage. Recent advances have also enhanced our understanding of effectors acting in specific plant organs and tissues. Effectors are excellent cellular probes that facilitate insight into biological processes as well as key points of vulnerability in plant immune signaling networks. PMID:27359369

  11. 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

  12. 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

  13. Homologous RXLR effectors from Hyaloperonospora arabidopsidis and Phytophthora sojae suppress immunity in distantly related plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diverse pathogens secrete effector proteins into plant cells to manipulate host cellular processes. Oomycete pathogens contain very large complements of predicted effector genes defined by an RXLR host cell entry motif. The genome of Hyaloperonospora arabidopsidis (Hpa, downy mildew of Arabidopsis) ...

  14. 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

  15. 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.

  16. Structural Basis for Rab1 De-AMPylation by the Legionella pneumophila Effector SidD

    PubMed Central

    Neunuebel, M. Ramona; Pallara, Chiara; Brady, Jacqueline; Kinch, Lisa N.; Fernández-Recio, Juan; Rojas, Adriana L.; Machner, Matthias P.; Hierro, Aitor

    2013-01-01

    The covalent attachment of adenosine monophosphate (AMP) to proteins, a process called AMPylation (adenylylation), has recently emerged as a novel theme in microbial pathogenesis. Although several AMPylating enzymes have been characterized, the only known virulence protein with de-AMPylation activity is SidD from the human pathogen Legionella pneumophila. SidD de-AMPylates mammalian Rab1, a small GTPase involved in secretory vesicle transport, thereby targeting the host protein for inactivation. The molecular mechanisms underlying Rab1 recognition and de-AMPylation by SidD are unclear. Here, we report the crystal structure of the catalytic region of SidD at 1.6 Å resolution. The structure reveals a phosphatase-like fold with additional structural elements not present in generic PP2C-type phosphatases. The catalytic pocket contains a binuclear metal-binding site characteristic of hydrolytic metalloenzymes, with strong dependency on magnesium ions. Subsequent docking and molecular dynamics simulations between SidD and Rab1 revealed the interface contacts and the energetic contribution of key residues to the interaction. In conjunction with an extensive structure-based mutational analysis, we provide in vivo and in vitro evidence for a remarkable adaptation of SidD to its host cell target Rab1 which explains how this effector confers specificity to the reaction it catalyses. PMID:23696742

  17. Structural basis for Rab1 de-AMPylation by the Legionella pneumophila effector SidD.

    PubMed

    Chen, Yang; Tascón, Igor; Neunuebel, M Ramona; Pallara, Chiara; Brady, Jacqueline; Kinch, Lisa N; Fernández-Recio, Juan; Rojas, Adriana L; Machner, Matthias P; Hierro, Aitor

    2013-01-01

    The covalent attachment of adenosine monophosphate (AMP) to proteins, a process called AMPylation (adenylylation), has recently emerged as a novel theme in microbial pathogenesis. Although several AMPylating enzymes have been characterized, the only known virulence protein with de-AMPylation activity is SidD from the human pathogen Legionella pneumophila. SidD de-AMPylates mammalian Rab1, a small GTPase involved in secretory vesicle transport, thereby targeting the host protein for inactivation. The molecular mechanisms underlying Rab1 recognition and de-AMPylation by SidD are unclear. Here, we report the crystal structure of the catalytic region of SidD at 1.6 Å resolution. The structure reveals a phosphatase-like fold with additional structural elements not present in generic PP2C-type phosphatases. The catalytic pocket contains a binuclear metal-binding site characteristic of hydrolytic metalloenzymes, with strong dependency on magnesium ions. Subsequent docking and molecular dynamics simulations between SidD and Rab1 revealed the interface contacts and the energetic contribution of key residues to the interaction. In conjunction with an extensive structure-based mutational analysis, we provide in vivo and in vitro evidence for a remarkable adaptation of SidD to its host cell target Rab1 which explains how this effector confers specificity to the reaction it catalyses.

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. Legionella pneumophila, armed to the hilt: justifying the largest arsenal of effectors in the bacterial world.

    PubMed

    Ensminger, Alexander W

    2016-02-01

    Many bacterial pathogens use dedicated translocation systems to deliver arsenals of effector proteins to their hosts. Once inside the host cytosol, these effectors modulate eukaryotic cell biology to acquire nutrients, block microbial degradation, subvert host defenses, and enable pathogen transmission to other hosts. Among all bacterial pathogens studied to date, the gram-negative pathogen, Legionella pneumophila, maintains the largest arsenal of effectors, with over 330 effector proteins translocated by the Dot/Icm type IVB translocation system. In this review, I will discuss some of the recent work on understanding the consequences of this large arsenal. I will also present several models that seek to explain how L. pneumophila has acquired and subsequently maintained so many more effectors than its peers.

  6. Effectors from Wheat Rust Fungi Suppress Multiple Plant Defense Responses.

    PubMed

    Ramachandran, Sowmya R; Yin, Chuntao; Kud, Joanna; Tanaka, Kiwamu; Mahoney, Aaron K; Xiao, Fangming; Hulbert, Scot H

    2017-01-01

    Fungi that cause cereal rust diseases (genus Puccinia) are important pathogens of wheat globally. Upon infection, the fungus secretes a number of effector proteins. Although a large repository of putative effectors has been predicted using bioinformatic pipelines, the lack of available high-throughput effector screening systems has limited functional studies on these proteins. In this study, we mined the available transcriptomes of Puccinia graminis and P. striiformis to look for potential effectors that suppress host hypersensitive response (HR). Twenty small (<300 amino acids), secreted proteins, with no predicted functions were selected for the HR suppression assay using Nicotiana benthamiana, in which each of the proteins were transiently expressed and evaluated for their ability to suppress HR caused by four cytotoxic effector-R gene combinations (Cp/Rx, ATR13/RPP13, Rpt2/RPS-2, and GPA/RBP-1) and one mutated R gene-Pto(Y207D). Nine out of twenty proteins, designated Shr1 to Shr9 (suppressors of hypersensitive response), were found to suppress HR in N. benthamiana. These effectors varied in the effector-R gene defenses they suppressed, indicating these pathogens can interfere with a variety of host defense pathways. In addition to HR suppression, effector Shr7 also suppressed PAMP-triggered immune response triggered by flg22. Finally, delivery of Shr7 through Pseudomonas fluorescens EtHAn suppressed nonspecific HR induced by Pseudomonas syringae DC3000 in wheat, confirming its activity in a homologous system. Overall, this study provides the first evidence for the presence of effectors in Puccinia species suppressing multiple plant defense responses.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  8. 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

  9. Parallel Evolution of a Type IV Secretion System in Radiating Lineages of the Host-Restricted Bacterial Pathogen Bartonella

    PubMed Central

    Engel, Philipp; Salzburger, Walter; Liesch, Marius; Chang, Chao-Chin; Maruyama, Soichi; Lanz, Christa; Calteau, Alexandra; Lajus, Aurélie; Médigue, Claudine; Schuster, Stephan C.; Dehio, Christoph

    2011-01-01

    Adaptive radiation is the rapid origination of multiple species from a single ancestor as the result of concurrent adaptation to disparate environments. This fundamental evolutionary process is considered to be responsible for the genesis of a great portion of the diversity of life. Bacteria have evolved enormous biological diversity by exploiting an exceptional range of environments, yet diversification of bacteria via adaptive radiation has been documented in a few cases only and the underlying molecular mechanisms are largely unknown. Here we show a compelling example of adaptive radiation in pathogenic bacteria and reveal their genetic basis. Our evolutionary genomic analyses of the α-proteobacterial genus Bartonella uncover two parallel adaptive radiations within these host-restricted mammalian pathogens. We identify a horizontally-acquired protein secretion system, which has evolved to target specific bacterial effector proteins into host cells as the evolutionary key innovation triggering these parallel adaptive radiations. We show that the functional versatility and adaptive potential of the VirB type IV secretion system (T4SS), and thereby translocated Bartonella effector proteins (Beps), evolved in parallel in the two lineages prior to their radiations. Independent chromosomal fixation of the virB operon and consecutive rounds of lineage-specific bep gene duplications followed by their functional diversification characterize these parallel evolutionary trajectories. Whereas most Beps maintained their ancestral domain constitution, strikingly, a novel type of effector protein emerged convergently in both lineages. This resulted in similar arrays of host cell-targeted effector proteins in the two lineages of Bartonella as the basis of their independent radiation. The parallel molecular evolution of the VirB/Bep system displays a striking example of a key innovation involved in independent adaptive processes and the emergence of bacterial pathogens

  10. Parallel evolution of a type IV secretion system in radiating lineages of the host-restricted bacterial pathogen Bartonella.

    PubMed

    Engel, Philipp; Salzburger, Walter; Liesch, Marius; Chang, Chao-Chin; Maruyama, Soichi; Lanz, Christa; Calteau, Alexandra; Lajus, Aurélie; Médigue, Claudine; Schuster, Stephan C; Dehio, Christoph

    2011-02-10

    Adaptive radiation is the rapid origination of multiple species from a single ancestor as the result of concurrent adaptation to disparate environments. This fundamental evolutionary process is considered to be responsible for the genesis of a great portion of the diversity of life. Bacteria have evolved enormous biological diversity by exploiting an exceptional range of environments, yet diversification of bacteria via adaptive radiation has been documented in a few cases only and the underlying molecular mechanisms are largely unknown. Here we show a compelling example of adaptive radiation in pathogenic bacteria and reveal their genetic basis. Our evolutionary genomic analyses of the α-proteobacterial genus Bartonella uncover two parallel adaptive radiations within these host-restricted mammalian pathogens. We identify a horizontally-acquired protein secretion system, which has evolved to target specific bacterial effector proteins into host cells as the evolutionary key innovation triggering these parallel adaptive radiations. We show that the functional versatility and adaptive potential of the VirB type IV secretion system (T4SS), and thereby translocated Bartonella effector proteins (Beps), evolved in parallel in the two lineages prior to their radiations. Independent chromosomal fixation of the virB operon and consecutive rounds of lineage-specific bep gene duplications followed by their functional diversification characterize these parallel evolutionary trajectories. Whereas most Beps maintained their ancestral domain constitution, strikingly, a novel type of effector protein emerged convergently in both lineages. This resulted in similar arrays of host cell-targeted effector proteins in the two lineages of Bartonella as the basis of their independent radiation. The parallel molecular evolution of the VirB/Bep system displays a striking example of a key innovation involved in independent adaptive processes and the emergence of bacterial pathogens

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. Identification and Characterisation CRN Effectors in Phytophthora capsici Shows Modularity and Functional Diversity

    PubMed Central

    Stam, Remco; Jupe, Julietta; Howden, Andrew J. M.; Morris, Jenny A.; Boevink, Petra C.; Hedley, Pete E.; Huitema, Edgar

    2013-01-01

    Phytophthora species secrete a large array of effectors during infection of their host plants. The Crinkler (CRN) gene family encodes a ubiquitous but understudied class of effectors with possible but as of yet unknown roles in infection. To appreciate CRN effector function in Phytophthora, we devised a simple Crn gene identification and annotation pipeline to improve effector prediction rates. We predicted 84 full-length CRN coding genes and assessed CRN effector domain diversity in sequenced Oomycete genomes. These analyses revealed evidence of CRN domain innovation in Phytophthora and expansion in the Peronosporales. We performed gene expression analyses to validate and define two classes of CRN effectors, each possibly contributing to infection at different stages. CRN localisation studies revealed that P. capsici CRN effector domains target the nucleus and accumulate in specific sub-nuclear compartments. Phenotypic analyses showed that few CRN domains induce necrosis when expressed in planta and that one cell death inducing effector, enhances P. capsici virulence on Nicotiana benthamiana. These results suggest that the CRN protein family form an important class of intracellular effectors that target the host nucleus during infection. These results combined with domain expansion in hemi-biotrophic and necrotrophic pathogens, suggests specific contributions to pathogen lifestyles. This work will bolster CRN identification efforts in other sequenced oomycete species and set the stage for future functional studies towards understanding CRN effector functions. PMID:23536880

  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. 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

  18. Differential expression of candidate salivary effector proteins in field collections of Hessian fly, Mayetiola destructor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Evidence is emerging that proteins secreted by gall forming plant-parasites are the effectors responsible for systemic changes in the host plant, such as galling and nutrient tissue formation. A large number of secreted salivary gland proteins (SSGPs) that are hypothesized to be the effectors respon...

  19. Subcellular Localization of Pseudomonas syringae pv. tomato Effector Proteins in Plants.

    PubMed

    Aung, Kyaw; Xin, Xiufang; Mecey, Christy; He, Sheng Yang

    2017-01-01

    Animal and plant pathogenic bacteria use type III secretion systems to translocate proteinaceous effectors to subvert innate immunity of their host organisms. Type III secretion/effector systems are a crucial pathogenicity factor in many bacterial pathogens of plants and animals. Pseudomonas syringae pv. tomato (Pst) DC3000 injects a total of 36 protein effectors that target a variety of host proteins. Studies of a subset of Pst DC3000 effectors demonstrated that bacterial effectors, once inside the host cell, are localized to different subcellular compartments, including plasma membrane, cytoplasm, mitochondria, chloroplast, and Trans-Golgi network, to carry out their virulence functions. Identifying the subcellular localization of bacterial effector proteins in host cells could provide substantial clues to understanding the molecular and cellular basis of the virulence activities of effector proteins. In this chapter, we present methods for transient or stable expression of bacterial effector proteins in tobacco and/or Arabidopsis thaliana for live cell imaging as well as confirming the subcellular localization in plants using fluorescent organelle markers or chemical treatment.

  20. Molecular determinants of resistance activation and suppression by Phytophthora infestans effector IPI-O

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The potato late blight pathogen, Phytophthora infestans, is able to rapidly evolve to overcome resistance genes. The pathogen accomplishes this by secreting an arsenal of proteins, termed effectors, that function to modify host cells. Although hundreds of candidate effectors have been identified in ...

  1. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. ...

  3. 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.

  4. Microbe-independent entry of oomycete RxLR effectors and fungal RxLR-like effectors into plant and animal cells is specific and reproducible.

    PubMed

    Tyler, Brett M; Kale, Shiv D; Wang, Qunqing; Tao, Kai; Clark, Helen R; Drews, Kelly; Antignani, Vincenzo; Rumore, Amanda; Hayes, Tristan; Plett, Jonathan M; Fudal, Isabelle; Gu, Biao; Chen, Qinghe; Affeldt, Katharyn J; Berthier, Erwin; Fischer, Gregory J; Dou, Daolong; Shan, Weixing; Keller, Nancy P; Martin, Francis; Rouxel, Thierry; Lawrence, Christopher B

    2013-06-01

    A wide diversity of pathogens and mutualists of plant and animal hosts, including oomycetes and fungi, produce effector proteins that enter the cytoplasm of host cells. A major question has been whether or not entry by these effectors can occur independently of the microbe or requires machinery provided by the microbe. Numerous publications have documented that oomycete RxLR effectors and fungal RxLR-like effectors can enter plant and animal cells independent of the microbe. A recent reexamination of whether the RxLR domain of oomycete RxLR effectors is sufficient for microbe-independent entry into host cells concluded that the RxLR domains of Phytophthora infestans Avr3a and of P. sojae Avr1b alone are NOT sufficient to enable microbe-independent entry of proteins into host and nonhost plant and animal cells. Here, we present new, more detailed data that unambiguously demonstrate that the RxLR domain of Avr1b does show efficient and specific entry into soybean root cells and also into wheat leaf cells, at levels well above background nonspecific entry. We also summarize host cell entry experiments with a wide diversity of oomycete and fungal effectors with RxLR or RxLR-like motifs that have been independently carried out by the seven different labs that coauthored this letter. Finally we discuss possible technical reasons why specific cell entry may have been not detected by Wawra et al. (2013).

  5. 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.

  6. MorTAL Kombat: the story of defense against TAL effectors through loss-of-susceptibility

    PubMed Central

    Hutin, Mathilde; Pérez-Quintero, Alvaro L.; Lopez, Camilo; Szurek, Boris

    2015-01-01

    Many plant-pathogenic xanthomonads rely on Transcription Activator-Like (TAL) effectors to colonize their host. This particular family of type III effectors functions as specific plant transcription factors via a programmable DNA-binding domain. Upon binding to the promoters of plant disease susceptibility genes in a sequence-specific manner, the expression of these host genes is induced. However, plants have evolved specific strategies to counter the action of TAL effectors and confer resistance. One mechanism is to avoid the binding of TAL effectors by mutations of their DNA binding sites, resulting in resistance by loss-of-susceptibility. This article reviews our current knowledge of the susceptibility hubs targeted by Xanthomonas TAL effectors, possible evolutionary scenarios for plants to combat the pathogen with loss-of-function alleles, and how this knowledge can be used overall to develop new pathogen-informed breeding strategies and improve crop resistance. PMID:26236326

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  9. Hacker within! Ehrlichia chaffeensis Effector Driven Phagocyte Reprogramming Strategy

    PubMed Central

    Lina, Taslima T.; Farris, Tierra; Luo, Tian; Mitra, Shubhajit; Zhu, Bing; McBride, Jere W.

    2016-01-01

    Ehrlichia chaffeensis is a small, gram negative, obligately intracellular bacterium that preferentially infects mononuclear phagocytes. It is the etiologic agent of human monocytotropic ehrlichiosis (HME), an emerging life-threatening tick-borne zoonosis. Mechanisms by which E. chaffeensis establishes intracellular infection, and avoids host defenses are not well understood, but involve functionally relevant host-pathogen interactions associated with tandem and ankyrin repeat effector proteins. In this review, we discuss the recent advances in our understanding of the molecular and cellular mechanisms that underlie Ehrlichia host cellular reprogramming strategies that enable intracellular survival. PMID:27303657

  10. 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.

  11. 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.

  12. Elucidating the Role of Effectors in Plant-Fungal Interactions: Progress and Challenges

    PubMed Central

    Selin, Carrie; de Kievit, Teresa R.; Belmonte, Mark F.; Fernando, W. G. Dilantha

    2016-01-01

    Pathogenic fungi have diverse growth lifestyles that support fungal colonization on plants. Successful colonization and infection for all lifestyles depends upon the ability to modify living host plants to sequester the necessary nutrients required for growth and reproduction. Secretion of virulence determinants referred to as “effectors” is assumed to be the key governing factor that determines host infection and colonization. Effector proteins are capable of suppressing plant defense responses and alter plant physiology to accommodate fungal invaders. This review focuses on effector molecules of biotrophic and hemibiotrophic plant pathogenic fungi, and the mechanism required for the release and uptake of effector molecules by the fungi and plant cells, respectively. We also place emphasis on the discovery of effectors, difficulties associated with predicting the effector repertoire, and fungal genomic features that have helped promote effector diversity leading to fungal evolution. We discuss the role of specific effectors found in biotrophic and hemibiotrophic fungi and examine how CRISPR/Cas9 technology may provide a new avenue for accelerating our ability in the discovery of fungal effector function. PMID:27199930

  13. Studying the Mechanism of Plasmopara viticola RxLR Effectors on Suppressing Plant Immunity

    PubMed Central

    Xiang, Jiang; Li, Xinlong; Wu, Jiao; Yin, Ling; Zhang, Yali; Lu, Jiang

    2016-01-01

    The RxLR effector family, produced by oomycete pathogens, may manipulate host physiological and biochemical events inside host cells. A group of putative RxLR effectors from Plasmopara viticola have been recently identified by RNA-Seq analysis in our lab. However, their roles in pathogenesis are poorly understood. In this study, we attempted to characterize 23 PvRxLR effector candidates identified from a P. viticola isolate “ZJ-1-1.” During host infection stages, expression patterns of the effector genes were varied and could be categorized into four different groups. By using transient expression assays in Nicotiana benthamiana, we found that 17 of these effector candidates fully suppressed programmed cell death elicited by a range of cell death-inducing proteins, including BAX, INF1, PsCRN63, PsojNIP, PvRxLR16 and R3a/Avr3a. We also discovered that all these PvRxLRs could target the plant cell nucleus, except for PvRxLR55 that localized to the membrane. Furthermore, we identified a single effector, PvRxLR28, that showed the highest expression level at 6 hpi. Functional analysis revealed that PvRxLR28 could significantly enhance susceptibilities of grapevine and tobacco to pathogens. These results suggest that most P. viticola effectors tested in this study may act as broad suppressors of cell death to manipulate immunity in plant. PMID:27242731

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. SnTox1, a Parastagonospora nodorum necrotrophic effector, is a dual function protein that facilitates infection while protecting from wheat-produced chitinases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    All fungal plant pathogens produce effectors to manipulate the plant immune system to colonize and gain nutrients from the plant cell. Much is known about how fungal pathogens classified as biotrophs use effectors to interact with their hosts and how the host responds, however, less is known about ...

  19. 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

  20. Tomato immune receptor Ve1 recognizes effector of multiple fungal pathogens uncovered by genome and RNA sequencing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fungal plant pathogens secrete effector molecules to establish disease on their hosts, while plants in turn utilize immune receptors to intercept these effectors. The tomato immune receptor Ve1 governs resistance to race 1 strains of the soil-borne vascular wilt fungi Verticillium dahliae and V. alb...

  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. Structure of NS1A effector domain from the influenza A/Udorn/72 virus

    SciTech Connect

    Xia, Shuangluo; Monzingo, Arthur F.; Robertus, Jon D.

    2009-01-01

    The structure of the effector domain of the influenza protein NS1, a validated antiviral drug target, has been solved in two space groups. The nonstructural protein NS1A from influenza virus is a multifunctional virulence factor and a potent inhibitor of host immunity. It has two functional domains: an N-terminal 73-amino-acid RNA-binding domain and a C-terminal effector domain. Here, the crystallographic structure of the NS1A effector domain of influenza A/Udorn/72 virus is presented. Structure comparison with the NS1 effector domain from mouse-adapted influenza A/Puerto Rico/8/34 (PR8) virus strain reveals a similar monomer conformation but a different dimer interface. Further analysis and evaluation shows that the dimer interface observed in the structure of the PR8 NS1 effector domain is likely to be a crystallographic packing effect. A hypothetical model of the intact NS1 dimer is presented.

  3. Using Hierarchical Clustering of Secreted Protein Families to Classify and Rank Candidate Effectors of Rust Fungi

    PubMed Central

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

    2012-01-01

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

  4. Effector-triggered post-translational modifications and their role in suppression of plant immunity

    PubMed Central

    Howden, Andrew J. M.; Huitema, Edgar

    2012-01-01

    Plant–pathogen interactions feature complex signaling exchanges between host and microbes that ultimately determine association outcomes. Plants deploy pattern recognition receptors to perceive pathogen-associated molecular patterns, mount pattern-triggered immunity (PTI), and fend off potential pathogens. In recent years an increasing number of defense-signaling components have been identified along with a mechanistic understanding of their regulation during immune responses. Post-translational modifications (PTMs) are now thought to play a crucial role in regulating defense signaling. In a bid to suppress PTI and infect their host, pathogens have evolved large repertoires of effectors that trigger susceptibility and allow colonization of host tissues. While great progress has been made in elucidating defense-signaling networks in plants and the activities of effectors in immune suppression, a critical gap exists in our understanding of effector mechanism-of-action. Given the importance of PTMs in the regulation of defense signaling, we will explore the question: how do effectors modify the post-translational status of host proteins and thus interfere with host processes required for immunity? We will consider how emerging proteomics-based experimental strategies may help us answer this important question and ultimately open the pathogens’ effector black box. PMID:22811685

  5. Adaptive evolution has targeted the C-terminal domain of the RXLR effectors of plant pathogenic oomycetes.

    PubMed

    Win, Joe; Morgan, William; Bos, Jorunn; Krasileva, Ksenia V; Cano, Liliana M; Chaparro-Garcia, Angela; Ammar, Randa; Staskawicz, Brian J; Kamoun, Sophien

    2007-08-01

    Oomycete plant pathogens deliver effector proteins inside host cells to modulate plant defense circuitry and to enable parasitic colonization. These effectors are defined by a conserved motif, termed RXLR (for Arg, any amino acid, Leu, Arg), that is located downstream of the signal peptide and that has been implicated in host translocation. Because the phenotypes of RXLR effectors extend to plant cells, their genes are expected to be the direct target of the evolutionary forces that drive the antagonistic interplay between pathogen and host. We used the draft genome sequences of three oomycete plant pathogens, Phytophthora sojae, Phytophthora ramorum, and Hyaloperonospora parasitica, to generate genome-wide catalogs of RXLR effector genes and determine the extent to which these genes are under positive selection. These analyses revealed that the RXLR sequence is overrepresented and positionally constrained in the secretome of Phytophthora relative to other eukaryotes. The three examined plant pathogenic oomycetes carry complex and diverse sets of RXLR effector genes that have undergone relatively rapid birth and death evolution. We obtained robust evidence of positive selection in more than two-thirds of the examined paralog families of RXLR effectors. Positive selection has acted for the most part on the C-terminal region, consistent with the view that RXLR effectors are modular, with the N terminus involved in secretion and host translocation and the C-terminal domain dedicated to modulating host defenses inside plant cells.

  6. Diverse evolutionary mechanisms shape the type III effector virulence factor repertoire in the plant pathogen Pseudomonas syringae.

    PubMed Central

    Rohmer, Laurence; Guttman, David S; Dangl, Jeffery L

    2004-01-01

    Many gram-negative pathogenic bacteria directly translocate effector proteins into eukaryotic host cells via type III delivery systems. Type III effector proteins are determinants of virulence on susceptible plant hosts; they are also the proteins that trigger specific disease resistance in resistant plant hosts. Evolution of type III effectors is dominated by competing forces: the likely requirement for conservation of virulence function, the avoidance of host defenses, and possible adaptation to new hosts. To understand the evolutionary history of type III effectors in Pseudomonas syringae, we searched for homologs to 44 known or candidate P. syringae type III effectors and two effector chaperones. We examined 24 gene families for distribution among bacterial species, amino acid sequence diversity, and features indicative of horizontal transfer. We assessed the role of diversifying and purifying selection in the evolution of these gene families. While some P. syringae type III effectors were acquired recently, others have evolved predominantly by descent. The majority of codons in most of these genes were subjected to purifying selection, suggesting selective pressure to maintain presumed virulence function. However, members of 7 families had domains subject to diversifying selection. PMID:15280247

  7. Two Fis Regulators Directly Repress the Expression of Numerous Effector-Encoding Genes in Legionella pneumophila

    PubMed Central

    Zusman, Tal; Speiser, Yariv

    2014-01-01

    Legionella pneumophila is an intracellular human pathogen that utilizes the Icm/Dot type IVB secretion system to translocate a large repertoire of effectors into host cells. For most of these effectors, there is no information regarding their regulation. Therefore, the aim of this study was to examine the involvement of the three L. pneumophila Fis homologs in the regulation of effector-encoding genes. Deletion mutants constructed in the genes encoding the three Fis regulators revealed that Fis1 (lpg0542 gene) and Fis3 (lpg1743) but not Fis2 (lpg1370) are partially required for intracellular growth of L. pneumophila in Acanthamoeba castellanii. To identify pathogenesis-related genes directly regulated by Fis, we established a novel in vivo system which resulted in the discovery of numerous effector-encoding genes directly regulated by Fis. Further examination of these genes revealed that Fis1 and Fis3 repress the level of expression of effector-encoding genes during exponential phase. Three groups of effector-encoding genes were identified: (i) effectors regulated mainly by Fis1, (ii) effectors regulated mainly by Fis3, and (iii) effectors regulated by both Fis1 and Fis3. Examination of the upstream regulatory region of all of these effector-encoding genes revealed multiple putative Fis regulatory elements, and site-directed mutagenesis confirmed that a few of these sites constitute part of a repressor binding element. Furthermore, gel mobility shift assays demonstrated the direct relation between the Fis1 and Fis3 regulators and these regulatory elements. Collectively, our results demonstrate for the first time that two of the three L. pneumophila Fis regulators directly repress the expression of Icm/Dot effector-encoding genes. PMID:25225276

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

    DOE PAGES

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

    2014-12-08

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

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

    SciTech Connect

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

    2014-12-08

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

  10. Deployment of the Burkholderia glumae type III secretion system as an efficient tool for translocating pathogen effectors to monocot cells.

    PubMed

    Sharma, Shailendra; Sharma, Shiveta; Hirabuchi, Akiko; Yoshida, Kentaro; Fujisaki, Koki; Ito, Akiko; Uemura, Aiko; Terauchi, Ryohei; Kamoun, Sophien; Sohn, Kee Hoon; Jones, Jonathan D G; Saitoh, Hiromasa

    2013-05-01

    Genome sequences of plant fungal pathogens have enabled the identification of effectors that cooperatively modulate the cellular environment for successful fungal growth and suppress host defense. Identification and characterization of novel effector proteins are crucial for understanding pathogen virulence and host-plant defense mechanisms. Previous reports indicate that the Pseudomonas syringae pv. tomato DC3000 type III secretion system (T3SS) can be used to study how non-bacterial effectors manipulate dicot plant cell function using the effector detector vector (pEDV) system. Here we report a pEDV-based effector delivery system in which the T3SS of Burkholderia glumae, an emerging rice pathogen, is used to translocate the AVR-Pik and AVR-Pii effectors of the fungal pathogen Magnaporthe oryzae to rice cytoplasm. The translocated AVR-Pik and AVR-Pii showed avirulence activity when tested in rice cultivars containing the cognate R genes. AVR-Pik reduced and delayed the hypersensitive response triggered by B. glumae in the non-host plant Nicotiana benthamiana, indicative of an immunosuppressive virulence activity. AVR proteins fused with fluorescent protein and nuclear localization signal were delivered by B. glumae T3SS and observed in the nuclei of infected cells in rice, wheat, barley and N. benthamiana. Our bacterial T3SS-enabled eukaryotic effector delivery and subcellular localization assays provide a useful method for identifying and studying effector functions in monocot plants.

  11. 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.

  12. The genome sequence and effector complement of the flax rust pathogen Melampsora lini

    PubMed Central

    Nemri, Adnane; Saunders, Diane G. O.; Anderson, Claire; Upadhyaya, Narayana M.; Win, Joe; Lawrence, Gregory J.; Jones, David A.; Kamoun, Sophien; Ellis, Jeffrey G.; Dodds, Peter N.

    2014-01-01

    Rust fungi cause serious yield reductions on crops, including wheat, barley, soybean, coffee, and represent real threats to global food security. Of these fungi, the flax rust pathogen Melampsora lini has been developed most extensively over the past 80 years as a model to understand the molecular mechanisms that underpin pathogenesis. During infection, M. lini secretes virulence effectors to promote disease. The number of these effectors, their function and their degree of conservation across rust fungal species is unknown. To assess this, we sequenced and assembled de novo the genome of M. lini isolate CH5 into 21,130 scaffolds spanning 189 Mbp (scaffold N50 of 31 kbp). Global analysis of the DNA sequence revealed that repetitive elements, primarily retrotransposons, make up at least 45% of the genome. Using ab initio predictions, transcriptome data and homology searches, we identified 16,271 putative protein-coding genes. An analysis pipeline was then implemented to predict the effector complement of M. lini and compare it to that of the poplar rust, wheat stem rust and wheat stripe rust pathogens to identify conserved and species-specific effector candidates. Previous knowledge of four cloned M. lini avirulence effector proteins and two basidiomycete effectors was used to optimize parameters of the effector prediction pipeline. Markov clustering based on sequence similarity was performed to group effector candidates from all four rust pathogens. Clusters containing at least one member from M. lini were further analyzed and prioritized based on features including expression in isolated haustoria and infected leaf tissue and conservation across rust species. Herein, we describe 200 of 940 clusters that ranked highest on our priority list, representing 725 flax rust candidate effectors. Our findings on this important model rust species provide insight into how effectors of rust fungi are conserved across species and how they may act to promote infection on their

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

    SciTech Connect

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

    2013-08-12

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

  14. 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

  15. Cancer stem cell targeted therapy: progress amid controversies.

    PubMed

    Wang, Tao; Shigdar, Sarah; Gantier, Michael P; Hou, Yingchun; Wang, Li; Li, Yong; Shamaileh, Hadi Al; Yin, Wang; Zhou, Shu-Feng; Zhao, Xinhan; Duan, Wei

    2015-12-29

    Although cancer stem cells have been well characterized in numerous malignancies, the fundamental characteristics of this group of cells, however, have been challenged by some recent observations: cancer stem cells may not necessary to be rare within tumors; cancer stem cells and non-cancer stem cells may undergo reversible phenotypic changes; and the cancer stem cells phenotype can vary substantially between patients. Here the current status and progresses of cancer stem cells theory is illustrated and via providing a panoramic view of cancer therapy, we addressed the recent controversies regarding the feasibility of cancer stem cells targeted anti-cancer therapy.

  16. Cancer stem cell targeted therapy: progress amid controversies

    PubMed Central

    Wang, Tao; Shigdar, Sarah; Gantier, Michael P.; Hou, Yingchun; Wang, Li; Li, Yong; Shamaileh, Hadi Al; Yin, Wang; Zhou, Shu-Feng; Zhao, Xinhan; Duan, Wei

    2015-01-01

    Although cancer stem cells have been well characterized in numerous malignancies, the fundamental characteristics of this group of cells, however, have been challenged by some recent observations: cancer stem cells may not necessary to be rare within tumors; cancer stem cells and non-cancer stem cells may undergo reversible phenotypic changes; and the cancer stem cells phenotype can vary substantially between patients. Here the current status and progresses of cancer stem cells theory is illustrated and via providing a panoramic view of cancer therapy, we addressed the recent controversies regarding the feasibility of cancer stem cells targeted anti-cancer therapy. PMID:26496035

  17. 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.

