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Sample records for suppress host protein

  1. A secretory protein of necrotrophic fungus Sclerotinia sclerotiorum that suppresses host resistance.

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    Wenjun Zhu

    Full Text Available SSITL (SS1G_14133 of Sclerotinia sclerotiorum encodes a protein with 302 amino acid residues including a signal peptide, its secretion property was confirmed with immunolocalization and immunofluorescence techniques. SSITL was classified in the integrin alpha N-terminal domain superfamily, and its 3D structure is similar to those of human integrin α4-subunit and a fungal integrin-like protein. When S. sclerotiorum was inoculated to its host, high expression of SSITL was detected during the initial stages of infection (1.5-3.0 hpi. Targeted silencing of SSITL resulted in a significant reduction in virulence; on the other hand, inoculation of SSITL silenced transformant A10 initiated strong and rapid defense response in Arabidopsis, the highest expressions of defense genes PDF1.2 and PR-1 appeared at 3 hpi which was 9 hr earlier than that time when plants were inoculated with the wild-type strain of S. sclerotiorum. Systemic resistance induced by A10 was detected by analysis of the expression of PDF1.2 and PR-1, and confirmed following inoculation with Botrytis cinerea. A10 induced much larger lesions on Arabidopsis mutant ein2 and jar1, and slightly larger lesions on mutant pad4 and NahG in comparison with the wild-type plants. Furthermore, both transient and constitutive expression of SSITL in Arabidopsis suppressed the expression of PDF1.2 and led to be more susceptible to A10 and the wild-type strain of S. sclerotiorum and B. cinerea. Our results suggested that SSITL is an effector possibly and plays significant role in the suppression of jasmonic/ethylene (JA/ET signal pathway mediated resistance at the early stage of infection.

  2. Multifunctional roles of leader protein of foot-and-mouth disease viruses in suppressing host antiviral responses.

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    Liu, Yingqi; Zhu, Zixiang; Zhang, Miaotao; Zheng, Haixue

    2015-10-28

    Foot-and-mouth disease virus (FMDV) leader protein (L(pro)) is a papain-like proteinase, which plays an important role in FMDV pathogenesis. L(pro) exists as two forms, Lab and Lb, due to translation being initiated from two different start codons separated by 84 nucleotides. L(pro) self-cleaves from the nascent viral polyprotein precursor as the first mature viral protein. In addition to its role as a viral proteinase, L(pro) also has the ability to antagonize host antiviral effects. To promote FMDV replication, L(pro) can suppress host antiviral responses by three different mechanisms: (1) cleavage of eukaryotic translation initiation factor 4 γ (eIF4G) to shut off host protein synthesis; (2) inhibition of host innate immune responses through restriction of interferon-α/β production; and (3) L(pro) can also act as a deubiquitinase and catalyze deubiquitination of innate immune signaling molecules. In the light of recent functional and biochemical findings regarding L(pro), this review introduces the basic properties of L(pro) and the mechanisms by which it antagonizes host antiviral responses.

  3. Tofacitinib Suppresses Antibody Responses to Protein Therapeutics in Murine Hosts1

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    Onda, Masanori; Ghoreschi, Kamran; Steward-Tharp, Scott; Thomas, Craig; O’Shea, John J.; Pastan, Ira H.; FitzGerald, David J.

    2014-01-01

    Immunogenicity remains the ‘Achilles’ heel’ of protein-based therapeutics. Anti-drug antibodies produced in response to protein therapeutics can severely limit both the safety and efficacy of this expanding class of agent. Here we report that monotherapy of mice with tofacitinib (the Janus kinase inhibitor) quells antibody responses to an immunotoxin derived from the bacterial protein, Pseudomonas exotoxin A, as well as to the model antigen, keyhole limpet hemocyanin. Thousandfold reductions in IgG1 titers to both antigens were observed 21 days post-immunization. In fact, suppression was evident for all IgG isotypes and IgM. A reduction in IgG3 production was also noted with a thymus-independent type II antigen. Mechanistic investigations revealed that tofacitinib treatment led to reduced numbers of CD127+ pro-B cells. Furthermore, we observed fewer germinal center B cells and the impaired formation of germinal centers of mice treated with tofacitinib. Since normal immunoglobulin levels were still present during the tofacitinib treatment, this agent specifically reduced anti-drug antibodies, thus preserving the potential efficacy of biological therapeutics, including those that are used as cancer therapeutics. PMID:24890727

  4. La Crosse bunyavirus nonstructural protein NSs serves to suppress the type I interferon system of mammalian hosts.

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    Blakqori, Gjon; Delhaye, Sophie; Habjan, Matthias; Blair, Carol D; Sánchez-Vargas, Irma; Olson, Ken E; Attarzadeh-Yazdi, Ghassem; Fragkoudis, Rennos; Kohl, Alain; Kalinke, Ulrich; Weiss, Siegfried; Michiels, Thomas; Staeheli, Peter; Weber, Friedemann

    2007-05-01

    La Crosse virus (LACV) is a mosquito-transmitted member of the Bunyaviridae family that causes severe encephalitis in children. For the LACV nonstructural protein NSs, previous overexpression studies with mammalian cells had suggested two different functions, namely induction of apoptosis and inhibition of RNA interference (RNAi). Here, we demonstrate that mosquito cells persistently infected with LACV do not undergo apoptosis and mount a specific RNAi response. Recombinant viruses that either express (rLACV) or lack (rLACVdelNSs) the NSs gene similarly persisted and were prone to the RNAi-mediated resistance to superinfection. Furthermore, in mosquito cells overexpressed LACV NSs was unable to inhibit RNAi against Semliki Forest virus. In mammalian cells, however, the rLACVdelNSs mutant virus strongly activated the antiviral type I interferon (IFN) system, whereas rLACV as well as overexpressed NSs suppressed IFN induction. Consequently, rLACVdelNSs was attenuated in IFN-competent mouse embryo fibroblasts and animals but not in systems lacking the type I IFN receptor. In situ analyses of mouse brains demonstrated that wild-type and mutant LACV mainly infect neuronal cells and that NSs is able to suppress IFN induction in the central nervous system. Thus, our data suggest little relevance of the NSs-induced apoptosis or RNAi inhibition for growth or pathogenesis of LACV in the mammalian host and indicate that NSs has no function in the insect vector. Since deletion of the viral NSs gene can be fully complemented by inactivation of the host's IFN system, we propose that the major biological function of NSs is suppression of the mammalian innate immune response.

  5. Analysis of putative apoplastic effectors from the nematode, Globodera rostochiensis, and identification of an expansin-like protein that can induce and suppress host defenses.

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    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.

  6. Analysis of putative apoplastic effectors from the nematode, Globodera rostochiensis, and identification of an expansin-like protein that can induce and suppress host defenses.

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    Shawkat Ali

    Full Text Available 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.

  7. Seneca Valley Virus Suppresses Host Type I Interferon Production by Targeting Adaptor Proteins MAVS, TRIF, and TANK for Cleavage.

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    Qian, Suhong; Fan, Wenchun; Liu, Tingting; Wu, Mengge; Zhang, Huawei; Cui, Xiaofang; Zhou, Yun; Hu, Junjie; Wei, Shaozhong; Chen, Huanchun; Li, Xiangmin; Qian, Ping

    2017-08-15

    Seneca Valley virus (SVV) is an oncolytic RNA virus belonging to the Picornaviridae family. Its nucleotide sequence is highly similar to those of members of the Cardiovirus genus. SVV is also a neuroendocrine cancer-selective oncolytic picornavirus that can be used for anticancer therapy. However, the interaction between SVV and its host is yet to be fully characterized. In this study, SVV inhibited antiviral type I interferon (IFN) responses by targeting different host adaptors, including mitochondrial antiviral signaling (MAVS), Toll/interleukin 1 (IL-1) receptor domain-containing adaptor inducing IFN-β (TRIF), and TRAF family member-associated NF-κB activator (TANK), via viral 3C protease (3C pro ). SVV 3C pro mediated the cleavage of MAVS, TRIF, and TANK at specific sites, which required its protease activity. The cleaved MAVS, TRIF, and TANK lost the ability to regulate pattern recognition receptor (PRR)-mediated IFN production. The cleavage of TANK also facilitated TRAF6-induced NF-κB activation. SVV was also found to be sensitive to IFN-β. Therefore, SVV suppressed antiviral IFN production to escape host antiviral innate immune responses by cleaving host adaptor molecules. IMPORTANCE Host cells have developed various defenses against microbial pathogen infection. The production of IFN is the first line of defense against microbial infection. However, viruses have evolved many strategies to disrupt this host defense. SVV, a member of the Picornavirus genus, is an oncolytic virus that shows potential functions in anticancer therapy. It has been demonstrated that IFN can be used in anticancer therapy for certain tumors. However, the relationship between oncolytic virus and innate immune response in anticancer therapy is still not well known. In this study, we showed that SVV has evolved as an effective mechanism to inhibit host type I IFN production by using its 3C pro to cleave the molecules MAVS, TRIF, and TANK directly. These molecules are crucial for

  8. Label-free Proteomic Reveals that Cowpea Severe Mosaic Virus Transiently Suppresses the Host Leaf Protein Accumulation During the Compatible Interaction with Cowpea (Vigna unguiculata [L.] Walp.).

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    Paiva, Ana L S; Oliveira, Jose T A; de Souza, Gustavo A; Vasconcelos, Ilka M

    2016-12-02

    Viruses are important plant pathogens that threaten diverse crops worldwide. Diseases caused by Cowpea severe mosaic virus (CPSMV) have drawn attention because of the serious damages they cause to economically important crops including cowpea. This work was undertaken to quantify and identify the responsive proteins of a susceptible cowpea genotype infected with CPSMV, in comparison with mock-inoculated controls, using label-free quantitative proteomics and databanks, aiming at providing insights on the molecular basis of this compatible interaction. Cowpea leaves were mock- or CPSMV-inoculated and 2 and 6 days later proteins were extracted and analyzed. More than 3000 proteins were identified (data available via ProteomeXchange, identifier PXD005025) and 75 and 55 of them differentially accumulated in response to CPSMV, at 2 and 6 DAI, respectively. At 2 DAI, 76% of the proteins decreased in amount and 24% increased. However, at 6 DAI, 100% of the identified proteins increased. Thus, CPSMV transiently suppresses the synthesis of proteins involved particularly in the redox homeostasis, protein synthesis, defense, stress, RNA/DNA metabolism, signaling, and other functions, allowing viral invasion and spread in cowpea tissues.

  9. HIV protein sequence hotspots for crosstalk with host hub proteins.

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    Mahdi Sarmady

    Full Text Available HIV proteins target host hub proteins for transient binding interactions. The presence of viral proteins in the infected cell results in out-competition of host proteins in their interaction with hub proteins, drastically affecting cell physiology. Functional genomics and interactome datasets can be used to quantify the sequence hotspots on the HIV proteome mediating interactions with host hub proteins. In this study, we used the HIV and human interactome databases to identify HIV targeted host hub proteins and their host binding partners (H2. We developed a high throughput computational procedure utilizing motif discovery algorithms on sets of protein sequences, including sequences of HIV and H2 proteins. We identified as HIV sequence hotspots those linear motifs that are highly conserved on HIV sequences and at the same time have a statistically enriched presence on the sequences of H2 proteins. The HIV protein motifs discovered in this study are expressed by subsets of H2 host proteins potentially outcompeted by HIV proteins. A large subset of these motifs is involved in cleavage, nuclear localization, phosphorylation, and transcription factor binding events. Many such motifs are clustered on an HIV sequence in the form of hotspots. The sequential positions of these hotspots are consistent with the curated literature on phenotype altering residue mutations, as well as with existing binding site data. The hotspot map produced in this study is the first global portrayal of HIV motifs involved in altering the host protein network at highly connected hub nodes.

  10. Insect Cells as Hosts for Recombinat Proteins

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    Murwani, Retno

    1997-01-01

    Since the development of recombinant baculovirus expression system, insect cell culture has rapidly gain popularity as the method of choice for production of a variety of biologically active proteins. Up to date tens of recombinant protein have been produced by this method commercially or non-commercially and have been widely used for research. This review describes the basic concept of baculovirus expression vector and the use of insect cells as host for recombinant proteins. Examples of the...

  11. Virus-mediated suppression of host non-self recognition facilitates horizontal transmission of heterologous viruses.

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    Songsong Wu

    2017-03-01

    Full Text Available Non-self recognition is a common phenomenon among organisms; it often leads to innate immunity to prevent the invasion of parasites and maintain the genetic polymorphism of organisms. Fungal vegetative incompatibility is a type of non-self recognition which often induces programmed cell death (PCD and restricts the spread of molecular parasites. It is not clearly known whether virus infection could attenuate non-self recognition among host individuals to facilitate its spread. Here, we report that a hypovirulence-associated mycoreovirus, named Sclerotinia sclerotiorum mycoreovirus 4 (SsMYRV4, could suppress host non-self recognition and facilitate horizontal transmission of heterologous viruses. We found that cell death in intermingled colony regions between SsMYRV4-infected Sclerotinia sclerotiorum strain and other tested vegetatively incompatible strains was markedly reduced and inhibition barrage lines were not clearly observed. Vegetative incompatibility, which involves Heterotrimeric guanine nucleotide-binding proteins (G proteins signaling pathway, is controlled by specific loci termed het (heterokaryon incompatibility loci. Reactive oxygen species (ROS plays a key role in vegetative incompatibility-mediated PCD. The expression of G protein subunit genes, het genes, and ROS-related genes were significantly down-regulated, and cellular production of ROS was suppressed in the presence of SsMYRV4. Furthermore, SsMYRV4-infected strain could easily accept other viruses through hyphal contact and these viruses could be efficiently transmitted from SsMYRV4-infected strain to other vegetatively incompatible individuals. Thus, we concluded that SsMYRV4 is capable of suppressing host non-self recognition and facilitating heterologous viruses transmission among host individuals. These findings may enhance our understanding of virus ecology, and provide a potential strategy to utilize hypovirulence-associated mycoviruses to control fungal diseases.

  12. Host DNA synthesis-suppressing factor in culture fluid of tissue cultures infected with measles virus

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    Minagawa, T.; Nakaya, C.; Iida, H.

    1974-01-01

    Host DNA synthesis is suppressed by the culture fluid of cell cultures infected with measles virus. This activity in the culture fluid is initiated somewhat later than the growth of infectious virus. Ninety percent of host DNA synthesis in HeLa cells is inhibited by culture fluid of 3-day-old cell cultures of Vero or HeLa cells infected with measles virus. This suppressing activity is not a property of the virion, but is due to nonvirion-associated componentnent which shows none of the activities of measles virus such as hemagglutination, hemolysis, or cell fusion nor does it have the antigenicity of measles virus as tested by complement-fixation or hemagglutination-inhibiting antibody blocking tests. Neutralization of the activity of this component is not attained with the pooled sera of convalescent measles patients. This component has molecular weights of about 45,000, 20,000, and 3,000 and appears to be a heat-stable protein. The production of host DNA suppressing factor (DSF) is blocked by cycloheximide. Neither uv-inactivated nor antiserum-neutralized measles virus produce DSF. Furthermore, such activity of nonvirion-associated component is not detected in the culture fluid of cultures infected with other RNA viruses such as poliovirus, vesicular stomatitis virus, or Sindbis virus. (auth)

  13. Demonstration and suppression of a radioresistant host-versus-graft reaction

    International Nuclear Information System (INIS)

    Thierfelder, S.; Roessler, R. v.; Ruppelt, W.

    1975-01-01

    The possibilities of suppressing and measuring a radioresistant host-versus-graft reaction are described. According to the authors, the host-versus-graft immune reaction may outlast whole-body irradiation at high doses. (BSC/AK) [de

  14. Suppression of matrix protein synthesis in endothelial cells by herpes simplex virus is not dependent on viral protein synthesis

    International Nuclear Information System (INIS)

    Kefalides, N.A.

    1986-01-01

    The synthesis of matrix proteins by human endothelial cells (EC) in vitro was studied before and at various times after infection with Herpes Simplex virus Type 1 (HSV-1) or 2 (HSV-2). Monolayers of EC were either mock-infected or infected with virus for 1 hr at a multiplicity infection (MOI) of 5 to 20 at 37 0 C. Control and infected cultures were pulse-labeled for 1 or 2 hrs with either [ 14 C]proline or [ 35 S]methionine. Synthesis of labeled matrix proteins was determined by SDS-gel electrophoresis. Suppression of synthesis of fibronectin, Type IV collagen and thrombospondin began as early as 2 hrs and became almost complete by 10 hrs post-infection. The degree of suppression varied with the protein and the virus dose. Suppression of Type IV collagen occurred first followed by that of fibronectin and then thrombospondin. Infection of EC with UV irradiated HSV-1 or HSV-2 resulted in suppression of host-cell protein synthesis as well as viral protein synthesis. Infection with intact virus in the presence of actinomycin-D resulted in suppression of both host-cell and viral protein synthesis. The data indicate that infection of EC with HSV leads to suppression of matrix protein synthesis which does not depend on viral protein synthesis

  15. Tipping the balance: Sclerotinia sclerotiorum secreted oxalic acid suppresses host defenses by manipulating the host redox environment.

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    Brett Williams

    2011-06-01

    Full Text Available Sclerotinia sclerotiorum is a necrotrophic ascomycete fungus with an extremely broad host range. This pathogen produces the non-specific phytotoxin and key pathogenicity factor, oxalic acid (OA. Our recent work indicated that this fungus and more specifically OA, can induce apoptotic-like programmed cell death (PCD in plant hosts, this induction of PCD and disease requires generation of reactive oxygen species (ROS in the host, a process triggered by fungal secreted OA. Conversely, during the initial stages of infection, OA also dampens the plant oxidative burst, an early host response generally associated with plant defense. This scenario presents a challenge regarding the mechanistic details of OA function; as OA both suppresses and induces host ROS during the compatible interaction. In the present study we generated transgenic plants expressing a redox-regulated GFP reporter. Results show that initially, Sclerotinia (via OA generates a reducing environment in host cells that suppress host defense responses including the oxidative burst and callose deposition, akin to compatible biotrophic pathogens. Once infection is established however, this necrotroph induces the generation of plant ROS leading to PCD of host tissue, the result of which is of direct benefit to the pathogen. In contrast, a non-pathogenic OA-deficient mutant failed to alter host redox status. The mutant produced hypersensitive response-like features following host inoculation, including ROS induction, callose formation, restricted growth and cell death. These results indicate active recognition of the mutant and further point to suppression of defenses by the wild type necrotrophic fungus. Chemical reduction of host cells with dithiothreitol (DTT or potassium oxalate (KOA restored the ability of this mutant to cause disease. Thus, Sclerotinia uses a novel strategy involving regulation of host redox status to establish infection. These results address a long-standing issue

  16. The Ustilago maydis effector Pep1 suppresses plant immunity by inhibition of host peroxidase activity.

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    Christoph Hemetsberger

    Full Text Available The corn smut Ustilago maydis establishes a biotrophic interaction with its host plant maize. This interaction requires efficient suppression of plant immune responses, which is attributed to secreted effector proteins. Previously we identified Pep1 (Protein essential during penetration-1 as a secreted effector with an essential role for U. maydis virulence. pep1 deletion mutants induce strong defense responses leading to an early block in pathogenic development of the fungus. Using cytological and functional assays we show that Pep1 functions as an inhibitor of plant peroxidases. At sites of Δpep1 mutant penetrations, H₂O₂ strongly accumulated in the cell walls, coinciding with a transcriptional induction of the secreted maize peroxidase POX12. Pep1 protein effectively inhibited the peroxidase driven oxidative burst and thereby suppresses the early immune responses of maize. Moreover, Pep1 directly inhibits peroxidases in vitro in a concentration-dependent manner. Using fluorescence complementation assays, we observed a direct interaction of Pep1 and the maize peroxidase POX12 in vivo. Functional relevance of this interaction was demonstrated by partial complementation of the Δpep1 mutant defect by virus induced gene silencing of maize POX12. We conclude that Pep1 acts as a potent suppressor of early plant defenses by inhibition of peroxidase activity. Thus, it represents a novel strategy for establishing a biotrophic interaction.

  17. Insecticidal activity of the metalloprotease AprA occurs through suppression of host cellular and humoral immunity.

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    Lee, Seung Ah; Jang, Seong Han; Kim, Byung Hyun; Shibata, Toshio; Yoo, Jinwook; Jung, Yunjin; Kawabata, Shun-Ichiro; Lee, Bok Luel

    2018-04-01

    The biochemical characterization of virulence factors from entomopathogenic bacteria is important to understand entomopathogen-insect molecular interactions. Pseudomonas entomophila is a typical entomopathogenic bacterium that harbors virulence factors against several insects. However, the molecular actions of these factors against host innate immune responses are not clearly elucidated. In this study, we observed that bean bugs (Riptortus pedestris) that were injected with P. entomophila were highly susceptible to this bacterium. To determine how P. entomophila counteracts the host innate immunity to survive within the insect, we purified a highly enriched protein with potential host insect-killing activity from the culture supernatant of P. entomophila. Then, a 45-kDa protein was purified to homogeneity and identified as AprA which is an alkaline zinc metalloprotease of the genus Pseudomonas by liquid chromatography mass spectrometry (LC-MS). Purified AprA showed a pronounced killing effect against host insects and suppressed both host cellular and humoral innate immunity. Furthermore, to show that AprA is an important insecticidal protein of P. entomophila, we used an aprA-deficient P. entomophila mutant strain (ΔaprA). When ΔaprA mutant cells were injected to host insects, this mutant exhibited extremely attenuated virulence. In addition, the cytotoxicity against host hemocytes and the antimicrobial peptide-degrading ability of the ΔaprA mutant were greatly decreased. These findings suggest that AprA functions as an important insecticidal protein of P. entomophila via suppression of host cellular and humoral innate immune responses. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Trans-suppression of defense DEFB1 gene in intestinal epithelial cells following Cryptosporidium parvum infection is associated with host delivery of parasite Cdg7_FLc_1000 RNA.

