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Sample records for host cell-targeted effectors

  1. Common and contrasting themes in host cell-targeted effectors from bacterial, fungal, oomycete and nematode plant symbionts described using the Gene Ontology.

    PubMed

    Torto-Alalibo, Trudy; Collmer, Candace W; Lindeberg, Magdalen; Bird, David; Collmer, Alan; Tyler, Brett M

    2009-01-01

    A wide diversity of plant-associated symbionts, including microbes, produce proteins that can enter host cells, or are injected into host cells in order to modify the physiology of the host to promote colonization. These molecules, termed effectors, commonly target the host defense signaling pathways in order to suppress the defense response. Others target the gene expression machinery or trigger specific modifications to host morphology or physiology that promote the nutrition and proliferation of the symbiont. When recognized by the host's surveillance machinery, which includes cognate resistance (R) gene products, defense responses are engaged to restrict pathogen proliferation. Effectors from diverse symbionts may be delivered into plant cells via varied mechanisms, including whole organism cellular entry (viruses, some bacteria and fungi), type III and IV secretion (in bacteria), physical injection (nematodes and insects) and protein translocation signal sequences (oomycetes and fungi). This mini-review will summarize both similarities and differences in effectors and effector delivery systems found in diverse plant-associated symbionts as well as how these are described with Plant-Associated Microbe Gene Ontology (PAMGO) terms. PMID:19278551

  2. 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

  3. 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. PMID:26467026

  4. 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

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

    PubMed

    Pha, Khavong; Navarro, Lorena

    2016-02-26

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

  6. The T6SSs of Pseudomonas aeruginosa Strain PAO1 and Their Effectors: Beyond Bacterial-Cell Targeting

    PubMed Central

    Sana, Thibault G.; Berni, Benjamin; Bleves, Sophie

    2016-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen responsible for many diseases such as chronic lung colonization in cystic fibrosis patients and acute infections in hospitals. The capacity of P. aeruginosa to be pathogenic toward several hosts is notably due to different secretion systems. Amongst them, P. aeruginosa encodes three Type Six Secretion Systems (T6SS), named H1- to H3-T6SS, that act against either prokaryotes and/or eukaryotic cells. They are independent from each other and inject diverse toxins that interact with different components in the host cell. Here we summarize the roles of these T6SSs in the PAO1 strain, as well as the toxins injected and their targets. While H1-T6SS is only involved in antiprokaryotic activity through at least seven different toxins, H2-T6SS and H3-T6SS are also able to target prokaryotic as well as eukaryotic cells. Moreover, recent studies proposed that H2- and H3-T6SS have a role in epithelial cells invasion by injecting at least three different toxins. The diversity of T6SS effectors is astounding and other effectors still remain to be discovered. In this review, we present a table with other putative P. aeruginosa strain PAO1 T6SS-dependent effectors. Altogether, the T6SSs of P. aeruginosa are important systems that help fight other bacteria for their ecological niche, and are important in the pathogenicity process. PMID:27376031

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

    PubMed

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

    2015-01-01

    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. PMID:26484613

  8. Experimental approaches to investigate effector translocation into host cells in the Ustilago maydis/maize pathosystem.

    PubMed

    Tanaka, Shigeyuki; Djamei, Armin; Presti, Libera Lo; Schipper, Kerstin; Winterberg, Sarah; Amati, Simone; Becker, Dirk; Büchner, Heike; Kumlehn, Jochen; Reissmann, Stefanie; Kahmann, Regine

    2015-01-01

    The fungus Ustilago maydis is a pathogen that establishes a biotrophic interaction with Zea mays. The interaction with the plant host is largely governed by more than 300 novel, secreted protein effectors, of which only four have been functionally characterized. Prerequisite to examine effector function is to know where effectors reside after secretion. Effectors can remain in the extracellular space, i.e. the plant apoplast (apoplastic effectors), or can cross the plant plasma membrane and exert their function inside the host cell (cytoplasmic effectors). The U. maydis effectors lack conserved motifs in their primary sequences that could allow a classification of the effectome into apoplastic/cytoplasmic effectors. This represents a significant obstacle in functional effector characterization. Here we describe our attempts to establish a system for effector classification into apoplastic and cytoplasmic members, using U. maydis for effector delivery. PMID:26118724

  9. Ehrlichia chaffeensis TRP32 Interacts with Host Cell Targets That Influence Intracellular Survival

    PubMed Central

    Luo, Tian

    2012-01-01

    Ehrlichia chaffeensis is an obligately intracellular bacterium that exhibits tropism for mononuclear phagocytes and survives by evading host cell defense mechanisms. Recently, molecular interactions of E. chaffeensis tandem repeat proteins 47 and 120 (TRP47 and -120) and the eukaryotic host cell have been described. In this investigation, yeast two-hybrid analysis demonstrated that an E. chaffeensis type 1 secretion system substrate, TRP32, interacts with a diverse group of human proteins associated with major biological processes of the host cell, including protein synthesis, trafficking, degradation, immune signaling, cell signaling, iron metabolism, and apoptosis. Eight target proteins, including translation elongation factor 1 alpha 1 (EF1A1), deleted in azoospermia (DAZ)-associated protein 2 (DAZAP2), ferritin light polypeptide (FTL), CD63, CD14, proteasome subunit beta type 1 (PSMB1), ring finger and CCCH-type domain 1 (RC3H1), and tumor protein p53-inducible protein 11 (TP53I11) interacted with TRP32 as determined by coimmunoprecipitation assays, colocalization with TRP32 in HeLa and THP-1 cells, and/or RNA interference. Interactions between TRP32 and host targets localized to the E. chaffeensis morulae or in the host cell cytoplasm adjacent to morulae. Common or closely related interacting partners of E. chaffeensis TRP32, TRP47, and TRP120 demonstrate a molecular convergence on common cellular processes and molecular cross talk between Ehrlichia TRPs and host targets. These findings further support the role of TRPs as effectors that promote intracellular survival. PMID:22547548

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

    PubMed Central

    Dunphy, Paige Selvy; Luo, Tian

    2014-01-01

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

  11. Effector triggered manipulation of host immune response elicited by different pathotypes of Escherichia coli

    PubMed Central

    Jayamani, Elamparithi; Mylonakis, Eleftherios

    2014-01-01

    Effectors are virulence factors that are secreted by bacteria during an infection in order to subvert cellular processes or induce the surveillance system of the host. Pathogenic microorganisms encode effectors, toxins and components of secretion systems that inject the effectors to the host. Escherichia coli is part of the innocuous commensal microbial flora of the gastrointestinal tract. However, pathogenic E. coli can cause diarrheal and extraintestinal diseases. Pathogenic E. coli uses secretion systems to inject an array of effector proteins directly into the host cells. Herein, we discuss the effectors secreted by different pathotypes of E. coli and provide an overview of strategies employed by effectors to target the host cellular and subcellular processes as well as their role in triggering host immune response. PMID:25513774

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

    PubMed Central

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

    2016-01-01

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

  13. A Chlamydia effector recruits CEP170 to reprogram host microtubule organization

    PubMed Central

    Dumoux, Maud; Menny, Anais; Delacour, Delphine; Hayward, Richard D.

    2015-01-01

    ABSTRACT The obligate intracellular bacterial pathogen Chlamydia trachomatis deploys virulence effectors to subvert host cell functions enabling its replication within a specialized membrane-bound compartment termed an inclusion. The control of the host cytoskeleton is crucial for Chlamydia uptake, inclusion biogenesis and cell exit. Here, we demonstrate how a Chlamydia effector rearranges the microtubule (MT) network by initiating organization of the MTs at the inclusion surface. We identified an inclusion-localized effector that is sufficient to interfere with MT assembly, which we named inclusion protein acting on MTs (IPAM). We established that IPAM recruits and stimulates the centrosomal protein 170 kDa (CEP170) to hijack the MT organizing functions of the host cell. We show that CEP170 is essential for chlamydial control of host MT assembly, and is required for inclusion morphogenesis and bacterial infectivity. Together, we demonstrate how a pathogen effector reprograms the host MT network to support its intracellular development. PMID:26220855

  14. Bacterial effectors target the plant cell nucleus to subvert host transcription

    PubMed Central

    Canonne, Joanne; Rivas, Susana

    2012-01-01

    In order to promote virulence, Gram-negative bacteria have evolved the ability to inject so-called type III effector proteins into host cells. The plant cell nucleus appears to be a subcellular compartment repeatedly targeted by bacterial effectors. In agreement with this observation, mounting evidence suggests that manipulation of host transcription is a major strategy developed by bacteria to counteract plant defense responses. It has been suggested that bacterial effectors may adopt at least three alternative, although not mutually exclusive, strategies to subvert host transcription. T3Es may (1) act as transcription factors that directly activate transcription in host cells, (2) affect histone packing and chromatin configuration, and/or (3) directly target host transcription factor activity. Here, we provide an overview on how all these strategies may lead to host transcriptional re-programming and, as a result, to improved bacterial multiplication inside plant cells. PMID:22353865

  15. 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

  16. Exploitation of Host Polyubiquitination Machinery through Molecular Mimicry by Eukaryotic-Like Bacterial F-Box Effectors

    PubMed Central

    Price, Christopher T. D.; Kwaik, Yousef Abu

    2010-01-01

    Microbial pathogens have evolved exquisite mechanisms to interfere and intercept host biological processes, often through molecular mimicry of specific host proteins. Ubiquitination is a highly conserved eukaryotic post-translational modification essential in determining protein fate, and is often hijacked by pathogenic bacteria. The conserved SKP1/CUL1/F-box (SCF) E3 ubiquitin ligase complex plays a key role in ubiquitination of proteins in eukaryotic cells. The F-box protein component of the SCF complex provides specificity to ubiquitination by binding to specific cellular proteins, targeting them to be ubiquitinated by the SCF complex. The bacterial pathogens. Legionella pneumophila, Agrobacterium tumefaciens, and Ralstonia solanacearum utilize type III or IV translocation systems to inject into the host cell eukaryotic-like F-box effectors that interact with the host SKP1 component of the SCF complex to trigger ubiquitination of specific host cells targets, which is essential to promote proliferation of these pathogens. Our bioinformatic analyses have identified at least 74 genes encoding putative F-box proteins belonging to 22 other bacterial species, including human pathogens, plant pathogens, and amebal endosymbionts. Therefore, subversion of the host ubiquitination machinery by bacterial F-box proteins may be a widespread strategy amongst pathogenic bacteria. The findings that bacterial F-box proteins harbor Ankyrin repeats as protein–protein interaction domains, which are present in F-box proteins of primitive but not higher eukaryotes, suggest acquisition of many bacterial F-box proteins from primitive eukaryotic hosts rather than the mammalian host. PMID:21687758

  17. Rust fungal effectors mimic host transit peptides to translocate into chloroplasts.

    PubMed

    Petre, Benjamin; Lorrain, Cécile; Saunders, Diane G O; Win, Joe; Sklenar, Jan; Duplessis, Sébastien; Kamoun, Sophien

    2016-04-01

    Parasite effector proteins target various host cell compartments to alter host processes and promote infection. How effectors cross membrane-rich interfaces to reach these compartments is a major question in effector biology. Growing evidence suggests that effectors use molecular mimicry to subvert host cell machinery for protein sorting. We recently identified chloroplast-targeted protein 1 (CTP1), a candidate effector from the poplar leaf rust fungus Melampsora larici-populina that carries a predicted transit peptide and accumulates in chloroplasts and mitochondria. Here, we show that the CTP1 transit peptide is necessary and sufficient for accumulation in the stroma of chloroplasts. CTP1 is part of a Melampsora-specific family of polymorphic secreted proteins. Two members of that family, CTP2 and CTP3, also translocate in chloroplasts in an N-terminal signal-dependent manner. CTP1, CTP2 and CTP3 are cleaved when they accumulate in chloroplasts, while they remain intact when they do not translocate into chloroplasts. Our findings reveal that fungi have evolved effector proteins that mimic plant-specific sorting signals to traffic within plant cells. PMID:26426202

  18. 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 ...

  19. 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...

  20. 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...

  1. The Salmonella effector SteA binds phosphatidylinositol 4-phosphate for subcellular targeting within host cells.

    PubMed

    Domingues, Lia; Ismail, Ahmad; Charro, Nuno; Rodríguez-Escudero, Isabel; Holden, David W; Molina, María; Cid, Víctor J; Mota, Luís Jaime

    2016-07-01

    Many bacterial pathogens use specialized secretion systems to deliver virulence effector proteins into eukaryotic host cells. The function of these effectors depends on their localization within infected cells, but the mechanisms determining subcellular targeting of each effector are mostly elusive. Here, we show that the Salmonella type III secretion effector SteA binds specifically to phosphatidylinositol 4-phosphate [PI(4)P]. Ectopically expressed SteA localized at the plasma membrane (PM) of eukaryotic cells. However, SteA was displaced from the PM of Saccharomyces cerevisiae in mutants unable to synthesize the local pool of PI(4)P and from the PM of HeLa cells after localized depletion of PI(4)P. Moreover, in infected cells, bacterially translocated or ectopically expressed SteA localized at the membrane of the Salmonella-containing vacuole (SCV) and to Salmonella-induced tubules; using the PI(4)P-binding domain of the Legionella type IV secretion effector SidC as probe, we found PI(4)P at the SCV membrane and associated tubules throughout Salmonella infection of HeLa cells. Both binding of SteA to PI(4)P and the subcellular localization of ectopically expressed or bacterially translocated SteA were dependent on a lysine residue near the N-terminus of the protein. Overall, this indicates that binding of SteA to PI(4)P is necessary for its localization within host cells. PMID:26676327

  2. Characterization of cell death inducing Phytophthora capsici CRN effectors suggests diverse activities in the host nucleus

    PubMed Central

    Stam, Remco; Howden, Andrew J. M.; Delgado-Cerezo, Magdalena; M. M. Amaro, Tiago M.; Motion, Graham B.; Pham, Jasmine; Huitema, Edgar

    2013-01-01

    Plant-Microbe interactions are complex associations that feature recognition of Pathogen Associated Molecular Patterns by the plant immune system and dampening of subsequent responses by pathogen encoded secreted effectors. With large effector repertoires now identified in a range of sequenced microbial genomes, much attention centers on understanding their roles in immunity or disease. These studies not only allow identification of pathogen virulence factors and strategies, they also provide an important molecular toolset suited for studying immunity in plants. The Phytophthora intracellular effector repertoire encodes a large class of proteins that translocate into host cells and exclusively target the host nucleus. Recent functional studies have implicated the CRN protein family as an important class of diverse effectors that target distinct subnuclear compartments and modify host cell signaling. Here, we characterized three necrosis inducing CRNs and show that there are differences in the levels of cell death. We show that only expression of CRN20_624 has an additive effect on PAMP induced cell death but not AVR3a induced ETI. Given their distinctive phenotypes, we assessed localization of each CRN with a set of nuclear markers and found clear differences in CRN subnuclear distribution patterns. These assays also revealed that expression of CRN83_152 leads to a distinct change in nuclear chromatin organization, suggesting a distinct series of events that leads to cell death upon over-expression. Taken together, our results suggest diverse functions carried by CRN C-termini, which can be exploited to identify novel processes that take place in the host nucleus and are required for immunity or susceptibility. PMID:24155749

  3. Structural insight into effector proteins of Gram-negative bacterial pathogens that modulate the phosphoproteome of their host

    PubMed Central

    Grishin, Andrey M; Beyrakhova, Ksenia A; Cygler, Miroslaw

    2015-01-01

    Invading pathogens manipulate cellular process of the host cell to establish a safe replicative niche. To this end they secrete a spectrum of proteins called effectors that modify cellular environment through a variety of mechanisms. One of the most important mechanisms is the manipulation of cellular signaling through modifications of the cellular phosphoproteome. Phosphorylation/dephosphorylation plays a pivotal role in eukaryotic cell signaling, with ∼500 different kinases and ∼130 phosphatases in the human genome. Pathogens affect the phosphoproteome either directly through the action of bacterial effectors, and/or indirectly through downstream effects of host proteins modified by the effectors. Here we review the current knowledge of the structure, catalytic mechanism and function of bacterial effectors that modify directly the phosphorylation state of host proteins. These effectors belong to four enzyme classes: kinases, phosphatases, phospholyases and serine/threonine acetylases. PMID:25565677

  4. Chemical Genetics Reveals Bacterial and Host Cell Functions Critical for Type IV Effector Translocation by Legionella pneumophila

    PubMed Central

    Charpentier, Xavier; Gabay, Joëlle E.; Reyes, Moraima; Zhu, Jing W.; Weiss, Arthur; Shuman, Howard A.

    2009-01-01

    Delivery of effector proteins is a process widely used by bacterial pathogens to subvert host cell functions and cause disease. Effector delivery is achieved by elaborate injection devices and can often be triggered by environmental stimuli. However, effector export by the L. pneumophila Icm/Dot Type IVB secretion system cannot be detected until the bacterium encounters a target host cell. We used chemical genetics, a perturbation strategy that utilizes small molecule inhibitors, to determine the mechanisms critical for L. pneumophila Icm/Dot activity. From a collection of more than 2,500 annotated molecules we identified specific inhibitors of effector translocation. We found that L. pneumophila effector translocation in macrophages requires host cell factors known to be involved in phagocytosis such as phosphoinositide 3-kinases, actin and tubulin. Moreover, we found that L. pneumophila phagocytosis and effector translocation also specifically require the receptor protein tyrosine phosphate phosphatases CD45 and CD148. We further show that phagocytosis is required to trigger effector delivery unless intimate contact between the bacteria and the host is artificially generated. In addition, real-time analysis of effector translocation suggests that effector export is rate-limited by phagocytosis. We propose a model in which L. pneumophila utilizes phagocytosis to initiate an intimate contact event required for the translocation of pre-synthesized effector molecules. We discuss the need for host cell participation in the initial step of the infection and its implications in the L. pneumophila lifestyle. Chemical genetic screening provides a novel approach to probe the host cell functions and factors involved in host–pathogen interactions. PMID:19578436

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

    PubMed

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

    2016-05-01

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

  6. Interactions between the Powdery Mildew Effector BEC1054 and Barley Proteins Identify Candidate Host Targets.

    PubMed

    Pennington, Helen G; Gheorghe, Dana M; Damerum, Annabelle; Pliego, Clara; Spanu, Pietro D; Cramer, Rainer; Bindschedler, Laurence V

    2016-03-01

    There are over 500 candidate secreted effector proteins (CSEPs) or Blumeria effector candidates (BECs) specific to the barley powdery mildew pathogen Blumeria graminis f.sp. hordei. The CSEP/BEC proteins are expressed and predicted to be secreted by biotrophic feeding structures called haustoria. Eight BECs are required for the formation of functional haustoria. These include the RNase-like effector BEC1054 (synonym CSEP0064). In order to identify host proteins targeted by BEC1054, recombinant BEC1054 was expressed in E. coli, solubilized, and used in pull-down assays from barley protein extracts. Many putative interactors were identified by LC-MS/MS after subtraction of unspecific binders in negative controls. Therefore, a directed yeast-2-hybrid assay, developed to measure the effectiveness of the interactions in yeast, was used to validate putative interactors. We conclude that BEC1054 may target several host proteins, including a glutathione-S-transferase, a malate dehydrogenase, and a pathogen-related-5 protein isoform, indicating a possible role for BEC1054 in compromising well-known key players of defense and response to pathogens. In addition, BEC1054 interacts with an elongation factor 1 gamma. This study already suggests that BEC1054 plays a central role in barley powdery mildew virulence by acting at several levels. PMID:26813582

  7. 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. PMID:26827929

  8. 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

  9. 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

  10. A Legionella effector modulates host cytoskeletal structure by inhibiting actin polymerization

    PubMed Central

    Guo, Zhenhua; Stephenson, Robert; Qiu, Jiazhang; Zheng, Shijun; Luo, Zhao-Qing

    2014-01-01

    Successful infection by the opportunistic pathogen Legionella pneumophila requires the collective activity of hundreds of virulence proteins delivered into the host cell by the Dot/Icm type IV secretion system. These virulence proteins, also called effectors modulate distinct host cellular processes to create a membrane-bound niche called the Legionella containing vacuole (LCV) supportive of bacterial growth. We found that Ceg14(Lpg0437), a Dot/Icm substrate is toxic to yeast and such toxicity can be alleviated by overexpression of profilin, a protein involved in cytoskeletal structure in eukaryotes. We further showed that mutations in profilin affect actin binding but not other functions such as interactions with poly-L-proline or phosphatidylinositol, abolish its suppressor activity. Consistent with the fact the profilin suppresses its toxicity, expression of Ceg14 but not its non-toxic mutants in yeast affects actin distribution and budding of daughter cells. Although Ceg14 does not detectably interact with profilin, it co-sediments with filamentous actin and inhibits actin polymerization, causing the accumulation of short actin filaments. These results reveal that multiple L. pneumophila effectors target components of the host cytoskeleton. PMID:24286927

  11. 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

  12. 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.

  13. 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. PMID:26228744

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

    PubMed

    Orlovskis, Zigmunds; Hogenhout, Saskia A

    2016-01-01

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

  15. 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

  16. 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

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

    PubMed

    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

  18. 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

  19. Effector-Triggered Immunity Determines Host Genotype-Specific Incompatibility in Legume-Rhizobium Symbiosis.

    PubMed

    Yasuda, Michiko; Miwa, Hiroki; Masuda, Sachiko; Takebayashi, Yumiko; Sakakibara, Hitoshi; Okazaki, Shin

    2016-08-01

    Symbiosis between legumes and rhizobia leads to the formation of N2-fixing root nodules. In soybean, several host genes, referred to as Rj genes, control nodulation. Soybean cultivars carrying the Rj4 gene restrict nodulation by specific rhizobia such as Bradyrhizobium elkanii We previously reported that the restriction of nodulation was caused by B. elkanii possessing a functional type III secretion system (T3SS), which is known for its delivery of virulence factors by pathogenic bacteria. In the present study, we investigated the molecular basis for the T3SS-dependent nodulation restriction in Rj4 soybean. Inoculation tests revealed that soybean cultivar BARC-2 (Rj4/Rj4) restricted nodulation by B. elkanii USDA61, whereas its nearly isogenic line BARC-3 (rj4/rj4) formed nitrogen-fixing nodules with the same strain. Root-hair curling and infection threads were not observed in the roots of BARC-2 inoculated with USDA61, indicating that Rj4 blocked B. elkanii infection in the early stages. Accumulation of H2O2 and salicylic acid (SA) was observed in the roots of BARC-2 inoculated with USDA61. Transcriptome analyses revealed that inoculation of USDA61, but not its T3SS mutant in BARC-2, induced defense-related genes, including those coding for hypersensitive-induced responsive protein, which act in effector-triggered immunity (ETI) in Arabidopsis. These findings suggest that B. elkanii T3SS triggers the SA-mediated ETI-type response in Rj4 soybean, which consequently blocks symbiotic interactions. This study revealed a common molecular mechanism underlying both plant-pathogen and plant-symbiont interactions, and suggests that establishment of a root nodule symbiosis requires the evasion or suppression of plant immune responses triggered by rhizobial effectors. PMID:27373538

  20. Host-Mediated Post-Translational Prenylation of Novel Dot/Icm-Translocated Effectors of Legionella Pneumophila

    PubMed Central

    Price, Christopher T. D.; Jones, Snake C.; Amundson, Karen E.; Kwaik, Yousef Abu

    2010-01-01

    The Dot/Icm type IV translocated Ankyrin B (AnkB) effector of Legionella pneumophila is modified by the host prenylation machinery that anchors it into the outer leaflet of the Legionella-containing vacuole (LCV), which is essential for biological function of the effector in vitro and in vivo. Prenylation involves the covalent linkage of an isoprenoid lipid moiety to a C-terminal CaaX motif in eukaryotic proteins enabling their anchoring into membranes. We show here that the LCV harboring an ankB null mutant is decorated with prenylated proteins in a Dot/Icm-dependent manner, indicating that other LCV membrane-anchored proteins are prenylated. In silico analyses of four sequenced L. pneumophila genomes revealed the presence of eleven other genes that encode proteins with a C-terminal eukaryotic CaaX prenylation motif. Of these eleven designated Prenylated effectors of Legionella (Pel), seven are also found in L. pneumophila AA100. We show that six L. pneumophila AA100 Pel proteins exhibit distinct cellular localization when ectopically expressed in mammalian cells and this is dependent on action of the host prenylation machinery and the conserved cysteine residue of the CaaX motif. Although inhibition of the host prenylation machinery completely blocks intra-vacuolar proliferation of L. pneumophila, it only had a modest effect on intracellular trafficking of the LCV. Five of the Pel proteins are injected into human macrophages by the Dot/Icm type IV translocation system of L. pneumophila. Taken together, the Pel proteins are novel Dot/Icm-translocated effectors of L. pneumophila that are post-translationally modified by the host prenylation machinery, which enables their anchoring into cellular membranes, and the prenylated effectors contribute to evasion of lysosomal fusion by the LCV. PMID:21687755

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

    PubMed

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

    2016-08-01

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

  2. Thermodynamic properties of the effector domains of MARTX toxins suggest their unfolding for translocation across the host membrane.

    PubMed

    Kudryashova, Elena; Heisler, David; Zywiec, Andrew; Kudryashov, Dmitri S

    2014-06-01

    MARTX (multifunctional autoprocessing repeats-in-toxin) family toxins are produced by Vibrio cholerae, Vibrio vulnificus, Aeromonas hydrophila and other Gram-negative bacteria. Effector domains of MARTX toxins cross the cytoplasmic membrane of a host cell through a putative pore formed by the toxin's glycine-rich repeats. The structure of the pore is unknown and the translocation mechanism of the effector domains is poorly understood. We examined the thermodynamic stability of the effector domains of V. cholerae and A. hydrophila MARTX toxins to elucidate the mechanism of their translocation. We found that all but one domain in each toxin are thermodynamically unstable and several acquire a molten globule state near human physiological temperatures. Fusion of the most stable cysteine protease domain to the adjacent effector domain reduces its thermodynamic stability ∼ 1.4-fold (from D G H 2 O 21.8 to 16.1 kJ mol(-1) ). Precipitation of several individual domains due to thermal denaturation is reduced upon their fusion into multi-domain constructs. We speculate that low thermostability of the MARTX effector domains correlates with that of many other membrane-penetrating toxins and implies their unfolding for cell entry. This study extends the list of thermolabile bacterial toxins, suggesting that this quality is essential and could be susceptible for selective targeting of pathogenic toxins. PMID:24724536

  3. Vibrio effector protein, VopQ, forms a lysosomal gated channel that disrupts host ion homeostasis and autophagic flux

    PubMed Central

    Sreelatha, Anju; Bennett, Terry L.; Zheng, Hui; Jiang, Qiu-Xing; Orth, Kim; Starai, Vincent J.

    2013-01-01

    Defects in normal autophagic pathways are implicated in numerous human diseases—such as neurodegenerative diseases, cancer, and cardiomyopathy—highlighting the importance of autophagy and its proper regulation. Herein we show that Vibrio parahaemolyticus uses the type III effector VopQ (Vibrio outer protein Q) to alter autophagic flux by manipulating the partitioning of small molecules and ions in the lysosome. This effector binds to the conserved Vo domain of the vacuolar-type H+-ATPase and causes deacidification of the lysosomes within minutes of entering the host cell. VopQ forms a gated channel ∼18 Å in diameter that facilitates outward flux of ions across lipid bilayers. The electrostatic interactions of this type 3 secretion system effector with target membranes dictate its preference for host vacuolar-type H+-ATPase–containing membranes, indicating that its pore-forming activity is specific and not promiscuous. As seen with other effectors, VopQ is exploiting a eukaryotic mechanism, in this case manipulating lysosomal homeostasis and autophagic flux through transmembrane permeation. PMID:23798441

  4. 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

  5. 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

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

    PubMed

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

    2016-08-01

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

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

    PubMed Central

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

    2011-01-01

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

  8. 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. PMID:22435635

  9. 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...

  10. The role of effectors and host immunity in plant–necrotrophic fungal interactions

    PubMed Central

    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. PMID:25513773

  11. The Salmonella SPI2 Effector SseI Mediates Long-Term Systemic Infection by Modulating Host Cell Migration

    PubMed Central

    Gerke, Christiane; Gopinath, Smita; Peng, Kaitian; Laidlaw, Grace; Chien, Yueh-Hsiu; Jeong, Ha-Won; Li, Zhigang; Brown, Matthew D.; Sacks, David B.; Monack, Denise

    2009-01-01

    Host-adapted strains of Salmonella enterica cause systemic infections and have the ability to persist systemically for long periods of time despite the presence of a robust immune response. Chronically infected hosts are asymptomatic and transmit disease to naïve hosts via fecal shedding of bacteria, thereby serving as a critical reservoir for disease. We show that the bacterial effector protein SseI (also called SrfH), which is translocated into host cells by the Salmonella Pathogenicity Island 2 (SPI2) type III secretion system (T3SS), is required for Salmonella typhimurium to maintain a long-term chronic systemic infection in mice. SseI inhibits normal cell migration of primary macrophages and dendritic cells (DC) in vitro, and such inhibition requires the host factor IQ motif containing GTPase activating protein 1 (IQGAP1), an important regulator of cell migration. SseI binds directly to IQGAP1 and co-localizes with this factor at the cell periphery. The C-terminal domain of SseI is similar to PMT/ToxA, a bacterial toxin that contains a cysteine residue (C1165) that is critical for activity. Mutation of the corresponding residue in SseI (C178A) eliminates SseI function in vitro and in vivo, but not binding to IQGAP1. In addition, infection with wild-type (WT) S. typhimurium suppressed DC migration to the spleen in vivo in an SseI-dependent manner. Correspondingly, examination of spleens from mice infected with WT S. typhimurium revealed fewer DC and CD4+ T lymphocytes compared to mice infected with ΔsseI S. typhimurium. Taken together, our results demonstrate that SseI inhibits normal host cell migration, which ultimately counteracts the ability of the host to clear systemic bacteria. PMID:19956712

  12. Dissection of effector-induced host susceptibility pathways in Stagonospora nodorum blotch of wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The necrotrophic Stagonospora nodorum-wheat interaction is characterized by several pathogen-derived proteinaceous host-selective toxins (SnToxA, SnTox1, SnTox2, SnTox3 and SnTox4) that induce diseases in the host carrying a corresponding dominant susceptibility gene (Tsn1, Snn1, Snn2, Snn3 and Snn4...

  13. Understanding Interactions between Phytopathogenic Phytophthora Effector IpiO and the Host Resistance Protein RB

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Species of phytopathogenic Phytophthora are well known for their ability to cause disease on economically important crops, with almost 100 recognized species targeting close to 300 different hosts. The host resistance protein RB, isolated from wild potato, specifically recognizes the P. infestans Ip...

  14. Propofol Increases Host Susceptibility to Microbial Infection by Reducing Subpopulations of Mature Immune Effector Cells at Sites of Infection

    PubMed Central

    Visvabharathy, Lavanya; Xayarath, Bobbi; Weinberg, Guy; Shilling, Rebecca A.; Freitag, Nancy E.

    2015-01-01

    Anesthetics are known to modulate host immune responses, but separating the variables of surgery from anesthesia when analyzing hospital acquired infections is often difficult. Here, the bacterial pathogen Listeria monocytogenes (Lm) was used to assess the impact of the common anesthetic propofol on host susceptibility to infection. Brief sedation of mice with physiologically relevant concentrations of propofol increased bacterial burdens in target organs by more than 10,000-fold relative to infected control animals. The adverse effects of propofol sedation on immune clearance of Lm persisted after recovery from sedation, as animals given the drug remained susceptible to infection for days following anesthesia. In contrast to propofol, sedation with alternative anesthetics such as ketamine/xylazine or pentobarbital did not increase susceptibility to systemic Lm infection. Propofol altered systemic cytokine and chemokine expression during infection, and prevented effective bacterial clearance by inhibiting the recruitment and/or activity of immune effector cells at sites of infection. Propofol exposure induced a marked reduction in marginal zone macrophages in the spleens of Lm infected mice, resulting in bacterial dissemination into deep tissue. Propofol also significantly increased mouse kidney abscess formation following infection with the common nosocomial pathogen Staphylococcus aureus. Taken together, these data indicate that even brief exposure to propofol severely compromises host resistance to microbial infection for days after recovery from sedation. PMID:26381144

  15. 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

    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

  16. 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

  17. Novel necrotrophic effectors from Stagonospora nodorum and corresponding host sensitivities in winter wheat germplasm in the southeastern United States.

    PubMed

    Crook, A D; Friesen, T L; Liu, Z H; Ojiambo, P S; Cowger, C

    2012-05-01

    Stagonospora nodorum blotch (SNB), caused by the necrotrophic fungus Stagonospora nodorum (teleomorph: Phaeosphaeria nodorum), is among the most common diseases of winter wheat in the United States. New opportunities in resistance breeding have arisen from the recent discovery of several necrotrophic effectors (NEs, also known as host-selective toxins) produced by S. nodorum, along with their corresponding host sensitivity (Snn) genes. Thirty-nine isolates of S. nodorum collected from wheat debris or grain from seven states in the southeastern United States were used to investigate the production of NEs in the region. Twenty-nine cultivars with varying levels of resistance to SNB, representing 10 eastern-U.S. breeding programs, were infiltrated with culture filtrates from the S. nodorum isolates in a randomized complete block design. Three single-NE Pichia pastoris controls, two S. nodorum isolate controls, and six Snn-differential wheat controls were also used. Cultivar-isolate interactions were visually evaluated for sensitivity at 7 days after infiltration. Production of NEs was detected in isolates originating in each sampled state except Maryland. Of the 39 isolates, 17 produced NEs different from those previously characterized in the upper Great Plains region. These novel NEs likely correspond to unidentified Snn genes in Southeastern wheat cultivars, because NEs are thought to arise under selection pressure from genes for resistance to biotrophic pathogens of wheat cultivars that differ by geographic region. Only 3, 0, and 23% of the 39 isolates produced SnToxA, SnTox1, and SnTox3, respectively, by the culture-filtrate test. A Southern dot-blot test showed that 15, 74, and 39% of the isolates carried the genes for those NEs, respectively; those percentages were lower than those found previously in larger international samples. Only two cultivars appeared to contain known Snn genes, although half of the cultivars displayed sensitivity to culture filtrates

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-03-01

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

  20. Compatibility in the Ustilago maydis–Maize Interaction Requires Inhibition of Host Cysteine Proteases by the Fungal Effector Pit2

    PubMed Central

    Mueller, André N.; Ziemann, Sebastian; Treitschke, Steffi; Aßmann, Daniela; Doehlemann, Gunther

    2013-01-01

    The basidiomycete Ustilago maydis causes smut disease in maize, with large plant tumors being formed as the most prominent disease symptoms. During all steps of infection, U. maydis depends on a biotrophic interaction, which requires an efficient suppression of plant immunity. In a previous study, we identified the secreted effector protein Pit2, which is essential for maintenance of biotrophy and induction of tumors. Deletion mutants for pit2 successfully penetrate host cells but elicit various defense responses, which stops further fungal proliferation. We now show that Pit2 functions as an inhibitor of a set of apoplastic maize cysteine proteases, whose activity is directly linked with salicylic-acid-associated plant defenses. Consequently, protease inhibition by Pit2 is required for U. maydis virulence. Sequence comparisons with Pit2 orthologs from related smut fungi identified a conserved sequence motif. Mutation of this sequence caused loss of Pit2 function. Consequently, expression of the mutated protein in U. maydis could not restore virulence of the pit2 deletion mutant, indicating that the protease inhibition by Pit2 is essential for fungal virulence. Moreover, synthetic peptides of the conserved sequence motif showed full activity as protease inhibitor, which identifies this domain as a new, minimal protease inhibitor domain in plant-pathogenic fungi. PMID:23459172

  1. An effector gene hopA1 influences on virulence, host specificity, and lifestyles of Pseudomonas cichorii JBC1.

    PubMed

    Hung, Nguyen Bao; Ramkumar, Gandhimani; Lee, Yong Hoon

    2014-10-01

    Pseudomonas cichorii is a devastating pathogen which infects a wide range of ornamental as well as agricultural crops worldwide. Characterization of virulence genes helps to understand pathogens' infection processes, which may lead to development of resistant crops. For functional validation of novel genes, we re-constructed pUCP18 vector with λ phage red operon and sacB gene (pUCP18_RedS), which simplified conventional marker exchange system. The effector gene hopA1 of P. cichorii JBC1 was marker exchanged with PCR product of kanamycin gene flanked by hopA1 flanking region using pUCP18_RedS. The virulence and internal growth of hopA1 defective mutant (ΔhopA1) in tomato seedlings was significantly reduced compared to wild type (WT) and hopA1 complemented strain (ΔhopA1::phopA1). The analysis on role of hopA1 in host range revealed that P. cichorii was hopA1-dependent to infect cabbage, tomato, soybean, hot pepper, and cucumber, but not melon and eggplant. Despite the similarity in growth pattern, the biofilm formation and swarming motility of ΔhopA1 were significantly reduced compared to WT and ΔhopA1::phopA1. The results of this study indicate that hopA1 plays a significant role not only in virulence and host specificity, but also motility and biofilm formation of P. cichorii which may influence the infection processes. PMID:25127676

  2. Effector triggered immunity

    PubMed Central

    Rajamuthiah, Rajmohan; Mylonakis, Eleftherios

    2014-01-01

    Pathogenic bacteria produce virulence factors called effectors, which are important components of the infection process. Effectors aid in pathogenesis by facilitating bacterial attachment, pathogen entry into or exit from the host cell, immunoevasion, and immunosuppression. Effectors also have the ability to subvert host cellular processes, such as hijacking cytoskeletal machinery or blocking protein translation. However, host cells possess an evolutionarily conserved innate immune response that can sense the pathogen through the activity of its effectors and mount a robust immune response. This “effector triggered immunity” (ETI) was first discovered in plants but recent evidence suggest that the process is also well conserved in metazoans. We will discuss salient points of the mechanism of ETI in metazoans from recent studies done in mammalian cells and invertebrate model hosts. PMID:25513770

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

    PubMed Central

    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.