  18. 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

  19. 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

  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. Type III secretion chaperones of Pseudomonas syringae protect effectors from Lon-associated degradation.

    PubMed

    Losada, Liliana C; Hutcheson, Steven W

    2005-02-01

    The hrp type III secretion system (TTSS) of Pseudomonas syringae translocates effector proteins into the cytoplasm of host cells. Proteolysis of HrpR by Lon has been shown to negatively regulate the hrp TTSS. The inability to bypass Lon-associated effects on the regulatory system by ectopic expression of the known regulators suggested a second site of action for Lon in TTSS-dependent effector secretion. In this study we report that TTSS-dependent effectors are subject to the proteolytic degradation that appears to be rate-limiting to secretion. The half-lives of the effectors AvrPto, AvrRpt2, HopPsyA, HopPsyB1, HopPtoB2, HopPsyV1, HopPtoG and HopPtoM were substantially higher in bacteria lacking Lon. TTSS-dependent secretion of several effectors was enhanced from Lon mutants. A primary role for chaperones appears to be protection of effectors from Lon-associated degradation prior to secretion. When coexpressed with their cognate chaperone, HopPsyB1, HopPsyV1 and HopPtoM were at least 10 times more stable in strains expressing Lon. Distinct Lon-targeting and chaperone-binding domains were identified in HopPtoM. The results imply that Lon is involved at two distinct levels in the regulation of the P. syringae TTSS: regulation of assembly of the secreton and modulation of effector secretion.

  3. 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

  4. 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.

  5. 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.

  6. Identification and Characterisation of a Hyper-Variable Apoplastic Effector Gene Family of the Potato Cyst Nematodes

    PubMed Central

    Eves-van den Akker, Sebastian; Lilley, Catherine J.; Jones, John T.; Urwin, Peter E.

    2014-01-01

    Sedentary endoparasitic nematodes are obligate biotrophs that modify host root tissues, using a suite of effector proteins to create and maintain a feeding site that is their sole source of nutrition. Using assumptions about the characteristics of genes involved in plant-nematode biotrophic interactions to inform the identification strategy, we provide a description and characterisation of a novel group of hyper-variable extracellular effectors termed HYP, from the potato cyst nematode Globodera pallida. HYP effectors comprise a large gene family, with a modular structure, and have unparalleled diversity between individuals of the same population: no two nematodes tested had the same genetic complement of HYP effectors. Individuals vary in the number, size, and type of effector subfamilies. HYP effectors are expressed throughout the biotrophic stages in large secretory cells associated with the amphids of parasitic stage nematodes as confirmed by in situ hybridisation. The encoded proteins are secreted into the host roots where they are detectable by immunochemistry in the apoplasm, between the anterior end of the nematode and the feeding site. We have identified HYP effectors in three genera of plant parasitic nematodes capable of infecting a broad range of mono- and dicotyledon crop species. In planta RNAi targeted to all members of the effector family causes a reduction in successful parasitism. PMID:25255291

  7. Human immune cell targeting of protein nanoparticles - caveospheres

    NASA Astrophysics Data System (ADS)

    Glass, Joshua J.; Yuen, Daniel; Rae, James; Johnston, Angus P. R.; Parton, Robert G.; Kent, Stephen J.; de Rose, Robert

    2016-04-01

    Nanotechnology has the power to transform vaccine and drug delivery through protection of payloads from both metabolism and off-target effects, while facilitating specific delivery of cargo to immune cells. However, evaluation of immune cell nanoparticle targeting is conventionally restricted to monocultured cell line models. We generated human caveolin-1 nanoparticles, termed caveospheres, which were efficiently functionalized with monoclonal antibodies. Using this platform, we investigated CD4+ T cell and CD20+ B cell targeting within physiological mixtures of primary human blood immune cells using flow cytometry, imaging flow cytometry and confocal microscopy. Antibody-functionalization enhanced caveosphere binding to targeted immune cells (6.6 to 43.9-fold) within mixed populations and in the presence of protein-containing fluids. Moreover, targeting caveospheres to CCR5 enabled caveosphere internalization by non-phagocytic CD4+ T cells--an important therapeutic target for HIV treatment. This efficient and flexible system of immune cell-targeted caveosphere nanoparticles holds promise for the development of advanced immunotherapeutics and vaccines.

  8. From GFP to β-lactamase: advancing intact cell imaging for toxins and effectors

    PubMed Central

    Zuverink, Madison; Barbieri, Joseph T.

    2015-01-01

    Canonical reporters such as green fluorescent protein (GFP) and luciferase have assisted researchers in probing cellular pathways and processes. Prior research in pathogenesis depended on sensitivity of biochemical and biophysical techniques to identify effectors and elucidate entry mechanisms. Recently, the β-lactamase (βlac) reporter system has advanced toxin and effector reporting by permitting measurement of βlac delivery into the cytosol or host βlac expression in intact cells. βlac measurement in cells was facilitated by the development of the fluorogenic substrate, CCF2-AM, to identify novel effectors, target cells, and domains involved in bacterial pathogenesis. The assay is also adaptable for high-throughput screening of small molecule inhibitors against toxins, providing information on mechanism and potential therapeutic agents. The versatility and limitations of the βlac reporter system as applied to toxins and effectors are discussed in this review. PMID:26500183

  9. The role of effectors in nonhost resistance to filamentous plant pathogens.

    PubMed

    Stam, Remco; Mantelin, Sophie; McLellan, Hazel; Thilliez, Gaëtan

    2014-01-01

    In nature, most plants are resistant to a wide range of phytopathogens. However, mechanisms contributing to this so-called nonhost resistance (NHR) are poorly understood. Besides constitutive defenses, plants have developed two layers of inducible defense systems. Plant innate immunity relies on recognition of conserved pathogen-associated molecular patterns (PAMPs). In compatible interactions, pathogenicity effector molecules secreted by the invader can suppress host defense responses and facilitate the infection process. Additionally, plants have evolved pathogen-specific resistance mechanisms based on recognition of these effectors, which causes secondary defense responses. The current effector-driven hypothesis is that NHR in plants that are distantly related to the host plant is triggered by PAMP recognition that cannot be efficiently suppressed by the pathogen, whereas in more closely related species, nonhost recognition of effectors would play a crucial role. In this review we give an overview of current knowledge of the role of effector molecules in host and NHR and place these findings in the context of the model. We focus on examples from filamentous pathogens (fungi and oomycetes), discuss their implications for the field of plant-pathogen interactions and relevance in plant breeding strategies for development of durable resistance in crops.

  10. Genetic analysis of the individual contribution to virulence of the type III effector inventory of Pseudomonas syringae pv. phaseolicola.

    PubMed

    Macho, Alberto P; Zumaquero, Adela; Gonzalez-Plaza, Juan J; Ortiz-Martín, Inmaculada; Rufián, José S; Beuzón, Carmen R

    2012-01-01

    Several reports have recently contributed to determine the effector inventory of the sequenced strain Pseudomonas syringae pv. phaseolicola (Pph) 1448a. However, the contribution to virulence of most of these effectors remains to be established. Genetic analysis of the contribution to virulence of individual P. syringae effectors has been traditionally hindered by the lack of phenotypes of the corresponding knockout mutants, largely attributed to a high degree of functional redundancy within their effector inventories. In support of this notion, effectors from Pseudomonas syringae pv. tomato (Pto) DC3000 have been classified into redundant effector groups (REGs), analysing virulence of polymutants in the model plant Nicotiana benthamiana. However, using competitive index (CI) as a virulence assay, we were able to establish the individual contribution of AvrPto1(Pto) (DC3000) to Pto DC3000 virulence in tomato, its natural host, even though typically, contribution to virulence of AvrPto1 is only shown in strains also lacking AvrPtoB (also called HopAB2), a member of its REG. This report raised the possibility that even effectors targeting the same defence signalling pathway may have an individual contribution to virulence, and pointed out to CI assays as the means to establish such a contribution for individual effectors. In this work, we have analysed the individual contribution to virulence of the majority of previously uncharacterised Pph 1448a effectors, by monitoring the development of disease symptoms and determining the CI of single knockout mutants at different stages of growth within bean, its natural host. Despite their potential functional redundancy, we have found individual contributions to virulence for six out of the fifteen effectors analysed. In addition, we have analysed the functional relationships between effectors displaying individual contribution to virulence, highlighting the diversity that these relationships may present, and the interest of

  11. Genetic Analysis of the Individual Contribution to Virulence of the Type III Effector Inventory of Pseudomonas syringae pv. phaseolicola

    PubMed Central

    Gonzalez-Plaza, Juan J.; Ortiz-Martín, Inmaculada; Rufián, José S.; Beuzón, Carmen R.

    2012-01-01

    Several reports have recently contributed to determine the effector inventory of the sequenced strain Pseudomonas syringae pv. phaseolicola (Pph) 1448a. However, the contribution to virulence of most of these effectors remains to be established. Genetic analysis of the contribution to virulence of individual P. syringae effectors has been traditionally hindered by the lack of phenotypes of the corresponding knockout mutants, largely attributed to a high degree of functional redundancy within their effector inventories. In support of this notion, effectors from Pseudomonas syringae pv. tomato (Pto) DC3000 have been classified into redundant effector groups (REGs), analysing virulence of polymutants in the model plant Nicotiana benthamiana. However, using competitive index (CI) as a virulence assay, we were able to establish the individual contribution of AvrPto1PtoDC3000 to Pto DC3000 virulence in tomato, its natural host, even though typically, contribution to virulence of AvrPto1 is only shown in strains also lacking AvrPtoB (also called HopAB2), a member of its REG. This report raised the possibility that even effectors targeting the same defence signalling pathway may have an individual contribution to virulence, and pointed out to CI assays as the means to establish such a contribution for individual effectors. In this work, we have analysed the individual contribution to virulence of the majority of previously uncharacterised Pph 1448a effectors, by monitoring the development of disease symptoms and determining the CI of single knockout mutants at different stages of growth within bean, its natural host. Despite their potential functional redundancy, we have found individual contributions to virulence for six out of the fifteen effectors analysed. In addition, we have analysed the functional relationships between effectors displaying individual contribution to virulence, highlighting the diversity that these relationships may present, and the interest of

  12. Intrinsic Disorder in Pathogen Effectors: Protein Flexibility as an Evolutionary Hallmark in a Molecular Arms Race[W

    PubMed Central

    Marín, Macarena; Uversky, Vladimir N.; Ott, Thomas

    2013-01-01

    Effector proteins represent a refined mechanism of bacterial pathogens to overcome plants’ innate immune systems. These modular proteins often manipulate host physiology by directly interfering with immune signaling of plant cells. Even if host cells have developed efficient strategies to perceive the presence of pathogenic microbes and to recognize intracellular effector activity, it remains an open question why only few effectors are recognized directly by plant resistance proteins. Based on in-silico genome-wide surveys and a reevaluation of published structural data, we estimated that bacterial effectors of phytopathogens are highly enriched in long-disordered regions (>50 residues). These structurally flexible segments have no secondary structure under physiological conditions but can fold in a stimulus-dependent manner (e.g., during protein–protein interactions). The high abundance of intrinsic disorder in effectors strongly suggests positive evolutionary selection of this structural feature and highlights the dynamic nature of these proteins. We postulate that such structural flexibility may be essential for (1) effector translocation, (2) evasion of the innate immune system, and (3) host function mimicry. The study of these dynamical regions will greatly complement current structural approaches to understand the molecular mechanisms of these proteins and may help in the prediction of new effectors. PMID:24038649

  13. Telepresence Master Glove Controller For Dexterous Robotic End-Effectors

    NASA Astrophysics Data System (ADS)

    Fowler, A. M.; Joyce, R. R.; Britt, J. P.

    1987-03-01

    This paper describes recent research in the Aerospace Human Factors Research Division at NASA's Ames Research Center to develop a glove-like, control and data-recording device (DataGlove) that records and transmits to a host computerin real time, and at appropriate resolution, a numeric data-record of a user's hand/finger shape and dynamics. System configuration and performance specifications are detailed, and current research is discussed investigating its applications in operator control of dexterous robotic end-effectors and for use as a human factors research tool in evaluation of operator hand function requirements and performance in other specialized task environments.

  14. Telepresence master glove controller for dexterous robotic end-effectors

    NASA Technical Reports Server (NTRS)

    Fisher, Scott S.

    1987-01-01

    This paper describes recent research in the Aerospace Human Factors Research Division at NASA's Ames Research Center to develop a glove-like, control and data-recording device (DataGlove) that records and transmits to a host computer in real time, and at appropriate resolution, a numeric data-record of a user's hand/finger shape and dynamics. System configuration and performance specifications are detailed, and current research is discussed investigating its applications in operator control of dexterous robotic end-effectors and for use as a human factors research tool in evaluation of operator hand function requirements and performance in other specialized task environments.

  15. Complement--tapping into new sites and effector systems.

    PubMed

    Kolev, Martin; Le Friec, Gaelle; Kemper, Claudia

    2014-12-01

    Complement is traditionally known to be a system of serum proteins that provide protection against pathogens through direct cell lysis and the mobilization of innate and adaptive immunity. However, recent work indicates that the complement system has additional physiological roles beyond those in host defence. In this Opinion article, we describe the new modes and locations of complement activation that enable it to interact with other cell effector systems, such as growth factor receptors, inflammasomes and metabolic pathways. We propose that the location of complement activation dictates its function.

  16. 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).

  17. Pathogen effectors and plant immunity determine specialization of the blast fungus to rice subspecies

    PubMed Central

    Liao, Jingjing; Huang, Huichuan; Meusnier, Isabelle; Adreit, Henri; Ducasse, Aurélie; Bonnot, François; Pan, Lei; He, Xiahong; Kroj, Thomas; Fournier, Elisabeth; Tharreau, Didier; Gladieux, Pierre; Morel, Jean-Benoit

    2016-01-01

    Understanding how fungi specialize on their plant host is crucial for developing sustainable disease control. A traditional, centuries-old rice agro-system of the Yuanyang terraces was used as a model to show that virulence effectors of the rice blast fungus Magnaporthe oryzaeh play a key role in its specialization on locally grown indica or japonica local rice subspecies. Our results have indicated that major differences in several components of basal immunity and effector-triggered immunity of the japonica and indica rice varieties are associated with specialization of M. oryzae. These differences thus play a key role in determining M. oryzae host specificity and may limit the spread of the pathogen within the Yuanyang agro-system. Specifically, the AVR-Pia effector has been identified as a possible determinant of the specialization of M. oryzae to local japonica rice. DOI: http://dx.doi.org/10.7554/eLife.19377.001 PMID:28008850

  18. Electroporation of functional bacterial effectors into mammalian cells.

    PubMed

    Sontag, Ryan L; Mihai, Cosmin; Orr, Galya; Savchenko, Alexei; Skarina, Tatiana; Cui, Hong; Cort, John R; Adkins, Joshua N; Brown, Roslyn N

    2015-01-19

    The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high

  19. Electroporation of Functional Bacterial Effectors into Mammalian Cells

    PubMed Central

    Sontag, Ryan L.; Mihai, Cosmin; Orr, Galya; Savchenko, Alexei; Skarina, Tatiana; Cui, Hong; Cort, John R.; Adkins, Joshua N.; Brown, Roslyn N.

    2015-01-01

    The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high

  20. Generating Cell Targeting Aptamers for Nanotheranostics Using Cell-SELEX

    PubMed Central

    Lyu, Yifan; Chen, Guang; Shangguan, Dihua; Zhang, Liqin; Wan, Shuo; Wu, Yuan; Zhang, Hui; Duan, Lian; Liu, Chao; You, Mingxu; Wang, Jie; Tan, Weihong

    2016-01-01

    Detecting and understanding changes in cell conditions on the molecular level is of great importance for the accurate diagnosis and timely therapy of diseases. Cell-based SELEX (Systematic Evolution of Ligands by EXponential enrichment), a foundational technology used to generate highly-specific, cell-targeting aptamers, has been increasingly employed in studies of molecular medicine, including biomarker discovery and early diagnosis/targeting therapy of cancer. In this review, we begin with a mechanical description of the cell-SELEX process, covering aptamer selection, identification and identification, and aptamer characterization; following this introduction is a comprehensive discussion of the potential for aptamers as targeting moieties in the construction of various nanotheranostics. Challenges and prospects for cell-SELEX and aptamer-based nanotheranostic are also discussed. PMID:27375791

  1. The wheat Snn7 gene confers susceptibility upon recognition of the Parastagonospora nodorum necrotrophic effector SnTox7

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Parastagonospora nodorum is a necrotrophic fungal pathogen that causes the disease Septoria nodorum blotch (SNB) on wheat. The fungus produces necrotrophic effectors (NEs), that when recognized by corresponding host genes, cause cell death, which ultimately leads to disease. To date, eight host ge...

  2. A perspective on B-cell-targeting therapy for SLE

    PubMed Central

    Looney, R. John; Anolik, Jennifer; Sanz, Inaki

    2014-01-01

    In recent years, large controlled trials have tested several new agents for systemic lupus erythematosus (SLE). Unfortunately, none of these trials has met its primary outcome. This does not mean progress has not been made. In fact, a great deal has been learned about doing clinical trials in lupus and about the biological and clinical effects of the drugs being tested. Many of these drugs were designed to target B cells directly, e.g., rituximab, belimumab, epratuzumab, and transmembrane activator and calcium modulator and cyclophilin ligand interactor–immunoglobulin (TACI–Ig). The enthusiasm for targeting B cells derives from substantial evidence showing the critical role of B cells in murine models of SLE, as well promising results from multiple open trials with rituximab, a chimeric anti-CD20 monoclonal antibody that specifically depletes B cells (Martin and Chan in Immunity 20(5):517–527, 2004; Sobel et al. in J Exp Med 173:1441–1449, 1991; Silverman and Weisman in Arthritis Rheum 48:1484–1492, 2003; Silverman in Arthritis Rheum 52(4):1342, 2005; Shlomchik et al. in Nat Rev Immunol 1:147–153, 2001; Looney et al. in Arthritis Rheum 50:2580–2589, 2004; Lu et al. in Arthritis Rheum 61(4):482–487, 2009; Saito et al. in Lupus 12(10):798–800, 2003; van Vollenhoven et al. in Scand J Rheumatol 33(6):423–427, 2004; Sfikakis et al. Arthritis Rheum 52(2):501–513, 2005). Why have the controlled trials of B-cell-targeting therapies failed to demonstrate efficacy? Were there flaws in design or execution of these trials? Or, were promising animal studies and open trials misleading, as so often happens? This perspective discusses the current state of B-cell-targeting therapies for human lupus and the future development of these therapies. PMID:19669389

  3. Code-assisted discovery of TAL effector targets in bacterial leaf streak of rice reveals contrast with bacterial blight and a novel susceptibility gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transcription activator-like (TAL) effectors found in Xanthomonas spp. promote bacterial growth and plant susceptibility by binding specific DNA sequences or, effector-binding elements (EBEs), and inducing host gene expression. In this study, we have found substantially different transcriptional pro...

  4. In planta processing and glycosylation of a nematode CLE effector and its interaction with a CLV2-like receptor to promote parasitism

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Like other biotrophic plant pathogens, plant-parasitic nematodes secrete effector proteins into host cells to facilitate infection. Effector proteins that mimic plant CLAVATA3/ESR (CLE)-like proteins have been identified in several cyst nematodes including the potato cyst nematode (PCN); however, th...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  7. Identification of candidate effector proteins potentially involved in Fusarium graminearum-wheat interactions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pathogen-derived small secreted cysteine-rich proteins (SSCPs) are known to be a common source of fungal effectors that trigger resistance or susceptibility in specific host plants. This group of proteins has not been well studied in Fusarium graminearum, the primary cause of Fusarium head blight (...

  8. Targeting of plant pattern recognition receptor-triggered immunity by bacterial type-III secretion system effectors.

    PubMed

    Macho, Alberto P; Zipfel, Cyril

    2015-02-01

    During infection, microbes are detected by surface-localized pattern recognition receptors (PRRs), leading to an innate immune response that prevents microbial ingress. Therefore, successful pathogens must evade or inhibit PRR-triggered immunity to cause disease. In the past decade, a number of type-III secretion system effector (T3Es) proteins from plant pathogenic bacteria have been shown to suppress this layer of innate immunity. More recently, the detailed mechanisms of action have been defined for several of these effectors. Interestingly, effectors display a wide array of virulence targets, being able to prevent activation of immune receptors and to hijack immune signaling pathways. Besides being a fascinating example of pathogen-host co-evolution, effectors have also emerged as valuable tools to dissect important biological processes in host cells.

  9. Targeted Disruption of Chlamydia trachomatis Invasion by in Trans Expression of Dominant Negative Tarp Effectors.

    PubMed

    Parrett, Christopher J; Lenoci, Robert V; Nguyen, Brenda; Russell, Lauren; Jewett, Travis J

    2016-01-01

    Chlamydia trachomatis invasion of eukaryotic host cells is facilitated, in part, by the type III secreted effector protein, Tarp. The role of Tarp in chlamydiae entry of host cells is supported by molecular approaches that examined recombinant Tarp or Tarp effectors expressed within heterologous systems. A major limitation in the ability to study the contribution of Tarp to chlamydial invasion of host cells was the prior absence of genetic tools for chlamydiae. Based on our knowledge of Tarp domain structure and function along with the introduction of genetic approaches in C. trachomatis, we hypothesized that Tarp function could be disrupted in vivo by the introduction of dominant negative mutant alleles. We provide evidence that transformed C. trachomatis produced epitope tagged Tarp, which was secreted into the host cell during invasion. We examined the effects of domain specific Tarp mutations on chlamydial invasion and growth and demonstrate that C. trachomatis clones harboring engineered Tarp mutants lacking either the actin binding domain or the phosphorylation domain had reduced levels of invasion into host cells. These data provide the first in vivo evidence for the critical role of Tarp in C. trachomatis pathogenesis and indicate that chlamydial invasion of host cells can be attenuated via the introduction of engineered dominant negative type three effectors.

  10. Targeted Disruption of Chlamydia trachomatis Invasion by in Trans Expression of Dominant Negative Tarp Effectors

    PubMed Central

    Parrett, Christopher J.; Lenoci, Robert V.; Nguyen, Brenda; Russell, Lauren; Jewett, Travis J.

    2016-01-01

    Chlamydia trachomatis invasion of eukaryotic host cells is facilitated, in part, by the type III secreted effector protein, Tarp. The role of Tarp in chlamydiae entry of host cells is supported by molecular approaches that examined recombinant Tarp or Tarp effectors expressed within heterologous systems. A major limitation in the ability to study the contribution of Tarp to chlamydial invasion of host cells was the prior absence of genetic tools for chlamydiae. Based on our knowledge of Tarp domain structure and function along with the introduction of genetic approaches in C. trachomatis, we hypothesized that Tarp function could be disrupted in vivo by the introduction of dominant negative mutant alleles. We provide evidence that transformed C. trachomatis produced epitope tagged Tarp, which was secreted into the host cell during invasion. We examined the effects of domain specific Tarp mutations on chlamydial invasion and growth and demonstrate that C. trachomatis clones harboring engineered Tarp mutants lacking either the actin binding domain or the phosphorylation domain had reduced levels of invasion into host cells. These data provide the first in vivo evidence for the critical role of Tarp in C. trachomatis pathogenesis and indicate that chlamydial invasion of host cells can be attenuated via the introduction of engineered dominant negative type three effectors. PMID:27602332

  11. 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

  12. Intervention of Phytohormone Pathways by Pathogen Effectors[OPEN

    PubMed Central

    Kazan, Kemal; Lyons, Rebecca

    2014-01-01

    The constant struggle between plants and microbes has driven the evolution of multiple defense strategies in the host as well as offense strategies in the pathogen. To defend themselves from pathogen attack, plants often rely on elaborate signaling networks regulated by phytohormones. In turn, pathogens have adopted innovative strategies to manipulate phytohormone-regulated defenses. Tactics frequently employed by plant pathogens involve hijacking, evading, or disrupting hormone signaling pathways and/or crosstalk. As reviewed here, this is achieved mechanistically via pathogen-derived molecules known as effectors, which target phytohormone receptors, transcriptional activators and repressors, and other components of phytohormone signaling in the host plant. Herbivores and sap-sucking insects employ obligate pathogens such as viruses, phytoplasma, or symbiotic bacteria to intervene with phytohormone-regulated defenses. Overall, an improved understanding of phytohormone intervention strategies employed by pests and pathogens during their interactions with plants will ultimately lead to the development of new crop protection strategies. PMID:24920334

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

    PubMed

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

    2016-07-01

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

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

    PubMed Central

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

    2016-01-01

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

  15. Transient Expression of Candidatus Liberibacter Asiaticus Effector Induces Cell Death in Nicotiana benthamiana

    PubMed Central

    Pitino, Marco; Armstrong, Cheryl M.; Cano, Liliana M.; Duan, Yongping

    2016-01-01

    Candidatus Liberibacter asiaticus “Las” is a phloem-limited bacterial plant pathogen, and the most prevalent species of Liberibacter associated with citrus huanglongbing (HLB), a devastating disease of citrus worldwide. Although, the complete sequence of the Las genome provides the basis for studying functional genomics of Las and molecular mechanisms of Las-plant interactions, the functional characterization of Las effectors remains a slow process since remains to be cultured. Like other plant pathogens, Las may deliver effector proteins into host cells and modulate a variety of host cellular functions for their infection progression. In this study, we identified 16 putative Las effectors via bioinformatics, and transiently expressed them in Nicotiana benthamiana. Diverse subcellular localization with different shapes and aggregation patterns of the effector candidates were revealed by UV- microscopy after transient expression in leaf tissue. Intriguingly, one of the 16 candidates, Las5315mp (mature protein), was localized in the chloroplast and induced cell death at 3 days post inoculation (dpi) in N. benthamiana. Moreover, Las5315mp induced strong callose deposition in plant cells. This study provides new insights into the localizations and potential roles of these Las effectors in planta. PMID:27458468

  16. The Barley Powdery Mildew Effector Candidates CSEP0081 and CSEP0254 Promote Fungal Infection Success

    PubMed Central

    Ahmed, Ali Abdurehim; Pedersen, Carsten

    2016-01-01

    Effectors play significant roles in the success of pathogens. Recent advances in genome sequencing have revealed arrays of effectors and effector candidates from a wide range of plant pathogens. Yet, the vast majority of them remain uncharacterized. Among the ~500 Candidate Secreted Effector Proteins (CSEPs) predicted from the barley powdery mildew fungal genome, only a few have been studied and shown to have a function in virulence. Here, we provide evidence that CSEP0081 and CSEP0254 contribute to infection by the fungus. This was studied using Host-Induced Gene Silencing (HIGS), where independent silencing of the transcripts for these CSEPs significantly reduced the fungal penetration and haustoria formation rate. Both CSEPs are likely required during and after the formation of haustoria, in which their transcripts were found to be differentially expressed, rather than in epiphytic tissue. When expressed in barley leaf epidermal cells, both CSEPs appears to move freely between the cytosol and the nucleus, suggesting that their host targets locate in these cellular compartments. Collectively, our data suggest that, in addition to the previously reported effectors, the barley powdery mildew fungus utilizes these two CSEPs as virulence factors to enhance infection. PMID:27322386

  17. Shigella IpaH Family Effectors as a Versatile Model for Studying Pathogenic Bacteria.

    PubMed

    Ashida, Hiroshi; Sasakawa, Chihiro

    2015-01-01

    Shigella spp. are highly adapted human pathogens that cause bacillary dysentery (shigellosis). Via the type III secretion system (T3SS), Shigella deliver a subset of virulence proteins (effectors) that are responsible for pathogenesis, with functions including pyroptosis, invasion of the epithelial cells, intracellular survival, and evasion of host immune responses. Intriguingly, T3SS effector activity and strategies are not unique to Shigella, but are shared by many other bacterial pathogens, including Salmonella, Yersinia, and enteropathogenic Escherichia coli (EPEC). Therefore, studying Shigella T3SS effectors will not only improve our understanding of bacterial infection systems, but also provide a molecular basis for developing live bacterial vaccines and antibacterial drugs. One of Shigella T3SS effectors, IpaH family proteins, which have E3 ubiquitin ligase activity and are widely conserved among other bacterial pathogens, are very relevant because they promote bacterial survival by triggering cell death and modulating the host immune responses. Here, we describe selected examples of Shigella pathogenesis, with particular emphasis on the roles of IpaH family effectors, which shed new light on bacterial survival strategies and provide clues about how to overcome bacterial infections.

  18. Using Population and Comparative Genomics to Understand the Genetic Basis of Effector-Driven Fungal Pathogen Evolution

    PubMed Central

    Plissonneau, Clémence; Benevenuto, Juliana; Mohd-Assaad, Norfarhan; Fouché, Simone; Hartmann, Fanny E.; Croll, Daniel

    2017-01-01

    Epidemics caused by fungal plant pathogens pose a major threat to agro-ecosystems and impact global food security. High-throughput sequencing enabled major advances in understanding how pathogens cause disease on crops. Hundreds of fungal genomes are now available and analyzing these genomes highlighted the key role of effector genes in disease. Effectors are small secreted proteins that enhance infection by manipulating host metabolism. Fungal genomes carry 100s of putative effector genes, but the lack of homology among effector genes, even for closely related species, challenges evolutionary and functional analyses. Furthermore, effector genes are often found in rapidly evolving chromosome compartments which are difficult to assemble. We review how population and comparative genomics toolsets can be combined to address these challenges. We highlight studies that associated genome-scale polymorphisms with pathogen lifestyles and adaptation to different environments. We show how genome-wide association studies can be used to identify effectors and other pathogenicity-related genes underlying rapid adaptation. We also discuss how the compartmentalization of fungal genomes into core and accessory regions shapes the evolution of effector genes. We argue that an understanding of genome evolution provides important insight into the trajectory of host-pathogen co-evolution. PMID:28217138

  19. Using Population and Comparative Genomics to Understand the Genetic Basis of Effector-Driven Fungal Pathogen Evolution.

    PubMed

    Plissonneau, Clémence; Benevenuto, Juliana; Mohd-Assaad, Norfarhan; Fouché, Simone; Hartmann, Fanny E; Croll, Daniel

    2017-01-01

    Epidemics caused by fungal plant pathogens pose a major threat to agro-ecosystems and impact global food security. High-throughput sequencing enabled major advances in understanding how pathogens cause disease on crops. Hundreds of fungal genomes are now available and analyzing these genomes highlighted the key role of effector genes in disease. Effectors are small secreted proteins that enhance infection by manipulating host metabolism. Fungal genomes carry 100s of putative effector genes, but the lack of homology among effector genes, even for closely related species, challenges evolutionary and functional analyses. Furthermore, effector genes are often found in rapidly evolving chromosome compartments which are difficult to assemble. We review how population and comparative genomics toolsets can be combined to address these challenges. We highlight studies that associated genome-scale polymorphisms with pathogen lifestyles and adaptation to different environments. We show how genome-wide association studies can be used to identify effectors and other pathogenicity-related genes underlying rapid adaptation. We also discuss how the compartmentalization of fungal genomes into core and accessory regions shapes the evolution of effector genes. We argue that an understanding of genome evolution provides important insight into the trajectory of host-pathogen co-evolution.

  20. Effector-Mining in the Poplar Rust Fungus Melampsora larici-populina Secretome

    PubMed Central

    Lorrain, Cécile; Hecker, Arnaud; Duplessis, Sébastien

    2015-01-01

    The poplar leaf rust fungus, Melampsora larici-populina has been established as a tree-microbe interaction model. Understanding the molecular mechanisms controlling infection by pathogens appears essential for durable management of tree plantations. In biotrophic plant-parasites, effectors are known to condition host cell colonization. Thus, investigation of candidate secreted effector proteins (CSEPs) is a major goal in the poplar–poplar rust interaction. Unlike oomycetes, fungal effectors do not share conserved motifs and candidate prediction relies on a set of a priori criteria established from reported bona fide effectors. Secretome prediction, genome-wide analysis of gene families and transcriptomics of M. larici-populina have led to catalogs of more than a thousand secreted proteins. Automatized effector-mining pipelines hold great promise for rapid and systematic identification and prioritization of CSEPs for functional characterization. In this review, we report on and discuss the current status of the poplar rust fungus secretome and prediction of candidate effectors from this species. PMID:26697026

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

    SciTech Connect

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

    2011-01-01

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

  2. 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

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

    SciTech Connect

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

    2004-11-23

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

  4. 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.

  5. Salmonellae interactions with host processes.

    PubMed

    LaRock, Doris L; Chaudhary, Anu; Miller, Samuel I

    2015-04-01

    Salmonellae invasion and intracellular replication within host cells result in a range of diseases, including gastroenteritis, bacteraemia, enteric fever and focal infections. In recent years, considerable progress has been made in our understanding of the molecular mechanisms that salmonellae use to alter host cell physiology; through the delivery of effector proteins with specific activities and through the modulation of defence and stress response pathways. In this Review, we summarize our current knowledge of the complex interplay between bacterial and host factors that leads to inflammation, disease and, in most cases, control of the infection by its animal hosts, with a particular focus on Salmonella enterica subsp. enterica serovar Typhimurium. We also highlight gaps in our knowledge of the contributions of salmonellae and the host to disease pathogenesis, and we suggest future avenues for further study.

  6. Cell-targeting antibodies in immunity to Ebola

    PubMed Central

    Schmaljohn, Alan; Lewis, George K.