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    Ming, Zhenping; Gong, Ai-Yu; Wang, Yang; Zhang, Xin-Tian; Li, Min; Dolata, Courtney E; Chen, Xian-Ming

    2018-03-01

    To counteract host immunity, Cryptosporidium parvum has evolved multiple strategies to suppress host antimicrobial defense. One such strategy is to reduce the production of the antimicrobial peptide beta-defensin 1 (DEFB1) by host epithelial cells but the underlying mechanisms remain unclear. Recent studies demonstrate that a panel of parasite RNA transcripts of low protein-coding potential are delivered into infected host cells and may modulate host gene transcription. Using in vitro models of intestinal cryptosporidiosis, in this study, we analyzed the expression profile of host beta-defensin genes in host cells following infection. We found that C. parvum infection caused a significant downregulation of the DEFB1 gene. Interestingly, downregulation of DEFB1 gene was associated with host delivery of Cdg7_FLc_1000 RNA transcript, a C. parvum RNA that has previously demonstrated to be delivered into the nuclei of infected host cells. Knockdown of Cdg7_FLc_1000 in host cells could attenuate the trans-suppression of host DEFB1 gene and decreased the parasite burden. Therefore, our data suggest that trans-suppression of DEFB1 gene in intestinal epithelial cells following C. parvum infection involves host delivery of parasite Cdg7_FLc_1000 RNA, a process that may be relevant to the epithelial defense evasion by C. parvum at the early stage of infection.

  19. Rapamycin delays growth of Wnt-1 tumors in spite of suppression of host immunity

    International Nuclear Information System (INIS)

    Svirshchevskaya, Elena V; Mariotti, Jacopo; Wright, Mollie H; Viskova, Natalia Y; Telford, William; Fowler, Daniel H; Varticovski, Lyuba

    2008-01-01

    Rapamycin, an inhibitor of mammalian target of Rapamycin (mTOR), is an immunosuppressive agent that has anti-proliferative effects on some tumors. However, the role of Rapamycin-induced immune suppression on tumor progression has not been examined. We developed a transplantation model for generation of mammary tumors in syngeneic recipients that can be used to address the role of the immune system on tumor progression. We examined the effect of Rapamycin on the immune system and growth of MMTV-driven Wnt-1 mammary tumors which were transplanted into irradiated and bone marrow-reconstituted, or naïve mice. Rapamycin induced severe immunosuppression and significantly delayed the growth of Wnt-1 tumors. T cell depletion in spleen and thymus and reduction in T cell cytokine secretion were evident within 7 days of therapy. By day 20, splenic but not thymic T cell counts, and cytokine secretion recovered. We determined whether adoptive T cell therapy enhances the anti-cancer effect using ex vivo generated Rapamycin-resistant T cells. However, T cell transfer during Rapamycin therapy did not improve the outcome relative to drug therapy alone. Thus, we could not confirm that suppression of T cell immunity contributes to tumor growth in this model. Consistent with suppression of the mTOR pathway, decreased 4E-BP1, p70 S6-kinase, and S6 protein phosphorylation correlated with a decrease in Wnt-1 tumor cell proliferation. Rapamycin has a direct anti-tumor effect on Wnt-1 breast cancer in vivo that involves inhibition of the mTOR pathway at doses that also suppress host immune responses

  20. Methods for production of proteins in host cells

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    Donnelly, Mark; Joachimiak, Andrzej

    2004-01-13

    The present invention provides methods for the production of proteins, particularly toxic proteins, in host cells. The invention provides methods which use a fusion protein comprising a chaperonin binding domain in host cells induced or regulated to have increased levels of chaperonin which binds the chaperonin binding domain.

  1. Trans-suppression of host CDH3 and LOXL4 genes during Cryptosporidium parvum infection involves nuclear delivery of parasite Cdg7_FLc_1000 RNA.

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    Ming, Zhenping; Gong, Ai-Yu; Wang, Yang; Zhang, Xin-Tian; Li, Min; Li, Yao; Pang, Jing; Dong, Stephanie; Strauss-Soukup, Juliane K; Chen, Xian-Ming

    2018-05-01

    Intestinal infection by Cryptosporidium parvum causes significant alterations in the gene expression profile in host epithelial cells. Previous studies demonstrate that a panel of parasite RNA transcripts of low protein-coding potential are delivered into infected host cells and may modulate host gene transcription. Using in vitro models of human intestinal cryptosporidiosis, we report here that trans-suppression of the cadherin 3 (CDH3) and lysyl oxidase like 4 (LOXL4) genes in human intestinal epithelial cells following C. parvum infection involves host delivery of the Cdg7_FLc_1000 RNA, a C. parvum RNA that has been previously demonstrated to be delivered into the nuclei of infected host cells. Downregulation of CDH3 and LOXL4 genes was detected in host epithelial cells following C. parvum infection or in cells expressing the parasite Cdg7_FLc_1000 RNA. Knockdown of Cdg7_FLc_1000 attenuated the trans-suppression of CDH3 and LOXL4 genes in host cells induced by infection. Interestingly, Cdg7_FLc_1000 was detected to be recruited to the promoter regions of both CDH3 and LOXL4 gene loci in host cells following C. parvum infection. Host delivery of Cdg7_FLc_1000 promoted the PH domain zinc finger protein 1 (PRDM1)-mediated H3K9 methylation associated with trans-suppression in the CDH3 gene locus, but not the LOXL4 gene. Therefore, our data suggest that host delivery of Cdg7_FLc_1000 causes CDH3 trans-suppression in human intestinal epithelial cells following C. parvum infection through PRDM1-mediated H3K9 methylation in the CDH3 gene locus, whereas Cdg7_FLc_1000 induces trans-suppression of the host LOXL4 gene through H3K9/H3K27 methylation-independent mechanisms. Copyright © 2018 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Georgina Fabro

    2011-11-01

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

  3. Plant pathology: monitoring a pathogen-targeted host protein.

    Science.gov (United States)

    Ellis, Jeff; Dodds, Peter

    2003-05-13

    A plant protein RIN4 is targeted and modified by bacterial pathogens as part of the disease process. At least two host resistance proteins monitor this pathogen interference and trigger the plant's defence responses.

  4. Pocket Proteins Suppress Head and Neck Cancer

    Science.gov (United States)

    Shin, Myeong-Kyun; Pitot, Henry C.; Lambert, Paul F.

    2012-01-01

    Head and neck squamous cell carcinomas (HNSCC) is a common cancer in humans long known to be caused by tobacco and alcohol use, but now an increasing percentage of HNSCC is recognized to be caused by the same human papillomaviruses (HPVs) that cause cervical and other anogenital cancers. HPV-positive HNSCCs differ remarkably from HPV-negative HNSCCs in their clinical response and molecular properties. From studies in mice, we know that E7 is the dominant HPV oncoprotein in head and neck cancer. E7 is best known for its ability to inactivate pRb, the product of the retinoblastoma tumor susceptibility gene. However loss of pRb function does not fully account for E7’s potency in causing head and neck cancer. In this study, we characterized the cancer susceptibility of mice deficient in the expression of pRb and either of two related “pocket” proteins, p107 and p130, that are also inactivated by E7. pRb/p107 deficient mice developed head and neck cancer as frequently as do HPV16 E7 transgenic mice. The head and neck epithelia of the pRb/p107 deficient mice also displayed the same acute phenotypes and biomarker readouts as observed in the epithelia of E7 transgenic mice. Mice deficient for pRb and p130 in their head and neck epithelia showed intermediate acute and tumor phenotypes. We conclude that pRb and p107 act together to efficiently suppress head and neck cancer, and are therefore highly relevant targets of HPV16 E7 in its contribution to HPV-positive HNSCC. PMID:22237625

  5. Protein prenylation: a new mode of host-pathogen interaction.

    Science.gov (United States)

    Amaya, Moushimi; Baranova, Ancha; van Hoek, Monique L

    2011-12-09

    Post translational modifications are required for proteins to be fully functional. The three step process, prenylation, leads to farnesylation or geranylgeranylation, which increase the hydrophobicity of the prenylated protein for efficient anchoring into plasma membranes and/or organellar membranes. Prenylated proteins function in a number of signaling and regulatory pathways that are responsible for basic cell operations. Well characterized prenylated proteins include Ras, Rac and Rho. Recently, pathogenic prokaryotic proteins, such as SifA and AnkB, have been shown to be prenylated by eukaryotic host cell machinery, but their functions remain elusive. The identification of other bacterial proteins undergoing this type of host-directed post-translational modification shows promise in elucidating host-pathogen interactions to develop new therapeutics. This review incorporates new advances in the study of protein prenylation into a broader aspect of biology with a focus on host-pathogen interaction. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. Differences in the effects of host suppression on the adoptive immunotherapy of subcutaneous and visceral tumors

    International Nuclear Information System (INIS)

    Chang, A.E.; Shu, S.Y.; Chou, T.; Lafreniere, R.; Rosenberg, S.A.

    1986-01-01

    A syngeneic transplantable sarcoma induced in C57BL/6 mice, MCA 105, was used in studies to examine host suppression on the adoptive immunotherapy of established intradermal and experimentally induced pulmonary and hepatic metastases. Fresh immune splenocytes were generated from mice immunized to the MCA 105 tumor by a mixture of viable tumor cells and Corynebacterium parvum. The adoptive immunotherapy of intradermal MCA 105 tumor with immune cells required prior immunosuppression of the recipient by sublethal irradiation with 500 R or T-cell depletion. The effect of whole-body sublethal irradiation appeared to eliminate a systemic host suppression mechanism, since partialbody irradiation involving the tumor-bearing area did not permit successful immunotherapy. Host irradiation was not required to achieve successful immunotherapy of experimentally induced pulmonary or hepatic metastases. In nonirradiated recipients bearing both intradermal and pulmonary tumors, host suppression did not affect the function of transferred immune cells to induce regression of pulmonary metastases. Thus, suppression of adoptive immunotherapy appears to be relevant to tumors confined to the skin and subcutaneous tissue but not to tumor in visceral sites, such as the lung and liver

  7. Influenza A Virus-Host Protein Interactions Control Viral Pathogenesis.

    Science.gov (United States)

    Zhao, Mengmeng; Wang, Lingyan; Li, Shitao

    2017-08-01

    The influenza A virus (IAV), a member of the Orthomyxoviridae family, is a highly transmissible respiratory pathogen and represents a continued threat to global health with considerable economic and social impact. IAV is a zoonotic virus that comprises a plethora of strains with different pathogenic profiles. The different outcomes of viral pathogenesis are dependent on the engagement between the virus and the host cellular protein interaction network. The interactions may facilitate virus hijacking of host molecular machinery to fulfill the viral life cycle or trigger host immune defense to eliminate the virus. In recent years, much effort has been made to discover the virus-host protein interactions and understand the underlying mechanisms. In this paper, we review the recent advances in our understanding of IAV-host interactions and how these interactions contribute to host defense and viral pathogenesis.

  8. Impact of High-Level Expression of Heterologous Protein on Lactococcus lactis Host.

    Science.gov (United States)

    Kim, Mina; Jin, Yerin; An, Hyun-Joo; Kim, Jaehan

    2017-07-28

    The impact of overproduction of a heterologous protein on the metabolic system of host Lactococcus lactis was investigated. The protein expression profiles of L. lactis IL1403 containing two near-identical plasmids that expressed high- and low-level of the green fluorescent protein (GFP) were examined via shotgun proteomics. Analysis of the two strains via high-throughput LC-MS/MS proteomics identified the expression of 294 proteins. The relative amount of each protein in the proteome of both strains was determined by label-free quantification using the spectral counting method. Although expression level of most proteins were similar, several significant alterations in metabolic network were identified in the high GFP-producing strain. These changes include alterations in the pyruvate fermentation pathway, oxidative pentose phosphate pathway, and de novo synthesis pathway for pyrimidine RNA. Expression of enzymes for the synthesis of dTDP-rhamnose and N -acetylglucosamine from glucose was suppressed in the high GFP strain. In addition, enzymes involved in the amino acid synthesis or interconversion pathway were downregulated. The most noticeable changes in the high GFP-producing strain were a 3.4-fold increase in the expression of stress response and chaperone proteins and increase of caseinolytic peptidase family proteins. Characterization of these host expression changes witnessed during overexpression of GFP was might suggested the metabolic requirements and networks that may limit protein expression, and will aid in the future development of lactococcal hosts to produce more heterologous protein.

  9. Suppression of Plant Defenses by Herbivorous Mites Is Not Associated with Adaptation to Host Plants

    Directory of Open Access Journals (Sweden)

    Jéssica T. Paulo

    2018-06-01

    Full Text Available Some herbivores suppress plant defenses, which may be viewed as a result of the coevolutionary arms race between plants and herbivores. However, this ability is usually studied in a one-herbivore-one-plant system, which hampers comparative studies that could corroborate this hypothesis. Here, we extend this paradigm and ask whether the herbivorous spider-mite Tetranychus evansi, which suppresses the jasmonic-acid pathway in tomato plants, is also able to suppress defenses in other host plants at different phylogenetic distances from tomatoes. We test this using different plants from the Solanales order, namely tomato, jimsonweed, tobacco, and morning glory (three Solanaceae and one Convolvulaceae, and bean plants (Fabales. First, we compare the performance of T. evansi to that of the other two most-commonly found species of the same genus, T. urticae and T. ludeni, on several plants. We found that the performance of T. evansi is higher than that of the other species only on tomato plants. We then showed, by measuring trypsin inhibitor activity and life history traits of conspecific mites on either clean or pre-infested plants, that T. evansi can suppress plant defenses on all plants except tobacco. This study suggests that the suppression of plant defenses may occur on host plants other than those to which herbivores are adapted.

  10. Nuclear imprisonment of host cellular mRNA by nsp1β protein of porcine reproductive and respiratory syndrome virus

    International Nuclear Information System (INIS)

    Han, Mingyuan; Ke, Hanzhong; Zhang, Qingzhan; Yoo, Dongwan

    2017-01-01

    Positive-strand RNA genomes function as mRNA for viral protein synthesis which is fully reliant on host cell translation machinery. Competing with cellular protein translation apparatus needs to ensure the production of viral proteins, but this also stifles host innate defense. In the present study, we showed that porcine reproductive and respiratory syndrome virus (PRRSV), whose replication takes place in the cytoplasm, imprisoned host cell mRNA in the nucleus, which suggests a novel mechanism to enhance translation of PRRSV genome. PRRSV nonstructural protein (nsp) 1β was identified as the nuclear protein playing the role for host mRNA nuclear retention and subversion of host protein synthesis. A SAP (SAF-A/B, Acinus, and PIAS) motif was identified in nsp1β with the consensus sequence of 126 -LQxxLxxxGL- 135 . In situ hybridization unveiled that SAP mutants were unable to cause nuclear retention of host cell mRNAs and did not suppress host protein synthesis. In addition, these SAP mutants reverted PRRSV-nsp1β-mediated suppression of interferon (IFN) production, IFN signaling, and TNF-α production pathway. Using reverse genetics, a series of SAP mutant PRRS viruses, vK124A, vL126A, vG134A, and vL135A were generated. No mRNA nuclear retention was observed during vL126A and vL135A infections. Importantly, vL126A and vL135A did not suppress IFN production. For other arteriviruses, mRNA nuclear accumulation was also observed for LDV-nsp1β and SHFV-nsp1β. EAV-nsp1 was exceptional and did not block the host mRNA nuclear export. - Highlights: •PRRS virus blocks host mRNA nuclear export to the cytoplasm. •PRRSV nsp1β is the viral protein responsible for host mRNA nuclear retention. •SAP domain in nsp1β is essential for host mRNA nuclear retention and type I interferon suppression. •Mutation in the SAP domain of nsp1β causes the loss of function. •Host mRNA nuclear retention by nsp1β is common in the family Arteriviridae, except equine arteritis virus.

  11. Nuclear imprisonment of host cellular mRNA by nsp1β protein of porcine reproductive and respiratory syndrome virus

    Energy Technology Data Exchange (ETDEWEB)

    Han, Mingyuan, E-mail: hanming@umich.edu; Ke, Hanzhong; Zhang, Qingzhan; Yoo, Dongwan, E-mail: dyoo@illinois.edu

    2017-05-15

    Positive-strand RNA genomes function as mRNA for viral protein synthesis which is fully reliant on host cell translation machinery. Competing with cellular protein translation apparatus needs to ensure the production of viral proteins, but this also stifles host innate defense. In the present study, we showed that porcine reproductive and respiratory syndrome virus (PRRSV), whose replication takes place in the cytoplasm, imprisoned host cell mRNA in the nucleus, which suggests a novel mechanism to enhance translation of PRRSV genome. PRRSV nonstructural protein (nsp) 1β was identified as the nuclear protein playing the role for host mRNA nuclear retention and subversion of host protein synthesis. A SAP (SAF-A/B, Acinus, and PIAS) motif was identified in nsp1β with the consensus sequence of {sub 126}-LQxxLxxxGL-{sub 135}. In situ hybridization unveiled that SAP mutants were unable to cause nuclear retention of host cell mRNAs and did not suppress host protein synthesis. In addition, these SAP mutants reverted PRRSV-nsp1β-mediated suppression of interferon (IFN) production, IFN signaling, and TNF-α production pathway. Using reverse genetics, a series of SAP mutant PRRS viruses, vK124A, vL126A, vG134A, and vL135A were generated. No mRNA nuclear retention was observed during vL126A and vL135A infections. Importantly, vL126A and vL135A did not suppress IFN production. For other arteriviruses, mRNA nuclear accumulation was also observed for LDV-nsp1β and SHFV-nsp1β. EAV-nsp1 was exceptional and did not block the host mRNA nuclear export. - Highlights: •PRRS virus blocks host mRNA nuclear export to the cytoplasm. •PRRSV nsp1β is the viral protein responsible for host mRNA nuclear retention. •SAP domain in nsp1β is essential for host mRNA nuclear retention and type I interferon suppression. •Mutation in the SAP domain of nsp1β causes the loss of function. •Host mRNA nuclear retention by nsp1β is common in the family Arteriviridae, except equine

  12. Protein A Suppresses Immune Responses during Staphylococcus aureus Bloodstream Infection in Guinea Pigs

    Science.gov (United States)

    Kim, Hwan Keun; Falugi, Fabiana; Thomer, Lena; Missiakas, Dominique M.

    2015-01-01

    ABSTRACT   Staphylococcus aureus infection is not associated with the development of protective immunity, and disease relapses occur frequently. We hypothesize that protein A, a factor that binds immunoglobulin Fcγ and cross-links VH3 clan B cell receptors (IgM), is the staphylococcal determinant for host immune suppression. To test this, vertebrate IgM was examined for protein A cross-linking. High VH3 binding activity occurred with human and guinea immunoglobulin, whereas mouse and rabbit immunoglobulins displayed little and no binding, respectively. Establishing a guinea pig model of S. aureus bloodstream infection, we show that protein A functions as a virulence determinant and suppresses host B cell responses. Immunization with SpAKKAA, which cannot bind immunoglobulin, elicits neutralizing antibodies that enable guinea pigs to develop protective immunity. Importance  Staphylococcus aureus is the leading cause of soft tissue and bloodstream infections; however, a vaccine with clinical efficacy is not available. Using mice to model staphylococcal infection, earlier work identified protective antigens; however, corresponding human clinical trials did not reach their endpoints. We show that B cell receptor (IgM) cross-linking by protein A is an important immune evasion strategy of S. aureus that can be monitored in a guinea pig model of bloodstream infection. Further, immunization with nontoxigenic protein A enables infected guinea pigs to elicit antibody responses that are protective against S. aureus. Thus, the guinea pig model may support preclinical development of staphylococcal vaccines. PMID:25564466

  13. Dual functions of Rift Valley fever virus NSs protein: inhibition of host mRNA transcription and post-transcriptional downregulation of protein kinase PKR.

    Science.gov (United States)

    Ikegami, Tetsuro; Narayanan, Krishna; Won, Sungyong; Kamitani, Wataru; Peters, C J; Makino, Shinji

    2009-09-01

    Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus, family Bunyaviridae, is a negative-stranded RNA virus carrying a single-stranded, tripartite RNA genome. RVFV is an important zoonotic pathogen transmitted by mosquitoes and causes large outbreaks among ruminants and humans in Africa and the Arabian Peninsula. Human patients develop an acute febrile illness, followed by a fatal hemorrhagic fever, encephalitis, or ocular diseases. A viral nonstructural protein, NSs, is a major viral virulence factor. Past studies showed that NSs suppresses the transcription of host mRNAs, including interferon-beta mRNAs. Here we demonstrated that the NSs protein induced post-transcriptional downregulation of dsRNA-dependent protein kinase (PKR), to prevent phosphorylation of eIF2alpha and promoted viral translation in infected cells. These two biological activities of the NSs most probably have a synergistic effect in suppressing host innate immune functions and facilitate efficient viral replication in infected mammalian hosts.

  14. Bioinformatic Prediction of WSSV-Host Protein-Protein Interaction

    Directory of Open Access Journals (Sweden)

    Zheng Sun

    2014-01-01

    Full Text Available WSSV is one of the most dangerous pathogens in shrimp aquaculture. However, the molecular mechanism of how WSSV interacts with shrimp is still not very clear. In the present study, bioinformatic approaches were used to predict interactions between proteins from WSSV and shrimp. The genome data of WSSV (NC_003225.1 and the constructed transcriptome data of F. chinensis were used to screen potentially interacting proteins by searching in protein interaction databases, including STRING, Reactome, and DIP. Forty-four pairs of proteins were suggested to have interactions between WSSV and the shrimp. Gene ontology analysis revealed that 6 pairs of these interacting proteins were classified into “extracellular region” or “receptor complex” GO-terms. KEGG pathway analysis showed that they were involved in the “ECM-receptor interaction pathway.” In the 6 pairs of interacting proteins, an envelope protein called “collagen-like protein” (WSSV-CLP encoded by an early virus gene “wsv001” in WSSV interacted with 6 deduced proteins from the shrimp, including three integrin alpha (ITGA, two integrin beta (ITGB, and one syndecan (SDC. Sequence analysis on WSSV-CLP, ITGA, ITGB, and SDC revealed that they possessed the sequence features for protein-protein interactions. This study might provide new insights into the interaction mechanisms between WSSV and shrimp.