    2010-01-01

    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: AVR3aKI, which is detected by potato resistance protein R3a, strongly suppresses infestin 1 (INF1)-triggered cell death (ICD), whereas AVR3aEM, 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, AVR3aKI/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 Avr3aKI or Avr3aEM but not the Avr3aKI/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. PMID:20457921

  4. 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. PMID:26077032

  5. An Effector-Targeted Protease Contributes to Defense against Phytophthora infestans and Is under Diversifying Selection in Natural Hosts1[W

    PubMed Central

    Kaschani, Farnusch; Shabab, Mohammed; Bozkurt, Tolga; Shindo, Takayuki; Schornack, Sebastian; Gu, Christian; Ilyas, Muhammad; Win, Joe; Kamoun, Sophien; van der Hoorn, Renier A.L.

    2010-01-01

    Since the leaf apoplast is a primary habitat for many plant pathogens, apoplastic proteins are potent, ancient targets for apoplastic effectors secreted by plant pathogens. So far, however, only a few apoplastic effector targets have been identified and characterized. Here, we discovered that the papain-like cysteine protease C14 is a new common target of EPIC1 and EPIC2B, two apoplastic, cystatin-like proteins secreted by the potato (Solanum tuberosum) late blight pathogen Phytophthora infestans. C14 is a secreted protease of tomato (Solanum lycopersicum) and potato typified by a carboxyl-terminal granulin domain. The EPIC-C14 interaction occurs at a wide pH range and is stronger than the previously described interactions of EPICs with tomato defense proteases PIP1 and RCR3. The selectivity of the EPICs is also different when compared with the AVR2 effector of the fungal tomato pathogen Cladosporium fulvum, which targets PIP1 and RCR3, and only at apoplastic pH. Importantly, silencing of C14 increased susceptibility to P. infestans, demonstrating that this protease plays a role in pathogen defense. Although C14 is under conservative selection in tomato, it is under diversifying selection in wild potato species (Solanum demissum, Solanum verrucosum, and Solanum stoliniferum) that are the natural hosts of P. infestans. These data reveal a novel effector target in the apoplast that contributes to immunity and is under diversifying selection, but only in the natural host of the pathogen. PMID:20940351

  6. 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

  7. Transient activation of mucosal effector immune responses by resident intestinal bacteria in normal hosts is regulated by interleukin-10 signalling.

    PubMed

    Wu, Cong; Sartor, R Balfour; Huang, Kehe; Tonkonogy, Susan L

    2016-07-01

    Interleukin-10 (IL-10) is a key regulator of mucosal homeostasis. In the current study we investigated the early events after monoassociating germ-free (GF) wild-type (WT) mice with an Escherichia coli strain that we isolated previously from the caecal contents of a normal mouse housed under specific pathogen-free conditions. Our results show that interferon-γ (IFN-γ) secreted by mesenteric lymph node (MLN) cells from both IL-10 deficient mice and WT mice, stimulated ex vivo with E. coli lysate, was dramatically higher at day 4 after monoassociation compared with IFN-γ secreted by cells from GF mice without E. coli colonization. Production of IFN-γ rapidly and progressively declined after colonization of WT but not IL-10-deficient mice. The E. coli lysate-stimulated WT MLN cells also produced IL-10 that peaked at day 4 and subsequently declined, but not as precipitously as IFN-γ. WT cells that express CD4, CD8 and NKp46 produced IFN-γ; WT CD4-positive cells and B cells produced IL-10. Recombinant IL-10 added to E. coli-stimulated MLN cell cultures inhibited IFN-γ secretion in a dose-dependent fashion. MLN cells from WT mice treated in vivo with neutralizing anti-IL-10 receptor antibody produced more IFN-γ compared with MLN cells from isotype control antibody-treated mice. These findings show that a resident E. coli that induces chronic colitis in monoassociated IL-10-deficient mice rapidly but transiently activates the effector immune system in normal hosts, in parallel with induction of protective IL-10 produced by B cells and CD4(+) cells that subsequently suppresses this response to mediate mucosal homeostasis. PMID:27147411

  8. Overexpression of a novel biotrophy-specific Colletotrichum truncatum effector, CtNUDIX, in hemibiotrophic fungal phytopathogens causes incompatibility with their host plants.

    PubMed

    Bhadauria, Vijai; Banniza, Sabine; Vandenberg, Albert; Selvaraj, Gopalan; Wei, Yangdou

    2013-01-01

    The hemibiotrophic fungus Colletotrichum truncatum causes anthracnose disease on lentils and a few other grain legumes. It shows initial symptomless intracellular growth, where colonized host cells remain viable (biotrophy), and then switches to necrotrophic growth, killing the colonized host plant tissues. Here, we report a novel effector gene, CtNUDIX, from C. truncatum that is exclusively expressed during the late biotrophic phase (before the switch to necrotrophy) and elicits a hypersensitive response (HR)-like cell death in tobacco leaves transiently expressing the effector. CtNUDIX homologs, which contain a signal peptide and a Nudix hydrolase domain, may be unique to hemibiotrophic fungal and fungus-like plant pathogens. CtNUDIX lacking a signal peptide or a Nudix motif failed to induce cell death in tobacco. Expression of CtNUDIX:eGFP in tobacco suggested that the fusion protein might act on the host cell plasma membrane. Overexpression of CtNUDIX in C. truncatum and the rice blast pathogen, Magnaporthe oryzae, resulted in incompatibility with the hosts lentil and barley, respectively, by causing an HR-like response in infected host cells associated with the biotrophic invasive hyphae. These results suggest that C. truncatum and possibly M. oryzae elicit cell death to signal the transition from biotrophy to necrotrophy. PMID:22962277

  9. SseK3 Is a Salmonella Effector That Binds TRIM32 and Modulates the Host's NF-κB Signalling Activity.

    PubMed

    Yang, Zhe; Soderholm, Amelia; Lung, Tania Wong Fok; Giogha, Cristina; Hill, Michelle M; Brown, Nathaniel F; Hartland, Elizabeth; Teasdale, Rohan D

    2015-01-01

    Salmonella Typhimurium employs an array of type III secretion system effectors that facilitate intracellular survival and replication during infection. The Salmonella effector SseK3 was originally identified due to amino acid sequence similarity with NleB; an effector secreted by EPEC/EHEC that possesses N-acetylglucoasmine (GlcNAc) transferase activity and modifies death domain containing proteins to block extrinsic apoptosis. In this study, immunoprecipitation of SseK3 defined a novel molecular interaction between SseK3 and the host protein, TRIM32, an E3 ubiquitin ligase. The conserved DxD motif within SseK3, which is essential for the GlcNAc transferase activity of NleB, was required for TRIM32 binding and for the capacity of SseK3 to suppress TNF-stimulated activation of NF-κB pathway. However, we did not detect GlcNAc modification of TRIM32 by SseK3, nor did the SseK3-TRIM32 interaction impact on TRIM32 ubiquitination that is associated with its activation. In addition, lack of sseK3 in Salmonella had no effect on production of the NF-κB dependent cytokine, IL-8, in HeLa cells even though TRIM32 knockdown suppressed TNF-induced NF-κB activity. Ectopically expressed SseK3 partially co-localises with TRIM32 at the trans-Golgi network, but SseK3 is not recruited to Salmonella induced vacuoles or Salmonella induced filaments during Salmonella infection. Our study has identified a novel effector-host protein interaction and suggests that SseK3 may influence NF-κB activity. However, the lack of GlcNAc modification of TRIM32 suggests that SseK3 has further, as yet unidentified, host targets. PMID:26394407

  10. Comparison of gene activation by two TAL effectors from Xanthomonas axonopodis pv. manihotis reveals candidate host susceptibility genes in cassava.

    PubMed

    Cohn, Megan; Morbitzer, Robert; Lahaye, Thomas; Staskawicz, Brian J

    2016-08-01

    Xanthomonas axonopodis pv. manihotis (Xam) employs transcription activator-like (TAL) effectors to promote bacterial growth and symptom formation during infection of cassava. TAL effectors are secreted via the bacterial type III secretion system into plant cells, where they are directed to the nucleus, bind DNA in plant promoters and activate the expression of downstream genes. The DNA-binding activity of TAL effectors is carried out by a central domain which contains a series of repeat variable diresidues (RVDs) that dictate the sequence of bound nucleotides. TAL14Xam668 promotes virulence in Xam strain Xam668 and has been shown to activate multiple cassava genes. In this study, we used RNA sequencing to identify the full target repertoire of TAL14Xam668 in cassava, which includes over 50 genes. A subset of highly up-regulated genes was tested for activation by TAL14CIO151 from Xam strain CIO151. Although TAL14CIO151 and TAL14Xam668 differ by only a single RVD, they display differential activation of gene targets. TAL14CIO151 complements the TAL14Xam668 mutant defect, implying that shared target genes are important for TAL14Xam668 -mediated disease susceptibility. Complementation with closely related TAL effectors is a novel approach to the narrowing down of biologically relevant susceptibility genes of TAL effectors with multiple targets. This study provides an example of how TAL effector target activation by two strains within a single species of Xanthomonas can be dramatically affected by a small change in RVD-nucleotide affinity at a single site, and reflects the parameters of RVD-nucleotide interaction determined using designer TAL effectors in transient systems. PMID:26575863

  11. 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. PMID:25938194

  12. Dynamic regulation of effector IFN-γ-producing and IL-17-producing T cell subsets in the development of acute graft-versus-host disease.

    PubMed

    Zhao, Kai; Ruan, Suhong; Yin, Lingling; Zhao, Dongmei; Chen, Chong; Pan, Bin; Zeng, Lingyu; Li, Zhenyu; Xu, Kailin

    2016-02-01

    Graft-versus-host disease (GVHD) as the predominant complication of allogeneic hematopoietic stem cell transplantation remains to be fully understood. It is known that the cytokines produced by allogeneic reactive effector CD4+ and CD8+ T cells are involved in GVHD. However, the regulation and coordination of IFN-γ-producing and IL-17-producing effector T cells remain unclear. The present study aimed to investigate the dynamic changes of alloantigen-specific effector CD4+ T and CD8+ T cell subsets by flow cytometry, which produce inflammatory cytokines involved in the multistep GVHD pathogenesis progress. The results demonstrated that IL-17-producing CD8+ T (Tc17) cells and IFN-γ+CD8+ T (Tc1) cells were detected in the early stage of GVHD. The differentiation of CD4+ T cells into Th1 cell (IFN-γ+CD4+ T) and Th17 (IL-17+CD4+ T) cells was later than that of the Tc1 and Tc17 cells. The effector CD4+ T and CD8+ T cell subsets either became exhausted or became memory cells, exhibiting a CD62L-CD44+ phenotype following marked expansion during GVHD. Furthermore, T cell-associated type I (IL-2 and IFN-γ) and type II (IL-4 and IL-10) classical cytokines exhibited coordinated dynamic regulation. It was concluded that the differentiation of cytokine-producing Tc1 and Tc17 cells may be the key step in the initiation of GVHD, whereas CD4+ effector Th1 and Th17 cells are considered to be pathophysiological factors leading to the continuous aggravation of GVHD. PMID:26647759

  13. A Host KH RNA-Binding Protein Is a Susceptibility Factor Targeted by an RXLR Effector to Promote Late Blight Disease☆

    PubMed Central

    Wang, Xiaodan; Boevink, Petra; McLellan, Hazel; Armstrong, Miles; Bukharova, Tatyana; Qin, Zhiwei; Birch, Paul R.J.

    2015-01-01

    Plant pathogens deliver effector proteins that alter host processes to create an environment conducive to colonization. Attention has focused on identifying the targets of effectors and how their manipulation facilitates disease. RXLR effector Pi04089 from the potato blight pathogen Phytophthora infestans accumulates in the host nucleus and enhances colonization when transiently expressed in planta. Its nuclear localization is required for enhanced P. infestans colonization. Pi04089 interacts in yeast and in planta with a putative potato K-homology (KH) RNA-binding protein, StKRBP1. Co-localization of Pi04089 and StKRBP1, and bimolecular fluorescence complementation between them, indicate they associate at nuclear speckles. StKRBP1 protein levels increased when it was co-expressed with Pi04089. Indeed, such accumulation of StKRBP1 was observed also on the first day of leaf colonization by the pathogen. Remarkably, overexpression of StKRBP1 significantly enhances P. infestans infection. Mutation of the nucleotide-binding motif GxxG to GDDG in all three KH domains of StKRBP1 abolishes its interaction with Pi04089, its localization to nuclear speckles, and its increased accumulation when co-expressed with the effector. Moreover, the mutant StKRBP1 protein no longer enhances leaf colonization by P. infestans, implying that nucleotide binding is likely required for this activity. We thus argue that StKRBP1 can be regarded as a susceptibility factor, as its activity is beneficial to the pathogen. PMID:25936676

  14. Identification of a host 14-3-3 Protein that Interacts with Xanthomonas effector AvrRxv

    Technology Transfer Automated Retrieval System (TEKTRAN)

    AvrRxv is a member of a family of pathogen effectors from both plant and mammalian pathogens. Using a yeast two hybrid screen, we identified a 14-3-3 protein from tomato that interacts with AvrRxv called AvrRxv Interactor 1 (ARI1). The interaction was confirmed in vitro with affinity chromatograph...

  15. 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...

  16. 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. PMID:26700936

  17. 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

  18. The cyst nematode effector protein 10A07 targets and recruits host posttranslational machinery to mediate its nuclear trafficking and to promote parasitism in Arabidopsis.

    PubMed

    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

    2015-03-01

    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

  19. 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

  20. 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. PMID:26723843

  1. A «Repertoire for Repertoire» Hypothesis: Repertoires of Type Three Effectors are Candidate Determinants of Host Specificity in Xanthomonas

    PubMed Central

    Hajri, Ahmed; Brin, Chrystelle; Hunault, Gilles; Lardeux, Frédéric; Lemaire, Christophe; Manceau, Charles

    2009-01-01

    Background The genetic basis of host specificity for animal and plant pathogenic bacteria remains poorly understood. For plant pathogenic bacteria, host range is restricted to one or a few host plant species reflecting a tight adaptation to specific hosts. Methodology/Principal Findings Two hypotheses can be formulated to explain host specificity: either it can be explained by the phylogenetic position of the strains, or by the association of virulence genes enabling a pathological convergence of phylogenically distant strains. In this latter hypothesis, host specificity would result from the interaction between repertoires of bacterial virulence genes and repertoires of genes involved in host defences. To challenge these two hypotheses, we selected 132 Xanthomonas axonopodis strains representative of 18 different pathovars which display different host range. First, the phylogenetic position of each strain was determined by sequencing the housekeeping gene rpoD. This study showed that many pathovars of Xanthomonas axonopodis are polyphyletic. Second, we investigated the distribution of 35 type III effector genes (T3Es) in these strains by both PCR and hybridization methods. Indeed, for pathogenic bacteria T3Es were shown to trigger and to subvert host defences. Our study revealed that T3E repertoires comprise core and variable gene suites that likely have distinct roles in pathogenicity and different evolutionary histories. Our results showed a correspondence between composition of T3E repertoires and pathovars of Xanthomonas axonopodis. For polyphyletic pathovars, this suggests that T3E genes might explain a pathological convergence of phylogenetically distant strains. We also identified several DNA rearrangements within T3E genes, some of which correlate with host specificity of strains. Conclusions/Significance These data provide insight into the potential role played by T3E genes for pathogenic bacteria and support a “repertoire for repertoire” hypothesis

  2. 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.

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

    PubMed

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

    2008-09-01

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

  4. Live Cell Imaging Reveals Novel Functions of Salmonella enterica SPI2-T3SS Effector Proteins in Remodeling of the Host Cell Endosomal System

    PubMed Central

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

    2014-01-01

    Intracellular Salmonella enterica induce a massive remodeling of the endosomal system in infected host cells. One dramatic consequence of this interference is the induction of various extensive tubular aggregations of membrane vesicles, and tubules positive for late endosomal/lysosomal markers are referred to as Salmonella-induced filaments or SIF. SIF are highly dynamic in nature with extension and collapse velocities of 0.4–0.5 µm x sec−1. The induction of SIF depends on the function of the Salmonella Pathogenicity Island 2 (SPI2) encoded type III secretion system (T3SS) and a subset of effector proteins. In this study, we applied live cell imaging and electron microscopy to analyze the role of individual effector proteins in SIF morphology and dynamic properties of SIF. SIF in cells infected with sifB, sseJ, sseK1, sseK2, sseI, sseL, sspH1, sspH2, slrP, steC, gogB or pipB mutant strains showed a morphology and dynamics comparable to SIF induced by WT Salmonella. SIF were absent in cells infected with the sifA-deficient strain and live cell analyses allowed tracking of the loss of the SCV membrane of intracellular sifA Salmonella. In contrast to analyses in fixed cells, in living host cells SIF induced by sseF- or sseG-deficient strains were not discontinuous, but rather continuous and thinner in diameter. A very dramatic phenotype was observed for the pipB2-deficient strain that induced very bulky, non-dynamic aggregations of membrane vesicles. Our study underlines the requirement of the study of Salmonella-host interaction in living systems and reveals new phenotypes due to the intracellular activities of Salmonella. PMID:25522146

  5. PLZF confers effector functions to donor T cells that preserve graft-versus-tumor effects while attenuating graft-versus-host-disease

    PubMed Central

    Ghosh, Arnab; Holland, Amanda M.; Dogan, Yildirim; Yim, Nury L.; Rao, Uttam K.; Young, Lauren F.; West, Mallory L.; Singer, Natalie V.; Lee, Hae; Na, Il-Kang; Tsai, Jennifer J.; Jenq, Robert R.; Penack, Olaf; Hanash, Alan M.; Lezcano, Cecilia; Murphy, George; Liu, Chen; Sadelain, Michel; Sauer, Martin G.; Sant’Angelo, Derek; van den Brink, Marcel R.M.

    2013-01-01

    Efforts to limit graft-versus-host disease (GVHD) mediated by alloreactive donor T cells after allogeneic bone marrow transplantation (allo-BMT) are limited by a concomitant decrease in graft-versus-tumor (GVT) activity and increased possibilities of tumor relapse. Using a novel approach, we adoptively transferred conventional T cells expressing the transcription factor promyelocytic leukemia zinc finger (PLZF), which confers effector properties resembling invariant natural killer T cells (iNKT cells), such as copious production of cytokines under suboptimal stimulation. PLZF expression in T cell allografts attenuates expansion of alloreactive T cells, leading to lower GVHD. Intact alloreactivity-driven antitumor cytokine responses result in preserved GVT effects leading to improved survival. Our findings suggest that therapy with PLZF-overexpressing T cells would result in overall improved outcomes due to less GVHD and intact GVT effects. PMID:23733752

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

    PubMed

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

    2012-02-01

    The type III effector HsvG of the gall-forming Pantoea agglomerans pv. gypsophilae is a DNA-binding protein that is imported to the host nucleus and involved in host specificity. The DNA-binding region of HsvG was delineated to 266 amino acids located within a secondary structure region near the N-terminus of the protein but did not display any homology to canonical DNA-binding motifs. A binding site selection procedure was used to isolate a target gene of HsvG, named HSVGT, in Gypsophila paniculata. HSVGT is a predicted acidic protein of the DnaJ family with 244 amino acids. It harbors characteristic conserved motifs of a eukaryotic transcription factor, including a bipartite nuclear localization signal, zinc finger, and leucine zipper DNA-binding motifs. Quantitative real-time polymerase chain reaction analysis demonstrated that HSVGT transcription is specifically induced in planta within 2 h after inoculation with the wild-type P. agglomerans pv. gypsophilae compared with the hsvG mutant. Induction of HSVGT reached a peak of sixfold at 4 h after inoculation and progressively declined thereafter. Gel-shift assay demonstrated that HsvG binds to the HSVGT promoter, indicating that HSVGT is a direct target of HsvG. Our results support the hypothesis that HsvG functions as a transcription factor in gypsophila. PMID:21995766

  7. Hammondia hammondi Harbors Functional Orthologs of the Host-Modulating Effectors GRA15 and ROP16 but Is Distinguished from Toxoplasma gondii by a Unique Transcriptional Profile

    PubMed Central

    Walzer, Katelyn A.; Wier, Gregory M.; Dam, Rachel A.; Srinivasan, Ananth R.; Borges, Adair L.; English, Elizabeth D.; Herrmann, Daland C.; Schares, Gereon; Dubey, Jitender P.

    2014-01-01

    Toxoplasma gondii and its nearest extant relative, Hammondia hammondi, are phenotypically distinct despite their remarkable similarity in gene content, synteny, and functionality. To begin to identify genetic differences that might drive distinct infection phenotypes of T. gondii and H. hammondi, in the present study we (i) determined whether two known host-interacting proteins, dense granule protein 15 (GRA15) and rhoptry protein 16 (ROP16), were functionally conserved in H. hammondi and (ii) performed the first comparative transcriptional analysis of H. hammondi and T. gondii sporulated oocysts. We found that GRA15 and ROP16 from H. hammondi (HhGRA15 and HhROP16) modulate the host NF-κB and STAT6 pathways, respectively, when expressed heterologously in T. gondii. We also found the transcriptomes of H. hammondi and T. gondii to be highly distinct. Consistent with the spontaneous conversion of H. hammondi tachyzoites into bradyzoites both in vitro and in vivo, H. hammondi high-abundance transcripts are enriched for genes that are of greater abundance in T. gondii bradyzoites. We also identified genes that are of high transcript abundance in H. hammondi but are poorly expressed in multiple T. gondii life stages, suggesting that these genes are uniquely expressed in H. hammondi. Taken together, these data confirm the functional conservation of known T. gondii virulence effectors in H. hammondi and point to transcriptional differences as a potential source of the phenotypic differences between these species. PMID:25280815

  8. 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 Arabidopsis.

    PubMed

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

    2015-09-01

    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

  9. Lactobacilli as effectors of host functions: no influence on the activities of enzymes in enterocytes of mice.

    PubMed Central

    Whitt, D D; Savage, D C

    1987-01-01

    Preparations of lactobacilli are often used as dietary supplements to improve the growth and efficiency in utilizing food of animals of commercial value. We tested in an experimental model whether the effects of lactobacilli on growth of and food utilization by animals may be due to alteration of the activities of absorptive enzymes in the small bowel. Germfree mice housed in isolators under tightly controlled conditions were monoassociated with one of four strains of indigenous Lactobacillus spp. From 1 to 5 weeks later, the activity of alkaline phosphatase was assayed in homogenates of segments of the upper small intestines of the associated animals and of matched germfree controls. The specific activity of the enzyme was the same in the mice in the two groups. In other experiments, epithelial cells were isolated from the upper small intestines of mice associated with eight Lactobacillus strains (octa-associated) and from those of matched germfree mice and assayed for alkaline phosphatase, phosphodiesterase, and thymidine kinase activities. The epithelial cells were harvested sequentially from the tips of the villi toward the crypts of Lieberkühn of the intestines. In all preparations, mice of both types yielded an equivalent mass (wet weight) of cells. The protein content of the cells reflected the mass. The activities of the microvillous membrane enzymes alkaline phosphatase and phosphodiesterase and the cytosol enzyme thymidine kinase were the same whether or not the animals contained the bacteria. Therefore, any effects on animal growth and food utilization observed when lactobacilli are used as dietary supplements may not be due to a direct alteration by the bacteria of the absorptive enzymes of the host animal.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:3032101

  10. 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

  11. 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. PMID:26680733

  12. In planta processing and glycosylation of a nematode CLAVATA3/ENDOSPERM SURROUNDING REGION-like effector and its interaction with a host CLAVATA2-like receptor to promote parasitism.

    PubMed

    Chen, Shiyan; Lang, Ping; Chronis, Demosthenis; Zhang, Sheng; De Jong, Walter S; Mitchum, Melissa G; Wang, Xiaohong

    2015-01-01

    Like other biotrophic plant pathogens, plant-parasitic nematodes secrete effector proteins into host cells to facilitate infection. Effector proteins that mimic plant CLAVATA3/ENDOSPERM SURROUNDING REGION-related (CLE) proteins have been identified in several cyst nematodes, including the potato cyst nematode (PCN); however, the mechanistic details of this cross-kingdom mimicry are poorly understood. Plant CLEs are posttranslationally modified and proteolytically processed to function as bioactive ligands critical to various aspects of plant development. Using ectopic expression coupled with nanoliquid chromatography-tandem mass spectrometry analysis, we show that the in planta mature form of proGrCLE1, a multidomain CLE effector secreted by PCN during infection, is a 12-amino acid arabinosylated glycopeptide (named GrCLE1-1Hyp4,7g) with striking structural similarity to mature plant CLE peptides. This glycopeptide is more resistant to hydrolytic degradation and binds with higher affinity to a CLAVATA2-like receptor (StCLV2) from potato (Solanum tuberosum) than its nonglycosylated forms. We further show that StCLV2 is highly up-regulated at nematode infection sites and that transgenic potatoes with reduced StCLV2 expression are less susceptible to PCN infection, indicating that interference of the CLV2-mediated signaling pathway confers nematode resistance in crop plants. These results strongly suggest that phytonematodes have evolved to utilize host cellular posttranslational modification and processing machinery for the activation of CLE effectors following secretion into plant cells and highlight the significance of arabinosylation in regulating nematode CLE effector activity. Our finding also provides evidence that multidomain CLEs are modified and processed similarly to single-domain CLEs, adding new insight into CLE maturation in plants. PMID:25416475

  13. A mutant with aberrant extracellular LcrV-YscF interactions fails to form pores and translocate Yop effector proteins but retains the ability to trigger Yop secretion in response to host cell contact.

    PubMed

    Harmon, Dana E; Murphy, Julia L; Davis, Alison J; Mecsas, Joan

    2013-05-01

    The plasmid-encoded type three secretion system (TTSS) of Yersinia spp. is responsible for the delivery of effector proteins into cells of the innate immune system, where these effectors disrupt the target cells' activity. Successful translocation of effectors into mammalian cells requires Yersinia to both insert a translocon into the host cell membrane and sense contact with host cells. To probe the events necessary for translocation, we investigated protein-protein interactions among TTSS components of the needle-translocon complex using a chemical cross-linking-based approach. We detected extracellular protein complexes containing YscF, LcrV, and YopD that were dependent upon needle formation. The formation of these complexes was evaluated in a secretion-competent but translocation-defective mutant, the YscFD28AD46A strain (expressing YscF with the mutations D28A and D46A). We found that one of the YscF and most of the LcrV and YopD cross-linked complexes were nearly absent in this mutant. Furthermore, the YscFD28AD46A strain did not support YopB insertion into mammalian membranes, supporting the idea that the LcrV tip complex is required for YopB insertion and translocon formation. However, the YscFD28AD46A strain did secrete Yops in the presence of host cells, indicating that a translocation-competent tip complex is not required to sense contact with host cells to trigger Yop secretion. In conclusion, in the absence of cross-linkable LcrV-YscF interactions, translocon insertion is abolished, but Yersinia still retains the ability to sense cell contact. PMID:23475976

  14. 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

  15. Effector Glycosyltransferases in Legionella

    PubMed Central

    Belyi, Yury; Jank, Thomas; Aktories, Klaus

    2011-01-01

    Legionella causes severe pneumonia in humans. The pathogen produces an array of effectors, which interfere with host cell functions. Among them are the glucosyltransferases Lgt1, Lgt2 and Lgt3 from L. pneumophila. Lgt1 and Lgt2 are produced predominately in the post-exponential phase of bacterial growth, while synthesis of Lgt3 is induced mainly in the lag-phase before intracellular replication of bacteria starts. Lgt glucosyltransferases are structurally similar to clostridial glucosylating toxins. The enzymes use UDP–glucose as a donor substrate and modify eukaryotic elongation factor eEF1A at serine-53. This modification results in inhibition of protein synthesis and death of target cells.In addition to Lgts, Legionella genomes disclose several genes, coding for effector proteins likely to possess glycosyltransferase activities, including SetA (subversion of eukaryotic vesicle trafficking A), which influences vesicular trafficking in the yeast model system and displays tropism for late endosomal/lysosomal compartments of mammalian cells. This review mainly discusses recent results on the structure–function relationship of Lgt glucosyltransferases. PMID:21833323

  16. 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

  17. 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 Central

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

    2016-01-01

    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. PMID:27126515

  18. 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. PMID:27126515

  19. 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 ...

  20. Evolution of Linked Avirulence Effectors in Leptosphaeria maculans Is Affected by Genomic Environment and Exposure to Resistance Genes in Host Plants

    PubMed Central

    Van de Wouw, Angela P.; Cozijnsen, Anton J.; Hane, James K.; Brunner, Patrick C.; McDonald, Bruce A.; Oliver, Richard P.; Howlett, Barbara J.

    2010-01-01

    Brassica napus (canola) cultivars and isolates of the blackleg fungus, Leptosphaeria maculans interact in a ‘gene for gene’ manner whereby plant resistance (R) genes are complementary to pathogen avirulence (Avr) genes. Avirulence genes encode proteins that belong to a class of pathogen molecules known as effectors, which includes small secreted proteins that play a role in disease. In Australia in 2003 canola cultivars with the Rlm1 resistance gene suffered a breakdown of disease resistance, resulting in severe yield losses. This was associated with a large increase in the frequency of virulence alleles of the complementary avirulence gene, AvrLm1, in fungal populations. Surprisingly, the frequency of virulence alleles of AvrLm6 (complementary to Rlm6) also increased dramatically, even though the cultivars did not contain Rlm6. In the L. maculans genome, AvrLm1 and AvrLm6 are linked along with five other genes in a region interspersed with transposable elements that have been degenerated by Repeat-Induced Point (RIP) mutations. Analyses of 295 Australian isolates showed deletions, RIP mutations and/or non-RIP derived amino acid substitutions in the predicted proteins encoded by these seven genes. The degree of RIP mutations within single copy sequences in this region was proportional to their proximity to the degenerated transposable elements. The RIP alleles were monophyletic and were present only in isolates collected after resistance conferred by Rlm1 broke down, whereas deletion alleles belonged to several polyphyletic lineages and were present before and after the resistance breakdown. Thus, genomic environment and exposure to resistance genes in B. napus has affected the evolution of these linked avirulence genes in L. maculans. PMID:21079787

  1. 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. PMID:26270241

  2. 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

  3. Transgenic expression of Erwinia amylovora effectors eopB1 and hopCEa in apple

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Erwinia amylovora (Ea), the causal agent of fire blight, uses a type three secretion system (TTSS) to deliver effector proteins into plant host cells. Once inside the host cell, these effector proteins are thought to be involved with suppressing host defense responses, redirecting normal host metab...

  4. 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...

  5. TGF-β-induced CD4+Foxp3+ T cells attenuate acute graft-versus-host disease by suppressing expansion and killing of effector CD8+ cells.

    PubMed

    Gu, Jian; Lu, Ling; Chen, Maogen; Xu, Lili; Lan, Qin; Li, Qiang; Liu, Zhongmin; Chen, Guihua; Wang, Ping; Wang, Xuehao; Brand, David; Olsen, Nancy; Zheng, Song Guo

    2014-10-01

    The use of TGF-β-induced CD4(+)Foxp3(+) T cells (induced regulatory T cells [iTregs]) is an important prevention and treatment strategy in autoimmune diseases and other disorders. However, the potential use of iTregs as a treatment modality for acute graft-versus-host disease (aGVHD) has not been realized because they may be unstable and less suppressive in this disease. We restudied the ability of iTregs to prevent and treat aGVHD in two mouse models. Our results showed that, as long as an appropriate iTreg-generation protocol is used, these iTregs consistently displayed a potent ability to control aGVHD development and reduce mortality in the aGVHD animal models. iTreg infusion markedly suppressed the engraftment of donor CD8(+) cells and CD4(+) cells, the expression of granzyme A and B, the cytotoxic effect of donor CD8(+) cells, and the production of T cell cytokines in aGVHD. Therefore, we conclude that as long as the correct methods for generating iTregs are used, they can prevent and even treat aGVHD. PMID:25156367

  6. Nematode effector proteins: an emerging paradigm of parasitism

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  7. Functional Analysis of Hyaloperonospora arabidopsidis RXLR Effectors

    PubMed Central

    Pel, Michiel J. C.; Wintermans, Paul C. A.; Cabral, Adriana; Robroek, Bjorn J. M.; Seidl, Michael F.; Bautor, Jaqueline; Parker, Jane E.; Van den Ackerveken, Guido; Pieterse, Corné M. J.