    2016-01-01

    As the 2014–15 Ebola virus epidemic in West Africa evolved from emergency to lesson, developers of both vaccines and therapeutic antibodies were left with the puzzlement of what kinds of anti-Ebola antibodies are predictably desirable in treating the afflicted, and what antibodies might account for the specific and lasting protection elicited by the more effective vaccines. The facile answer in virology is that neutralizing antibody (NAb) is desired and required. However, with Ebola and other filoviruses (as with many prior viral examples), there are multiple discordances in which neutralizing antibodies fail to protect animals, and others in which antibody-mediated protection is observed in the absence of measured virus neutralization. Explanation presumably resides in the protective role of antibodies that bind and functionally ‘target’ virus-infected cells, here called ‘cell-targeting antibody’, or CTAb. To be clear, many NAbs are also CTAbs, and in the case of Ebola the great majority of NAbs are likely CTAbs. Isotype, glycosylation, and other features of CTAbs are likely crucial in their capacity to mediate protection. Overall, results and analysis invite an increasingly complex view of antibody-mediated immunity to enveloped viruses. PMID:27005312

  7. Surface-modified gold nanorods for specific cell targeting

    NASA Astrophysics Data System (ADS)

    Wang, Chan-Ung; Arai, Yoshie; Kim, Insun; Jang, Wonhee; Lee, Seonghyun; Hafner, Jason H.; Jeoung, Eunhee; Jung, Deokho; Kwon, Youngeun

    2012-05-01

    Gold nanoparticles (GNPs) have unique properties that make them highly attractive materials for developing functional reagents for various biomedical applications including photothermal therapy, targeted drug delivery, and molecular imaging. For in vivo applications, GNPs need to be prepared with very little or negligible cytotoxicitiy. Most GNPs are, however, prepared using growth-directing surfactants such as cetyl trimethylammonium bromide (CTAB), which are known to have considerable cytotoxicity. In this paper, we describe an approach to remove CTAB to a non-toxic concentration. We optimized the conditions for surface modification with methoxypolyethylene glycol thiol (mPEG), which replaced CTAB and formed a protective layer on the surface of gold nanorods (GNRs). The cytotoxicities of pristine and surface-modified GNRs were measured in primary human umbilical vein endothelial cells and human cell lines derived from hepatic carcinoma cells, embryonic kidney cells, and thyroid papillary carcinoma cells. Cytotoxicity assays revealed that treating cells with GNRs did not significantly affect cell viability except for thyroid papillary carcinoma cells. Thyroid cancer cells were more susceptible to residual CTAB, so CTAB had to be further removed by dialysis in order to use GNRs for thyroid cell targeting. PEGylated GNRs are further modified to present monoclonal antibodies that recognize a specific surface marker, Na-I symporter, for thyroid cells. Antibody-conjugated GNRs specifically targeted human thyroid cells in vitro.

  8. Identification of Phakopsora pachyrhizi Candidate Effectors with Virulence Activity in a Distantly Related Pathosystem

    PubMed Central

    Kunjeti, Sridhara G.; Iyer, Geeta; Johnson, Ebony; Li, Eric; Broglie, Karen E.; Rauscher, Gilda; Rairdan, Gregory J.

    2016-01-01

    Phakopsora pachyrhizi is the causal agent of Asian Soybean Rust, a disease that causes enormous economic losses, most markedly in South America. P. pachyrhizi is a biotrophic pathogen that utilizes specialized feeding structures called haustoria to colonize its hosts. In rusts and other filamentous plant pathogens, haustoria have been shown to secrete effector proteins into their hosts to permit successful completion of their life cycle. We have constructed a cDNA library from P. pachyrhizi haustoria using paramagnetic bead-based methodology and have identified 35 P. pachyrhizi candidate effector (CE) genes from this library which are described here. In addition, we quantified the transcript expression pattern of six of these genes and show that two of these CEs are able to greatly increase the susceptibility of Nicotiana benthamiana to Phytophthora infestans. This strongly suggests that these genes play an important role in P. pachyrhizi virulence on its hosts. PMID:27014295

  9. 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.

  10. 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.

  11. Code-Assisted Discovery of TAL Effector Targets in Bacterial Leaf Streak of Rice Reveals Contrast with Bacterial Blight and a Novel Susceptibility Gene

    PubMed Central

    Cernadas, Raul A.; Doyle, Erin L.; Niño-Liu, David O.; Wilkins, Katherine E.; Bancroft, Timothy; Wang, Li; Schmidt, Clarice L.; Caldo, Rico; Yang, Bing; White, Frank F.; Nettleton, Dan; Wise, Roger P.; Bogdanove, Adam J.

    2014-01-01

    Bacterial leaf streak of rice, caused by Xanthomonas oryzae pv. oryzicola (Xoc) is an increasingly important yield constraint in this staple crop. A mesophyll colonizer, Xoc differs from X. oryzae pv. oryzae (Xoo), which invades xylem to cause bacterial blight of rice. Both produce multiple distinct TAL effectors, type III-delivered proteins that transactivate effector-specific host genes. A TAL effector finds its target(s) via a partially degenerate code whereby the modular effector amino acid sequence identifies nucleotide sequences to which the protein binds. Virulence contributions of some Xoo TAL effectors have been shown, and their relevant targets, susceptibility (S) genes, identified, but the role of TAL effectors in leaf streak is uncharacterized. We used host transcript profiling to compare leaf streak to blight and to probe functions of Xoc TAL effectors. We found that Xoc and Xoo induce almost completely different host transcriptional changes. Roughly one in three genes upregulated by the pathogens is preceded by a candidate TAL effector binding element. Experimental analysis of the 44 such genes predicted to be Xoc TAL effector targets verified nearly half, and identified most others as false predictions. None of the Xoc targets is a known bacterial blight S gene. Mutational analysis revealed that Tal2g, which activates two genes, contributes to lesion expansion and bacterial exudation. Use of designer TAL effectors discriminated a sulfate transporter gene as the S gene. Across all targets, basal expression tended to be higher than genome-average, and induction moderate. Finally, machine learning applied to real vs. falsely predicted targets yielded a classifier that recalled 92% of the real targets with 88% precision, providing a tool for better target prediction in the future. Our study expands the number of known TAL effector targets, identifies a new class of S gene, and improves our ability to predict functional targeting. PMID:24586171

  12. Code-assisted discovery of TAL effector targets in bacterial leaf streak of rice reveals contrast with bacterial blight and a novel susceptibility gene.

    PubMed

    Cernadas, Raul A; Doyle, Erin L; Niño-Liu, David O; Wilkins, Katherine E; Bancroft, Timothy; Wang, Li; Schmidt, Clarice L; Caldo, Rico; Yang, Bing; White, Frank F; Nettleton, Dan; Wise, Roger P; Bogdanove, Adam J

    2014-02-01

    Bacterial leaf streak of rice, caused by Xanthomonas oryzae pv. oryzicola (Xoc) is an increasingly important yield constraint in this staple crop. A mesophyll colonizer, Xoc differs from X. oryzae pv. oryzae (Xoo), which invades xylem to cause bacterial blight of rice. Both produce multiple distinct TAL effectors, type III-delivered proteins that transactivate effector-specific host genes. A TAL effector finds its target(s) via a partially degenerate code whereby the modular effector amino acid sequence identifies nucleotide sequences to which the protein binds. Virulence contributions of some Xoo TAL effectors have been shown, and their relevant targets, susceptibility (S) genes, identified, but the role of TAL effectors in leaf streak is uncharacterized. We used host transcript profiling to compare leaf streak to blight and to probe functions of Xoc TAL effectors. We found that Xoc and Xoo induce almost completely different host transcriptional changes. Roughly one in three genes upregulated by the pathogens is preceded by a candidate TAL effector binding element. Experimental analysis of the 44 such genes predicted to be Xoc TAL effector targets verified nearly half, and identified most others as false predictions. None of the Xoc targets is a known bacterial blight S gene. Mutational analysis revealed that Tal2g, which activates two genes, contributes to lesion expansion and bacterial exudation. Use of designer TAL effectors discriminated a sulfate transporter gene as the S gene. Across all targets, basal expression tended to be higher than genome-average, and induction moderate. Finally, machine learning applied to real vs. falsely predicted targets yielded a classifier that recalled 92% of the real targets with 88% precision, providing a tool for better target prediction in the future. Our study expands the number of known TAL effector targets, identifies a new class of S gene, and improves our ability to predict functional targeting.

  13. Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing.

    PubMed

    Newton, Patrice; Latomanski, Eleanor A; Newton, Hayley J

    2016-07-06

    Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on β-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

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

    PubMed Central

    Dubytska, Lidiya P.; Rogge, Matthew L.

    2016-01-01

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

  15. 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

  16. EssD, a Nuclease Effector of the Staphylococcus aureus ESS Pathway.

    PubMed

    Ohr, Ryan Jay; Anderson, Mark; Shi, Miaomiao; Schneewind, Olaf; Missiakas, Dominique

    2017-01-01

    Specialized secretion systems of bacteria evolved for selective advantage, either killing microbial competitors or implementing effector functions during parasitism. Earlier work characterized the ESAT-6 secretion system (ESS) of Staphylococcus aureus and demonstrated its contribution to persistent staphylococcal infection of vertebrate hosts. Here, we identify a novel secreted effector of the ESS pathway, EssD, that functions as a nuclease and cleaves DNA but not RNA. EssI, a protein of the DUF600 family, binds EssD to block its nuclease activity in the staphylococcal cytoplasm. An essD knockout mutant or a variant lacking nuclease activity, essD(L546P), elicited a diminished interleukin-12 (IL-12) cytokine response following bloodstream infection of mice, suggesting that the effector function of EssD stimulates immune signaling to support the pathogenesis of S. aureus infections.

  17. 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.

  18. Mining the human gut microbiota for effector strains that shape the immune system.

    PubMed

    Ahern, Philip P; Faith, Jeremiah J; Gordon, Jeffrey I

    2014-06-19

    The gut microbiota codevelops with the immune system beginning at birth. Mining the microbiota for bacterial strains responsible for shaping the structure and dynamic operations of the innate and adaptive arms of the immune system represents a formidable combinatorial problem but one that needs to be overcome to advance mechanistic understanding of microbial community and immune system coregulation and to develop new diagnostic and therapeutic approaches that promote health. Here, we discuss a scalable, less biased approach for identifying effector strains in complex microbial communities that impact immune function. The approach begins by identifying uncultured human fecal microbiota samples that transmit immune phenotypes to germ-free mice. Clonally arrayed sequenced collections of bacterial strains are constructed from representative donor microbiota. If the collection transmits phenotypes, effector strains are identified by testing randomly generated subsets with overlapping membership in individually housed germ-free animals. Detailed mechanistic studies of effector strain-host interactions can then be performed.

  19. Xanthomonas euvesicatoria type III effector XopQ interacts with tomato and pepper 14-3-3 isoforms to suppress effector-triggered immunity.

    PubMed

    Teper, Doron; Salomon, Dor; Sunitha, Sukumaran; Kim, Jung-Gun; Mudgett, Mary Beth; Sessa, Guido

    2014-01-01

    Effector-triggered immunity (ETI) to host-adapted pathogens is associated with rapid cell death at the infection site. The plant-pathogenic bacterium Xanthomonas euvesicatoria (Xcv) interferes with plant cellular processes by injecting effector proteins into host cells through the type III secretion system. Here, we show that the Xcv effector XopQ suppresses cell death induced by components of the ETI-associated MAP kinase cascade MAPKKKα MEK2/SIPK and by several R/avr gene pairs. Inactivation of xopQ by insertional mutagenesis revealed that this effector inhibits ETI-associated cell death induced by avirulent Xcv in resistant pepper (Capsicum annuum), and enhances bacterial growth in resistant pepper and tomato (Solanum lycopersicum). Using protein-protein interaction studies in yeast (Saccharomyces cerevisiae) and in planta, we identified the tomato 14-3-3 isoform SlTFT4 and homologs from other plant species as XopQ interactors. A mutation in the putative 14-3-3 binding site of XopQ impaired interaction of the effector with CaTFT4 in yeast and its virulence function in planta. Consistent with a role in ETI, TFT4 mRNA abundance increased during the incompatible interaction of tomato and pepper with Xcv. Silencing of NbTFT4 in Nicotiana benthamiana significantly reduced cell death induced by MAPKKKα. In addition, silencing of CaTFT4 in pepper delayed the appearance of ETI-associated cell death and enhanced growth of virulent and avirulent Xcv, demonstrating the requirement of TFT4 for plant immunity to Xcv. Our results suggest that the XopQ virulence function is to suppress ETI and immunity-associated cell death by interacting with TFT4, which is an important component of ETI and a bona fide target of XopQ.

  20. Identification of putative TAL effector targets of the citrus canker pathogens shows functional convergence underlying disease development and defense response

    PubMed Central

    2014-01-01

    Background Transcriptional activator-like (TAL) effectors, formerly known as the AvrBs3/PthA protein family, are DNA-binding effectors broadly found in Xanthomonas spp. that transactivate host genes upon injection via the bacterial type three-secretion system. Biologically relevant targets of TAL effectors, i.e. host genes whose induction is vital to establish a compatible interaction, have been reported for xanthomonads that colonize rice and pepper; however, citrus genes modulated by the TAL effectors PthA“s” and PthC“s” of the citrus canker bacteria Xanthomonas citri (Xc) and Xanthomonas aurantifolii pathotype C (XaC), respectively, are poorly characterized. Of particular interest, XaC causes canker disease in its host lemon (Citrus aurantifolia), but triggers a defense response in sweet orange. Results Based on, 1) the TAL effector-DNA binding code, 2) gene expression data of Xc and XaC-infiltrated sweet orange leaves, and 3) citrus hypocotyls transformed with PthA2, PthA4 or PthC1, we have identified a collection of Citrus sinensis genes potentially targeted by Xc and XaC TAL effectors. Our results suggest that similar with other strains of Xanthomonas TAL effectors, PthA2 and PthA4, and PthC1 to some extent, functionally converge. In particular, towards induction of genes involved in the auxin and gibberellin synthesis and response, cell division, and defense response. We also present evidence indicating that the TAL effectors act as transcriptional repressors and that the best scoring predicted DNA targets of PthA“s” and PthC“s” in citrus promoters predominantly overlap with or localize near to TATA boxes of core promoters, supporting the idea that TAL effectors interact with the host basal transcriptional machinery to recruit the RNA pol II and start transcription. Conclusions The identification of PthA“s” and PthC“s” targets, such as the LOB (LATERAL ORGAN BOUNDARY) and CCNBS genes that we report here, is key for the understanding

  1. Transcription Factors Encoded on Core and Accessory Chromosomes of Fusarium oxysporum Induce Expression of Effector Genes

    PubMed Central

    van der Does, H. Charlotte; Schmidt, Sarah M.; Langereis, Léon; Hughes, Timothy R.

    2016-01-01

    Proteins secreted by pathogens during host colonization largely determine the outcome of pathogen-host interactions and are commonly called ‘effectors’. In fungal plant pathogens, coordinated transcriptional up-regulation of effector genes is a key feature of pathogenesis and effectors are often encoded in genomic regions with distinct repeat content, histone code and rate of evolution. In the tomato pathogen Fusarium oxysporum f. sp. lycopersici (Fol), effector genes reside on one of four accessory chromosomes, known as the ‘pathogenicity’ chromosome, which can be exchanged between strains through horizontal transfer. The three other accessory chromosomes in the Fol reference strain may also be important for virulence towards tomato. Expression of effector genes in Fol is highly up-regulated upon infection and requires Sge1, a transcription factor encoded on the core genome. Interestingly, the pathogenicity chromosome itself contains 13 predicted transcription factor genes and for all except one, there is a homolog on the core genome. We determined DNA binding specificity for nine transcription factors using oligonucleotide arrays. The binding sites for homologous transcription factors were highly similar, suggesting that extensive neofunctionalization of DNA binding specificity has not occurred. Several DNA binding sites are enriched on accessory chromosomes, and expression of FTF1, its core homolog FTF2 and SGE1 from a constitutive promoter can induce expression of effector genes. The DNA binding sites of only these three transcription factors are enriched among genes up-regulated during infection. We further show that Ftf1, Ftf2 and Sge1 can activate transcription from their binding sites in yeast. RNAseq analysis revealed that in strains with constitutive expression of FTF1, FTF2 or SGE1, expression of a similar set of plant-responsive genes on the pathogenicity chromosome is induced, including most effector genes. We conclude that the Fol

  2. Phytoplasma Effector SAP54 Induces Indeterminate Leaf-Like Flower Development in Arabidopsis Plants1[C][W][OA

    PubMed Central

    MacLean, Allyson M.; Sugio, Akiko; Makarova, Olga V.; Findlay, Kim C.; Grieve, Victoria M.; Tóth, Réka; Nicolaisen, Mogens; Hogenhout, Saskia A.

    2011-01-01

    Phytoplasmas are insect-transmitted bacterial plant pathogens that cause considerable damage to a diverse range of agricultural crops globally. Symptoms induced in infected plants suggest that these phytopathogens may modulate developmental processes within the plant host. We report herein that Aster Yellows phytoplasma strain Witches’ Broom (AY-WB) readily infects the model plant Arabidopsis (Arabidopsis thaliana) ecotype Columbia, inducing symptoms that are characteristic of phytoplasma infection, such as the production of green leaf-like flowers (virescence and phyllody) and increased formation of stems and branches (witches’ broom). We found that the majority of genes encoding secreted AY-WB proteins (SAPs), which are candidate effector proteins, are expressed in Arabidopsis and the AY-WB insect vector Macrosteles quadrilineatus (Hemiptera; Cicadellidae). To identify which of these effector proteins induce symptoms of phyllody and virescence, we individually expressed the effector genes in Arabidopsis. From this screen, we have identified a novel AY-WB effector protein, SAP54, that alters floral development, resulting in the production of leaf-like flowers that are similar to those produced by plants infected with this phytoplasma. This study offers novel insight into the effector profile of an insect-transmitted plant pathogen and reports to our knowledge the first example of a microbial pathogen effector protein that targets flower development in a host. PMID:21849514

  3. Infection assays in Arabidopsis reveal candidate effectors from the poplar rust fungus that promote susceptibility to bacteria and oomycete pathogens.

    PubMed

    Germain, Hugo; Joly, David L; Mireault, Caroline; Plourde, Mélodie B; Letanneur, Claire; Stewart, Donald; Morency, Marie-Josée; Petre, Benjamin; Duplessis, Sébastien; Séguin, Armand

    2016-11-21

    Fungi of the Pucciniales order cause rust diseases which, altogether, affect thousands of plant species worldwide and pose a major threat to several crops. How rust effectors-virulence proteins delivered into infected tissues to modulate host functions-contribute to pathogen virulence remains poorly understood. Melampsora larici-populina is a devastating and widespread rust pathogen of poplar, and its genome encodes 1184 identified small secreted proteins that could potentially act as effectors. Here, following specific criteria, we selected 16 candidate effector proteins and characterized their virulence activities and subcellular localizations in the leaf cells of Arabidopsis thaliana. Infection assays using bacterial (Pseudomonas syringae) and oomycete (Hyaloperonospora arabidopsidis) pathogens revealed subsets of candidate effectors that enhanced or decreased pathogen leaf colonization. Confocal imaging of green fluorescent protein-tagged candidate effectors constitutively expressed in stable transgenic plants revealed that some protein fusions specifically accumulate in nuclei, chloroplasts, plasmodesmata and punctate cytosolic structures. Altogether, our analysis suggests that rust fungal candidate effectors target distinct cellular components in host cells to promote parasitic growth.

  4. Identification of novel Xanthomonas euvesicatoria type III effector proteins by a machine-learning approach.

    PubMed

    Teper, Doron; Burstein, David; Salomon, Dor; Gershovitz, Michael; Pupko, Tal; Sessa, Guido

    2016-04-01

    The Gram-negative bacterium Xanthomonas euvesicatoria (Xcv) is the causal agent of bacterial spot disease in pepper and tomato. Xcv pathogenicity depends on a type III secretion (T3S) system that delivers effector proteins into host cells to suppress plant immunity and promote disease. The pool of known Xcv effectors includes approximately 30 proteins, most identified in the 85-10 strain by various experimental and computational techniques. To identify additional Xcv 85-10 effectors, we applied a genome-wide machine-learning approach, in which all open reading frames (ORFs) were scored according to their propensity to encode effectors. Scoring was based on a large set of features, including genomic organization, taxonomic dispersion, hypersensitive response and pathogenicity (hrp)-dependent expression, 5' regulatory sequences, amino acid composition bias and GC content. Thirty-six predicted effectors were tested for translocation into plant cells using the hypersensitive response (HR)-inducing domain of AvrBs2 as a reporter. Seven proteins (XopAU, XopAV, XopAW, XopAP, XopAX, XopAK and XopAD) harboured a functional translocation signal and their translocation relied on the HrpF translocon, indicating that they are bona fide T3S effectors. Remarkably, four belong to novel effector families. Inactivation of the xopAP gene reduced the severity of disease symptoms in infected plants. A decrease in cell death and chlorophyll content was observed in pepper leaves inoculated with the xopAP mutant when compared with the wild-type strain. However, populations of the xopAP mutant in infected leaves were similar in size to those of wild-type bacteria, suggesting that the reduction in virulence was not caused by impaired bacterial growth.

  5. Bacterial Internalization, Localization, and Effectors Shape the Epithelial Immune Response during Shigella flexneri Infection

    PubMed Central

    Lippmann, Juliane; Gwinner, Frederik; Rey, Camille; Tamir, Uyanga; Law, Helen K. W.

    2015-01-01

    Intracellular pathogens are differentially sensed by the compartmentalized host immune system. Nevertheless, gene expression studies of infected cells commonly average the immune responses, neglecting the precise pathogen localization. To overcome this limitation, we dissected the transcriptional immune response to Shigella flexneri across different infection stages in bulk and single cells. This identified six distinct transcriptional profiles characterizing the dynamic, multilayered host response in both bystander and infected cells. These profiles were regulated by external and internal danger signals, as well as whether bacteria were membrane bound or cytosolic. We found that bacterial internalization triggers a complex, effector-independent response in bystander cells, possibly to compensate for the undermined host gene expression in infected cells caused by bacterial effectors, particularly OspF. Single-cell analysis revealed an important bacterial strategy to subvert host responses in infected cells, demonstrating that OspF disrupts concomitant gene expression of proinflammatory, apoptosis, and stress pathways within cells. This study points to novel mechanisms through which bacterial internalization, localization, and injected effectors orchestrate immune response transcriptional signatures. PMID:26123804

  6. 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

  7. 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.

  8. 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.

  9. 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.

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

    PubMed Central

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

    2015-01-01

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

  11. Effector Polymorphisms of the Sunflower Downy Mildew Pathogen Plasmopara halstedii and Their Use to Identify Pathotypes from Field Isolates.

    PubMed

    Gascuel, Quentin; Bordat, Amandine; Sallet, Erika; Pouilly, Nicolas; Carrere, Sébastien; Roux, Fabrice; Vincourt, Patrick; Godiard, Laurence

    2016-01-01

    The obligate biotroph oomycete Plasmopara halstedii causes downy mildew on sunflower crop, Helianthus annuus. The breakdown of several Pl resistance genes used in sunflower hybrids over the last 25 years came along with the appearance of new Pl. halstedii isolates showing modified virulence profiles. In oomycetes, two classes of effector proteins, key players of pathogen virulence, are translocated into the host: RXLR and CRN effectors. We identified 54 putative CRN or RXLR effector genes from transcriptomic data and analyzed their genetic diversity in seven Pl. halstedii pathotypes representative of the species variability. Pl. halstedii effector genes were on average more polymorphic at both the nucleic and protein levels than random non-effector genes, suggesting a potential adaptive dynamics of pathogen virulence over the last 25 years. Twenty-two KASP (Competitive Allele Specific PCR) markers designed on polymorphic effector genes were genotyped on 35 isolates belonging to 14 Pl. halstedii pathotypes. Polymorphism analysis based on eight KASP markers aims at proposing a determination key suitable to classify the eight multi-isolate pathotypes into six groups. This is the first report of a molecular marker set able to discriminate Pl. halstedii pathotypes based on the polymorphism of pathogenicity effectors. Compared to phenotypic tests handling living spores used until now to discriminate Pl. halstedii pathotypes, this set of molecular markers constitutes a first step in faster pathotype diagnosis of Pl. halstedii isolates. Hence, emerging sunflower downy mildew isolates could be more rapidly characterized and thus, assessment of plant resistance breakdown under field conditions should be improved.

  12. Fluorescent reporter analysis revealed the timing and localization of AVR-Pia expression, an avirulence effector of Magnaporthe oryzae.

    PubMed

    Sornkom, Worawan; Miki, Shinsuke; Takeuchi, Saori; Abe, Ayumi; Asano, Kozo; Sone, Teruo

    2016-08-16

    In order to facilitate infection, the rice blast pathogen Magnaporthe oryzae secretes an abundance of proteins, including avirulence effectors, to diminish its host's defences. Avirulence effectors are recognized by host resistance proteins and trigger the host's hypersensitive response, which is a rapid and effective form of innate plant immunity. An understanding of the underlying molecular mechanisms of such interactions is crucial for the development of strategies to control disease. However, the expression and secretion of certain effector proteins, such as AVR-Pia, have yet to be reported. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that AVR-Pia was only expressed during infection. Fluorescently labelled AVR-Pia indicated that AVR-Pia expression was induced during appressorial differentiation in the cells of both rice and onion, as well as in a penetration-deficient (Δpls1) mutant capable of developing melanized appressoria, but unable to penetrate host cells, suggesting that AVR-Pia expression is independent of fungal penetration. Using live-cell imaging, we also documented the co-localization of green fluorescent protein (GFP)-labelled AVR-Pia and monomeric red fluorescent protein (mRFP)-labelled PWL2, which indicates that AVR-Pia accumulates in biotrophic interfacial complexes before being delivered to the plant cytosol. Together, these results suggest that AVR-Pia is a cytoplasmic effector that is expressed at the onset of appressorial differentiation and is translocated to the biotrophic interfacial complex, and then into the host's cytoplasm.

  13. Brucella Modulates Secretory Trafficking via Multiple Type IV Secretion Effector Proteins

    PubMed Central

    Myeni, Sebenzile; Child, Robert; Ng, Tony W.; Kupko, John J.; Wehrly, Tara D.; Porcella, Stephen F.; Knodler, Leigh A.; Celli, Jean

    2013-01-01

    The intracellular pathogenic bacterium Brucella generates a replicative vacuole (rBCV) derived from the endoplasmic reticulum via subversion of the host cell secretory pathway. rBCV biogenesis requires the expression of the Type IV secretion system (T4SS) VirB, which is thought to translocate effector proteins that modulate membrane trafficking along the endocytic and secretory pathways. To date, only a few T4SS substrates have been identified, whose molecular functions remain unknown. Here, we used an in silico screen to identify putative T4SS effector candidate proteins using criteria such as limited homology in other bacterial genera, the presence of features similar to known VirB T4SS effectors, GC content and presence of eukaryotic-like motifs. Using β-lactamase and CyaA adenylate cyclase reporter assays, we identified eleven proteins translocated into host cells by Brucella, five in a VirB T4SS-dependent manner, namely BAB1_0678 (BspA), BAB1_0712 (BspB), BAB1_0847 (BspC), BAB1_1671 (BspE) and BAB1_1948 (BspF). A subset of the translocated proteins targeted secretory pathway compartments when ectopically expressed in HeLa cells, and the VirB effectors BspA, BspB and BspF inhibited protein secretion. Brucella infection also impaired host protein secretion in a process requiring BspA, BspB and BspF. Single or combined deletions of bspA, bspB and bspF affected Brucella ability to replicate in macrophages and persist in the liver of infected mice. Taken together, these findings demonstrate that Brucella modulates secretory trafficking via multiple T4SS effector proteins that likely act coordinately to promote Brucella pathogenesis. PMID:23950720

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

    PubMed Central

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

    2016-01-01

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

  15. SecretEPDB: a comprehensive web-based resource for secreted effector proteins of the bacterial types III, IV and VI secretion systems

    PubMed Central

    An, Yi; Wang, Jiawei; Li, Chen; Revote, Jerico; Zhang, Yang; Naderer, Thomas; Hayashida, Morihiro; Akutsu, Tatsuya; Webb, Geoffrey I.; Lithgow, Trevor; Song, Jiangning

    2017-01-01

    Bacteria translocate effector molecules to host cells through highly evolved secretion systems. By definition, the function of these effector proteins is to manipulate host cell biology and the sequence, structural and functional annotations of these effector proteins will provide a better understanding of how bacterial secretion systems promote bacterial survival and virulence. Here we developed a knowledgebase, termed SecretEPDB (Bacterial Secreted Effector Protein DataBase), for effector proteins of type III secretion system (T3SS), type IV secretion system (T4SS) and type VI secretion system (T6SS). SecretEPDB provides enriched annotations of the aforementioned three classes of effector proteins by manually extracting and integrating structural and functional information from currently available databases and the literature. The database is conservative and strictly curated to ensure that every effector protein entry is supported by experimental evidence that demonstrates it is secreted by a T3SS, T4SS or T6SS. The annotations of effector proteins documented in SecretEPDB are provided in terms of protein characteristics, protein function, protein secondary structure, Pfam domains, metabolic pathway and evolutionary details. It is our hope that this integrated knowledgebase will serve as a useful resource for biological investigation and the generation of new hypotheses for research efforts aimed at bacterial secretion systems. PMID:28112271

  16. 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.

  17. Dendritic cell targeting vaccine for HPV-associated cancer

    PubMed Central

    Yin, Wenjie; Duluc, Dorothée; Joo, HyeMee; Oh, SangKon

    2017-01-01

    Dendritic cells (DCs) are major antigen presenting cells that can efficiently prime and activate cellular immune responses. Delivering antigens to in vivo DCs has thus been considered as a promising strategy that could allow us to mount T cell-mediated therapeutic immunity against cancers in patients. Successful development of such types of cancer vaccines that can target in vivo DCs, however, requires a series of outstanding questions that need to be addressed. These include the proper selection of which DC surface receptors, specific DC subsets and DC activators that can further enhance the efficacy of vaccines by promoting effector T cell infiltration and retention in tumors and their actions against tumors. Supplementing these areas of research with additional strategies that can counteract tumor immune evasion mechanisms is also expected to enhance the efficacy of such therapeutic vaccines against cancers. After more than a decade of study, we have concluded that antigen targeting to DCs via CD40 to evoke cellular responses is more efficient than targeting antigens to the same types of DCs via eleven other DC surface receptors tested. In recent work, we have further demonstrated that a prototype vaccine (anti-CD40-HPV16.E6/7, a recombinant fusion protein of anti-human CD40 and HPV16.E6/7 protein) for HPV16-associated cancers can efficiently activate HPV16.E6/7-specific T cells, particularly CD8+ T cells, from the blood of HPV16+ head-and-neck cancer patients. Moreover, anti-CD40-HPV16.E6/7 plus poly(I:C) can mount potent therapeutic immunity against TC-1 tumor expressing HPV16.E6/7 protein in human CD40 transgenic mice. In this manuscript, we thus highlight our recent findings for the development of novel CD40 targeting immunotherapeutic vaccines for HPV16-associated malignancies. In addition, we further discuss several of key questions that still remain to be addressed for enhancing therapeutic immunity elicited by our prototype vaccine against HPV16

  18. NOD-like receptor cooperativity in effector-triggered immunity.

    PubMed

    Griebel, Thomas; Maekawa, Takaki; Parker, Jane E

    2014-11-01

    Intracellular nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are basic elements of innate immunity in plants and animals. Whereas animal NLRs react to conserved microbe- or damage-associated molecular patterns, plant NLRs intercept the actions of diverse pathogen virulence factors (effectors). In this review, we discuss recent genetic and molecular evidence for functional NLR pairs, and discuss the significance of NLR self-association and heteromeric NLR assemblies in the triggering of downstream signaling pathways. We highlight the versatility and impact of cooperating NLR pairs that combine pathogen sensing with the initiation of defense signaling in both plant and animal immunity. We propose that different NLR receptor molecular configurations provide opportunities for fine-tuning resistance pathways and enhancing the host's pathogen recognition spectrum to keep pace with rapidly evolving microbial populations.

  19. BtpB, a novel Brucella TIR-containing effector protein with immune modulatory functions.

    PubMed

    Salcedo, Suzana P; Marchesini, María I; Degos, Clara; Terwagne, Matthieu; Von Bargen, Kristine; Lepidi, Hubert; Herrmann, Claudia K; Santos Lacerda, Thais L; Imbert, Paul R C; Pierre, Philippe; Alexopoulou, Lena; Letesson, Jean-Jacques; Comerci, Diego J; Gorvel, Jean-Pierre

    2013-01-01

    Several bacterial pathogens have TIR domain-containing proteins that contribute to their pathogenesis. We identified a second TIR-containing protein in Brucella spp. that we have designated BtpB. We show it is a potent inhibitor of TLR signaling, probably via MyD88. BtpB is a novel Brucella effector that is translocated into host cells and interferes with activation of dendritic cells. In vivo mouse studies revealed that BtpB is contributing to virulence and control of local inflammatory responses with relevance in the establishment of chronic brucellosis. Together, our results show that BtpB is a novel Brucella effector that plays a major role in the modulation of host innate immune response during infection.