  15. Analysis of Tospovirus NSs Proteins in Suppression of Systemic Silencing.

    Science.gov (United States)

    Hedil, Marcio; Sterken, Mark G; de Ronde, Dryas; Lohuis, Dick; Kormelink, Richard

    2015-01-01

    RNA silencing is a sequence-specific gene regulation mechanism that in plants also acts antiviral. In order to counteract antiviral RNA silencing, viruses have evolved RNA silencing suppressors (RSS). In the case of tospoviruses, the non-structural NSs protein has been identified as the RSS. Although the tomato spotted wilt virus (TSWV) tospovirus NSs protein has been shown to exhibit affinity to long and small dsRNA molecules, its ability to suppress the non-cell autonomous part of RNA silencing has only been studied to a limited extent. Here, the NSs proteins of TSWV, groundnut ringspot virus (GRSV) and tomato yellow ring virus (TYRV), representatives for three distinct tospovirus species, have been studied on their ability and strength to suppress local and systemic silencing. A system has been developed to quantify suppression of GFP silencing in Nicotiana benthamiana 16C lines, to allow a comparison of relative RNA silencing suppressor strength. It is shown that NSs of all three tospoviruses are suppressors of local and systemic silencing. Unexpectedly, suppression of systemic RNA silencing by NSsTYRV was just as strong as those by NSsTSWV and NSsGRSV, even though NSsTYRV was expressed in lower amounts. Using the system established, a set of selected NSsTSWV gene constructs mutated in predicted RNA binding domains, as well as NSs from TSWV isolates 160 and 171 (resistance breakers of the Tsw resistance gene), were analyzed for their ability to suppress systemic GFP silencing. The results indicate another mode of RNA silencing suppression by NSs that acts further downstream the biogenesis of siRNAs and their sequestration. The findings are discussed in light of the affinity of NSs for small and long dsRNA, and recent mutant screen of NSsTSWV to map domains required for RSS activity and triggering of Tsw-governed resistance.

  16. The nucleocapsid protein of measles virus blocks host interferon response

    International Nuclear Information System (INIS)

    Takayama, Ikuyo; Sato, Hiroki; Watanabe, Akira; Omi-Furutani, Mio; Sugai, Akihiro; Kanki, Keita; Yoneda, Misako; Kai, Chieko

    2012-01-01

    Measles virus (MV) belongs to the genus Morbillivirus of the family Paramyxoviridae. A number of paramyxoviruses inhibit host interferon (IFN) signaling pathways in host immune systems by various mechanisms. Inhibition mechanisms have been described for many paramyxoviruses. Although there are inconsistencies among previous reports concerning MV, it appears that P/V/C proteins interfere with the pathways. In this study, we confirmed the effects of MV P gene products of a wild MV strain on IFN pathways and examined that of other viral proteins on it. Interestingly, we found that N protein acts as an IFN-α/β and γ-antagonist as strong as P gene products. We further investigated the mechanisms of MV-N inhibition, and revealed that MV-N blocks the nuclear import of activated STAT without preventing STAT and Jak activation or STAT degradation, and that the nuclear translocation of MV-N is important for the inhibition. The inhibitory effect of the N protein was observed as a common feature of other morbilliviruses. The results presented in this report suggest that N protein of MV as well as P/V/C proteins is involved in the inhibition of host IFN signaling pathways.

  17. The nucleocapsid protein of measles virus blocks host interferon response

    Energy Technology Data Exchange (ETDEWEB)

    Takayama, Ikuyo; Sato, Hiroki; Watanabe, Akira; Omi-Furutani, Mio; Sugai, Akihiro; Kanki, Keita; Yoneda, Misako; Kai, Chieko, E-mail: ckai@ims.u-tokyo.ac.jp

    2012-03-01

    Measles virus (MV) belongs to the genus Morbillivirus of the family Paramyxoviridae. A number of paramyxoviruses inhibit host interferon (IFN) signaling pathways in host immune systems by various mechanisms. Inhibition mechanisms have been described for many paramyxoviruses. Although there are inconsistencies among previous reports concerning MV, it appears that P/V/C proteins interfere with the pathways. In this study, we confirmed the effects of MV P gene products of a wild MV strain on IFN pathways and examined that of other viral proteins on it. Interestingly, we found that N protein acts as an IFN-{alpha}/{beta} and {gamma}-antagonist as strong as P gene products. We further investigated the mechanisms of MV-N inhibition, and revealed that MV-N blocks the nuclear import of activated STAT without preventing STAT and Jak activation or STAT degradation, and that the nuclear translocation of MV-N is important for the inhibition. The inhibitory effect of the N protein was observed as a common feature of other morbilliviruses. The results presented in this report suggest that N protein of MV as well as P/V/C proteins is involved in the inhibition of host IFN signaling pathways.

  18. Fusion protein is the main determinant of metapneumovirus host tropism.

    Science.gov (United States)

    de Graaf, Miranda; Schrauwen, Eefje J A; Herfst, Sander; van Amerongen, Geert; Osterhaus, Albert D M E; Fouchier, Ron A M

    2009-06-01

    Human metapneumovirus (HMPV) and avian metapneumovirus subgroup C (AMPV-C) infect humans and birds, respectively. This study confirmed the difference in host range in turkey poults, and analysed the contribution of the individual metapneumovirus genes to host range in an in vitro cell-culture model. Mammalian Vero-118 cells supported replication of both HMPV and AMPV-C in contrast to avian quail fibroblast (QT6) cells in which only AMPV-C replicated to high titres. Inoculation of Vero-118 and QT6 cells with recombinant HMPV in which genes were exchanged with those of AMPV-C revealed that the metapneumovirus fusion (F) protein is the main determinant for host tropism. Chimeric viruses in which polymerase complex proteins were exchanged between HMPV and AMPV-C replicated less efficiently compared with HMPV in QT6 cells. Using mini-genome systems, it was shown that exchanging these polymerase proteins resulted in reduced replication and transcription efficiency in QT6 cells. Examination of infected Vero-118 and QT6 cells revealed that viruses containing the F protein of AMPV-C yielded larger syncytia compared with viruses containing the HMPV F protein. Cell-content mixing assays revealed that the F protein of AMPV-C was more fusogenic compared with the F protein of HMPV, and that the F2 region is responsible for the difference observed between AMPV-C and HMPV F-promoted fusion in QT6 and Vero-118 cells. This study provides insight into the determinants of host tropism and membrane fusion of metapneumoviruses.

  19. Extractable Bacterial Surface Proteins in Probiotic–Host Interaction

    Directory of Open Access Journals (Sweden)

    Fillipe L. R. do Carmo

    2018-04-01

    Full Text Available Some Gram-positive bacteria, including probiotic ones, are covered with an external proteinaceous layer called a surface-layer. Described as a paracrystalline layer and formed by the self-assembly of a surface-layer-protein (Slp, this optional structure is peculiar. The surface layer per se is conserved and encountered in many prokaryotes. However, the sequence of the corresponding Slp protein is highly variable among bacterial species, or even among strains of the same species. Other proteins, including surface layer associated proteins (SLAPs, and other non-covalently surface-bound proteins may also be extracted with this surface structure. They can be involved a various functions. In probiotic Gram-positives, they were shown by different authors and experimental approaches to play a role in key interactions with the host. Depending on the species, and sometime on the strain, they can be involved in stress tolerance, in survival within the host digestive tract, in adhesion to host cells or mucus, or in the modulation of intestinal inflammation. Future trends include the valorization of their properties in the formation of nanoparticles, coating and encapsulation, and in the development of new vaccines.

  20. SUPPRESSION OF STAR FORMATION IN THE HOSTS OF LOW-EXCITATION RADIO GALAXIES

    Energy Technology Data Exchange (ETDEWEB)

    Pace, Cameron; Salim, Samir, E-mail: cameronpace@suu.edu, E-mail: salims@indiana.edu [Indiana University, Department of Astronomy, Swain Hall West 319, Bloomington, IN 47405-7105 (United States)

    2016-02-10

    The feedback from radio-loud active galactic nuclei (R-AGNs) may help maintain low star-formation (SF) rates in their early-type hosts, but the observational evidence for this mechanism has been inconclusive. We study systematic differences of aggregate spectral energy distributions (SEDs) of various subsets of ∼4000 low-redshift R-AGNs from Best and Heckman with respect to (currently) inactive control samples selected to have matching redshift, stellar mass, population age, axis ratio, and environment. Aggregate SEDs, ranging from the ultraviolet (UV) through mid-infrared (mid-IR, 22 μm), were constructed using a Bayesian method that eliminates biases from non-detections in Galaxy Evolution Explorer and Wide-field Infrared Survey Explorer. We study rare high-excitation sources separately from low-excitation ones, which we split by environment and host properties. We find that both the UV and mid-IR emission of non-cluster R-AGNs (80% of sample) are suppressed by ∼0.2 dex relative to that of the control group, especially for moderately massive galaxies (log M{sub *} ≲ 11). The difference disappears for high-mass R-AGNs and for R-AGNs in clusters, where other, non-AGN quenching/maintenance mechanisms may dominate, or where the suppression of SF due to AGNs may persist between active phases of the central engine, perhaps because of the presence of a hot gaseous halo storing AGN energy. High-excitation (high accretion rate) sources, which make up 2% of the R-AGN sample, do not show any evidence of SF suppression (their UV is the same as in controls), but they exhibit a strong mid-IR excess due to AGN dust heating.

  1. Characterization of host proteins interacting with the lymphocytic choriomeningitis virus L protein.

    Science.gov (United States)

    Khamina, Kseniya; Lercher, Alexander; Caldera, Michael; Schliehe, Christopher; Vilagos, Bojan; Sahin, Mehmet; Kosack, Lindsay; Bhattacharya, Anannya; Májek, Peter; Stukalov, Alexey; Sacco, Roberto; James, Leo C; Pinschewer, Daniel D; Bennett, Keiryn L; Menche, Jörg; Bergthaler, Andreas

    2017-12-01

    RNA-dependent RNA polymerases (RdRps) play a key role in the life cycle of RNA viruses and impact their immunobiology. The arenavirus lymphocytic choriomeningitis virus (LCMV) strain Clone 13 provides a benchmark model for studying chronic infection. A major genetic determinant for its ability to persist maps to a single amino acid exchange in the viral L protein, which exhibits RdRp activity, yet its functional consequences remain elusive. To unravel the L protein interactions with the host proteome, we engineered infectious L protein-tagged LCMV virions by reverse genetics. A subsequent mass-spectrometric analysis of L protein pulldowns from infected human cells revealed a comprehensive network of interacting host proteins. The obtained LCMV L protein interactome was bioinformatically integrated with known host protein interactors of RdRps from other RNA viruses, emphasizing interconnected modules of human proteins. Functional characterization of selected interactors highlighted proviral (DDX3X) as well as antiviral (NKRF, TRIM21) host factors. To corroborate these findings, we infected Trim21-/- mice with LCMV and found impaired virus control in chronic infection. These results provide insights into the complex interactions of the arenavirus LCMV and other viral RdRps with the host proteome and contribute to a better molecular understanding of how chronic viruses interact with their host.

  2. Analysis of Tospovirus NSs Proteins in Suppression of Systemic Silencing

    OpenAIRE

    Hedil, Marcio; Sterken, Mark G.; de Ronde, Dryas; Lohuis, Dick; Kormelink, Richard

    2015-01-01

    RNA silencing is a sequence-specific gene regulation mechanism that in plants also acts antiviral. In order to counteract antiviral RNA silencing, viruses have evolved RNA silencing suppressors (RSS). In the case of tospoviruses, the non-structural NSs protein has been identified as the RSS. Although the tomato spotted wilt virus (TSWV) tospovirus NSs protein has been shown to exhibit affinity to long and small dsRNA molecules, its ability to suppress the non-cell autonomous part of RNA silen...

  3. Rift Valley fever virus NSs inhibits host transcription independently of the degradation of dsRNA-dependent Protein Kinase PKR

    OpenAIRE

    Kalveram, Birte; Lihoradova, Olga; Indran, Sabarish V.; Lokugamage, Nandadeva; Head, Jennifer A.; Ikegami, Tetsuro

    2012-01-01

    Rift Valley fever virus (RVFV) encodes one major virulence factor, the NSs protein. NSs suppresses host general transcription, including interferon (IFN)-β mRNA synthesis, and promotes degradation of the dsRNA-dependent protein kinase (PKR). We generated a novel RVFV mutant (rMP12-NSsR173A) specifically lacking the function to promote PKR degradation. rMP12-NSsR173A infection induces early phosphorylation of eIF2α through PKR activation, while retaining the function to inhibit host general tr...

  4. Metal binding proteins, recombinant host cells and methods

    Science.gov (United States)

    Summers, Anne O.; Caguiat, Jonathan J.

    2004-06-15

    The present disclosure provides artificial heavy metal binding proteins termed chelons by the inventors. These chelons bind cadmium and/or mercuric ions with relatively high affinity. Also disclosed are coding sequences, recombinant DNA molecules and recombinant host cells comprising those recombinant DNA molecules for expression of the chelon proteins. In the recombinant host cells or transgenic plants, the chelons can be used to bind heavy metals taken up from contaminated soil, groundwater or irrigation water and to concentrate and sequester those ions. Recombinant enteric bacteria can be used within the gastrointestinal tracts of animals or humans exposed to toxic metal ions such as mercury and/or cadmium, where the chelon recombinantly expressed in chosen in accordance with the ion to be rededicated. Alternatively, the chelons can be immobilized to solid supports to bind and concentrate heavy metals from a contaminated aqueous medium including biological fluids.

  5. Rift Valley fever virus NSs inhibits host transcription independently of the degradation of dsRNA-dependent protein kinase PKR.

    Science.gov (United States)

    Kalveram, Birte; Lihoradova, Olga; Indran, Sabarish V; Lokugamage, Nandadeva; Head, Jennifer A; Ikegami, Tetsuro

    2013-01-20

    Rift Valley fever virus (RVFV) encodes one major virulence factor, the NSs protein. NSs suppresses host general transcription, including interferon (IFN)-β mRNA synthesis, and promotes degradation of the dsRNA-dependent protein kinase (PKR). We generated a novel RVFV mutant (rMP12-NSsR173A) specifically lacking the function to promote PKR degradation. rMP12-NSsR173A infection induces early phosphorylation of eIF2α through PKR activation, while retaining the function to inhibit host general transcription including IFN-β gene inhibition. MP-12 NSs but not R173A NSs binds to wt PKR. R173A NSs formed filamentous structure in nucleus in a mosaic pattern, which was distinct from MP-12 NSs filament pattern. Due to early phosphorylation of eIF2α, rMP12-NSsR173A could not efficiently accumulate viral proteins. Our results suggest that NSs-mediated host general transcription suppression occurs independently of PKR degradation, while the PKR degradation is important to inhibit the phosphorylation of eIF2α in infected cells undergoing host general transcription suppression. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Viral RNA Silencing Suppression: The Enigma of Bunyavirus NSs Proteins

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    Marcio Hedil

    2016-07-01

    Full Text Available The Bunyaviridae is a family of arboviruses including both plant- and vertebrate-infecting representatives. The Tospovirus genus accommodates plant-infecting bunyaviruses, which not only replicate in their plant host, but also in their insect thrips vector during persistent propagative transmission. For this reason, they are generally assumed to encounter antiviral RNA silencing in plants and insects. Here we present an overview on how tospovirus nonstructural NSs protein counteracts antiviral RNA silencing in plants and what is known so far in insects. Like tospoviruses, members of the related vertebrate-infecting bunyaviruses classified in the genera Orthobunyavirus, Hantavirus and Phlebovirus also code for a NSs protein. However, for none of them RNA silencing suppressor activity has been unambiguously demonstrated in neither vertebrate host nor arthropod vector. The second part of this review will briefly describe the role of these NSs proteins in modulation of innate immune responses in mammals and elaborate on a hypothetical scenario to explain if and how NSs proteins from vertebrate-infecting bunyaviruses affect RNA silencing. If so, why this discovery has been hampered so far.

  7. Viral RNA Silencing Suppression: The Enigma of Bunyavirus NSs Proteins.

    Science.gov (United States)

    Hedil, Marcio; Kormelink, Richard

    2016-07-23

    The Bunyaviridae is a family of arboviruses including both plant- and vertebrate-infecting representatives. The Tospovirus genus accommodates plant-infecting bunyaviruses, which not only replicate in their plant host, but also in their insect thrips vector during persistent propagative transmission. For this reason, they are generally assumed to encounter antiviral RNA silencing in plants and insects. Here we present an overview on how tospovirus nonstructural NSs protein counteracts antiviral RNA silencing in plants and what is known so far in insects. Like tospoviruses, members of the related vertebrate-infecting bunyaviruses classified in the genera Orthobunyavirus, Hantavirus and Phlebovirus also code for a NSs protein. However, for none of them RNA silencing suppressor activity has been unambiguously demonstrated in neither vertebrate host nor arthropod vector. The second part of this review will briefly describe the role of these NSs proteins in modulation of innate immune responses in mammals and elaborate on a hypothetical scenario to explain if and how NSs proteins from vertebrate-infecting bunyaviruses affect RNA silencing. If so, why this discovery has been hampered so far.

  8. Functional analysis of virion host shutoff protein of pseudorabies virus

    International Nuclear Information System (INIS)

    Lin, H.-W.; Chang, Y.-Y.; Wong, M.-L.; Lin, J.-W.; Chang, T.-J.

    2004-01-01

    During lytic infection, the virion host shutoff (vhs) protein of alphaherpesviruses causes the degradation of mRNAs nonspecifically. In this work, we cloned the vhs gene (UL41 open reading frame) of pseudorabies virus (PRV; TNL strain) by PCR, and its nucleotide sequences were determined. The PCR product of vhs gene was subcloned into the prokaryotic pET32b expression vector, and production of the recombinant vhs protein was examined by SDS-PAGE. Result of Western blotting demonstrated that our recombinant vhs protein reacted with antiserum against a synthetic peptide of 17 amino acids of the vhs protein. After purification with nickel-chelate affinity chromatography, the purified recombinant vhs protein exhibited in vitro ribonuclease activity as expected. We further cloned the vhs gene into eukaryotic expression vectors and investigated the intracellular function of vhs protein by DNA transfection. By transient trasfection and CAT assay, we found the CAT activity was reduced in the presence of vhs, indicating that degradation of mRNA of the CAT gene was caused by the vhs. Furthermore, our results showed that the plaque formation of pseudorabies virus was blocked by exogenous vhs. Taken together, we have cloned the vhs gene of pseudorabies virus (TNL strain) and conducted functional analysis of the recombinant vhs protein in vitro as well as in vivo

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

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    Peter eMoffett

    2015-08-01

    Full Text Available 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 genera 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 response in N. tabacum, and tobacco 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.

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

    KAUST Repository

    Ali, Shawkat

    2015-08-11

    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.

  11. Staphylococcal Immune Evasion Proteins: Structure, Function, and Host Adaptation.

    Science.gov (United States)

    Koymans, Kirsten J; Vrieling, Manouk; Gorham, Ronald D; van Strijp, Jos A G

    2017-01-01

    Staphylococcus aureus is a successful human and animal pathogen. Its pathogenicity is linked to its ability to secrete a large amount of virulence factors. These secreted proteins interfere with many critical components of the immune system, both innate and adaptive, and hamper proper immune functioning. In recent years, numerous studies have been conducted in order to understand the molecular mechanism underlying the interaction of evasion molecules with the host immune system. Structural studies have fundamentally contributed to our understanding of the mechanisms of action of the individual factors. Furthermore, such studies revealed one of the most striking characteristics of the secreted immune evasion molecules: their conserved structure. Despite high-sequence variability, most immune evasion molecules belong to a small number of structural categories. Another remarkable characteristic is that S. aureus carries most of these virulence factors on mobile genetic elements (MGE) or ex-MGE in its accessory genome. Coevolution of pathogen and host has resulted in immune evasion molecules with a highly host-specific function and prevalence. In this review, we explore how these shared structures and genomic locations relate to function and host specificity. This is discussed in the context of therapeutic options for these immune evasion molecules in infectious as well as in inflammatory diseases.

  12. Plum pox virus capsid protein suppresses plant pathogen-associated molecular pattern (PAMP)-triggered immunity.

    Science.gov (United States)

    Nicaise, Valerie; Candresse, Thierry

    2017-08-01

    The perception of pathogen-associated molecular patterns (PAMPs) by immune receptors launches defence mechanisms referred to as PAMP-triggered immunity (PTI). Successful pathogens must suppress PTI pathways via the action of effectors to efficiently colonize their hosts. So far, plant PTI has been reported to be active against most classes of pathogens, except viruses, although this defence layer has been hypothesized recently as an active part of antiviral immunity which needs to be suppressed by viruses for infection success. Here, we report that Arabidopsis PTI genes are regulated upon infection by viruses and contribute to plant resistance to Plum pox virus (PPV). Our experiments further show that PPV suppresses two early PTI responses, the oxidative burst and marker gene expression, during Arabidopsis infection. In planta expression of PPV capsid protein (CP) was found to strongly impair these responses in Nicotiana benthamiana and Arabidopsis, revealing its PTI suppressor activity. In summary, we provide the first clear evidence that plant viruses acquired the ability to suppress PTI mechanisms via the action of effectors, highlighting a novel strategy employed by viruses to escape plant defences. © 2016 BSPP AND JOHN WILEY & SONS LTD.

  13. Inhibition of host cell translation elongation by Legionella pneumophila blocks the host cell unfolded protein response.