    2014-01-01

    The biotrophic plant pathogen Hyaloperonospora arabidopsidis produces a set of putative effector proteins that contain the conserved RXLR motif. For most of these RXLR proteins the role during infection is unknown. Thirteen RXLR proteins from H. arabidopsidis strain Waco9 were analyzed for sequence similarities and tested for a role in virulence. The thirteen RXLR proteins displayed conserved N-termini and this N-terminal conservation was also found in the 134 predicted RXLR genes from the genome of H. arabidopsidis strain Emoy2. To investigate the effects of single RXLR effector proteins on plant defense responses, thirteen H. arabidopsidis Waco9 RXLR genes were expressed in Arabidopsis thaliana. Subsequently, these plants were screened for altered susceptibility to the oomycetes H. arabidopsidis and Phytophthora capsici, and the bacterial pathogen Pseudomonas syringae. Additionally, the effect of the RXLR proteins on flg22-triggered basal immune responses was assessed. Multifactorial analysis of results collated from all experiments revealed that, except for RXLR20, all RXLR effector proteins tested affected plant immunity. For RXLR9 this was confirmed using a P. syringae ΔCEL-mediated effector delivery system. Together, the results show that many H. arabidopsidis RXLR effectors have small effects on the plant immune response, suggesting that suppression of host immunity by this biotrophic pathogen is likely to be caused by the combined actions of effectors. PMID:25375163

  8. 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

  9. Bioinformatic analysis of expression data to identify effector candidates.

    PubMed

    Reid, Adam J; Jones, John T

    2014-01-01

    Pathogens produce effectors that manipulate the host to the benefit of the pathogen. These effectors are often secreted proteins that are upregulated during the early phases of infection. These properties can be used to identify candidate effectors from genomes and transcriptomes of pathogens. Here we describe commonly used bioinformatic approaches that (1) allow identification of genes encoding predicted secreted proteins within a genome and (2) allow the identification of genes encoding predicted secreted proteins that are upregulated at important stages of the life cycle. Other approaches for bioinformatic identification of effector candidates, including OrthoMCL analysis to identify expanded gene families, are also described. PMID:24643549

  10. 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.

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

    PubMed

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

    2015-12-01

    Fungal plant pathogens, such as Zymoseptoria tritici (formerly known as Mycosphaerella graminicola), secrete repertoires of effectors to facilitate infection or trigger host defence mechanisms. The discovery and functional characterization of effectors provides valuable knowledge that can contribute to the design of new and effective disease management strategies. Here, we combined bioinformatics approaches with expression profiling during pathogenesis to identify candidate effectors of Z. tritici. In addition, a genetic approach was conducted to map quantitative trait loci (QTLs) carrying putative effectors, enabling the validation of both complementary strategies for effector discovery. In planta expression profiling revealed that candidate effectors were up-regulated in successive waves corresponding to consecutive stages of pathogenesis, contrary to candidates identified by QTL mapping that were, overall, expressed at low levels. Functional analyses of two top candidate effectors (SSP15 and SSP18) showed their dispensability for Z. tritici pathogenesis. These analyses reveal that generally adopted criteria, such as protein size, cysteine residues and expression during pathogenesis, may preclude an unbiased effector discovery. Indeed, genetic mapping of genomic regions involved in specificity render alternative effector candidates that do not match the aforementioned criteria, but should nevertheless be considered as promising new leads for effectors that are crucial for the Z. tritici-wheat pathosystem. PMID:25727413

  12. 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...

  13. TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction

    PubMed Central

    Doyle, Erin L.; Booher, Nicholas J.; Standage, Daniel S.; Voytas, Daniel F.; Brendel, Volker P.; VanDyk, John K.; Bogdanove, Adam J.

    2012-01-01

    Transcription activator-like (TAL) effectors are repeat-containing proteins used by plant pathogenic bacteria to manipulate host gene expression. Repeats are polymorphic and individually specify single nucleotides in the DNA target, with some degeneracy. A TAL effector-nucleotide binding code that links repeat type to specified nucleotide enables prediction of genomic binding sites for TAL effectors and customization of TAL effectors for use in DNA targeting, in particular as custom transcription factors for engineered gene regulation and as site-specific nucleases for genome editing. We have developed a suite of web-based tools called TAL Effector-Nucleotide Targeter 2.0 (TALE-NT 2.0; https://boglab.plp.iastate.edu/) that enables design of custom TAL effector repeat arrays for desired targets and prediction of TAL effector binding sites, ranked by likelihood, in a genome, promoterome or other sequence of interest. Search parameters can be set by the user to work with any TAL effector or TAL effector nuclease architecture. Applications range from designing highly specific DNA targeting tools and identifying potential off-target sites to predicting effector targets important in plant disease. PMID:22693217

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

    PubMed

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

    2016-08-01

    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

  15. 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) ...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  17. Antibodies as effectors.

    PubMed

    Corbeil, L B

    2002-09-10

    Antibodies are critical in protection against extracellular microbial pathogens. Although antibodies also play a role in transplant/tumor rejection and in autoimmune disease, this paper focuses on defense against bovine infections. Effector mechanisms of different bovine isotypes, subisotypes and allotypes are discussed. The importance of antigen specificity is also stressed. PMID:12072231

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

    PubMed

    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

  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. pH sensing by intracellular Salmonella induces effector translocation.

    PubMed

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

    2010-05-21

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

  2. 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.

  3. Robotic end effector

    DOEpatents

    Minichan, Richard L.

    1993-01-01

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

  4. Robotic end effector

    DOEpatents

    Minichan, R.L.

    1993-10-05

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

  5. Whole-Genome Sequencing of Erwinia amylovora Strains from Mexico Detects Single Nucleotide Polymorphisms in rpsL Conferring Streptomycin Resistance and in the avrRpt2 Effector Altering Host Interactions.

    PubMed

    Smits, Theo H M; Guerrero-Prieto, Víctor M; Hernández-Escarcega, Germán; Blom, Jochem; Goesmann, Alexander; Rezzonico, Fabio; Duffy, Brion; Stockwell, Virginia O

    2014-01-01

    We report draft genome sequences of three Mexican Erwinia amylovora strains. A novel plasmid, pEA78, was identified. Comparative genomics revealed an rpsL chromosomal mutation conferring high-level streptomycin resistance in two strains. In the effector gene avrRpt2, a single nucleotide polymorphism was detected that overcomes fire blight disease resistance in Malus × robusta 5. PMID:24459281

  6. Effector-triggered defence against apoplastic fungal pathogens.

    PubMed

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

    2014-08-01

    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

  7. 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. PMID:26709975

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  10. 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 ...

  11. Two-axis angular effector

    DOEpatents

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

    1997-01-21

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

  12. The chemically inducible expression of Erwinia amylovora bacterial effectors EopB1 and HopCEa in apple

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Erwinia amylovora, the causal agent of fire blight disease, utilizes a type three secretion system to deliver effector proteins into plant host cells. To investigate the role of individual bacterial effector proteins, we have engineered an apple host that transgenically expresses the bacterial effe...

  13. 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...

  14. 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 ...

  15. 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

  16. 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. ...

  17. The Type VI secretion system - a widespread and versatile cell targeting system.

    PubMed

    Coulthurst, Sarah J

    2013-01-01

    The Type VI secretion system (T6SS) is the most recently described of the Gram-negative bacterial secretion systems and is widely distributed amongst diverse species. T6SSs are currently believed to be complex molecular machines which inject effector proteins into target cells and which incorporate a bacteriophage-like cell-puncturing device. T6SSs have been implicated in eukaryotic cell targeting and virulence in a range of important pathogens. More recently, 'antibacterial' T6SSs have been reported, which are used to efficiently target competitor bacterial cells by the injection of antibacterial toxins. Although it is clear that T6SSs can be deployed as versatile weapons to compete with other bacteria or attack simple or higher eukaryotes, much remains to be determined about this intriguing system. PMID:23542428

  18. The Functions of Effector Proteins in Yersinia Virulence.

    PubMed

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

    2016-01-01

    Yersinia species are bacterial pathogens that can cause plague and intestinal diseases after invading into human cells through the Three Secretion System (TTSS). The effect of pathogenesis is mediated by Yersinia outer proteins (Yop) and manifested as down-regulation of the cytokine genes expression by inhibiting nuclear factor-κ-gene binding (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. In addition, its pathogenesis can also manipulate the disorder of host innate immune system and cell death such as apoptosis, pyroptosis, and autophagy. Among the Yersinia effector proteins, YopB and YopD assist the injection of other virulence effectors into the host cytoplasm, while YopE, YopH, YopJ, YopO, and YopT target on disrupting host cell signaling pathways in the host cytosols. Many efforts have been applied to reveal that intracellular proteins such as Rho-GTPase, and transmembrane receptors such as Toll-like receptors (TLRs) both play critical roles in Yersinia pathogenesis, establishing a connection between the pathogenic process and the signaling response. This review will mainly focus on how the effector proteins of Yersinia modulate the intrinsic signals in host cells and disturb the innate immunity of hosts through TTSS. PMID:27281989

  19. The Shigella flexneri OspB effector: an early immunomodulator.

    PubMed

    Ambrosi, Cecilia; Pompili, Monica; Scribano, Daniela; Limongi, Dolores; Petrucca, Andrea; Cannavacciuolo, Sonia; Schippa, Serena; Zagaglia, Carlo; Grossi, Milena; Nicoletti, Mauro

    2015-01-01

    Through the action of the type three secretion system (T3SS) Shigella flexneri delivers several effectors into host cells to promote cellular invasion, multiplication and to exploit host-cell signaling pathways to modulate the host innate immune response. Although much progress has been made in the understanding of many type III effectors, the molecular and cellular mechanism of the OspB effector is still poorly characterized. In this study we present new evidence that better elucidates the role of OspB as pro-inflammatory factor at very early stages of infection. Indeed, we demonstrate that, during the first hour of infection, OspB is required for full activation of ERK1/2 and p38 MAPKs and the cytosolic phospholipase A(2) (cPLA(2)). Activation of cPLA(2) ultimately leads to the production and secretion of PMN chemoattractant metabolite(s) uncoupled with release of IL-8. Moreover, we also present evidence that OspB is required for the development of the full and promptly inflammatory reaction characteristic of S. flexneri wild-type infection in vivo. Based on OspB and OspF similarity (both effectors share similar transcription regulation, temporal secretion into host cells and nuclear localization) we hypothesized that OspB and OspF effectors may form a pair aimed at modulating the host cell response throughout the infection process, with opposite effects. A model is presented to illustrate how OspB activity would promote S. flexneri invasion and bacterial dissemination at early critical phases of infection. PMID:25434600

  20. Orbital maneuvering end effectors

    NASA Technical Reports Server (NTRS)

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

    1986-01-01

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

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

    PubMed Central

    2014-01-01

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

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

    PubMed

    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

  3. Cancer-cell-targeted theranostic cubosomes.

    PubMed

    Caltagirone, Claudia; Falchi, Angela Maria; Lampis, Sandrina; Lippolis, Vito; Meli, Valeria; Monduzzi, Maura; Prodi, Luca; Schmidt, Judith; Sgarzi, Massimo; Talmon, Yeshayahu; Bizzarri, Ranieri; Murgia, Sergio

    2014-06-01

    This work was devoted to the development of a new type of lipid-based (cubosome) theranostic nanoparticle able to simultaneously host camptothecin, a potent anticancer drug, and a squarain-based NIR-emitting fluorescent probe. Furthermore, to confer targeting abilities on these nanoparticles, they were dispersed using mixtures of Pluronic F108 and folate-conjugated Pluronic F108 in appropriate ratios. The physicochemical characterization, performed via SAXS, DLS, and cryo-TEM techniques, proved that aqueous dispersions of such cubosomes can be effectively prepared, while the photophysical characterization demonstrated that these nanoparticles may be used for in vivo imaging purposes. The superior ability of these innovative nanoparticles in targeting cancer cells was emphasized by investigating the lipid droplet alterations induced in HeLa cells upon exposure to targeted and nontargeted cubosomes. PMID:24815031

  4. 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

  5. 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

  6. 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

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

    PubMed

    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

  8. Domain switching and host recognition.

    PubMed

    Tian, Miaoying; Day, Brad

    2006-09-01

    The collective function of secreted pathogen effector molecules is to enhance the virulence and avirulence activity of the pathogen during the infection of its host. While the activity of a majority of pathogen effectors is unknown, several classes of effector molecules have been well characterized. Among these include proteins which function to modulate host defences either through proteolysis, post-translational modifications, or by directly manipulating the host transcriptional machinery that regulates the induction of defence responses. In recent years, several key advances have been made in the characterization of the latter class of effector molecules. Among these include research characterizing the processes associated with host nuclear import and the targeting of host transcriptional defences. While current research is beginning to reveal the biochemical and genetic mechanisms controlling the induction of host resistance, the signalling events that control host specificity remain largely unknown. In this issue of Molecular Microbiology, work by Nissan et al. sheds light onto the molecular-genetic patterns involved in determining host specificity and pathogen virulence in the Pantoea-gypsophila interaction. PMID:16879410

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

    PubMed Central

    Hallstrom, Kelly N.; McCormick, Beth A.

    2016-01-01

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

  10. Apicomplexans pulling the strings: manipulation of the host cell cytoskeleton dynamics.

    PubMed

    Cardoso, Rita; Soares, Helena; Hemphill, Andrew; Leitão, Alexandre

    2016-07-01

    Invasive stages of apicomplexan parasites require a host cell to survive, proliferate and advance to the next life cycle stage. Once invasion is achieved, apicomplexans interact closely with the host cell cytoskeleton, but in many cases the different species have evolved distinct mechanisms and pathways to modulate the structural organization of cytoskeletal filaments. The host cell cytoskeleton is a complex network, largely, but not exclusively, composed of microtubules, actin microfilaments and intermediate filaments, all of which are modulated by associated proteins, and it is involved in diverse functions including maintenance of cell morphology and mechanical support, migration, signal transduction, nutrient uptake, membrane and organelle trafficking and cell division. The ability of apicomplexans to modulate the cytoskeleton to their own advantage is clearly beneficial. We here review different aspects of the interactions of apicomplexans with the three main cytoskeletal filament types, provide information on the currently known parasite effector proteins and respective host cell targets involved, and how these interactions modulate the host cell physiology. Some of these findings could provide novel targets that could be exploited for the development of preventive and/or therapeutic strategies. PMID:27041483

  11. Current activities of the Yersinia effector protein YopM.

    PubMed

    Höfling, Sabrina; Grabowski, Benjamin; Norkowski, Stefanie; Schmidt, M Alexander; Rüter, Christian

    2015-05-01

    Yersinia outer protein M (YopM) belongs to the group of Yop effector proteins, which are highly conserved among pathogenic Yersinia species. During infection, the effectors are delivered into the host cell cytoplasm via the type 3 secretion system to subvert the host immune response and support the survival of Yersinia. In contrast to the other Yop effectors, YopM does not possess a known enzymatic activity and its molecular mechanism(s) of action remain(s) poorly understood. However, YopM was shown to promote colonization and dissemination of Yersinia, thus being crucial for the pathogen's virulence in vivo. Moreover, YopM interacts with several host cell proteins and might utilize them to execute its anti-inflammatory activities. The results obtained so far indicate that YopM is a multifunctional protein that counteracts the host immune defense by multiple activities, which are at least partially independent of each other. Finally, its functions seem to be also influenced by differences between the specific YopM isoforms expressed by Yersinia subspecies. In this review, we focus on the global as well as more specific contribution of YopM to virulence of Yersinia during infection and point out the various extra- and intracellular molecular functions of YopM. In addition, the novel cell-penetrating ability of recombinant YopM and its potential applications as a self-delivering immunomodulatory therapeutic will be discussed. PMID:25865799

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

    PubMed

    Ehrbar, Kristin; Hardt, Wolf-Dietrich

    2005-01-01

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

  14. 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

  15. Subversion of Retrograde Trafficking by Translocated Pathogen Effectors.

    PubMed

    Personnic, Nicolas; Bärlocher, Kevin; Finsel, Ivo; Hilbi, Hubert

    2016-06-01

    Intracellular bacterial pathogens subvert the endocytic bactericidal pathway to form specific replication-permissive compartments termed pathogen vacuoles or inclusions. To this end, the pathogens employ type III or type IV secretion systems, which translocate dozens, if not hundreds, of different effector proteins into their host cells, where they manipulate vesicle trafficking and signaling pathways in favor of the intruders. While the distinct cocktail of effectors defines the specific processes by which a pathogen vacuole is formed, the different pathogens commonly target certain vesicle trafficking routes, including the endocytic or secretory pathway. Recently, the retrograde transport pathway from endosomal compartments to the trans-Golgi network emerged as an important route affecting pathogen vacuole formation. Here, we review current insight into the host cell's retrograde trafficking pathway and how vacuolar pathogens of the genera Legionella, Coxiella, Salmonella, Chlamydia, and Simkania employ mechanistically distinct strategies to subvert this pathway, thus promoting intracellular survival and replication. PMID:26924068

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

    PubMed

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

    2011-10-01

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

  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. PMID:26443209

  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. RAR1, a Central Player in Plant Immunity, is Targeted by Pseudomonas syringae Effector AvrB

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pathogenic bacterial effectors suppress Pathogen-Associated Molecular Pattern (PAMP)-triggered host immunity, thereby promoting parasitism. In the presence of cognate resistance genes, it is proposed that plants detect the virulence activity of bacterial effectors and trigger a defense response, ref...

  1. 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...

  2. TAL effectors: highly adaptable phytobacterial virulence factors and readily engineered DNA targeting proteins

    PubMed Central

    Doyle, Erin L.; Stoddard, Barry L.; Voytas, Daniel F.; Bogdanove, Adam J.

    2013-01-01

    Transcription activator-like (TAL) effectors are transcription factors injected into plant cells by pathogenic bacteria in the genus Xanthomonas. They function as virulence factors by activating host genes important for disease, or as avirulence factors by turning on genes that provide resistance. DNA binding specificity is encoded by polymorphic repeats in each protein that correspond one-to-one with different nucleotides. This code has facilitated target identification and opened new avenues for engineering disease resistance. It has also enabled TAL effector customization for targeted gene control, genome editing, and other applications. This article reviews the structural basis for TAL effector-DNA specificity, the impact of the TAL effector-DNA code on plant pathology and engineered resistance, and recent accomplishments and future challenges in TAL effector-based DNA targeting. PMID:23707478

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

    PubMed Central

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

    2014-01-01

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

  4. 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.

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

    PubMed Central

    Alcoforado Diniz, Juliana; Liu, Yi‐Chia

    2015-01-01

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

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

    PubMed

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

    2012-01-01

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

  7. B-cell targeted therapies in human autoimmune diseases: an updated perspective.

    PubMed

    Townsend, Michael J; Monroe, John G; Chan, Andrew C

    2010-09-01

    The advent of therapies that specifically target the B-lymphocyte lineage in human disease has rejuvenated interest in the mechanistic biology by which B cells mediate autoimmunity. B cells have a multitude of effector functions including production of self-reactive antibodies, ability to present antigen to T lymphocytes in the context of costimulation, involvement in generation and maintenance of neo-organogenesis at sites of disease, and opposing function through production of both immunostimulatory and immunomodulatory cytokines. In this review, we first discuss the role of B cells in driving autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, and Sjögren's syndrome, and discuss how studies in these diseases have revealed differentially important roles for the multiple B-cell effector functions. These data reveal the complex and interrelated roles of B cells working in concert with other components of the innate and adaptive immune system to drive pathogenesis. We then focus on data from mouse and human in which B cells in the setting of disease have been targeted with drugs directed against CD20, CD22, and the BAFF (B-cell activating factor belonging to the tumor necrosis factor family)/APRIL (a proliferation inducing ligand) pathways. Pre-clinical studies in animal models in addition to and clinical trials targeting B cells have added further to the understanding of the differential roles B cells play in disease both through demonstration of clinical efficacy in the context of B-cell depletion or modulation, and also by failure of B-cell targeting in some diseases and disease patient subgroups. Moving forward, it will be imperative to apply these lessons to new interventional trials to ensure better targeting of the B-cell lineage and concomitantly better selection of patients most likely to benefit from these therapies. PMID:20727041

  8. Translocation of Magnaporthe oryzae effectors into rice cells and their subsequent cell-to-cell movement.

    PubMed

    Khang, Chang Hyun; Berruyer, Romain; Giraldo, Martha C; Kankanala, Prasanna; Park, Sook-Young; Czymmek, Kirk; Kang, Seogchan; Valent, Barbara

    2010-04-01

    Knowledge remains limited about how fungal pathogens that colonize living plant cells translocate effector proteins inside host cells to regulate cellular processes and neutralize defense responses. To cause the globally important rice blast disease, specialized invasive hyphae (IH) invade successive living rice (Oryza sativa) cells while enclosed in host-derived extrainvasive hyphal membrane. Using live-cell imaging, we identified a highly localized structure, the biotrophic interfacial complex (BIC), which accumulates fluorescently labeled effectors secreted by IH. In each newly entered rice cell, effectors were first secreted into BICs at the tips of the initially filamentous hyphae in the cell. These tip BICs were left behind beside the first-differentiated bulbous IH cells as the fungus continued to colonize the host cell. Fluorescence recovery after photobleaching experiments showed that the effector protein PWL2 (for prevents pathogenicity toward weeping lovegrass [Eragrostis curvula]) continued to accumulate in BICs after IH were growing elsewhere. PWL2 and BAS1 (for biotrophy-associated secreted protein 1), BIC-localized secreted proteins, were translocated into the rice cytoplasm. By contrast, BAS4, which uniformly outlines the IH, was not translocated into the host cytoplasm. Fluorescent PWL2 and BAS1 proteins that reached the rice cytoplasm moved into uninvaded neighbors, presumably preparing host cells before invasion. We report robust assays for elucidating the molecular mechanisms that underpin effector secretion into BICs, translocation to the rice cytoplasm, and cell-to-cell movement in rice. PMID:20435900

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

    DOE PAGESBeta

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

    2014-12-08

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

  10. 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.

  11. Do CD8 effector cells need IL-7R expression to become resting memory cells?

    PubMed

    Buentke, Eva; Mathiot, Anne; Tolaini, Mauro; Di Santo, James; Zamoyska, Rose; Seddon, Benedict

    2006-09-15

    The role for IL-7R expression in the differentiation of effector T cells into resting memory remains controversial. Here, using a conditional IL-7R transgenic model, we were able to test directly whether CD8 effector T cells require IL-7R expression for their differentiation into resting memory cells. In the absence of IL-7R expression, effector cells transferred into "full" hosts underwent a protracted and unremitting contraction compared with IL-7R-expressing control cells and were unable to develop into long-term resting memory cells. Surprisingly, when the same effector cells were transferred into empty T-cell-deficient hosts, they could generate long-lived fully functional resting memory cells independently of IL-7R expression. Formation of these latter cells was found to be dependent on IL-15, because the same IL-7R-deficient effector cells were rapidly lost from IL-15-deficient hosts, having a half-life of less than 40 hours. Therefore, our data suggest that, under physiological conditions, both IL-7 and IL-15 synergize to promote the formation of memory cells directly by limiting the contraction of effectors that occurs following an immune response and that reexpression of IL-7R is a key checkpoint in the regulation of this process. PMID:16705084

  12. Effector proteins support the asymmetric apportioning of Salmonella during cytokinesis.

    PubMed

    Zhao, Yaya; Gorvel, Jean-Pierre; Méresse, Stéphane

    2016-08-17

    Salmonella-infected cells are characterized by the presence of intra-cellular membranous tubules that emerge from bacterial vacuoles and extend along microtubules. The formation of Salmonella-induced tubules depends on the Salmonella pathogenicity island 2-encoded type III secretion system (T3SS-2) that translocates bacterial effector proteins inside host cells. Effector proteins have enzymatic activities or allow for hijacking of cellular functions. The role of Salmonella-induced tubules in virulence remains unclear but their absence is correlated with virulence defects. This study describes the presence of inter-cellular tubules that arise between daughter cells during cytokinesis. Inter-cellular tubules connect bacterial vacuoles originally present in the parent cell and that have been apportioned between daughters. Their formation requires a functional T3SS-2 and effector proteins. Our data establish a correlation between the formation of inter-cellular tubules and the asymmetric distribution of bacterial vacuoles in daughters. Thus, by manipulating the distribution of bacteria in cytokinetic cells, Salmonella T3SS-2 effector proteins may increase bacterial spreading and the systemic character of the infection. PMID:27046257

  13. 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. PMID:25494461

  14. Effector proteins support the asymmetric apportioning of Salmonella during cytokinesis

    PubMed Central

    Zhao, Yaya; Gorvel, Jean-Pierre; Méresse, Stéphane

    2016-01-01

    ABSTRACT Salmonella-infected cells are characterized by the presence of intra-cellular membranous tubules that emerge from bacterial vacuoles and extend along microtubules. The formation of Salmonella-induced tubules depends on the Salmonella pathogenicity island 2-encoded type III secretion system (T3SS-2) that translocates bacterial effector proteins inside host cells. Effector proteins have enzymatic activities or allow for hijacking of cellular functions. The role of Salmonella-induced tubules in virulence remains unclear but their absence is correlated with virulence defects. This study describes the presence of inter-cellular tubules that arise between daughter cells during cytokinesis. Inter-cellular tubules connect bacterial vacuoles originally present in the parent cell and that have been apportioned between daughters. Their formation requires a functional T3SS-2 and effector proteins. Our data establish a correlation between the formation of inter-cellular tubules and the asymmetric distribution of bacterial vacuoles in daughters. Thus, by manipulating the distribution of bacteria in cytokinetic cells, Salmonella T3SS-2 effector proteins may increase bacterial spreading and the systemic character of the infection. PMID:27046257

  15. Dexterous end effector flight demonstration

    NASA Technical Reports Server (NTRS)

    Carter, Edward L.; Monford, Leo G.

    1994-01-01

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

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

    PubMed Central

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

    2011-01-01

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

  17. The Salmonella effector protein SpvC, a phosphothreonine lyase is functional in plant cells

    PubMed Central

    Neumann, Christina; Fraiture, Malou; Hernàndez-Reyes, Casandra; Akum, Fidele N.; Virlogeux-Payant, Isabelle; Chen, Ying; Pateyron, Stephanie; Colcombet, Jean; Kogel, Karl-Heinz; Hirt, Heribert; Brunner, Frédéric; Schikora, Adam

    2014-01-01

    Salmonella is one of the most prominent causes of food poisoning and growing evidence indicates that contaminated fruits and vegetables are an increasing concern for human health. Successful infection demands the suppression of the host immune system, which is often achieved via injection of bacterial effector proteins into host cells. In this report we present the function of Salmonella effector protein in plant cell, supporting the new concept of trans-kingdom competence of this bacterium. We screened a range of Salmonella Typhimurium effector proteins for interference with plant immunity. Among these, the phosphothreonine lyase SpvC attenuated the induction of immunity-related genes when present in plant cells. Using in vitro and in vivo systems we show that this effector protein interacts with and dephosphorylates activated Arabidopsis Mitogen-activated Protein Kinase 6 (MPK6), thereby inhibiting defense signaling. Moreover, the requirement of Salmonella SpvC was shown by the decreased proliferation of the ΔspvC mutant in Arabidopsis plants. These results suggest that some Salmonella effector proteins could have a conserved function during proliferation in different hosts. The fact that Salmonella and other Enterobacteriaceae use plants as hosts strongly suggests that plants represent a much larger reservoir for animal pathogens than so far estimated. PMID:25368608

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  20. 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.

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

    PubMed

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

    2016-08-01

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

  2. 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

  3. 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.

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

    PubMed

    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

  5. 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

  6. The Commonalities in Bacterial Effector Inhibition of Apoptosis.

    PubMed

    Robinson, Keith S; Aw, Rochelle

    2016-08-01

    Antiapoptotic pathways of the host cell play integral roles in bacterial pathogenesis, with inhibition of those pathways resulting in halted disease pathology. Certain pathogens have developed elegant mechanisms to modulate the fate of the host cell, many of which target novel pathways that are poorly understood in the context of the cell biology. Bacterial pathogenesis research not only promotes the understanding of the role of antiapoptotic pathways in bacterial infection, but has a broader context in understanding the epitome of human disease, that is, developing the understanding of tumorigenic or inflammatory pathways. Here we review host antiapoptotic signalling pathways manipulated by translocated bacterial effectors that propagate the disease state, drawing common parallels and showing the novel differences. PMID:27117049

  7. A massive expansion of effector genes underlies gall-formation in the wheat pest Mayetiola destructor.

    PubMed

    Zhao, Chaoyang; Escalante, Lucio Navarro; Chen, Hang; Benatti, Thiago R; Qu, Jiaxin; Chellapilla, Sanjay; Waterhouse, Robert M; Wheeler, David; Andersson, Martin N; Bao, Riyue; Batterton, Matthew; Behura, Susanta K; Blankenburg, Kerstin P; Caragea, Doina; Carolan, James C; Coyle, Marcus; El-Bouhssini, Mustapha; Francisco, Liezl; Friedrich, Markus; Gill, Navdeep; Grace, Tony; Grimmelikhuijzen, Cornelis J P; Han, Yi; Hauser, Frank; Herndon, Nicolae; Holder, Michael; Ioannidis, Panagiotis; Jackson, LaRonda; Javaid, Mehwish; Jhangiani, Shalini N; Johnson, Alisha J; Kalra, Divya; Korchina, Viktoriya; Kovar, Christie L; Lara, Fremiet; Lee, Sandra L; Liu, Xuming; Löfstedt, Christer; Mata, Robert; Mathew, Tittu; Muzny, Donna M; Nagar, Swapnil; Nazareth, Lynne V; Okwuonu, Geoffrey; Ongeri, Fiona; Perales, Lora; Peterson, Brittany F; Pu, Ling-Ling; Robertson, Hugh M; Schemerhorn, Brandon J; Scherer, Steven E; Shreve, Jacob T; Simmons, DeNard; Subramanyam, Subhashree; Thornton, Rebecca L; Xue, Kun; Weissenberger, George M; Williams, Christie E; Worley, Kim C; Zhu, Dianhui; Zhu, Yiming; Harris, Marion O; Shukle, Richard H; Werren, John H; Zdobnov, Evgeny M; Chen, Ming-Shun; Brown, Susan J; Stuart, Jeffery J; Richards, Stephen

    2015-03-01

    Gall-forming arthropods are highly specialized herbivores that, in combination with their hosts, produce extended phenotypes with unique morphologies [1]. Many are economically important, and others have improved our understanding of ecology and adaptive radiation [2]. However, the mechanisms that these arthropods use to induce plant galls are poorly understood. We sequenced the genome of the Hessian fly (Mayetiola destructor; Diptera: Cecidomyiidae), a plant parasitic gall midge and a pest of wheat (Triticum spp.), with the aim of identifying genic modifications that contribute to its plant-parasitic lifestyle. Among several adaptive modifications, we discovered an expansive reservoir of potential effector proteins. Nearly 5% of the 20,163 predicted gene models matched putative effector gene transcripts present in the M. destructor larval salivary gland. Another 466 putative effectors were discovered among the genes that have no sequence similarities in other organisms. The largest known arthropod gene family (family SSGP-71) was also discovered within the effector reservoir. SSGP-71 proteins lack sequence homologies to other proteins, but their structures resemble both ubiquitin E3 ligases in plants and E3-ligase-mimicking effectors in plant pathogenic bacteria. SSGP-71 proteins and wheat Skp proteins interact in vivo. Mutations in different SSGP-71 genes avoid the effector-triggered immunity that is directed by the wheat resistance genes H6 and H9. Results point to effectors as the agents responsible for arthropod-induced plant gall formation. PMID:25660540

  8. Space Station end effector strategy study

    NASA Technical Reports Server (NTRS)

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

    1987-01-01

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

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

    PubMed

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

    2014-01-01

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

  10. Identification and characterisation of a hyper-variable apoplastic effector gene family of the potato cyst nematodes.

    PubMed

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

    2014-09-01

    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

  11. 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

    2016-01-01

    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

  12. 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

  13. Human Urinary Exosomes as Innate Immune Effectors

    PubMed Central

    Hiemstra, Thomas F.; Charles, Philip D.; Gracia, Tannia; Hester, Svenja S.; Gatto, Laurent; Al-Lamki, Rafia; Floto, R. Andres; Su, Ya; Skepper, Jeremy N.

    2014-01-01

    Exosomes are small extracellular vesicles, approximately 50 nm in diameter, derived from the endocytic pathway and released by a variety of cell types. Recent data indicate a spectrum of exosomal functions, including RNA transfer, antigen presentation, modulation of apoptosis, and shedding of obsolete protein. Exosomes derived from all nephron segments are also present in human urine, where their function is unknown. Although one report suggested in vitro uptake of exosomes by renal cortical collecting duct cells, most studies of human urinary exosomes have focused on biomarker discovery rather than exosome function. Here, we report results from in-depth proteomic analyses and EM showing that normal human urinary exosomes are significantly enriched for innate immune proteins that include antimicrobial proteins and peptides and bacterial and viral receptors. Urinary exosomes, but not the prevalent soluble urinary protein uromodulin (Tamm–Horsfall protein), potently inhibited growth of pathogenic and commensal Escherichia coli and induced bacterial lysis. Bacterial killing depended on exosome structural integrity and occurred optimally at the acidic pH typical of urine from omnivorous humans. Thus, exosomes are innate immune effectors that contribute to host defense within the urinary tract. PMID:24700864

  14. Bacterial secreted effectors and caspase-3 interactions

    PubMed Central

    Wall, Daniel M; McCormick, Beth A

    2014-01-01

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

  15. 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

  16. Cell Targeting in Anti-Cancer Gene Therapy

    PubMed Central

    Lila, Mohd Azmi Mohd; Siew, John Shia Kwong; Zakaria, Hayati; Saad, Suria Mohd; Ni, Lim Shen; Abdullah, Jafri Malin

    2004-01-01

    Gene therapy is a promising approach towards cancer treatment. The main aim of the therapy is to destroy cancer cells, usually by apoptotic mechanisms, and preserving others. However, its application has been hindered by many factors including poor cellular uptake, non-specific cell targeting and undesirable interferences with other genes or gene products. A variety of strategies exist to improve cellular uptake efficiency of gene-based therapies. This paper highlights advancements in gene therapy research and its application in relation to anti-cancer treatment. PMID:22977356

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

    PubMed Central

    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. PMID:25426123

  18. 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.

  19. Human immune cell targeting of protein nanoparticles - caveospheres.

    PubMed

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

    2016-04-14

    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. PMID:27031090

  20. 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.

  1. Smooth Muscle Cell-targeted RNA Aptamer Inhibits Neointimal Formation.

    PubMed

    Thiel, William H; Esposito, Carla L; Dickey, David D; Dassie, Justin P; Long, Matthew E; Adam, Joshua; Streeter, Jennifer; Schickling, Brandon; Takapoo, Maysam; Flenker, Katie S; Klesney-Tait, Julia; Franciscis, Vittorio de; Miller, Francis J; Giangrande, Paloma H

    2016-04-01

    Inhibition of vascular smooth muscle cell (VSMC) proliferation by drug eluting stents has markedly reduced intimal hyperplasia and subsequent in-stent restenosis. However, the effects of antiproliferative drugs on endothelial cells (EC) contribute to delayed re-endothelialization and late stent thrombosis. Cell-targeted therapies to inhibit VSMC remodeling while maintaining EC health are necessary to allow vascular healing while preventing restenosis. We describe an RNA aptamer (Apt 14) that functions as a smart drug by preferentially targeting VSMCs as compared to ECs and other myocytes. Furthermore, Apt 14 inhibits phosphatidylinositol 3-kinase/protein kinase-B (PI3K/Akt) and VSMC migration in response to multiple agonists by a mechanism that involves inhibition of platelet-derived growth factor receptor (PDGFR)-β phosphorylation. In a murine model of carotid injury, treatment of vessels with Apt 14 reduces neointimal formation to levels similar to those observed with paclitaxel. Importantly, we confirm that Apt 14 cross-reacts with rodent and human VSMCs, exhibits a half-life of ~300 hours in human serum, and does not elicit immune activation of human peripheral blood mononuclear cells. We describe a VSMC-targeted RNA aptamer that blocks cell migration and inhibits intimal formation. These findings provide the foundation for the translation of cell-targeted RNA therapeutics to vascular disease. PMID:26732878

  2. 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).