  20. 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

  1. Glycan-Based Cell Targeting To Modulate Immune Responses.

    PubMed

    Johannssen, Timo; Lepenies, Bernd

    2017-04-01

    Glycosylation is an integral post-translational modification present in more than half of all eukaryotic proteins. It affects key protein functions, including folding, stability, and immunogenicity. Glycoengineering approaches, such as the use of bacterial N-glycosylation systems, or expression systems, including yeasts, insect cells, and mammalian cells, have enabled access to defined and homogenous glycoproteins. Given that glycan structures on proteins can be recognized by host lectin receptors, they may facilitate cell-specific targeting and immune modulation. Myeloid C-type lectin receptors (CLRs) expressed by antigen-presenting cells are attractive targets to shape immune responses. Multivalent glycan display on nanoparticles, liposomes, or dendrimers has successfully enabled CLR targeting. In this review, we discuss novel strategies to access defined glycan structures and highlight CLR targeting approaches for immune modulation.

  2. 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.

  3. 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.

  4. 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.

  5. 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…

  6. Duplications and losses in gene families of rust pathogens highlight putative effectors

    PubMed Central

    Pendleton, Amanda L.; Smith, Katherine E.; Feau, Nicolas; Martin, Francis M.; Grigoriev, Igor V.; Hamelin, Richard; Nelson, C. Dana; Burleigh, J. Gordon; Davis, John M.

    2014-01-01

    Rust fungi are a group of fungal pathogens that cause some of the world's most destructive diseases of trees and crops. A shared characteristic among rust fungi is obligate biotrophy, the inability to complete a lifecycle without a host. This dependence on a host species likely affects patterns of gene expansion, contraction, and innovation within rust pathogen genomes. The establishment of disease by biotrophic pathogens is reliant upon effector proteins that are encoded in the fungal genome and secreted from the pathogen into the host's cell apoplast or within the cells. This study uses a comparative genomic approach to elucidate putative effectors and determine their evolutionary histories. We used OrthoMCL to identify nearly 20,000 gene families in proteomes of 16 diverse fungal species, which include 15 basidiomycetes and one ascomycete. We inferred patterns of duplication and loss for each gene family and identified families with distinctive patterns of expansion/contraction associated with the evolution of rust fungal genomes. To recognize potential contributors for the unique features of rust pathogens, we identified families harboring secreted proteins that: (i) arose or expanded in rust pathogens relative to other fungi, or (ii) contracted or were lost in rust fungal genomes. While the origin of rust fungi appears to be associated with considerable gene loss, there are many gene duplications associated with each sampled rust fungal genome. We also highlight two putative effector gene families that have expanded in Cqf that we hypothesize have roles in pathogenicity. PMID:25018762

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

    PubMed

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

    2013-09-01

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

  8. Coevolution between a family of parasite virulence effectors and a class of LINE-1 retrotransposons.

    PubMed

    Sacristán, Soledad; Vigouroux, Marielle; Pedersen, Carsten; Skamnioti, Pari; Thordal-Christensen, Hans; Micali, Cristina; Brown, James K M; Ridout, Christopher J

    2009-10-15

    Parasites are able to evolve rapidly and overcome host defense mechanisms, but the molecular basis of this adaptation is poorly understood. Powdery mildew fungi (Erysiphales, Ascomycota) are obligate biotrophic parasites infecting nearly 10,000 plant genera. They obtain their nutrients from host plants through specialized feeding structures known as haustoria. We previously identified the AVR(k1) powdery mildew-specific gene family encoding effectors that contribute to the successful establishment of haustoria. Here, we report the extensive proliferation of the AVR(k1) gene family throughout the genome of B. graminis, with sequences diverging in formae speciales adapted to infect different hosts. Also, importantly, we have discovered that the effectors have coevolved with a particular family of LINE-1 retrotransposons, named TE1a. The coevolution of these two entities indicates a mutual benefit to the association, which could ultimately contribute to parasite adaptation and success. We propose that the association would benefit 1) the powdery mildew fungus, by providing a mechanism for amplifying and diversifying effectors and 2) the associated retrotransposons, by providing a basis for their maintenance through selection in the fungal genome.

  9. Genes conferring sensitivity to stagonospora nodorum necrotrophic effectors in stagonospora nodorum blotch-susceptible U.S. wheat cultivars

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Stagonospora nodorum is a necrotrophic fungal pathogen that causes Stagonospora nodorum blotch (SNB), a yield- and quality-reducing disease of wheat. S. nodorum produces a set of necrotrophic effectors (NEs) that interact with the products of host sensitivity genes to cause cell death and increased...

  10. Analysis of Globodera rostochiensis effectors reveals conserved functions of SPRYSEC proteins in suppressing and eliciting plant immune responses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Potato cyst nematodes (PCNs), including Globodera rostochiensis (Woll.), are important pests of potato. Plant parasitic nematodes produce multiple effector proteins, secreted from their stylets, to successfully infect their hosts. These include proteins that are delivered to the apoplast, as well as...

  11. SnTox3 Acts in Effector Triggered Susceptibility to Induce Disease on Wheat Carrying the Snn3 Gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The necrotrophic fungus Stagonospora nodorum produces multiple proteinaceous host-selective toxins (HSTs) which act as effectors of pathogenicity. Here, we report the molecular cloning and functional characterization of the SnTox3-producing gene, designated SnTox3, as well as the initial characteriz...

  12. Avian host defense peptides.

    PubMed

    Cuperus, Tryntsje; Coorens, Maarten; van Dijk, Albert; Haagsman, Henk P

    2013-11-01

    Host defense peptides (HDPs) are important effector molecules of the innate immune system of vertebrates. These antimicrobial peptides are also present in invertebrates, plants and fungi. HDPs display broad-spectrum antimicrobial activities and fulfill an important role in the first line of defense of many organisms. It is becoming increasingly clear that in the animal kingdom the functions of HDPs are not confined to direct antimicrobial actions. Research in mammals has indicated that HDPs have many immunomodulatory functions and are also involved in other physiological processes ranging from development to wound healing. During the past five years our knowledge about avian HDPs has increased considerably. This review addresses our current knowledge on the evolution, regulation and biological functions of HDPs of birds.

  13. Mathematical analysis of cell-target encounter rates in three dimensions. Effect of chemotaxis.

    PubMed

    Charnick, S B; Lauffenburger, D A

    1990-05-01

    Efficient and rapid immune response upon challenge by an infectious agent is vital to host defense. The encounter of leukocytes (white blood cells of the immune system) with their targets is the first step in this response. Analysis of the kinetics of this process is essential not only to understanding dynamic behavior of the immune response, but also to elucidating the consequences of many leukocyte functional abnormalities. The motion of leukocytes in the presence of targets typically involves a directed, or chemotactic component. These immune cells orient the direction of their motion in the presence of gradients in chemical attractants generated by pathogens. Fisher and Lauffenburger (1987. Biophys. J. 51:705-716) developed a model for macrophage/bacterium encounter in two dimensions which includes chemotaxis, and applied it to the particular system of alveolar macrophages (phagocytic leukocytes on the lung surface). Their model showed that macrophage/target encounter is likely the rate-limiting step in clearance of bacteria from the lung surface (Fisher, E. S., D. A. Lauffenburger, and R. P. Daniele. 1988. Am. Rev. Resp. Dis. 137:1129-1134). We have extended this model to analyze the effects of cell motility properties and geometric parameters on cell-target encounter in three dimensions. The differential equation governing encounter time in three dimensions is essentially the same as that in two dimensions, except for changed probability values. Our results show that more highly directed motion is necessary in three dimensions to achieve substantially decreased encounter times than in two dimensions, because of the increased search dimensionality. These general results were applied to the particular system of neutrophils operating in three dimensions in response to a bacterial challenge in connective tissue. Our results provide a plausible rationalization for both the chemotactic and chemokinetic behavior observed in neutrophils. That is, these cells exhibit

  14. Functionally Redundant RXLR Effectors from Phytophthora infestans Act at Different Steps to Suppress Early flg22-Triggered Immunity

    PubMed Central

    Fraiture, Malou; Liu, Xiaoyu; Boevink, Petra C.; Gilroy, Eleanor M.; Chen, Ying; Kandel, Kabindra; Sessa, Guido; Birch, Paul R. J.; Brunner, Frédéric

    2014-01-01

    Genome sequences of several economically important phytopathogenic oomycetes have revealed the presence of large families of so-called RXLR effectors. Functional screens have identified RXLR effector repertoires that either compromise or induce plant defense responses. However, limited information is available about the molecular mechanisms underlying the modes of action of these effectors in planta. The perception of highly conserved pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs), such as flg22, triggers converging signaling pathways recruiting MAP kinase cascades and inducing transcriptional re-programming, yielding a generic anti-microbial response. We used a highly synchronizable, pathogen-free protoplast-based assay to identify a set of RXLR effectors from Phytophthora infestans (PiRXLRs), the causal agent of potato and tomato light blight that manipulate early stages of flg22-triggered signaling. Of thirty-three tested PiRXLR effector candidates, eight, called Suppressor of early Flg22-induced Immune response (SFI), significantly suppressed flg22-dependent activation of a reporter gene under control of a typical MAMP-inducible promoter (pFRK1-Luc) in tomato protoplasts. We extended our analysis to Arabidopsis thaliana, a non-host plant species of P. infestans. From the aforementioned eight SFI effectors, three appeared to share similar functions in both Arabidopsis and tomato by suppressing transcriptional activation of flg22-induced marker genes downstream of post-translational MAP kinase activation. A further three effectors interfere with MAMP signaling at, or upstream of, the MAP kinase cascade in tomato, but not in Arabidopsis. Transient expression of the SFI effectors in Nicotiana benthamiana enhances susceptibility to P. infestans and, for the most potent effector, SFI1, nuclear localization is required for both suppression of MAMP signaling and virulence function. The present study provides a framework to decipher the molecular

  15. Functionally redundant RXLR effectors from Phytophthora infestans act at different steps to suppress early flg22-triggered immunity.

    PubMed

    Zheng, Xiangzi; McLellan, Hazel; Fraiture, Malou; Liu, Xiaoyu; Boevink, Petra C; Gilroy, Eleanor M; Chen, Ying; Kandel, Kabindra; Sessa, Guido; Birch, Paul R J; Brunner, Frédéric

    2014-04-01

    Genome sequences of several economically important phytopathogenic oomycetes have revealed the presence of large families of so-called RXLR effectors. Functional screens have identified RXLR effector repertoires that either compromise or induce plant defense responses. However, limited information is available about the molecular mechanisms underlying the modes of action of these effectors in planta. The perception of highly conserved pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs), such as flg22, triggers converging signaling pathways recruiting MAP kinase cascades and inducing transcriptional re-programming, yielding a generic anti-microbial response. We used a highly synchronizable, pathogen-free protoplast-based assay to identify a set of RXLR effectors from Phytophthora infestans (PiRXLRs), the causal agent of potato and tomato light blight that manipulate early stages of flg22-triggered signaling. Of thirty-three tested PiRXLR effector candidates, eight, called Suppressor of early Flg22-induced Immune response (SFI), significantly suppressed flg22-dependent activation of a reporter gene under control of a typical MAMP-inducible promoter (pFRK1-Luc) in tomato protoplasts. We extended our analysis to Arabidopsis thaliana, a non-host plant species of P. infestans. From the aforementioned eight SFI effectors, three appeared to share similar functions in both Arabidopsis and tomato by suppressing transcriptional activation of flg22-induced marker genes downstream of post-translational MAP kinase activation. A further three effectors interfere with MAMP signaling at, or upstream of, the MAP kinase cascade in tomato, but not in Arabidopsis. Transient expression of the SFI effectors in Nicotiana benthamiana enhances susceptibility to P. infestans and, for the most potent effector, SFI1, nuclear localization is required for both suppression of MAMP signaling and virulence function. The present study provides a framework to decipher the molecular

  16. Metabolic effectors secreted by bacterial pathogens: essential facilitators of plastid endosymbiosis?

    PubMed

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

    2013-01-01

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

  17. 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

  18. 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.

  19. 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

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

    PubMed Central

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

    2011-01-01

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

  1. Salmonella - at home in the host cell.

    PubMed

    Malik-Kale, Preeti; Jolly, Carrie E; Lathrop, Stephanie; Winfree, Seth; Luterbach, Courtney; Steele-Mortimer, Olivia

    2011-01-01

    The Gram-negative bacterium Salmonella enterica has developed an array of sophisticated tools to manipulate the host cell and establish an intracellular niche, for successful propagation as a facultative intracellular pathogen. While Salmonella exerts diverse effects on its host cell, only the cell biology of the classic "trigger"-mediated invasion process and the subsequent development of the Salmonella-containing vacuole have been investigated extensively. These processes are dependent on cohorts of effector proteins translocated into host cells by two type III secretion systems (T3SS), although T3SS-independent mechanisms of entry may be important for invasion of certain host cell types. Recent studies into the intracellular lifestyle of Salmonella have provided new insights into the mechanisms used by this pathogen to modulate its intracellular environment. Here we discuss current knowledge of Salmonella-host interactions including invasion and establishment of an intracellular niche within the host.

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

    PubMed

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

    2016-05-01

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

  3. Evolutionary selection of new breast cancer cell-targeting peptides and phages with the cell-targeting peptides fully displayed on the major coat and their effects on actin dynamics during cell internalization

    PubMed Central

    Abbineni, Gopal; Modali, Sita; Safiejko-Mroczka, Barbara; Petrenko, Valery A.; Mao, Chuanbin

    2010-01-01

    Filamentous phage as a bacteria-specific virus can be conjugated with an anti-cancer drug and has been proposed to serve as a carrier to deliver drugs to cancer cells for targeted therapy. However, how cell-targeting filamentous phage alone affects cancer cell biology is unclear. Phage libraries provide an inexhaustible reservoir of new ligands against tumor cells and tissues that have potential therapeutic and diagnostic applications in cancer treatment. Some of these identified ligands might stimulate various cell responses. Here we identified new cell internalizing peptides (and the phages with such peptides fused to each of ~3900 copies of their major coat protein) using landscape phage libraries and for the first time investigated the actin dynamics when selected phages are internalized into the SKBR-3 breast cancer cells. Our results show that phages harboring VSSTQDFP and DGSIPWST peptides could selectively internalize into the SKBR-3 breast cancer cells with high affinity, and also show rapid involvement of membrane ruffling and re-arrangements of actin cytoskeleton during the phage entry. The actin dynamics was studied by using live cell and fluorescence imaging. The cell-targeting phages were found to enter breast cancer cells through energy dependent mechanism and phage entry interferes with actin dynamics, resulting in reorganization of actin filaments and increased membrane rufflings in SKBR-3 cells. These results suggest that, when phage enters epithelial cells, it triggers transient changes in the host cell actin cytoskeleton. This study also shows that using multivalent phage libraries considerably increases the repertoire of available cell-internalizing ligands with potential applications in targeted drug delivery, imaging, molecular monitoring and profiling of breast cancer cells. PMID:20735141

  4. 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

  5. Alternative Splicing of a Multi-Drug Transporter from Pseudoperonospora cubensis Generates an RXLR Effector Protein That Elicits a Rapid Cell Death

    PubMed Central

    Savory, Elizabeth A.; Zou, Cheng; Adhikari, Bishwo N.; Hamilton, John P.; Buell, C. Robin; Shiu, Shin-Han; Day, Brad

    2012-01-01

    Pseudoperonospora cubensis, an obligate oomycete pathogen, is the causal agent of cucurbit downy mildew, a foliar disease of global economic importance. Similar to other oomycete plant pathogens, Ps. cubensis has a suite of RXLR and RXLR-like effector proteins, which likely function as virulence or avirulence determinants during the course of host infection. Using in silico analyses, we identified 271 candidate effector proteins within the Ps. cubensis genome with variable RXLR motifs. In extending this analysis, we present the functional characterization of one Ps. cubensis effector protein, RXLR protein 1 (PscRXLR1), and its closest Phytophthora infestans ortholog, PITG_17484, a member of the Drug/Metabolite Transporter (DMT) superfamily. To assess if such effector-non-effector pairs are common among oomycete plant pathogens, we examined the relationship(s) among putative ortholog pairs in Ps. cubensis and P. infestans. Of 271 predicted Ps. cubensis effector proteins, only 109 (41%) had a putative ortholog in P. infestans and evolutionary rate analysis of these orthologs shows that they are evolving significantly faster than most other genes. We found that PscRXLR1 was up-regulated during the early stages of infection of plants, and, moreover, that heterologous expression of PscRXLR1 in Nicotiana benthamiana elicits a rapid necrosis. More interestingly, we also demonstrate that PscRXLR1 arises as a product of alternative splicing, making this the first example of an alternative splicing event in plant pathogenic oomycetes transforming a non-effector gene to a functional effector protein. Taken together, these data suggest a role for PscRXLR1 in pathogenicity, and, in total, our data provide a basis for comparative analysis of candidate effector proteins and their non-effector orthologs as a means of understanding function and evolutionary history of pathogen effectors. PMID:22496844

  6. 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

  7. From pathogen genomes to host plant processes: the power of plant parasitic oomycetes

    PubMed Central

    2013-01-01

    Recent pathogenomic research on plant parasitic oomycete effector function and plant host responses has resulted in major conceptual advances in plant pathology, which has been possible thanks to the availability of genome sequences. PMID:23809564

  8. From pathogen genomes to host plant processes: the power of plant parasitic oomycetes.

    PubMed

    Pais, Marina; Win, Joe; Yoshida, Kentaro; Etherington, Graham J; Cano, Liliana M; Raffaele, Sylvain; Banfield, Mark J; Jones, Alex; Kamoun, Sophien; Saunders, Diane G O

    2013-06-28

    Recent pathogenomic research on plant parasitic oomycete effector function and plant host responses has resulted in major conceptual advances in plant pathology, which has been possible thanks to the availability of genome sequences.

  9. 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.

  10. 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.

  11. 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.

  12. Phytophthora effector targets a novel component of small RNA pathway in plants to promote infection

    PubMed Central

    Qiao, Yongli; Shi, Jinxia; Zhai, Yi; Hou, Yingnan; Ma, Wenbo

    2015-01-01

    A broad range of parasites rely on the functions of effector proteins to subvert host immune response and facilitate disease development. The notorious Phytophthora pathogens evolved effectors with RNA silencing suppression activity to promote infection in plant hosts. Here we report that the Phytophthora Suppressor of RNA Silencing 1 (PSR1) can bind to an evolutionarily conserved nuclear protein containing the aspartate–glutamate–alanine–histidine-box RNA helicase domain in plants. This protein, designated PSR1-Interacting Protein 1 (PINP1), regulates the accumulation of both microRNAs and endogenous small interfering RNAs in Arabidopsis. A null mutation of PINP1 causes embryonic lethality, and silencing of PINP1 leads to developmental defects and hypersusceptibility to Phytophthora infection. These phenotypes are reminiscent of transgenic plants expressing PSR1, supporting PINP1 as a direct virulence target of PSR1. We further demonstrate that the localization of the Dicer-like 1 protein complex is impaired in the nucleus of PINP1-silenced or PSR1-expressing cells, indicating that PINP1 may facilitate small RNA processing by affecting the assembly of dicing complexes. A similar function of PINP1 homologous genes in development and immunity was also observed in Nicotiana benthamiana. These findings highlight PINP1 as a previously unidentified component of RNA silencing that regulates distinct classes of small RNAs in plants. Importantly, Phytophthora has evolved effectors to target PINP1 in order to promote infection. PMID:25902521

  13. A type III effector antagonises death receptor signalling during bacterial gut infection

    PubMed Central

    Pearson, Jaclyn S; Giogha, Cristina; Ong, Sze Ying; Kennedy, Catherine L; Kelly, Michelle; Robinson, Keith S; Wong, Tania; Mansell, Ashley; Riedmaier, Patrice; Oates, Clare VL; Zaid, Ali; Mühlen, Sabrina; Crepin, Valerie F; Marches, Olivier; Ang, Ching-Seng; Williamson, Nicholas A; O’Reilly, Lorraine A; Bankovacki, Aleksandra; Nachbur, Ueli; Infusini, Giuseppe; Webb, Andrew I; Silke, John; Strasser, Andreas; Frankel, Gad; Hartland, Elizabeth L

    2013-01-01

    Successful infection by enteric bacterial pathogens depends on the ability of the bacteria to colonise the gut, replicate in host tissues and disseminate to other hosts. Pathogens such as Salmonella, Shigella and enteropathogenic and enterohaemorrhagic E. coli (EPEC and EHEC), utilise a type III secretion system (T3SS) to deliver virulence effector proteins into host cells during infection that promote colonisation and interfere with antimicrobial host responses 1-3. Here we report that the T3SS effector NleB1 from EPEC binds to host cell death domain containing proteins and thereby inhibits death receptor signalling. Protein interaction studies identified FADD, TRADD and RIPK1 as binding partners of NleB1. NleB1 expressed ectopically or injected by the bacterial T3SS prevented Fas ligand or TNF-induced formation of the canonical death inducing signalling complex (DISC) and proteolytic activation of caspase-8, an essential step in death receptor induced apoptosis. This inhibition depended on the N-GlcNAc transferase activity of NleB1, which specifically modified Arg117 in the death domain of FADD. The importance of the death receptor apoptotic pathway to host defence was demonstrated using mice deficient in the FAS signalling pathway, which showed delayed clearance of the EPEC-like mouse pathogen Citrobacter rodentium and reversion to virulence of an nleB mutant. The activity of NleB suggests that EPEC and other attaching and effacing (A/E) pathogens antagonise death receptor induced apoptosis of infected cells, thereby blocking a major antimicrobial host response. PMID:24025841

  14. 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

  15. 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

  16. 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

  17. Focal effector accumulation in a biotrophic interface at the primary invasion sites of Colletotrichum orbiculare in multiple susceptible plants.

    PubMed

    Irieda, Hiroki; Ogawa, Suthitar; Takano, Yoshitaka

    2016-01-01

    We identified virulence-related effectors of a hemibiotrophic fungal pathogen Colletotrichum orbiculare, and found that a novel interface was generated by a biotrophic interaction between C. orbiculare and the host cucumber, in which the effectors secreted from the pathogen accumulated preferentially. The interface was located around the biotrophic primary hyphal neck. Here, we showed that C. orbiculare also developed this interface in a biotrophic interaction with melon, which belongs to Cucurbitaceae. Furthermore, C. orbiculare developed interface in the interaction with a susceptible plant, Nicotiana benthamiana, which is distantly related to Cucurbitaceae, suggesting that the spatial regulation strategy for effectors in C. orbiculare is not specific to cucumber; rather, it is conserved among the various plants that are susceptible to this pathogen.

  18. Focal effector accumulation in a biotrophic interface at the primary invasion sites of Colletotrichum orbiculare in multiple susceptible plants

    PubMed Central

    Irieda, Hiroki; Ogawa, Suthitar; Takano, Yoshitaka

    2016-01-01

    Abstract We identified virulence-related effectors of a hemibiotrophic fungal pathogen Colletotrichum orbiculare, and found that a novel interface was generated by a biotrophic interaction between C. orbiculare and the host cucumber, in which the effectors secreted from the pathogen accumulated preferentially. The interface was located around the biotrophic primary hyphal neck. Here, we showed that C. orbiculare also developed this interface in a biotrophic interaction with melon, which belongs to Cucurbitaceae. Furthermore, C. orbiculare developed interface in the interaction with a susceptible plant, Nicotiana benthamiana, which is distantly related to Cucurbitaceae, suggesting that the spatial regulation strategy for effectors in C. orbiculare is not specific to cucumber; rather, it is conserved among the various plants that are susceptible to this pathogen. PMID:26829249

  19. 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.

  20. Designer TAL effectors induce disease susceptibility and resistance to Xanthomonas oryzae pv. oryzae in rice.

    PubMed

    Li, Ting; Huang, Sheng; Zhou, Junhui; Yang, Bing

    2013-05-01

    TAL (transcription activator-like) effectors from Xanthomonas bacteria activate the cognate host genes, leading to disease susceptibility or resistance dependent on the genetic context of host target genes. The modular nature and DNA recognition code of TAL effectors enable custom-engineering of designer TAL effectors (dTALE) for gene activation. However, the feasibility of dTALEs as transcription activators for gene functional analysis has not been demonstrated. Here, we report the use of dTALEs, as expressed and delivered by the pathogenic Xanthomonas oryzae pv. oryzae (Xoo), in revealing the new function of two previously identified disease-related genes and the potential of one developmental gene for disease susceptibility in rice/Xoo interactions. The dTALE gene dTALE-xa27, designed to target the susceptible allele of the resistance gene Xa27, elicited a resistant reaction in the otherwise susceptible rice cultivar IR24. Four dTALE genes were made to induce the four annotated Xa27 homologous genes in rice cultivar Nipponbare, but none of the four induced Xa27-like genes conferred resistance to the dTALE-containing Xoo strains. A dTALE gene was also generated to activate the recessive resistance gene xa13, an allele of the disease-susceptibility gene Os8N3 (also named Xa13 or OsSWEET11, a member of sucrose efflux transporter SWEET gene family). The induction of xa13 by the dTALE rendered the resistant rice IRBB13 (xa13/xa13) susceptible to Xoo. Finally, OsSWEET12, an as-yet uncharacterized SWEET gene with no corresponding naturally occurring TAL effector identified, conferred susceptibility to the Xoo strains expressing the corresponding dTALE genes. Our results demonstrate that dTALEs can be delivered through the bacterial secretion system to activate genes of interest for functional analysis in plants.

  1. EST mining and functional expression assays identify extracellular effector proteins from the plant pathogen Phytophthora.

    PubMed

    Torto, Trudy A; Li, Shuang; Styer, Allison; Huitema, Edgar; Testa, Antonino; Gow, Neil A R; van West, Pieter; Kamoun, Sophien

    2003-07-01

    Plant pathogenic microbes have the remarkable ability to manipulate biochemical, physiological, and morphological processes in their host plants. These manipulations are achieved through a diverse array of effector molecules that can either promote infection or trigger defense responses. We describe a general functional genomics approach aimed at identifying extracellular effector proteins from plant pathogenic microorganisms by combining data mining of expressed sequence tags (ESTs) with virus-based high-throughput functional expression assays in plants. PexFinder, an algorithm for automated identification of extracellular proteins from EST data sets, was developed and applied to 2147 ESTs from the oomycete plant pathogen Phytophthora infestans. The program identified 261 ESTs (12.2%) corresponding to a set of 142 nonredundant Pex (Phytophthora extracellular protein) cDNAs. Of these, 78 (55%) Pex cDNAs were novel with no significant matches in public databases. Validation of PexFinder was performed using proteomic analysis of secreted protein of P. infestans. To identify which of the Pex cDNAs encode effector proteins that manipulate plant processes, high-throughput functional expression assays in plants were performed on 63 of the identified cDNAs using an Agrobacterium tumefaciens binary vector carrying the potato virus X (PVX) genome. This led to the discovery of two novel necrosis-inducing cDNAs, crn1 and crn2, encoding extracellular proteins that belong to a large and complex protein family in Phytophthora. Further characterization of the crn genes indicated that they are both expressed in P. infestans during colonization of the host plant tomato and that crn2 induced defense-response genes in tomato. Our results indicate that combining data mining using PexFinder with PVX-based functional assays can facilitate the discovery of novel pathogen effector proteins. In principle, this strategy can be applied to a variety of eukaryotic plant pathogens, including

  2. Effector Polymorphisms of the Sunflower Downy Mildew Pathogen Plasmopara halstedii and Their Use to Identify Pathotypes from Field Isolates

    PubMed Central

    Gascuel, Quentin; Bordat, Amandine; Sallet, Erika; Pouilly, Nicolas; Carrere, Sébastien; Roux, Fabrice

    2016-01-01

    The obligate biotroph oomycete Plasmopara halstedii causes downy mildew on sunflower crop, Helianthus annuus. The breakdown of several Pl resistance genes used in sunflower hybrids over the last 25 years came along with the appearance of new Pl. halstedii isolates showing modified virulence profiles. In oomycetes, two classes of effector proteins, key players of pathogen virulence, are translocated into the host: RXLR and CRN effectors. We identified 54 putative CRN or RXLR effector genes from transcriptomic data and analyzed their genetic diversity in seven Pl. halstedii pathotypes representative of the species variability. Pl. halstedii effector genes were on average more polymorphic at both the nucleic and protein levels than random non-effector genes, suggesting a potential adaptive dynamics of pathogen virulence over the last 25 years. Twenty-two KASP (Competitive Allele Specific PCR) markers designed on polymorphic effector genes were genotyped on 35 isolates belonging to 14 Pl. halstedii pathotypes. Polymorphism analysis based on eight KASP markers aims at proposing a determination key suitable to classify the eight multi-isolate pathotypes into six groups. This is the first report of a molecular marker set able to discriminate Pl. halstedii pathotypes based on the polymorphism of pathogenicity effectors. Compared to phenotypic tests handling living spores used until now to discriminate Pl. halstedii pathotypes, this set of molecular markers constitutes a first step in faster pathotype diagnosis of Pl. halstedii isolates. Hence, emerging sunflower downy mildew isolates could be more rapidly characterized and thus, assessment of plant resistance breakdown under field conditions should be improved. PMID:26845339

  3. Analysis of Globodera rostochiensis effectors reveals conserved functions of SPRYSEC proteins in suppressing and eliciting plant immune responses

    PubMed Central

    Ali, Shawkat; Magne, Maxime; Chen, Shiyan; Obradovic, Natasa; Jamshaid, Lubna; Wang, Xiaohong; Bélair, Guy; Moffett, Peter

    2015-01-01

    Potato cyst nematodes (PCNs), including Globodera rostochiensis (Woll.), are important pests of potato. Plant parasitic nematodes produce multiple effector proteins, secreted from their stylets, to successfully infect their hosts. These include proteins delivered to the apoplast and to the host cytoplasm. A number of effectors from G. rostochiensis predicted to be delivered to the host cytoplasm have been identified, including several belonging to the secreted SPRY domain (SPRYSEC) family. SPRYSEC proteins are unique to members of the genus Globodera and have been implicated in both the induction and the repression of host defense responses. We have tested the properties of six different G. rostochiensis SPRYSEC proteins by expressing them in Nicotiana benthamiana and N. tabacum. We have found that all SPRYSEC proteins tested are able to suppress defense responses induced by NB-LRR proteins as well as cell death induced by elicitors, suggesting that defense repression is a common characteristic of members of this effector protein family. At the same time, GrSPRYSEC-15 elicited a defense responses in N. tabacum, which was found to be resistant to a virus expressing GrSPRYSEC-15. These results suggest that SPRYSEC proteins may possess characteristics that allow them to be recognized by the plant immune system. PMID:26322064

  4. Chlamydia trachomatis Slc1 is a type III secretion chaperone that enhances the translocation of its invasion effector substrate TARP.

    PubMed

    Brinkworth, Amanda J; Malcolm, Denise S; Pedrosa, António T; Roguska, Katarzyna; Shahbazian, Sevanna; Graham, James E; Hayward, Richard D; Carabeo, Rey A

    2011-10-01

    Bacterial type III secretion system (T3SS) chaperones pilot substrates to the export apparatus in a secretion-competent state, and are consequently central to the translocation of effectors into target cells. Chlamydia trachomatis is a genetically intractable obligate intracellular pathogen that utilizes T3SS effectors to trigger its entry into mammalian cells. The only well-characterized T3SS effector is TARP (translocated actin recruitment protein), but its chaperone is unknown. Here we exploited a known structural signature to screen for putative type III secretion chaperones encoded within the C. trachomatis genome. Using bacterial two-hybrid, co-precipitation, cross-linking and size exclusion chromatography we show that Slc1 (SycE-like chaperone 1; CT043) specifically interacts with a 200-amino-acid residue N-terminal region of TARP (TARP¹⁻²⁰⁰). Slc1 formed homodimers in vitro, as shown in cross-linking and gel filtration experiments. Biochemical analysis of an isolated Slc1-TARP¹⁻²⁰⁰ complex was consistent with a characteristic 2:1 chaperone-effector stoichiometry. Furthermore, Slc1 was co-immunoprecipitated with TARP from C. trachomatis elementary bodies. Also, coexpression of Slc1 specifically enhanced host cell translocation of TARP by a heterologous Yersinia enterocolitica T3SS. Taken together, we propose Slc1 as a chaperone of the C. trachomatis T3SS effector TARP.

  5. Transcription Factor Bcl11b Controls Effector and Memory CD8 T cell Fate Decision and Function during Poxvirus Infection

    PubMed Central

    Abboud, Georges; Stanfield, Jessica; Tahiliani, Vikas; Desai, Pritesh; Hutchinson, Tarun E.; Lorentsen, Kyle J.; Cho, Jonathan J.; Avram, Dorina; Salek-Ardakani, Shahram

    2016-01-01

    CD8+ T cells play an important role in host resistance to many viral infections, but the underlying transcriptional mechanisms governing their differentiation and functionality remain poorly defined. By using a highly virulent systemic and respiratory poxvirus infection in mice, we show that the transcription factor Bcl11b provides a dual trigger that sustains the clonal expansion of virus-specific effector CD8+ T cells, while simultaneously suppressing the expression of surface markers associated with short-lived effector cell (SLEC) differentiation. Additionally, we demonstrate that Bcl11b supports the acquisition of memory precursor effector cell (MPEC) phenotype and, thus, its absence causes near complete loss of lymphoid and lung-resident memory cells. Interestingly, despite having normal levels of T-bet and Eomesodermin, Bcl11b-deficient CD8+ T cells failed to execute effector differentiation needed for anti-viral cytokine production and degranulation, suggesting a non-redundant role of Bcl11b in regulation of this program. Thus, Bcl11b is a critical player in fate decision of SLECs and MPECs, as well as effector function and memory formation. PMID:27790219

  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. 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.