    Science.gov (United States)

    Hempstead, Andrew D; Isberg, Ralph R

    2015-12-08

    Cells of the innate immune system recognize bacterial pathogens by detecting common microbial patterns as well as pathogen-specific activities. One system that responds to these stimuli is the IRE1 branch of the unfolded protein response (UPR), a sensor of endoplasmic reticulum (ER) stress. Activation of IRE1, in the context of Toll-like receptor (TLR) signaling, induces strong proinflammatory cytokine induction. We show here that Legionella pneumophila, an intravacuolar pathogen that replicates in an ER-associated compartment, blocks activation of the IRE1 pathway despite presenting pathogen products that stimulate this response. L. pneumophila TLR ligands induced the splicing of mRNA encoding XBP1s, the main target of IRE1 activity. L. pneumophila was able to inhibit both chemical and bacterial induction of XBP1 splicing via bacterial translocated proteins that interfere with host protein translation. A strain lacking five translocated translation elongation inhibitors was unable to block XBP1 splicing, but this could be rescued by expression of a single such inhibitor, consistent with limitation of the response by translation elongation inhibitors. Chemical inhibition of translation elongation blocked pattern recognition receptor-mediated XBP1 splicing, mimicking the effects of the bacterial translation inhibitors. In contrast, host cell-promoted inhibition of translation initiation in response to the pathogen was ineffective in blocking XBP1 splicing, demonstrating the need for the elongation inhibitors for protection from the UPR. The inhibition of host translation elongation may be a common strategy used by pathogens to limit the innate immune response by interfering with signaling via the UPR.

  14. A bacterial cysteine protease effector protein interferes with photosynthesis to suppress plant innate immune responses.

    Science.gov (United States)

    Rodríguez-Herva, José J; González-Melendi, Pablo; Cuartas-Lanza, Raquel; Antúnez-Lamas, María; Río-Alvarez, Isabel; Li, Ziduo; López-Torrejón, Gema; Díaz, Isabel; Del Pozo, Juan C; Chakravarthy, Suma; Collmer, Alan; Rodríguez-Palenzuela, Pablo; López-Solanilla, Emilia

    2012-05-01

    The bacterial pathogen Pseudomonas syringae pv tomato DC3000 suppresses plant innate immunity with effector proteins injected by a type III secretion system (T3SS). The cysteine protease effector HopN1, which reduces the ability of DC3000 to elicit programmed cell death in non-host tobacco, was found to also suppress the production of defence-associated reactive oxygen species (ROS) and callose when delivered by Pseudomonas fluorescens heterologously expressing a P. syringae T3SS. Purified His(6) -tagged HopN1 was used to identify tomato PsbQ, a member of the oxygen evolving complex of photosystem II (PSII), as an interacting protein. HopN1 localized to chloroplasts and both degraded PsbQ and inhibited PSII activity in chloroplast preparations, whereas a HopN1(D299A) non-catalytic mutant lost these abilities. Gene silencing of NtPsbQ in tobacco compromised ROS production and programmed cell death by DC3000. Our data reveal PsbQ as a contributor to plant immunity responses and a target for pathogen suppression. © 2012 Blackwell Publishing Ltd.

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

    Science.gov (United States)

    2017-01-01

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

  16. An Aphid Effector Targets Trafficking Protein VPS52 in a Host-Specific Manner to Promote Virulence.

    Science.gov (United States)

    Rodriguez, Patricia A; Escudero-Martinez, Carmen; Bos, Jorunn I B

    2017-03-01

    Plant- and animal-feeding insects secrete saliva inside their hosts, containing effectors, which may promote nutrient release and suppress immunity. Although for plant pathogenic microbes it is well established that effectors target host proteins to modulate host cell processes and promote disease, the host cell targets of herbivorous insects remain elusive. Here, we show that the existing plant pathogenic microbe effector paradigm can be extended to herbivorous insects in that effector-target interactions inside host cells modify critical host processes to promote plant susceptibility. We showed that the effector Mp1 from Myzus persicae associates with the host Vacuolar Protein Sorting Associated Protein52 (VPS52). Using natural variants, we provide a strong link between effector virulence activity and association with VPS52, and show that the association is highly specific to M persicae -host interactions. Also, coexpression of Mp1, but not Mp1-like variants, specifically with host VPS52s resulted in effector relocalization to vesicle-like structures that associate with prevacuolar compartments. We show that high VPS52 levels negatively impact virulence, and that aphids are able to reduce VPS52 levels during infestation, indicating that VPS52 is an important virulence target. Our work is an important step forward in understanding, at the molecular level, how a major agricultural pest promotes susceptibility during infestation of crop plants. We give evidence that an herbivorous insect employs effectors that interact with host proteins as part of an effective virulence strategy, and that these effectors likely function in a species-specific manner. © 2017 American Society of Plant Biologists. All Rights Reserved.

  17. Protein Disulfide Isomerase and Host-Pathogen Interaction

    Directory of Open Access Journals (Sweden)

    Beatriz S. Stolf

    2011-01-01

    Full Text Available Reactive oxygen species (ROS production by immunological cells is known to cause damage to pathogens. Increasing evidence accumulated in the last decade has shown, however, that ROS (and redox signals functionally regulate different cellular pathways in the host-pathogen interaction. These especially affect (i pathogen entry through protein redox switches and redox modification (i.e., intra- and interdisulfide and cysteine oxidation and (ii phagocytic ROS production via Nox family NADPH oxidase enzyme and the control of phagolysosome function with key implications for antigen processing. The protein disulfide isomerase (PDI family of redox chaperones is closely involved in both processes and is also implicated in protein unfolding and trafficking across the endoplasmic reticulum (ER and towards the cytosol, a thiol-based redox locus for antigen processing. Here, we summarise examples of the cellular association of host PDI with different pathogens and explore the possible roles of pathogen PDIs in infection. A better understanding of these complex regulatory steps will provide insightful information on the redox role and coevolutional biological process, and assist the development of more specific therapeutic strategies in pathogen-mediated infections.

  18. Sequence- and interactome-based prediction of viral protein hotspots targeting host proteins: a case study for HIV Nef.

    Directory of Open Access Journals (Sweden)

    Mahdi Sarmady

    Full Text Available Virus proteins alter protein pathways of the host toward the synthesis of viral particles by breaking and making edges via binding to host proteins. In this study, we developed a computational approach to predict viral sequence hotspots for binding to host proteins based on sequences of viral and host proteins and literature-curated virus-host protein interactome data. We use a motif discovery algorithm repeatedly on collections of sequences of viral proteins and immediate binding partners of their host targets and choose only those motifs that are conserved on viral sequences and highly statistically enriched among binding partners of virus protein targeted host proteins. Our results match experimental data on binding sites of Nef to host proteins such as MAPK1, VAV1, LCK, HCK, HLA-A, CD4, FYN, and GNB2L1 with high statistical significance but is a poor predictor of Nef binding sites on highly flexible, hoop-like regions. Predicted hotspots recapture CD8 cell epitopes of HIV Nef highlighting their importance in modulating virus-host interactions. Host proteins potentially targeted or outcompeted by Nef appear crowding the T cell receptor, natural killer cell mediated cytotoxicity, and neurotrophin signaling pathways. Scanning of HIV Nef motifs on multiple alignments of hepatitis C protein NS5A produces results consistent with literature, indicating the potential value of the hotspot discovery in advancing our understanding of virus-host crosstalk.

  19. A bacterial E3 ubiquitin ligase targets a host protein kinase to disrupt plant immunity.

    Science.gov (United States)

    Rosebrock, Tracy R; Zeng, Lirong; Brady, Jennifer J; Abramovitch, Robert B; Xiao, Fangming; Martin, Gregory B

    2007-07-19

    Many bacterial pathogens of plants and animals use a type III secretion system to deliver diverse virulence-associated 'effector' proteins into the host cell. The mechanisms by which these effectors act are mostly unknown; however, they often promote disease by suppressing host immunity. One type III effector, AvrPtoB, expressed by the plant pathogen Pseudomonas syringae pv. tomato, has a carboxy-terminal domain that is an E3 ubiquitin ligase. Deletion of this domain allows an amino-terminal region of AvrPtoB (AvrPtoB(1-387)) to be detected by certain tomato varieties leading to immunity-associated programmed cell death. Here we show that a host kinase, Fen, physically interacts with AvrPtoB(1-387 )and is responsible for activating the plant immune response. The AvrPtoB E3 ligase specifically ubiquitinates Fen and promotes its degradation in a proteasome-dependent manner. This degradation leads to disease susceptibility in Fen-expressing tomato lines. Various wild species of tomato were found to exhibit immunity in response to AvrPtoB(1-387 )and not to full-length AvrPtoB. Thus, by acquiring an E3 ligase domain, AvrPtoB has thwarted a highly conserved host resistance mechanism.

  20. Expression of immune-response genes in lepidopteran host is suppressed by venom from an endoparasitoid, Pteromalus puparum

    Directory of Open Access Journals (Sweden)

    Fang Qi

    2010-09-01

    Full Text Available Abstract Background The relationships between parasitoids and their insect hosts have attracted attention at two levels. First, the basic biology of host-parasitoid interactions is of fundamental interest. Second, parasitoids are widely used as biological control agents in sustainable agricultural programs. Females of the gregarious endoparasitoid Pteromalus puparum (Hymenoptera: Pteromalidae inject venom along with eggs into their hosts. P. puparum does not inject polydnaviruses during oviposition. For this reason, P. puparum and its pupal host, the small white butterfly Pieris rapae (Lepidoptera: Pieridae, comprise an excellent model system for studying the influence of an endoparasitoid venom on the biology of the pupal host. P. puparum venom suppresses the immunity of its host, although the suppressive mechanisms are not fully understood. In this study, we tested our hypothesis that P. puparum venom influences host gene expression in the two main immunity-conferring tissues, hemocytes and fat body. Results At 1 h post-venom injection, we recorded significant decreases in transcript levels of 217 EST clones (revealing 113 genes identified in silico, including 62 unknown contigs derived from forward subtractive libraries of host hemocytes and in transcript levels of 288 EST clones (221 genes identified in silico, including 123 unknown contigs from libraries of host fat body. These genes are related to insect immune response, cytoskeleton, cell cycle and apoptosis, metabolism, transport, stress response and transcriptional and translational regulation. We verified the reliability of the suppression subtractive hybridization (SSH data with semi-quantitative RT-PCR analysis of a set of randomly selected genes. This analysis showed that most of the selected genes were down-regulated after venom injection. Conclusions Our findings support our hypothesis that P. puparum venom influences gene expression in host hemocytes and fat body. Specifically

  1. Proteomics in the investigation of HIV-1 interactions with host proteins.

    Science.gov (United States)

    Li, Ming

    2015-02-01

    Productive HIV-1 infection depends on host machinery, including a broad array of cellular proteins. Proteomics has played a significant role in the discovery of HIV-1 host proteins. In this review, after a brief survey of the HIV-1 host proteins that were discovered by proteomic analyses, I focus on analyzing the interactions between the virion and host proteins, as well as the technologies and strategies used in those proteomic studies. With the help of proteomics, the identification and characterization of HIV-1 host proteins can be translated into novel antiretroviral therapeutics. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Use of Host-like Peptide Motifs in Viral Proteins Is a Prevalent Strategy in Host-Virus Interactions

    Directory of Open Access Journals (Sweden)

    Tzachi Hagai

    2014-06-01

    Full Text Available Viruses interact extensively with host proteins, but the mechanisms controlling these interactions are not well understood. We present a comprehensive analysis of eukaryotic linear motifs (ELMs in 2,208 viral genomes and reveal that viruses exploit molecular mimicry of host-like ELMs to possibly assist in host-virus interactions. Using a statistical genomics approach, we identify a large number of potentially functional ELMs and observe that the occurrence of ELMs is often evolutionarily conserved but not uniform across virus families. Some viral proteins contain multiple types of ELMs, in striking similarity to complex regulatory modules in host proteins, suggesting that ELMs may act combinatorially to assist viral replication. Furthermore, a simple evolutionary model suggests that the inherent structural simplicity of ELMs often enables them to tolerate mutations and evolve quickly. Our findings suggest that ELMs may allow fast rewiring of host-virus interactions, which likely assists rapid viral evolution and adaptation to diverse environments.

  3. Measles virus C protein suppresses gamma-activated factor formation and virus-induced cell growth arrest

    International Nuclear Information System (INIS)

    Yokota, Shin-ichi; Okabayashi, Tamaki; Fujii, Nobuhiro

    2011-01-01

    Measles virus (MeV) produces two accessory proteins, V and C, from the P gene. These accessory proteins have been reported to contribute to efficient virus proliferation through the modulation of host cell events. Our previous paper described that Vero cell-adapted strains of MeV led host cells to growth arrest through the upregulation of interferon regulatory factor 1 (IRF-1), and wild strains did not. In the present study, we found that C protein expression levels varied among MeV strains in infected SiHa cells. C protein levels were inversely correlated with IRF-1 expression levels and with cell growth arrest. Forced expression of C protein released cells from growth arrest. C-deficient recombinant virus efficiently upregulated IRF-1 and caused growth arrest more efficiently than the wild-type virus. C protein preferentially bound to phosphorylated STAT1 and suppressed STAT1 dimer formation. We conclude that MeV C protein suppresses IFN-γ signaling pathway via inhibition of phosphorylated STAT1 dimerization.

  4. Direct identification of the Meloidogyne incognita secretome reveals proteins with host cell reprogramming potential.

    Directory of Open Access Journals (Sweden)

    Stéphane Bellafiore

    2008-10-01

    Full Text Available The root knot nematode, Meloidogyne incognita, is an obligate parasite that causes significant damage to a broad range of host plants. Infection is associated with secretion of proteins surrounded by proliferating cells. Many parasites are known to secrete effectors that interfere with plant innate immunity, enabling infection to occur; they can also release pathogen-associated molecular patterns (PAMPs, e.g., flagellin that trigger basal immunity through the nematode stylet into the plant cell. This leads to suppression of innate immunity and reprogramming of plant cells to form a feeding structure containing multinucleate giant cells. Effectors have generally been discovered using genetics or bioinformatics, but M. incognita is non-sexual and its genome sequence has not yet been reported. To partially overcome these limitations, we have used mass spectrometry to directly identify 486 proteins secreted by M. incognita. These proteins contain at least segmental sequence identity to those found in our 3 reference databases (published nematode proteins; unpublished M. incognita ESTs; published plant proteins. Several secreted proteins are homologous to plant proteins, which they may mimic, and they contain domains that suggest known effector functions (e.g., regulating the plant cell cycle or growth. Others have regulatory domains that could reprogram cells. Using in situ hybridization we observed that most secreted proteins were produced by the subventral glands, but we found that phasmids also secreted proteins. We annotated the functions of the secreted proteins and classified them according to roles they may play in the development of root knot disease. Our results show that parasite secretomes can be partially characterized without cognate genomic DNA sequence. We observed that the M. incognita secretome overlaps the reported secretome of mammalian parasitic nematodes (e.g., Brugia malayi, suggesting a common parasitic behavior and a possible

  5. Structural basis for antagonizing a host restriction factor by C7 family of poxvirus host-range proteins

    OpenAIRE

    Meng, Xiangzhi; Krumm, Brian; Li, Yongchao; Deng, Junpeng; Xiang, Yan

    2015-01-01

    Productive viral replication requires overcoming many barriers posed by the host innate immune system. Human sterile alpha motif domain-containing 9 (SAMD9) is a newly identified antiviral factor that is specifically targeted by poxvirus proteins belonging to the C7 family of host-range factors. Here we provide the first, to our knowledge, atomic view of two functionally divergent proteins from the C7 family and determine the molecular basis that dictates whether they can target SAMD9 effecti...

  6. Artificial Metalloenzymes through Chemical Modification of Engineered Host Proteins

    KAUST Repository

    Zernickel, Anna

    2014-10-01

    With a few exceptions, all organisms are restricted to the 20 canonical amino acids for ribosomal protein biosynthesis. Addition of new amino acids to the genetic code can introduce novel functionalities to proteins, broadening the diversity of biochemical as well as chemical reactions and providing new tools to study protein structure, reactivity, dynamics and protein-protein-interactions. The site directed in vivo incorporation developed by P. G. SCHULTZ and coworkers, using an archeal orthogonal tRNA/aaRS (aminoacyl-tRNA synthase) pair, allows site-specifically insertion of a synthetic unnatural amino acid (UAA) by reprogramming the amber TAG stop codon. A variety of over 80 different UAAs can be introduced by this technique. However by now a very limited number can form kinetically stable bonds to late transition metals. This thesis aims to develop new catalytically active unnatural amino acids or strategies for a posttranslational modification of site-specific amino acids in order to achieve highly enantioselective metallorganic enzyme hybrids (MOEH). As a requirement a stable protein host has to be established, surviving the conditions for incorporation, posttranslational modification and the final catalytic reactions. mTFP* a fluorescent protein was genetically modified by excluding any exposed Cys, His and Met forming a variant mTFP*, which fulfills the required specifications. Posttranslational chemical modification of mTFP* allow the introduction of single site metal chelating moieties. For modification on exposed cysteines different maleiimid containing ligand structures were synthesized. In order to perform copper catalyzed click reactions, suitable unnatural amino acids (para-azido-(L)-phenylalanine, para-ethynyl-(L)-phenylalanine) were synthesized and a non-cytotoxic protocol was established. The triazole ring formed during this reaction may contribute as a moderate σ-donor/π-acceptor ligand to the metal binding site. Since the cell limits the

  7. Two Novel Motifs of Watermelon Silver Mottle Virus NSs Protein Are Responsible for RNA Silencing Suppression and Pathogenicity.

    Science.gov (United States)

    Huang, Chung-Hao; Hsiao, Weng-Rong; Huang, Ching-Wen; Chen, Kuan-Chun; Lin, Shih-Shun; Chen, Tsung-Chi; Raja, Joseph A J; Wu, Hui-Wen; Yeh, Shyi-Dong

    2015-01-01

    The NSs protein of Watermelon silver mottle virus (WSMoV) is the RNA silencing suppressor and pathogenicity determinant. In this study, serial deletion and point-mutation mutagenesis of conserved regions (CR) of NSs protein were performed, and the silencing suppression function was analyzed through agroinfiltration in Nicotiana benthamiana plants. We found two amino acid (aa) residues, H113 and Y398, are novel functional residues for RNA silencing suppression. Our further analyses demonstrated that H113 at the common epitope (CE) ((109)KFTMHNQ(117)), which is highly conserved in Asia type tospoviruses, and the benzene ring of Y398 at the C-terminal β-sheet motif ((397)IYFL(400)) affect NSs mRNA stability and protein stability, respectively, and are thus critical for NSs RNA silencing suppression. Additionally, protein expression of other six deleted (ΔCR1-ΔCR6) and five point-mutated (Y15A, Y27A, G180A, R181A and R212A) mutants were hampered and their silencing suppression ability was abolished. The accumulation of the mutant mRNAs and proteins, except Y398A, could be rescued or enhanced by co-infiltration with potyviral suppressor HC-Pro. When assayed with the attenuated Zucchini yellow mosaic virus vector in squash plants, the recombinants carrying individual seven point-mutated NSs proteins displayed symptoms much milder than the recombinant carrying the wild type NSs protein, suggesting that these aa residues also affect viral pathogenicity by suppressing the host silencing mechanism.

  8. Involvement of Cryptosporidium parvum Cdg7_FLc_1000 RNA in the Attenuation of Intestinal Epithelial Cell Migration via Trans-Suppression of Host Cell SMPD3.

    Science.gov (United States)

    Ming, Zhenping; Gong, Ai-Yu; Wang, Yang; Zhang, Xin-Tian; Li, Min; Mathy, Nicholas W; Strauss-Soukup, Juliane K; Chen, Xian-Ming

    2017-12-27

    Intestinal infection by Cryptosporidium parvum causes inhibition of epithelial turnover, but underlying mechanisms are unclear. Previous studies demonstrate that a panel of parasite RNA transcripts of low protein-coding potential are delivered into infected epithelial cells. Using in vitro and in vivo models of intestinal cryptosporidiosis, we report here that host delivery of parasite Cdg7_FLc_1000 RNA results in inhibition of epithelial cell migration through suppression of the gene encoding sphingomyelinase 3 (SMPD3). Delivery of Cdg7_FLc_1000 into infected cells promotes the histone methyltransferase G9a-mediated H3K9 methylation in the SMPD3 locus. The DNA-binding transcriptional repressor, PR domain zinc finger protein 1, is required for the assembly of Cdg7_FLc_1000 into the G9a complex and associated with the enrichment of H3K9 methylation at the gene locus. Pathologically, nuclear transfer of Cryptosporidium parvum Cdg7_FLc_1000 RNA is involved in the attenuation of intestinal epithelial cell migration via trans-suppression of host cell SMPD3. © The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  9. A protein interaction mechanism for suppressing the mechanosensitive Piezo channels.

    Science.gov (United States)

    Zhang, Tingxin; Chi, Shaopeng; Jiang, Fan; Zhao, Qiancheng; Xiao, Bailong

    2017-11-27

    Piezo proteins are bona fide mammalian mechanotransduction channels for various cell types including endothelial cells. The mouse Piezo1 of 2547 residues forms a three-bladed, propeller-like homo-trimer comprising a central pore-module and three propeller-structures that might serve as mechanotransduction-modules. However, the mechanogating and regulation of Piezo channels remain unclear. Here we identify the sarcoplasmic /endoplasmic-reticulum Ca 2+ ATPase (SERCA), including the widely expressed SERCA2, as Piezo interacting proteins. SERCA2 strategically suppresses Piezo1 via acting on a 14-residue-constituted intracellular linker connecting the pore-module and mechanotransduction-module. Mutating the linker impairs mechanogating and SERCA2-mediated modulation of Piezo1. Furthermore, the synthetic linker-peptide disrupts the modulatory effects of SERCA2, demonstrating the key role of the linker in mechanogating and regulation. Importantly, the SERCA2-mediated regulation affects Piezo1-dependent migration of endothelial cells. Collectively, we identify SERCA-mediated regulation of Piezos and the functional significance of the linker, providing important insights into the mechanogating and regulation mechanisms of Piezo channels.