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

    PubMed

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

    2014-01-01

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

  4. 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

  5. Exploiting the ubiquitin and phosphoinositide pathways by the Legionella pneumophila effector, SidC.

    PubMed

    Wasilko, David J; Mao, Yuxin

    2016-02-01

    Intracellular bacterial pathogens use secreted effector proteins to alter host cellular processes, with the goal of subverting host defenses and allowing the infection to progress. One such pathogen, Legionella pneumophila, secretes ~300 proteins into its host to alter a number of pathways including intracellular trafficking, phosphoinositide metabolism, and cell signaling. The Legionella effector SidC was previously found to bind to PI(4)P and was responsible for the enrichment of ER proteins and ubiquitinated species on the Legionella-containing vacuoles. Through our recent work, we have discovered that SidC contains a unique N-terminal E3 ubiquitin ligase domain and a C-terminal novel PI(4)P-binding domain. Our results demonstrate that SidC serves to link two distinct cellular pathways, ubiquitin and phosphoinositide. However, how the ubiquitin ligase activity regulates host membrane trafficking events remains to be investigated. PMID:26433729

  6. Structural and Functional Studies Indicate That the EPEC Effector, EspG, Directly Binds p21-Activated Kinase

    SciTech Connect

    Germane, Katherine L.; Spiller, Benjamin W.

    2011-09-20

    Bacterial pathogens secrete effectors into their hosts that subvert host defenses and redirect host processes. EspG is a type three secretion effector with a disputed function that is found in enteropathogenic Escherichia coli. Here we show that EspG is structurally similar to VirA, a Shigella virulence factor; EspG has a large, conserved pocket on its surface; EspG binds directly to the amino-terminal inhibitory domain of human p21-activated kinase (PAK); and mutations to conserved residues in the surface pocket disrupt the interaction with PAK.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  8. 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...

  9. 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...

  10. 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...

  11. 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...

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

    PubMed

    Poirier, Valérie; Av-Gay, Yossef

    2015-12-01

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

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

    PubMed Central

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

    2015-01-01

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

  14. 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...

  15. 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 (...

  16. 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

  17. 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

  18. 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. PMID:27602332

  19. 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

  20. 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

  1. Proteogenomic analysis of the Venturia pirina (Pear Scab Fungus) secretome reveals potential effectors.

    PubMed

    Cooke, Ira R; Jones, Dan; Bowen, Joanna K; Deng, Cecilia; Faou, Pierre; Hall, Nathan E; Jayachandran, Vignesh; Liem, Michael; Taranto, Adam P; Plummer, Kim M; Mathivanan, Suresh

    2014-08-01

    A proteogenomic analysis is presented for Venturia pirina, a fungus that causes scab disease on European pear (Pyrus communis). V. pirina is host-specific, and the infection is thought to be mediated by secreted effector proteins. Currently, only 36 V. pirina proteins are catalogued in GenBank, and the genome sequence is not publicly available. To identify putative effectors, V. pirina was grown in vitro on and in cellophane sheets mimicking its growth in infected leaves. Secreted extracts were analyzed by tandem mass spectrometry, and the data (ProteomeXchange identifier PXD000710) was queried against a protein database generated by combining in silico predicted transcripts with six frame translations of a whole genome sequence of V. pirina (GenBank Accession JEMP00000000 ). We identified 1088 distinct V. pirina protein groups (FDR 1%) including 1085 detected for the first time. Thirty novel (not in silico predicted) proteins were found, of which 14 were identified as potential effectors based on characteristic features of fungal effector protein sequences. We also used evidence from semitryptic peptides at the protein N-terminus to corroborate in silico signal peptide predictions for 22 proteins, including several potential effectors. The analysis highlights the utility of proteogenomics in the study of secreted effectors. PMID:24965097

  2. 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.

  3. 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

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

    PubMed

    Ahmed, Ali Abdurehim; Pedersen, Carsten; Thordal-Christensen, Hans

    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

  5. 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. PMID:26779450

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

    PubMed

    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

  7. 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

  8. Integrated decoys and effector traps: how to catch a plant pathogen.

    PubMed

    Ellis, Jeffrey G

    2016-01-01

    Plant immune receptors involved in disease resistance and crop protection are related to the animal Nod-like receptor (NLR) class, and recognise the virulence effectors of plant pathogens, whereby they arm the plant's defensive response. Although plant NLRs mainly contain three protein domains, about 10% of these receptors identified by extensive cross-plant species data base searches have now been shown to include novel and highly variable integrated domains, some of which have been shown to detect pathogen effectors by direct interaction. Sarris et al. have identified a large number of integrated domains that can be used to detect effector targets in host plant proteomes and identify unknown pathogen effectors.Please see related Research article: Comparative analysis of plant immune receptor architectures uncovers host proteins likely targeted by pathogens, http://dx.doi.org/10.1186/s12915-016-0228-7 Since the time of writing, a closely related paper has been released: Kroj T, Chanclud E, Michel-Romiti C, Grand X, Morel J-B. Integration of decoy domains derived from protein targets of pathogen effectors into plant immune receptors is widespread. New Phytol. 2016 (ahead of print). PMID:26896088

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

    PubMed Central

    Ashida, Hiroshi; Sasakawa, Chihiro

    2016-01-01

    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. PMID:26779450

  10. 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

  11. 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

  12. 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

  13. 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.

  14. RXLR effector reservoir in two Phytophthora species is dominated by a single rapidly evolving superfamily with more than 700 members

    PubMed Central

    Jiang, Rays H. Y.; Tripathy, Sucheta; Govers, Francine; Tyler, Brett M.

    2008-01-01

    Pathogens secrete effector molecules that facilitate the infection of their hosts. A number of effectors identified in plant pathogenic Phytophthora species possess N-terminal motifs (RXLR-dEER) required for targeting these effectors into host cells. Here, we bioinformatically identify >370 candidate effector genes in each of the genomes of P. sojae and P. ramorum. A single superfamily, termed avirulence homolog (Avh) genes, accounts for most of the effectors. The Avh proteins show extensive sequence divergence but are all related and likely evolved from a common ancestor by rapid duplication and divergence. More than half of the Avh proteins contain conserved C-terminal motifs (termed W, Y, and L) that are usually arranged as a module that can be repeated up to eight times. The Avh genes belong to the most rapidly evolving part of the genome, and they are nearly always located at synteny breakpoints. The superfamily includes all experimentally identified oomycete effector and avirulence genes, and its rapid pace of evolution is consistent with a role for Avh proteins in interaction with plant hosts. PMID:18344324

  15. Phytophthora suppressor of RNA silencing 2 is a conserved RxLR effector that promotes infection in soybean and Arabidopsis thaliana.

    PubMed

    Xiong, Qin; Ye, Wenwu; Choi, Duseok; Wong, James; Qiao, Yongli; Tao, Kai; Wang, Yuanchao; Ma, Wenbo

    2014-12-01

    The genus Phytophthora consists of notorious and emerging pathogens of economically important crops. Each Phytophthora genome encodes several hundreds of cytoplasmic effectors, which are believed to manipulate plant immune response inside the host cells. However, the majority of Phytophthora effectors remain functionally uncharacterized. We recently discovered two effectors from the soybean stem and root rot pathogen Phytophthora sojae with the activity to suppress RNA silencing in plants. These effectors are designated Phytophthora suppressor of RNA silencing (PSRs). Here, we report that the P. sojae PSR2 (PsPSR2) belongs to a conserved and widespread effector family in Phytophthora. A PsPSR2-like effector produced by P. infestans (PiPSR2) can also suppress RNA silencing in plants and promote Phytophthora infection, suggesting that the PSR2 family effectors have conserved functions in plant hosts. Using Agrobacterium rhizogenes-mediated hairy roots induction, we demonstrated that the expression of PsPSR2 rendered hypersusceptibility of soybean to P. sojae. Enhanced susceptibility was also observed in PsPSR2-expressing Arabidopsis thaliana plants during Phytophthora but not bacterial infection. These experiments provide strong evidence that PSR2 is a conserved Phytophthora effector family that performs important virulence functions specifically during Phytophthora infection of various plant hosts. PMID:25387135

  16. Structural and Functional Investigations of the Effector Protein LpiR1 from Legionella pneumophila.

    PubMed

    Beyrakhova, Ksenia A; van Straaten, Karin; Li, Lei; Boniecki, Michal T; Anderson, Deborah H; Cygler, Miroslaw

    2016-07-22

    Legionella pneumophila is a causative agent of a severe pneumonia, known as Legionnaires' disease. Legionella pathogenicity is mediated by specific virulence factors, called bacterial effectors, which are injected into the invaded host cell by the bacterial type IV secretion system. Bacterial effectors are involved in complex interactions with the components of the host cell immune and signaling pathways, which eventually lead to bacterial survival and replication inside the mammalian cell. Structural and functional studies of bacterial effectors are, therefore, crucial for elucidating the mechanisms of Legionella virulence. Here we describe the crystal structure of the LpiR1 (Lpg0634) effector protein and investigate the effects of its overexpression in mammalian cells. LpiR1 is an α-helical protein that consists of two similar domains aligned in an antiparallel fashion. The hydrophilic cleft between the domains might serve as a binding site for a potential host cell interaction partner. LpiR1 binds the phosphate group at a conserved site and is stabilized by Mn(2+), Ca(2+), or Mg(2+) ions. When overexpressed in mammalian cells, a GFP-LpiR1 fusion protein is localized in the cytoplasm. Intracellular signaling antibody array analysis revealed small changes in the phosphorylation state of several components of the Akt signaling pathway in HEK293T cells overexpressing LpiR1. PMID:27226543

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

    PubMed Central

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

    2013-01-01

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

  18. Lysine11-Linked Polyubiquitination of the AnkB F-Box Effector of Legionella pneumophila

    PubMed Central

    Bruckert, William M.

    2015-01-01

    The fate of the polyubiquitinated protein is determined by the lysine linkages involved in the polymerization of the ubiquitin monomers, which has seven lysine residues (K6, K11, K27, K29, K33, K48, and K63). The translocated AnkB effector of the intravacuolar pathogen Legionella pneumophila is a bona fide F-box protein, which is localized to the cytosolic side of the Legionella-containing vacuole (LCV) and is essential for intravacuolar proliferation within macrophages and amoebae. The F-box domain of AnkB interacts with the host SCF1 E3 ubiquitin ligase that triggers the decoration of the LCV with K48-linked polyubiquitinated proteins that are targeted for proteasomal degradation. Here we report that AnkB becomes rapidly polyubiquitinated within the host cell, and this modification is independent of the F-box domain of AnkB, indicating host-mediated polyubiquitination. We show that the AnkB effector interacts specifically with the host E3 ubiquitin ligase Trim21. Mass spectrometry analyses have shown that AnkB is modified by K11-linked polyubiquitination, which has no effect on its stability. This work shows the first example of K11-linked polyubiquitination of a bacterial effector and its interaction with the host Trim21 ubiquitin ligase. PMID:26483404

  19. 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

  20. 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.

  1. 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.

  2. Spiral lead platen robotic end effector

    NASA Technical Reports Server (NTRS)

    Beals, David C. (Inventor)

    1990-01-01

    A robotic end effector is disclosed which makes use of a rotating platen with spiral leads used to impact lateral motion to gripping fingers. Actuation is provided by the contact of rolling pins with the walls of the leads. The use of the disclosed method of actuation avoids jamming and provides excellent mechanical advantage while remaining light in weight and durable. The entire end effector is compact and easily adapted for attachment to robotic arms currently in use.

  3. 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

  4. 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

    2016-01-01

    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. PMID:27501079

  5. Cell-targeting antibodies in immunity to Ebola.

    PubMed

    Schmaljohn, Alan; Lewis, George K

    2016-06-01

    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

  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. Cell-targeting aptamers act as intracellular delivery vehicles.

    PubMed

    Gopinath, Subash C B; Lakshmipriya, Thangavel; Chen, Yeng; Arshad, M K Md; Kerishnan, Jesinda P; Ruslinda, A R; Al-Douri, Yarub; Voon, C H; Hashim, Uda

    2016-08-01

    Aptamers are single-stranded nucleic acids or peptides identified from a randomized combinatorial library through specific interaction with the target of interest. Targets can be of any size, from small molecules to whole cells, attesting to the versatility of aptamers for binding a wide range of targets. Aptamers show drug properties that are analogous to antibodies, with high specificity and affinity to their target molecules. Aptamers can penetrate disease-causing microbial and mammalian cells. Generated aptamers that target surface biomarkers act as cell-targeting agents and intracellular delivery vehicles. Within this context, the "cell-internalizing aptamers" are widely investigated via the process of cell uptake with selective binding during in vivo systematic evolution of ligands by exponential enrichment (SELEX) or by cell-internalization SELEX, which targets cell surface antigens to be receptors. These internalizing aptamers are highly preferable for the localization and functional analyses of multiple targets. In this overview, we discuss the ways by which internalizing aptamers are generated and their successful applications. Furthermore, theranostic approaches featuring cell-internalized aptamers are discussed with the purpose of analyzing and diagnosing disease-causing pathogens. PMID:27350620

  8. 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.

  9. Porcine circovirus type 2 displays pluripotency in cell targeting

    SciTech Connect

    Steiner, Esther Balmelli, Carole Herrmann, Brigitte; Summerfield, Artur; McCullough, Kenneth

    2008-09-01

    Porcine circovirus type 2 (PCV2) is the causative agent of a multifactorial disease associated with immunocompromisation and co-infections. In vivo, viral DNA and antigens are found in monocytic, epithelial and endothelial cells. Of these, PCV2 replication has only been studied in monocytic cells, in which little or no replication was identified. Accordingly, PCV2 infection was studied in the endothelial cell line PEDSV.15, aortic endothelial cells, gut epithelial cells, fibrocytes and dendritic cells (DC). In all cells except DC PCV2 replication was detectable, with an increase in the levels of capsid and replicase protein. Variations in endocytic activity, virus binding and uptake did not relate to the replication efficiency in a particular cell. Furthermore, replication did not correlate to cell proliferation, although a close association of viral proteins with chromatin in dividing cells was observed. No alteration in the division rate of PCV2-infected cultures was measurable, relating to replicase expression in only a small minority of the cells. In conclusion, the broad cell targeting of PCV2 offers an explanation for its widespread tissue distribution.

  10. Selective Cell Targeting with Light-Absorbing Microparticles and Nanoparticles

    PubMed Central

    Pitsillides, Costas M.; Joe, Edwin K.; Wei, Xunbin; Anderson, R. Rox; Lin, Charles P.

    2003-01-01

    We describe a new method for selective cell targeting based on the use of light-absorbing microparticles and nanoparticles that are heated by short laser pulses to create highly localized cell damage. The method is closely related to chromophore-assisted laser inactivation and photodynamic therapy, but is driven solely by light absorption, without the need for photochemical intermediates (particularly singlet oxygen). The mechanism of light-particle interaction was investigated by nanosecond time-resolved microscopy and by thermal modeling. The extent of light-induced damage was investigated by cell lethality, by cell membrane permeability, and by protein inactivation. Strong particle size dependence was found for these interactions. A technique based on light to target endogenous particles is already being exploited to treat pigmented cells in dermatology and ophthalmology. With exogenous particles, phamacokinetics and biodistribution studies are needed before the method can be evaluated against photodynamic therapy for cancer treatment. However, particles are unique, unlike photosensitizers, in that they can remain stable and inert in cells for extended periods. Thus they may be particularly useful for prelabeling cells in engineered tissue before implantation. Subsequent irradiation with laser pulses will allow control of the implanted cells (inactivation or modulation) in a noninvasive manner. PMID:12770906

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

    PubMed

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

    2016-01-01

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

  12. 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

  13. Myeloid cells - targets of medication in multiple sclerosis.

    PubMed

    Mishra, Manoj K; Yong, V Wee

    2016-09-01

    Discussions of multiple sclerosis (MS) pathophysiology tend to focus on T cells and B cells of the adaptive immune response. The innate immune system is less commonly considered in this context, although dendritic cells, monocytes, macrophages and microglia - collectively referred to as myeloid cells - have prominent roles in MS pathogenesis. These populations of myeloid cells function as antigen-presenting cells and effector cells in neuroinflammation. Furthermore, a vicious cycle of interactions between T cells and myeloid cells exacerbates pathology. Several disease-modifying therapies are now available to treat MS, and insights into their mechanisms of action have largely focused on the adaptive immune system, but these therapies also have important effects on myeloid cells. In this Review, we discuss the evidence for the roles of myeloid cells in MS and the experimental autoimmune encephalomyelitis model of MS, and consider how interactions between myeloid cells and T cells and/or B cells promote MS pathology. Finally, we discuss the direct and indirect effects of existing MS medications on myeloid cells. PMID:27514287

  14. Immune Cell Targets of Infection at the Tick-Skin Interface during Powassan Virus Transmission

    PubMed Central

    Hermance, Meghan E.; Santos, Rodrigo I.; Kelly, Brent C.; Valbuena, Gustavo; Thangamani, Saravanan

    2016-01-01

    Powassan virus (POWV) is a tick-borne flavivirus that can result in a severe neuroinvasive disease with 50% of survivors displaying long-term neurological sequelae. Human POWV cases have been documented in Canada, the United States, and Russia. Although the number of reported POWV human cases has increased in the past fifteen years, POWV remains one of the less studied human pathogenic flaviviruses. Ixodes ticks are the vectors for POWV, and the virus is transmitted to a host’s skin very early during the tick feeding process. Central to the successful transmission of a tick-borne pathogen are complex interactions between the host immune response and early tick-mediated immunomodulation, all of which initially occur at the skin interface. In our prior work, we examined the cutaneous immune gene expression during the early stages of POWV-infected Ixodes scapularis feeding. The present study serves to further investigate the skin interface by identifying early cell targets of infection at the POWV-infected tick feeding site. An in vivo infection model consisting of POWV-infected ticks feeding on mice for short durations was used in this study. Skin biopsies from the tick feeding sites were harvested at various early time points, enabling us to examine the skin histopathology and detect POWV viral antigen in immune cells present at the tick feeding site. The histopathology from the present study demonstrates that neutrophil and mononuclear cell infiltrates are recruited earlier to the feeding site of a POWV-infected tick versus an uninfected tick. This is the first report demonstrating that macrophages and fibroblasts contain POWV antigens, which suggests that they are early cellular targets of infection at the tick feeding site. These data provide key insights towards defining the complex interactions between the host immune response and early tick-mediated immunomodulation. PMID:27203436

  15. 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

  16. Modulation of host microtubule dynamics by pathogenic bacteria

    PubMed Central

    Radhakrishnan, Girish K.; Splitter, Gary A.

    2013-01-01

    The eukaryotic cytoskeleton is a vulnerable target of many microbial pathogens during the course of infection. Rearrangements of host cytoskeleton benefit microbes in various stages of their infection cycle such as invasion, motility, and persistence. Bacterial pathogens deliver a number of effector proteins into host cells for modulating the dynamics of actin and microtubule cytoskeleton. Alteration of the actin cytoskeleton is generally achieved by bacterial effectors that target the small GTPases of the host. Modulation of microtubule dynamics involves direct interaction of effector proteins with the subunits of microtubules or recruiting cellular proteins that affect microtubule dynamics. This review will discuss effector proteins from animal and human bacterial pathogens that either destabilize or stabilize host micro-tubules to advance the infectious process. A compilation of these research findings will provide an overview of known and unknown strategies used by various bacterial effectors to modulate the host microtubule dynamics. The present review will undoubtedly help direct future research to determine the mechanisms of action of many bacterial effector proteins and contribute to understanding the survival strategies of diverse adherent and invasive bacterial pathogens. PMID:23585820

  17. Coxiella burnetii Effector Proteins That Localize to the Parasitophorous Vacuole Membrane Promote Intracellular Replication

    PubMed Central

    Larson, Charles L.; Beare, Paul A.; Voth, Daniel E.; Howe, Dale; Cockrell, Diane C.; Bastidas, Robert J.; Valdivia, Raphael H.

    2014-01-01

    The intracellular bacterial pathogen Coxiella burnetii directs biogenesis of a parasitophorous vacuole (PV) that acquires host endolysosomal components. Formation of a PV that supports C. burnetii replication requires a Dot/Icm type 4B secretion system (T4BSS) that delivers bacterial effector proteins into the host cell cytosol. Thus, a subset of T4BSS effectors are presumed to direct PV biogenesis. Recently, the PV-localized effector protein CvpA was found to promote C. burnetii intracellular growth and PV expansion. We predict additional C. burnetii effectors localize to the PV membrane and regulate eukaryotic vesicle trafficking events that promote pathogen growth. To identify these vacuolar effector proteins, a list of predicted C. burnetii T4BSS substrates was compiled using bioinformatic criteria, such as the presence of eukaryote-like coiled-coil domains. Adenylate cyclase translocation assays revealed 13 proteins were secreted in a Dot/Icm-dependent fashion by C. burnetii during infection of human THP-1 macrophages. Four of the Dot/Icm substrates, termed Coxiella vacuolar protein B (CvpB), CvpC, CvpD, and CvpE, labeled the PV membrane and LAMP1-positive vesicles when ectopically expressed as fluorescently tagged fusion proteins. C. burnetii ΔcvpB, ΔcvpC, ΔcvpD, and ΔcvpE mutants exhibited significant defects in intracellular replication and PV formation. Genetic complementation of the ΔcvpD and ΔcvpE mutants rescued intracellular growth and PV generation, whereas the growth of C. burnetii ΔcvpB and ΔcvpC was rescued upon cohabitation with wild-type bacteria in a common PV. Collectively, these data indicate C. burnetii encodes multiple effector proteins that target the PV membrane and benefit pathogen replication in human macrophages. PMID:25422265

  18. Effectors of root sedentary nematodes target diverse plant cell compartments to manipulate plant functions and promote infection

    PubMed Central

    Jaouannet, Maëlle; Rosso, Marie-Noëlle

    2013-01-01

    Sedentary plant-parasitic nematodes maintain a biotrophic relationship with their hosts over a period of several weeks and induce the differentiation of root cells into specialized feeding cells. Nematode effectors, which are synthesized in the esophageal glands and injected into the plant tissue through the syringe-like stylet, play a central role in these processes. Previous work on nematode effectors has shown that the apoplasm is targeted during invasion of the host while the cytoplasm is targeted during the induction and the maintenance of the feeding site. A large number of candidate effectors potentially secreted by the nematode into the plant tissues to promote infection have now been identified. This work has shown that the targeting and the role of effectors are more complex than previously thought. This review will not cover the prolific recent findings in nematode effector function but will instead focus on recent selected examples that illustrate the variety of plant cell compartments that effectors are addressed to in order reach their plant targets. PMID:23857349

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

    PubMed

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

    2014-03-01

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

  20. 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

  1. 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.

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

    PubMed Central

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

    2015-01-01

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

  3. 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

  4. 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

    2015-01-01

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

  5. A Diverse Repertoire of CD4 T Cells Targets the Immediate-Early 1 Protein of Human Cytomegalovirus

    PubMed Central

    Ameres, Stefanie; Liang, Xiaoling; Wiesner, Martina; Mautner, Josef; Moosmann, Andreas

    2015-01-01

    T-cell responses to the immediate-early 1 (IE-1) protein of human cytomegalovirus (HCMV) are associated with protection from viral disease. Thus, IE-1 is a promising target for immunotherapy. CD8 T-cell responses to IE-1 are generally strong. In contrast, CD4 T-cell responses to IE-1 were described to be comparatively infrequent or undetectable in HCMV carriers, and information on their target epitopes and their function has been limited. To analyze the repertoire of IE-1-specific CD4 T cells, we expanded them from healthy donors with autologous IE-1-expressing mini-Epstein–Barr virus-transformed B-cell lines and established IE-1-specific CD4 T-cell clones. Clones from seven out of seven HCMV-positive donors recognized endogenously processed IE-1 epitopes restricted through HLA-DR, DQ, or DP. Three to seven IE-1 epitopes were recognized per donor. Cumulatively, about 27 different HLA/peptide class II complexes were recognized by 117 IE-1-specific clones. Our results suggest that a highly diversified repertoire of IE-1-specific CD4 T cells targeting multiple epitopes is usually present in healthy HCMV carriers. Therefore, multiepitope approaches to immunomonitoring and immunotherapy will make optimal use of this potentially important class of HCMV-specific effector cells. PMID:26635812

  6. 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

  7. Rho GTPases and their effector proteins.

    PubMed Central

    Bishop, A L; Hall, A

    2000-01-01

    Rho GTPases are molecular switches that regulate many essential cellular processes, including actin dynamics, gene transcription, cell-cycle progression and cell adhesion. About 30 potential effector proteins have been identified that interact with members of the Rho family, but it is still unclear which of these are responsible for the diverse biological effects of Rho GTPases. This review will discuss how Rho GTPases physically interact with, and regulate the activity of, multiple effector proteins and how specific effector proteins contribute to cellular responses. To date most progress has been made in the cytoskeleton field, and several biochemical links have now been established between GTPases and the assembly of filamentous actin. The main focus of this review will be Rho, Rac and Cdc42, the three best characterized mammalian Rho GTPases, though the genetic analysis of Rho GTPases in lower eukaryotes is making increasingly important contributions to this field. PMID:10816416

  8. Cell-autonomous effector mechanisms against mycobacterium tuberculosis.

    PubMed

    MacMicking, John D

    2014-10-01

    Few pathogens run the gauntlet of sterilizing immunity like Mycobacterium tuberculosis (Mtb). This organism infects mononuclear phagocytes and is also ingested by neutrophils, both of which possess an arsenal of cell-intrinsic effector mechanisms capable of eliminating it. Here Mtb encounters acid, oxidants, nitrosylating agents, and redox congeners, often exuberantly delivered under low oxygen tension. Further pressure is applied by withholding divalent Fe²⁺, Mn²⁺, Cu²⁺, and Zn²⁺, as well as by metabolic privation in the form of carbon needed for anaplerosis and aromatic amino acids for growth. Finally, host E3 ligases ubiquinate, cationic peptides disrupt, and lysosomal enzymes digest Mtb as part of the autophagic response to this particular pathogen. It is a testament to the evolutionary fitness of Mtb that sterilization is rarely complete, although sufficient to ensure most people infected with this airborne bacterium remain disease-free. PMID:25081628

  9. Immune Effector Mechanisms Implicated in Atherosclerosis: From Mice to Humans

    PubMed Central

    Libby, Peter; Lichtman, Andrew H.; Hansson, Göran K.

    2013-01-01

    According to the traditional view, atherosclerosis results from a passive buildup of cholesterol in the artery wall. Yet, burgeoning evidence implicates inflammation and immune effector mechanisms in the pathogenesis of this disease. Both innate and adaptive immunity operate during atherogenesis and link many traditional risk factors to altered arterial functions. Inflammatory pathways have become targets in the quest for novel preventive and therapeutic strategies against cardiovascular disease, a growing contributor to morbidity and mortality worldwide. Here we review current experimental and clinical knowledge of the pathogenesis of atherosclerosis through an immunological lens and how host defense mechanisms essential for survival of the species actually contribute to this chronic disease but also present new opportunities for its mitigation. PMID:23809160

  10. Cell-Autonomous Effector Mechanisms against Mycobacterium tuberculosis

    PubMed Central

    MacMicking, John D.

    2014-01-01

    Few pathogens run the gauntlet of sterilizing immunity like Mycobacterium tuberculosis (Mtb). This organism infects mononuclear phagocytes and is also ingested by neutrophils, both of which possess an arsenal of cell-intrinsic effector mechanisms capable of eliminating it. Here Mtb encounters acid, oxidants, nitrosylating agents, and redox congeners, often exuberantly delivered under low oxygen tension. Further pressure is applied by withholding divalent Fe2+, Mn2+, Cu2+, and Zn2+, as well as by metabolic privation in the form of carbon needed for anaplerosis and aromatic amino acids for growth. Finally, host E3 ligases ubiquinate, cationic peptides disrupt, and lysosomal enzymes digest Mtb as part of the autophagic response to this particular pathogen. It is a testament to the evolutionary fitness of Mtb that sterilization is rarely complete, although sufficient to ensure most people infected with this airborne bacterium remain disease-free. PMID:25081628

  11. Role of mTOR Downstream Effector Signaling Molecules in Francisella Tularensis Internalization by Murine Macrophages

    PubMed Central

    Edwards, Michael W.; Aultman, James A.; Harber, Gregory; Bhatt, Jay M.; Sztul, Elizabeth; Xu, Qingan; Zhang, Ping; Michalek, Suzanne M.; Katz, Jannet

    2013-01-01

    Francisella tularensis is an infectious, gram-negative, intracellular microorganism, and the cause of tularemia. Invasion of host cells by intracellular pathogens like Francisella is initiated by their interaction with different host cell membrane receptors and the rapid phosphorylation of different downstream signaling molecules. PI3K and Syk have been shown to be involved in F. tularensis host cell entry, and both of these signaling molecules are associated with the master regulator serine/threonine kinase mTOR; yet the involvement of mTOR in F. tularensis invasion of host cells has not been assessed. Here, we report that infection of macrophages with F. tularensis triggers the phosphorylation of mTOR downstream effector molecules, and that signaling via TLR2 is necessary for these events. Inhibition of mTOR or of PI3K, ERK, or p38, but not Akt signaling, downregulates the levels of phosphorylation of mTOR downstream targets, and significantly reduces the number of F. tularensis cells invading macrophages. Moreover, while phosphorylation of mTOR downstream effectors occurs via the PI3K pathway, it also involves PLCγ1 and Ca2+ signaling. Furthermore, abrogation of PLC or Ca2+ signaling revealed their important role in the ability of F. tularensis to invade host cells. Together, these findings suggest that F. tularensis invasion of primary macrophages utilize a myriad of host signaling pathways to ensure effective cell entry. PMID:24312679

  12. IFNγR signaling in non-T cell targets regulates T cell-mediated intestinal inflammation through multiple mechanisms

    PubMed Central

    Do, Jeong-su; Asosingh, Kewal; Baldwin, William M.; Min, Booki

    2014-01-01

    Naïve CD4 T cells transferred into lymphopenic mice undergo spontaneous proliferation and induce chronic inflammation in the intestine. Cellular mechanisms regulating the proliferative and inflammatory processes are not fully understood. In this study, we report that IFNγ signaling in host cells plays a major role in limiting both T cell expansion and T cell-induced intestinal inflammation. However, the role for IFNγ appears to be distinct depending on the target cells. IFNγ signaling in DCs controls T cell expansion, while IFNγ signaling in neutrophils seems to regulate both T cell expansion and inflammation. IFNγ signaling in non-hematopoietic cells may control inflammation. Therefore, our results suggest novel immunoregulatory functions for IFNγ to orchestrate colitogenic T cell responses through its distinct action on different non-T cell target cells. PMID:24523506

  13. 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…

  14. 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.

  15. New Insights into Molecular Ehrlichia chaffeensis-Host Interactions

    PubMed Central

    Wakeel, Abdul; Zhu, Bing; Yu, Xue-jie; McBride, Jere W.

    2010-01-01

    Ehrlichia chaffeensis is an obligately intracellular bacterium that exhibits tropism for mononuclear phagocytes and survives by reprogramming the host cell. Here we review new information regarding the newly characterized effector molecules and the complex network of molecular host-pathogen interactions that the organism exploits enabling it to thrive and persist intracellularly. PMID:20116446

  16. Variable expression of the Stagonospora nodorum effector SnToxA among isolates is correlated with levels of disease susceptibility in wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Most research on host-pathogen interactions is focused on mechanisms of resistance, but less is known regarding mechanisms of susceptibility. The wheat-Stagonospora nodorum pathosystem involves pathogen-produced effectors, also known as host-selective toxins, that interact with corresponding dominan...

  17. Two Putatively Homoeologous Wheat Genes Mediate Recognition of SnTox3 to Confer Effector-triggered Susceptibility to Stagonospora nodorum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The pathogen Stagonospora nodorum produces multiple virulence effectors, also known as host-selective toxins (HSTs), that interact with corresponding host sensitivity genes in an inverse gene-for-gene manner to cause the disease Stagonospora nodorum blotch (SNB) in wheat. In this study, a novel sens...

  18. Prevalence and importance of sensitivity to the Stagonospora nodorum necrotrophic effector SnTox3, in current Western Australian wheat cultivars

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Stagonospora nodorum is a major pathogen of wheat in many parts of the world and particularly in Western Australia. The pathosystem is characterised by interactions of multiple pathogen necrotrophic effectors (NEs) (formerly host-specific toxins) with corresponding dominant host sensitivity loci. T...

  19. 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

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

    PubMed

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

    2013-09-01

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

  1. 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...

  2. 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...

  3. Bacterial Effector Nanoparticles as Breast Cancer Therapeutics.

    PubMed

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

    2016-03-01

    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. PMID:26800341

  4. 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

  5. Pseudomonas syringae Effector HopF2 Suppresses Arabidopsis Immunity by Targeting BAK1

    PubMed Central

    Zhou, Jinggeng; Wu, Shujing; Chen, Xin; Liu, Chenglong; Sheen, Jen; Shan, Libo; He, Ping

    2014-01-01

    Summary Pseudomonas syringae delivers a plethora of effector proteins into host cells to sabotage immune responses and modulate physiology to favor infection. We have previously shown that P. syringae pv. tomato DC3000 effector HopF2 suppresses Arabidopsis innate immunity triggered by multiple microbe-associated molecular patterns (MAMP) at the plasma membrane. We show here that HopF2 possesses distinct mechanisms in the suppression of two branches of MAMP-activated MAP kinase (MPK) cascades. Besides blocking MKK5 (MPK kinase 5) activation in the MEKK1/MEKKs-MKK4/5-MPK3/6 cascade, HopF2 targets additional component(s) upstream of MEKK1 in the MEKK1-MKK1/2-MPK4 cascade and plasma membrane-localized receptor-like cytoplasmic kinase BIK1 and its homologs. We further show that HopF2 directly targets BAK1, a plasma membrane-localized receptor-like kinase involved in multiple MAMP signaling. The interaction between BAK1 and HopF2 or two other P. syringae effectors AvrPto and AvrPtoB, was confirmed in vivo and in vitro. Consistent with BAK1 as a physiological target of AvrPto, AvrPtoB and HopF2, the strong growth defects or lethality associated with ectopic expression of these effectors in wild-type Arabidopsis transgenic plants were largely alleviated in bak1 mutant plants. Thus, our results provide genetic evidence to further support that BAK1 is a physiological target of AvrPto, AvrPtoB and HopF2. Identification of BAK1 as an additional target of HopF2 virulence not only explains HopF2 suppression of multiple MAMP signaling at the plasma membrane, but also supports the notion that pathogen virulence effectors act through multiple targets in host cells. PMID:24237140

  6. The Pseudomonas syringae effector HopF2 suppresses Arabidopsis immunity by targeting BAK1.

    PubMed

    Zhou, Jinggeng; Wu, Shujing; Chen, Xin; Liu, Chenglong; Sheen, Jen; Shan, Libo; He, Ping

    2014-01-01

    Pseudomonas syringae delivers a plethora of effector proteins into host cells to sabotage immune responses and modulate physiology to favor infection. The P. syringae pv. tomato DC3000 effector HopF2 suppresses Arabidopsis innate immunity triggered by multiple microbe-associated molecular patterns (MAMP) at the plasma membrane. We show here that HopF2 possesses distinct mechanisms for suppression of two branches of MAMP-activated MAP kinase (MAPK) cascades. In addition to blocking MKK5 (MAPK kinase 5) activation in the MEKK1 (MAPK kinase kinase 1)/MEKKs-MKK4/5-MPK3/6 cascade, HopF2 targets additional component(s) upstream of MEKK1 in the MEKK1-MKK1/2-MPK4 cascade and the plasma membrane-localized receptor-like cytoplasmic kinase BIK1 and its homologs. We further show that HopF2 directly targets BAK1, a plasma membrane-localized receptor-like kinase that is involved in multiple MAMP signaling. The interaction between BAK1 and HopF2 and between two other P. syringae effectors, AvrPto and AvrPtoB, was confirmed in vivo and in vitro. Consistent with BAK1 as a physiological target of AvrPto, AvrPtoB and HopF2, the strong growth defects or lethality associated with ectopic expression of these effectors in wild-type Arabidopsis transgenic plants were largely alleviated in bak1 mutant plants. Thus, our results provide genetic evidence to show that BAK1 is a physiological target of AvrPto, AvrPtoB and HopF2. Identification of BAK1 as an additional target of HopF2 virulence not only explains HopF2 suppression of multiple MAMP signaling at the plasma membrane, but also supports the notion that pathogen virulence effectors act through multiple targets in host cells. PMID:24237140

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

    PubMed Central

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

    2013-01-01

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

  8. 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

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

    PubMed Central

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

    2016-01-01

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

  10. Diversifying selection in the wheat stem rust fungus acts predominantly on pathogen-associated gene families and reveals candidate effectors.