  8. Autoimmune effector memory T cells: the bad and the good

    PubMed Central

    Devarajan, Priyadharshini; Chen, Zhibin

    2014-01-01

    Immunological memory is a hallmark of adaptive immunity, a defense mechanism endowed to vertebrates during evolution. However, an autoimmune pathogenic role of memory lymphocytes is also emerging with accumulating evidence, despite reasonable skepticism on their existence in a chronic setting of autoimmune damage. It is conceivable that autoimmune memory would be particularly harmful since memory cells would constantly “remember” and attack the body's healthy tissues. It is even more detrimental given the resistance of memory T cells to immunomodulatory therapies. In this review, we focus on self-antigen-reactive CD4+ effector memory T (TEM) cells, surveying the evidence for the role of the TEM compartment in autoimmune pathogenesis. We will also discuss the role of TEM cells in chronic and acute infectious disease settings and how they compare to their counterparts in autoimmune diseases. With their long-lasting potency, the autoimmune TEM cells could also play a critical role in anti-tumor immunity, which may be largely based on their reactivity to self-antigens. Therefore, although autoimmune TEM cells are “bad” due to their role in relentless perpetration of tissue damage in autoimmune disease settings, they are unlikely a by-product of industrial development along the modern surge of autoimmune disease prevalence. Rather, they may be a product of evolution for their “good” in clearing damaged host cells in chronic infections and malignant cells in cancer settings. PMID:24203440

  9. RipAY, a Plant Pathogen Effector Protein, Exhibits Robust γ-Glutamyl Cyclotransferase Activity When Stimulated by Eukaryotic Thioredoxins*

    PubMed Central

    Fujiwara, Shoko; Kawazoe, Tomoki; Ohnishi, Kouhei; Kitagawa, Takao; Popa, Crina; Valls, Marc; Genin, Stéphane; Nakamura, Kazuyuki; Kuramitsu, Yasuhiro; Tanaka, Naotaka; Tabuchi, Mitsuaki

    2016-01-01

    The plant pathogenic bacterium Ralstonia solanacearum injects more than 70 effector proteins (virulence factors) into the host plant cells via the needle-like structure of a type III secretion system. The type III secretion system effector proteins manipulate host regulatory networks to suppress defense responses with diverse molecular activities. Uncovering the molecular function of these effectors is essential for a mechanistic understanding of R. solanacearum pathogenicity. However, few of the effectors from R. solanacearum have been functionally characterized, and their plant targets remain largely unknown. Here, we show that the ChaC domain-containing effector RipAY/RSp1022 from R. solanacearum exhibits γ-glutamyl cyclotransferase (GGCT) activity to degrade the major intracellular redox buffer, glutathione. Heterologous expression of RipAY, but not other ChaC family proteins conserved in various organisms, caused growth inhibition of yeast Saccharomyces cerevisiae, and the intracellular glutathione level was decreased to ∼30% of the normal level following expression of RipAY in yeast. Although active site mutants of GGCT activity were non-toxic, the addition of glutathione did not reverse the toxicity, suggesting that the toxicity might be a consequence of activity against other γ-glutamyl compounds. Intriguingly, RipAY protein purified from a bacterial expression system did not exhibit any GGCT activity, whereas it exhibited robust GGCT activity upon its interaction with eukaryotic thioredoxins, which are important for intracellular redox homeostasis during bacterial infection in plants. Our results suggest that RipAY has evolved to sense the host intracellular redox environment, which triggers its enzymatic activity to create a favorable environment for R. solanacearum infection. PMID:26823466

  10. 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.

  11. 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.

  12. 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.

  13. A transcription activator-like effector from Xanthomonas oryzae pv. oryzicola elicits dose-dependent resistance in rice.

    PubMed

    Hummel, Aaron W; Wilkins, Katherine E; Wang, Li; Cernadas, R Andres; Bogdanove, Adam J

    2017-01-01

    Xanthomonas spp. reduce crop yields and quality worldwide. During infection of their plant hosts, many strains secrete transcription activator-like (TAL) effectors, which enter the host cell nucleus and activate specific corresponding host genes at effector binding elements (EBEs) in the promoter. TAL effectors may contribute to disease by activating the expression of susceptibility genes or trigger resistance associated with the hypersensitive reaction (HR) by activating an executor resistance (R) gene. The rice bacterial leaf streak pathogen X. oryzae pv. oryzicola (Xoc) is known to suppress host resistance, and no host R gene has been identified against it, despite considerable effort. To further investigate Xoc suppression of host resistance, we conducted a screen of effectors from BLS256 and identified Tal2a as an HR elicitor in rice when delivered heterologously by a strain of the closely related rice bacterial blight pathogen X. oryzae pv. oryzae (Xoo) or by the soybean pathogen X. axonopodis pv. glycines. The HR required the Tal2a activation domain, suggesting an executor R gene. Tal2a activity was differentially distributed among geographically diverse Xoc isolates, being largely conserved among Asian isolates. We identified four genes induced by Tal2a in next-generation RNA sequencing experiments and confirmed them using quantitative real-time reverse transcription-polymerase chain reaction (qPCR). However, neither individual nor collective activation of these genes by designer TAL effectors resulted in HR. A tal2a knockout mutant of BLS256 showed virulence comparable with the wild-type, but plasmid-based overexpression of tal2a at different levels in the wild-type reduced virulence in a directly corresponding way. Overall, the results reveal that host resistance suppression by Xoc plays a critical role in pathogenesis. Further, the dose-dependent avirulence activity of Tal2a and the apparent lack of a single canonical target that accounts for HR point to

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. Identification of Novel Coxiella burnetii Icm/Dot Effectors and Genetic Analysis of Their Involvement in Modulating a Mitogen-Activated Protein Kinase Pathway

    PubMed Central

    Lifshitz, Ziv; Burstein, David; Schwartz, Kierstyn; Shuman, Howard A.; Pupko, Tal

    2014-01-01

    Coxiella burnetii, the causative agent of Q fever, is a human intracellular pathogen that utilizes the Icm/Dot type IVB secretion system to translocate effector proteins into host cells. To identify novel C. burnetii effectors, we applied a machine-learning approach to predict C. burnetii effectors, and examination of 20 such proteins resulted in the identification of 13 novel effectors. To determine whether these effectors, as well as several previously identified effectors, modulate conserved eukaryotic pathways, they were expressed in Saccharomyces cerevisiae. The effects on yeast growth were examined under regular growth conditions and in the presence of caffeine, a known modulator of the yeast cell wall integrity (CWI) mitogen-activated protein (MAP) kinase pathway. In the presence of caffeine, expression of the effectors CBU0885 and CBU1676 caused an enhanced inhibition of yeast growth, and the growth inhibition of CBU0388 was suppressed. Furthermore, analysis of synthetic lethality effects and examination of the activity of the CWI MAP kinase transcription factor Rlm1 indicated that CBU0388 enhances the activation of this MAP kinase pathway in yeast, while CBU0885 and CBU1676 abolish this activation. Additionally, coexpression of CBU1676 and CBU0388 resulted in mutual suppression of their inhibition of yeast growth. These results strongly indicate that these three effectors modulate the CWI MAP kinase pathway in yeast. Moreover, both CBU1676 and CBU0885 were found to contain a conserved haloacid dehalogenase (HAD) domain, which was found to be required for their activity. Collectively, our results demonstrate that MAP kinase pathways are most likely targeted by C. burnetii Icm/Dot effectors. PMID:24958706

  20. The Type IV Secretion System Effector Protein CirA Stimulates the GTPase Activity of RhoA and Is Required for Virulence in a Mouse Model of Coxiella burnetii Infection

    PubMed Central

    Weber, Mary M.; Faris, Robert; van Schaik, Erin J.; McLachlan, Juanita Thrasher; Wright, William U.; Tellez, Andres; Roman, Victor A.; Rowin, Kristina; Case, Elizabeth Di Russo; Luo, Zhao-Qing

    2016-01-01

    Coxiella burnetii, the etiological agent of Q fever in humans, is an intracellular pathogen that replicates in an acidified parasitophorous vacuole derived from host lysosomes. Generation of this replicative compartment requires effectors delivered into the host cell by the Dot/Icm type IVb secretion system. Several effectors crucial for C. burnetii intracellular replication have been identified, but the host pathways coopted by these essential effectors are poorly defined, and very little is known about how spacious vacuoles are formed and maintained. Here we demonstrate that the essential type IVb effector, CirA, stimulates GTPase activity of RhoA. Overexpression of CirA in mammalian cells results in cell rounding and stress fiber disruption, a phenotype that is rescued by overexpression of wild-type or constitutively active RhoA. Unlike other effector proteins that subvert Rho GTPases to modulate uptake, CirA is the first effector identified that is dispensable for uptake and instead recruits Rho GTPase to promote biogenesis of the bacterial vacuole. Collectively our results highlight the importance of CirA in coopting host Rho GTPases for establishment of Coxiella burnetii infection and virulence in mammalian cell culture and mouse models of infection. PMID:27324482

  1. The Type IV Secretion System Effector Protein CirA Stimulates the GTPase Activity of RhoA and Is Required for Virulence in a Mouse Model of Coxiella burnetii Infection.

    PubMed

    Weber, Mary M; Faris, Robert; van Schaik, Erin J; McLachlan, Juanita Thrasher; Wright, William U; Tellez, Andres; Roman, Victor A; Rowin, Kristina; Case, Elizabeth Di Russo; Luo, Zhao-Qing; Samuel, James E

    2016-09-01

    Coxiella burnetii, the etiological agent of Q fever in humans, is an intracellular pathogen that replicates in an acidified parasitophorous vacuole derived from host lysosomes. Generation of this replicative compartment requires effectors delivered into the host cell by the Dot/Icm type IVb secretion system. Several effectors crucial for C. burnetii intracellular replication have been identified, but the host pathways coopted by these essential effectors are poorly defined, and very little is known about how spacious vacuoles are formed and maintained. Here we demonstrate that the essential type IVb effector, CirA, stimulates GTPase activity of RhoA. Overexpression of CirA in mammalian cells results in cell rounding and stress fiber disruption, a phenotype that is rescued by overexpression of wild-type or constitutively active RhoA. Unlike other effector proteins that subvert Rho GTPases to modulate uptake, CirA is the first effector identified that is dispensable for uptake and instead recruits Rho GTPase to promote biogenesis of the bacterial vacuole. Collectively our results highlight the importance of CirA in coopting host Rho GTPases for establishment of Coxiella burnetii infection and virulence in mammalian cell culture and mouse models of infection.

  2. The necrotrophic effector SnToxA induces the synthesis of a novel phytoalexin in wheat.

    PubMed

    Du Fall, Lauren A; Solomon, Peter S

    2013-10-01

    Stagonospora nodorum and Pyrenophora tritici-repentis produce the effector ToxA that interacts with the dominant susceptibility gene in wheat, Tsn1. However, the way in which ToxA induces cell death and causes disease is unclear. Here, we performed comprehensive metabolite profiling of ToxA-infiltrated wheat (Triticum aestivum) to observe the secondary metabolite response to this effector. A strong induction of secondary metabolism subsequent to SnToxA infiltration was observed, including the monoamine serotonin. We established a novel role for serotonin as a phytoalexin in wheat and demonstrated that serotonin strongly inhibited sporulation of S. nodorum. Microscopy revealed that serotonin interferes with spore formation and maturation within pycnidial structures of the fungus. Subsequent analysis of S. nodorum exposed to serotonin revealed metabolites changes previously associated with sporulation, including trehalose and alternariol. Furthermore, we identified significantly lower concentrations of serotonin during infection compared with infiltration with ToxA, providing evidence that S. nodorum may suppress plant defence. This is the first study demonstrating induction of plant secondary metabolites in response to a necrotrophic effector that have significant antifungal potential against the pathogen. While it is generally accepted that necrotrophs exploit host cell responses, the current research strengthens the notion that necrotrophs require mechanisms to overcome plant defence to survive initial stages of infection.

  3. Differential expression of candidate salivary effector proteins in field collections of Hessian fly, Mayetiola destructor

    PubMed Central

    Johnson, A J; Shukle, R H; Chen, M-S; Srivastava, S; Subramanyam, S; Schemerhorn, B J; Weintraub, P G; Abdel Moniem, H E M; Flanders, K L; Buntin, G D; Williams, C E

    2015-01-01

    Evidence is emerging that some proteins secreted by gall-forming parasites of plants act as effectors responsible for systemic changes in the host plant, such as galling and nutrient tissue formation. A large number of secreted salivary gland proteins (SSGPs) that are the putative effectors responsible for the physiological changes elicited in susceptible seedling wheat by Hessian fly, Mayetiola destructor (Say), larvae have been documented. However, how the genes encoding these candidate effectors might respond under field conditions is unknown. The goal of this study was to use microarray analysis to investigate variation in SSGP transcript abundance amongst field collections from different geographical regions (southeastern USA, central USA, and the Middle East). Results revealed significant variation in SSGP transcript abundance amongst the field collections studied. The field collections separated into three distinct groups that corresponded to the wheat classes grown in the different geographical regions as well as to recently described Hessian fly populations. These data support previous reports correlating Hessian fly population structure with micropopulation differences owing to agro-ecosystem parameters such as cultivation of regionally adapted wheat varieties, deployment of resistance genes and variation in climatic conditions. PMID:25528896

  4. Dendritic Cell Targeting of Bacillus anthracis Protective Antigen Expressed by Lactobacillus acidophilus Protects Mice from Lethal Challenge

    DTIC Science & Technology

    2008-10-28

    Dendritic cell targeting of Bacillus anthracis protective antigen expressed by Lactobacillus acidophilus protects mice from lethal challenge M...lethal chal- lenge. A vaccine strategy was established by using Lactobacillus acidophilus to deliver Bacillus anthracis protective antigen (PA) via...4. TITLE AND SUBTITLE Dendritic cell targeting of Bacillus anthracis protective antigen expressed by Lactobacillus acidophilus protects mice

  5. Type III secretion and effectors shape the survival and growth pattern of Pseudomonas syringae on leaf surfaces.

    PubMed

    Lee, Jiyoung; Teitzel, Gail M; Munkvold, Kathy; del Pozo, Olga; Martin, Gregory B; Michelmore, Richard W; Greenberg, Jean T

    2012-04-01

    The bacterium Pseudomonas syringae pv syringae B728a (PsyB728a) uses a type III secretion system (T3SS) to inject effector proteins into plant cells, a process that modulates the susceptibility of different plants to infection. Analysis of GREEN FLUORESCENT PROTEIN-expressing PsyB728a after spray inoculation without additives under moderate relative humidity conditions permitted (1) a detailed analysis of this strain's survival and growth pattern on host (Nicotiana benthamiana) and nonhost (tomato [Solanum lycopersicum]) leaf surfaces, (2) an assessment of the role of plant defenses in affecting PsyB728a leaf surface (epiphytic) growth, and (3) the contribution of the T3SS and specific effectors to PsyB728a epiphytic survival and growth. On host leaf surfaces, PsyB728a cells initially persist without growing, and show an increased population only after 48 h, unless plants are pretreated with the defense-inducing chemical benzothiazole. During the persistence period, some PsyB728a cells induce a T3SS reporter, whereas a T3SS-deficient mutant shows reduced survival. By 72 h, rare invasion by PsyB728a to the mesophyll region of host leaves occurs, but endophytic and epiphytic bacterial growths are not correlated. The effectors HopZ3 and HopAA1 delay the onset of epiphytic growth of PsyB728a on N. benthamiana, whereas they promote epiphytic survival/growth on tomato. These effectors localize to distinct sites in plant cells and likely have different mechanisms of action. HopZ3 may enzymatically modify host targets, as it requires residues important for the catalytic activity of other proteins in its family of proteases. Thus, the T3SS, HopAA1, HopZ3, and plant defenses strongly influence epiphytic survival and/or growth of PsyB728a.

  6. Gene targeting by the TAL effector PthXo2 reveals cryptic resistance gene for bacterial blight of rice.

    PubMed

    Zhou, Junhui; Peng, Zhao; Long, Juying; Sosso, Davide; Liu, Bo; Eom, Joon-Seob; Huang, Sheng; Liu, Sanzhen; Vera Cruz, Casiana; Frommer, Wolf B; White, Frank F; Yang, Bing

    2015-05-01

    Bacterial blight of rice is caused by the γ-proteobacterium Xanthomonas oryzae pv. oryzae, which utilizes a group of type III TAL (transcription activator-like) effectors to induce host gene expression and condition host susceptibility. Five SWEET genes are functionally redundant to support bacterial disease, but only two were experimentally proven targets of natural TAL effectors. Here, we report the identification of the sucrose transporter gene OsSWEET13 as the disease-susceptibility gene for PthXo2 and the existence of cryptic recessive resistance to PthXo2-dependent X. oryzae pv. oryzae due to promoter variations of OsSWEET13 in japonica rice. PthXo2-containing strains induce OsSWEET13 in indica rice IR24 due to the presence of an unpredicted and undescribed effector binding site not present in the alleles in japonica rice Nipponbare and Kitaake. The specificity of effector-associated gene induction and disease susceptibility is attributable to a single nucleotide polymorphism (SNP), which is also found in a polymorphic allele of OsSWEET13 known as the recessive resistance gene xa25 from the rice cultivar Minghui 63. The mutation of OsSWEET13 with CRISPR/Cas9 technology further corroborates the requirement of OsSWEET13 expression for the state of PthXo2-dependent disease susceptibility to X. oryzae pv. oryzae. Gene profiling of a collection of 104 strains revealed OsSWEET13 induction by 42 isolates of X. oryzae pv. oryzae. Heterologous expression of OsSWEET13 in Nicotiana benthamiana leaf cells elevates sucrose concentrations in the apoplasm. The results corroborate a model whereby X. oryzae pv. oryzae enhances the release of sucrose from host cells in order to exploit the host resources.

  7. 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.

  8. 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.

  9. E2~Ub conjugates regulate the kinase activity of Shigella effector OspG during pathogenesis

    SciTech Connect

    Pruneda, Jonathan N.; Smith, F. Donelson; Daurie, Angela; Swaney, Danielle L.; Villén, Judit; Scott, John D.; Stadnyk, Andrew W.; Le Trong, Isolde; Stenkamp, Ronald E.; Klevit, Rachel E.; Rohde, John R.; Brzovic, Peter S.

    2014-01-20

    Pathogenic bacteria introduce effector proteins directly into the cytosol of eukaryotic cells to promote invasion and colonization. OspG, a Shigella spp. effector kinase, plays a role in this process by helping to suppress the host inflammatory response. OspG has been reported to bind host E2 ubiquitin-conjugating enzymes activated with ubiquitin (E2~Ub), a key enzyme complex in ubiquitin transfer pathways. A cocrystal structure of the OspG/UbcH5c~Ub complex reveals that complex formation has important ramifications for the activity of both OspG and the UbcH5c~Ub conjugate. OspG is a minimal kinase domain containing only essential elements required for catalysis. UbcH5c~Ub binding stabilizes an active conformation of the kinase, greatly enhancing OspG kinase activity. In contrast, interaction with OspG stabilizes an extended, less reactive form of UbcH5c~Ub. Recognizing conserved E2 features, OspG can interact with at least ten distinct human E2s~Ub. Mouse oral infection studies indicate that E2~Ub conjugates act as novel regulators of OspG effector kinase function in eukaryotic host cells.

  10. 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.

  11. 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.

  12. 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.

  13. [B-cell targeted therapy for children and adolescents with rheumatic diseases].

    PubMed

    Morbach, H; Girschick, H J

    2013-05-01

    The introduction of cytokine-targeted therapies has significantly improved the treatment options of rheumatic diseases; however, some patients are also refractory to these treatment measures. The B cells play a central role in the pathogenesis of many rheumatic diseases and B-cell targeted therapies are a promising option as second-line medication for treating patients with a refractory disease course. Randomized controlled trials analyzing the efficacy of B-cell directed therapies for childhood rheumatic diseases have not yet been performed. The use of the B-cell depleting antibody rituximab showed positive results in non-controlled case series of juvenile systemic lupus erythematosus (SLE) patients. Patients with a refractory disease course of oligoarticular or polyarticular juvenile idiopathic arthritis might also benefit from B-cell depletion using rituximab. The B cell-targeting therapies for the treatment of childhood rheumatic diseases should be initiated and closely supervised by a pediatric rheumatologist.

  14. Analysis of the role of the type III effector inventory of Pseudomonas syringae pv. phaseolicola 1448a in interaction with the plant.

    PubMed

    Zumaquero, Adela; Macho, Alberto P; Rufián, José S; Beuzón, Carmen R

    2010-09-01

    In Pseudomonas syringae, the type III secretion system (T3SS) is essential for disease in compatible hosts and for eliciting the hypersensitive response in incompatible hosts. P. syringae pathovars secrete a variable number of type III effectors that form their secretomes. The secretome of Pseudomonas syringae pv. phaseolicola 1448a (Pph1448a) currently includes 22 experimentally validated effectors, one HrpL-regulated candidate for which translocation results have been inconsistent, two translocated candidates for which in planta expression has not been established, one bioinformatically identified candidate, and six candidates that have been experimentally discarded. We analyzed the translocation and/or expression of these and other candidates to complete the Pph1448a effector inventory, bringing this inventory to 27 bona fide effectors, including a new one that does not belong to any of the previously described effector families. We developed a simple process for rapidly making single and double knockout mutants and apply it to the generation of an effector mutant collection that includes single knockouts for the majority of the Pph1448a effector inventory. We also generated two double mutant strains containing effectors with potentially redundant functions and analyzed the virulence of the single and double mutant strains as well as strains expressing each of the effectors from a plasmid. We demonstrate that AvrB4-1 and AvrB4-2, as well as HopW1-1 and HopW1-2, are fully redundant and contribute to virulence in bean plants, thus validating this approach for dissecting the contribution of the Pph1448a type III effector inventory to virulence. We also analyzed the effect that the expression of these four effectors from Pseudomonas syringae pv. tomato DC3000 (PtoDC3000) has during its interaction with Arabidopsis thaliana, establishing that AvrB4-1, but not the others, determines a restriction of bacterial growth that takes place mostly independently of the

  15. Analysis of the Role of the Type III Effector Inventory of Pseudomonas syringae pv. phaseolicola 1448a in Interaction with the Plant ▿

    PubMed Central

    Zumaquero, Adela; Macho, Alberto P.; Rufián, José S.; Beuzón, Carmen R.

    2010-01-01

    In Pseudomonas syringae, the type III secretion system (T3SS) is essential for disease in compatible hosts and for eliciting the hypersensitive response in incompatible hosts. P. syringae pathovars secrete a variable number of type III effectors that form their secretomes. The secretome of Pseudomonas syringae pv. phaseolicola 1448a (Pph1448a) currently includes 22 experimentally validated effectors, one HrpL-regulated candidate for which translocation results have been inconsistent, two translocated candidates for which in planta expression has not been established, one bioinformatically identified candidate, and six candidates that have been experimentally discarded. We analyzed the translocation and/or expression of these and other candidates to complete the Pph1448a effector inventory, bringing this inventory to 27 bona fide effectors, including a new one that does not belong to any of the previously described effector families. We developed a simple process for rapidly making single and double knockout mutants and apply it to the generation of an effector mutant collection that includes single knockouts for the majority of the Pph1448a effector inventory. We also generated two double mutant strains containing effectors with potentially redundant functions and analyzed the virulence of the single and double mutant strains as well as strains expressing each of the effectors from a plasmid. We demonstrate that AvrB4-1 and AvrB4-2, as well as HopW1-1 and HopW1-2, are fully redundant and contribute to virulence in bean plants, thus validating this approach for dissecting the contribution of the Pph1448a type III effector inventory to virulence. We also analyzed the effect that the expression of these four effectors from Pseudomonas syringae pv. tomato DC3000 (PtoDC3000) has during its interaction with Arabidopsis thaliana, establishing that AvrB4-1, but not the others, determines a restriction of bacterial growth that takes place mostly independently of the

  16. Genome-wide analysis of small secreted cysteine-rich proteins identifies candidate effector proteins potentially involved in Fusarium graminearum-wheat interactions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pathogen-derived, small secreted cysteine-rich proteins (SSCPs) are known to be a common source of fungal effectors that trigger resistance or susceptibility in specific host plants. This group of proteins has not been well studied in Fusarium graminearum, the primary cause of Fusarium head blight ...

  17. Breaking the DNA-binding code of Ralstonia solanacearum TAL effectors provides new possibilities to generate plant resistance genes against bacterial wilt disease.

    PubMed

    de Lange, Orlando; Schreiber, Tom; Schandry, Niklas; Radeck, Jara; Braun, Karl Heinz; Koszinowski, Julia; Heuer, Holger; Strauß, Annett; Lahaye, Thomas

    2013-08-01

    Ralstonia solanacearum is a devastating bacterial phytopathogen with a broad host range. Ralstonia solanacearum injected effector proteins (Rips) are key to the successful invasion of host plants. We have characterized Brg11(hrpB-regulated 11), the first identified member of a class of Rips with high sequence similarity to the transcription activator-like (TAL) effectors of Xanthomonas spp., collectively termed RipTALs. Fluorescence microscopy of in planta expressed RipTALs showed nuclear localization. Domain swaps between Brg11 and Xanthomonas TAL effector (TALE) AvrBs3 (avirulence protein triggering Bs3 resistance) showed the functional interchangeability of DNA-binding and transcriptional activation domains. PCR was used to determine the sequence of brg11 homologs from strains infecting phylogenetically diverse host plants. Brg11 localizes to the nucleus and activates promoters containing a matching effector-binding element (EBE). Brg11 and homologs preferentially activate promoters containing EBEs with a 5' terminal guanine, contrasting with the TALE preference for a 5' thymine. Brg11 and other RipTALs probably promote disease through the transcriptional activation of host genes. Brg11 and the majority of homologs identified in this study were shown to activate similar or identical target sequences, in contrast to TALEs, which generally show highly diverse target preferences. This information provides new options for the engineering of plants resistant to R. solanacearum.

  18. Effector-Triggered Immune Response in Arabidopsis thaliana Is a Quantitative Trait

    PubMed Central

    Iakovidis, Michail; Teixeira, Paulo J. P. L.; Exposito-Alonso, Moises; Cowper, Matthew G.; Law, Theresa F.; Liu, Qingli; Vu, Minh Chau; Dang, Troy Minh; Corwin, Jason A.; Weigel, Detlef; Dangl, Jeffery L.; Grant, Sarah R.

    2016-01-01

    We identified loci responsible for natural variation in Arabidopsis thaliana (Arabidopsis) responses to a bacterial pathogen virulence factor, HopAM1. HopAM1 is a type III effector protein secreted by the virulent Pseudomonas syringae strain Pto DC3000. Delivery of HopAM1 from disarmed Pseudomonas strains leads to local cell death, meristem chlorosis, or both, with varying intensities in different Arabidopsis accessions. These phenotypes are not associated with differences in bacterial growth restriction. We treated the two phenotypes as quantitative traits to identify host loci controlling responses to HopAM1. Genome-wide association (GWA) of 64 Arabidopsis accessions identified independent variants highly correlated with response to each phenotype. Quantitative trait locus (QTL) mapping in a recombinant inbred population between Bur-0 and Col-0 accessions revealed genetic linkage to regions distinct from the top GWA hits. Two major QTL associated with HopAM1-induced cell death were also associated with HopAM1-induced chlorosis. HopAM1-induced changes in Arabidopsis gene expression showed that rapid HopAM1-dependent cell death in Bur-0 is correlated with effector-triggered immune responses. Studies of the effect of mutations in known plant immune system genes showed, surprisingly, that both cell death and chlorosis phenotypes are enhanced by loss of EDS1, a regulatory hub in the plant immune-signaling network. Our results reveal complex genetic architecture for response to this particular type III virulence effector, in contrast to the typical monogenic control of cell death and disease resistance triggered by most type III effectors. PMID:27412712

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

    PubMed Central

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

    2016-01-01

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

  20. Metabolic crosstalk between host and pathogen: sensing, adapting and competing.

    PubMed

    Olive, Andrew J; Sassetti, Christopher M

    2016-04-01

    Our understanding of bacterial pathogenesis is dominated by the cell biology of the host-pathogen interaction. However, the majority of metabolites that are used in prokaryotic and eukaryotic physiology and signalling are chemically similar or identical. Therefore, the metabolic crosstalk between pathogens and host cells may be as important as the interactions between bacterial effector proteins and their host targets. In this Review we focus on host-pathogen interactions at the metabolic level: chemical signalling events that enable pathogens to sense anatomical location and the local physiology of the host; microbial metabolic pathways that are dedicated to circumvent host immune mechanisms; and a few metabolites as central points of competition between the host and bacterial pathogens.

  1. 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

  2. Toxoplasma exports dense granule proteins beyond the vacuole to the host cell nucleus and rewires the host genome expression.

    PubMed

    Bougdour, Alexandre; Tardieux, Isabelle; Hakimi, Mohamed-Ali

    2014-03-01

    Toxoplasma gondii is the most widespread apicomplexan parasite and occupies a large spectrum of niches by infecting virtually any warm-blooded animals. As an obligate intracellular parasite, Toxoplasma has evolved a repertoire of strategies to fine-tune the cellular environment in an optimal way to promote growth and persistence in host tissues hence increasing the chance to be transmitted to new hosts. Short and long-term intracellular survival is associated with Toxoplasma ability to both evade the host deleterious immune defences and to stimulate a beneficial immune balance by governing host cell gene expression. It is only recently that parasite proteins responsible for driving these transcriptional changes have been identified. While proteins contained in the apical secretory Rhoptry organelle have already been identified as bona fide secreted effectors that divert host signalling pathways, recent findings revealed that dense granule proteins should be added to the growing list of effectors as they reach the host cell cytoplasm and nucleus and target various host cell pathways in the course of cell infection. Herein, we emphasize on a novel subfamily of dense granule residentproteins, exemplified with the GRA16 and GRA24 members we recently discovered as both are exported beyond the vacuole-containing parasites and reach the host cell nucleus to reshape the host genome expression.

  3. WASp identity theft by a bacterial effector.

    PubMed

    Daugherty-Clarke, Karen; Goode, Bruce L

    2008-09-01

    EspF(U), a protein secreted by pathogenic enterohaemorrhagic E. coli (EHEC), activates N-WASp/WASp to induce actin pedestal formation in host cells. Two recent papers in Nature show that EspF(U) exploits a WASp activation strategy so extreme that it may effectively sequester WASp, blinding it to both autoinhibition and cellular regulation.

  4. Two Cytoplasmic Effectors of Phytophthora sojae Regulate Plant Cell Death via Interactions with Plant Catalases1

    PubMed Central

    Zhang, Meixiang; Li, Qi; Liu, Tingli; Liu, Li; Shen, Danyu; Zhu, Ye; Liu, Peihan; Zhou, Jian-Min; Dou, Daolong

    2015-01-01

    Plant pathogenic oomycetes, such as Phytophthora sojae, secrete an arsenal of host cytoplasmic effectors to promote infection. We have shown previously that P. sojae PsCRN63 (for crinkling- and necrosis-inducing proteins) induces programmed cell death (PCD) while PsCRN115 blocks PCD in planta; however, they are jointly required for full pathogenesis. Here, we find that PsCRN63 alone or PsCRN63 and PsCRN115 together might suppress the immune responses of Nicotiana benthamiana and demonstrate that these two cytoplasmic effectors interact with catalases from N. benthamiana and soybean (Glycine max). Transient expression of PsCRN63 increases hydrogen peroxide (H2O2) accumulation, whereas PsCRN115 suppresses this process. Transient overexpression of NbCAT1 (for N. benthamiana CATALASE1) or GmCAT1 specifically alleviates PsCRN63-induced PCD. Suppression of the PsCRN63-induced PCD by PsCRN115 is compromised when catalases are silenced in N. benthamiana. Interestingly, the NbCAT1 is recruited into the plant nucleus in the presence of PsCRN63 or PsCRN115; NbCAT1 and GmCAT1 are destabilized when PsCRN63 is coexpressed, and PsCRN115 inhibits the processes. Thus, PsCRN63/115 manipulates plant PCD through interfering with catalases and perturbing H2O2 homeostasis. Furthermore, silencing of catalase genes enhances susceptibility to Phytophthora capsici, indicating that catalases are essential for plant resistance. Taken together, we suggest that P. sojae secretes these two effectors to regulate plant PCD and H2O2 homeostasis through direct interaction with catalases and, therefore, overcome host immune responses. PMID:25424308

  5. Inhibition of antiskin allograft immunity induced by infusions with photoinactivated effector T lymphocytes (PET cells).

    PubMed Central

    Perez, M. I.; Edelson, R. L.; John, L.; Laroche, L.; Berger, C. L.