  10. Leptospiral outer membrane protein microarray, a novel approach to identification of host ligand-binding proteins.

    Science.gov (United States)

    Pinne, Marija; Matsunaga, James; Haake, David A

    2012-11-01

    Leptospirosis is a zoonosis with worldwide distribution caused by pathogenic spirochetes belonging to the genus Leptospira. The leptospiral life cycle involves transmission via freshwater and colonization of the renal tubules of their reservoir hosts. Infection requires adherence to cell surfaces and extracellular matrix components of host tissues. These host-pathogen interactions involve outer membrane proteins (OMPs) expressed on the bacterial surface. In this study, we developed an Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 OMP microarray containing all predicted lipoproteins and transmembrane OMPs. A total of 401 leptospiral genes or their fragments were transcribed and translated in vitro and printed on nitrocellulose-coated glass slides. We investigated the potential of this protein microarray to screen for interactions between leptospiral OMPs and fibronectin (Fn). This approach resulted in the identification of the recently described fibronectin-binding protein, LIC10258 (MFn8, Lsa66), and 14 novel Fn-binding proteins, denoted Microarray Fn-binding proteins (MFns). We confirmed Fn binding of purified recombinant LIC11612 (MFn1), LIC10714 (MFn2), LIC11051 (MFn6), LIC11436 (MFn7), LIC10258 (MFn8, Lsa66), and LIC10537 (MFn9) by far-Western blot assays. Moreover, we obtained specific antibodies to MFn1, MFn7, MFn8 (Lsa66), and MFn9 and demonstrated that MFn1, MFn7, and MFn9 are expressed and surface exposed under in vitro growth conditions. Further, we demonstrated that MFn1, MFn4 (LIC12631, Sph2), and MFn7 enable leptospires to bind fibronectin when expressed in the saprophyte, Leptospira biflexa. Protein microarrays are valuable tools for high-throughput identification of novel host ligand-binding proteins that have the potential to play key roles in the virulence mechanisms of pathogens.

  11. Essential multimeric enzymes in kinetoplastid parasites: A host of potentially druggable protein-protein interactions.

    Science.gov (United States)

    Wachsmuth, Leah M; Johnson, Meredith G; Gavenonis, Jason

    2017-06-01

    Parasitic diseases caused by kinetoplastid parasites of the genera Trypanosoma and Leishmania are an urgent public health crisis in the developing world. These closely related species possess a number of multimeric enzymes in highly conserved pathways involved in vital functions, such as redox homeostasis and nucleotide synthesis. Computational alanine scanning of these protein-protein interfaces has revealed a host of potentially ligandable sites on several established and emerging anti-parasitic drug targets. Analysis of interfaces with multiple clustered hotspots has suggested several potentially inhibitable protein-protein interactions that may have been overlooked by previous large-scale analyses focusing solely on secondary structure. These protein-protein interactions provide a promising lead for the development of new peptide and macrocycle inhibitors of these enzymes.

  12. Disease susceptibiliy in the zig-zag model of host-microbe Interactions: only a consequence of immune suppression?

    OpenAIRE

    Keller, Harald; Boyer, Laurent; Abad, Pierre

    2016-01-01

    For almost ten years, the Zig-Zag model has provided a convenient framework for explaining the molecular bases of compatibility and incompatibility in plant-microbe interactions (Jones and Dangl, 2006). According to the Zig-Zag model, disease susceptibility is a consequence of the suppression of host immunity during the evolutionary arms race between plants and pathogens. The Zig-Zag model thus fits well with biotrophic interactions, but is less applicable to interactions involving pathogens ...

  13. Cross-Species Virus-Host Protein-Protein Interactions Inhibiting Innate Immunity

    Science.gov (United States)

    2016-07-01

    diseases are a regular occurrence globally (Figure 1). The Zika virus is the latest example gaining widespread attention. Many of the (re-)emerging...for establishing infection and/or modulating pathogenesis (Figures 2 and 3). 3 Figure 2. Schematic of several virus -host protein interactions within...8725 John J. Kingman Road, MS 6201 Fort Belvoir, VA 22060-6201 T E C H N IC A L R E P O R T DTRA-TR-16-79 Cross-species virus -host

  14. Kluyveromyces marxianus as a host for heterologous protein synthesis.

    Science.gov (United States)

    Gombert, Andreas K; Madeira, José Valdo; Cerdán, María-Esperanza; González-Siso, María-Isabel

    2016-07-01

    The preferentially respiring and thermotolerant yeast Kluyveromyces marxianus is an emerging host for heterologous protein synthesis, surpassing the traditional preferentially fermenting yeast Saccharomyces cerevisiae in some important aspects: K . marxianus can grow at temperatures 10 °C higher than S. cerevisiae, which may result in decreased costs for cooling bioreactors and reduced contamination risk; has ability to metabolize a wider variety of sugars, such as lactose and xylose; is the fastest growing eukaryote described so far; and does not require special cultivation techniques (such as fed-batch) to avoid fermentative metabolism. All these advantages exist together with a high secretory capacity, performance of eukaryotic post-translational modifications, and with a generally regarded as safe (GRAS) status. In the last years, replication origins from several Kluyveromyces spp. have been used for the construction of episomal vectors, and also integrative strategies have been developed based on the tendency for non-homologous recombination displayed by K. marxianus. The recessive URA3 auxotrophic marker and the dominant Kan(R) are mostly used for selection of transformed cells, but other markers have been made available. Homologous and heterologous promoters and secretion signals have been characterized, with the K. marxianus INU1 expression and secretion system being of remarkable functionality. The efficient synthesis of roughly 50 heterologous proteins has been demonstrated, including one thermophilic enzyme. In this mini-review, we summarize the physiological characteristics of K. marxianus relevant for its use in the efficient synthesis of heterologous proteins, the efforts performed hitherto in the development of a molecular toolbox for this purpose, and some successful examples.

  15. Visualization of Host-Polerovirus Interaction Topologies Using Protein Interaction Reporter Technology.

    Science.gov (United States)

    DeBlasio, Stacy L; Chavez, Juan D; Alexander, Mariko M; Ramsey, John; Eng, Jimmy K; Mahoney, Jaclyn; Gray, Stewart M; Bruce, James E; Cilia, Michelle

    2016-02-15

    Demonstrating direct interactions between host and virus proteins during infection is a major goal and challenge for the field of virology. Most protein interactions are not binary or easily amenable to structural determination. Using infectious preparations of a polerovirus (Potato leafroll virus [PLRV]) and protein interaction reporter (PIR), a revolutionary technology that couples a mass spectrometric-cleavable chemical cross-linker with high-resolution mass spectrometry, we provide the first report of a host-pathogen protein interaction network that includes data-derived, topological features for every cross-linked site that was identified. We show that PLRV virions have hot spots of protein interaction and multifunctional surface topologies, revealing how these plant viruses maximize their use of binding interfaces. Modeling data, guided by cross-linking constraints, suggest asymmetric packing of the major capsid protein in the virion, which supports previous epitope mapping studies. Protein interaction topologies are conserved with other species in the Luteoviridae and with unrelated viruses in the Herpesviridae and Adenoviridae. Functional analysis of three PLRV-interacting host proteins in planta using a reverse-genetics approach revealed a complex, molecular tug-of-war between host and virus. Structural mimicry and diversifying selection-hallmarks of host-pathogen interactions-were identified within host and viral binding interfaces predicted by our models. These results illuminate the functional diversity of the PLRV-host protein interaction network and demonstrate the usefulness of PIR technology for precision mapping of functional host-pathogen protein interaction topologies. The exterior shape of a plant virus and its interacting host and insect vector proteins determine whether a virus will be transmitted by an insect or infect a specific host. Gaining this information is difficult and requires years of experimentation. We used protein interaction

  16. Structural basis for antagonizing a host restriction factor by C7 family of poxvirus host-range proteins.

    Science.gov (United States)

    Meng, Xiangzhi; Krumm, Brian; Li, Yongchao; Deng, Junpeng; Xiang, Yan

    2015-12-01

    Human sterile alpha motif domain-containing 9 (SAMD9) protein is a host restriction factor for poxviruses, but it can be overcome by some poxvirus host-range proteins that share homology with vaccinia virus C7 protein. To understand the mechanism of action for this important family of host-range factors, we determined the crystal structures of C7 and myxoma virus M64, a C7 family member that is unable to antagonize SAMD9. Despite their different functions and only 23% sequence identity, the two proteins have very similar overall structures, displaying a previously unidentified fold comprised of a compact 12-stranded antiparallel β-sandwich wrapped in two short α helices. Extensive structure-guided mutagenesis of C7 identified three loops clustered on one edge of the β sandwich as critical for viral replication and binding with SAMD9. The loops are characterized with functionally important negatively charged, positively charged, and hydrophobic residues, respectively, together forming a unique "three-fingered molecular claw." The key residues of the claw are not conserved in two C7 family members that do not antagonize SAMD9 but are conserved in distantly related C7 family members from four poxvirus genera that infect diverse mammalian species. Indeed, we found that all in the latter group of proteins bind SAMD9. Taken together, our data indicate that diverse mammalian poxviruses use a conserved molecular claw in a C7-like protein to target SAMD9 and overcome host restriction.

  17. The Polerovirus F box protein P0 targets ARGONAUTE1 to suppress RNA silencing.

    Science.gov (United States)

    Bortolamiol, Diane; Pazhouhandeh, Maghsoud; Marrocco, Katia; Genschik, Pascal; Ziegler-Graff, Véronique

    2007-09-18

    Plants employ post-transcriptional gene silencing (PTGS) as an antiviral defense response. In this mechanism, viral-derived small RNAs are incorporated into the RNA-induced silencing complex (RISC) to guide degradation of the corresponding viral RNAs. ARGONAUTE1 (AGO1) is a key component of RISC: it carries the RNA slicer activity. As a counter-defense, viruses have evolved various proteins that suppress PTGS. Recently, we showed that the Polerovirus P0 protein carries an F box motif required to form an SCF-like complex, which is also essential for P0's silencing suppressor function. Here, we investigate the molecular mechanism by which P0 impairs PTGS. First we show that P0's expression does not affect the biogenesis of primary siRNAs in an inverted repeat-PTGS assay, but it does affect their activity. Moreover, P0's expression in transformed Arabidopsis plants leads to various developmental abnormalities reminiscent of mutants affected in miRNA pathways, which is accompanied by enhanced levels of several miRNA-target transcripts, suggesting that P0 acts at the level of RISC. Interestingly, ectopic expression of P0 triggered AGO1 protein decay in planta. Finally, we provide evidence that P0 physically interacts with AGO1. Based on these results, we propose that P0 hijacks the host SCF machinery to modulate gene silencing by destabilizing AGO1.

  18. Translational Control of Host Gene Expression by a Cys-Motif Protein Encoded in a Bracovirus.

    Directory of Open Access Journals (Sweden)

    Eunseong Kim

    Full Text Available Translational control is a strategy that various viruses use to manipulate their hosts to suppress acute antiviral response. Polydnaviruses, a group of insect double-stranded DNA viruses symbiotic to some endoparasitoid wasps, are divided into two genera: ichnovirus (IV and bracovirus (BV. In IV, some Cys-motif genes are known as host translation-inhibitory factors (HTIF. The genome of endoparasitoid wasp Cotesia plutellae contains a Cys-motif gene (Cp-TSP13 homologous to an HTIF known as teratocyte-secretory protein 14 (TSP14 of Microplitis croceipes. Cp-TSP13 consists of 129 amino acid residues with a predicted molecular weight of 13.987 kDa and pI value of 7.928. Genomic DNA region encoding its open reading frame has three introns. Cp-TSP13 possesses six conserved cysteine residues as other Cys-motif genes functioning as HTIF. Cp-TSP13 was expressed in Plutella xylostella larvae parasitized by C. plutellae. C. plutellae bracovirus (CpBV was purified and injected into non-parasitized P. xylostella that expressed Cp-TSP13. Cp-TSP13 was cloned into a eukaryotic expression vector and used to infect Sf9 cells to transiently express Cp-TSP13. The synthesized Cp-TSP13 protein was detected in culture broth. An overlaying experiment showed that the purified Cp-TSP13 entered hemocytes. It was localized in the cytosol. Recombinant Cp-TSP13 significantly inhibited protein synthesis of secretory proteins when it was added to in vitro cultured fat body. In addition, the recombinant Cp-TSP13 directly inhibited the translation of fat body mRNAs in in vitro translation assay using rabbit reticulocyte lysate. Moreover, the recombinant Cp-TSP13 significantly suppressed cellular immune responses by inhibiting hemocyte-spreading behavior. It also exhibited significant insecticidal activities by both injection and feeding routes. These results indicate that Cp-TSP13 is a viral HTIF.

  19. Suppression of type I interferon production by porcine epidemic diarrhea virus and degradation of CREB-binding protein by nsp1

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Qingzhan; Shi, Kaichuang; Yoo, Dongwan, E-mail: dyoo@illinois.edu

    2016-02-15

    Type I interferons (IFN-α/β) are the major components of the innate immune response of hosts, and in turn many viruses have evolved to modulate the host response during infection. We found that the IFN-β production was significantly suppressed during PEDV infection in cells. To identify viral IFN antagonists and to study their suppressive function, viral coding sequences for the entire structural and nonstructural proteins were cloned and expressed. Of 16 PEDV nonstructural proteins (nsps), nsp1, nsp3, nsp7, nsp14, nsp15 and nsp16 were found to inhibit the IFN-β and IRF3 promoter activities. The sole accessory protein ORF3, structure protein envelope (E), membrane (M), and nucleocapsid (N) protein were also shown to inhibit such activities. PEDV nsp1 did not interfere the IRF3 phosphorylation and nuclear translocation but interrupted the enhanceosome assembly of IRF3 and CREB-binding protein (CBP) by degrading CBP. A further study showed that the CBP degradation by nsp1 was proteasome-dependent. Our data demonstrate that PEDV modulates the host innate immune responses by degrading CBP and suppressing ISGs expression. - Highlights: • PEDV modulates the host innate immune system by suppressing the type I interferon production and ISGs expression. • Ten viral proteins were identified as IFN antagonists, and nsp1 was the most potent viral IFN antagonist. • PEDV nsp1 did not interfere the IRF3 phosphorylation and nuclear translocation but interrupted the enhanceosome assembly of IRF3 and CREB-binding protein (CBP). • PEDV nsp1 caused the CBP degradation in the nucleus, which may be the key mechanism for PEDV-mediated IFN downregulation.

  20. Mycobacterium tuberculosis co-operonic PE32/PPE65 proteins alter host immune responses by hampering Th1 response

    Directory of Open Access Journals (Sweden)

    Mohd eKhubaib

    2016-05-01

    Full Text Available PE/PPE genes, present in cluster with ESAT-6 like genes, are suspected to have a role in antigenic variation and virulence of Mycobacterium tuberculosis. Their roles in immune evasion and immune modulation of host are also well documented. We present evidence that PE32/PPE65 present within the RD8 region are co-operonic, co-transcribed and co-translated, and play role in modulating host immune responses. Experiments with macrophage cell lines revealed that this protein complex suppresses pro-inflammatory cytokines such as TNF-α and IL-6 whereas also inducing high expression of anti-inflammatory IL-10. Immunization of mice with these recombinant proteins dampens an effective Th1 response as evident from reduced frequency of IFN-g and IL-2 producing CD4+ and CD8+ T cells. IgG sub-typing from serum of immunized mice revealed high levels of IgG1 when compared with IgG2a and IgG2b. Further IgG1/IgG2a ratio clearly demonstrated that the protein complex manipulates the host immune response favourable to the pathogen. Our results demonstrate that the co-transcribed and co-translated PE32 and PPE65 antigens are involved specifically in modulating anti-mycobacterial host immune response by hampering Th1 response.

  1. Identification and monitoring of host cell proteins by mass spectrometry combined with high performance immunochemistry testing.

    Directory of Open Access Journals (Sweden)

    Katrin Bomans

    Full Text Available Biotherapeutics are often produced in non-human host cells like Escherichia coli, yeast, and various mammalian cell lines. A major focus of any therapeutic protein purification process is to reduce host cell proteins to an acceptable low level. In this study, various E. coli host cell proteins were identified at different purifications steps by HPLC fractionation, SDS-PAGE analysis, and tryptic peptide mapping combined with online liquid chromatography mass spectrometry (LC-MS. However, no host cell proteins could be verified by direct LC-MS analysis of final drug substance material. In contrast, the application of affinity enrichment chromatography prior to comprehensive LC-MS was adequate to identify several low abundant host cell proteins at the final drug substance level. Bacterial alkaline phosphatase (BAP was identified as being the most abundant host cell protein at several purification steps. Thus, we firstly established two different assays for enzymatic and immunological BAP monitoring using the cobas® technology. By using this strategy we were able to demonstrate an almost complete removal of BAP enzymatic activity by the established therapeutic protein purification process. In summary, the impact of fermentation, purification, and formulation conditions on host cell protein removal and biological activity can be conducted by monitoring process-specific host cell proteins in a GMP-compatible and high-throughput (> 1000 samples/day manner.

  2. An ANNEXIN-like protein from the cereal cyst nematode Heterodera avenae suppresses plant defense.

    Directory of Open Access Journals (Sweden)

    Changlong Chen

    Full Text Available Parasitism genes encoding secreted effector proteins of plant-parasitic nematodes play important roles in facilitating parasitism. An annexin-like gene was isolated from the cereal cyst nematode Heterodera avenae (termed Ha-annexin and had high similarity to annexin 2, which encodes a secreted protein of Globodera pallida. Ha-annexin encodes a predicted 326 amino acid protein containing four conserved annexin domains. Southern blotting revealed that there are at least two homologies in the H. avenae genome. Ha-annexin transcripts were expressed within the subventral gland cells of the pre-parasitic second-stage juveniles by in situ hybridization. Additionally, expression of these transcripts were relatively higher in the parasitic second-stage juveniles by quantitative real-time RT-PCR analysis, coinciding with the time when feeding cell formation is initiated. Knockdown of Ha-annexin by method of barley stripe mosaic virus-based host-induced gene silencing (BSMV-HIGS caused impaired nematode infections at 7 dpi and reduced females at 40 dpi, indicating important roles of the gene in parasitism at least in early stage in vivo. Transiently expression of Ha-ANNEXIN in onion epidermal cells and Nicotiana benthamiana leaf cells showed the whole cell-localization. Using transient expression assays in N. benthamiana, we found that Ha-ANNEXIN could suppress programmed cell death triggered by the pro-apoptotic mouse protein BAX and the induction of marker genes of PAMP-triggered immunity (PTI in N. benthamiana. In addition, Ha-ANNEXIN targeted a point in the mitogen-activated protein kinase (MAPK signaling pathway downstream of two kinases MKK1 and NPK1 in N. benthamiana.

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

    Directory of Open Access Journals (Sweden)

    Suayib Üstün

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

  4. Serratia marcescens Suppresses Host Cellular Immunity via the Production of an Adhesion-inhibitory Factor against Immunosurveillance Cells*

    Science.gov (United States)

    Ishii, Kenichi; Adachi, Tatsuo; Hamamoto, Hiroshi; Sekimizu, Kazuhisa

    2014-01-01

    Injection of a culture supernatant of Serratia marcescens into the bloodstream of the silkworm Bombyx mori increased the number of freely circulating immunosurveillance cells (hemocytes). Using a bioassay with live silkworms, serralysin metalloprotease was purified from the culture supernatant and identified as the factor responsible for this activity. Serralysin inhibited the in vitro attachment of both silkworm hemocytes and murine peritoneal macrophages. Incubation of silkworm hemocytes or murine macrophages with serralysin resulted in degradation of the cellular immune factor BmSPH-1 or calreticulin, respectively. Furthermore, serralysin suppressed in vitro phagocytosis of bacteria by hemocytes and in vivo bacterial clearance in silkworms. Disruption of the ser gene in S. marcescens attenuated its host killing ability in silkworms and mice. These findings suggest that serralysin metalloprotease secreted by S. marcescens suppresses cellular immunity by decreasing the adhesive properties of immunosurveillance cells, thereby contributing to bacterial pathogenesis. PMID:24398686

  5. Serratia marcescens suppresses host cellular immunity via the production of an adhesion-inhibitory factor against immunosurveillance cells.

    Science.gov (United States)

    Ishii, Kenichi; Adachi, Tatsuo; Hamamoto, Hiroshi; Sekimizu, Kazuhisa

    2014-02-28

    Injection of a culture supernatant of Serratia marcescens into the bloodstream of the silkworm Bombyx mori increased the number of freely circulating immunosurveillance cells (hemocytes). Using a bioassay with live silkworms, serralysin metalloprotease was purified from the culture supernatant and identified as the factor responsible for this activity. Serralysin inhibited the in vitro attachment of both silkworm hemocytes and murine peritoneal macrophages. Incubation of silkworm hemocytes or murine macrophages with serralysin resulted in degradation of the cellular immune factor BmSPH-1 or calreticulin, respectively. Furthermore, serralysin suppressed in vitro phagocytosis of bacteria by hemocytes and in vivo bacterial clearance in silkworms. Disruption of the ser gene in S. marcescens attenuated its host killing ability in silkworms and mice. These findings suggest that serralysin metalloprotease secreted by S. marcescens suppresses cellular immunity by decreasing the adhesive properties of immunosurveillance cells, thereby contributing to bacterial pathogenesis.

  6. Comparative Analyses of Tomato yellow leaf curl virus C4 Protein-Interacting Host Proteins in Healthy and Infected Tomato Tissues

    Directory of Open Access Journals (Sweden)

    Namgyu Kim

    2016-10-01

    Full Text Available Tomato yellow leaf curl virus (TYLCV, a member of the genus Begomovirus, is one of the most important viruses of cultivated tomatoes worldwide, mainly causing yellowing and curling of leaves with stunting in plants. TYLCV causes severe problems in sub-tropical and tropical countries, as well as in Korea. However, the mechanism of TYLCV infection remains unclear, although the function of each viral component has been identified. TYLCV C4 codes for a small protein involved in various cellular functions, including symptom determination, gene silencing, viral movement, and induction of the plant defense response. In this study, through yeast-two hybrid screenings, we identified TYLCV C4-interacting host proteins from both healthy and symptom-exhibiting tomato tissues, to determine the role of TYLCV C4 proteins in the infection processes. Comparative analyses of 28 proteins from healthy tissues and 36 from infected tissues showing interactions with TYLCV C4 indicated that TYLCV C4 mainly interacts with host proteins involved in translation, ubiquitination, and plant defense, and most interacting proteins differed between the two tissues but belong to similar molecular functional categories. Four proteins—two ribosomal proteins, S-adenosyl-L-homocysteine hydrolase, and 14-3-3 family protein—were detected in both tissues. Furthermore, the identified proteins in symptom-exhibiting tissues showed greater involvement in plant defenses. Some are key regulators, such as receptor-like kinases and pathogenesis-related proteins, of plant defenses. Thus, TYLCV C4 may contribute to the suppression of host defense during TYLCV infection and be involved in ubiquitination for viral infection.