    PubMed

    Sperschneider, Jana; Ying, Hua; Dodds, Peter N; Gardiner, Donald M; Upadhyaya, Narayana M; Singh, Karam B; Manners, John M; Taylor, Jennifer M

    2014-01-01

    Plant pathogens cause severe losses to crop plants and threaten global food production. One striking example is the wheat stem rust fungus, Puccinia graminis f. sp. tritici, which can rapidly evolve new virulent pathotypes in response to resistant host lines. Like several other filamentous fungal and oomycete plant pathogens, its genome features expanded gene families that have been implicated in host-pathogen interactions, possibly encoding effector proteins that interact directly with target host defense proteins. Previous efforts to understand virulence largely relied on the prediction of secreted, small and cysteine-rich proteins as candidate effectors and thus delivered an overwhelming number of candidates. Here, we implement an alternative analysis strategy that uses the signal of adaptive evolution as a line of evidence for effector function, combined with comparative information and expression data. We demonstrate that in planta up-regulated genes that are rapidly evolving are found almost exclusively in pathogen-associated gene families, affirming the impact of host-pathogen co-evolution on genome structure and the adaptive diversification of specialized gene families. In particular, we predict 42 effector candidates that are conserved only across pathogens, induced during infection and rapidly evolving. One of our top candidates has recently been shown to induce genotype-specific hypersensitive cell death in wheat. This shows that comparative genomics incorporating the evolutionary signal of adaptation is powerful for predicting effector candidates for laboratory verification. Our system can be applied to a wide range of pathogens and will give insight into host-pathogen dynamics, ultimately leading to progress in strategies for disease control. PMID:25225496

  11. 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

  12. Alternative splicing of a multi-drug transporter from Pseudoperonospora cubensis generates an RXLR effector protein that elicits a rapid cell death.

    PubMed

    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

  13. 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

  14. TAL effector-mediated genome visualization (TGV).

    PubMed

    Miyanari, Yusuke

    2014-09-01

    The three-dimensional remodeling of chromatin within nucleus is being recognized as determinant for genome regulation. Recent technological advances in live imaging of chromosome loci begun to explore the biological roles of the movement of the chromatin within the nucleus. To facilitate better understanding of the functional relevance and mechanisms regulating genome architecture, we applied transcription activator-like effector (TALE) technology to visualize endogenous repetitive genomic sequences in mouse cells. The application, called TAL effector-mediated genome visualization (TGV), allows us to label specific repetitive sequences and trace nuclear remodeling in living cells. Using this system, parental origin of chromosomes was specifically traced by distinction of single-nucleotide polymorphisms (SNPs). This review will present our approaches to monitor nuclear dynamics of target sequences and highlights key properties and potential uses of TGV. PMID:24704356

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

    PubMed Central

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

    2012-01-01

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

  16. 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.

  17. Exploiting viral cell-targeting abilities in a single polypeptide, non-infectious, recombinant vehicle for integrin-mediated DNA delivery and gene expression.

    PubMed

    Arís, A; Feliu, J X; Knight, A; Coutelle, C; Villaverde, A

    2000-06-20

    A recombinant, multifunctional protein has been designed for optimized, cell-targeted DNA delivery and gene expression in mammalian cells. This hybrid construct comprises a viral peptide ligand for integrin alpha(V)beta(3) binding, a DNA-condensing poly-L-lysine domain, and a complete, functional beta-galactosidase protein that serves simultaneously as purification tag and DNA-shielding agent. This recombinant protein is stable; it has been produced successfully in Escherichia coli and can be purified in a single step by affinity chromatography. At optimal molar ratios, mixtures of this vector and a luciferase-reporter plasmid form stable complexes that transfect cultured cells. After exposure to these cell-targeted complexes, steady levels of gene expression are observed for more than 3 days after transfection, representing between 20 and 40% of those achieved with untargeted, lipid-based DNA-condensing agents. The principle to include viral motifs for cell infection in single polypeptide recombinant proteins represents a promising approach towards the design of non-viral modular DNA transfer vectors that conserve the cell-target- ing specificity of native viruses and that do not need further processing after bioproduction in a recombinant host. PMID:10799995

  18. 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.

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

    PubMed Central

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

    2012-01-01

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

  20. Overexpression of a Phytophthora Cytoplasmic CRN Effector Confers Resistance to Disease, Salinity and Drought in Nicotiana benthamiana.

    PubMed

    Rajput, Nasir Ahmed; Zhang, Meixiang; Shen, Danyu; Liu, Tingli; Zhang, Qimeng; Ru, Yanyan; Sun, Peng; Dou, Daolong

    2015-12-01

    The Crinkler (CRN) effector family is produced by oomycete pathogens and may manipulate host physiological and biochemical events inside host cells. Here, PsCRN161 was identified from Phytophthora sojae based on its broad and strong cell death suppression activities. The effector protein contains two predicted nuclear localization signals and localized to nuclei of plant cells, indicating that it may target plant nuclei to modify host cell physiology and function. The chimeric gene GFP:PsCRN161 driven by the Cauliflower mosaic virus (CaMV) 35S promoter was introduced into Nicotiana benthamiana. The four independent PsCRN161-transgenic lines exhibited increased resistance to two oomycete pathogens (P. parasitica and P. capsici) and showed enhanced tolerance to salinity and drought stresses. Digital gene expression profiling analysis showed that defense-related genes, including ABC transporters, Cyt P450 and receptor-like kinases (RLKs), were significantly up-regulated in PsCRN161-transgenic plants compared with GFP (green fluorescent protein) lines, implying that PsCRN161 expression may protect plants from biotic and abiotic stresses by up-regulation of many defense-related genes. The results reveal previously unknown functions of the oomycete effectors, suggesting that the pathogen effectors could be directly used as functional genes for plant molecular breeding for enhancement of tolerance to biotic and abiotic stresses. PMID:26546319

  1. The Salmonella effector protein SifA plays a dual role in virulence

    PubMed Central

    Zhao, Weidong; Moest, Thomas; Zhao, Yaya; Guilhon, Aude-Agnès; Buffat, Christophe; Gorvel, Jean-Pierre; Méresse, Stéphane

    2015-01-01

    The virulence of Salmonella relies on the expression of effector proteins that the bacterium injects inside infected cells. Salmonella enters eukaryotic cells and resides in a vacuolar compartment on which a number of effector proteins such as SifA are found. SifA plays an essential role in Salmonella virulence. It is made of two distinct domains. The N-terminal domain of SifA interacts with the host protein SKIP. This interaction regulates vacuolar membrane dynamics. The C-terminal has a fold similar to other bacterial effector domains having a guanine nucleotide exchange factor activity. Although SifA interacts with RhoA, it does not stimulate the dissociation of GDP and the activation of this GTPase. Hence it remains unknown whether the C-terminal domain contributes to the function of SifA in virulence. We used a model of SKIP knockout mice to show that this protein mediates the host susceptibility to salmonellosis and to establish that SifA also contributes to Salmonella virulence independently of its interaction with SKIP. We establish that the C-terminal domain of SifA mediates this SKIP-independent contribution. Moreover, we show that the two domains of SifA are functionally linked and participate to the same signalling cascade that supports Salmonella virulence. PMID:26268777

  2. 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

  3. MARTX effector cross kingdom activation by Golgi-associated ADP-ribosylation factors.

    PubMed

    Kim, Byoung Sik; Satchell, Karla J F

    2016-08-01

    Vibrio vulnificus infects humans and causes lethal septicemia. The primary virulence factor is a multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin consisting of conserved repeats-containing regions and various effector domains. Recent genomic analyses for the newly emerged V. vulnificus biotype 3 strain revealed that its MARTX toxin has two previously unknown effector domains. Herein, we characterized one of these domains, Domain X (DmXVv ). A structure-based homology search revealed that DmXVv belongs to the C58B cysteine peptidase subfamily. When ectopically expressed in cells, DmXVv was autoprocessed and induced cytopathicity including Golgi dispersion. When the catalytic cysteine or the region flanking the scissile bond was mutated, both autoprocessing and cytopathicity were significantly reduced indicating that DmXVv cytopathicity is activated by amino-terminal autoprocessing. Consistent with this, host cell protein export was affected by Vibrio cells producing a toxin with wild-type, but not catalytically inactive, DmXVv . DmXVv was found to localize to Golgi and to directly interact with Golgi-associated ADP-ribosylation factors ARF1, ARF3 and ARF4, although ARF binding was not necessary for the subcellular localization. Rather, this interaction was found to induce autoprocessing of DmXVv . These data demonstrate that the V. vulnificus hijacks the host ARF proteins to activate the cytopathic DmXVv effector domain of MARTX toxin. PMID:26780191

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

    PubMed Central

    Armentrout, Erin I.; Rietsch, Arne

    2016-01-01

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

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

    PubMed

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

    2013-11-01

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

  6. 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

  7. 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. PMID:26829249

  8. Specific Conformational States of Ras GTPase upon Effector Binding

    PubMed Central

    2012-01-01

    To uncover the structural and dynamical determinants involved in the highly specific binding of Ras GTPase to its effectors, the conformational states of Ras in uncomplexed form and complexed to the downstream effectors Byr2, PI3Kγ, PLCε, and RalGDS were investigated using molecular dynamics and cross-comparison of the trajectories. The subtle changes in the dynamics and conformations of Ras upon effector binding require an analysis that targets local changes independent of global motions. Using a structural alphabet, a computational procedure is proposed to quantify local conformational changes. Positions detected by this approach were characterized as either specific for a particular effector, specific for an effector domain type, or as effector unspecific. A set of nine structurally connected residues (Ras residues 5–8, 32–35, 39–42, 55–59, 73–78, and 161–165), which link the effector binding site to the distant C-terminus, changed dynamics upon effector binding, indicating a potential effector-unspecific signaling route within the Ras structure. Additional conformational changes were detected along the N-terminus of the central β-sheet. Besides the Ras residues at the effector interface (e.g., D33, E37, D38, and Y40), which adopt effector-specific local conformations, the binding signal propagates from the interface to distant hot-spot residues, in particular to Y5 and D57. The results of this study reveal possible conformational mechanisms for the stabilization of the active state of Ras upon downstream effector binding and for the structural determinants responsible for effector specificity. PMID:23316125

  9. 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.

  10. 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. PMID:26845339

  11. 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

  12. Tricholoma vaccinum host communication during ectomycorrhiza formation.

    PubMed

    Wagner, Katharina; Linde, Jörg; Krause, Katrin; Gube, Matthias; Koestler, Tina; Sammer, Dominik; Kniemeyer, Olaf; Kothe, Erika

    2015-11-01

    The genome sequence of Tricholoma vaccinum was obtained to predict its secretome in order to elucidate communication of T. vaccinum with its host tree spruce (Picea abies) in interkingdom signaling. The most prominent protein domains within the 206 predicted secreted proteins belong to energy and nutrition (52%), cell wall degradation (19%) and mycorrhiza establishment (9%). Additionally, we found small secreted proteins that show typical features of effectors potentially involved in host communication. From the secretome, 22 proteins could be identified, two of which showed higher protein abundances after spruce root exudate exposure, while five were downregulated in this treatment. The changes in T. vaccinum protein excretion with first recognition of the partner were used to identify small secreted proteins with the potential to act as effectors in the mutually beneficial symbiosis. Our observations support the hypothesis of a complex communication network including a cocktail of communication molecules induced long before physical contact of the partners. PMID:26449385

  13. Separation of effector cells mediating antibody-dependent cellular cytotoxicity (ADC) to erythrocyte targets from those mediating ADC to tumor targets.

    PubMed

    Pollack, S B; Nelson, K; Grausz, J D

    1976-04-01

    Murine spleen cells mediate antibody-dependent cellular cytotoxicity (ADC) both to erythrocyte targets in a 51Cr release assay and to syngeneic tumor targets in a microcytotoxicity assay. The effector cells active in the two ADC assays can be separated by passage of the spleen cells through columns of Sephadex G-10 at 37 degrees C. Cells mediating ADC to sarcoma cells did not adhere to the G-10 and were recovered in the column effluent. These nonadherent cells were not cytotoxic to antibody-coated chicken red blood cells. Spleen cells which mediated ADC in a 51Cr release assay to the red cell targets adhered to G-10. Adherent effector cells could subsequently be recovered from the columns by elution with 5 X 10(-4) M EDTA. PMID:815438

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

    PubMed

    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

  15. 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

  16. CD8+ Effector T Cell Migration to Pancreatic Islet Grafts Is Dependent on Cognate Antigen Presentation by Donor Graft Cells.

    PubMed

    Zhang, Qianqian; Dai, Hehua; Yatim, Karim M; Abou-Daya, Khodor; Williams, Amanda L; Oberbarnscheidt, Martin H; Camirand, Geoffrey; Rudd, Christopher E; Lakkis, Fadi G

    2016-08-15

    Pancreatic islet transplantation is a promising therapy for diabetes, but acute rejection of the islets by host effector T cells has hindered clinical application. In this study, we addressed the mechanisms of CD8(+) effector T cell migration to islet grafts because interrupting this step is key to preventing rejection. We found that effector T cell migration to revascularized islet transplants in mice is dependent on non-self Ag recognition rather than signaling via Gαi-coupled chemokine receptors. Presentation of non-self Ag by donor cells was necessary for migration, whereas Ag presentation by recipient cells was dispensable. We also observed that deficiency of SKAP1, an immune cell adaptor downstream of the TCR and important for integrin activation, prolongs allograft survival but does not reduce effector T cell migration to the graft. Therefore, effector T cell migration to transplanted islets is Ag driven, not chemokine driven, but SKAP1 does not play a critical role in this process. PMID:27357151

  17. Formins as effector proteins of Rho GTPases

    PubMed Central

    Kühn, Sonja; Geyer, Matthias

    2014-01-01

    Formin proteins were recognized as effectors of Rho GTPases some 15 years ago. They contribute to different cellular actin cytoskeleton structures by their ability to polymerize straight actin filaments at the barbed end. While not all formins necessarily interact with Rho GTPases, a subgroup of mammalian formins, termed Diaphanous-related formins or DRFs, were shown to be activated by small GTPases of the Rho superfamily. DRFs are autoinhibited in the resting state by an N- to C-terminal interaction that renders the central actin polymerization domain inactive. Upon the interaction with a GTP-bound Rho, Rac, or Cdc42 GTPase, the C-terminal autoregulation domain is displaced from its N-terminal recognition site and the formin becomes active to polymerize actin filaments. In this review we discuss the current knowledge on the structure, activation, and function of formin-GTPase interactions for the mammalian formin families Dia, Daam, FMNL, and FHOD. We describe both direct and indirect interactions of formins with GTPases, which lead to formin activation and cytoskeletal rearrangements. The multifaceted function of formins as effector proteins of Rho GTPases thus reflects the diversity of the actin cytoskeleton in cells. PMID:24914801

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

    PubMed

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

    2016-03-25

    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

  19. Structural Analysis of an Avr4 Effector Ortholog Offers Insight into Chitin Binding and Recognition by the Cf-4 Receptor.

    PubMed

    Kohler, Amanda C; Chen, Li-Hung; Hurlburt, Nicholas; Salvucci, Anthony; Schwessinger, Benjamin; Fisher, Andrew J; Stergiopoulos, Ioannis

    2016-08-01

    Chitin is a key component of fungal cell walls and a potent inducer of innate immune responses. Consequently, fungi may secrete chitin-binding lectins, such as the Cf-Avr4 effector protein from the tomato pathogen Cladosporium fulvum, to shield chitin from host-derived chitinases during infection. Homologs of Cf-Avr4 are found throughout Dothideomycetes, and despite their modest primary sequence identity, many are perceived by the cognate tomato immune receptor Cf-4. Here, we determined the x-ray crystal structure of Pf-Avr4 from the tomato pathogen Pseudocercospora fuligena, thus providing a three-dimensional model of an Avr4 effector protein. In addition, we explored structural, biochemical, and functional aspects of Pf-Avr4 and Cf-Avr4 to further define the biology of core effector proteins and outline a conceptual framework for their pleiotropic recognition by single immune receptors. We show that Cf-Avr4 and Pf-Avr4 share functional specificity in binding (GlcNAc)6 and in providing protection against plant- and microbial-derived chitinases, suggesting a broader role beyond deregulation of host immunity. Furthermore, structure-guided site-directed mutagenesis indicated that residues in Pf-Avr4 important for binding chitin do not directly influence recognition by Cf-4 and further suggested that the property of recognition is structurally separated or does not fully overlap with the virulence function of the effector. PMID:27401545

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

    PubMed

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

    2016-05-01

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

  1. Escape from suppression: tumor-specific effector cells outcompete regulatory T cells following stem-cell transplantation

    PubMed Central

    Mirmonsef, Paria; Tan, Gladys; Zhou, Gang; Morino, Tricia; Noonan, Kimberly; Borrello, Ivan

    2008-01-01

    Immune reconstitution of autologous hematopoietic stem-cell transplant recipients with the progeny of mature T cells in the graft leads to profound changes in the emerging functional T-cell repertoire. In the steady state, the host is frequently tolerant to tumor antigens, reflecting dominant suppression of naive and effector T cells by regulatory T cells (Tregs). We examined the relative frequency and function of these 3 components within the tumor-specific T-cell compartment during immune reconstitution. Grafts from tumor-bearing donors exerted a significant antitumor effect in irradiated, syngeneic tumor-bearing recipients. This was associated with dramatic clonal expansion and interferon-γ (IFNγ) production by previously tolerant tumor-specific T cells. While donor-derived Tregs expanded in recipients, they did not inhibit the antigen-driven expansion of effector T cells in the early posttransplantation period. Indeed, the repopulation of tumor-specific effector T cells significantly exceeded that of Tregs, the expansion of which was limited by IL-2 availability. Although the intrinsic suppressive capacity of Tregs remained intact, their diminished frequency was insufficient to suppress effector cell function. These findings provide an explanation for the reversal of tolerance leading to tumor rejection in transplant recipients and likely contribute to the efficacy of adoptive T-cell therapies in lymphopenic hosts. PMID:18063750

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

    PubMed

    Shi, Xingqi; Halder, Partho; Yavuz, Halenur; Jahn, Reinhard; Shuman, Howard A

    2016-08-01

    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

  3. The FTF gene family regulates virulence and expression of SIX effectors in Fusarium oxysporum.

    PubMed

    Niño-Sánchez, Jonathan; Casado-Del Castillo, Virginia; Tello, Vega; De Vega-Bartol, José J; Ramos, Brisa; Sukno, Serenella A; Díaz Mínguez, José María

    2016-09-01

    The FTF (Fusarium transcription factor) gene family comprises a single copy gene, FTF2, which is present in all the filamentous ascomycetes analysed, and several copies of a close relative, FTF1, which is exclusive to Fusarium oxysporum. An RNA-mediated gene silencing system was developed to target mRNA produced by all the FTF genes, and tested in two formae speciales: F. oxysporum f. sp. phaseoli (whose host is common bean) and F. oxysporum f. sp. lycopersici (whose host is tomato). Quantification of the mRNA levels showed knockdown of FTF1 and FTF2 in randomly isolated transformants of both formae speciales. The attenuation of FTF expression resulted in a marked reduction in virulence, a reduced expression of several SIX (Secreted In Xylem) genes, the best studied family of effectors in F. oxysporum, and lower levels of SGE1 (Six Gene Expression 1) mRNA, the presumptive regulator of SIX expression. Moreover, the knockdown mutants showed a pattern of colonization of the host plant similar to that displayed by strains devoid of FTF1 copies (weakly virulent strains). Gene knockout of FTF2 also resulted in a reduction in virulence, but to a lesser extent. These results demonstrate the role of the FTF gene expansion, mostly the FTF1 paralogues, as a regulator of virulence in F. oxysporum and suggest that the control of effector expression is the mechanism involved. PMID:26817616

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

    PubMed Central

    Havey, James C.

    2015-01-01

    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. PMID:26099583

  5. 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.

  6. 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. PMID:26874289

  7. 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.

  8. 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. PMID:27324482

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

    PubMed Central

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

    2014-01-01

    Summary The gut microbiota co-develops 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-immune system co-regulation, and in order 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. PMID:24950201

  10. The Molecular Basis for Ubiquitin and Ubiquitin-like Specificities in Bacterial Effector Proteases.

    PubMed

    Pruneda, Jonathan N; Durkin, Charlotte H; Geurink, Paul P; Ovaa, Huib; Santhanam, Balaji; Holden, David W; Komander, David

    2016-07-21

    Pathogenic bacteria rely on secreted effector proteins to manipulate host signaling pathways, often in creative ways. CE clan proteases, specific hydrolases for ubiquitin-like modifications (SUMO and NEDD8) in eukaryotes, reportedly serve as bacterial effector proteins with deSUMOylase, deubiquitinase, or, even, acetyltransferase activities. Here, we characterize bacterial CE protease activities, revealing K63-linkage-specific deubiquitinases in human pathogens, such as Salmonella, Escherichia, and Shigella, as well as ubiquitin/ubiquitin-like cross-reactive enzymes in Chlamydia, Rickettsia, and Xanthomonas. Five crystal structures, including ubiquitin/ubiquitin-like complexes, explain substrate specificities and redefine relationships across the CE clan. Importantly, this work identifies novel family members and provides key discoveries among previously reported effectors, such as the unexpected deubiquitinase activity in Xanthomonas XopD, contributed by an unstructured ubiquitin binding region. Furthermore, accessory domains regulate properties such as subcellular localization, as exemplified by a ubiquitin-binding domain in Salmonella Typhimurium SseL. Our work both highlights and explains the functional adaptations observed among diverse CE clan proteins. PMID:27425412

  11. 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

  12. Acquisition of effector-specific and effector-independent components of sequencing skill.

    PubMed

    Berner, Michael P; Hoffman, Joachim

    2009-01-01

    In a serial reaction time task, participants practiced a repeating sequence with 1 hand. In interleaved blocks, they responded to random sequences with the other hand. Experiment 1 was composed of 5 sessions, each consisting of 30 blocks. Intermanual transfer, reflecting a hand-independent component of sequence knowledge, increased across session. A smaller but significant, nontransferable, and hand-specific component was evident in each session and did not increase with practice. Experiment 2 comprised only 1 session. Uninterrupted practice (no interleaved random blocks) improved hand-independent sequence learning in comparison with interrupted practice (as implemented in Experiment 1), whereas hand-specific sequence learning was unaffected by this between-subjects manipulation. These findings suggest separate mechanisms for effector-independent sequence learning and effector-specific acquisition of optimized response coarticulation. PMID:19073469

  13. 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.

  14. Tissue Specific Heterogeneity in Effector Immune Cell Response

    PubMed Central

    Tufail, Saba; Badrealam, Khan Farheen; Sherwani, Asif; Gupta, Umesh D.; Owais, Mohammad

    2013-01-01

    Post pathogen invasion, migration of effector T-cell subsets to specific tissue locations is of prime importance for generation of robust immune response. Effector T cells are imprinted with distinct “homing codes” (adhesion molecules and chemokine receptors) during activation which regulate their targeted trafficking to specific tissues. Internal cues in the lymph node microenvironment along with external stimuli from food (vitamin A) and sunlight (vitamin D3) prime dendritic cells, imprinting them to play centre stage in the induction of tissue tropism in effector T cells. B cells as well, in a manner similar to effector T cells, exhibit tissue-tropic migration. In this review, we have focused on the factors regulating the generation and migration of effector T cells to various tissues along with giving an overview of tissue tropism in B cells. PMID:23986763

  15. The FonSIX6 gene acts as an avirulence effector in the Fusarium oxysporum f. sp. niveum - watermelon pathosystem.

    PubMed

    Niu, Xiaowei; Zhao, Xiaoqiang; Ling, Kai-Shu; Levi, Amnon; Sun, Yuyan; Fan, Min

    2016-01-01

    When infecting a host plant, the fungus Fusarium oxysporum secretes several effector proteins into the xylem tissue to promote virulence. However, in a host plant with an innate immune system involving analogous resistance proteins, the fungus effector proteins may trigger resistance, rather than promoting virulence. Identity of the effector genes of Fusarium oxysporum f. sp. niveum (Fon) races that affect watermelon (Citrullus lanatus) are currently unknown. In this study, the SIX6 (secreted in xylem protein 6) gene was identified in Fon races 0 and 1 but not in the more virulent Fon race 2. Disrupting the FonSIX6 gene in Fon race 1 did not affect the sporulation or growth rate of the fungus but significantly enhanced Fon virulence in watermelon, suggesting that the mutant ΔFon1SIX6 protein allowed evasion of R protein-mediated host resistance. Complementation of the wild-type race 2 (which lacks FonSIX6) with FonSIX6 reduced its virulence. These results provide evidence supporting the hypothesis that FonSIX6 is an avirulence gene. The identification of FonSix6 as an avirulence factor may be a first step in understanding the mechanisms of Fon virulence and resistance in watermelon and further elucidating the role of Six6 in Fusarium-plant interactions. PMID:27320044

  16. The FonSIX6 gene acts as an avirulence effector in the Fusarium oxysporum f. sp. niveum - watermelon pathosystem

    PubMed Central

    Niu, Xiaowei; Zhao, Xiaoqiang; Ling, Kai-Shu; Levi, Amnon; Sun, Yuyan; Fan, Min

    2016-01-01

    When infecting a host plant, the fungus Fusarium oxysporum secretes several effector proteins into the xylem tissue to promote virulence. However, in a host plant with an innate immune system involving analogous resistance proteins, the fungus effector proteins may trigger resistance, rather than promoting virulence. Identity of the effector genes of Fusarium oxysporum f. sp. niveum (Fon) races that affect watermelon (Citrullus lanatus) are currently unknown. In this study, the SIX6 (secreted in xylem protein 6) gene was identified in Fon races 0 and 1 but not in the more virulent Fon race 2. Disrupting the FonSIX6 gene in Fon race 1 did not affect the sporulation or growth rate of the fungus but significantly enhanced Fon virulence in watermelon, suggesting that the mutant ΔFon1SIX6 protein allowed evasion of R protein-mediated host resistance. Complementation of the wild-type race 2 (which lacks FonSIX6) with FonSIX6 reduced its virulence. These results provide evidence supporting the hypothesis that FonSIX6 is an avirulence gene. The identification of FonSix6 as an avirulence factor may be a first step in understanding the mechanisms of Fon virulence and resistance in watermelon and further elucidating the role of Six6 in Fusarium-plant interactions. PMID:27320044

  17. E2∼Ub conjugates regulate the kinase activity of Shigella effector OspG during pathogenesis

    PubMed Central

    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

    2014-01-01

    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 co-crystal 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. PMID:24446487

  18. 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.

  19. 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.

  20. 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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  3. 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

  4. Is B Cell-Targeted Therapy Effective in Systemic Lupus Erythematosus?

    PubMed

    Paran, Daphna; Naparstek, Yaakov

    2015-02-01

    In the past decade we have witnessed a dramatic change in the management of autoimmune diseases, such as rheumatoid arthritis, due to the development of new biologic drugs designed to target key mediators in the autoimmune process. However, the development of similar target-specific drugs for the management of SLE has not been as successful. The B cell has long been considered central to the pathogenesis of SLE and has been regarded as an important target for biologic drugs. Several B cell-targeted drugs have been developed and although the mechanisms seem promising, most of the studies published to date have failed to achieve their primary endpoints, leading to an ongoing debate regarding the role of B cell therapy in SLE. The present report discusses the pros and cons of B cell-targeted therapy in SLE, reviews the clinical studies, and offers possible explanations forthe discrepancies between randomized control studies and real-life experience. PMID:26223085

  5. Post-translational cleavage and self-interaction of the phytoplasma effector SAP11.

    PubMed

    Lu, Yen-Ting; Cheng, Kai-Tan; Jiang, Shin-Ying; Yang, Jun-Yi

    2014-04-28

    Phytoplasmas are insect-transmitted intracellular plant bacterial pathogens that secrete effector molecules into host cells that interfere with the host's developmental or metabolic processes. Recently, the secreted Aster Yellows phytoplasma strain Witches' Broom protein11 (SAP11) has been shown to act as a virulence factor that alters the development, hormone biosynthesis, phosphate (Pi) homeostasis, and defense responses in the affected plants. We found that SAP11 undergoes proteolytic processing in planta and self-interaction in vitro. These biochemical studies provide foundational insights necessary for the functional characterization of SAP11; however, the biological relevance of post-translational cleavage and self-interaction of SAP11 to its role as a virulence factor warrants further investigation. PMID:24776784

  6. Specific in planta recognition of two GKLR proteins of the downy mildew Bremia lactucae revealed in a large effector screen in lettuce.

    PubMed

    Stassen, Joost H M; den Boer, Erik; Vergeer, Pim W J; Andel, Annemiek; Ellendorff, Ursula; Pelgrom, Koen; Pel, Mathieu; Schut, Johan; Zonneveld, Olaf; Jeuken, Marieke J W; Van den Ackerveken, Guido

    2013-11-01

    Breeding lettuce (Lactuca sativa) for resistance to the downy mildew pathogen Bremia lactucae is mainly achieved by introgression of dominant downy mildew resistance (Dm) genes. New Bremia races quickly render Dm genes ineffective, possibly by mutation of recognized host-translocated effectors or by suppression of effector-triggered immunity. We have previously identified 34 potential RXLR(-like) effector proteins of B. lactucae that were here tested for specific recognition within a collection of 129 B. lactucae-resistant Lactuca lines. Two effectors triggered a hypersensitive response: BLG01 in 52 lines, predominantly L. saligna, and BLG03 in two L. sativa lines containing Dm2 resistance. The N-terminal sequences of BLG01 and BLG03, containing the signal peptide and GKLR variant of the RXLR translocation motif, are not required for in planta recognition but function in effector delivery. The locus responsible for BLG01 recognition maps to the bottom of lettuce chromosome 9, whereas recognition of BLG03 maps in the RGC2 cluster on chromosome 2. Lactuca lines that recognize the BLG effectors are not resistant to Bremia isolate Bl:24 that expresses both BLG genes, suggesting that Bl:24 can suppress the triggered immune responses. In contrast, lettuce segregants displaying Dm2-mediated resistance to Bremia isolate Bl:5 are responsive to BLG03, suggesting that BLG03 is a candidate Avr2 protein. PMID:23883357

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2009-06-01

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

  9. 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. PMID:18804428

  10. 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. PMID:26143263

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

    PubMed

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

    2014-04-01

    Plant pathogens secrete effectors to manipulate their host and facilitate colonization. Fusarium oxysporum f. sp. lycopersici is the causal agent of Fusarium wilt disease in tomato. Upon infection, F. oxysporum f. sp. lycopersici secretes numerous small proteins into the xylem sap (Six proteins). Most Six proteins are unique to F. oxysporum, but Six6 is an exception; a homolog is also present in two Colletotrichum spp. SIX6 expression was found to require living host cells and a knockout of SIX6 in F. oxysporum f. sp. lycopersici compromised virulence, classifying it as a genuine effector. Heterologous expression of SIX6 did not affect growth of Agrobacterium tumefaciens in Nicotiana benthamiana leaves or susceptibility of Arabidopsis thaliana toward Verticillium dahliae, Pseudomonas syringae, or F. oxysporum, suggesting a specific function for F. oxysporum f. sp. lycopersici Six6 in the F. oxysporum f. sp. lycopersici- tomato pathosystem. Remarkably, Six6 was found to specifically suppress I-2-mediated cell death (I2CD) upon transient expression in N. benthamiana, whereas it did not compromise the activity of other cell-death-inducing genes. Still, this I2CD suppressing activity of Six6 does not allow the fungus to overcome I-2 resistance in tomato, suggesting that I-2-mediated resistance is independent from cell death. PMID:24313955

  12. The Legionella pneumophila Effector Protein, LegC7, Alters Yeast Endosomal Trafficking

    PubMed Central

    O’Brien, Kevin M.; Lindsay, Elizabeth L.; Starai, Vincent J.

    2015-01-01

    The intracellular pathogen, Legionella pneumophila, relies on numerous secreted effector proteins to manipulate host endomembrane trafficking events during pathogenesis, thereby preventing fusion of the bacteria-laden phagosome with host endolysosomal compartments, and thus escaping degradation. Upon expression in the surrogate eukaryotic model Saccharomyces cerevisiae, we find that the L. pneumophila LegC7/YlfA effector protein disrupts the delivery of both biosynthetic and endocytic cargo to the yeast vacuole. We demonstrate that the effects of LegC7 are specific to the endosome:vacuole delivery pathways; LegC7 expression does not disrupt other known vacuole-directed pathways. Deletions of the ESCRT-0 complex member, VPS27, provide resistance to the LegC7 toxicity, providing a possible target for LegC7 function in vivo. Furthermore, a single amino acid substitution in LegC7 abrogates both its toxicity and ability to alter endosomal traffic in vivo, thereby identifying a critical functional domain. LegC7 likely inhibits endosomal trafficking during L. pneumophila pathogenesis to prevent entry of the phagosome into the endosomal maturation pathway and eventual fusion with the lysosome. PMID:25643265

  13. Subversion of Cell-Autonomous Immunity and Cell Migration by Legionella pneumophila Effectors

    PubMed Central

    Simon, Sylvia; Hilbi, Hubert

    2015-01-01

    Bacteria trigger host defense and inflammatory processes, such as cytokine production, pyroptosis, and the chemotactic migration of immune cells toward the source of infection. However, a number of pathogens interfere with these immune functions by producing specific so-called “effector” proteins, which are delivered to host cells via dedicated secretion systems. Air-borne Legionella pneumophila bacteria trigger an acute and potential fatal inflammation in the lung termed Legionnaires’ disease. The opportunistic pathogen L. pneumophila is a natural parasite of free-living amoebae, but also replicates in alveolar macrophages and accidentally infects humans. The bacteria employ the intracellular multiplication/defective for organelle trafficking (Icm/Dot) type IV secretion system and as many as 300 different effector proteins to govern host–cell interactions and establish in phagocytes an intracellular replication niche, the Legionella-containing vacuole. Some Icm/Dot-translocated effector proteins target cell-autonomous immunity or cell migration, i.e., they interfere with (i) endocytic, secretory, or retrograde vesicle trafficking pathways, (ii) organelle or cell motility, (iii) the inflammasome and programed cell death, or (iv) the transcription factor NF-κB. Here, we review recent mechanistic insights into the subversion of cellular immune functions by L. pneumophila. PMID:26441958

  14. 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

  15. 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.