    1989-01-01

    Induction of tolerance for skin allotransplantation requires selective suppression of the host response to foreign histocompatibility antigens. This report describes a new approach which employs pre-treatment with 8-methoxypsoralen (8-MOP) and ultraviolet A light (UVA) to render the effector cells of graft rejection immunogenic for the syngeneic recipient. Eight days after BALB/c mice received CBA/j skin grafts, their splenocytes were treated with 100 ng/ml 8-MOP and 1 J/cm2 UVA prior to reinfusion into naive BALB/c recipients. Recipient mice were tested for tolerance to alloantigens in mixed leukocyte culture (MLC), cytotoxicity (CTL), delayed-type hypersensitivity assays (DTH), and challenge with a fresh CBA/j graft. Splenocytes from BALB/c recipients of photoinactivated splenocytes containing the effector cells of CBA/j alloantigen rejection proliferated poorly in MLC and generated lower cytotoxic T-cell responses to CBA/j alloantigens in comparison with sensitized and naive controls and suppressed the MLC and CTL response to alloantigen from sensitized and naive BALB/c mice. In vivo, the DTH response was specifically suppressed to the relevant alloantigen in comparison with controls. BALB/c mice treated in this fashion retained a CBA/j skin graft for up to 42 days post-transplantation without visual evidence of rejection. These results showed that reinfusion of photoinactivated effector cells resulted in an immunosuppressive host response which specifically inhibited in vitro and in vivo responses that correlate with allograft rejection and permitted prolonged retention of histoincompatible skin grafts. PMID:2636801

  6. 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.

  7. Ubiquitination independent of E1 and E2 enzymes by bacterial effectors

    SciTech Connect

    Qiu, Jiazhang; Sheedlo, Michael J.; Yu, Kaiwen; Tan, Yunhao; Nakayasu, Ernesto S.; Das, Chittaranjan; Liu, Xiaoyun; Luo, Zhao-Qing

    2016-04-06

    Signaling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalyzed by the E1, E2 and E3 three-enzyme cascade 1, which links the C terminus of ubiquitin via an isopeptide bond mostly to the ε-amino group of a lysine of the substrate. Given the essential roles of ubiquitination in the regulation of the immune system, it is not surprising that the ubiquitination network is a common target for diverse infectious agents 2. For example, many bacterial pathogens exploit ubiquitin signaling using virulence factors that function as E3 ligases, deubiquitinases 3 or as enzymes that directly attack ubiquitin 4. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a niche permissive for its replication in phagocytes 5. Here we demonstrate that members of the SidE effector family (SidEs) of L. pneumophila ubiquitinate multiple Rab small GTPases associated with the endoplasmic reticulum (ER). Moreover, we show that these proteins are capable of catalyzing ubiquitination without the need for the E1 and E2 enzymes. The E1/E2-independent ubiquitination catalyzed by these enzymes requires NAD but not ATP and Mg2+. A putative mono ADP-ribosyltransferase (mART) motif critical for the ubiquitination activity is also essential for the role of SidEs in intracellular bacterial replication in a protozoan host. These results establish that ubiquitination can be catalyzed by a single enzyme.

  8. Direct targeting of membrane fusion by SNARE mimicry: Convergent evolution of Legionella effectors

    PubMed Central

    Shi, Xingqi; Halder, Partho; Yavuz, Halenur; Shuman, Howard A.

    2016-01-01

    Legionella pneumophila, the Gram-negative pathogen causing Legionnaires’ disease, infects host cells by hijacking endocytic pathways and forming a Legionella-containing vacuole (LCV) in which the bacteria replicate. To promote LCV expansion and prevent lysosomal targeting, effector proteins are translocated into the host cell where they alter membrane traffic. Here we show that three of these effectors [LegC2 (Legionella eukaryotic-like gene C2)/YlfB (yeast lethal factor B), LegC3, and LegC7/YlfA] functionally mimic glutamine (Q)-SNARE proteins. In infected cells, the three proteins selectively form complexes with the endosomal arginine (R)-SNARE vesicle-associated membrane protein 4 (VAMP4). When reconstituted in proteoliposomes, these proteins avidly fuse with liposomes containing VAMP4, resulting in a stable complex with properties resembling canonical SNARE complexes. Intriguingly, however, the LegC/SNARE hybrid complex cannot be disassembled by N-ethylmaleimide-sensitive factor. We conclude that LegCs use SNARE mimicry to divert VAMP4-containing vesicles for fusion with the LCV, thus promoting its expansion. In addition, the LegC/VAMP4 complex avoids the host’s disassembly machinery, thus effectively trapping VAMP4 in an inactive state. PMID:27436892

  9. The secreted effector protein EspZ is essential for virulence of rabbit enteropathogenic Escherichia coli.

    PubMed

    Wilbur, John Scott; Byrd, Wyatt; Ramamurthy, Shylaja; Ledvina, Hannah E; Khirfan, Khaldoon; Riggs, Michael W; Boedeker, Edgar C; Vedantam, Gayatri; Viswanathan, V K

    2015-03-01

    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

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. Host-targeting protein 1 (SpHtp1) from the oomycete Saprolegnia parasitica translocates specifically into fish cells in a tyrosine-O-sulphate-dependent manner.

    PubMed

    Wawra, Stephan; Bain, Judith; Durward, Elaine; de Bruijn, Irene; Minor, Kirsty L; Matena, Anja; Löbach, Lars; Whisson, Stephen C; Bayer, Peter; Porter, Andrew J; Birch, Paul R J; Secombes, Chris J; van West, Pieter

    2012-02-07

    The eukaryotic oomycetes, or water molds, contain several species that are devastating pathogens of plants and animals. During infection, oomycetes translocate effector proteins into host cells, where they interfere with host-defense responses. For several oomycete effectors (i.e., the RxLR-effectors) it has been shown that their N-terminal polypeptides are important for the delivery into the host. Here we demonstrate that the putative RxLR-like effector, host-targeting protein 1 (SpHtp1), from the fish pathogen Saprolegnia parasitica translocates specifically inside host cells. We further demonstrate that cell-surface binding and uptake of this effector protein is mediated by an interaction with tyrosine-O-sulfate-modified cell-surface molecules and not via phospholipids, as has been reported for RxLR-effectors from plant pathogenic oomycetes. These results reveal an effector translocation route based on tyrosine-O-sulfate binding, which could be highly relevant for a wide range of host-microbe interactions.

  16. Hijacking Host Cell Highways: Manipulation of the Host Actin Cytoskeleton by Obligate Intracellular Bacterial Pathogens

    PubMed Central

    Colonne, Punsiri M.; Winchell, Caylin G.; Voth, Daniel E.

    2016-01-01

    Intracellular bacterial pathogens replicate within eukaryotic cells and display unique adaptations that support key infection events including invasion, replication, immune evasion, and dissemination. From invasion to dissemination, all stages of the intracellular bacterial life cycle share the same three-dimensional cytosolic space containing the host cytoskeleton. For successful infection and replication, many pathogens hijack the cytoskeleton using effector proteins introduced into the host cytosol by specialized secretion systems. A subset of effectors contains eukaryotic-like motifs that mimic host proteins to exploit signaling and modify specific cytoskeletal components such as actin and microtubules. Cytoskeletal rearrangement promotes numerous events that are beneficial to the pathogen, including internalization of bacteria, structural support for bacteria-containing vacuoles, altered vesicular trafficking, actin-dependent bacterial movement, and pathogen dissemination. This review highlights a diverse group of obligate intracellular bacterial pathogens that manipulate the host cytoskeleton to thrive within eukaryotic cells and discusses underlying molecular mechanisms that promote these dynamic host-pathogen interactions. PMID:27713866

  17. 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.

  18. 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.

  19. Isolation of whole esophageal gland cells from plant-parasitic nematodes for transcriptome analyses and effector identification.

    PubMed

    Maier, Tom R; Hewezi, Tarek; Peng, Jiqing; Baum, Thomas J

    2013-01-01

    Esophageal glands of plant-parasitic nematodes are highly specialized cells whose gene expression products include secreted effector proteins, which govern nematode parasitism of host plants. Therefore, elucidating the transcriptomes of esophageal glands with the goal of identifying nematode effectors is a promising avenue to understanding nematode parasitism and its evolutionary origins as well as to devising nematode control strategies. We have developed a method to separate and isolate individual esophageal gland cells from multiple species of plant-parasitic nematodes while preserving RNA quality. We have used such isolated gland cells for transcriptome analysis via high-throughput DNA sequencing. This method relies on the differential histochemical staining of the gland cells after homogenization of phytonematode tissues. Total RNA was extracted from whole gland cells isolated from eight different plant-parasitic nematode species. To validate this approach, the isolated RNA from three plant-parasitic nematode species-Globodera rostochiensis, Pratylenchus penetrans, and Radopholus similis-was amplified, gel purified, and used for 454 sequencing. We obtained 456,801 total reads with an average read length of 409 bp. Sequence analyses revealed the presence of homologs of previously known nematode effectors in these libraries, thus validating our approach. These data provide compelling evidence that this technical advance can be used to relatively easily and expediently discover effector repertoires of plant-parasitic nematodes.

  20. 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

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

    PubMed

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

    2016-02-01

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

  2. Bartonella entry mechanisms into mammalian host cells.

    PubMed

    Eicher, Simone C; Dehio, Christoph

    2012-08-01

    The Gram-negative genus Bartonella comprises arthropod-borne pathogens that typically infect mammals in a host-specific manner. Bartonella bacilliformis and Bartonella quintana are human-specific pathogens, while several zoonotic bartonellae specific for diverse animal hosts infect humans as an incidental host. Clinical manifestations of Bartonella infections range from mild symptoms to life-threatening disease. Following transmission by blood-sucking arthropods or traumatic contact with infected animals, bartonellae display sequential tropisms towards endothelial and possibly other nucleated cells and erythrocytes, the latter in a host-specific manner. Attachment to the extracellular matrix (ECM) and to nucleated cells is mediated by surface-exposed bacterial adhesins, in particular trimeric autotransporter adhesins (TAAs). The subsequent engulfment of the pathogen into a vacuolar structure follows a unique series of events whereby the pathogen avoids the endolysosomal compartments. For Bartonella henselae and assumingly most other species, the infection process is aided at different steps by Bartonella effector proteins (Beps). They are injected into host cells through the type IV secretion system (T4SS) VirB/D4 and subvert host cellular functions to favour pathogen uptake. Bacterial binding to erythrocytes is mediated by Trw, another T4SS, in a strictly host-specific manner, followed by pathogen-forced uptake involving the IalB invasin and subsequent replication and persistence within a membrane-bound intra-erythrocytic compartment.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. Endothelial cell targeted molecular imaging in tumor angiogenesis: strategies and current status.

    PubMed

    Xu, Ye; Zeng, Yun; Liu, Yanhong; Liu, Gang; Ai, Hua

    2013-01-01

    Angiogenesis plays crucial roles in tumor growth, progression and metastasis. Non-invasive in vivo imaging of tumor neovasculature is a fundamental prerequisite for effective therapeutic intervention, particularly anti-angiogenic treatment regimens. Emerging molecular imaging techniques now allow recognition of cellular/molecular processes before gross pathological changes, leading to better understanding of fundamental biological processes of tumor angiogenesis. In this review, we will summarize recent progresses on molecular imaging of attractive biochemical epitopes in tumor angiogenesis, especially the endothelial cell targets-based imaging probes.

  8. 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

  9. 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

  10. 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

  11. 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.

  12. 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).

  13. Genome analyses of the wheat yellow (stripe) rust pathogen Puccinia striiformis f. sp. tritici reveal polymorphic and haustorial expressed secreted proteins as candidate effectors

    PubMed Central

    2013-01-01

    Background Wheat yellow (stripe) rust caused by Puccinia striiformis f. sp. tritici (PST) is one of the most devastating diseases of wheat worldwide. To design effective breeding strategies that maximize the potential for durable disease resistance it is important to understand the molecular basis of PST pathogenicity. In particular, the characterisation of the structure, function and evolutionary dynamics of secreted effector proteins that are detected by host immune receptors can help guide and prioritize breeding efforts. However, to date, our knowledge of the effector repertoire of cereal rust pathogens is limited. Results We re-sequenced genomes of four PST isolates from the US and UK to identify effector candidates and relate them to their distinct virulence profiles. First, we assessed SNP frequencies between all isolates, with heterokaryotic SNPs being over tenfold more frequent (5.29 ± 2.23 SNPs/kb) than homokaryotic SNPs (0.41 ± 0.28 SNPs/kb). Next, we implemented a bioinformatics pipeline to integrate genomics, transcriptomics, and effector-focused annotations to identify and classify effector candidates in PST. RNAseq analysis highlighted transcripts encoding secreted proteins that were significantly enriched in haustoria compared to infected tissue. The expression of 22 candidate effector genes was characterised using qRT-PCR, revealing distinct temporal expression patterns during infection in wheat. Lastly, we identified proteins that displayed non-synonymous substitutions specifically between the two UK isolates PST-87/7 and PST-08/21, which differ in virulence to two wheat varieties. By focusing on polymorphic variants enriched in haustoria, we identified five polymorphic effector candidates between PST-87/7 and PST-08/21 among 2,999 secreted proteins. These allelic variants are now a priority for functional validation as virulence/avirulence effectors in the corresponding wheat varieties. Conclusions Integration of genomics, transcriptomics, and

  14. Modulation of membrane phosphoinositide dynamics by the phosphatidylinositide 4-kinase activity of the Legionella LepB effector.

    PubMed

    Dong, Na; Niu, Miao; Hu, Liyan; Yao, Qing; Zhou, Rui; Shao, Feng

    2016-12-12

    Legionella pneumophila, the causative bacterium for Legionnaires' disease, hijacks host membrane trafficking for the maturation of the Legionella-containing vacuole (LCV). The LCV membrane mainly contains PtdIns4P, which is important for anchoring many secreted Legionella effectors onto the LCV. Here, we identify a cryptic functional domain (LepB_NTD) preceding the well-characterized RabGAP domain in the Legionella Dot/Icm type IV secretion system effector LepB. LepB_NTD alone is toxic to yeast and can disrupt the Golgi in mammalian cells. The crystal structure reveals an unexpected kinase fold and catalytic motif important for LepB_NTD function in eukaryotes. Cell biology-guided biochemical analyses uncovered a lipid kinase activity in LepB_NTD that specifically converts PtdIns3P into PtdIns(3,4)P2. PtdIns(3,4)P2 is efficiently hydrolysed into PtdIns4P by another Dot/Icm effector SidF that is known to possess phosphoinositide phosphatase activity. Consistently, SidF is capable of counteracting the cellular functions of LepB_NTD. Genetic analyses show a requirement for LepB kinase activity as well as lipid phosphatase activity of SidF for PtdIns4P biosynthesis on the LCV membrane. Our study identifies an unprecedented phosphatidylinositide 4-kinase activity from bacteria and highlights a sophisticated manipulation of host phosphoinositide metabolism by a bacterial pathogen.

  15. 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.

  16. Molecular Characterization of A Novel Effector Expansin-like Protein from Heterodera avenae that Induces Cell Death in Nicotiana benthamiana

    PubMed Central

    Liu, Jing; Peng, Huan; Cui, Jiangkuan; Huang, Wenkun; Kong, Lingan; Clarke, Jihong Liu; Jian, Heng; Wang, Guo Liang; Peng, Deliang

    2016-01-01

    Cereal cyst nematodes are sedentary biotrophic endoparasites that maintain a complex interaction with their host plants. Nematode effector proteins are synthesized in the oesophageal glands and are secreted into plant tissues through the stylet. To understand the function of nematode effectors in parasitic plants, we cloned predicted effectors genes from Heterodera avenae and transiently expressed them in Nicotiana benthamiana. Infiltration assays showed that HaEXPB2, a predicted expansin-like protein, caused cell death in N. benthamiana. In situ hybridization showed that HaEXPB2 transcripts were localised within the subventral gland cells of the pre-parasitic second-stage nematode. HaEXPB2 had the highest expression levels in parasitic second-stage juveniles. Subcellular localization assays revealed that HaEXPB2 could be localized in the plant cell wall after H. avenae infection.This The cell wall localization was likely affected by its N-terminal and C-terminal regions. In addition, we found that HaEXPB2 bound to cellulose and its carbohydrate-binding domain was required for this binding. The infectivity of H. avenae was significantly reduced when HaEXPB2 was knocked down by RNA interference in vitro. This study indicates that HaEXPB2 may play an important role in the parasitism of H. avenae through targeting the host cell wall. PMID:27808156

  17. 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.

  18. 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

  19. Host-targeting protein 1 (SpHtp1) from the oomycete Saprolegnia parasitica translocates specifically into fish cells in a tyrosine-O-sulphate–dependent manner

    PubMed Central

    Wawra, Stephan; Bain, Judith; Durward, Elaine; de Bruijn, Irene; Minor, Kirsty L.; Matena, Anja; Löbach, Lars; Whisson, Stephen C.; Bayer, Peter; Porter, Andrew J.; Birch, Paul R. J.; Secombes, Chris J.; van West, Pieter

    2012-01-01

    The eukaryotic oomycetes, or water molds, contain several species that are devastating pathogens of plants and animals. During infection, oomycetes translocate effector proteins into host cells, where they interfere with host-defense responses. For several oomycete effectors (i.e., the RxLR-effectors) it has been shown that their N-terminal polypeptides are important for the delivery into the host. Here we demonstrate that the putative RxLR-like effector, host-targeting protein 1 (SpHtp1), from the fish pathogen Saprolegnia parasitica translocates specifically inside host cells. We further demonstrate that cell-surface binding and uptake of this effector protein is mediated by an interaction with tyrosine-O-sulfate–modified cell-surface molecules and not via phospholipids, as has been reported for RxLR-effectors from plant pathogenic oomycetes. These results reveal an effector translocation route based on tyrosine-O-sulfate binding, which could be highly relevant for a wide range of host–microbe interactions. PMID:22308362

  20. Mathematical analysis of cell-target encounter rates in two dimensions. The effect of chemotaxis.

    PubMed Central

    Fisher, E S; Lauffenburger, D A

    1987-01-01

    The process by which cells encounter their targets is the first step of a number of cell functions involved in the immune response, such as cell-mediated cytotoxicity and phagocytic ingestion of foreign material. In many instances, this encounter may be rate-limiting, and therefore it is important to understand what factors influence the encounter rate. One key aspect of cell-target encounter is the motility behavior of the cell in the vicinity of a target. This movement may be entirely random, or there may be a directed, or chemotactic, component to it. In this paper we focus on the effects of cell motility properties, and particularly the chemotactic directional bias, on the rate of cell-target encounter. Specifically, we derive an expression for the mean encounter time of cells that meet targets in two dimensions as a function of the cells' directional orientation bias. We show that a modest degree of bias can reduce the mean encounter time by orders of magnitude, while nearly perfect directional bias offers little additional benefit. We illustrate the application of these results to a particular example system: alveolar macrophages removing inhaled particles and bacteria from the lung surface. PMID:3593868

  1. Genetically Programmed Clusters of Gold Nanoparticles for Cancer Cell-Targeted Photothermal Therapy.

    PubMed

    Oh, Mi Hwa; Yu, Jeong Heon; Kim, Insu; Nam, Yoon Sung

    2015-10-14

    Interpretations of the interactions of nanocarriers with biological cells are often complicated by complex synthesis of materials, broad size distribution, and heterogeneous surface chemistry. Herein, the major capsid proteins of an icosahedral T7 phage (55 nm in diameter) are genetically engineered to display a gold-binding peptide and a prostate cancer cell-binding peptide in a tandem sequence. The genetically modified phage attracts gold nanoparticles (AuNPs) to form a cluster of gold nanoparticles (about 70 nanoparticles per phage). The cluster of AuNPs maintains cell-targeting functionality and exhibits excellent dispersion stability in serum. Under a very low light irradiation (60 mW cm(-2)), only targeted AuNP clusters kill the prostate cancer cells in minutes (not in other cell types), whereas neither nontargeted AuNP clusters nor citrate-stabilized AuNPs cause any significant cell death. The result suggests that the prostate cancer cell-targeted clusters of AuNPs are targeted to only prostate cancer cells and, when illuminated, generate local heating to more efficiently and selectively kill the targeted cancer cells. Our strategy can be generalized to target other types of cells and assemble other kinds of nanoparticles for a broad range of applications.

  2. Semiconductor nanocrystal-aptamer bioconjugate probes for specific prostate carcinoma cell targeting

    NASA Astrophysics Data System (ADS)

    Shieh, Felice; Lavery, Laura; Chu, Chitai T.; Richards-Kortum, Rebecca; Ellington, Andrew D.; Korgel, Brian A.

    2005-04-01

    Cancer of the prostate affects approximately 1 in 11 men. Current early screening for prostate cancer utilizes digital rectal examinations to detect anomalies in the prostate gland and blood test screenings for upregulated levels of prostate specific antigen (PSA). Many of these tests are invasive and can often be inconclusive as PSA levels may be heightened due to benign factors. Prostate specific membrane antigen (PSMA), a well-characterized integral membrane protein, is expressed in virtually all prostate cancers and often correlates with cancer aggressiveness. Therefore, it may be used as an indicator of cancer growth and metastases. PSMA-specific antibodies have been identified and conjugated to fluorescent markers for cancer cell targeting; however, both the antibodies and markers possess significant limitations in their pharmaceutical and diagnostic value. Here we report the use of semiconductor nanocrystals bioconjugated to PSMA-specific aptamer recognition molecules for prostate carcinoma cell targeting. The nanocrystal/aptamer bioconjugates are small biocompatible probes with the potential for color-tunability for multicolor imaging. Ongoing in vitro and in vivo research seeks to introduce these nanoparticle bioconjugates into medical diagnostics.

  3. Selective cell targeting and lineage tracing of human induced pluripotent stem cells using recombinant avian retroviruses.

    PubMed

    Hildebrand, Laura; Seemann, Petra; Kurtz, Andreas; Hecht, Jochen; Contzen, Jörg; Gossen, Manfred; Stachelscheid, Harald

    2015-12-01

    Human induced pluripotent stem cells (hiPSC) differentiate into multiple cell types. Selective cell targeting is often needed for analyzing gene function by overexpressing proteins in a distinct population of hiPSC-derived cell types and for monitoring cell fate in response to stimuli. However, to date, this has not been possible, as commonly used viruses enter the hiPSC via ubiquitously expressed receptors. Here, we report for the first time the application of a heterologous avian receptor, the tumor virus receptor A (TVA), to selectively transduce TVA(+) cells in a mixed cell population. Expression of the TVA surface receptor via genetic engineering renders cells susceptible for infection by avian leucosis virus (ALV). We generated hiPSC lines with this stably integrated, ectopic TVA receptor gene that expressed the receptor while retaining pluripotency. The undifferentiated hiPSC(TVA+) as well as their differentiating progeny could be infected by recombinant ALV (so-called RCAS virus) with high efficiency. Due to incomplete receptor blocking, even sequential infection of differentiating or undifferentiated TVA(+) cells was possible. In conclusion, the TVA/RCAS system provides an efficient and gentle gene transfer system for hiPSC and extends our possibilities for selective cell targeting and lineage tracing studies.

  4. Graft-infiltrating host dendritic cells play a key role in organ transplant rejection

    PubMed Central

    Zhuang, Quan; Liu, Quan; Divito, Sherrie J.; Zeng, Qiang; Yatim, Karim M.; Hughes, Andrew D.; Rojas-Canales, Darling M.; Nakao, A.; Shufesky, William J.; Williams, Amanda L.; Humar, Rishab; Hoffman, Rosemary A.; Shlomchik, Warren D.; Oberbarnscheidt, Martin H.; Lakkis, Fadi G.; Morelli, Adrian E.

    2016-01-01

    Successful engraftment of organ transplants has traditionally relied on preventing the activation of recipient (host) T cells. Once T-cell activation has occurred, however, stalling the rejection process becomes increasingly difficult, leading to graft failure. Here we demonstrate that graft-infiltrating, recipient (host) dendritic cells (DCs) play a key role in driving the rejection of transplanted organs by activated (effector) T cells. We show that donor DCs that accompany heart or kidney grafts are rapidly replaced by recipient DCs. The DCs originate from non-classical monocytes and form stable, cognate interactions with effector T cells in the graft. Eliminating recipient DCs reduces the proliferation and survival of graft-infiltrating T cells and abrogates ongoing rejection or rejection mediated by transferred effector T cells. Therefore, host DCs that infiltrate transplanted organs sustain the alloimmune response after T-cell activation has already occurred. Targeting these cells provides a means for preventing or treating rejection. PMID:27554168

  5. A novel nematode effector suppresses plant immunity by activating host reactuve oxygen species-scavenging system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Oxidative burst is a hallmark event of the pathogen-associated molecular pattern (PAMP) triggered immunity (PTI), which is the first line of plant defense mechanisms, but it remains unclear how nematodes can overcome this defense mechanism. In this study, we show that plant-parasitic nematode Meloid...

  6. Three New Pierce's Disease Pathogenicity Effectors Identified Using Xylella fastidiosa Biocontrol Strain EB92-1

    PubMed Central

    Zhang, Shujian; Chakrabarty, Pranjib K.; Fleites, Laura A.; Rayside, Patricia A.; Hopkins, Donald L.; Gabriel, Dean W.

    2015-01-01

    Xylella fastidiosa (X. fastidiosa) infects a wide range of plant hosts and causes economically serious diseases, including Pierce's Disease (PD) of grapevines. X. fastidiosa biocontrol strain EB92-1 was isolated from elderberry and is infectious and persistent in grapevines but causes only very slight symptoms under ideal conditions. The draft genome of EB92-1 revealed that it appeared to be missing genes encoding 10 potential PD pathogenicity effectors found in Temecula1. Subsequent PCR and sequencing analyses confirmed that EB92-1 was missing the following predicted effectors found in Temecula1: two type II secreted enzymes, including a lipase (LipA; PD1703) and a serine protease (PD0956); two identical genes encoding proteins similar to Zonula occludens toxins (Zot; PD0915 and PD0928), and at least one relatively short, hemagglutinin-like protein (PD0986). Leaves of tobacco and citrus inoculated with cell-free, crude protein extracts of E. coli BL21(DE3) overexpressing PD1703 exhibited a hypersensitive response (HR) in less than 24 hours. When cloned into shuttle vector pBBR1MCS-5, PD1703 conferred strong secreted lipase activity to Xanthomonas citri, E. coli and X. fastidiosa EB92-1 in plate assays. EB92-1/PD1703 transformants also showed significantly increased disease symptoms on grapevines, characteristic of PD. Genes predicted to encode PD0928 (Zot) and a PD0986 (hemagglutinin) were also cloned into pBBR1MCS-5 and moved into EB92-1; both transformants also showed significantly increased symptoms on V. vinifera vines, characteristic of PD. Together, these results reveal that PD effectors include at least a lipase, two Zot-like toxins and a possibly redundant hemagglutinin, none of which are necessary for parasitic survival of X. fastidiosa populations in grapevines or elderberry. PMID:26218423

  7. Legionella pneumophila Effector LpdA Is a Palmitoylated Phospholipase D Virulence Factor.

    PubMed

    Schroeder, Gunnar N; Aurass, Philipp; Oates, Clare V; Tate, Edward W; Hartland, Elizabeth L; Flieger, Antje; Frankel, Gad

    2015-10-01

    Legionella pneumophila is a bacterial pathogen that thrives in alveolar macrophages, causing a severe pneumonia. The virulence of L. pneumophila depends on its Dot/Icm type IV secretion system (T4SS), which delivers more than 300 effector proteins into the host, where they rewire cellular signaling to establish a replication-permissive niche, the Legionella-containing vacuole (LCV). Biogenesis of the LCV requires substantial redirection of vesicle trafficking and remodeling of intracellular membranes. In order to achieve this, several T4SS effectors target regulators of membrane trafficking, while others resemble lipases. Here, we characterized LpdA, a phospholipase D effector, which was previously proposed to modulate the lipid composition of the LCV. We found that ectopically expressed LpdA was targeted to the plasma membrane and Rab4- and Rab14-containing vesicles. Subcellular targeting of LpdA required a C-terminal motif, which is posttranslationally modified by S-palmitoylation. Substrate specificity assays showed that LpdA hydrolyzed phosphatidylinositol, -inositol-3- and -4-phosphate, and phosphatidylglycerol to phosphatidic acid (PA) in vitro. In HeLa cells, LpdA generated PA at vesicles and the plasma membrane. Imaging of different phosphatidylinositol phosphate (PIP) and organelle markers revealed that while LpdA did not impact on membrane association of various PIP probes, it triggered fragmentation of the Golgi apparatus. Importantly, although LpdA is translocated inefficiently into cultured cells, an L. pneumophila ΔlpdA mutant displayed reduced replication in murine lungs, suggesting that it is a virulence factor contributing to L. pneumophila infection in vivo.

  8. Three New Pierce's Disease Pathogenicity Effectors Identified Using Xylella fastidiosa Biocontrol Strain EB92-1.

    PubMed

    Zhang, Shujian; Chakrabarty, Pranjib K; Fleites, Laura A; Rayside, Patricia A; Hopkins, Donald L; Gabriel, Dean W

    2015-01-01

    Xylella fastidiosa (X. fastidiosa) infects a wide range of plant hosts and causes economically serious diseases, including Pierce's Disease (PD) of grapevines. X. fastidiosa biocontrol strain EB92-1 was isolated from elderberry and is infectious and persistent in grapevines but causes only very slight symptoms under ideal conditions. The draft genome of EB92-1 revealed that it appeared to be missing genes encoding 10 potential PD pathogenicity effectors found in Temecula1. Subsequent PCR and sequencing analyses confirmed that EB92-1 was missing the following predicted effectors found in Temecula1: two type II secreted enzymes, including a lipase (LipA; PD1703) and a serine protease (PD0956); two identical genes encoding proteins similar to Zonula occludens toxins (Zot; PD0915 and PD0928), and at least one relatively short, hemagglutinin-like protein (PD0986). Leaves of tobacco and citrus inoculated with cell-free, crude protein extracts of E. coli BL21(DE3) overexpressing PD1703 exhibited a hypersensitive response (HR) in less than 24 hours. When cloned into shuttle vector pBBR1MCS-5, PD1703 conferred strong secreted lipase activity to Xanthomonas citri, E. coli and X. fastidiosa EB92-1 in plate assays. EB92-1/PD1703 transformants also showed significantly increased disease symptoms on grapevines, characteristic of PD. Genes predicted to encode PD0928 (Zot) and a PD0986 (hemagglutinin) were also cloned into pBBR1MCS-5 and moved into EB92-1; both transformants also showed significantly increased symptoms on V. vinifera vines, characteristic of PD. Together, these results reveal that PD effectors include at least a lipase, two Zot-like toxins and a possibly redundant hemagglutinin, none of which are necessary for parasitic survival of X. fastidiosa populations in grapevines or elderberry.

  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. The TAL effector PthA4 interacts with nuclear factors involved in RNA-dependent processes including a HMG protein that selectively binds poly(U) RNA.

    PubMed

    de Souza, Tiago Antonio; Soprano, Adriana Santos; de Lira, Nayara Patricia Vieira; Quaresma, Alexandre José Christino; Pauletti, Bianca Alves; Paes Leme, Adriana Franco; Benedetti, Celso Eduardo

    2012-01-01

    Plant pathogenic bacteria utilize an array of effector proteins to cause disease. Among them, transcriptional activator-like (TAL) effectors are unusual in the sense that they modulate transcription in the host. Although target genes and DNA specificity of TAL effectors have been elucidated, how TAL proteins control host transcription is poorly understood. Previously, we showed that the Xanthomonas citri TAL effectors, PthAs 2 and 3, preferentially targeted a citrus protein complex associated with transcription control and DNA repair. To extend our knowledge on the mode of action of PthAs, we have identified new protein targets of the PthA4 variant, required to elicit canker on citrus. Here we show that all the PthA4-interacting proteins are DNA and/or RNA-binding factors implicated in chromatin remodeling and repair, gene regulation and mRNA stabilization/modification. The majority of these proteins, including a structural maintenance of chromosomes protein (CsSMC), a translin-associated factor X (CsTRAX), a VirE2-interacting protein (CsVIP2), a high mobility group (CsHMG) and two poly(A)-binding proteins (CsPABP1 and 2), interacted with each other, suggesting that they assemble into a multiprotein complex. CsHMG was shown to bind DNA and to interact with the invariable leucine-rich repeat region of PthAs. Surprisingly, both CsHMG and PthA4 interacted with PABP1 and 2 and showed selective binding to poly(U) RNA, a property that is novel among HMGs and TAL effectors. Given that homologs of CsHMG, CsPABP1, CsPABP2, CsSMC and CsTRAX in other organisms assemble into protein complexes to regulate mRNA stability and translation, we suggest a novel role of TAL effectors in mRNA processing and translational control.

  11. Shared weapons of blood- and plant-feeding insects: Surprising commonalities for manipulating hosts.

    PubMed

    Guiguet, Antoine; Dubreuil, Géraldine; Harris, Marion O; Appel, Heidi M; Schultz, Jack C; Pereira, Marcos H; Giron, David

    2016-01-01

    Insects that reprogram host plants during colonization remind us that the insect side of plant-insect story is just as interesting as the plant side. Insect effectors secreted by the salivary glands play an important role in plant reprogramming. Recent discoveries point to large numbers of salivary effectors being produced by a single herbivore species. Since genetic and functional characterization of effectors is an arduous task, narrowing the field of candidates is useful. We present ideas about types and functions of effectors from research on blood-feeding parasites and their mammalian hosts. Because of their importance for human health, blood-feeding parasites have more tools from genomics and other - omics than plant-feeding parasites. Four themes have emerged: (1) mechanical damage resulting from attack by blood-feeding parasites triggers "early danger signals" in mammalian hosts, which are mediated by eATP, calcium, and hydrogen peroxide, (2) mammalian hosts need to modulate their immune responses to the three "early danger signals" and use apyrases, calreticulins, and peroxiredoxins, respectively, to achieve this, (3) blood-feeding parasites, like their mammalian hosts, rely on some of the same "early danger signals" and modulate their immune responses using the same proteins, and (4) blood-feeding parasites deploy apyrases, calreticulins, and peroxiredoxins in their saliva to manipulate the "danger signals" of their mammalian hosts. We review emerging evidence that plant-feeding insects also interfere with "early danger signals" of their hosts by deploying apyrases, calreticulins and peroxiredoxins in saliva. Given emerging links between these molecules, and plant growth and defense, we propose that these effectors interfere with phytohormone signaling, and therefore have a special importance for gall-inducing and leaf-mining insects, which manipulate host-plants to create better food and shelter.