  7. Genome wide expression profiling reveals suppression of host defence responses during colonisation by Neisseria meningitides but not N. lactamica.

    Directory of Open Access Journals (Sweden)

    Hazel En En Wong

    Full Text Available Both Neisseria meningitidis and the closely related bacterium Neisseria lactamica colonise human nasopharyngeal mucosal surface, but only N. meningitidis invades the bloodstream to cause potentially life-threatening meningitis and septicaemia. We have hypothesised that the two neisserial species differentially modulate host respiratory epithelial cell gene expression reflecting their disease potential. Confluent monolayers of 16HBE14 human bronchial epithelial cells were exposed to live and/or dead N. meningitidis (including capsule and pili mutants and N. lactamica, and their transcriptomes were compared using whole genome microarrays. Changes in expression of selected genes were subsequently validated using Q-RT-PCR and ELISAs. Live N. meningitidis and N. lactamica induced genes involved in host energy production processes suggesting that both bacterial species utilise host resources. N. meningitidis infection was associated with down-regulation of host defence genes. N. lactamica, relative to N. meningitidis, initiates up-regulation of proinflammatory genes. Bacterial secreted proteins alone induced some of the changes observed. The results suggest N. meningitidis and N. lactamica differentially regulate host respiratory epithelial cell gene expression through colonisation and/or protein secretion, and that this may contribute to subsequent clinical outcomes associated with these bacteria.

  8. Bacterial Serine/Threonine Protein Kinases in Host-Pathogen Interactions*

    Science.gov (United States)

    Canova, Marc J.; Molle, Virginie

    2014-01-01

    In bacterial pathogenesis, monitoring and adapting to the dynamically changing environment in the host and an ability to disrupt host immune responses are critical. The virulence determinants of pathogenic bacteria include the sensor/signaling proteins of the serine/threonine protein kinase (STPK) family that have a dual role of sensing the environment and subverting specific host defense processes. STPKs can sense a wide range of signals and coordinate multiple cellular processes to mount an appropriate response. Here, we review some of the well studied bacterial STPKs that are essential virulence factors and that modify global host responses during infection. PMID:24554701

  9. Bacterial serine/threonine protein kinases in host-pathogen interactions.

    Science.gov (United States)

    Canova, Marc J; Molle, Virginie

    2014-04-04

    In bacterial pathogenesis, monitoring and adapting to the dynamically changing environment in the host and an ability to disrupt host immune responses are critical. The virulence determinants of pathogenic bacteria include the sensor/signaling proteins of the serine/threonine protein kinase (STPK) family that have a dual role of sensing the environment and subverting specific host defense processes. STPKs can sense a wide range of signals and coordinate multiple cellular processes to mount an appropriate response. Here, we review some of the well studied bacterial STPKs that are essential virulence factors and that modify global host responses during infection.

  10. Artificial Metalloenzymes through Chemical Modification of Engineered Host Proteins

    KAUST Repository

    Zernickel, Anna

    2014-01-01

    With a few exceptions, all organisms are restricted to the 20 canonical amino acids for ribosomal protein biosynthesis. Addition of new amino acids to the genetic code can introduce novel functionalities to proteins, broadening the diversity

  11. Reviewing host proteins of Rhabdoviridae: possible leads for lesser studied viruses.

    Science.gov (United States)

    Guleria, A; Kiranmayi, M; Sreejith, R; Kumar, K; Sharma, S K; Gupta, S

    2011-12-01

    Rhabdoviridae, characterized by bullet-shaped viruses, is known for its diverse host range, which includes plants, arthropods, fishes and humans. Understanding the viral-host interactions of this family can prove beneficial in developing effective therapeutic strategies. The host proteins interacting with animal rhabdoviruses have been reviewed in this report. Several important host proteins commonly interacting with animal rhabdoviruses are being reported, some of which, interestingly, have molecular features, which can serve as potential antiviral targets. This review not only provides the generalized importance of the functions of animal rhabdovirus-associated host proteins for the first time but also compares them among the two most studied viruses, i.e. Rabies virus (RV) and Vesicular Stomatitis virus (VSV). The comparative data can be used for studying emerging viruses such as Chandipura virus (CHPV) and the lesser studied viruses such as Piry virus (PIRYV) and Isfahan virus (ISFV) of the Rhabdoviridae family.

  12. A novel Meloidogyne graminicola effector, MgMO237, interacts with multiple host defence-related proteins to manipulate plant basal immunity and promote parasitism.

    Science.gov (United States)

    Chen, Jiansong; Hu, Lili; Sun, Longhua; Lin, Borong; Huang, Kun; Zhuo, Kan; Liao, Jinling

    2018-02-27

    Plant-parasitic nematodes can secrete effector proteins into the host tissue to facilitate their parasitism. In this study, we report a novel effector protein, MgMO237, from Meloidogyne graminicola, which is exclusively expressed within the dorsal oesophageal gland cell and markedly up-regulated in parasitic third-/fourth-stage juveniles of M. graminicola. Transient expression of MgMO237 in protoplasts from rice roots showed that MgMO237 was localized in the cytoplasm and nucleus of the host cells. Rice plants overexpressing MgMO237 showed an increased susceptibility to M. graminicola. In contrast, rice plants expressing RNA interference vectors targeting MgMO237 showed an increased resistance to M. graminicola. In addition, yeast two-hybrid and co-immunoprecipitation assays showed that MgMO237 interacted specifically with three rice endogenous proteins, i.e. 1,3-β-glucan synthase component (OsGSC), cysteine-rich repeat secretory protein 55 (OsCRRSP55) and pathogenesis-related BetvI family protein (OsBetvI), which are all related to host defences. Moreover, MgMO237 can suppress host defence responses, including the expression of host defence-related genes, cell wall callose deposition and the burst of reactive oxygen species. These results demonstrate that the effector MgMO237 probably promotes the parasitism of M. graminicola by interacting with multiple host defence-related proteins and suppressing plant basal immunity in the later parasitic stages of nematodes. © 2018 BSPP AND JOHN WILEY & SONS LTD.

  13. Host Proteins Determine MRSA Biofilm Structure and Integrity

    DEFF Research Database (Denmark)

    Dreier, Cindy; Nielsen, Astrid; Jørgensen, Nis Pedersen

    Human extracellular matrix (hECM) proteins aids the initial attachment and initiation of an infection, by specific binding to bacterial cell surface proteins. However, the importance of hECM proteins in structure, integrity and antibiotic resilience of a biofilm is unknown. This study aims...... to determine how specific hECM proteins affect S. aureus USA300 JE2 biofilms. Biofilms were grown in the presence of synovial fluid from rheumatoid arteritis patients to mimic in vivo conditions, where bacteria incorporate hECM proteins into the biofilm matrix. Difference in biofilm structure, with and without...... addition of hECM to growth media, was visualized by confocal laser scanning microscopy. Two enzymatic degradation experiments were used to study biofilm matrix composition and importance of hECM proteins: enzymatic removal of specific hECM proteins from growth media, before biofilm formation, and enzymatic...

  14. Host cell proteins in biotechnology-derived products: A risk assessment framework.

    Science.gov (United States)

    de Zafra, Christina L Zuch; Quarmby, Valerie; Francissen, Kathleen; Vanderlaan, Martin; Zhu-Shimoni, Judith

    2015-11-01

    To manufacture biotechnology products, mammalian or bacterial cells are engineered for the production of recombinant therapeutic human proteins including monoclonal antibodies. Host cells synthesize an entire repertoire of proteins which are essential for their own function and survival. Biotechnology manufacturing processes are designed to produce recombinant therapeutics with a very high degree of purity. While there is typically a low residual level of host cell protein in the final drug product, under some circumstances a host cell protein(s) may copurify with the therapeutic protein and, if it is not detected and removed, it may become an unintended component of the final product. The purpose of this article is to enumerate and discuss factors to be considered in an assessment of risk of residual host cell protein(s) detected and identified in the drug product. The consideration of these factors and their relative ranking will lead to an overall risk assessment that informs decision-making around how to control the levels of host cell proteins. © 2015 Wiley Periodicals, Inc.

  15. Mild hypothermic culture conditions affect residual host cell protein composition post-Protein A chromatography.

    Science.gov (United States)

    Goey, Cher Hui; Bell, David; Kontoravdi, Cleo

    2018-04-01

    Host cell proteins (HCPs) are endogenous impurities, and their proteolytic and binding properties can compromise the integrity, and, hence, the stability and efficacy of recombinant therapeutic proteins such as monoclonal antibodies (mAbs). Nonetheless, purification of mAbs currently presents a challenge because they often co-elute with certain HCP species during the capture step of protein A affinity chromatography. A Quality-by-Design (QbD) strategy to overcome this challenge involves identifying residual HCPs and tracing their source to the harvested cell culture fluid (HCCF) and the corresponding cell culture operating parameters. Then, problematic HCPs in HCCF may be reduced by cell engineering or culture process optimization. Here, we present experimental results linking cell culture temperature and post-protein A residual HCP profile. We had previously reported that Chinese hamster ovary cell cultures conducted at standard physiological temperature and with a shift to mild hypothermia on day 5 produced HCCF of comparable product titer and HCP concentration, but with considerably different HCP composition. In this study, we show that differences in HCP variety at harvest cascaded to downstream purification where different residual HCPs were present in the two sets of samples post-protein A purification. To detect low-abundant residual HCPs, we designed a looping liquid chromatography-mass spectrometry method with continuous expansion of a preferred, exclude, and targeted peptide list. Mild hypothermic cultures produced 20% more residual HCP species, especially cell membrane proteins, distinct from the control. Critically, we identified that half of the potentially immunogenic residual HCP species were different between the two sets of samples.

  16. Predicting the subcellular localization of viral proteins within a mammalian host cell

    Directory of Open Access Journals (Sweden)

    Thomas DY

    2006-04-01

    Full Text Available Abstract Background The bioinformatic prediction of protein subcellular localization has been extensively studied for prokaryotic and eukaryotic organisms. However, this is not the case for viruses whose proteins are often involved in extensive interactions at various subcellular localizations with host proteins. Results Here, we investigate the extent of utilization of human cellular localization mechanisms by viral proteins and we demonstrate that appropriate eukaryotic subcellular localization predictors can be used to predict viral protein localization within the host cell. Conclusion Such predictions provide a method to rapidly annotate viral proteomes with subcellular localization information. They are likely to have widespread applications both in the study of the functions of viral proteins in the host cell and in the design of antiviral drugs.

  17. Reflects the coat protein variability of apple mosaic virus host preference?

    Czech Academy of Sciences Publication Activity Database

    Grimová, L.; Winkowska, L.; Ryšánek, P.; Svoboda, P.; Petrzik, Karel

    2013-01-01

    Roč. 47, č. 1 (2013), s. 119-125 ISSN 0920-8569 Institutional support: RVO:60077344 Keywords : Positive selection tests * capsid protein * algae host Subject RIV: EE - Microbiology, Virology Impact factor: 1.837, year: 2013

  18. Identification of proteins involved in the functioning of Riftia pachyptila symbiosis by Subtractive Suppression Hybridization

    Directory of Open Access Journals (Sweden)

    Lallier François H

    2007-09-01

    Full Text Available Abstract Background Since its discovery around deep sea hydrothermal vents of the Galapagos Rift about 30 years ago, the chemoautotrophic symbiosis between the vestimentiferan tubeworm Riftia pachyptila and its symbiotic sulfide-oxidizing γ-proteobacteria has been extensively studied. However, studies on the tubeworm host were essentially targeted, biochemical approaches. We decided to use a global molecular approach to identify new proteins involved in metabolite exchanges and assimilation by the host. We used a Subtractive Suppression Hybridization approach (SSH in an unusual way, by comparing pairs of tissues from a single individual. We chose to identify the sequences preferentially expressed in the branchial plume tissue (the only organ in contact with the sea water and in the trophosome (the organ housing the symbiotic bacteria using the body wall as a reference tissue because it is supposedly not involved in metabolite exchanges in this species. Results We produced four cDNA libraries: i body wall-subtracted branchial plume library (BR-BW, ii and its reverse library, branchial plume-subtracted body wall library (BW-BR, iii body wall-subtracted trophosome library (TR-BW, iv and its reverse library, trophosome-subtracted body wall library (BW-TR. For each library, we sequenced about 200 clones resulting in 45 different sequences on average in each library (58 and 59 cDNAs for BR-BW and TR-BW libraries respectively. Overall, half of the contigs matched records found in the databases with good E-values. After quantitative PCR analysis, it resulted that 16S, Major Vault Protein, carbonic anhydrase (RpCAbr, cathepsin and chitinase precursor transcripts were highly represented in the branchial plume tissue compared to the trophosome and the body wall tissues, whereas carbonic anhydrase (RpCAtr, myohemerythrin, a putative T-Cell receptor and one non identified transcript were highly specific of the trophosome tissue. Conclusion Quantitative PCR

  19. Identification of proteins involved in the functioning of Riftia pachyptila symbiosis by Subtractive Suppression Hybridization.

    Science.gov (United States)

    Sanchez, Sophie; Hourdez, Stéphane; Lallier, François H

    2007-09-24

    Since its discovery around deep sea hydrothermal vents of the Galapagos Rift about 30 years ago, the chemoautotrophic symbiosis between the vestimentiferan tubeworm Riftia pachyptila and its symbiotic sulfide-oxidizing gamma-proteobacteria has been extensively studied. However, studies on the tubeworm host were essentially targeted, biochemical approaches. We decided to use a global molecular approach to identify new proteins involved in metabolite exchanges and assimilation by the host. We used a Subtractive Suppression Hybridization approach (SSH) in an unusual way, by comparing pairs of tissues from a single individual. We chose to identify the sequences preferentially expressed in the branchial plume tissue (the only organ in contact with the sea water) and in the trophosome (the organ housing the symbiotic bacteria) using the body wall as a reference tissue because it is supposedly not involved in metabolite exchanges in this species. We produced four cDNA libraries: i) body wall-subtracted branchial plume library (BR-BW), ii) and its reverse library, branchial plume-subtracted body wall library (BW-BR), iii) body wall-subtracted trophosome library (TR-BW), iv) and its reverse library, trophosome-subtracted body wall library (BW-TR). For each library, we sequenced about 200 clones resulting in 45 different sequences on average in each library (58 and 59 cDNAs for BR-BW and TR-BW libraries respectively). Overall, half of the contigs matched records found in the databases with good E-values. After quantitative PCR analysis, it resulted that 16S, Major Vault Protein, carbonic anhydrase (RpCAbr), cathepsin and chitinase precursor transcripts were highly represented in the branchial plume tissue compared to the trophosome and the body wall tissues, whereas carbonic anhydrase (RpCAtr), myohemerythrin, a putative T-Cell receptor and one non identified transcript were highly specific of the trophosome tissue. Quantitative PCR analyses were congruent with our libraries

  20. Inhibition of host cell protein synthesis by UV-inactivated poliovirus

    International Nuclear Information System (INIS)

    Helentjaris, T.; Ehrenfeld, E.

    1977-01-01

    The ability of poliovirus that was irradiated with UV light at energies up to 2,160 ergs/mm 2 to subsequently inhibit host cell protein synthesis was measured. The inactivation of the host cell shutoff function followed one-hit kinetics. Increasing irradiation did not affect the rate of inhibition until the multiplicity of infection after irradiation was reduced to approximately 1 PFU/cell. At higher functional multiplicities, the rate was unchanged, but an increasing lag before the onset of inhibition was observed with increasing irradiation. The energy levels required to inactivate virus-induced inhibition of host cell protein synthesis suggest that damage to virus RNA rather than to virus capsid proteins is responsible for the loss of function. When the inactivation of host cell shutoff was compared with the inactivation of other viral functions by UV irradiation, it correlated exactly with the loss of infectivity but not with other viral functions measured. Guanidine treatment, which prevents detectable viral RNA and protein synthesis, completely inhibited host cell shutoff by low multiplicities of unirradiated virus infection but not higher multiplicities. When a high multiplicity of virus was first reduced to a low titer by irradiation, host cell shutoff was still evident in the presence of guanidine. The results demonstrate that the complete inhibition of host cell protein synthesis can be accomplished by one infectious viral genome per cell

  1. Host-derived, pore-forming toxin-like protein and trefoil factor complex protects the host against microbial infection.

    Science.gov (United States)

    Xiang, Yang; Yan, Chao; Guo, Xiaolong; Zhou, Kaifeng; Li, Sheng'an; Gao, Qian; Wang, Xuan; Zhao, Feng; Liu, Jie; Lee, Wen-Hui; Zhang, Yun

    2014-05-06

    Aerolysins are virulence factors belonging to the bacterial β-pore-forming toxin superfamily. Surprisingly, numerous aerolysin-like proteins exist in vertebrates, but their biological functions are unknown. βγ-CAT, a complex of an aerolysin-like protein subunit (two βγ-crystallin domains followed by an aerolysin pore-forming domain) and two trefoil factor subunits, has been identified in frogs (Bombina maxima) skin secretions. Here, we report the rich expression of this protein, in the frog blood and immune-related tissues, and the induction of its presence in peritoneal lavage by bacterial challenge. This phenomena raises the possibility of its involvement in antimicrobial infection. When βγ-CAT was administrated in a peritoneal infection model, it greatly accelerated bacterial clearance and increased the survival rate of both frogs and mice. Meanwhile, accelerated Interleukin-1β release and enhanced local leukocyte recruitments were determined, which may partially explain the robust and effective antimicrobial responses observed. The release of interleukin-1β was potently triggered by βγ-CAT from the frog peritoneal cells and murine macrophages in vitro. βγ-CAT was rapidly endocytosed and translocated to lysosomes, where it formed high molecular mass SDS-stable oligomers (>170 kDa). Lysosomal destabilization and cathepsin B release were detected, which may explain the activation of caspase-1 inflammasome and subsequent interleukin-1β maturation and release. To our knowledge, these results provide the first functional evidence of the ability of a host-derived aerolysin-like protein to counter microbial infection by eliciting rapid and effective host innate immune responses. The findings will also largely help to elucidate the possible involvement and action mechanisms of aerolysin-like proteins and/or trefoil factors widely existing in vertebrates in the host defense against pathogens.

  2. The Protein Interaction Network of Bacteriophage Lambda with Its Host, Escherichia coli

    Science.gov (United States)

    Blasche, Sonja; Wuchty, Stefan; Rajagopala, Seesandra V.

    2013-01-01

    Although most of the 73 open reading frames (ORFs) in bacteriophage λ have been investigated intensively, the function of many genes in host-phage interactions remains poorly understood. Using yeast two-hybrid screens of all lambda ORFs for interactions with its host Escherichia coli, we determined a raw data set of 631 host-phage interactions resulting in a set of 62 high-confidence interactions after multiple rounds of retesting. These links suggest novel regulatory interactions between the E. coli transcriptional network and lambda proteins. Targeted host proteins and genes required for lambda infection are enriched among highly connected proteins, suggesting that bacteriophages resemble interaction patterns of human viruses. Lambda tail proteins interact with both bacterial fimbrial proteins and E. coli proteins homologous to other phage proteins. Lambda appears to dramatically differ from other phages, such as T7, because of its unusually large number of modified and processed proteins, which reduces the number of host-virus interactions detectable by yeast two-hybrid screens. PMID:24049175

  3. Computational Approaches for Prediction of Pathogen-Host Protein-Protein Interactions

    Directory of Open Access Journals (Sweden)

    Esmaeil eNourani

    2015-02-01

    Full Text Available Infectious diseases are still among the major and prevalent health problems, mostly because of the drug resistance of novel variants of pathogens. Molecular interactions between pathogens and their hosts are the key part of the infection mechanisms. Novel antimicrobial therapeutics to fight drug resistance is only possible in case of a thorough understanding of pathogen-host interaction (PHI systems. Existing databases, which contain experimentally verified PHI data, suffer from scarcity of reported interactions due to the technically challenging and time consuming process of experiments. This has motivated many researchers to address the problem by proposing computational approaches for analysis and prediction of PHIs. The computational methods primarily utilize sequence information, protein structure and known interactions. Classic machine learning techniques are used when there are sufficient known interactions to be used as training data. On the opposite case, transfer and multi task learning methods are preferred. Here, we present an overview of these computational approaches for PHI prediction, discussing their weakness and abilities, with future directions.

  4. A hydrophobic anchor mechanism defines a deacetylase family that suppresses host response against YopJ effectors.

    Science.gov (United States)

    Bürger, Marco; Willige, Björn C; Chory, Joanne

    2017-12-19

    Several Pseudomonas and Xanthomonas species are plant pathogens that infect the model organism Arabidopsis thaliana and important crops such as Brassica. Resistant plants contain the infection by rapid cell death of the infected area through the hypersensitive response (HR). A family of highly related α/β hydrolases is involved in diverse processes in all domains of life. Functional details of their catalytic machinery, however, remained unclear. We report the crystal structures of α/β hydrolases representing two different clades of the family, including the protein SOBER1, which suppresses AvrBsT-incited HR in Arabidopsis. Our results reveal a unique hydrophobic anchor mechanism that defines a previously unknown family of protein deacetylases. Furthermore, this study identifies a lid-loop as general feature for substrate turnover in acyl-protein thioesterases and the described family of deacetylases. Furthermore, we found that SOBER1's biological function is not restricted to Arabidopsis thaliana and not limited to suppress HR induced by AvrBsT.