  16. Modes of TAL effector-mediated repression

    PubMed Central

    Werner, Jeannette; Gossen, Manfred

    2014-01-01

    Engineered transcription activator-like effectors, or TALEs, have emerged as a new class of designer DNA-binding proteins. Their DNA recognition sites can be specified with great flexibility. When fused to appropriate transcriptional regulatory domains, they can serve as designer transcription factors, modulating the activity of targeted promoters. We created tet operator (tetO)-specific TALEs (tetTALEs), with an identical DNA-binding site as the Tet repressor (TetR) and the TetR-based transcription factors that are extensively used in eukaryotic transcriptional control systems. Different constellations of tetTALEs and tetO modified chromosomal transcription units were analyzed for their efficacy in mammalian cells. We find that tetTALE-silencers can entirely abrogate expression from the strong human EF1α promoter when binding upstream of the transcriptional control sequence. Remarkably, the DNA-binding domain of tetTALE alone can effectively counteract trans-activation mediated by the potent tettrans-activator and also directly interfere with RNA polymerase II transcription initiation from the strong CMV promoter. Our results demonstrate that TALEs can act as highly versatile tools in genetic engineering, serving as trans-activators, trans-silencers and also competitive repressors. PMID:25389273

  17. 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. PMID:26949049

  18. 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.

  19. 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.

  20. 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. PMID:22876962

  1. 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

  2. Potato NPH3/RPT2-Like Protein StNRL1, Targeted by a Phytophthora infestans RXLR Effector, Is a Susceptibility Factor.

    PubMed

    Yang, Lina; McLellan, Hazel; Naqvi, Shaista; He, Qin; Boevink, Petra C; Armstrong, Miles; Giuliani, Licida M; Zhang, Wei; Tian, Zhendong; Zhan, Jiasui; Gilroy, Eleanor M; Birch, Paul R J

    2016-05-01

    Plant pathogens deliver effectors to manipulate host processes. We know little about how fungal and oomycete effectors target host proteins to promote susceptibility, yet such knowledge is vital to understand crop disease. We show that either transient expression in Nicotiana benthamiana, or stable transgenic expression in potato (Solanum tuberosum), of the Phytophthora infestans RXLR effector Pi02860 enhances leaf colonization by the pathogen. Expression of Pi02860 also attenuates cell death triggered by the P. infestans microbe-associated molecular pattern INF1, indicating that the effector suppresses pattern-triggered immunity. However, the effector does not attenuate cell death triggered by Cf4/Avr4 coexpression, showing that it does not suppress all cell death activated by cell surface receptors. Pi02860 interacts in yeast two-hybrid assays with potato NPH3/RPT2-LIKE1 (NRL1), a predicted CULLIN3-associated ubiquitin E3 ligase. Interaction of Pi02860 in planta was confirmed by coimmunoprecipitation and bimolecular fluorescence complementation assays. Virus-induced gene silencing of NRL1 in N. benthamiana resulted in reduced P. infestans colonization and accelerated INF1-mediated cell death, indicating that this host protein acts as a negative regulator of immunity. Moreover, whereas NRL1 virus-induced gene silencing had no effect on the ability of the P. infestans effector Avr3a to suppress INF1-mediated cell death, such suppression by Pi02860 was significantly attenuated, indicating that this activity of Pi02860 is mediated by NRL1. Transient overexpression of NRL1 resulted in the suppression of INF1-mediated cell death and enhanced P. infestans leaf colonization, demonstrating that NRL1 acts as a susceptibility factor to promote late blight disease. PMID:26966171

  3. Potato NPH3/RPT2-Like Protein StNRL1, Targeted by a Phytophthora infestans RXLR Effector, Is a Susceptibility Factor1[OPEN

    PubMed Central

    Yang, Lina; Naqvi, Shaista; Boevink, Petra C.; Armstrong, Miles; Giuliani, Licida M.; Zhan, Jiasui; Birch, Paul R.J.

    2016-01-01

    Plant pathogens deliver effectors to manipulate host processes. We know little about how fungal and oomycete effectors target host proteins to promote susceptibility, yet such knowledge is vital to understand crop disease. We show that either transient expression in Nicotiana benthamiana, or stable transgenic expression in potato (Solanum tuberosum), of the Phytophthora infestans RXLR effector Pi02860 enhances leaf colonization by the pathogen. Expression of Pi02860 also attenuates cell death triggered by the P. infestans microbe-associated molecular pattern INF1, indicating that the effector suppresses pattern-triggered immunity. However, the effector does not attenuate cell death triggered by Cf4/Avr4 coexpression, showing that it does not suppress all cell death activated by cell surface receptors. Pi02860 interacts in yeast two-hybrid assays with potato NPH3/RPT2-LIKE1 (NRL1), a predicted CULLIN3-associated ubiquitin E3 ligase. Interaction of Pi02860 in planta was confirmed by coimmunoprecipitation and bimolecular fluorescence complementation assays. Virus-induced gene silencing of NRL1 in N. benthamiana resulted in reduced P. infestans colonization and accelerated INF1-mediated cell death, indicating that this host protein acts as a negative regulator of immunity. Moreover, whereas NRL1 virus-induced gene silencing had no effect on the ability of the P. infestans effector Avr3a to suppress INF1-mediated cell death, such suppression by Pi02860 was significantly attenuated, indicating that this activity of Pi02860 is mediated by NRL1. Transient overexpression of NRL1 resulted in the suppression of INF1-mediated cell death and enhanced P. infestans leaf colonization, demonstrating that NRL1 acts as a susceptibility factor to promote late blight disease. PMID:26966171

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

    PubMed

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

    2005-08-30

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

  5. 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. PMID:25989853

  6. 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. PMID:2733012

  7. Robotic end-effector for rewaterproofing shuttle tiles

    NASA Technical Reports Server (NTRS)

    Manouchehri, Davoud; Hansen, Joseph M.; Wu, Cheng M.; Yamamoto, Brian S.; Graham, Todd

    1992-01-01

    This paper summarizes work by Rockwell International's Space Systems Division's Robotics Group at Downey, California. The work is part of a NASA-led team effort to automate Space Shuttle rewaterproofing in the Orbiter Processing Facility at the Kennedy Space Center and the ferry facility at the Ames-Dryden Flight Research Facility. Rockwell's effort focuses on the rewaterproofing end-effector, whose function is to inject hazardous dimethylethyloxysilane into thousands of ceramic tiles on the underside of the orbiter after each flight. The paper has five sections. First, it presents background on the present manual process. Second, end-effector requirements are presented, including safety and interface control. Third, a design is presented for the five end-effector systems: positioning, delivery, containment, data management, and command and control. Fourth, end-effector testing and integrating to the total system are described. Lastly, future applications for this technology are discussed.

  8. 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.

  9. Host-pathogen reorganisation during host cell entry by Chlamydia trachomatis.

    PubMed

    Nans, Andrea; Ford, Charlotte; Hayward, Richard D

    2015-01-01

    Chlamydia trachomatis is obligate intracellular bacterial pathogen that remains a significant public health burden worldwide. A critical early event during infection is chlamydial entry into non-phagocytic host epithelial cells. Like other Gram-negative bacteria, C. trachomatis uses a type III secretion system (T3SS) to deliver virulence effector proteins into host cells. These effectors trigger bacterial uptake and promote bacterial survival and replication within the host cell. In this review, we highlight recent cryo-electron tomography that has provided striking insights into the initial interactions between Chlamydia and its host. We describe the polarised structure of extracellular C. trachomatis elementary bodies (EBs), and the supramolecular organisation of T3SS complexes on the EB surface, in addition to the changes in host and pathogen architecture that accompany bacterial internalisation and EB encapsulation into early intracellular vacuoles. Finally, we consider the implications for further understanding the mechanism of C. trachomatis entry and how this might relate to those of other bacteria and viruses. PMID:26320027

  10. Host-pathogen reorganisation during host cell entry by Chlamydia trachomatis

    PubMed Central

    Nans, Andrea; Ford, Charlotte; Hayward, Richard D.

    2015-01-01

    Chlamydia trachomatis is obligate intracellular bacterial pathogen that remains a significant public health burden worldwide. A critical early event during infection is chlamydial entry into non-phagocytic host epithelial cells. Like other Gram-negative bacteria, C. trachomatis uses a type III secretion system (T3SS) to deliver virulence effector proteins into host cells. These effectors trigger bacterial uptake and promote bacterial survival and replication within the host cell. In this review, we highlight recent cryo-electron tomography that has provided striking insights into the initial interactions between Chlamydia and its host. We describe the polarised structure of extracellular C. trachomatis elementary bodies (EBs), and the supramolecular organisation of T3SS complexes on the EB surface, in addition to the changes in host and pathogen architecture that accompany bacterial internalisation and EB encapsulation into early intracellular vacuoles. Finally, we consider the implications for further understanding the mechanism of C. trachomatis entry and how this might relate to those of other bacteria and viruses. PMID:26320027

  11. Epigenetic Control of Effector Gene Expression in the Plant Pathogenic Fungus Leptosphaeria maculans

    PubMed Central

    Soyer, Jessica L.; El Ghalid, Mennat; Glaser, Nicolas; Ollivier, Bénédicte; Linglin, Juliette; Grandaubert, Jonathan; Balesdent, Marie-Hélène; Connolly, Lanelle R.; Freitag, Michael; Rouxel, Thierry; Fudal, Isabelle

    2014-01-01

    Plant pathogens secrete an arsenal of small secreted proteins (SSPs) acting as effectors that modulate host immunity to facilitate infection. SSP-encoding genes are often located in particular genomic environments and show waves of concerted expression at diverse stages of plant infection. To date, little is known about the regulation of their expression. The genome of the Ascomycete Leptosphaeria maculans comprises alternating gene-rich GC-isochores and gene-poor AT-isochores. The AT-isochores harbor mosaics of transposable elements, encompassing one-third of the genome, and are enriched in putative effector genes that present similar expression patterns, namely no expression or low-level expression during axenic cultures compared to strong induction of expression during primary infection of oilseed rape (Brassica napus). Here, we investigated the involvement of one specific histone modification, histone H3 lysine 9 methylation (H3K9me3), in epigenetic regulation of concerted effector gene expression in L. maculans. For this purpose, we silenced the expression of two key players in heterochromatin assembly and maintenance, HP1 and DIM-5 by RNAi. By using HP1-GFP as a heterochromatin marker, we observed that almost no chromatin condensation is visible in strains in which LmDIM5 was silenced by RNAi. By whole genome oligoarrays we observed overexpression of 369 or 390 genes, respectively, in the silenced-LmHP1 and -LmDIM5 transformants during growth in axenic culture, clearly favouring expression of SSP-encoding genes within AT-isochores. The ectopic integration of four effector genes in GC-isochores led to their overexpression during growth in axenic culture. These data strongly suggest that epigenetic control, mediated by HP1 and DIM-5, represses the expression of at least part of the effector genes located in AT-isochores during growth in axenic culture. Our hypothesis is that changes of lifestyle and a switch toward pathogenesis lift chromatin

  12. Epigenetic control of effector gene expression in the plant pathogenic fungus Leptosphaeria maculans.

    PubMed

    Soyer, Jessica L; El Ghalid, Mennat; Glaser, Nicolas; Ollivier, Bénédicte; Linglin, Juliette; Grandaubert, Jonathan; Balesdent, Marie-Hélène; Connolly, Lanelle R; Freitag, Michael; Rouxel, Thierry; Fudal, Isabelle

    2014-03-01

    Plant pathogens secrete an arsenal of small secreted proteins (SSPs) acting as effectors that modulate host immunity to facilitate infection. SSP-encoding genes are often located in particular genomic environments and show waves of concerted expression at diverse stages of plant infection. To date, little is known about the regulation of their expression. The genome of the Ascomycete Leptosphaeria maculans comprises alternating gene-rich GC-isochores and gene-poor AT-isochores. The AT-isochores harbor mosaics of transposable elements, encompassing one-third of the genome, and are enriched in putative effector genes that present similar expression patterns, namely no expression or low-level expression during axenic cultures compared to strong induction of expression during primary infection of oilseed rape (Brassica napus). Here, we investigated the involvement of one specific histone modification, histone H3 lysine 9 methylation (H3K9me3), in epigenetic regulation of concerted effector gene expression in L. maculans. For this purpose, we silenced the expression of two key players in heterochromatin assembly and maintenance, HP1 and DIM-5 by RNAi. By using HP1-GFP as a heterochromatin marker, we observed that almost no chromatin condensation is visible in strains in which LmDIM5 was silenced by RNAi. By whole genome oligoarrays we observed overexpression of 369 or 390 genes, respectively, in the silenced-LmHP1 and -LmDIM5 transformants during growth in axenic culture, clearly favouring expression of SSP-encoding genes within AT-isochores. The ectopic integration of four effector genes in GC-isochores led to their overexpression during growth in axenic culture. These data strongly suggest that epigenetic control, mediated by HP1 and DIM-5, represses the expression of at least part of the effector genes located in AT-isochores during growth in axenic culture. Our hypothesis is that changes of lifestyle and a switch toward pathogenesis lift chromatin

  13. 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.

  14. 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).

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

    PubMed Central

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

    2011-01-01

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

  16. The RalGEF-Ral Effector Signaling Network

    PubMed Central

    Neel, Nicole F.; Martin, Timothy D.; Stratford, Jeran K.; Zand, Tanya P.; Reiner, David J.; Der, Channing J.

    2011-01-01

    The high frequency of RAS mutations in human cancers (33%) has stimulated intense interest in the development of anti-Ras inhibitors for cancer therapy. Currently, the major focus of these efforts is centered on inhibitors of components involved in Ras downstream effector signaling. In particular, more than 40 inhibitors of the Raf-MEK-ERK mitogen-activated protein kinase cascade and phosphoinositide 3-kinase-AKT-mTOR effector signaling networks are currently under clinical evaluation. However, these efforts are complicated by the fact that Ras can utilize at least 9 additional functionally distinct effectors, with at least 3 additional effectors with validated roles in Ras-mediated oncogenesis. Of these, the guanine nucleotide exchange factors of the Ras-like (Ral) small GTPases (RalGEFs) have emerged as important effectors of mutant Ras in pancreatic, colon, and other cancers. In this review, we summarize the evidence for the importance of this effector pathway in cancer and discuss possible directions for therapeutic inhibition of aberrant Ral activation and signaling. PMID:21779498

  17. Characterization of the largest effector gene cluster of Ustilago maydis.

    PubMed

    Brefort, Thomas; Tanaka, Shigeyuki; Neidig, Nina; Doehlemann, Gunther; Vincon, Volker; Kahmann, Regine

    2014-07-01

    In the genome of the biotrophic plant pathogen Ustilago maydis, many of the genes coding for secreted protein effectors modulating virulence are arranged in gene clusters. The vast majority of these genes encode novel proteins whose expression is coupled to plant colonization. The largest of these gene clusters, cluster 19A, encodes 24 secreted effectors. Deletion of the entire cluster results in severe attenuation of virulence. Here we present the functional analysis of this genomic region. We show that a 19A deletion mutant behaves like an endophyte, i.e. is still able to colonize plants and complete the infection cycle. However, tumors, the most conspicuous symptoms of maize smut disease, are only rarely formed and fungal biomass in infected tissue is significantly reduced. The generation and analysis of strains carrying sub-deletions identified several genes significantly contributing to tumor formation after seedling infection. Another of the effectors could be linked specifically to anthocyanin induction in the infected tissue. As the individual contributions of these genes to tumor formation were small, we studied the response of maize plants to the whole cluster mutant as well as to several individual mutants by array analysis. This revealed distinct plant responses, demonstrating that the respective effectors have discrete plant targets. We propose that the analysis of plant responses to effector mutant strains that lack a strong virulence phenotype may be a general way to visualize differences in effector function. PMID:24992561

  18. A family of conserved bacterial effectors inhibits salicylic acid-mediated basal immunity and promotes disease necrosis in plants.

    PubMed

    DebRoy, Sruti; Thilmony, Roger; Kwack, Yong-Bum; Nomura, Kinya; He, Sheng Yang

    2004-06-29

    Salicylic acid (SA)-mediated host immunity plays a central role in combating microbial pathogens in plants. Inactivation of SA-mediated immunity, therefore, would be a critical step in the evolution of a successful plant pathogen. It is known that mutations in conserved effector loci (CEL) in the plant pathogens Pseudomonas syringae (the Delta CEL mutation), Erwinia amylovora (the dspA/E mutation), and Pantoea stewartii subsp. stewartii (the wtsE mutation) exert particularly strong negative effects on bacterial virulence in their host plants by unknown mechanisms. We found that the loss of virulence in Delta CEL and dspA/E mutants was linked to their inability to suppress cell wall-based defenses and to cause normal disease necrosis in Arabidopsis and apple host plants. The Delta CEL mutant activated SA-dependent callose deposition in wild-type Arabidopsis but failed to elicit high levels of callose-associated defense in Arabidopsis plants blocked in SA accumulation or synthesis. This mutant also multiplied more aggressively in SA-deficient plants than in wild-type plants. The hopPtoM and avrE genes in the CEL of P. syringae were found to encode suppressors of this SA-dependent basal defense. The widespread conservation of the HopPtoM and AvrE families of effectors in various bacteria suggests that suppression of SA-dependent basal immunity and promotion of host cell death are important virulence strategies for bacterial infection of plants. PMID:15210989

  19. The Salmonella Typhimurium effector protein SopE transiently localizes to the early SCV and contributes to intracellular replication.

    PubMed

    Vonaesch, Pascale; Sellin, Mikael E; Cardini, Steven; Singh, Vikash; Barthel, Manja; Hardt, Wolf-Dietrich

    2014-12-01

    Salmonella enterica serovar Typhimurium (S. Tm) is a facultative intracellular pathogen that induces entry into non-phagocytic cells by a Type III secretion system (TTSS) and cognate effector proteins. Upon host cell entry, S. Tm expresses a second TTSS and subverts intracellular trafficking to create a replicative niche - the Salmonella-containing vacuole (SCV). SopE, a guanidyl exchange factor (GEF) for Rac1 and Cdc42, is translocated by the TTSS-1 upon host cell contact and promotes entry through triggering of actin-dependent ruffles. After host cell entry, the bulk of SopE undergoes proteasomal degradation. Here we show that a subfraction is however detectable on the nascent SCV membrane up to ∼ 6 h post infection. Membrane localization of SopE and the closely related SopE2 differentially depend on the Rho-GTPase-binding GEF domain, and to some extent involves also the unstructured N-terminus. SopE localizes transiently to the early SCV, dependent on continuous synthesis and secretion by the TTSS-1 during the intracellular state. Mutant strains lacking SopE or SopE2 are attenuated in early intracellular replication, while complementation restores this defect. Hence, the present study reveals an unanticipated role for SopE and SopE2 in establishing the Salmonella replicative niche, and further emphasizes the importance of entry effectors in later stages of host-cell manipulation. PMID:25052734

  20. A magnetic vehicle realized tumor cell-targeted radiotherapy using low-dose radiation.

    PubMed

    Chen, Hsiao-Ping; Tung, Fu-I; Chen, Ming-Hong; Liu, Tse-Ying

    2016-03-28

    Radiotherapy, a common cancer treatment, often adversely affects the surrounding healthy tissue and/or cells. Some tumor tissue-focused radiation therapies have been developed to lower radiation-induced lesion formation; however, achieving tumor cell-targeted radiotherapy (i.e., precisely focusing the radiation efficacy to tumor cells) remains a challenge. In the present study, we developed a novel tumor cell-targeted radiotherapy, named targeted sensitization-enhanced radiotherapy (TSER), that exploits tumor-specific folic acid-conjugated carboxymethyl lauryl chitosan/superparamagnetic iron oxide (FA-CLC/SPIO) micelles to effectively deliver chlorin e6 (Ce6, a sonosensitizer) to mitochondria of HeLa cells under magnetic guidance. For the in vitro tests, the sensitization of Ce6 induced by ultrasound, that could weaken the radiation resistant ability of tumor cells, occurred only in Ce6-internalizing tumor cells. Therefore, low-dose X-ray irradiation, that was not harmful to normal cells, could exert high tumor cell-specific killing ability. The ratio of viable normal cells to tumor cells was increased considerably, from 7.8 (at 24h) to 97.1 (at 72h), after they had received TSER treatment. Our data suggest that TSER treatment significantly weakens tumor cells, resulting in decreased viability in vitro as well as decreased in vivo subcutaneous tumor growth in nude mice, while the adverse effects were minimal. Taken together, TSER treatment appears to be an effective, clinically feasible tumor cell-targeted radiotherapy that can solve the problems of traditional radiotherapy and photodynamic therapy. PMID:26892750

  1. Smart Cancer Cell Targeting Imaging and Drug Delivery System by Systematically Engineering Periodic Mesoporous Organosilica Nanoparticles.

    PubMed

    Lu, Nan; Tian, Ying; Tian, Wei; Huang, Peng; Liu, Ying; Tang, Yuxia; Wang, Chunyan; Wang, Shouju; Su, Yunyan; Zhang, Yunlei; Pan, Jing; Teng, Zhaogang; Lu, Guangming

    2016-02-10

    The integration of diagnosis and therapy into one nanoplatform, known as theranostics, has attracted increasing attention in the biomedical areas. Herein, we first present a cancer cell targeting imaging and drug delivery system based on engineered thioether-bridged periodic mesoporous organosilica nanoparticles (PMOs). The PMOs are stably and selectively conjugated with near-infrared fluorescence (NIRF) dye Cyanine 5.5 (Cy5.5) and anti-Her2 affibody on the outer surfaces to endow them with excellent NIRF imaging and cancer targeting properties. Also, taking the advantage of the thioether-group-incorporated mesopores, the release of chemotherapy drug doxorubicin (DOX) loaded in the PMOs is responsive to the tumor-related molecule glutathione (GSH). The drug release percentage reaches 84.8% in 10 mM of GSH solution within 24 h, which is more than 2-fold higher than that without GSH. In addition, the drug release also exhibits pH-responsive, which reaches 53.6% at pH 5 and 31.7% at pH 7.4 within 24 h. Confocal laser scanning microscopy and flow cytometry analysis demonstrate that the PMOs-based theranostic platforms can efficiently target to and enter Her2 positive tumor cells. Thus, the smart imaging and drug delivery nanoplatforms induce high tumor cell growth inhibition. Meanwhile, the Cy5.5 conjugated PMOs perform great NIRF imaging ability, which could monitor the intracellular distribution, delivery and release of the chemotherapy drug. In addition, cell viability and histological assessments show the engineered PMOs have good biocompatibility, further encouraging the following biomedical applications. Over all, the systemically engineered PMOs can serve as a novel cancer cell targeting imaging and drug delivery platform with NIRF imaging, GSH and pH dual-responsive drug release, and high tumor cell targeting ability. PMID:26767305

  2. Reconsidering mutualistic plant-fungal interactions through the lens of effector biology.

    PubMed

    Plett, Jonathan M; Martin, Francis

    2015-08-01

    Mutualistic mycorrhizal plant-fungal interactions have shaped the evolution of plant life on land. In these intimate associations, fungal hyphae grow invasively within plant tissues. Despite this invasion, these mycorrhizal fungi are not repulsed leading to a great deal of research focused on the signals exchanged between mutualistic fungi and their host plants in an effort to understand how these relationships are established. In this review, we focus on one type of signal used by mutualistic fungi during symbiosis: effector proteins. These small secreted proteins have recently been found to be used by a range of beneficial fungi to alter the physiological status of the plant host such that symbiosis is favoured. We discuss how the role of these novel proteins has altered our vision of how the 'mutualistic' lifestyle evolved in fungi: rather than being perceived as beneficial by their plant hosts, these microbes currently viewed as 'beneficial' may actually be overcoming the defences of their plant hosts in a mechanism originally thought to be unique to pathogenic microbes. PMID:26116975

  3. Oligoperoxide Based Physically Detectable Nanocomposites for Cell Targeting, Visualization and Treatment

    NASA Astrophysics Data System (ADS)

    Zaichenko, A.; Mitina, N.; Shevchuk, O.; Shapoval, O.; Boiko, N.; Bilyy, R.; Stoika, R.; Voloshinovskii, A.; Horak, D.

    2010-10-01

    Novel promising routes for the obtaining luminescent, magnetic, plasmon resonance capable nanocomposites as well as drug delivery systems are proposed and studied. These routes are based on the synthesis and application of original type of functional oligoperoxides, their coordinating complexes of transition including rare earth metal cations for controlled synthesis of block, comb-like or nanogel oligoelectrolytes as well as for template synthesis of functional polymer and polymer-mineral nanoparticles. Developed nanoscale delivery systems were tested as medicines for cancer administration in vitro and in vivo. Physically detectable lectin-conjugated nanocomposites were studied as biomarkers for pathological cell targeting and visualization.

  4. Separable roles for Mycobacterium tuberculosis ESX-3 effectors in iron acquisition and virulence.

    PubMed

    Tufariello, JoAnn M; Chapman, Jessica R; Kerantzas, Christopher A; Wong, Ka-Wing; Vilchèze, Catherine; Jones, Christopher M; Cole, Laura E; Tinaztepe, Emir; Thompson, Victor; Fenyö, David; Niederweis, Michael; Ueberheide, Beatrix; Philips, Jennifer A; Jacobs, William R

    2016-01-19

    Mycobacterium tuberculosis (Mtb) encodes five type VII secretion systems (T7SS), designated ESX-1-ESX-5, that are critical for growth and pathogenesis. The best characterized is ESX-1, which profoundly impacts host cell interactions. In contrast, the ESX-3 T7SS is implicated in metal homeostasis, but efforts to define its function have been limited by an inability to recover deletion mutants. We overcame this impediment using medium supplemented with various iron complexes to recover mutants with deletions encompassing select genes within esx-3 or the entire operon. The esx-3 mutants were defective in uptake of siderophore-bound iron and dramatically accumulated cell-associated mycobactin siderophores. Proteomic analyses of culture filtrate revealed that secretion of EsxG and EsxH was codependent and that EsxG-EsxH also facilitated secretion of several members of the proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) protein families (named for conserved PE and PPE N-terminal motifs). Substrates that depended on EsxG-EsxH for secretion included PE5, encoded within the esx-3 locus, and the evolutionarily related PE15-PPE20 encoded outside the esx-3 locus. In vivo characterization of the mutants unexpectedly showed that the ESX-3 secretion system plays both iron-dependent and -independent roles in Mtb pathogenesis. PE5-PPE4 was found to be critical for the siderophore-mediated iron-acquisition functions of ESX-3. The importance of this iron-acquisition function was dependent upon host genotype, suggesting a role for ESX-3 secretion in counteracting host defense mechanisms that restrict iron availability. Further, we demonstrate that the ESX-3 T7SS secretes certain effectors that are important for iron uptake while additional secreted effectors modulate virulence in an iron-independent fashion. PMID:26729876

  5. Separable roles for Mycobacterium tuberculosis ESX-3 effectors in iron acquisition and virulence

    PubMed Central

    Tufariello, JoAnn M.; Chapman, Jessica R.; Kerantzas, Christopher A.; Wong, Ka-Wing; Vilchèze, Catherine; Jones, Christopher M.; Cole, Laura E.; Tinaztepe, Emir; Thompson, Victor; Fenyö, David; Niederweis, Michael; Ueberheide, Beatrix; Philips, Jennifer A.; Jacobs, William R.

    2016-01-01

    Mycobacterium tuberculosis (Mtb) encodes five type VII secretion systems (T7SS), designated ESX-1–ESX-5, that are critical for growth and pathogenesis. The best characterized is ESX-1, which profoundly impacts host cell interactions. In contrast, the ESX-3 T7SS is implicated in metal homeostasis, but efforts to define its function have been limited by an inability to recover deletion mutants. We overcame this impediment using medium supplemented with various iron complexes to recover mutants with deletions encompassing select genes within esx-3 or the entire operon. The esx-3 mutants were defective in uptake of siderophore-bound iron and dramatically accumulated cell-associated mycobactin siderophores. Proteomic analyses of culture filtrate revealed that secretion of EsxG and EsxH was codependent and that EsxG–EsxH also facilitated secretion of several members of the proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) protein families (named for conserved PE and PPE N-terminal motifs). Substrates that depended on EsxG–EsxH for secretion included PE5, encoded within the esx-3 locus, and the evolutionarily related PE15–PPE20 encoded outside the esx-3 locus. In vivo characterization of the mutants unexpectedly showed that the ESX-3 secretion system plays both iron-dependent and -independent roles in Mtb pathogenesis. PE5–PPE4 was found to be critical for the siderophore-mediated iron-acquisition functions of ESX-3. The importance of this iron-acquisition function was dependent upon host genotype, suggesting a role for ESX-3 secretion in counteracting host defense mechanisms that restrict iron availability. Further, we demonstrate that the ESX-3 T7SS secretes certain effectors that are important for iron uptake while additional secreted effectors modulate virulence in an iron-independent fashion. PMID:26729876

  6. Effector kinase coupling enables high-throughput screens for direct HIV-1 Nef antagonists with antiretroviral activity.

    PubMed

    Emert-Sedlak, Lori A; Narute, Purushottam; Shu, Sherry T; Poe, Jerrod A; Shi, Haibin; Yanamala, Naveena; Alvarado, John Jeff; Lazo, John S; Yeh, Joanne I; Johnston, Paul A; Smithgall, Thomas E

    2013-01-24

    HIV-1 Nef, a critical AIDS progression factor, represents an important target protein for antiretroviral drug discovery. Because Nef lacks intrinsic enzymatic activity, we developed an assay that couples Nef to the activation of Hck, a Src family member and Nef effector protein. Using this assay, we screened a large, diverse chemical library and identified small molecules that block Nef-dependent Hck activity with low micromolar potency. Of these, a diphenylpyrazolo compound demonstrated submicromolar potency in HIV-1 replication assays against a broad range of primary Nef variants. This compound binds directly to Nef via a pocket formed by the Nef dimerization interface and disrupts Nef dimerization in cells. Coupling of nonenzymatic viral accessory factors to host cell effector proteins amenable to high-throughput screening may represent a general strategy for the discovery of new antimicrobial agents. PMID:23352142

  7. Serine Protease Inhibitor-6 Differentially Affects the Survival of Effector and Memory Alloreactive CD8-T Cells

    PubMed Central

    Azzi, J.; Ohori, S.; Ting, C.; Uehara, M.; Abdoli, R.; Smith, B. D.; Safa, K.; Solhjou, Z.; Lukyanchykov, P.; Patel, J.; McGrath, M.; Abdi, R.