  12. How Biofilms Evade Host Defenses.

    PubMed

    Roilides, Emmanuel; Simitsopoulou, Maria; Katragkou, Aspasia; Walsh, Thomas J

    2015-06-01

    The steps involved during the biofilm growth cycle include attachment to a substrate followed by more permanent adherence of the microorganisms, microcolony arrangement, and cell detachment required for the dissemination of single or clustered cells to other organ systems. Various methods have been developed for biofilm detection and quantitation. Biofilm-producing microorganisms can be detected in tissue culture plates, using silicone tubes and staining methods, and by visual assessment using scanning electron microscopy or confocal scanning laser microscopy. Quantitative measurement of biofilm growth is determined by using methods that include dry cell weight assays, colony-forming-unit counting, DNA quantification, or XTT 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide reduction assay. Upon infection, innate immune defense strategies are able to establish an immediate response through effector mechanisms mediated by immune cells, receptors, and several humoral factors. We present an overview of the life cycle of biofilms and their diversity, detection methods for biofilm development, and host immune responses to pathogens. We then focus on current concepts in bacterial and fungal biofilm immune evasion mechanisms. This appears to be of particular importance because the use of host immune responses may represent a novel therapeutic approach against biofilms.

  13. Emerging functions as host cell factors - an encyclopedia of annexin-pathogen interactions.

    PubMed

    Kuehnl, Alexander; Musiol, Agnes; Raabe, Carsten A; Rescher, Ursula

    2016-10-01

    Emerging infectious diseases and drug-resistant infectious agents call for the development of innovative antimicrobial strategies. With pathogenicity now considered to arise from the complex and bi-directional interplay between a microbe and the host, host cell factor targeting has emerged as a promising approach that might overcome the limitations of classical antimicrobial drug development and could open up novel and efficient therapeutic strategies. Interaction with and modulation of host cell membranes is a recurrent theme in the host-microbe relationship. In this review, we provide an overview of what is currently known about the role of the Ca2+ dependent, membrane-binding annexin protein family in pathogen-host interactions, and discuss their emerging functions as host cell derived auxiliary proteins in microbe-host interactions and host cell targets.

  14. Addition of transcription activator-like effector binding sites to a pathogen strain-specific rice bacterial blight resistance gene makes it effective against additional strains and against bacterial leaf streak.

    PubMed

    Hummel, Aaron W; Doyle, Erin L; Bogdanove, Adam J

    2012-09-01

    Xanthomonas transcription activator-like (TAL) effectors promote disease in plants by binding to and activating host susceptibility genes. Plants counter with TAL effector-activated executor resistance genes, which cause host cell death and block disease progression. We asked whether the functional specificity of an executor gene could be broadened by adding different TAL effector binding elements (EBEs) to it. We added six EBEs to the rice Xa27 gene, which confers resistance to strains of the bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) that deliver the TAL effector AvrXa27. The EBEs correspond to three other effectors from Xoo strain PXO99(A) and three from strain BLS256 of the bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc). Stable integration into rice produced healthy lines exhibiting gene activation by each TAL effector, and resistance to PXO99(A) , a PXO99(A) derivative lacking AvrXa27, and BLS256, as well as two other Xoo and 10 Xoc strains virulent toward wildtype Xa27 plants. Transcripts initiated primarily at a common site. Sequences in the EBEs were found to occur nonrandomly in rice promoters, suggesting an overlap with endogenous regulatory sequences. Thus, executor gene specificity can be broadened by adding EBEs, but caution is warranted because of the possible coincident introduction of endogenous regulatory elements.

  15. The HopZ family of Pseudomonas syringae type III effectors require myristoylation for virulence and avirulence functions in Arabidopsis thaliana.

    PubMed

    Lewis, Jennifer D; Abada, Wasan; Ma, Wenbo; Guttman, David S; Desveaux, Darrell

    2008-04-01

    Pseudomonas syringae utilizes the type III secretion system to translocate effector proteins into plant cells, where they can contribute to the pathogen's ability to infect and cause disease. Recognition of these effectors by resistance proteins induces defense responses that typically include a programmed cell death reaction called the hypersensitive response. The YopJ/HopZ family of type III effector proteins is a common family of effector proteins found in animal- and plant-pathogenic bacteria. The HopZ family in P. syringae includes HopZ1a(PsyA2), HopZ1b(PgyUnB647), HopZ1c(PmaE54326), HopZ2(Ppi895A) and HopZ3(PsyB728a). HopZ1a is predicted to be most similar to the ancestral hopZ allele and causes a hypersensitive response in multiple plant species, including Arabidopsis thaliana. Therefore, it has been proposed that host defense responses have driven the diversification of this effector family. In this study, we further characterized the hypersensitive response induced by HopZ1a and demonstrated that it is not dependent on known resistance genes. Further, we identified a novel virulence function for HopZ2 that requires the catalytic cysteine demonstrated to be required for protease activity. Sequence analysis of the HopZ family revealed the presence of a predicted myristoylation sequence in all members except HopZ3. We demonstrated that the myristoylation site is required for membrane localization of this effector family and contributes to the virulence and avirulence activities of HopZ2 and HopZ1a, respectively. This paper provides insight into the selective pressures driving virulence protein evolution by describing a detailed functional characterization of the diverse HopZ family of type III effectors with the model plant Arabidopsis.

  16. The HopZ Family of Pseudomonas syringae Type III Effectors Require Myristoylation for Virulence and Avirulence Functions in Arabidopsis thaliana▿ †

    PubMed Central

    Lewis, Jennifer D.; Abada, Wasan; Ma, Wenbo; Guttman, David S.; Desveaux, Darrell

    2008-01-01

    Pseudomonas syringae utilizes the type III secretion system to translocate effector proteins into plant cells, where they can contribute to the pathogen's ability to infect and cause disease. Recognition of these effectors by resistance proteins induces defense responses that typically include a programmed cell death reaction called the hypersensitive response. The YopJ/HopZ family of type III effector proteins is a common family of effector proteins found in animal- and plant-pathogenic bacteria. The HopZ family in P. syringae includes HopZ1aPsyA2, HopZ1bPgyUnB647, HopZ1cPmaE54326, HopZ2Ppi895A and HopZ3PsyB728a. HopZ1a is predicted to be most similar to the ancestral hopZ allele and causes a hypersensitive response in multiple plant species, including Arabidopsis thaliana. Therefore, it has been proposed that host defense responses have driven the diversification of this effector family. In this study, we further characterized the hypersensitive response induced by HopZ1a and demonstrated that it is not dependent on known resistance genes. Further, we identified a novel virulence function for HopZ2 that requires the catalytic cysteine demonstrated to be required for protease activity. Sequence analysis of the HopZ family revealed the presence of a predicted myristoylation sequence in all members except HopZ3. We demonstrated that the myristoylation site is required for membrane localization of this effector family and contributes to the virulence and avirulence activities of HopZ2 and HopZ1a, respectively. This paper provides insight into the selective pressures driving virulence protein evolution by describing a detailed functional characterization of the diverse HopZ family of type III effectors with the model plant Arabidopsis. PMID:18263728

  17. 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.

  18. Optimal expression of a Fab-effector fusion protein in Escherichia coli by removing the cysteine residues responsible for an interchain disulfide bond of a Fab molecule.

    PubMed

    Kang, Hyeon-Ju; Kim, Hye-Jin; Jung, Mun-Sik; Han, Jae-Kyu; Cha, Sang-Hoon

    2017-04-01

    Development of novel bi-functional or even tri-functional Fab-effector fusion proteins would have a great potential in the biomedical sciences. However, the expression of Fab-effector fusion proteins in Escherichia coli is problematic especially when a eukaryotic effector moiety is genetically linked to a Fab due to the lack of proper chaperone proteins and an inappropriate physicochemical environment intrinsic to the microbial hosts. We previously reported that a human Fab molecule, referred to as SL335, reactive to human serum albumin has a prolonged in vivo serum half-life in rats. We, herein, tested six discrete SL335-human growth hormone (hGH) fusion constructs as a model system to define an optimal Fab-effector fusion format for E. coli expression. We found that one variant, referred to as HserG/Lser, outperformed the others in terms of a soluble expression yield and functionality in that HserG/Lser has a functional hGH bioactivity and possesses an serum albumin-binding affinity comparable to SL335. Our results clearly demonstrated that the genetic linkage of an effector domain to the C-terminus of Fd (VH+CH1) and the removal of cysteine (Cys) residues responsible for an interchain disulfide bond (IDB) ina Fab molecule optimize the periplasmic expression of a Fab-effector fusion protein in E. coli. We believe that our approach can contribute the development of diverse bi-functional Fab-effector fusion proteins by providing a simple strategy that enables the reliable expression of a functional fusion proteins in E. coli.

  19. 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.

  20. 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.

  1. Characterization of the membrane-associated HaRxL17 Hpa effector candidate

    PubMed Central

    Caillaud, Marie-Cécile; Piquerez, Sophie J. M.; Jones, Jonathan D. G.

    2012-01-01

    We examined changes to subcellular architecture during the compatible interaction between the biotroph pathogen Hyaloperonospora arabidopsidis (Hpa) and its host Arabidopsis. Live-cell imaging highlighted rearrangements in plant cell membranes upon infection. In particular, the tonoplast appeared close to the extrahaustorial membrane surrounding the haustorium. We investigated the subcellular localization patterns of Hpa RxLR effector candidates (HaRxLs) in planta. This subcellular localization screening led to the identification of an extrahaustorial membrane-localized effector, HaRxL17 that when stably expressed in Arabidopsis increased plant susceptibility to Hpa during compatible and incompatible interactions. Here, we report that the N-terminal part of HaRxL17 is sufficient to target the plant cell membranes. We showed that both C- or N-terminal fluorescent-tagged HaRxL17 localizes around Hpa haustoria, in early and in late stages of infection. As with Hpa infection, GFP-HaRxL17 also localizes around haustoria during infection with Albugo laibachii. Thus, HaRxL17 that increases plant susceptibility to Hpa during both compatible and incompatible interactions, localizes around oomycete haustoria when stably expressed in Arabidopsis. PMID:22301983

  2. 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.

  3. 'Drugs from bugs': bacterial effector proteins as promising biological (immune-) therapeutics.

    PubMed

    Rüter, Christian; Hardwidge, Philip R

    2014-02-01

    Immune system malfunctions cause many of the most severe human diseases. The immune system has evolved primarily to control bacterial, viral, fungal, and parasitic infections. In turn, over millions of years of coevolution, microbial pathogens have evolved various mechanisms to control and modulate the host immune system for their own benefit and survival. For example, many bacterial pathogens use virulence proteins to modulate and exploit target cell mechanisms. Our understanding of these bacterial strategies opens novel possibilities to exploit 'microbial knowledge' to control excessive immune reactions. Gaining access to strategies of microbial pathogens could lead to potentially huge benefits for the therapy of inflammatory diseases. Most work on bacterial pathogen effector proteins has the long-term aim of neutralizing the infectious capabilities of the pathogen. However, attenuated pathogens and microbial products have been used for over a century with overwhelming success in the form of vaccines to induce specific immune responses that protect against the respective infectious diseases. In this review, we focus on bacterial effector and virulence proteins capable of modulating and suppressing distinct signaling pathways with potentially desirable immune-modulating effects for treating unrelated inflammatory diseases.

  4. 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.

  5. 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.

  6. Combination of chemotherapy and cancer stem cell targeting agents: Preclinical and clinical studies.

    PubMed

    Li, Yanyan; Atkinson, Katharine; Zhang, Tao

    2017-03-12

    The cancer stem cell model claims that the initiation, maintenance, and growth of a tumor are driven by a small population of cancer cells termed cancer stem cells. Cancer stem cells possess a variety of phenotypes associated with therapeutic resistance and often cause recurrence of the diseases. Several strategies have been investigated to target cancer stem cells in a variety of cancers, such as blocking one or more self-renewal signaling pathways, reducing the expression of drug efflux and ATP-binding cassette efflux transporters, modulating epigenetic aberrations, and promoting cancer stem cell differentiation. A number of cell and animal studies strongly support the potential benefits of combining chemotherapeutic drugs with cancer stem cell targeting agents. Clinical trials are still underway to address the pharmacokinetics, safety, and efficacy of combination treatment. This mini-review provides an updated discussion of these preclinical and clinical studies.

  7. Solvent-Free Click-Mechanochemistry for the Preparation of Cancer Cell Targeting Graphene Oxide

    PubMed Central

    2015-01-01

    Polyethylene glycol-functionalized nanographene oxide (PEGylated n-GO) was synthesized from alkyne-modified n-GO, using solvent-free click-mechanochemistry, i.e., copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC). The modified n-GO was subsequently conjugated to a mucin 1 receptor immunoglobulin G antibody (anti-MUC1 IgG) via thiol–ene coupling reaction. n-GO derivatives were characterized with Fourier-transformed infrared (FT-IR) spectroscopy, thermogravimetric analysis (TGA), Bradford assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and atomic force microscopy (AFM). Cell targeting was confirmed in vitro in MDA-MB-231 cells, either expressing or lacking MUC1 receptors, using flow cytometry, confocal laser scanning microscopy (CLSM) and multiphoton (MP) fluorescence microscopy. Biocompatibility was assessed using the modified lactate dehydrongenase (mLDH) assay. PMID:26278410

  8. B-cell-targeted therapy for systemic lupus erythematosus: an update.

    PubMed

    Ding, Changhai; Foote, Simon; Jones, Graeme

    2008-01-01

    Systemic lupus erythematosus (SLE) is a classic autoimmune disease characterized by a myriad of immune system aberrations, most likely resulting from pathogenic autoantibody production, immune complex deposition, and subsequent end-organ damage. B cells play a key role in the pathogenesis; therefore, B-cell-targeted therapies, including B-cell depletion and blockage of B-cell survival factors such as B-lymphocyte stimulator (BLyS), are potential therapeutic targets for SLE. In uncontrolled clinical trials from approximately 20 studies, rituximab--a mouse-human chimeric anti-CD20 monoclonal antibody that effectively depletes B cells--has been demonstrated to reduce disease activity and decrease serum autoantibodies, with a clinical response of 86% in a case series of approximately 400 SLE patients with refractory disease, with or without concomitant use of cyclophosphamide. Epratuzumab, a humanized anti-CD22 monoclonal antibody that partially depletes B cells, has also been shown to reduce disease activity but not to decrease autoantibody levels in patients with moderately active SLE. Randomized controlled phase I/II trials in patients with active SLE have documented that belimumab, a humanized anti-BLyS monoclonal antibody, reduces B-cell numbers, inhibits disease activity and decreases anti-double-stranded DNA autoantibody in SLE patients. All these therapies are well tolerated, but accompanying infectious complications have been observed. Other B-cell-targeted therapies such as 'humanized' monoclonal antibodies to CD20 (e.g. ocrelizumab) and agents that interrupt B-cell/T-cell interactions also have potential, and the efficacy of these, along with rituximab, belimumab and epratuzumab, needs to be determined by randomized controlled trials.

  9. Fabrication of DNA-antibody-apatite composite layers for cell-targeted gene transfer

    NASA Astrophysics Data System (ADS)

    Yazaki, Yushin; Oyane, Ayako; Araki, Hiroko; Sogo, Yu; Ito, Atsuo; Yamazaki, Atsushi; Tsurushima, Hideo

    2012-12-01

    Surface-mediated gene transfer systems using apatite (Ap)-based composite layers have received increased attention in tissue engineering applications owing to their safety, biocompatibility and relatively high efficiency. In this study, DNA-antibody-apatite composite layers (DA-Ap layers), in which DNA and antibody molecules are immobilized within a matrix of apatite nanocrystals, were fabricated using a biomimetic coating process. They were then assayed for their gene transfer capability for application in a specific cell-targeted gene transfer. A DA-Ap layer that was fabricated with an anti-CD49f antibody showed a higher gene transfer capability to the CD49f-positive CHO-K1 cells than a DNA-apatite composite layer (D-Ap layer). The antibody facilitated the gene transfer capability of the DA-Ap layer only to the specific cells that were expressing corresponding antigens. When the DA-Ap layer was fabricated with an anti-N-cadherin antibody, a higher gene transfer capability compared with the D-Ap layer was found in the N-cadherin-positive P19CL6 cells, but not in the N-cadherin-negative UV♀2 cells or in the P19CL6 cells that were pre-blocked with anti-N-cadherin. Therefore, the antigen-antibody binding that takes place at the cell-layer interface should be responsible for the higher gene transfer capability of the DA-Ap than D-Ap layer. These results suggest that the DA-Ap layer works as a mediator in a specific cell-targeted gene transfer system.

  10. Fabrication of DNA-antibody-apatite composite layers for cell-targeted gene transfer.

    PubMed

    Yazaki, Yushin; Oyane, Ayako; Araki, Hiroko; Sogo, Yu; Ito, Atsuo; Yamazaki, Atsushi; Tsurushima, Hideo

    2012-12-01

    Surface-mediated gene transfer systems using apatite (Ap)-based composite layers have received increased attention in tissue engineering applications owing to their safety, biocompatibility and relatively high efficiency. In this study, DNA-antibody-apatite composite layers (DA-Ap layers), in which DNA and antibody molecules are immobilized within a matrix of apatite nanocrystals, were fabricated using a biomimetic coating process. They were then assayed for their gene transfer capability for application in a specific cell-targeted gene transfer. A DA-Ap layer that was fabricated with an anti-CD49f antibody showed a higher gene transfer capability to the CD49f-positive CHO-K1 cells than a DNA-apatite composite layer (D-Ap layer). The antibody facilitated the gene transfer capability of the DA-Ap layer only to the specific cells that were expressing corresponding antigens. When the DA-Ap layer was fabricated with an anti-N-cadherin antibody, a higher gene transfer capability compared with the D-Ap layer was found in the N-cadherin-positive P19CL6 cells, but not in the N-cadherin-negative UV♀2 cells or in the P19CL6 cells that were pre-blocked with anti-N-cadherin. Therefore, the antigen-antibody binding that takes place at the cell-layer interface should be responsible for the higher gene transfer capability of the DA-Ap than D-Ap layer. These results suggest that the DA-Ap layer works as a mediator in a specific cell-targeted gene transfer system.

  11. A Cell-targeted Photodynamic Nanomedicine Strategy for Head & Neck Cancers

    PubMed Central

    Master, Alyssa; Malamas, Anthony; Solanki, Rachna; Clausen, Dana M.; Eiseman, Julie L.; Gupta, Anirban Sen

    2013-01-01

    Photodynamic Therapy (PDT) holds great promise for the treatment of head and neck (H&N) carcinomas where repeated loco-regional therapy often becomes necessary due to the highly aggressive and recurrent nature of the cancers. While interstitial light delivery technologies are being refined for PDT of H&N and other cancers, a parallel clinically relevant research area is the formulation of photosensitizers in nanovehicles that allow systemic administration yet preferential enhanced uptake in the tumor. This approach can render dual-selectivity of PDT, by harnessing both the drug and the light delivery within the tumor. To this end, we report on a cell-targeted nanomedicine approach for the photosensitizer silicon phthalocyanine-4 (Pc 4), by packaging it within polymeric micelles that are surface-decorated with GE11-peptides to promote enhanced cell-selective binding and receptor-mediated internalization in EGFR-overexpressing H&N cancer cells. Using fluorescence spectroscopy and confocal microscopy, we demonstrate in vitro that the EGFR-targeted Pc 4-nanoformulation undergoes faster and higher uptake in EGFR-overexpressing H&N SCC-15 cells. We further demonstrate that this enhanced Pc 4 uptake results in significant cell-killing and drastically reduced post-PDT clonogenicity. Building on this in vitro data, we demonstrate that the EGFR-targeted Pc 4-nanoformulation results in significant intra-tumoral drug uptake and subsequent enhanced PDT response, in vivo, in SCC-15 xenografts in mice. Altogether our results show significant promise towards a cell-targeted photodynamic nanomedicine for effective treatment of H&N carcinomas. PMID:23531079

  12. A cell-targeted photodynamic nanomedicine strategy for head and neck cancers.

    PubMed

    Master, Alyssa; Malamas, Anthony; Solanki, Rachna; Clausen, Dana M; Eiseman, Julie L; Sen Gupta, Anirban

    2013-05-06

    Photodynamic therapy (PDT) holds great promise for the treatment of head and neck (H&N) carcinomas where repeated loco-regional therapy often becomes necessary due to the highly aggressive and recurrent nature of the cancers. While interstitial light delivery technologies are being refined for PDT of H&N and other cancers, a parallel clinically relevant research area is the formulation of photosensitizers in nanovehicles that allow systemic administration yet preferential enhanced uptake in the tumor. This approach can render dual-selectivity of PDT, by harnessing both the drug and the light delivery within the tumor. To this end, we report on a cell-targeted nanomedicine approach for the photosensitizer silicon phthalocyanine-4 (Pc 4), by packaging it within polymeric micelles that are surface-decorated with GE11-peptides to promote enhanced cell-selective binding and receptor-mediated internalization in EGFR-overexpressing H&N cancer cells. Using fluorescence spectroscopy and confocal microscopy, we demonstrate in vitro that the EGFR-targeted Pc 4-nanoformulation undergoes faster and higher uptake in EGFR-overexpressing H&N SCC-15 cells. We further demonstrate that this enhanced Pc 4 uptake results in significant cell-killing and drastically reduced post-PDT clonogenicity. Building on this in vitro data, we demonstrate that the EGFR-targeted Pc 4-nanoformulation results in significant intratumoral drug uptake and subsequent enhanced PDT response, in vivo, in SCC-15 xenografts in mice. Altogether our results show significant promise toward a cell-targeted photodynamic nanomedicine for effective treatment of H&N carcinomas.

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

    PubMed Central

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

    2015-01-01

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

  14. A Recent Expansion of the RXLR Effector Gene Avrblb2 Is Maintained in Global Populations of Phytophthora infestans Indicating Different Contributions to Virulence.

    PubMed

    Oliva, Ricardo F; Cano, Liliana M; Raffaele, Sylvain; Win, Joe; Bozkurt, Tolga O; Belhaj, Khaoula; Oh, Sang-Keun; Thines, Marco; Kamoun, Sophien

    2015-08-01

    The introgression of disease resistance (R) genes encoding immunoreceptors with broad-spectrum recognition into cultivated potato appears to be the most promising approach to achieve sustainable management of late blight caused by the oomycete pathogen Phytophthora infestans. Rpi-blb2 from Solanum bulbocastanum shows great potential for use in agriculture based on preliminary potato disease trials. Rpi-blb2 confers immunity by recognizing the P. infestans avirulence effector protein AVRblb2 after it is translocated inside the plant cell. This effector belongs to the RXLR class of effectors and is under strong positive selection. Structure-function analyses revealed a key polymorphic amino acid (position 69) in AVRblb2 effector that is critical for activation of Rpi-blb2. In this study, we reconstructed the evolutionary history of the Avrblb2 gene family and further characterized its genetic structure in worldwide populations. Our data indicate that Avrblb2 evolved as a single-copy gene in a putative ancestral species of P. infestans and has recently expanded in the Phytophthora spp. that infect solanaceous hosts. As a consequence, at least four variants of AVRblb2 arose in P. infestans. One of these variants, with a Phe residue at position 69, evades recognition by the cognate resistance gene. Surprisingly, all Avrblb2 variants are maintained in pathogen populations. This suggests a potential benefit for the pathogen in preserving duplicated versions of AVRblb2, possibly because the variants may have different contributions to pathogen fitness in a diversified solanaceous host environment.

  15. 3′-NADP and 3′-NAADP, Two Metabolites Formed by the Bacterial Type III Effector AvrRxo1*♦

    PubMed Central

    Schuebel, Felix; Rocker, Andrea; Edelmann, Daniel; Schessner, Julia; Brieke, Clara; Meinhart, Anton

    2016-01-01

    An arsenal of effector proteins is injected by bacterial pathogens into the host cell or its vicinity to increase virulence. The commonly used top-down approaches inferring the toxic mechanism of individual effector proteins from the host's phenotype are often impeded by multiple targets of different effectors as well as by their pleiotropic effects. Here we describe our bottom-up approach, showing that the bacterial type III effector AvrRxo1 of plant pathogens is an authentic phosphotransferase that produces two novel metabolites by phosphorylating nicotinamide/nicotinic acid adenine dinucleotide at the adenosine 3′-hydroxyl group. Both products of AvrRxo1, 3′-NADP and 3′-nicotinic acid adenine dinucleotide phosphate (3′-NAADP), are substantially different from the ubiquitous co-enzyme 2′-NADP and the calcium mobilizer 2′-NAADP. Interestingly, 3′-NADP and 3′-NAADP have previously been used as inhibitors or signaling molecules but were regarded as “artificial” compounds so far. Our findings now necessitate a shift in thinking about the biological importance of 3′-phosphorylated NAD derivatives. PMID:27621317

  16. Brucella T4SS: the VIP pass inside host cells.

    PubMed

    Lacerda, Thais Lourdes Santos; Salcedo, Suzana Pinto; Gorvel, Jean-Pierre

    2013-02-01

    For many Gram-negative bacteria, like Brucella, the type IV secretion system (T4SS) has a critical role in bacterial virulence. In Brucella, the VirB T4SS permits the injection of bacterial effectors inside host cells, leading to subversion of signaling pathways and favoring bacterial growth and pathogenesis. The virB operon promoter is tightly regulated by a combination of transcriptional activators and repressors that are expressed according to the environmental conditions encountered by Brucella. Recent advances have shed light on the Brucella T4SS regulatory mechanisms and also its substrates. Characterization of the targets and functions of these translocated effectors is underway and will help understand the role of the T4SS in the establishment of a replication niche inside host cells.

  17. Clinically Relevant Reactivation of Polyomavirus BK (BKPyV) in HLA-A02-Positive Renal Transplant Recipients Is Associated with Impaired Effector-Memory Differentiation of BKPyV-Specific CD8+ T Cells

    PubMed Central

    Remmerswaal, Ester B. M.; Heutinck, Kirstin M.; ten Brinke, Anja; Feltkamp, Mariet C. W.; van der Weerd, Neelke C.; van der Pant, Karlijn A. M. I.; Bemelman, Frederike J.; van Lier, René A. W.; ten Berge, Ineke J. M.

    2016-01-01

    Polyomavirus BK (BKPyV) frequently reactivates in immunosuppressed renal transplant recipients (RTRs) and may lead to graft loss due to BKPyV-induced interstitial nephritis (BKVN). Little is known on the differentiation of CD8+ T cells targeting BKPyV in RTRs. Here we investigated whether BKPyV-specific CD8+ T cell differentiation differs in RTRs with varying degrees of BKPyV reactivation and/or BKVN. Using combinatorial encoding with tetramers carrying BKPyV major capsid protein (VP1) and large T antigen protein (LTAG) epitopes, we investigated CD8+ T cell responses to BKPyV in longitudinally obtained PBMC samples from 46 HLA-A02-positive RTRs and 20 healthy adults. We were also able to isolate BKPyV-specific CD8+ T cells from five renal allografts, two of which were affected by BKVN. Before transplantation, BKPyV-specific CD8+ T cells targeting VP1 and LTAG epitopes appeared predominantly as central-memory and CD27+/CD28+ effector-memory (TEM), and naïve-like PD-1-expressing cells, respectively. After viral reactivation, BKPyV-specific CD8+ T cells assumed CD28− TEM and TEMRA states in patients who were able to control BKPyV, whereas differentiation lagged behind in patients with severe viral reactivation or BKVN. Furthermore, VP1-specific CD69+/CD103+ tissue-resident memory (TRM) cells accumulated in BKVN-affected allografts but lacked signs of effector differentiation. In contrast, granzyme B-expressing effector cells were detected in allografts not affected by BKVN. In conclusion, effector-memory differentiation of BKPyV-specific CD8+ T cells in patients with high viral load or BKVN is impaired. Further characterization of the specific mechanisms behind this altered cellular differentiation is necessary to develop therapies that can prevent the emergence of BKVN. PMID:27723787

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. Phytophthora infestans RXLR Effector AVR1 Interacts with Exocyst Component Sec5 to Manipulate Plant Immunity1[OPEN

    PubMed Central

    Du, Yu; Mpina, Mohamed H.; Birch, Paul R.J.; Bouwmeester, Klaas; Govers, Francine

    2015-01-01

    Phytophthora infestans secretes numerous RXLR effectors that modulate host defense and thereby pave the way for successful invasion. Here, we show that the RXLR effector AVR1 is a virulence factor that promotes colonization and suppresses callose deposition, a hallmark of basal defense. To identify host targets of AVR1, we performed yeast two-hybrid screens and selected Sec5 as a candidate. Sec5 is a subunit of the exocyst, a protein complex that is involved in vesicle trafficking. AVR1-like (A-L), a close homolog of AVR1, also acts as a virulence factor, but unlike AVR1, A-L does not suppress CRINKLER2 (CRN2)-induced cell death or interact with Sec5. Compared with AVR1, A-L is shorter and lacks the carboxyl-terminal tail, the T-region that is crucial for CRN2-induced cell death suppression and Sec5 interaction. In planta analyses revealed that AVR1 and Sec5 are in close proximity, and coimmunoprecipitation confirmed the interaction. Sec5 is required for secretion of the pathogenesis-related protein PR-1 and callose deposition and also plays a role in CRN2-induced cell death. Our findings show that P. infestans manipulates an exocyst subunit and thereby potentially disturbs vesicle trafficking, a cellular process that is important for basal defense. This is a novel strategy that oomycete pathogens exploit to modulate host defense. PMID:26336092

  3. Subversion of plant cellular functions by bacterial type-III effectors: beyond suppression of immunity.

    PubMed

    Macho, Alberto P

    2016-04-01

    Most bacterial plant pathogens employ a type-III secretion system to inject type-III effector (T3E) proteins directly inside plant cells. These T3Es manipulate host cellular processes in order to create a permissive niche for bacterial proliferation, allowing development of the disease. An important role of T3Es in plant pathogenic bacteria is the suppression of plant immune responses. However, in recent years, research has uncovered T3E functions different from direct immune suppression, including the modulation of plant hormone signaling, metabolism or organelle function. This insight article discusses T3E functions other than suppression of immunity, which may contribute to the modulation of plant cells in order to promote bacterial survival, nutrient release, and bacterial replication and dissemination.

  4. Phytophthora sojae Avirulence Effector Avr3b is a Secreted NADH and ADP-ribose Pyrophosphorylase that Modulates Plant Immunity

    PubMed Central

    Dong, Suomeng; Yin, Weixiao; Kong, Guanghui; Yang, Xinyu; Qutob, Dinah; Chen, Qinghe; Kale, Shiv D.; Sui, Yangyang; Zhang, Zhengguang; Dou, Daolong; Zheng, Xiaobo; Gijzen, Mark; M. Tyler, Brett; Wang, Yuanchao

    2011-01-01

    Plants have evolved pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) to protect themselves from infection by diverse pathogens. Avirulence (Avr) effectors that trigger plant ETI as a result of recognition by plant resistance (R) gene products have been identified in many plant pathogenic oomycetes and fungi. However, the virulence functions of oomycete and fungal Avr effectors remain largely unknown. Here, we combined bioinformatics and genetics to identify Avr3b, a new Avr gene from Phytophthora sojae, an oomycete pathogen that causes soybean root rot. Avr3b encodes a secreted protein with the RXLR host-targeting motif and C-terminal W and Nudix hydrolase motifs. Some isolates of P. sojae evade perception by the soybean R gene Rps3b through sequence mutation in Avr3b and lowered transcript accumulation. Transient expression of Avr3b in Nicotiana benthamiana increased susceptibility to P. capsici and P. parasitica, with significantly reduced accumulation of reactive oxygen species (ROS) around invasion sites. Biochemical assays confirmed that Avr3b is an ADP-ribose/NADH pyrophosphorylase, as predicted from the Nudix motif. Deletion of the Nudix motif of Avr3b abolished enzyme activity. Mutation of key residues in Nudix motif significantly impaired Avr3b virulence function but not the avirulence activity. Some Nudix hydrolases act as negative regulators of plant immunity, and thus Avr3b might be delivered into host cells as a Nudix hydrolase to impair host immunity. Avr3b homologues are present in several sequenced Phytophthora genomes, suggesting that Phytophthora pathogens might share similar strategies to suppress plant immunity. PMID:22102810

  5. 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

  6. Host Defense Peptides in Wound Healing

    PubMed Central

    Steinstraesser, Lars; Koehler, Till; Jacobsen, Frank; Daigeler, Adrien; Goertz, Ole; Langer, Stefan; Kesting, Marco; Steinau, Hans; Eriksson, Elof; Hirsch, Tobias

    2008-01-01

    Host defense peptides are effector molecules of the innate immune system. They show broad antimicrobial action against gram-positive and -negative bacteria, and they likely play a key role in activating and mediating the innate as well as adaptive immune response in infection and inflammation. These features make them of high interest for wound healing research. Non-healing and infected wounds are a major problem in patient care and health care spending. Increasing infection rates, growing bacterial resistance to common antibiotics, and the lack of effective therapeutic options for the treatment of problematic wounds emphasize the need for new approaches in therapy and pathophysiologic understanding. This review focuses on the current knowledge of host defense peptides affecting wound healing and infection. We discuss the current data and highlight the potential future developments in this field of research. PMID:18385817

  7. The Shigella flexneri effector OspI deamidates UBC13 to dampen the inflammatory response.

    PubMed

    Sanada, Takahito; Kim, Minsoo; Mimuro, Hitomi; Suzuki, Masato; Ogawa, Michinaga; Oyama, Akiho; Ashida, Hiroshi; Kobayashi, Taira; Koyama, Tomohiro; Nagai, Shinya; Shibata, Yuri; Gohda, Jin; Inoue, Jun-ichiro; Mizushima, Tsunehiro; Sasakawa, Chihiro

    2012-03-11

    Many bacterial pathogens can enter various host cells and then survive intracellularly, transiently evade humoral immunity, and further disseminate to other cells and tissues. When bacteria enter host cells and replicate intracellularly, the host cells sense the invading bacteria as damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) by way of various pattern recognition receptors. As a result, the host cells induce alarm signals that activate the innate immune system. Therefore, bacteria must modulate host inflammatory signalling and dampen these alarm signals. How pathogens do this after invading epithelial cells remains unclear, however. Here we show that OspI, a Shigella flexneri effector encoded by ORF169b on the large plasmid and delivered by the type ΙΙΙ secretion system, dampens acute inflammatory responses during bacterial invasion by suppressing the tumour-necrosis factor (TNF)-receptor-associated factor 6 (TRAF6)-mediated signalling pathway. OspI is a glutamine deamidase that selectively deamidates the glutamine residue at position 100 in UBC13 to a glutamic acid residue. Consequently, the E2 ubiquitin-conjugating activity required for TRAF6 activation is inhibited, allowing S. flexneri OspI to modulate the diacylglycerol-CBM (CARD-BCL10-MALT1) complex-TRAF6-nuclear-factor-κB signalling pathway. We determined the 2.0 Å crystal structure of OspI, which contains a putative cysteine-histidine-aspartic acid catalytic triad. A mutational analysis showed this catalytic triad to be essential for the deamidation of UBC13. Our results suggest that S. flexneri inhibits acute inflammatory responses in the initial stage of infection by targeting the UBC13-TRAF6 complex.