  5. Distinct Host Tropism Protein Signatures to Identify Possible Zoonotic Influenza A Viruses.

    Science.gov (United States)

    Eng, Christine L P; Tong, Joo Chuan; Tan, Tin Wee

    2016-01-01

    Zoonotic influenza A viruses constantly pose a health threat to humans as novel strains occasionally emerge from the avian population to cause human infections. Many past epidemic as well as pandemic strains have originated from avian species. While most viruses are restricted to their primary hosts, zoonotic strains can sometimes arise from mutations or reassortment, leading them to acquire the capability to escape host species barrier and successfully infect a new host. Phylogenetic analyses and genetic markers are useful in tracing the origins of zoonotic infections, but there are still no effective means to identify high risk strains prior to an outbreak. Here we show that distinct host tropism protein signatures can be used to identify possible zoonotic strains in avian species which have the potential to cause human infections. We have discovered that influenza A viruses can now be classified into avian, human, or zoonotic strains based on their host tropism protein signatures. Analysis of all influenza A viruses with complete proteome using the host tropism prediction system, based on machine learning classifications of avian and human viral proteins has uncovered distinct signatures of zoonotic strains as mosaics of avian and human viral proteins. This is in contrast with typical avian or human strains where they show mostly avian or human viral proteins in their signatures respectively. Moreover, we have found that zoonotic strains from the same influenza outbreaks carry similar host tropism protein signatures characteristic of a common ancestry. Our results demonstrate that the distinct host tropism protein signature in zoonotic strains may prove useful in influenza surveillance to rapidly identify potential high risk strains circulating in avian species, which may grant us the foresight in anticipating an impending influenza outbreak.

  6. quinolinium iodide in suppression of protein–protein interactions

    Indian Academy of Sciences (India)

    In searching for alternative ways to reduce protein–protein interactions or to inhibit the amyloid formation, the inhibitory effects ..... ing the exposure of hydrophobic surfaces mirrors the ... is well-supported by electrostatic interactions between.

  7. A viral suppressor protein inhibits host RNA silencing by hooking up with Argonautes

    KAUST Repository

    Jin, Hailing

    2010-05-01

    RNA viruses are particularly vulnerable to RNAi-based defenses in the host, and thus have evolved specific proteins, known as viral suppressors of RNA silencing (VSRs), as a counterdefense. In this issue of Genes & Development, Azevedo and colleagues (pp. 904-915) discovered that P38, the VSR of Turnip crinkle virus, uses its glycine/tryptophane (GW) motifs as an ARGONAUTE (AGO) hook to attract and disarm the host\\'s essential effector of RNA silencing. Several GW motif-containing cellular proteins are known to be important partners of AGOs in RNA silencing effector complexes in yeast, plants, and animals. The GW motif appears to be a versatile and effective tool for regulating the activities of RNA silencing pathways, and the use of GW mimicry to compete for and inhibit host AGOs may be a strategy used by many pathogens to counteract host RNAi-based defenses. © 2010 by Cold Spring Harbor Laboratory Press.

  8. Insight into bacterial virulence mechanisms against host immune response via the Yersinia pestis-human protein-protein interaction network.

    Science.gov (United States)

    Yang, Huiying; Ke, Yuehua; Wang, Jian; Tan, Yafang; Myeni, Sebenzile K; Li, Dong; Shi, Qinghai; Yan, Yanfeng; Chen, Hui; Guo, Zhaobiao; Yuan, Yanzhi; Yang, Xiaoming; Yang, Ruifu; Du, Zongmin

    2011-11-01

    A Yersinia pestis-human protein interaction network is reported here to improve our understanding of its pathogenesis. Up to 204 interactions between 66 Y. pestis bait proteins and 109 human proteins were identified by yeast two-hybrid assay and then combined with 23 previously published interactions to construct a protein-protein interaction network. Topological analysis of the interaction network revealed that human proteins targeted by Y. pestis were significantly enriched in the proteins that are central in the human protein-protein interaction network. Analysis of this network showed that signaling pathways important for host immune responses were preferentially targeted by Y. pestis, including the pathways involved in focal adhesion, regulation of cytoskeleton, leukocyte transendoepithelial migration, and Toll-like receptor (TLR) and mitogen-activated protein kinase (MAPK) signaling. Cellular pathways targeted by Y. pestis are highly relevant to its pathogenesis. Interactions with host proteins involved in focal adhesion and cytoskeketon regulation pathways could account for resistance of Y. pestis to phagocytosis. Interference with TLR and MAPK signaling pathways by Y. pestis reflects common characteristics of pathogen-host interaction that bacterial pathogens have evolved to evade host innate immune response by interacting with proteins in those signaling pathways. Interestingly, a large portion of human proteins interacting with Y. pestis (16/109) also interacted with viral proteins (Epstein-Barr virus [EBV] and hepatitis C virus [HCV]), suggesting that viral and bacterial pathogens attack common cellular functions to facilitate infections. In addition, we identified vasodilator-stimulated phosphoprotein (VASP) as a novel interaction partner of YpkA and showed that YpkA could inhibit in vitro actin assembly mediated by VASP.

  9. Identification of New Protein Interactions between Dengue Fever Virus and Its Hosts, Human and Mosquito

    Science.gov (United States)

    Mairiang, Dumrong; Zhang, Huamei; Sodja, Ann; Murali, Thilakam; Suriyaphol, Prapat; Malasit, Prida; Limjindaporn, Thawornchai; Finley, Russell L.

    2013-01-01

    The four divergent serotypes of dengue virus are the causative agents of dengue fever, dengue hemorrhagic fever and dengue shock syndrome. About two-fifths of the world's population live in areas where dengue is prevalent, and thousands of deaths are caused by the viruses every year. Dengue virus is transmitted from one person to another primarily by the yellow fever mosquito, Aedes aegypti. Recent studies have begun to define how the dengue viral proteins interact with host proteins to mediate viral replication and pathogenesis. A combined analysis of these studies, however, suggests that many virus-host protein interactions remain to be identified, especially for the mosquito host. In this study, we used high-throughput yeast two-hybrid screening to identify mosquito and human proteins that physically interact with dengue proteins. We tested each identified host protein against the proteins from all four serotypes of dengue to identify interactions that are conserved across serotypes. We further confirmed many of the interactions using co-affinity purification assays. As in other large-scale screens, we identified some previously detected interactions and many new ones, moving us closer to a complete host – dengue protein interactome. To help summarize and prioritize the data for further study, we combined our interactions with other published data and identified a subset of the host-dengue interactions that are now supported by multiple forms of evidence. These data should be useful for understanding the interplay between dengue and its hosts and may provide candidates for drug targets and vector control strategies. PMID:23326450

  10. Identification of new protein interactions between dengue fever virus and its hosts, human and mosquito.

    Science.gov (United States)

    Mairiang, Dumrong; Zhang, Huamei; Sodja, Ann; Murali, Thilakam; Suriyaphol, Prapat; Malasit, Prida; Limjindaporn, Thawornchai; Finley, Russell L

    2013-01-01

    The four divergent serotypes of dengue virus are the causative agents of dengue fever, dengue hemorrhagic fever and dengue shock syndrome. About two-fifths of the world's population live in areas where dengue is prevalent, and thousands of deaths are caused by the viruses every year. Dengue virus is transmitted from one person to another primarily by the yellow fever mosquito, Aedes aegypti. Recent studies have begun to define how the dengue viral proteins interact with host proteins to mediate viral replication and pathogenesis. A combined analysis of these studies, however, suggests that many virus-host protein interactions remain to be identified, especially for the mosquito host. In this study, we used high-throughput yeast two-hybrid screening to identify mosquito and human proteins that physically interact with dengue proteins. We tested each identified host protein against the proteins from all four serotypes of dengue to identify interactions that are conserved across serotypes. We further confirmed many of the interactions using co-affinity purification assays. As in other large-scale screens, we identified some previously detected interactions and many new ones, moving us closer to a complete host - dengue protein interactome. To help summarize and prioritize the data for further study, we combined our interactions with other published data and identified a subset of the host-dengue interactions that are now supported by multiple forms of evidence. These data should be useful for understanding the interplay between dengue and its hosts and may provide candidates for drug targets and vector control strategies.

  11. Identification of new protein interactions between dengue fever virus and its hosts, human and mosquito.

    Directory of Open Access Journals (Sweden)

    Dumrong Mairiang

    Full Text Available The four divergent serotypes of dengue virus are the causative agents of dengue fever, dengue hemorrhagic fever and dengue shock syndrome. About two-fifths of the world's population live in areas where dengue is prevalent, and thousands of deaths are caused by the viruses every year. Dengue virus is transmitted from one person to another primarily by the yellow fever mosquito, Aedes aegypti. Recent studies have begun to define how the dengue viral proteins interact with host proteins to mediate viral replication and pathogenesis. A combined analysis of these studies, however, suggests that many virus-host protein interactions remain to be identified, especially for the mosquito host. In this study, we used high-throughput yeast two-hybrid screening to identify mosquito and human proteins that physically interact with dengue proteins. We tested each identified host protein against the proteins from all four serotypes of dengue to identify interactions that are conserved across serotypes. We further confirmed many of the interactions using co-affinity purification assays. As in other large-scale screens, we identified some previously detected interactions and many new ones, moving us closer to a complete host - dengue protein interactome. To help summarize and prioritize the data for further study, we combined our interactions with other published data and identified a subset of the host-dengue interactions that are now supported by multiple forms of evidence. These data should be useful for understanding the interplay between dengue and its hosts and may provide candidates for drug targets and vector control strategies.

  12. Mapping Protein Interactions between Dengue Virus and Its Human and Insect Hosts

    Science.gov (United States)

    Doolittle, Janet M.; Gomez, Shawn M.

    2011-01-01

    Background Dengue fever is an increasingly significant arthropod-borne viral disease, with at least 50 million cases per year worldwide. As with other viral pathogens, dengue virus is dependent on its host to perform the bulk of functions necessary for viral survival and replication. To be successful, dengue must manipulate host cell biological processes towards its own ends, while avoiding elimination by the immune system. Protein-protein interactions between the virus and its host are one avenue through which dengue can connect and exploit these host cellular pathways and processes. Methodology/Principal Findings We implemented a computational approach to predict interactions between Dengue virus (DENV) and both of its hosts, Homo sapiens and the insect vector Aedes aegypti. Our approach is based on structural similarity between DENV and host proteins and incorporates knowledge from the literature to further support a subset of the predictions. We predict over 4,000 interactions between DENV and humans, as well as 176 interactions between DENV and A. aegypti. Additional filtering based on shared Gene Ontology cellular component annotation reduced the number of predictions to approximately 2,000 for humans and 18 for A. aegypti. Of 19 experimentally validated interactions between DENV and humans extracted from the literature, this method was able to predict nearly half (9). Additional predictions suggest specific interactions between virus and host proteins relevant to interferon signaling, transcriptional regulation, stress, and the unfolded protein response. Conclusions/Significance Dengue virus manipulates cellular processes to its advantage through specific interactions with the host's protein interaction network. The interaction networks presented here provide a set of hypothesis for further experimental investigation into the DENV life cycle as well as potential therapeutic targets. PMID:21358811

  13. Mapping protein interactions between Dengue virus and its human and insect hosts.

    Directory of Open Access Journals (Sweden)

    Janet M Doolittle

    Full Text Available BACKGROUND: Dengue fever is an increasingly significant arthropod-borne viral disease, with at least 50 million cases per year worldwide. As with other viral pathogens, dengue virus is dependent on its host to perform the bulk of functions necessary for viral survival and replication. To be successful, dengue must manipulate host cell biological processes towards its own ends, while avoiding elimination by the immune system. Protein-protein interactions between the virus and its host are one avenue through which dengue can connect and exploit these host cellular pathways and processes. METHODOLOGY/PRINCIPAL FINDINGS: We implemented a computational approach to predict interactions between Dengue virus (DENV and both of its hosts, Homo sapiens and the insect vector Aedes aegypti. Our approach is based on structural similarity between DENV and host proteins and incorporates knowledge from the literature to further support a subset of the predictions. We predict over 4,000 interactions between DENV and humans, as well as 176 interactions between DENV and A. aegypti. Additional filtering based on shared Gene Ontology cellular component annotation reduced the number of predictions to approximately 2,000 for humans and 18 for A. aegypti. Of 19 experimentally validated interactions between DENV and humans extracted from the literature, this method was able to predict nearly half (9. Additional predictions suggest specific interactions between virus and host proteins relevant to interferon signaling, transcriptional regulation, stress, and the unfolded protein response. CONCLUSIONS/SIGNIFICANCE: Dengue virus manipulates cellular processes to its advantage through specific interactions with the host's protein interaction network. The interaction networks presented here provide a set of hypothesis for further experimental investigation into the DENV life cycle as well as potential therapeutic targets.

  14. Characterization of the RNA silencing suppression activity of the Ebola virus VP35 protein in plants and mammalian cells.

    Science.gov (United States)

    Zhu, Yali; Cherukuri, Nil Celebi; Jackel, Jamie N; Wu, Zetang; Crary, Monica; Buckley, Kenneth J; Bisaro, David M; Parris, Deborah S

    2012-03-01

    Ebola virus (EBOV) causes a lethal hemorrhagic fever for which there is no approved effective treatment or prevention strategy. EBOV VP35 is a virulence factor that blocks innate antiviral host responses, including the induction of and response to alpha/beta interferon. VP35 is also an RNA silencing suppressor (RSS). By inhibiting microRNA-directed silencing, mammalian virus RSSs have the capacity to alter the cellular environment to benefit replication. A reporter gene containing specific microRNA target sequences was used to demonstrate that prior expression of wild-type VP35 was able to block establishment of microRNA silencing in mammalian cells. In addition, wild-type VP35 C-terminal domain (CTD) protein fusions were shown to bind small interfering RNA (siRNA). Analysis of mutant proteins demonstrated that reporter activity in RSS assays did not correlate with their ability to antagonize double-stranded RNA (dsRNA)-activated protein kinase R (PKR) or bind siRNA. The results suggest that enhanced reporter activity in the presence of VP35 is a composite of nonspecific translational enhancement and silencing suppression. Moreover, most of the specific RSS activity in mammalian cells is RNA binding independent, consistent with VP35's proposed role in sequestering one or more silencing complex proteins. To examine RSS activity in a system without interferon, VP35 was tested in well-characterized plant silencing suppression assays. VP35 was shown to possess potent plant RSS activity, and the activities of mutant proteins correlated strongly, but not exclusively, with RNA binding ability. The results suggest the importance of VP35-protein interactions in blocking silencing in a system (mammalian) that cannot amplify dsRNA.

  15. Co-suppression of sterol-regulatory element binding protein ...

    African Journals Online (AJOL)

    Administrator

    2011-06-22

    Jun 22, 2011 ... In Arabidopsis,. At5g35220 gene being sterol regulatory element-binding protein site 2, protease and metalloendopeptidase activity were required for chloroplast development and play a role in regulation of endodermal plastid size and number that are involved in ethylene-dependent gravitropism of light-.

  16. Analysis of Tospovirus NSs Proteins in Suppression of Systemic Silencing

    NARCIS (Netherlands)

    Hedil, M.; Sterken, M.G.; Ronde, de D.; Lohuis, D.; Kormelink, R.

    2015-01-01

    RNA silencing is a sequence-specific gene regulation mechanism that in plants also acts antiviral. In order to counteract antiviral RNA silencing, viruses have evolved RNA silencing suppressors (RSS). In the case of tospoviruses, the non-structural NSs protein has been identified as the RSS.

  17. Co-suppression of sterol-regulatory element binding protein ...

    African Journals Online (AJOL)

    Administrator

    2011-06-22

    Jun 22, 2011 ... protein mediates etiolation in Arabidopsis thaliana .... (A) The scheme of At5g35220 gene and pEGAD-At5g35220; .... stem length of 42-day-old plants; root length of 5-day-old seedlings grown on MS medium; fresh weight of ...

  18. Understanding protein–protein interactions by genetic suppression

    Indian Academy of Sciences (India)

    Unknown

    Protein–protein interactions influence many cellular processes and it is increasingly being felt that even a weak and ... In a bacterial system where the complete genome sequence is available, it is an arduous ... teins (primary mutations) are useful in these studies. ... of interaction of this antibiotic with the central enzyme.

  19. Small proteins of plant-pathogenic fungi secreted during host colonization.

    NARCIS (Netherlands)

    Rep, M.

    2005-01-01

    Small proteins secreted by plant pathogenic fungi in their hosts have been implicated in disease symptom development as well as in R-gene mediated disease resistance. Characteristically, this class of proteins shows very limited phylogenetic distribution, possibly due to accelerated evolution

  20. Prediction of host - pathogen protein interactions between Mycobacterium tuberculosis and Homo sapiens using sequence motifs.

    Science.gov (United States)

    Huo, Tong; Liu, Wei; Guo, Yu; Yang, Cheng; Lin, Jianping; Rao, Zihe

    2015-03-26

    Emergence of multiple drug resistant strains of M. tuberculosis (MDR-TB) threatens to derail global efforts aimed at reigning in the pathogen. Co-infections of M. tuberculosis with HIV are difficult to treat. To counter these new challenges, it is essential to study the interactions between M. tuberculosis and the host to learn how these bacteria cause disease. We report a systematic flow to predict the host pathogen interactions (HPIs) between M. tuberculosis and Homo sapiens based on sequence motifs. First, protein sequences were used as initial input for identifying the HPIs by 'interolog' method. HPIs were further filtered by prediction of domain-domain interactions (DDIs). Functional annotations of protein and publicly available experimental results were applied to filter the remaining HPIs. Using such a strategy, 118 pairs of HPIs were identified, which involve 43 proteins from M. tuberculosis and 48 proteins from Homo sapiens. A biological interaction network between M. tuberculosis and Homo sapiens was then constructed using the predicted inter- and intra-species interactions based on the 118 pairs of HPIs. Finally, a web accessible database named PATH (Protein interactions of M. tuberculosis and Human) was constructed to store these predicted interactions and proteins. This interaction network will facilitate the research on host-pathogen protein-protein interactions, and may throw light on how M. tuberculosis interacts with its host.

  1. Comparative Proteomics Identifies Host Immune System Proteins Affected by Infection with Mycobacterium bovis.

    Directory of Open Access Journals (Sweden)

    Vladimir López

    2016-03-01

    Full Text Available Mycobacteria of the Mycobacterium tuberculosis complex (MTBC greatly impact human and animal health worldwide. The mycobacterial life cycle is complex, and the mechanisms resulting in pathogen infection and survival in host cells are not fully understood. Eurasian wild boar (Sus scrofa are natural reservoir hosts for MTBC and a model for mycobacterial infection and tuberculosis (TB. In the wild boar TB model, mycobacterial infection affects the expression of innate and adaptive immune response genes in mandibular lymph nodes and oropharyngeal tonsils, and biomarkers have been proposed as correlates with resistance to natural infection. However, the mechanisms used by mycobacteria to manipulate host immune response are not fully characterized. Our hypothesis is that the immune system proteins under-represented in infected animals, when compared to uninfected controls, are used by mycobacteria to guarantee pathogen infection and transmission. To address this hypothesis, a comparative proteomics approach was used to compare host response between uninfected (TB- and M. bovis-infected young (TB+ and adult animals with different infection status [TB lesions localized in the head (TB+ or affecting multiple organs (TB++]. The results identified host immune system proteins that play an important role in host response to mycobacteria. Calcium binding protein A9, Heme peroxidase, Lactotransferrin, Cathelicidin and Peptidoglycan-recognition protein were under-represented in TB+ animals when compared to uninfected TB- controls, but protein levels were higher as infection progressed in TB++ animals when compared to TB- and/or TB+ adult wild boar. MHCI was the only protein over-represented in TB+ adult wild boar when compared to uninfected TB- controls. The results reported here suggest that M. bovis manipulates host immune response by reducing the production of immune system proteins. However, as infection progresses, wild boar immune response recovers to

  2. Comparative Proteomics Identifies Host Immune System Proteins Affected by Infection with Mycobacterium bovis.

    Science.gov (United States)

    López, Vladimir; Villar, Margarita; Queirós, João; Vicente, Joaquín; Mateos-Hernández, Lourdes; Díez-Delgado, Iratxe; Contreras, Marinela; Alves, Paulo C; Alberdi, Pilar; Gortázar, Christian; de la Fuente, José

    2016-03-01

    Mycobacteria of the Mycobacterium tuberculosis complex (MTBC) greatly impact human and animal health worldwide. The mycobacterial life cycle is complex, and the mechanisms resulting in pathogen infection and survival in host cells are not fully understood. Eurasian wild boar (Sus scrofa) are natural reservoir hosts for MTBC and a model for mycobacterial infection and tuberculosis (TB). In the wild boar TB model, mycobacterial infection affects the expression of innate and adaptive immune response genes in mandibular lymph nodes and oropharyngeal tonsils, and biomarkers have been proposed as correlates with resistance to natural infection. However, the mechanisms used by mycobacteria to manipulate host immune response are not fully characterized. Our hypothesis is that the immune system proteins under-represented in infected animals, when compared to uninfected controls, are used by mycobacteria to guarantee pathogen infection and transmission. To address this hypothesis, a comparative proteomics approach was used to compare host response between uninfected (TB-) and M. bovis-infected young (TB+) and adult animals with different infection status [TB lesions localized in the head (TB+) or affecting multiple organs (TB++)]. The results identified host immune system proteins that play an important role in host response to mycobacteria. Calcium binding protein A9, Heme peroxidase, Lactotransferrin, Cathelicidin and Peptidoglycan-recognition protein were under-represented in TB+ animals when compared to uninfected TB- controls, but protein levels were higher as infection progressed in TB++ animals when compared to TB- and/or TB+ adult wild boar. MHCI was the only protein over-represented in TB+ adult wild boar when compared to uninfected TB- controls. The results reported here suggest that M. bovis manipulates host immune response by reducing the production of immune system proteins. However, as infection progresses, wild boar immune response recovers to limit pathogen

  3. Bactericidal Permeability-Increasing Proteins Shape Host-Microbe Interactions

    Directory of Open Access Journals (Sweden)

    Fangmin Chen

    2017-04-01

    Full Text Available We characterized bactericidal permeability-increasing proteins (BPIs of the squid Euprymna scolopes, EsBPI2 and EsBPI4. They have molecular characteristics typical of other animal BPIs, are closely related to one another, and nest phylogenetically among invertebrate BPIs. Purified EsBPIs had antimicrobial activity against the squid’s symbiont, Vibrio fischeri, which colonizes light organ crypt epithelia. Activity of both proteins was abrogated by heat treatment and coincubation with specific antibodies. Pretreatment under acidic conditions similar to those during symbiosis initiation rendered V. fischeri more resistant to the antimicrobial activity of the proteins. Immunocytochemistry localized EsBPIs to the symbiotic organ and other epithelial surfaces interacting with ambient seawater. The proteins differed in intracellular distribution. Further, whereas EsBPI4 was restricted to epithelia, EsBPI2 also occurred in blood and in a transient juvenile organ that mediates hatching. The data provide evidence that these BPIs play different defensive roles early in the life of E. scolopes, modulating interactions with the symbiont.