    2016-01-01

    The clonal expansion of effector T cells and subsequent generation of memory T cells are critical in determining the outcome of transplantation. While cytotoxic T lymphocytes induce direct cytolysis of target cells through secretion of Granzyme-B (GrB), they also express cytoplasmic serine protease inhibitor-6 (Spi6) to protect themselves from GrB that has leaked from granules. Here, we studied the role of GrB/Spi6 axis in determining clonal expansion of alloreactive CD8-T cells and subsequent generation of memory CD8-T cells in transplantation. CD8-T cells from Spi6−/− mice underwent more GrB mediated apoptosis upon alloantigen stimulation in vitro and in vivo following adoptive transfer into an allogeneic host. Interestingly, while OT1.Spi6−/− CD8 T cells showed significantly lower clonal expansion following skin transplants from OVA mice, there was no difference in the size of the effector memory CD8-T cells long after transplantation. Furthermore, lack of Spi6 resulted in a decrease of short-lived-effector-CD8-cells but did not impact the pool of memory-precursor-effector-CD8-cells. Similar results were found in heart transplant models. Our findings suggest that the final alloreactive CD8-memory-pool-size is independent from the initial clonal-proliferation as memory precursors express low levels of GrB and therefore are independent of Spi6 for survival. These data advance our understanding of memory T cells generation in transplantation and provide basis for Spi6 based strategies to target effector T cells. PMID:25534448

  8. Salmonella – At Home in the Host Cell

    PubMed Central

    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. PMID:21687432

  9. Tyrosine phosphorylation of RAS by ABL allosterically enhances effector binding

    PubMed Central

    Ting, Pamela Y.; Johnson, Christian W.; Fang, Cong; Cao, Xiaoqing; Graeber, Thomas G.; Mattos, Carla; Colicelli, John

    2015-01-01

    RAS proteins are signal transduction gatekeepers that mediate cell growth, survival, and differentiation through interactions with multiple effector proteins. The RAS effector RAS- and RAB-interacting protein 1 (RIN1) activates its own downstream effectors, the small GTPase RAB5 and the tyrosine kinase Abelson tyrosine-protein kinase (ABL), to modulate endocytosis and cytoskeleton remodeling. To identify ABL substrates downstream of RAS-to-RIN1 signaling, we examined human HEK293T cells overexpressing components of this pathway. Proteomic analysis revealed several novel phosphotyrosine peptides, including Harvey rat sarcoma oncogene (HRAS)-pTyr137. Here we report that ABL phosphorylates tyrosine 137 of H-, K-, and NRAS. Increased RIN1 levels enhanced HRAS-Tyr137 phosphorylation by nearly 5-fold, suggesting that RAS-stimulated RIN1 can drive ABL-mediated RAS modification in a feedback circuit. Tyr137 is well conserved among RAS orthologs and is part of a transprotein H-bond network. Crystal structures of HRASY137F and HRASY137E revealed conformation changes radiating from the mutated residue. Although consistent with Tyr137 participation in allosteric control of HRAS function, the mutations did not alter intrinsic GTP hydrolysis rates in vitro. HRAS-Tyr137 phosphorylation enhanced HRAS signaling capacity in cells, however, as reflected by a 4-fold increase in the association of phosphorylated HRASG12V with its effector protein RAF proto-oncogene serine/threonine protein kinase 1 (RAF1). These data suggest that RAS phosphorylation at Tyr137 allosterically alters protein conformation and effector binding, providing a mechanism for effector-initiated modulation of RAS signaling.—Ting, P. Y., Johnson, C. W., Fang, C., Cao, X., Graeber, T. G., Mattos, C., Colicelli, J. Tyrosine phosphorylation of RAS by ABL allosterically enhances effector binding. PMID:25999467

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

    PubMed

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

    2012-10-01

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

  11. Salmonella Disrupts Host Endocytic Trafficking by SopD2-Mediated Inhibition of Rab7.

    PubMed

    D'Costa, Vanessa M; Braun, Virginie; Landekic, Marija; Shi, Rong; Proteau, Ariane; McDonald, Laura; Cygler, Miroslaw; Grinstein, Sergio; Brumell, John H

    2015-09-01

    Intracellular bacterial pathogens of a diverse nature share the ability to evade host immunity by impairing trafficking of endocytic cargo to lysosomes for degradation, a process that is poorly understood. Here, we show that the Salmonella enterica type 3 secreted effector SopD2 mediates this process by binding the host regulatory GTPase Rab7 and inhibiting its nucleotide exchange. Consequently, this limits Rab7 interaction with its dynein- and kinesin-binding effectors RILP and FYCO1 and thereby disrupts host-driven regulation of microtubule motors. Our study identifies a bacterial effector capable of directly binding and thereby modulating Rab7 activity and a mechanism of endocytic trafficking disruption that may provide insight into the pathogenesis of other bacteria. Additionally, we provide a powerful tool for the study of Rab7 function, and a potential therapeutic target. PMID:26299973

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

    PubMed

    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

  13. 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

  14. Cyclic RGD peptide-labeled upconversion nanophosphors for tumor cell-targeted imaging.

    PubMed

    Zako, Tamotsu; Nagata, Hiroyasu; Terada, Naofumi; Utsumi, Arata; Sakono, Masafumi; Yohda, Masafumi; Ueda, Hiroshi; Soga, Kohei; Maeda, Mizuo

    2009-03-27

    One of the great challenges of oncology is to improve methods for early tumor detection. Thus tumor cell-targeted optical imaging has been intensively studied. Bioimaging with upconversion (UC) phosphors (UCPs) is of considerable interest due to a variety of possible applications taking advantage of infrared-to-visible luminescence. Here we report for the first time tumor cell-targeted UC imaging using UCPs modified with cyclic RGD peptide (RGD-Y2O3). Cyclic RGD peptide binds specifically to integrin alphavbeta3 which is highly expressed in a tumor cell surface of certain cancer types but not in normal tissues. Since UC emission from RGD-Y2O3 was observed for U87MG cancer cell (high integrin alphavbeta3 expression), but not for MCF-7 cancer cell (low integrin alphavbeta3 expression), this UC imaging is considered to be integrin alphavbeta3 specific. The non-invasive imaging of integrin alphavbeta3 expression using UCP-based probes will have great potential in cancer imaging in general in living subjects. PMID:19351594

  15. 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. PMID:26218423

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

    PubMed Central

    2015-01-01

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

  17. Substrate recognition by the zinc metalloprotease effector NleC from enteropathogenic Escherichia coli.

    PubMed

    Giogha, Cristina; Lung, Tania Wong Fok; Mühlen, Sabrina; Pearson, Jaclyn S; Hartland, Elizabeth L

    2015-12-01

    Upon infection of epithelial cells, enteropathogenic Escherichia coli suppresses host cell inflammatory signalling in a type III secretion system (T3SS) dependent manner. Two key T3SS effector proteins involved in this response are NleE and NleC. NleC is a zinc metalloprotease effector that degrades the p65 subunit of NF-κB. Although the site of p65 cleavage by NleC is now well described, other areas of interaction have not been precisely defined. Here we constructed overlapping truncations of p65 to identify regions required for NleC cleavage. We determined that NleC cleaved both p65 and p50 within the Rel homology domain (RHD) and that two motifs, E22IIE25 and P177VLS180 , within the RHD of p65 were important for recognition and binding by NleC. Alanine substitution of one or both of these motifs protected p65 from binding and degradation by NleC. The E22IIE25 and P177VLS180 motifs were located within the structurally distinct N-terminal subdomain of the RHD involved in DNA binding by p65 on adjacent, parallel strands. Although these motifs have not been recognized previously, both were needed for the correct localization and function of p65. In summary, this work has identified two regions of p65 within the RHD needed for binding and cleavage by NleC and provides further insight into the molecular basis of substrate recognition by a T3SS effector. PMID:26096513

  18. 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

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

    PubMed Central

    Aurass, Philipp; Oates, Clare V.; Tate, Edward W.; Hartland, Elizabeth L.; Flieger, Antje

    2015-01-01

    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. PMID:26216420

  20. Bacterial Effector Binding to Ribosomal Protein S3 Subverts NF-κB Function

    PubMed Central

    Gao, Xiaofei; Wan, Fengyi; Mateo, Kristina; Callegari, Eduardo; Wang, Dan; Deng, Wanyin; Puente, Jose; Li, Feng; Chaussee, Michael S.; Finlay, B. Brett; Lenardo, Michael J.; Hardwidge, Philip R.

    2009-01-01

    Enteric bacterial pathogens cause food borne disease, which constitutes an enormous economic and health burden. Enterohemorrhagic Escherichia coli (EHEC) causes a severe bloody diarrhea following transmission to humans through various means, including contaminated beef and vegetable products, water, or through contact with animals. EHEC also causes a potentially fatal kidney disease (hemolytic uremic syndrome) for which there is no effective treatment or prophylaxis. EHEC and other enteric pathogens (e.g., enteropathogenic E. coli (EPEC), Salmonella, Shigella, Yersinia) utilize a type III secretion system (T3SS) to inject virulence proteins (effectors) into host cells. While it is known that T3SS effectors subvert host cell function to promote diarrheal disease and bacterial transmission, in many cases, the mechanisms by which these effectors bind to host proteins and disrupt the normal function of intestinal epithelial cells have not been completely characterized. In this study, we present evidence that the E. coli O157:H7 nleH1 and nleH2 genes encode T3SS effectors that bind to the human ribosomal protein S3 (RPS3), a subunit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcriptional complexes. NleH1 and NleH2 co-localized with RPS3 in the cytoplasm, but not in cell nuclei. The N-terminal region of both NleH1 and NleH2 was required for binding to the N-terminus of RPS3. NleH1 and NleH2 are autophosphorylated Ser/Thr protein kinases, but their binding to RPS3 is independent of kinase activity. NleH1, but not NleH2, reduced the nuclear abundance of RPS3 without altering the p50 or p65 NF-κB subunits or affecting the phosphorylation state or abundance of the inhibitory NF-κB chaperone IκBα NleH1 repressed the transcription of a RPS3/NF-κB-dependent reporter plasmid, but did not inhibit the transcription of RPS3-independent reporters. In contrast, NleH2 stimulated RPS3-dependent transcription, as well as an AP-1-dependent reporter

  1. 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...

  2. Influenza A virus ribonucleoproteins modulate host recycling by competing with Rab11 effectors.

    PubMed

    Vale-Costa, Sílvia; Alenquer, Marta; Sousa, Ana Laura; Kellen, Bárbara; Ramalho, José; Tranfield, Erin M; Amorim, Maria João

    2016-04-15

    Influenza A virus assembly is an unclear process, whereby individual virion components form an infectious particle. The segmented nature of the influenza A genome imposes a problem to assembly because it requires packaging of eight distinct RNA particles (vRNPs). It also allows genome mixing from distinct parental strains, events associated with influenza pandemic outbreaks. It is important to public health to understand how segmented genomes assemble, a process that is dependent on the transport of components to assembly sites. Previously, it has been shown that vRNPs are carried by recycling endosome vesicles, resulting in a change of Rab11 distribution. Here, we describe that vRNP binding to recycling endosomes impairs recycling endosome function, by competing for Rab11 binding with family-interacting proteins, and that there is a causal relationship between Rab11 ability to recruit family-interacting proteins and Rab11 redistribution. This competition reduces recycling sorting at an unclear step, resulting in clustering of single- and double-membraned vesicles. These morphological changes in Rab11 membranes are indicative of alterations in protein and lipid homeostasis during infection. Vesicular clustering creates hotspots of the vRNPs that need to interact to form an infectious particle. PMID:26940915

  3. Innate Immune Effectors in Mycobacterial Infection

    PubMed Central

    Saiga, Hiroyuki; Shimada, Yosuke; Takeda, Kiyoshi

    2011-01-01

    Tuberculosis, which is caused by infection with Mycobacterium tuberculosis (Mtb), remains one of the major bacterial infections worldwide. Host defense against Mtb is mediated by a combination of innate and adaptive immune responses. In the last 15 years, the mechanisms for activation of innate immunity have been elucidated. Toll-like receptors (TLRs) have been revealed to be critical for the recognition of pathogenic microorganisms including mycobacteria. Subsequent studies further revealed that NOD-like receptors and C-type lectin receptors are responsible for the TLR-independent recognition of mycobacteria. Several molecules, such as active vitamin D3, secretary leukocyte protease inhibitor, and lipocalin 2, all of which are induced by TLR stimulation, have been shown to direct innate immune responses to mycobacteria. In addition, Irgm1-dependent autophagy has recently been demonstrated to eliminate intracellular mycobacteria. Thus, our understanding of the mechanisms for the innate immune response to mycobacteria is developing. PMID:21274449

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

    PubMed Central

    Üstün, Suayib; Börnke, Frederik

    2014-01-01

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

  5. Target selection biases from recent experience transfer across effectors.

    PubMed

    Moher, Jeff; Song, Joo-Hyun

    2016-02-01

    Target selection is often biased by an observer's recent experiences. However, not much is known about whether these selection biases influence behavior across different effectors. For example, does looking at a red object make it easier to subsequently reach towards another red object? In the current study, we asked observers to find the uniquely colored target object on each trial. Randomly intermixed pre-trial cues indicated the mode of action: either an eye movement or a visually guided reach movement to the target. In Experiment 1, we found that priming of popout, reflected in faster responses following repetition of the target color on consecutive trials, occurred regardless of whether the effector was repeated from the previous trial or not. In Experiment 2, we examined whether an inhibitory selection bias away from a feature could transfer across effectors. While priming of popout reflects both enhancement of the repeated target features and suppression of the repeated distractor features, the distractor previewing effect isolates a purely inhibitory component of target selection in which a previewed color is presented in a homogenous display and subsequently inhibited. Much like priming of popout, intertrial suppression biases in the distractor previewing effect transferred across effectors. Together, these results suggest that biases for target selection driven by recent trial history transfer across effectors. This indicates that representations in memory that bias attention towards or away from specific features are largely independent from their associated actions. PMID:26563393

  6. Characterization of a chemoattractant for endothelium induced by angiogenesis effectors.

    PubMed

    Raju, K S; Alessandri, G; Gullino, P M

    1984-04-01

    The mechanism of neovascularization was further explored by the use of chemically defined angiogenesis effectors. The vascularization of the rabbit cornea was selected as an experimental approach that permits comparison of one cornea treated by the angiogenesis effector with the contralateral cornea of the same subject treated by the same molecule deprived of angiogenic capacity. Under these conditions, we observed that neovascularization was initiated by the appearance of a chemoattractant for the bovine capillary endothelium only in the cornea treated by the angiogenesis effector. The chemoattractant was purified about 150-fold by a single-step procedure, using gelatin:Sepharose affinity chromatography. Chemoattraction resulted from the combined effect of a chemotactic factor(s) and an activating factor(s). The association of the two enhanced 5- to 8-fold the motility of the capillary endothelium in a concentration-dependent manner with optimum at 0.2 mg/ml. The activating factor(s) does not have chemotactic capacity, but without it, chemotaxis is reduced to about one half. The chemotactic complex was present in the cornea regardless of the nature of the angiogenesis effector used as the triggering device. Heat and proteases eliminated chemotaxis and destroyed the chemotactic complex. Thus, neovascularization may be triggered by effectors able to induce in the cornea proteins, normally not present, that influence angiogenesis via mobilization of capillary endothelium. PMID:6200213

  7. Potency of Transgenic Effectors for Neurogenetic Manipulation in Drosophila Larvae

    PubMed Central

    Pauls, Dennis; von Essen, Alina; Lyutova, Radostina; van Giesen, Lena; Rosner, Ronny; Wegener, Christian; Sprecher, Simon G.

    2015-01-01

    Genetic manipulations of neuronal activity are a cornerstone of studies aimed to identify the functional impact of defined neurons for animal behavior. With its small nervous system, rapid life cycle, and genetic amenability, the fruit fly Drosophila melanogaster provides an attractive model system to study neuronal circuit function. In the past two decades, a large repertoire of elegant genetic tools has been developed to manipulate and study neural circuits in the fruit fly. Current techniques allow genetic ablation, constitutive silencing, or hyperactivation of neuronal activity and also include conditional thermogenetic or optogenetic activation or inhibition. As for all genetic techniques, the choice of the proper transgenic tool is essential for behavioral studies. Potency and impact of effectors may vary in distinct neuron types or distinct types of behavior. We here systematically test genetic effectors for their potency to alter the behavior of Drosophila larvae, using two distinct behavioral paradigms: general locomotor activity and directed, visually guided navigation. Our results show largely similar but not equal effects with different effector lines in both assays. Interestingly, differences in the magnitude of induced behavioral alterations between different effector lines remain largely consistent between the two behavioral assays. The observed potencies of the effector lines in aminergic and cholinergic neurons assessed here may help researchers to choose the best-suited genetic tools to dissect neuronal networks underlying the behavior of larval fruit flies. PMID:25359929

  8. Structural Analysis of Iac Repressor Bound to Allosteric Effectors

    SciTech Connect

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

    2007-01-01

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

  9. ICAM-1–expressing neutrophils exhibit enhanced effector functions in murine models of endotoxemia

    PubMed Central

    Woodfin, Abigail; Beyrau, Martina; Voisin, Mathieu-Benoit; Ma, Bin; Whiteford, James R.; Hordijk, Peter L.; Hogg, Nancy

    2016-01-01

    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. PMID:26647392

  10. Structural elucidation and functional characterization of the Hyaloperonospora arabidopsidis effector protein ATR13.

    PubMed

    Leonelli, Lauriebeth; Pelton, Jeffery; Schoeffler, Allyn; Dahlbeck, Douglas; Berger, James; Wemmer, David E; Staskawicz, Brian

    2011-12-01

    The oomycete Hyaloperonospora arabidopsidis (Hpa) is the causal agent of downy mildew on the model plant Arabidopsis thaliana and has been adapted as a model system to investigate pathogen virulence strategies and plant disease resistance mechanisms. Recognition of Hpa infection occurs when plant resistance proteins (R-genes) detect the presence or activity of pathogen-derived protein effectors delivered to the plant host. This study examines the Hpa effector ATR13 Emco5 and its recognition by RPP13-Nd, the cognate R-gene that triggers programmed cell death (HR) in the presence of recognized ATR13 variants. Herein, we use NMR to solve the backbone structure of ATR13 Emco5, revealing both a helical domain and a disordered internal loop. Additionally, we use site-directed and random mutagenesis to identify several amino acid residues involved in the recognition response conferred by RPP13-Nd. Using our structure as a scaffold, we map these residues to one of two surface-exposed patches of residues under diversifying selection. Exploring possible roles of the disordered region within the ATR13 structure, we perform domain swapping experiments and identify a peptide sequence involved in nucleolar localization. We conclude that ATR13 is a highly dynamic protein with no clear structural homologues that contains two surface-exposed patches of polymorphism, only one of which is involved in RPP13-Nd recognition specificity. PMID:22194684

  11. A Burkholderia Type VI Effector Deamidates Rho GTPases to Activate the Pyrin Inflammasome and Trigger Inflammation.

    PubMed

    Aubert, Daniel F; Xu, Hao; Yang, Jieling; Shi, Xuyan; Gao, Wenqing; Li, Lin; Bisaro, Fabiana; Chen, She; Valvano, Miguel A; Shao, Feng

    2016-05-11

    Burkholderia cenocepacia is an opportunistic pathogen of the cystic fibrosis lung that elicits a strong inflammatory response. B. cenocepacia employs a type VI secretion system (T6SS) to survive in macrophages by disarming Rho-type GTPases, causing actin cytoskeletal defects. Here, we identified TecA, a non-VgrG T6SS effector responsible for actin disruption. TecA and other bacterial homologs bear a cysteine protease-like catalytic triad, which inactivates Rho GTPases by deamidating a conserved asparagine in the GTPase switch-I region. RhoA deamidation induces caspase-1 inflammasome activation, which is mediated by the familial Mediterranean fever disease protein Pyrin. In mouse infection, the deamidase activity of TecA is necessary and sufficient for B. cenocepacia-triggered lung inflammation and also protects mice from lethal B. cenocepacia infection. Therefore, Burkholderia TecA is a T6SS effector that modifies a eukaryotic target through an asparagine deamidase activity, which in turn elicits host cell death and inflammation through activation of the Pyrin inflammasome. PMID:27133449

  12. Coxiella burnetii effector CvpB modulates phosphoinositide metabolism for optimal vacuole development.

    PubMed

    Martinez, Eric; Allombert, Julie; Cantet, Franck; Lakhani, Anissa; Yandrapalli, Naresh; Neyret, Aymeric; Norville, Isobel H; Favard, Cyril; Muriaux, Delphine; Bonazzi, Matteo

    2016-06-01

    The Q fever bacterium Coxiella burnetii replicates inside host cells within a large Coxiella-containing vacuole (CCV) whose biogenesis relies on the Dot/Icm-dependent secretion of bacterial effectors. Several membrane trafficking pathways contribute membranes, proteins, and lipids for CCV biogenesis. These include the endocytic and autophagy pathways, which are characterized by phosphatidylinositol 3-phosphate [PI(3)P]-positive membranes. Here we show that the C. burnetii secreted effector Coxiella vacuolar protein B (CvpB) binds PI(3)P and phosphatidylserine (PS) on CCVs and early endosomal compartments and perturbs the activity of the phosphatidylinositol 5-kinase PIKfyve to manipulate PI(3)P metabolism. CvpB association to early endosome triggers vacuolation and clustering, leading to the channeling of large PI(3)P-positive membranes to CCVs for vacuole expansion. At CCVs, CvpB binding to early endosome- and autophagy-derived PI(3)P and the concomitant inhibition of PIKfyve favor the association of the autophagosomal machinery to CCVs for optimal homotypic fusion of the Coxiella-containing compartments. The importance of manipulating PI(3)P metabolism is highlighted by mutations in cvpB resulting in a multivacuolar phenotype, rescuable by gene complementation, indicative of a defect in CCV biogenesis. Using the insect model Galleria mellonella, we demonstrate the in vivo relevance of defective CCV biogenesis by highlighting an attenuated virulence phenotype associated with cvpB mutations. PMID:27226300

  13. Persistent BCG bacilli perpetuate CD4 T effector memory and optimal protection against tuberculosis.

    PubMed

    Kaveh, Daryan A; Carmen Garcia-Pelayo, M; Hogarth, Philip J

    2014-12-01

    Tuberculosis (TB) remains one of the most important infectious diseases of man and animals, and the only available vaccine (BCG) requires urgent replacement or improvement. To facilitate this, the protective mechanisms induced by BCG require further understanding. As a live attenuated vaccine, persistence of BCG bacilli in the host may be a crucial mechanism. We have investigated the long term persistence of BCG following vaccination and the influence on the induced immune response and protection, using an established murine model. We sought to establish whether previously identified BCG-specific CD4 TEM cells represent genuine long-lived memory cells of a relatively high frequency, or are a consequence of continual priming by chronically persistent BCG vaccine bacilli. By clearing persistent bacilli, we have compared immune responses (spleen and lung CD4: cytokine producing T effector/TEM; TCR-specific) and BCG-induced protection, in the presence and absence of these persisting vaccine bacilli. Viable BCG bacilli persisted for at least 16 months post-vaccination, associated with specific CD4 T effector/TEM and tetramer-specific responses. Clearing these bacilli abrogated all BCG-specific CD4 T cells whilst only reducing protection by 1log10. BCG may induce two additive mechanisms of immunity: (i) dependant on the presence of viable bacilli and TEM; and (ii) independent of these factors. These data have crucial implications on the rational generation of replacement TB vaccines, and the interpretation of BCG induced immunity in animal models. PMID:25444816

  14. Effector Functions of Natural Killer Cell Subsets in the Control of Hematological Malignancies

    PubMed Central

    Gismondi, Angela; Stabile, Helena; Nisti, Paolo; Santoni, Angela

    2015-01-01

    Treatment of hematological malignant disorders has been improved over the last years, but high relapse rate mainly attributable to the presence of minimal residual disease still persists. Therefore, it is of great interest to explore novel therapeutic strategies to obtain long-term remission. Immune effector cells, and especially natural killer (NK) cells, play a crucial role in the control of hematological malignancies. In this regard, the efficiency of allogeneic stem cell transplantation clearly depends on the immune-mediated graft versus leukemia effect without the risk of inducing graft versus host disease. Alloreactive donor NK cells generated following hematopoietic stem cell transplantation ameliorate the outcome of leukemia patients; in addition, in vivo transfer of in vitro expanded NK cells represents a crucial tool for leukemia treatment. To improve NK cell effector functions against resistant leukemia cells, novel immunotherapeutic strategies are oriented to the identification, isolation, expansion, and administration of particular NK cell subsets endowed with multifunctional anti-tumor potential and tropism toward tumor sites. Moreover, the relationship between the emergence and persistence of distinct NK cell subsets during post-graft reconstitution and the maintenance of a remission state is still rather unclear. PMID:26594216

  15. 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. PMID:26647392

  16. Dynamic Imaging of the Effector Immune Response to Listeria Infection In Vivo

    PubMed Central

    Waite, Janelle C.; Leiner, Ingrid; Lauer, Peter; Rae, Chris S.; Barbet, Gaetan; Zheng, Huan; Portnoy, Daniel A.; Pamer, Eric G.; Dustin, Michael L.

    2011-01-01

    Host defense against the intracellular pathogen Listeria monocytogenes (Lm) requires innate and adaptive immunity. Here, we directly imaged immune cell dynamics at Lm foci established by dendritic cells in the subcapsular red pulp (scDC) using intravital microscopy. Blood borne Lm rapidly associated with scDC. Myelomonocytic cells (MMC) swarmed around non-motile scDC forming foci from which blood flow was excluded. The depletion of scDC after foci were established resulted in a 10-fold reduction in viable Lm, while graded depletion of MMC resulted in 30–1000 fold increase in viable Lm in foci with enhanced blood flow. Effector CD8+ T cells at sites of infection displayed a two-tiered reduction in motility with antigen independent and antigen dependent components, including stable interactions with infected and non-infected scDC. Thus, swarming MMC contribute to control of Lm prior to development of T cell immunity by direct killing and sequestration from blood flow, while scDC appear to promote Lm survival while preferentially interacting with CD8+ T cells in effector sites. PMID:21455492

  17. 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. PMID:26705897

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

    PubMed

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

    2015-01-01

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

  19. 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

  20. Hepatic effector CD8+ T-cell dynamics

    PubMed Central

    Iannacone, Matteo

    2015-01-01

    CD8+ T cells play a critical role in hepatitis B virus (HBV) pathogenesis. During acute, self-limited infections, these cells are instrumental to viral clearance; in chronic settings, they sustain repetitive cycles of hepatocellular necrosis that promote hepatocellular carcinoma development. Both CD8+ T-cell defensive and destructive functions are mediated by antigen-experienced effector cells and depend on the ability of these cells to migrate to the liver, recognize hepatocellular antigens and perform effector functions. Understanding the signals that modulate the spatiotemporal dynamics of CD8+ T cells in the liver, particularly in the context of antigen recognition, is therefore critical to gaining insight into the pathogenesis of acute and chronic HBV infection. Here, we highlight recent data on how effector CD8+ T cells traffic within the liver, and we discuss the potential for novel imaging techniques to shed light on this important aspect of HBV pathogenesis. PMID:25242274

  1. 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

  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. Development and testing of the cooling coil cleaning end effector

    SciTech Connect

    Johnson, K.I.; Mullen, O.D.; Powell, M.R.; Daly, D.S.; Engel, D.W.

    1997-09-30

    The Retrieval Process Development and Enhancement (KPD{ampersand}E) program has developed and tested an end effector to support the waste retrieval mission at the Idaho National Engineering and Environmental Laboratory (INEEL). The end effector was developed specifically to remove a sticky waste material from the cooling coils in the High Level Liquid Waste (HLLW) tank, and to vacuum up a sediment layer that has settled beneath the cooling coils. An extensive testing program was conducted in the hydraulic test bed (HTB) at the Pacific Northwest National Laboratory (PNNL) to evaluate the performance of the end effector under simulated in-tank conditions. A mock up of the cooling coils was installed in the test bed tank, and simulated waste materials were included to represent the sticky waste on the tubes and the particulate waste settled beneath them. The testing program focused on assessing long-duration mining strategies for cleaning the cooling coils and removing the particulate waste forms. The report describes the results of the end effector testing program at PNNL. Section 2 describes the physical characteristics of the HLLW tanks, including the layout of the cooling coils, and it also describes what is known of the waste forms in the tanks. Section 3 describes the cleaning and retrieval strategy that was used in developing the end effector design. Section 4 describes the cooling coil mockup in the hydraulic test bed. Section 5 discusses the rationale used in selecting the simulants for the tarry waste and particulate waste forms. Section 6 describes the tests that were performed to evaluate cleaning of the cooling coils and retrieval of the particulate simulant. Section 7 summarizes the cleaning and retrieval tests, assesses the relative importance of cleaning the cooling coils and retrieving the particulate waste, and suggests modifications that would simplify the end effector design.

  4. T Cell Signaling Targets for Enhancing Regulatory or Effector Function

    PubMed Central

    Pan, Fan; Fan, Huimin; Liu, Zhongmin; Jiang, Shuiping

    2015-01-01

    To respond to infection, resting or naïve T cells must undergo activation, clonal expansion, and differentiation into specialized functional subsets of effector T cells. However, to prevent excessive or self-destructive immune responses, regulatory T cells (Tregs) are instrumental in suppressing the activation and function of effector cells, including effector T cells. The transcription factor Forkhead box P3 (Foxp3) regulates the expression of genes involved in the development and function of Tregs. Foxp3 interacts with other transcription factors and with epigenetic elements such as histone deacetylases (HDACs) and histone acetyltransferases. Treg suppressive function can be increased by exposure to HDAC inhibitors. The individual contributions of different HDAC family members to Treg function and their respective mechanisms of action, however, remain unclear. A study showed that HDAC6, HDAC9, and Sirtuin-1 had distinct effects on Foxp3 expression and function, suggesting that selectively targeting HDACs individually or in combination may enhance Treg stability and suppressive function. Another study showed that the receptor programmed death 1 (PD-1), a well-known inhibitor of T cell activation, halted cell cycle progression in effector T cells by inhibiting the transcription of the gene encoding the substrate-recognition component (Skp2) of the ubiquitin ligase SCFSkp2. Together, these findings reveal new signaling targets for enhancing Treg or effector T cell function that may be helpful in designing future therapies, either to increase Treg suppressive function in transplantation and autoimmune diseases or to block PD-1 function, thus increasing the magnitude of antiviral or antitumor immune responses of effector T cells. PMID:22855503

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

    PubMed Central

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

    2011-01-01

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

  6. Regulation of effector and memory CD8(+) T cell function by inflammatory cytokines.

    PubMed

    Valbon, Stefanie F; Condotta, Stephanie A; Richer, Martin J

    2016-06-01

    Cells communicate with each other through the production and secretion of cytokines, which are integral to the host response to infection. Once recognized by specific cytokine receptors expressed on the cell surface, these exogenous signals direct the biological function of a cell in order to adapt to their microenvironment. CD8(+) T cells are critical immune cells that play an important role in the control and elimination of intracellular pathogens. Current findings have demonstrated that cytokines influence all aspects of the CD8(+) T cell response to infection or immunization. The cytokine milieu induced at the time of activation impacts the overall magnitude and function of the effector CD8(+) T cell response and the generation of functional memory CD8(+) T cells. This review will focus on the impact of inflammatory cytokines on different aspects of CD8(+) T cell biology. PMID:26688544

  7. General mechanism for modulating immunoglobulin effector function

    PubMed Central

    Sondermann, Peter; Pincetic, Andrew; Maamary, Jad; Lammens, Katja; Ravetch, Jeffrey V.

    2013-01-01

    Immunoglobulins recognize and clear microbial pathogens and toxins through the coupling of variable region specificity to Fc-triggered cellular activation. These proinflammatory activities are regulated, thus avoiding the pathogenic sequelae of uncontrolled inflammation by modulating the composition of the Fc-linked glycan. Upon sialylation, the affinities for Fcγ receptors are reduced, whereas those for alternative cellular receptors, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN)/CD23, are increased. We demonstrate that sialylation induces significant structural alterations in the Cγ2 domain and propose a model that explains the observed changes in ligand specificity and biological activity. By analogy to related complexes formed by IgE and its evolutionarily related Fc receptors, we conclude that this mechanism is general for the modulation of antibody-triggered immune responses, characterized by a shift between an “open” activating conformation and a “closed” anti-inflammatory state of antibody Fc fragments. This common mechanism has been targeted by pathogens to avoid host defense and offers targets for therapeutic intervention in allergic and autoimmune disorders. PMID:23697368

  8. Suppression of plant defenses by a Myzus persicae (green peach aphid) salivary effector protein

    PubMed Central

    Elzinga, Dezi A.; De Vos, Martin

    2014-01-01

    The complex interactions between aphids and their host plant are species-specific and involve multiple layers of recognition and defense. Aphid salivary proteins, which are released into the plant during phloem feeding, are a likely mediator of these interactions. In an approach to identify aphid effectors that facilitate feeding from host plants, eleven Myzus persicae (green peach aphid) salivary proteins and the GroEL protein of Buchnera aphidicola, a bacterial endosymbiont of this aphid species, were expressed transiently in Nicotiana tabacum (tobacco). Whereas two salivary proteins increased aphid reproduction, expression of three other aphid proteins and GroEL significantly decreased aphid reproduction on N. tabacum. These effects were recapitulated in stable transgenic Arabidopsis thaliana (Arabidopsis) plants. Further experiments with A. thaliana expressing Mp55, a salivary protein that increased aphid reproduction, showed lower accumulation of 4-methoxyindol-3-ylmethylglucosinolate, callose, and hydrogen peroxide in response to aphid feeding. Mp55-expressing plants also were more attractive for aphids in choice assays. Silencing Mp55 gene expression in M. persicae using RNA interference approaches reduced aphid reproduction on N. tabacum, A. thaliana, and Nicotiana benthamiana. Together, these results demonstrate a role for Mp55, a protein with as yet unknown molecular function, in the interaction of M. persicae with its host plants. PMID:24654979

  9. Suppression of plant defenses by a Myzus persicae (green peach aphid) salivary effector protein.

    PubMed

    Elzinga, Dezi A; De Vos, Martin; Jander, Georg

    2014-07-01

    The complex interactions between aphids and their host plant are species-specific and involve multiple layers of recognition and defense. Aphid salivary proteins, which are released into the plant during phloem feeding, are a likely mediator of these interactions. In an approach to identify aphid effectors that facilitate feeding from host plants, eleven Myzus persicae (green peach aphid) salivary proteins and the GroEL protein of Buchnera aphidicola, a bacterial endosymbiont of this aphid species, were expressed transiently in Nicotiana tabacum (tobacco). Whereas two salivary proteins increased aphid reproduction, expression of three other aphid proteins and GroEL significantly decreased aphid reproduction on N. tabacum. These effects were recapitulated in stable transgenic Arabidopsis thaliana plants. Further experiments with A. thaliana expressing Mp55, a salivary protein that increased aphid reproduction, showed lower accumulation of 4-methoxyindol-3-ylmethylglucosinolate, callose and hydrogen peroxide in response to aphid feeding. Mp55-expressing plants also were more attractive for aphids in choice assays. Silencing Mp55 gene expression in M. persicae using RNA interference approaches reduced aphid reproduction on N. tabacum, A. thaliana, and N. benthamiana. Together, these results demonstrate a role for Mp55, a protein with as-yet-unknown molecular function, in the interaction of M. persicae with its host plants. PMID:24654979

  10. VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor–effector complex

    PubMed Central

    Bondage, Devanand D.; Lin, Jer-Sheng; Ma, Lay-Sun; Kuo, Chih-Horng; Lai, Erh-Min

    2016-01-01

    Type VI secretion system (T6SS) is a macromolecular machine used by many Gram-negative bacteria to inject effectors/toxins into eukaryotic hosts or prokaryotic competitors for survival and fitness. To date, our knowledge of the molecular determinants and mechanisms underlying the transport of these effectors remains limited. Here, we report that two T6SS encoded valine-glycine repeat protein G (VgrG) paralogs in Agrobacterium tumefaciens C58 specifically control the secretion and interbacterial competition activity of the type VI DNase toxins Tde1 and Tde2. Deletion and domain-swapping analysis identified that the C-terminal extension of VgrG1 specifically confers Tde1 secretion and Tde1-dependent interbacterial competition activity in planta, and the C-terminal variable region of VgrG2 governs this specificity for Tde2. Functional studies of VgrG1 and VgrG2 variants with stepwise deletion of the C terminus revealed that the C-terminal 31 aa (C31) of VgrG1 and 8 aa (C8) of VgrG2 are the molecular determinants specifically required for delivery of each cognate Tde toxin. Further in-depth studies on Tde toxin delivery mechanisms revealed that VgrG1 interacts with the adaptor/chaperone–effector complex (Tap-1–Tde1) in the absence of proline-alanine-alanine-arginine (PAAR) and the VgrG1–PAAR complex forms independent of Tap-1 and Tde1. Importantly, we identified the regions involved in these interactions. Although the entire C31 segment is required for binding with the Tap-1–Tde1 complex, only the first 15 aa of this region are necessary for PAAR binding. These results suggest that the VgrG1 C terminus interacts sequentially or simultaneously with the Tap-1–Tde1 complex and PAAR to govern Tde1 translocation across bacterial membranes and delivery into target cells for antibacterial activity. PMID:27313214

  11. VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor-effector complex.

    PubMed

    Bondage, Devanand D; Lin, Jer-Sheng; Ma, Lay-Sun; Kuo, Chih-Horng; Lai, Erh-Min

    2016-07-01

    Type VI secretion system (T6SS) is a macromolecular machine used by many Gram-negative bacteria to inject effectors/toxins into eukaryotic hosts or prokaryotic competitors for survival and fitness. To date, our knowledge of the molecular determinants and mechanisms underlying the transport of these effectors remains limited. Here, we report that two T6SS encoded valine-glycine repeat protein G (VgrG) paralogs in Agrobacterium tumefaciens C58 specifically control the secretion and interbacterial competition activity of the type VI DNase toxins Tde1 and Tde2. Deletion and domain-swapping analysis identified that the C-terminal extension of VgrG1 specifically confers Tde1 secretion and Tde1-dependent interbacterial competition activity in planta, and the C-terminal variable region of VgrG2 governs this specificity for Tde2. Functional studies of VgrG1 and VgrG2 variants with stepwise deletion of the C terminus revealed that the C-terminal 31 aa (C31) of VgrG1 and 8 aa (C8) of VgrG2 are the molecular determinants specifically required for delivery of each cognate Tde toxin. Further in-depth studies on Tde toxin delivery mechanisms revealed that VgrG1 interacts with the adaptor/chaperone-effector complex (Tap-1-Tde1) in the absence of proline-alanine-alanine-arginine (PAAR) and the VgrG1-PAAR complex forms independent of Tap-1 and Tde1. Importantly, we identified the regions involved in these interactions. Although the entire C31 segment is required for binding with the Tap-1-Tde1 complex, only the first 15 aa of this region are necessary for PAAR binding. These results suggest that the VgrG1 C terminus interacts sequentially or simultaneously with the Tap-1-Tde1 complex and PAAR to govern Tde1 translocation across bacterial membranes and delivery into target cells for antibacterial activity. PMID:27313214

  12. 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

  13. 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.