  8. 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

  9. 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.

  10. Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy

    PubMed Central

    Rolando, Monica; Escoll, Pedro; Nora, Tamara; Botti, Joëlle; Boitez, Valérie; Daniels, Craig; Abraham, Gilu; Stogios, Peter J.; Skarina, Tatiana; Christophe, Charlotte; Dervins-Ravault, Delphine; Cazalet, Christel; Hilbi, Hubert; Rupasinghe, Thusitha W. T.; Tull, Dedreia; McConville, Malcolm J.; Ong, Sze Ying; Hartland, Elizabeth L.; Codogno, Patrice; Levade, Thierry; Naderer, Thomas; Savchenko, Alexei; Buchrieser, Carmen

    2016-01-01

    Autophagy is an essential component of innate immunity, enabling the detection and elimination of intracellular pathogens. Legionella pneumophila, an intracellular pathogen that can cause a severe pneumonia in humans, is able to modulate autophagy through the action of effector proteins that are translocated into the host cell by the pathogen’s Dot/Icm type IV secretion system. Many of these effectors share structural and sequence similarity with eukaryotic proteins. Indeed, phylogenetic analyses have indicated their acquisition by horizontal gene transfer from a eukaryotic host. Here we report that L. pneumophila translocates the effector protein sphingosine-1 phosphate lyase (LpSpl) to target the host sphingosine biosynthesis and to curtail autophagy. Our structural characterization of LpSpl and its comparison with human SPL reveals high structural conservation, thus supporting prior phylogenetic analysis. We show that LpSpl possesses S1P lyase activity that was abrogated by mutation of the catalytic site residues. L. pneumophila triggers the reduction of several sphingolipids critical for macrophage function in an LpSpl-dependent and -independent manner. LpSpl activity alone was sufficient to prevent an increase in sphingosine levels in infected host cells and to inhibit autophagy during macrophage infection. LpSpl was required for efficient infection of A/J mice, highlighting an important virulence role for this effector. Thus, we have uncovered a previously unidentified mechanism used by intracellular pathogens to inhibit autophagy, namely the disruption of host sphingolipid biosynthesis. PMID:26831115

  11. Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy.

    PubMed

    Rolando, Monica; Escoll, Pedro; Nora, Tamara; Botti, Joëlle; Boitez, Valérie; Bedia, Carmen; Daniels, Craig; Abraham, Gilu; Stogios, Peter J; Skarina, Tatiana; Christophe, Charlotte; Dervins-Ravault, Delphine; Cazalet, Christel; Hilbi, Hubert; Rupasinghe, Thusitha W T; Tull, Dedreia; McConville, Malcolm J; Ong, Sze Ying; Hartland, Elizabeth L; Codogno, Patrice; Levade, Thierry; Naderer, Thomas; Savchenko, Alexei; Buchrieser, Carmen

    2016-02-16

    Autophagy is an essential component of innate immunity, enabling the detection and elimination of intracellular pathogens. Legionella pneumophila, an intracellular pathogen that can cause a severe pneumonia in humans, is able to modulate autophagy through the action of effector proteins that are translocated into the host cell by the pathogen's Dot/Icm type IV secretion system. Many of these effectors share structural and sequence similarity with eukaryotic proteins. Indeed, phylogenetic analyses have indicated their acquisition by horizontal gene transfer from a eukaryotic host. Here we report that L. pneumophila translocates the effector protein sphingosine-1 phosphate lyase (LpSpl) to target the host sphingosine biosynthesis and to curtail autophagy. Our structural characterization of LpSpl and its comparison with human SPL reveals high structural conservation, thus supporting prior phylogenetic analysis. We show that LpSpl possesses S1P lyase activity that was abrogated by mutation of the catalytic site residues. L. pneumophila triggers the reduction of several sphingolipids critical for macrophage function in an LpSpl-dependent and -independent manner. LpSpl activity alone was sufficient to prevent an increase in sphingosine levels in infected host cells and to inhibit autophagy during macrophage infection. LpSpl was required for efficient infection of A/J mice, highlighting an important virulence role for this effector. Thus, we have uncovered a previously unidentified mechanism used by intracellular pathogens to inhibit autophagy, namely the disruption of host sphingolipid biosynthesis.

  12. 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

  13. Intracellular Voyeurism: Examining the Modulation of Host Cell Activities bySalmonella enterica Serovar Typhimurium.

    PubMed

    Szeto, Jason; Brumell, John H

    2005-11-01

    Salmonella spp. can infect host cells by gaining entry through phagocytosis or by inducing host cell membrane ruffling that facilitates bacterial uptake. With its wide host range, Salmonella enterica serovar Typhimurium has proven to be an important model organism for studying intracellular bacterial pathogenesis. Upon entry into host cells, serovar Typhimurium typically resides within a membrane-bound compartment termed the Salmonella-containing vacuole (SCV). From the SCV, serovar Typhimurium can inject several effector proteins that subvert many normal host cell systems, including endocytic trafficking, cytoskeletal rearrangements, lipid signaling and distribution, and innate and adaptive host defenses. The study of these intracellular events has been made possible through the use of various imaging techniques, ranging from classic methods of transmission electron microscopy to advanced livecell fluorescence confocal microscopy. In addition, DNA microarrays have now been used to provide a "snapshot" of global gene expression in serovar Typhimurium residing within the infected host cell. This review describes key aspects of Salmonella-induced subversion of host cell activities, providing examples of imaging that have been used to elucidate these events. Serovar Typhimurium engages specific host cell machinery from initial contact with the host cell to replication within the SCV. This continuous interaction with the host cell has likely contributed to the extensive arsenal that serovar Typhimurium now possesses, including two type III secretion systems, a range of ammunition in the form of TTSS effectors, and a complex genetic regulatory network that coordinates the expression of hundreds of virulence factors.

  14. Toxoplasma Co-opts Host Cells It Does Not Invade

    PubMed Central

    Koshy, Anita A.; Dietrich, Hans K.; Christian, David A.; Melehani, Jason H.; Shastri, Anjali J.; Hunter, Christopher A.; Boothroyd, John C.

    2012-01-01

    Like many intracellular microbes, the protozoan parasite Toxoplasma gondii injects effector proteins into cells it invades. One group of these effector proteins is injected from specialized organelles called the rhoptries, which have previously been described to discharge their contents only during successful invasion of a host cell. In this report, using several reporter systems, we show that in vitro the parasite injects rhoptry proteins into cells it does not productively invade and that the rhoptry effector proteins can manipulate the uninfected cell in a similar manner to infected cells. In addition, as one of the reporter systems uses a rhoptry:Cre recombinase fusion protein, we show that in Cre-reporter mice infected with an encysting Toxoplasma-Cre strain, uninfected-injected cells, which could be derived from aborted invasion or cell-intrinsic killing after invasion, are actually more common than infected-injected cells, especially in the mouse brain, where Toxoplasma encysts and persists. This phenomenon has important implications for how Toxoplasma globally affects its host and opens a new avenue for how other intracellular microbes may similarly manipulate the host environment at large. PMID:22910631

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

    SciTech Connect

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

    2010-11-08

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

  16. Transcriptome analysis reveals a complex interplay between resistance and effector genes during the compatible lentil-Colletotrichum lentis interaction

    PubMed Central

    Bhadauria, Vijai; Vijayan, Perumal; Wei, Yangdou; Banniza, Sabine

    2017-01-01

    Colletotrichum lentis is a hemibiotrophic pathogen and causes anthracnose on lentil. To understand the molecular mechanism underlying the symptomatic phase of infection, a cDNA plasmid library was developed from the susceptible lentil cultivar Eston infected with an isolate of the virulent race 0 of C. lentis. The library was sequenced on the Sanger sequencing platform, generating a total of 11,094 expressed sequence tags (ESTs) representing 3,488 unigenes. Mapping of unigenes onto the C. lentis and the L. culinaris genomes resulted in the identification of 2,418 unigenes of fungal origin and 1,070 unigenes of plant origin. Gene ontology term analysis of unigenes revealed that the transcriptome contained 22 candidate effectors, such as in planta induced ToxB and CyanoVirin-N, and 26 resistance genes, including suppressor of npr1-1 constitutive 1 and dirigent. Comparative genomics analyses revealed that three of the candidate effectors are likely located in the subtelomeric regions, and two of them show no synteny with the closely related species C. higginsianum, suggesting genomic rearrangements, such as translocation during speciation to colonize different niches. The data suggest a complex molecular interplay between disease resistance proteins and effectors during compatible interaction in which the pathogen exploits defense responses mounted by the host. PMID:28186158

  17. Induced autoprocessing of the cytopathic Makes Caterpillars Floppy-like effector domain of the Vibrio vulnificus MARTX toxin

    PubMed Central

    Agarwal, Shivangi; Agarwal, Shivani; Biancucci, Marco; Satchell, Karla J. F.

    2015-01-01

    Summary The multifunctional-autoprocessing repeats-in-toxin (MARTXVv) toxin that harbors a varied repertoire of effector domains is the primary virulence factor of Vibrio vulnificus. Although ubiquitously present among Biotype I toxin variants, the Makes Caterpillars Floppy-like effector domain (MCFVv) is previously unstudied. Using transient expression and protein delivery, MCFVv and MCFAh from the Aeromonas hydrophila MARTXAh toxin are shown for the first time to induce cell rounding. Alanine mutagenesis across the C-terminal subdomain of MCFVv identified an RCD tripeptide motif shown to comprise a cysteine protease catalytic site essential for autoprocessing of MCFVv. The autoprocessing could be recapitulated in vitro by addition of host cell lysate to recombinant MCFVv, indicating induced autoprocessing by cellular factors. The RCD motif is also essential for cytopathicity, suggesting autoprocessing is essential first to activate the toxin and then to process a cellular target protein resulting in cell rounding. Sequence homology places MCFVv within the C58 cysteine protease family that includes the type III secretion effectors YopT from Yersinia spp. and AvrPphB from Pseudomonas syringae. However, the catalytic site RCD motif is unique compared to other C58 peptidases and is here proposed to represent a new subgroup of autopeptidase found within a number of putative large bacterial toxins. PMID:25912102

  18. Transcriptome analysis reveals a complex interplay between resistance and effector genes during the compatible lentil-Colletotrichum lentis interaction.

    PubMed

    Bhadauria, Vijai; Vijayan, Perumal; Wei, Yangdou; Banniza, Sabine

    2017-02-10

    Colletotrichum lentis is a hemibiotrophic pathogen and causes anthracnose on lentil. To understand the molecular mechanism underlying the symptomatic phase of infection, a cDNA plasmid library was developed from the susceptible lentil cultivar Eston infected with an isolate of the virulent race 0 of C. lentis. The library was sequenced on the Sanger sequencing platform, generating a total of 11,094 expressed sequence tags (ESTs) representing 3,488 unigenes. Mapping of unigenes onto the C. lentis and the L. culinaris genomes resulted in the identification of 2,418 unigenes of fungal origin and 1,070 unigenes of plant origin. Gene ontology term analysis of unigenes revealed that the transcriptome contained 22 candidate effectors, such as in planta induced ToxB and CyanoVirin-N, and 26 resistance genes, including suppressor of npr1-1 constitutive 1 and dirigent. Comparative genomics analyses revealed that three of the candidate effectors are likely located in the subtelomeric regions, and two of them show no synteny with the closely related species C. higginsianum, suggesting genomic rearrangements, such as translocation during speciation to colonize different niches. The data suggest a complex molecular interplay between disease resistance proteins and effectors during compatible interaction in which the pathogen exploits defense responses mounted by the host.

  19. Candidate effector proteins of the necrotrophic apple canker pathogen Valsa mali can suppress BAX-induced PCD

    PubMed Central

    Li, Zhengpeng; Yin, Zhiyuan; Fan, Yanyun; Xu, Ming; Kang, Zhensheng; Huang, Lili

    2015-01-01

    Canker caused by the Ascomycete Valsa mali is the most destructive disease of apple in Eastern Asia, resulting in yield losses of up to 100%. This necrotrophic fungus induces severe necrosis on apple, eventually leading to the death of the whole tree. Identification of necrosis inducing factors may help to unravel the molecular bases for colonization of apple trees by V. mali. As a first step toward this goal, we identified and characterized the V. mali repertoire of candidate effector proteins (CEPs). In total, 193 secreted proteins with no known function were predicted from genomic data, of which 101 were V. mali-specific. Compared to non-CEPs predicted for the V. mali secretome, CEPs have shorter sequence length and a higher content of cysteine residues. Based on transient over-expression in Nicotiana benthamiana performed for 70 randomly selected CEPs, seven V. mali Effector Proteins (VmEPs) were shown to significantly suppress BAX-induced PCD. Furthermore, targeted deletion of VmEP1 resulted in a significant reduction of virulence. These results suggest that V. mali expresses secreted proteins that can suppress PCD usually associated with effector-triggered immunity (ETI). ETI in turn may play an important role in the V. mali–apple interaction. The ability of V. mali to suppress plant ETI sheds a new light onto the interaction of a necrotrophic fungus with its host plant. PMID:26284095

  20. Induced autoprocessing of the cytopathic Makes caterpillars floppy-like effector domain of the Vibrio vulnificus MARTX toxin.

    PubMed

    Agarwal, Shivangi; Agarwal, Shivani; Biancucci, Marco; Satchell, Karla J F

    2015-10-01

    The multifunctional-autoprocessing repeats-in-toxin (MARTX(Vv)) toxin that harbours a varied repertoire of effector domains is the primary virulence factor of Vibrio vulnificus. Although ubiquitously present among Biotype I toxin variants, the 'Makes caterpillars floppy-like' effector domain (MCF(Vv)) is previously unstudied. Using transient expression and protein delivery, MCF(Vv) and MCF(Ah) from the Aeromonas hydrophila MARTX(Ah)) toxin are shown for the first time to induce cell rounding. Alanine mutagenesis across the C-terminal subdomain of MCF(Vv) identified an Arg-Cys-Asp (RCD) tripeptide motif shown to comprise a cysteine protease catalytic site essential for autoprocessing of MCF(Vv). The autoprocessing could be recapitulated in vitro by the addition of host cell lysate to recombinant MCF(Vv), indicating induced autoprocessing by cellular factors. The RCD motif is also essential for cytopathicity, suggesting autoprocessing is essential first to activate the toxin and then to process a cellular target protein resulting in cell rounding. Sequence homology places MCF(Vv) within the C58 cysteine protease family that includes the type III secretion effectors YopT from Yersinia spp. and AvrPphB from Pseudomonas syringae. However, the catalytic site RCD motif is unique compared with other C58 peptidases and is here proposed to represent a new subgroup of autopeptidase found within a number of putative large bacterial toxins.

  1. 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

  2. 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

  3. Distinct Kinetics of Effector CD8+ Cytotoxic T Cells after Infection with Trypanosoma cruzi in Naïve or Vaccinated Mice

    PubMed Central

    Tzelepis, Fanny; de Alencar, Bruna C. G.; Penido, Marcus L. O.; Gazzinelli, Ricardo T.; Persechini, Pedro M.; Rodrigues, Mauricio M.

    2006-01-01

    The kinetics of effector CD8+-T-cell responses to specific Trypanosoma cruzi epitopes was investigated after challenge. Our results suggest that the delayed kinetics differs from that observed in other microbial infections and facilitates the establishment of the disease in naïve mice. In contrast, in vaccinated mice, the swift CD8+-T-cell response helps host survival after challenge. PMID:16552083

  4. Development of Viral Capsid DNA Aptamer Conjugates as Cell-Targeted Delivery Vehicles

    NASA Astrophysics Data System (ADS)

    Tong, Gary Jen-Wei

    The ability to generate semi-synthetic DNA-protein conjugates has become increasingly important in the fields of chemical biology and nanobiotechnology. As applications in these fields become more complex, there is also an increased need for methods of attaching synthetic DNA to protein substrates in a well-defined manner. This work outlines the development of new methods for site-specific DNA-protein bioconjugation, as well as the development of novel viral capsid DNA aptamer conjugates for cell-targeting purposes. In order to generate DNA-protein conjugates in a site-specific manner, chemistries orthogonal to native functional groups present on DNA and proteins were exploited. In one method, the attachment of DNA to proteins was achieved via oxime formation. This strategy involved the in situ deprotection of an allyloxycarbonyl-protected alkoxyamine-bearing DNA in the presence of a protein containing a single ketone group. The utility of this approach was demonstrated in the synthesis of a DNA-GFP conjugate. In addition to the oxime formation route, two oxidative coupling methods were also developed for DNA-protein bioconjugation. The first reaction coupled phenylenediamine-containing DNA to anilines, which had been site-specifically incorporated into proteins, in the presence of NaIO4. These reaction conditions were demonstrated on the proteins bacteriophage MS2 and GFP, and were mild enough for the components to retain both protein structure and DNA base-pairing capabilities. The second oxidative coupling reaction conjugated aniline-containing proteins to DNA bearing an o-aminophenol moiety. This reaction occurred under similarly mild conditions; however, higher coupling yields were achieved on MS2 at shorter reaction times by using this strategy. In all three of these methods, the generation of a singly-modified product was achieved. Using one of our oxidative coupling strategies, MS2-DNA aptamer conjugates were synthesized for the development of multivalent

  5. Stagonospora nodorum: from pathology to genomics and host resistance.

    PubMed

    Oliver, Richard P; Friesen, Timothy L; Faris, Justin D; Solomon, Peter S

    2012-01-01

    Stagonospora nodorum is a major necrotrophic pathogen of wheat that causes the diseases S. nodorum leaf and glume blotch. A series of tools and resources, including functional genomics, a genome sequence, proteomics and metabolomics, host-mapping populations, and a worldwide collection of isolates, have enabled the dissection of pathogenicity mechanisms. Metabolic and signaling genes required for pathogenicity have been defined. Interaction with the host is dominated by interplay of fungal effectors that induce necrosis on wheat lines carrying specific sensitivity loci. As such, the pathogen has emerged as a model for the Pleosporales group of pathogens.

  6. 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.

  7. 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…

  8. 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

  9. 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

  10. 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.

  11. Dissociating Visual Attention and Effector Selection in Spatial Precuing Tasks

    ERIC Educational Resources Information Center

    Adam, Jos J.; Pratt, Jay

    2004-01-01

    In this study the authors tested and rejected a recent proposal that response precuing effects depend on the spatial extent of the precues rather than on the number of response choices. Moreover, the authors tested and supported the hypothesis that the number of effectors in the response set is an important determinant of spatial precuing effects.…

  12. 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

  13. Whole-Genome Sequences of Xanthomonas euvesicatoria Strains Clarify Taxonomy and Reveal a Stepwise Erosion of Type 3 Effectors

    PubMed Central

    Barak, Jeri D.; Vancheva, Taca; Lefeuvre, Pierre; Jones, Jeffrey B.; Timilsina, Sujan; Minsavage, Gerald V.; Vallad, Gary E.; Koebnik, Ralf

    2016-01-01

    Multiple species of Xanthomonas cause bacterial spot of tomato (BST) and pepper. We sequenced five Xanthomonas euvesicatoria strains isolated from three continents (Africa, Asia, and South America) to provide a set of representative genomes with temporal and geographic diversity. LMG strains 667, 905, 909, and 933 were pathogenic on tomato and pepper, except LMG 918 elicited a hypersensitive reaction (HR) on tomato. Furthermore, LMG 667, 909, and 918 elicited a HR on Early Cal Wonder 30R containing Bs3. We examined pectolytic activity and starch hydrolysis, two tests which are useful in differentiating X. euvesicatoria from X. perforans, both causal agents of BST. LMG strains 905, 909, 918, and 933 were nonpectolytic while only LMG 918 was amylolytic. These results suggest that LMG 918 is atypical of X. euvesicatoria. Sequence analysis of all the publicly available X. euvesicatoria and X. perforans strains comparing seven housekeeping genes identified seven haplotypes with few polymorphisms. Whole genome comparison by average nucleotide identity (ANI) resulted in values of >99% among the LMG strains 667, 905, 909, 918, and 933 and X. euvesicatoria strains and >99.6% among the LMG strains and a subset of X. perforans strains. These results suggest that X. euvesicatoria and X. perforans should be considered a single species. ANI values between strains of X. euvesicatoria, X. perforans, X. allii, X. alfalfa subsp. citrumelonis, X. dieffenbachiae, and a recently described pathogen of rose were >97.8% suggesting these pathogens should be a single species and recognized as X. euvesicatoria. Analysis of the newly sequenced X. euvesicatoria strains revealed interesting findings among the type 3 (T3) effectors, relatively ancient stepwise erosion of some T3 effectors, additional X. euvesicatoria-specific T3 effectors among the causal agents of BST, orthologs of avrBs3 and avrBs4, and T3 effectors shared among xanthomonads pathogenic against various hosts. The results from

  14. GogB Is an Anti-Inflammatory Effector that Limits Tissue Damage during Salmonella Infection through Interaction with Human FBXO22 and Skp1

    PubMed Central

    Pilar, Ana Victoria C.; Reid-Yu, Sarah A.; Cooper, Colin A.; Mulder, David T.; Coombes, Brian K.

    2012-01-01

    Bacterial pathogens often manipulate host immune pathways to establish acute and chronic infection. Many Gram-negative bacteria do this by secreting effector proteins through a type III secretion system that alter the host response to the pathogen. In this study, we determined that the phage-encoded GogB effector protein in Salmonella targets the host SCF E3 type ubiquitin ligase through an interaction with Skp1 and the human F-box only 22 (FBXO22) protein. Domain mapping and functional knockdown studies indicated that GogB-containing bacteria inhibited IκB degradation and NFκB activation in macrophages, which required Skp1 and a eukaryotic-like F-box motif in the C-terminal domain of GogB. GogB-deficient Salmonella were unable to limit NFκB activation, which lead to increased proinflammatory responses in infected mice accompanied by extensive tissue damage and enhanced colonization in the gut during long-term chronic infections. We conclude that GogB is an anti-inflammatory effector that helps regulate inflammation-enhanced colonization by limiting tissue damage during infection. PMID:22761574

  15. The Pseudomonas syringae Type III Effector HopG1 Induces Actin Remodeling to Promote Symptom Development and Susceptibility during Infection1[OPEN

    PubMed Central

    Shimono, Masaki; Porter, Katie; Kvitko, Brian H.; Henty-Ridilla, Jessica; Creason, Allison; Chang, Jeff H.; Staiger, Christopher J.

    2016-01-01

    The plant cytoskeleton underpins the function of a multitude of cellular mechanisms, including those associated with developmental- and stress-associated signaling processes. In recent years, the actin cytoskeleton has been demonstrated to play a key role in plant immune signaling, including a recent demonstration that pathogens target actin filaments to block plant defense and immunity. Herein, we quantified spatial changes in host actin filament organization after infection with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), demonstrating that the type-III effector HopG1 is required for pathogen-induced changes to actin filament architecture and host disease symptom development during infection. Using a suite of pathogen effector deletion constructs, coupled with high-resolution microscopy, we found that deletion of hopG1 from Pst DC3000 resulted in a reduction in actin bundling and a concomitant increase in the density of filament arrays in Arabidopsis, both of which correlate with host disease symptom development. As a mechanism underpinning this activity, we further show that the HopG1 effector interacts with an Arabidopsis mitochondrial-localized kinesin motor protein. Kinesin mutant plants show reduced disease symptoms after pathogen infection, which can be complemented by actin-modifying agents. In total, our results support a model in which HopG1 induces changes in the organization of the actin cytoskeleton as part of its virulence function in promoting disease symptom development. PMID:27217495

  16. The Rhoptry Pseudokinase ROP54 Modulates Toxoplasma gondii Virulence and Host GBP2 Loading.

    PubMed

    Kim, Elliot W; Nadipuram, Santhosh M; Tetlow, Ashley L; Barshop, William D; Liu, Philip T; Wohlschlegel, James A; Bradley, Peter J

    2016-01-01

    Toxoplasma gondii uses unique secretory organelles called rhoptries to inject an array of effector proteins into the host cytoplasm that hijack host cell functions. We have discovered a novel rhoptry pseudokinase effector, ROP54, which is injected into the host cell upon invasion and traffics to the cytoplasmic face of the parasitophorous vacuole membrane (PVM). Disruption of ROP54 in a type II strain of T. gondii does not affect growth in vitro but results in a 100-fold decrease in virulence in vivo, suggesting that ROP54 modulates some aspect of the host immune response. We show that parasites lacking ROP54 are more susceptible to macrophage-dependent clearance, further suggesting that ROP54 is involved in evasion of innate immunity. To determine how ROP54 modulates parasite virulence, we examined the loading of two known innate immune effectors, immunity-related GTPase b6 (IRGb6) and guanylate binding protein 2 (GBP2), in wild-type and ∆rop54II mutant parasites. While no difference in IRGb6 loading was seen, we observed a substantial increase in GBP2 loading on the parasitophorous vacuole (PV) of ROP54-disrupted parasites. These results demonstrate that ROP54 is a novel rhoptry effector protein that promotes Toxoplasma infections by modulating GBP2 loading onto parasite-containing vacuoles. IMPORTANCE The interactions between intracellular microbes and their host cells can lead to the discovery of novel drug targets. During Toxoplasma infections, host cells express an array of immunity-related GTPases (IRGs) and guanylate binding proteins (GBPs) that load onto the parasite-containing vacuole to clear the parasite. To counter this mechanism, the parasite secretes effector proteins that traffic to the vacuole to disarm the immunity-related loading proteins and evade the immune response. While the interplay between host IRGs and Toxoplasma effector proteins is well understood, little is known about how Toxoplasma neutralizes the GBP response. We describe here a T

  17. The Rhoptry Pseudokinase ROP54 Modulates Toxoplasma gondii Virulence and Host GBP2 Loading

    PubMed Central

    Kim, Elliot W.; Nadipuram, Santhosh M.; Tetlow, Ashley L.; Barshop, William D.; Liu, Philip T.; Wohlschlegel, James A.

    2016-01-01

    ABSTRACT Toxoplasma gondii uses unique secretory organelles called rhoptries to inject an array of effector proteins into the host cytoplasm that hijack host cell functions. We have discovered a novel rhoptry pseudokinase effector, ROP54, which is injected into the host cell upon invasion and traffics to the cytoplasmic face of the parasitophorous vacuole membrane (PVM). Disruption of ROP54 in a type II strain of T. gondii does not affect growth in vitro but results in a 100-fold decrease in virulence in vivo, suggesting that ROP54 modulates some aspect of the host immune response. We show that parasites lacking ROP54 are more susceptible to macrophage-dependent clearance, further suggesting that ROP54 is involved in evasion of innate immunity. To determine how ROP54 modulates parasite virulence, we examined the loading of two known innate immune effectors, immunity-related GTPase b6 (IRGb6) and guanylate binding protein 2 (GBP2), in wild-type and ∆rop54II mutant parasites. While no difference in IRGb6 loading was seen, we observed a substantial increase in GBP2 loading on the parasitophorous vacuole (PV) of ROP54-disrupted parasites. These results demonstrate that ROP54 is a novel rhoptry effector protein that promotes Toxoplasma infections by modulating GBP2 loading onto parasite-containing vacuoles. IMPORTANCE The interactions between intracellular microbes and their host cells can lead to the discovery of novel drug targets. During Toxoplasma infections, host cells express an array of immunity-related GTPases (IRGs) and guanylate binding proteins (GBPs) that load onto the parasite-containing vacuole to clear the parasite. To counter this mechanism, the parasite secretes effector proteins that traffic to the vacuole to disarm the immunity-related loading proteins and evade the immune response. While the interplay between host IRGs and Toxoplasma effector proteins is well understood, little is known about how Toxoplasma neutralizes the GBP response. We describe

  18. Specificity and Dynamics of Effector and Memory CD8 T Cell Responses in Human Tick-Borne Encephalitis Virus Infection

    PubMed Central

    Blom, Kim; Braun, Monika; Pakalniene, Jolita; Dailidyte, Laura; Béziat, Vivien; Lampen, Margit H.; Klingström, Jonas; Lagerqvist, Nina; Kjerstadius, Torbjörn; Michaëlsson, Jakob; Lindquist, Lars; Ljunggren, Hans-Gustaf; Sandberg, Johan K.; Mickiene, Aukse; Gredmark-Russ, Sara

    2015-01-01

    Tick-borne encephalitis virus (TBEV) is transferred to humans by ticks. The virus causes tick-borne encephalitis (TBE) with symptoms such as meningitis and meningoencephalitis. About one third of the patients suffer from long-lasting sequelae after clearance of the infection. Studies of the immune response during TBEV-infection are essential to the understanding of host responses to TBEV-infection and for the development of therapeutics. Here, we studied in detail the primary CD8 T cell response to TBEV in patients with acute TBE. Peripheral blood CD8 T cells mounted a considerable response to TBEV-infection as assessed by Ki67 and CD38 co-expression. These activated cells showed a CD45RA-CCR7-CD127- phenotype at day 7 after hospitalization, phenotypically defining them as effector cells. An immunodominant HLA-A2-restricted TBEV epitope was identified and utilized to study the characteristics and temporal dynamics of the antigen-specific response. The functional profile of TBEV-specific CD8 T cells was dominated by variants of mono-functional cells as the effector response matured. Antigen-specific CD8 T cells predominantly displayed a distinct Eomes+Ki67+T-bet+ effector phenotype at the peak of the response, which transitioned to an Eomes-Ki67-T-bet+ phenotype as the infection resolved and memory was established. These transcription factors thus characterize and discriminate stages of the antigen-specific T cell response during acute TBEV-infection. Altogether, CD8 T cells responded strongly to acute TBEV infection and passed through an effector phase, prior to gradual differentiation into memory cells with distinct transcription factor expression-patterns throughout the different phases. PMID:25611738

  19. Galactosylated chitosan oligosaccharide nanoparticles for hepatocellular carcinoma cell-targeted delivery of adenosine triphosphate.

    PubMed

    Zhu, Xiu Liang; Du, Yong Zhong; Yu, Ri Sheng; Liu, Ping; Shi, Dan; Chen, Ying; Wang, Ying; Huang, Fang Fang

    2013-07-29

    Nanoparticles composed of galactosylated chitosan oligosaccharide (Gal-CSO) and adenosine triphosphate (ATP) were prepared for hepatocellular carcinoma cell-specific uptake, and the characteristics of Gal-CSO/ATP nanoparticles were evaluated. CSO/ATP nanoparticles were prepared as a control. The average diameter and zeta potential of Gal-CSO/ATP nanoparticles were 51.03 ± 3.26 nm and 30.50 ± 1.25 mV, respectively, suggesting suitable properties for a drug delivery system. Subsequently, the cytotoxicity of Gal-CSO/ATP nanoparticles were examined by the methyl tetrazolium (MTT) assay, and the half maximal inhibitory concentration (IC50) values were calculated with HepG2 (human hepatocellular carcinoma cell line) cells. The results showed that the cytotoxic effect of nanoparticles on HepG2 cells was low. In the meantime, it was also found that the Gal-CSO/ATP nanoparticles could be uptaken by HepG2 cells, due to expression of the asialoglycoprotein receptor (ASGP-R) on their surfaces. The presented results indicate that the Gal-CSO nanoparticles might be very attractive to be used as an intracellular drug delivery carrier for hepatocellular carcinoma c