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

    PubMed

    Rubio, Noelia; Mei, Kuo-Ching; Klippstein, Rebecca; Costa, Pedro M; Hodgins, Naomi; Wang, Julie Tzu-Wen; Festy, Frederic; Abbate, Vincenzo; Hider, Robert C; Chan, Ka Lung Andrew; Al-Jamal, Khuloud T

    2015-09-01

    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

  15. Synthesis of strongly fluorescent molybdenum disulfide nanosheets for cell-targeted labeling.

    PubMed

    Wang, Nan; Wei, Fang; Qi, Yuhang; Li, Hongxiang; Lu, Xin; Zhao, Guoqiang; Xu, Qun

    2014-11-26

    MoS2 nanosheets with polydispersity of the lateral dimensions from natural mineral molybdenite have been prepared in the emulsions microenvironment built by the water/surfactant/CO2 system. The size, thickness, and atomic structure are characterized by transmission electron microscopy (TEM), atomic force microscopy (AFM), and laser-scattering particle size analysis. Meanwhile, by the analysis of photoluminescence spectroscopy and microscope, the MoS2 nanosheets with smaller lateral dimensions exhibit extraordinary photoluminescence properties different from those with relatively larger lateral dimensions. The discovery of the excitation dependent photoluminescence for MoS2 nanosheets makes them potentially of interests for the applications in optoelectronics and biology. Moreover, we demonstrate that the fabricated MoS2 nanosheets can be a nontoxic fluorescent label for cell-targeted labeling application. PMID:25380411

  16. 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

  17. Structural Features Facilitating Tumor Cell Targeting and Internalization by Bleomycin and Its Disaccharide

    PubMed Central

    2016-01-01

    We have shown previously that the bleomycin (BLM) carbohydrate moiety can recapitulate the tumor cell targeting effects of the entire BLM molecule, that BLM itself is modular in nature consisting of a DNA-cleaving aglycone which is delivered selectively to the interior of tumor cells by its carbohydrate moiety, and that there are disaccharides structurally related to the BLM disaccharide which are more efficient than the natural disaccharide at tumor cell targeting/uptake. Because BLM sugars can deliver molecular cargoes selectively to tumor cells, and thus potentially form the basis for a novel antitumor strategy, it seemed important to consider additional structural features capable of affecting the efficiency of tumor cell recognition and delivery. These included the effects of sugar polyvalency and net charge (at physiological pH) on tumor cell recognition, internalization, and trafficking. Since these parameters have been shown to affect cell surface recognition, internalization, and distribution in other contexts, this study has sought to define the effects of these structural features on tumor cell recognition by bleomycin and its disaccharide. We demonstrate that both can have a significant effect on tumor cell binding/internalization, and present data which suggests that the metal ions normally bound by bleomycin following clinical administration may significantly contribute to the efficiency of tumor cell uptake, in addition to their characterized function in DNA cleavage. A BLM disaccharide-Cy5** conjugate incorporating the positively charged dipeptide d-Lys-d-Lys was found to associate with both the mitochondria and the nuclear envelope of DU145 cells, suggesting possible cellular targets for BLM disaccharide–cytotoxin conjugates. PMID:25905565

  18. 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

    2013-05-01

    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 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. PMID:23531079

  19. A cyst nematode effector binds to diverse plant proteins, increases nematode susceptibility and affects root morphology.

    PubMed

    Pogorelko, Gennady; Juvale, Parijat S; Rutter, William B; Hewezi, Tarek; Hussey, Richard; Davis, Eric L; Mitchum, Melissa G; Baum, Thomas J

    2016-08-01

    Cyst nematodes are plant-parasitic roundworms that are of significance in many cropping systems around the world. Cyst nematode infection is facilitated by effector proteins secreted from the nematode into the plant host. The cDNAs of the 25A01-like effector family are novel sequences that were isolated from the oesophageal gland cells of the soybean cyst nematode (Heterodera glycines). To aid functional characterization, we identified an orthologous member of this protein family (Hs25A01) from the closely related sugar beet cyst nematode H. schachtii, which infects Arabidopsis. Constitutive expression of the Hs25A01 CDS in Arabidopsis plants caused a small increase in root length, accompanied by up to a 22% increase in susceptibility to H. schachtii. A plant-expressed RNA interference (RNAi) construct targeting Hs25A01 transcripts in invading nematodes significantly reduced host susceptibility to H. schachtii. These data document that Hs25A01 has physiological functions in planta and a role in cyst nematode parasitism. In vivo and in vitro binding assays confirmed the specific interactions of Hs25A01 with an Arabidopsis F-box-containing protein, a chalcone synthase and the translation initiation factor eIF-2 β subunit (eIF-2bs), making these proteins probable candidates for involvement in the observed changes in plant growth and parasitism. A role of eIF-2bs in the mediation of Hs25A01 virulence function is further supported by the observation that two independent eIF-2bs Arabidopsis knock-out lines were significantly more susceptible to H. schachtii. PMID:26575318

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

    PubMed

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

    2016-05-01

    Signalling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalysed by the E1, E2 and E3 three-enzyme cascade, which links the carboxy terminus of ubiquitin to the ε-amino group of, in most cases, a lysine of the substrate via an isopeptide bond. 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. For example, many bacterial pathogens exploit ubiquitin signalling using virulence factors that function as E3 ligases, deubiquitinases or as enzymes that directly attack ubiquitin. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a permissive niche for its replication in phagocytes. Here we demonstrate that members of the SidE effector family of L. pneumophila ubiquitinate multiple Rab small GTPases associated with the endoplasmic reticulum. Moreover, we show that these proteins are capable of catalysing ubiquitination without the need for the E1 and E2 enzymes. A putative mono-ADP-ribosyltransferase motif critical for the ubiquitination activity is also essential for the role of the SidE family in intracellular bacterial replication in a protozoan host. The E1/E2-independent ubiquitination catalysed by these enzymes is energized by nicotinamide adenine dinucleotide, which activates ubiquitin by the formation of ADP-ribosylated ubiquitin. These results establish that ubiquitination can be catalysed by a single enzyme, the activity of which does not require ATP. PMID:27049943

  1. 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

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

    PubMed Central

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

    2012-01-01

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

  3. 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

  4. 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. PMID:26831115

  5. 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

  6. 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. PMID:25912102

  7. 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

  8. Electroporation of Functional Bacterial Effectors into Mammalian Cells

    DOE PAGESBeta

    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

    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.

  9. 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…

  10. Electroporation of Functional Bacterial Effectors into Mammalian Cells

    SciTech Connect

    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

    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. Developmental control of integrin expression regulates Th2 effector homing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Integrin CD18, a component of the LFA-1 complex that also includes CD11a, is essential for Th2, but not Th1, cell homing, but the explanation for this phenomenon remains obscure. In this study, we investigate the mechanism by which Th2 effector responses require the LFA-1 complex. CD11a-deficient T ...

  12. Robot End Effector To Place and Solder Solar Cells

    NASA Technical Reports Server (NTRS)

    Hagerty, J. J.

    1982-01-01

    Encapsulated in robot end effector is RF induction-heating coil for heating solar cell while in transit. Holes in encapsulant permit end of unit to act as vacuum pickup to grip solar cell. Use of RF induction heating allows cell to be heated without requiring direct mechanical and thermal contact of bonding tool such as soldering iron.

  13. 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

  14. The Coding and Effector Transfer of Movement Sequences

    ERIC Educational Resources Information Center

    Kovacs, Attila J.; Muhlbauer, Thomas; Shea, Charles H.

    2009-01-01

    Three experiments utilizing a 14-element arm movement sequence were designed to determine if reinstating the visual-spatial coordinates, which require movements to the same spatial locations utilized during acquisition, results in better effector transfer than reinstating the motor coordinates, which require the same pattern of homologous muscle…

  15. [PROBLEM OF END EFFECTOR OF ISCHEMIC POSTCONDITIONING OF THE HEART].

    PubMed

    Maslov, L N; Naryzhnaya, N V; Pei, J-M; Zhang, Y; Wang, H; Khaliulin, I J; Lishmanov, Yu B

    2015-06-01

    It is well known that cardiovascular disease and in particular acute myocardial infarction are a major cause of death among working-age population in Russia. Some of the patients die after successful recanalization of the infarct-related coronary artery as a result of ischemic and reperfusion injury of the heart. It is obvious that there is an urgent need to develop new approaches to prevention reoxygenation heart damages. In this regard the study of adaptive phenomenon postconditioning is of particular interest. This analysis of literature source preformed by authors of the article indicates that main pretenders to the role of end-effectors of ischemic postconditioning of the heart are: (1) Ca(2+)-dependent K+ channel of BK-type (big conductance K+ channel), (2) mitoKATp channel (mitochondrial ATP-sensitive K+ channel), (3) MPT pore (mitochondrial permeability transition pore). At the same time, some investigators consider that mitoK(ATP) channel is only an intermediate link in the series of signaling events ensured an increase in cardiac tolerance to impact of ischemia-reperfusion. The most likely end effector of these three structures is MPT pore. Alternatively, it is possible, that unique molecular complex appearing a single end effector of postconditioning does not exist. Perhaps, that there are several effectors ensured cardioprotective effect of an adaptive phenomenon of postconditioning. PMID:26470485

  16. RMS end effector waiting for command and SPAS-01 nearby

    NASA Technical Reports Server (NTRS)

    1983-01-01

    The end effector of the remote manipulator system (RMS) appears to be waiting for its next command at the top of this frame and the Shuttle pallet satellite (SPAS-01), in its free flying mode, appears nearby. The three letters legible on the SPAS stand for Messerschmitt-Boelkow-Blohm Gmbit, a West German firm.

  17. 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

  18. Toxoplasma co-opts host cells it does not invade.

    PubMed

    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

  19. 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

  20. The Pseudomonas syringae Type III Effector HopG1 Induces Actin Remodeling to Promote Symptom Development and Susceptibility during Infection.

    PubMed

    Shimono, Masaki; Lu, Yi-Ju; Porter, Katie; Kvitko, Brian H; Henty-Ridilla, Jessica; Creason, Allison; He, Sheng Yang; Chang, Jeff H; Staiger, Christopher J; Day, Brad

    2016-07-01

    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

  1. SnTox5-Snn5: A novel Stagonospora nodorum effector-wheat gene interaction and its relationship with the SnToxA-Tsn1 and SnTox3-Snn3-B1 interactions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Stagonospora nodorum-wheat interaction consists of multiple pathogen - produced necrotrophic effectors that interact directly or indirectly with specific host gene products to induce the disease Stagonospora nodorum blotch (SNB). Here, we used a tetraploid wheat mapping population to identify an...

  2. The Salmonella effector SopB prevents ROS-induced apoptosis of epithelial cells by retarding TRAF6 recruitment to mitochondria.

    PubMed

    Ruan, Haihua; Zhang, Zhen; Tian, Li; Wang, Suying; Hu, Shuangyan; Qiao, Jian-Jun

    2016-09-16

    Microbial pathogens enter host cells by injecting effector proteins of the Type III secretion system (T3SS), which facilitate pathogen translocation across the host cell membrane. These effector proteins exert their effects by modulating a variety of host innate immune responses, thereby facilitating bacterial replication and systemic infection. Salmonella enterica serovar typhimurium (S.typhimurium) is a clinically important pathogen that causes food poisoning and gastroenteritis. The SopB effector protein of S. typhimurium, encoded by Salmonella pathogenicity islands (SPI)-1 T3SS, protects host epithelial cells from infection-induced apoptosis. However, how SopB influences apoptosis induction remains unclear. Here, we investigated the mechanism of SopB action in host cells. We found that SopB inhibits infection-induced apoptosis by attenuating the production of reactive oxygen species (ROS) in mitochondria, the crucial organelles for apoptosis initiation. Further investigation revealed that SopB binds to cytosolic tumor necrosis factor receptor associated factor 6 (TRAF6) and forms a trap preventing the mitochondrial recruitment of TRAF6, an essential event for ROS generation within mitochondria. By studying the response of Traf6(+/+) and Traf6(-/-)mouse embryonic fibroblasts to S. typhimurium infection, we found that TRAF6 promoted apoptosis by increasing ROS accumulation, which led to increased Bax/Bcl-2 ratio, Bax recruitment to mitochondrial membrane, and release of Cyt c into the cytoplasm. These findings show that SopB suppresses host cell apoptosis by binding to TRAF6 and preventing mitochondrial ROS generation. PMID:27473656

  3. 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

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

    PubMed Central

    Hatanaka, Tadashi; Nakano, Masahito; Oda, Kenji

    2016-01-01

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

  6. Neem leaf glycoprotein promotes dual generation of central and effector memory CD8(+) T cells against sarcoma antigen vaccine to induce protective anti-tumor immunity.

    PubMed

    Ghosh, Sarbari; Sarkar, Madhurima; Ghosh, Tithi; Guha, Ipsita; Bhuniya, Avishek; Saha, Akata; Dasgupta, Shayani; Barik, Subhasis; Bose, Anamika; Baral, Rathindranath

    2016-03-01

    We have previously shown that Neem Leaf Glycoprotein (NLGP) mediates sustained tumor protection by activating host immune response. Now we report that adjuvant help from NLGP predominantly generates CD44(+)CD62L(high)CCR7(high) central memory (TCM; in lymph node) and CD44(+)CD62L(low)CCR7(low) effector memory (TEM; in spleen) CD8(+) T cells of Swiss mice after vaccination with sarcoma antigen (SarAg). Generated TCM and TEM participated either to replenish memory cell pool for sustained disease free states or in rapid tumor eradication respectively. TCM generated after SarAg+NLGP vaccination underwent significant proliferation and IL-2 secretion following SarAg re-stimulation. Furthermore, SarAg+NLGP vaccination helps in greater survival of the memory precursor effector cells at the peak of the effector response and their maintenance as mature memory cells, in comparison to single modality treatment. Such response is corroborated with the reduced phosphorylation of FOXO in the cytosol and increased KLF2 in the nucleus associated with enhanced CD62L, CCR7 expression of lymph node-resident CD8(+) T cells. However, spleen-resident CD8(+) T memory cells show superior efficacy for immediate memory-to-effector cell conversion. The data support in all aspects that SarAg+NLGP demonstrate superiority than SarAg vaccination alone that benefits the host by rapid effector functions whenever required, whereas, central-memory cells are thought to replenish the memory cell pool for ultimate sustained disease free survival till 60 days following post-vaccination tumor inoculation. PMID:26851529

  7. Operation and maintenance manual for the common video end effector system (CVEE) system 6260

    SciTech Connect

    Pardini, A.F., Westinghouse Hanford

    1996-07-24

    This document defines the requirements for the operation,maintenance, and storage of the Common Video End Effector System (CVEE) used with the video end effectors as part of the Light Duty Utility Arm (LDUA) system.

  8. Host-microbe interactions in the gut of Drosophila melanogaster

    PubMed Central

    Kuraishi, Takayuki; Hori, Aki; Kurata, Shoichiro

    2013-01-01

    Many insect species subsist on decaying and contaminated matter and are thus exposed to large quantities of microorganisms. To control beneficial commensals and combat infectious pathogens, insects must be armed with efficient systems for microbial recognition, signaling pathways, and effector molecules. The molecular mechanisms regulating these host-microbe interactions in insects have been largely clarified in Drosophila melanogaster with its powerful genetic and genomic tools. Here we review recent advances in this field, focusing mainly on the relationships between microbes and epithelial cells in the intestinal tract where the host exposure to the external environment is most frequent. PMID:24381562

  9. 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

  10. Early effector cells survive the contraction phase in malaria infection and generate both central and effector memory T cells.

    PubMed

    Opata, Michael M; Carpio, Victor H; Ibitokou, Samad A; Dillon, Brian E; Obiero, Joshua M; Stephens, Robin

    2015-06-01

    CD4 T cells orchestrate immunity against blood-stage malaria. However, a major challenge in designing vaccines to the disease is poor understanding of the requirements for the generation of protective memory T cells (Tmem) from responding effector T cells (Teff) in chronic parasite infection. In this study, we use a transgenic mouse model with T cells specific for the merozoite surface protein (MSP)-1 of Plasmodium chabaudi to show that activated T cells generate three distinct Teff subsets with progressive activation phenotypes. The earliest observed Teff subsets (CD127(-)CD62L(hi)CD27(+)) are less divided than CD62L(lo) Teff and express memory genes. Intermediate (CD62L(lo)CD27(+)) effector subsets include the most multicytokine-producing T cells, whereas fully activated (CD62L(lo)CD27(-)) late effector cells have a terminal Teff phenotype (PD-1(+), Fas(hi), AnnexinV(+)). We show that although IL-2 promotes expansion, it actually slows terminal effector differentiation. Using adoptive transfer, we show that only early Teff survive the contraction phase and generate the terminal late Teff subsets, whereas in uninfected recipients, they become both central and effector Tmem. Furthermore, we show that progression toward full Teff activation is promoted by increased duration of infection, which in the long-term promotes Tem differentiation. Therefore, we have defined markers of progressive activation of CD4 Teff at the peak of malaria infection, including a subset that survives the contraction phase to make Tmem, and show that Ag and cytokine levels during CD4 T cell expansion influence the proportion of activated cells that can survive contraction and generate memory in malaria infection. PMID:25911759

  11. Dual RNA-seq reveals Meloidogyne graminicola transcriptome and candidate effectors during the interaction with rice plants.

    PubMed

    Petitot, Anne-Sophie; Dereeper, Alexis; Agbessi, Mawusse; Da Silva, Corinne; Guy, Julie; Ardisson, Morgane; Fernandez, Diana

    2016-08-01

    Root-knot nematodes secrete proteinaceous effectors into plant tissues to facilitate infection by suppressing host defences and reprogramming the host metabolism to their benefit. Meloidogyne graminicola is a major pest of rice (Oryza sativa) in Asia and Latin America, causing important crop losses. The goal of this study was to identify M. graminicola pathogenicity genes expressed during the plant-nematode interaction. Using the dual RNA-sequencing (RNA-seq) strategy, we generated transcriptomic data of M. graminicola samples covering the pre-parasitic J2 stage and five parasitic stages in rice plants, from the parasitic J2 to the adult female. In the absence of a reference genome, a de novo M. graminicola transcriptome of 66 396 contigs was obtained from those reads that were not mapped on the rice genome. Gene expression profiling across the M. graminicola life cycle revealed key genes involved in nematode development and provided insights into the genes putatively associated with parasitism. The development of a 'secreted protein prediction' pipeline revealed a typical set of proteins secreted by nematodes, as well as a large number of cysteine-rich proteins and putative nuclear proteins. Combined with expression data, this pipeline enabled the identification of 15 putative effector genes, including two homologues of well-characterized effectors from cyst nematodes (CLE-like and VAP1) and a metallothionein. The localization of gene expression was assessed by in situ hybridization for a subset of candidates. All of these data represent important molecular resources for the elucidation of M. graminicola biology and for the selection of potential targets for the development of novel control strategies for this nematode species. PMID:26610268

  12. The dark side of mast cell-targeted therapy in prostate cancer.

    PubMed

    Pittoni, Paola; Colombo, Mario Paolo

    2012-02-15

    Tumor development requires accomplices among white blood cells. Other than macrophages, mast cells have been observed to support the outgrowth of certain neoplasias because of their proangiogenic properties. In some tumor settings, however, mast cells may have a protective role, exerted by their proinflammatory mediators. In prostate cancer, no conclusive data on mast cell function were available. Here, we discuss recent work on the role of mast cells in mouse and human prostate cancer, showing that mast cells can behave alternatively as dangerous promoters, innocent bystanders, or essential guardians of tumors, according to the stage and origin of transformed cells. In particular, mast cells are essential for the outgrowth of early-stage tumors due to their matrix metalloproteinase-9 production, become dispensable in advanced-stage, post-epithelial-to-mesenchymal transition, and are protective against neuroendocrine prostate tumor variants. The common expression of c-Kit by mast cells and neuroendocrine clones suggests a possible competition for the ligand Stem cell factor and offers the chance of curing early-stage disease while preventing neuroendocrine tumors using c-Kit-targeted therapy. This review discusses the implications of these findings on the advocated mast cell-targeted cancer therapy and considers future directions in the study of mast cells and their interactions with other c-Kit-expressing cells. PMID:22307838

  13. Mast Cell Targeted Chimeric Toxin Can Be Developed as an Adjunctive Therapy in Colon Cancer Treatment

    PubMed Central

    Wang, Shan; Li, Linmei; Shi, Renren; Liu, Xueting; Zhang, Junyan; Zou, Zehong; Hao, Zhuofang; Tao, Ailin

    2016-01-01

    The association of colitis with colorectal cancer has become increasingly clear with mast cells being identified as important inflammatory cells in the process. In view of the relationship between mast cells and cancer, we studied the effect and mechanisms of mast cells in the development of colon cancer. Functional and mechanistic insights were gained from ex vivo and in vivo studies of cell interactions between mast cells and CT26 cells. Further evidence was reversely obtained in studies of mast cell targeted Fcε-PE40 chimeric toxin. Experiments revealed mast cells could induce colon tumor cell proliferation and invasion. Cancer progression was found to be related to the density of mast cells in colonic submucosa. The activation of MAPK, Rho-GTPase, and STAT pathways in colon cancer cells was triggered by mast cells during cell-to-cell interaction. Lastly, using an Fcε-PE40 chimeric toxin we constructed, we confirmed the promoting effect of mast cells in development of colon cancer. Mast cells are a promoting factor of colon cancer and thus also a potential therapeutic target. The Fcε-PE40 chimeric toxin targeting mast cells could effectively prevent colon cancer in vitro and in vivo. Consequently, these data may demonstrate a novel immunotherapeutic approach for the treatment of tumors. PMID:26978404

  14. Discovery of NKT cells and development of NKT cell-targeted anti-tumor immunotherapy

    PubMed Central

    TANIGUCHI, Masaru; HARADA, Michishige; DASHTSOODOL, Nyambayar; KOJO, Satoshi

    2015-01-01

    Natural Killer T (NKT) cells are unique lymphocytes characterized by their expression of a single invariant antigen receptor encoded by Vα14Jα18 in mice and Vα24Jα18 in humans, which recognizes glycolipid antigens in association with the monomorphic CD1d molecule. NKT cells mediate adjuvant activity to activate both CD8T cells to kill MHC-positive tumor cells and NK cells to eliminate MHC-negative tumor at the same time in patients, resulting in the complete eradication of tumors without relapse. Therefore, the NKT cell-targeted therapy can be applied to any type of tumor and also to anyone individual, regardless of HLA type. Phase IIa clinical trials on advanced lung cancers and head and neck tumors have been completed and showed significantly prolonged median survival times with only the primary treatment. Another potential treatment option for the future is to use induced pluripotent stem cell (iPS)-derived NKT cells, which induced adjuvant effects on anti-tumor responses, inhibiting in vivo tumor growth in a mouse model. PMID:26194854

  15. Functional single-walled carbon nanotubes/chitosan conjugate for tumor cells targeting

    NASA Astrophysics Data System (ADS)

    Wu, Baoyan; Ou, Zhongmin; Xing, Da

    2009-08-01

    The application of single-walled carbon nanotubes (SWCNTs) in the field of biomedicine is becoming an exciting topic because of their flexible structure and propensity for chemical functionalization. In this assay, a novel noncovalently functional SWCNTs based on a natural biocompatible polymer chitosan has been developed for tumor cells targeting. First, SWCNTs were modified by chitosan (CHIT-SWCNT). Second, CHIT-SWCNT was coupled with fluorescein isothiocyanate (FITC), based on the reaction between the isothiocyanate group of FITC and the primary amino group of chitosan. Third, the FITC functionalized CHIT-SWCNT was conjugated with folic acid (FA) after activation with EDC/NHS, based on the reaction between the NHS group of FA and the primary free amino group of chitosan to construct the functional SWCNT/CHIT conjugate, CHIT-SWCNT-FA. The fluorescence CHIT-SWCNT-FA has been used to detect tumor cells with confocal microscopy imaging technology. Our experimental results indicate that the novel CHIT-SWCNT-FA is soluble and stable in PBS, and it can be readily transported inside tumor cells. Combining the intrinsic properties of carbon nanotubes and the versatility of chitosan, CHIT-SWCNT can be used as potential devices for targeted drug delivery and tumor cell sensing. The proposed assay could provide a feasible alternative to presently available functional SWCNTs in biological applications.

  16. Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles

    PubMed Central

    Unzueta, Ugutz; Céspedes, María Virtudes; Ferrer-Miralles, Neus; Casanova, Isolda; Cedano, Juan; Corchero, José Luis; Domingo-Espín, Joan; Villaverde, Antonio; Mangues, Ramón; Vázquez, Esther

    2012-01-01

    Background Cell-targeting peptides or proteins are appealing tools in nanomedicine and innovative medicines because they increase the local drug concentration and reduce potential side effects. CXC chemokine receptor 4 (CXCR4) is a cell surface marker associated with several severe human pathologies, including colorectal cancer, for which intracellular targeting agents are currently missing. Results Four different peptides that bind CXCR4 were tested for their ability to internalize a green fluorescent protein-based reporter nanoparticle into CXCR4+ cells. Among them, only the 18 mer peptide T22, an engineered segment derivative of polyphemusin II from the horseshoe crab, efficiently penetrated target cells via a rapid, receptor-specific endosomal route. This resulted in accumulation of the reporter nanoparticle in a fully fluorescent and stable form in the perinuclear region of the target cells, without toxicity either in cell culture or in an in vivo model of metastatic colorectal cancer. Conclusion Given the urgent demand for targeting agents in the research, diagnosis, and treatment of CXCR4-linked diseases, including colorectal cancer and human immunodeficiency virus infection, T22 appears to be a promising tag for the intracellular delivery of protein drugs, nanoparticles, and imaging agents. PMID:22923991

  17. Quantum dot surface chemistry and functionalization for cell targeting and imaging.

    PubMed

    Bilan, Regina; Fleury, Fabrice; Nabiev, Igor; Sukhanova, Alyona

    2015-04-15

    Quantum dots (QDs) are highly fluorescent nanoscale crystals with size-dependent emission spectra. Due to their excellent photophysical properties, QDs are a promising alternative to organic fluorescent dyes and fluorescent proteins for cell targeting, imaging, and drug delivery. For biomedical applications, QDs should be chemically modified to be stable in aqueous solutions and tagged with the recognition molecules or drugs. Here, we review surface modification approaches to, and strategies for, conjugation of bioactive molecules with QDs. There are a variety of methods of QD surface modification and QD incorporation into larger delivery systems that yield fluorescent nanocarriers from 10 nm to several micrometers. Conjugates of QDs with peptides, proteins, antibodies, oligonucleotides, and small molecules have been used for fluorescent targeting, tracking, and imaging both in vitro and in vivo. Due to an extremely high stability to photobleaching, QDs were used for long-term visualization. QD applications pave the way for new generations of ultrasensitive detection, diagnostic systems, as well as drug delivery approaches, combining accurate targeting, delivery, and imaging in a single assay. PMID:25710410

  18. Galactosylated Chitosan Oligosaccharide Nanoparticles for Hepatocellular Carcinoma Cell-Targeted Delivery of Adenosine Triphosphate

    PubMed Central

    Zhu, Xiu Liang; Du, Yong Zhong; Yu, Ri Sheng; Liu, Ping; Shi, Dan; Chen, Ying; Wang, Ying; Huang, Fang Fang

    2013-01-01

    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 cell targeting, thus warranting further in vivo or clinical investigations. PMID:23899789

  19. End-Effector Development for the PIP Puck Handling Robot

    SciTech Connect

    Fowley, M.D.

    2001-01-03

    It has been decided that excess, weapons-grade plutonium shall be immobilized to prevent nuclear proliferation. The method of immobilization is to encapsulate the plutonium in a ceramic puck, roughly the size of a hockey puck, using a sintering process. This method has been officially identified as the Plutonium Immobilization Process (PIP). A Can-in-Canister storage method will be used to further immobilize the plutonium. The Can-in-Canister method uses the existing design of a Defense Waste Processing Facility (DWPF) canister to house the plutonium pucks. the process begins with several pucks being stacked in a stainless steel can. Several of the stainless steel cans are stacked in a cage-like magazine. Several of the magazines are then placed in a DWPF canister. The DWPF canister is then filled with molten glass containing high-level, radioactive waste from the DWPF vitrification process. The Can-in-Canister method makes reclamation of plutonium from the pucks technically difficult and highly undesirable. The mechanical requirements of the Can-in-Canister process, in conjunction with the amount of time required to immobilize the vast quantities of weapons-grade plutonium, will expose personnel to unnecessarily high levels of radiation if the processes were completed manually, in glove boxes. Therefore, automated equipment is designed into the process to reduce or eliminate personnel exposure. Robots are used whenever the automated handling operations become complicated. There are two such operations in the initial stages of the Can-in-Canister process, which required a six-axis robot. The first operation is a press unloading process. The second operation is a tray transfer process. To successfully accomplish the operational tasks described in the two operations, the end-effector of the robot must be versatile, lightweight, and rugged. As a result of these demands, an extensive development process was undertaken to design the optimum end-effector for these puck

  20. End-Effector Development for the PIP Puck Handling Robot

    SciTech Connect

    Fowley, M.D.

    2001-01-31

    It has been decided that excess, weapons-grade plutonium shall be immobilized to prevent nuclear proliferation. The method of immobilization is to encapsulate the plutonium in a ceramic puck, roughly the size of a hockey puck, using a sintering process. This method has been officially identified as the Plutonium Immobilization Process (PIP). A Can-in-Canister storage method will be used to further immobilize the plutonium. The Can-in-Canister method uses the existing design of a Defense Waste Processing Facility (DWPF) canister to house the plutonium pucks. the process begins with several pucks being stacked in a stainless steel can. Several of the stainless steel cans are stacked in a cage-like magazine. Several of the magazines are then placed in a DWPF canister. The DWPF canister is then filled with molten glass containing high-level, radioactive waste from the DWPF vitrification process. The Can-in-Canister method makes reclamation of plutonium from the pucks technically difficult and highly undesirable. The mechanical requirements of the Can-in-Canister process, in conjunction with the amount of time required to immobilize the vast quantities of weapons-grade plutonium, will expose personnel to unnecessarily high levels of radiation if the processes were completed manually, in glove boxes. Therefore, automated equipment is designed into the process to reduce or eliminate personnel exposure. Robots are used whenever the automated handling operations become complicated. There are two such operations in the initial stages of the Can-in-Canister process, which required a six-axis robot. The first operation is a press unloading process. The second operation is a tray transfer process. To successfully accomplish the operational tasks described in the two operations, the end-effector of the robot must be versatile, lightweight, and rugged. As a result of these demands, an extensive development process was undertaken to design the optimum end-effector for these puck

  1. The enteropathogenic E. coli effector EspF targets and disrupts the nucleolus by a process regulated by mitochondrial dysfunction.

    PubMed

    Dean, Paul; Scott, Jon A; Knox, Andrew A; Quitard, Sabine; Watkins, Nicholas J; Kenny, Brendan

    2010-01-01

    The nucleolus is a multifunctional structure within the nucleus of eukaryotic cells and is the primary site of ribosome biogenesis. Almost all viruses target and disrupt the nucleolus--a feature exclusive to this pathogen group. Here, using a combination of bio-imaging, genetic and biochemical analyses, we demonstrate that the enteropathogenic E. coli (EPEC) effector protein EspF specifically targets the nucleolus and disrupts a subset of nucleolar factors. Driven by a defined N-terminal nucleolar targeting domain, EspF causes the complete loss from the nucleolus of nucleolin, the most abundant nucleolar protein. We also show that other bacterial species disrupt the nucleolus, dependent on their ability to deliver effector proteins into the host cell. Moreover, we uncover a novel regulatory mechanism whereby nucleolar targeting by EspF is strictly controlled by EPEC's manipulation of host mitochondria. Collectively, this work reveals that the nucleolus may be a common feature of bacterial pathogenesis and demonstrates that a bacterial pathogen has evolved a highly sophisticated mechanism to enable spatio-temporal control over its virulence proteins. PMID:20585567

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  4. Utilizing Gene Tree Variation to Identify Candidate Effector Genes in Zymoseptoria tritici

    PubMed Central

    McDonald, Megan C.; McGinness, Lachlan; Hane, James K.; Williams, Angela H.; Milgate, Andrew; Solomon, Peter S.

    2016-01-01

    Zymoseptoria tritici is a host-specific, necrotrophic pathogen of wheat. Infection by Z. tritici is characterized by its extended latent period, which typically lasts 2 wks, and is followed by extensive host cell death, and rapid proliferation of fungal biomass. This work characterizes the level of genomic variation in 13 isolates, for which we have measured virulence on 11 wheat cultivars with differential resistance genes. Between the reference isolate, IPO323, and the 13 Australian isolates we identified over 800,000 single nucleotide polymorphisms, of which ∼10% had an effect on the coding regions of the genome. Furthermore, we identified over 1700 probable presence/absence polymorphisms in genes across the Australian isolates using de novo assembly. Finally, we developed a gene tree sorting method that quickly identifies groups of isolates within a single gene alignment whose sequence haplotypes correspond with virulence scores on a single wheat cultivar. Using this method, we have identified < 100 candidate effector genes whose gene sequence correlates with virulence toward a wheat cultivar carrying a major resistance gene. PMID:26837952

  5. Msp40 effector of root-knot nematode manipulates plant immunity to facilitate parasitism

    PubMed Central

    Niu, Junhai; Liu, Pei; Liu, Qian; Chen, Changlong; Guo, Quanxin; Yin, Junmei; Yang, Guangsui; Jian, Heng

    2016-01-01

    Root-knot nematodes (RKNs) are obligate biotrophic parasites that invade plant roots and engage in prolonged and intimate relationships with their hosts. Nematode secretions, some of which have immunosuppressing activity, play essential roles in successful parasitism; however, their mechanisms of action remain largely unknown. Here, we show that the RKN-specific gene MiMsp40, cloned from Meloidogyne incognita, is expressed exclusively in subventral oesophageal gland cells and is strongly upregulated during early parasitic stages. Arabidopsis plants overexpressing MiMsp40 were more susceptible to nematode infection than were wild type plants. Conversely, the host-derived MiMsp40 RNAi suppressed nematode parasitism and/or reproduction. Moreover, overexpression of MiMsp40 in plants suppressed the deposition of callose and the expression of marker genes for bacterial elicitor elf18-triggered immunity. Transient expression of MiMsp40 prevented Bax-triggered defence-related programmed cell death. Co-agroinfiltration assays indicated that MiMsp40 also suppressed macroscopic cell death triggered by MAPK cascades or by the ETI cognate elicitors R3a/Avr3a. Together, these results demonstrate that MiMsp40 is a novel Meloidogyne-specific effector that is injected into plant cells by early parasitic stages of the nematode and that plays a role in suppressing PTI and/or ETI signals to facilitate RKN parasitism. PMID:26797310

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

    PubMed Central

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

    2007-01-01

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

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

    PubMed Central

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

    2015-01-01

    Coxiella burnetii is a highly infectious bacterium that promotes its own replication in macrophages by inhibiting several host cell responses. Here, we show that C. burnetii inhibits caspase-1 activation in primary mouse macrophages. By using co-infection experiments, we determine that the infection of macrophages with C. burnetii inhibits the caspase-11-mediated non-canonical activation of the NLRP3 inflammasome induced by subsequent infection with Escherichia coli or Legionella pneumophila. Genetic screening using flagellin mutants of L. pneumophila as a sur