  4. Lactococcus lactis as host for overproduction of functional membrane proteins

    NARCIS (Netherlands)

    Kunji, ERS; Slotboom, DJ; Poolman, B

    2003-01-01

    Lactococcus lactis has many properties that are ideal for enhanced expression of membrane proteins. The organism is easy and inexpensive to culture, has a single membrane and relatively mild proteolytic activity. Methods for genetic manipulation are fully established and a tightly controlled

  5. Suppression of expression of muscle-associated proteins by PPARα in brown adipose tissue

    International Nuclear Information System (INIS)

    Tong, Yuhong; Hara, Atsushi; Komatsu, Makiko; Tanaka, Naoki; Kamijo, Yuji; Gonzalez, Frank J.; Aoyama, Toshifumi

    2005-01-01

    Peroxisome proliferator-activated receptor α (PPARα) belongs to the steroid/nuclear receptor superfamily. Two-dimensional (2D) SDS-PAGE analysis of brown adipose tissue (BAT) unexpectedly revealed six spots that were present only in PPARα-null mice. Proteomic analysis indicated that these proteins were tropomyosin-1 α chain, tropomyosin β chain, myosin regulatory light chain 2, myosin light chain 3, and parvalbumin α. Analyses of mRNA have revealed that PPARα suppressed the genes encoding these proteins in a synchronous manner in adult wild-type mice. Histological and physiological analyses of BAT showed in adult wild-type mice, a marked suppression of BAT growth concurrent with a prominent decrease in lipolytic and thermogenesis activities. These results suggest that in adult mice, PPARα functions to suppress the expression of the proteins that may be involved in the architecture of BAT, and thus may function in keeping BAT in a quiescent state

  6. A protein interaction mechanism for suppressing the mechanosensitive Piezo channels

    OpenAIRE

    Zhang, Tingxin; Chi, Shaopeng; Jiang, Fan; Zhao, Qiancheng; Xiao, Bailong

    2017-01-01

    Piezo proteins are bona fide mammalian mechanotransduction channels for various cell types including endothelial cells. The mouse Piezo1 of 2547 residues forms a three-bladed, propeller-like homo-trimer comprising a central pore-module and three propeller-structures that might serve as mechanotransduction-modules. However, the mechanogating and regulation of Piezo channels remain unclear. Here we identify the sarcoplasmic /endoplasmic-reticulum Ca2+ ATPase (SERCA), including the widely expres...

  7. Convergent evolution and mimicry of protein linear motifs in host-pathogen interactions.

    Science.gov (United States)

    Chemes, Lucía Beatriz; de Prat-Gay, Gonzalo; Sánchez, Ignacio Enrique

    2015-06-01

    Pathogen linear motif mimics are highly evolvable elements that facilitate rewiring of host protein interaction networks. Host linear motifs and pathogen mimics differ in sequence, leading to thermodynamic and structural differences in the resulting protein-protein interactions. Moreover, the functional output of a mimic depends on the motif and domain repertoire of the pathogen protein. Regulatory evolution mediated by linear motifs can be understood by measuring evolutionary rates, quantifying positive and negative selection and performing phylogenetic reconstructions of linear motif natural history. Convergent evolution of linear motif mimics is widespread among unrelated proteins from viral, prokaryotic and eukaryotic pathogens and can also take place within individual protein phylogenies. Statistics, biochemistry and laboratory models of infection link pathogen linear motifs to phenotypic traits such as tropism, virulence and oncogenicity. In vitro evolution experiments and analysis of natural sequences suggest that changes in linear motif composition underlie pathogen adaptation to a changing environment. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Prediction of interactions between viral and host proteins using supervised machine learning methods.

    Directory of Open Access Journals (Sweden)

    Ranjan Kumar Barman

    Full Text Available BACKGROUND: Viral-host protein-protein interaction plays a vital role in pathogenesis, since it defines viral infection of the host and regulation of the host proteins. Identification of key viral-host protein-protein interactions (PPIs has great implication for therapeutics. METHODS: In this study, a systematic attempt has been made to predict viral-host PPIs by integrating different features, including domain-domain association, network topology and sequence information using viral-host PPIs from VirusMINT. The three well-known supervised machine learning methods, such as SVM, Naïve Bayes and Random Forest, which are commonly used in the prediction of PPIs, were employed to evaluate the performance measure based on five-fold cross validation techniques. RESULTS: Out of 44 descriptors, best features were found to be domain-domain association and methionine, serine and valine amino acid composition of viral proteins. In this study, SVM-based method achieved better sensitivity of 67% over Naïve Bayes (37.49% and Random Forest (55.66%. However the specificity of Naïve Bayes was the highest (99.52% as compared with SVM (74% and Random Forest (89.08%. Overall, the SVM and Random Forest achieved accuracy of 71% and 72.41%, respectively. The proposed SVM-based method was evaluated on blind dataset and attained a sensitivity of 64%, specificity of 83%, and accuracy of 74%. In addition, unknown potential targets of hepatitis B virus-human and hepatitis E virus-human PPIs have been predicted through proposed SVM model and validated by gene ontology enrichment analysis. Our proposed model shows that, hepatitis B virus "C protein" binds to membrane docking protein, while "X protein" and "P protein" interacts with cell-killing and metabolic process proteins, respectively. CONCLUSION: The proposed method can predict large scale interspecies viral-human PPIs. The nature and function of unknown viral proteins (HBV and HEV, interacting partners of host

  9. Host-derived viral transporter protein for nitrogen uptake in infected marine phytoplankton

    Science.gov (United States)

    Chambouvet, Aurélie; Milner, David S.; Attah, Victoria; Terrado, Ramón; Lovejoy, Connie; Moreau, Hervé; Derelle, Évelyne; Richards, Thomas A.

    2017-01-01

    Phytoplankton community structure is shaped by both bottom–up factors, such as nutrient availability, and top–down processes, such as predation. Here we show that marine viruses can blur these distinctions, being able to amend how host cells acquire nutrients from their environment while also predating and lysing their algal hosts. Viral genomes often encode genes derived from their host. These genes may allow the virus to manipulate host metabolism to improve viral fitness. We identify in the genome of a phytoplankton virus, which infects the small green alga Ostreococcus tauri, a host-derived ammonium transporter. This gene is transcribed during infection and when expressed in yeast mutants the viral protein is located to the plasma membrane and rescues growth when cultured with ammonium as the sole nitrogen source. We also show that viral infection alters the nature of nitrogen compound uptake of host cells, by both increasing substrate affinity and allowing the host to access diverse nitrogen sources. This is important because the availability of nitrogen often limits phytoplankton growth. Collectively, these data show that a virus can acquire genes encoding nutrient transporters from a host genome and that expression of the viral gene can alter the nutrient uptake behavior of host cells. These results have implications for understanding how viruses manipulate the physiology and ecology of phytoplankton, influence marine nutrient cycles, and act as vectors for horizontal gene transfer. PMID:28827361

  10. A viral suppressor protein inhibits host RNA silencing by hooking up with Argonautes

    KAUST Repository

    Jin, Hailing; Zhu, Jian-Kang

    2010-01-01

    RNA viruses are particularly vulnerable to RNAi-based defenses in the host, and thus have evolved specific proteins, known as viral suppressors of RNA silencing (VSRs), as a counterdefense. In this issue of Genes & Development, Azevedo and colleagues (pp. 904-915) discovered that P38, the VSR of Turnip crinkle virus, uses its glycine/tryptophane (GW) motifs as an ARGONAUTE (AGO) hook to attract and disarm the host's essential effector of RNA silencing. Several GW motif-containing cellular proteins are known to be important partners of AGOs in RNA silencing effector complexes in yeast, plants, and animals. The GW motif appears to be a versatile and effective tool for regulating the activities of RNA silencing pathways, and the use of GW mimicry to compete for and inhibit host AGOs may be a strategy used by many pathogens to counteract host RNAi-based defenses. © 2010 by Cold Spring Harbor Laboratory Press.

  11. Protein nutrition governs within-host race of honey bee pathogens.

    Science.gov (United States)

    Tritschler, Manuel; Vollmann, Jutta J; Yañez, Orlando; Chejanovsky, Nor; Crailsheim, Karl; Neumann, Peter

    2017-11-08

    Multiple infections are common in honey bees, Apis mellifera, but the possible role of nutrition in this regard is poorly understood. Microsporidian infections, which are promoted by protein-fed, can negatively correlate with virus infections, but the role of protein nutrition for the microsporidian-virus interface is unknown. Here, we challenged naturally deformed wing virus - B (DWV-B) infected adult honey bee workers fed with or without pollen ( = protein) in hoarding cages, with the microsporidian Nosema ceranae. Bee mortality was recorded for 14 days and N. ceranae spore loads and DWV-B titers were quantified. Amongst the groups inoculated with N. ceranae, more spores were counted in protein-fed bees. However, N. ceranae infected bees without protein-diet had reduced longevity compared to all other groups. N. ceranae infection had no effect on protein-fed bee's longevity, whereas bees supplied only with sugar-water showed reduced survival. Our data also support that protein-feeding can have a significant negative impact on virus infections in insects. The negative correlation between N. ceranae spore loads and DWV-B titers was stronger expressed in protein-fed hosts. Proteins not only enhance survival of infected hosts, but also significantly shape the microsporidian-virus interface, probably due to increased spore production and enhanced host immunity.

  12. Pathogen Trojan Horse Delivers Bioactive Host Protein to Alter Maize Anther Cell Behavior in Situ.

    Science.gov (United States)

    van der Linde, Karina; Timofejeva, Ljudmilla; Egger, Rachel L; Ilau, Birger; Hammond, Reza; Teng, Chong; Meyers, Blake C; Doehlemann, Gunther; Walbot, Virginia

    2018-03-01

    Small proteins are crucial signals during development, host defense, and physiology. The highly spatiotemporal restricted functions of signaling proteins remain challenging to study in planta. The several month span required to assess transgene expression, particularly in flowers, combined with the uncertainties from transgene position effects and ubiquitous or overexpression, makes monitoring of spatiotemporally restricted signaling proteins lengthy and difficult. This situation could be rectified with a transient assay in which protein deployment is tightly controlled spatially and temporally in planta to assess protein functions, timing, and cellular targets as well as to facilitate rapid mutagenesis to define functional protein domains. In maize ( Zea mays ), secreted ZmMAC1 (MULTIPLE ARCHESPORIAL CELLS1) was proposed to trigger somatic niche formation during anther development by participating in a ligand-receptor module. Inspired by Homer's Trojan horse myth, we engineered a protein delivery system that exploits the secretory capabilities of the maize smut fungus Ustilago maydis , to allow protein delivery to individual cells in certain cell layers at precise time points. Pathogen-supplied ZmMAC1 cell-autonomously corrected both somatic cell division and differentiation defects in mutant Zm mac1-1 anthers. These results suggest that exploiting host-pathogen interactions may become a generally useful method for targeting host proteins to cell and tissue types to clarify cellular autonomy and to analyze steps in cell responses. © 2018 American Society of Plant Biologists. All rights reserved.

  13. Identifying potential survival strategies of HIV-1 through virus-host protein interaction networks

    Directory of Open Access Journals (Sweden)

    Boucher Charles AB

    2010-07-01

    Full Text Available Abstract Background The National Institute of Allergy and Infectious Diseases has launched the HIV-1 Human Protein Interaction Database in an effort to catalogue all published interactions between HIV-1 and human proteins. In order to systematically investigate these interactions functionally and dynamically, we have constructed an HIV-1 human protein interaction network. This network was analyzed for important proteins and processes that are specific for the HIV life-cycle. In order to expose viral strategies, network motif analysis was carried out showing reoccurring patterns in virus-host dynamics. Results Our analyses show that human proteins interacting with HIV form a densely connected and central sub-network within the total human protein interaction network. The evaluation of this sub-network for connectivity and centrality resulted in a set of proteins essential for the HIV life-cycle. Remarkably, we were able to associate proteins involved in RNA polymerase II transcription with hubs and proteasome formation with bottlenecks. Inferred network motifs show significant over-representation of positive and negative feedback patterns between virus and host. Strikingly, such patterns have never been reported in combined virus-host systems. Conclusions HIV infection results in a reprioritization of cellular processes reflected by an increase in the relative importance of transcriptional machinery and proteasome formation. We conclude that during the evolution of HIV, some patterns of interaction have been selected for resulting in a system where virus proteins preferably interact with central human proteins for direct control and with proteasomal proteins for indirect control over the cellular processes. Finally, the patterns described by network motifs illustrate how virus and host interact with one another.

  14. Comparative proteomic analysis of differentially expressed proteins in the urine of reservoir hosts of leptospirosis.

    Directory of Open Access Journals (Sweden)

    Jarlath E Nally

    Full Text Available Rattus norvegicus is a natural reservoir host for pathogenic species of Leptospira. Experimentally infected rats remain clinically normal, yet persistently excrete large numbers of leptospires from colonized renal tubules via urine, despite a specific host immune response. Whilst persistent renal colonization and shedding is facilitated in part by differential antigen expression by leptospires to evade host immune responses, there is limited understanding of kidney and urinary proteins expressed by the host that facilitates such biological equilibrium. Urine pellets were collected from experimentally infected rats shedding leptospires and compared to urine from non-infected controls spiked with in vitro cultivated leptospires for analysis by 2-D DIGE. Differentially expressed host proteins include membrane metallo endopeptidase, napsin A aspartic peptidase, vacuolar H+ATPase, kidney aminopeptidase and immunoglobulin G and A. Loa22, a virulence factor of Leptospira, as well as the GroEL, were increased in leptospires excreted in urine compared to in vitro cultivated leptospires. Urinary IgG from infected rats was specific for leptospires. Results confirm differential protein expression by both host and pathogen during chronic disease and include markers of kidney function and immunoglobulin which are potential biomarkers of infection.

  15. Synthesis of protein in host-free reticulate bodies of Chlamydia psittaci and Chlamydia trachomatis

    International Nuclear Information System (INIS)

    Hatch, T.P.; Miceli, M.; Silverman, J.A.

    1985-01-01

    Synthesis of protein by the obligate intracellular parasitic bacteria Chlamydia psittaci (6BC) and Chlamydia trachomatis (serovar L2) isolated from host cells (host-free chlamydiae) was demonstrated for the first time. Incorporation of [ 35 S]methionine and [ 35 S]cysteine into trichloroacetic acid-precipitable material by reticulate bodies of chlamydiae persisted for 2 h and was dependent upon a exogenous source of ATP, an ATP-regenerating system, and potassium or sodium ions. Magnesium ions and amino acids stimulated synthesis; chloramphenicol, rifampin, oligomycin, and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (a proton ionophore) inhibited incorporation. Ribonucleoside triphosphates (other than ATP) had little stimulatory effect. The optimum pH for host-free synthesis was between 7.0 and 7.5. The molecular weights of proteins synthesized by host-free reticulate bodies closely resembled the molecular weights of proteins synthesized by reticulate bodies in an intracellular environment, and included outer membrane proteins. Elementary bodies of chlamydiae were unable to synthesize protein even when incubated in the presence of 10 mM dithiothreitol, a reducing agent which converted the highly disulfide bond cross-linked major outer membrane protein to monomeric form

  16. Coxiella burnetii Nine Mile II proteins modulate gene expression of monocytic host cells during infection

    Directory of Open Access Journals (Sweden)

    Shaw Edward I

    2010-09-01

    Full Text Available Abstract Background Coxiella burnetii is an intracellular bacterial pathogen that causes acute and chronic disease in humans. Bacterial replication occurs within enlarged parasitophorous vacuoles (PV of eukaryotic cells, the biogenesis and maintenance of which is dependent on C. burnetii protein synthesis. These observations suggest that C. burnetii actively subverts host cell processes, however little is known about the cellular biology mechanisms manipulated by the pathogen during infection. Here, we examined host cell gene expression changes specifically induced by C. burnetii proteins during infection. Results We have identified 36 host cell genes that are specifically regulated when de novo C. burnetii protein synthesis occurs during infection using comparative microarray analysis. Two parallel sets of infected and uninfected THP-1 cells were grown for 48 h followed by the addition of chloramphenicol (CAM to 10 μg/ml in one set. Total RNA was harvested at 72 hpi from all conditions, and microarrays performed using Phalanx Human OneArray™ slides. A total of 784 (mock treated and 901 (CAM treated THP-1 genes were up or down regulated ≥2 fold in the C. burnetii infected vs. uninfected cell sets, respectively. Comparisons between the complementary data sets (using >0 fold, eliminated the common gene expression changes. A stringent comparison (≥2 fold between the separate microarrays revealed 36 host cell genes modulated by C. burnetii protein synthesis. Ontological analysis of these genes identified the innate immune response, cell death and proliferation, vesicle trafficking and development, lipid homeostasis, and cytoskeletal organization as predominant cellular functions modulated by C. burnetii protein synthesis. Conclusions Collectively, these data indicate that C. burnetii proteins actively regulate the expression of specific host cell genes and pathways. This is in addition to host cell genes that respond to the presence of the

  17. Human Antiviral Protein IFIX Suppresses Viral Gene Expression during Herpes Simplex Virus 1 (HSV-1) Infection and Is Counteracted by Virus-induced Proteasomal Degradation.

    Science.gov (United States)

    Crow, Marni S; Cristea, Ileana M

    2017-04-01

    The interferon-inducible protein X (IFIX), a member of the PYHIN family, was recently recognized as an antiviral factor against infection with herpes simplex virus 1 (HSV-1). IFIX binds viral DNA upon infection and promotes expression of antiviral cytokines. How IFIX exerts its host defense functions and whether it is inhibited by the virus remain unknown. Here, we integrated live cell microscopy, proteomics, IFIX domain characterization, and molecular virology to investigate IFIX regulation and antiviral functions during HSV-1 infection. We find that IFIX has a dynamic localization during infection that changes from diffuse nuclear and nucleoli distribution in uninfected cells to discrete nuclear puncta early in infection. This is rapidly followed by a reduction in IFIX protein levels. Indeed, using immunoaffinity purification and mass spectrometry, we define IFIX interactions during HSV-1 infection, finding an association with a proteasome subunit and proteins involved in ubiquitin-proteasome processes. Using synchronized HSV-1 infection, microscopy, and proteasome-inhibition experiments, we demonstrate that IFIX co-localizes with nuclear proteasome puncta shortly after 3 h of infection and that its pyrin domain is rapidly degraded in a proteasome-dependent manner. We further demonstrate that, in contrast to several other host defense factors, IFIX degradation is not dependent on the E3 ubiquitin ligase activity of the viral protein ICP0. However, we show IFIX degradation requires immediate-early viral gene expression, suggesting a viral host suppression mechanism. The IFIX interactome also demonstrated its association with transcriptional regulatory proteins, including the 5FMC complex. We validate this interaction using microscopy and reciprocal isolations and determine it is mediated by the IFIX HIN domain. Finally, we show IFIX suppresses immediate-early and early viral gene expression during infection. Altogether, our study demonstrates that IFIX antiviral

  18. Vp130, a chloroviral surface protein that interacts with the host Chlorella cell wall

    International Nuclear Information System (INIS)

    Onimatsu, Hideki; Sugimoto, Ichiro; Fujie, Makoto; Usami, Shoji; Yamada, Takashi

    2004-01-01

    A protein, Vp130, that interacts with the host cell wall was isolated from Chlorovirus CVK2. From its peptide sequence, the gene for Vp130 was identified on the PBCV-1 genomic sequence as an ORF combining A140R and A145R. In Vp130, the N-terminus was somehow modified and the C-terminus was occupied by 23-26 tandem repeats of a PAPK motif. In the internal region, Vp130 contained seven repeats of 70-73 amino acids, each copy of which was separated by PAPK sequences. This protein was well conserved among NC64A viruses. A recombinant rVp130N protein formed in Escherichia coli was shown not only to bind directly to the host cell wall in vitro but also to specifically bind to the host cells, as demonstrated by fluorescence microscopy. Because externally added rVp130N competed with CVK2 to bind to host cells, Vp130 is most likely to be a host-recognizing protein on the virion

  19. Structure homology and interaction redundancy for discovering virus–host protein interactions

    Science.gov (United States)

    de Chassey, Benoît; Meyniel-Schicklin, Laurène; Aublin-Gex, Anne; Navratil, Vincent; Chantier, Thibaut; André, Patrice; Lotteau, Vincent

    2013-01-01

    Virus–host interactomes are instrumental to understand global perturbations of cellular functions induced by infection and discover new therapies. The construction of such interactomes is, however, technically challenging and time consuming. Here we describe an original method for the prediction of high-confidence interactions between viral and human proteins through a combination of structure and high-quality interactome data. Validation was performed for the NS1 protein of the influenza virus, which led to the identification of new host factors that control viral replication. PMID:24008843

  20. Structure homology and interaction redundancy for discovering virus-host protein interactions.

    Science.gov (United States)

    de Chassey, Benoît; Meyniel-Schicklin, Laurène; Aublin-Gex, Anne; Navratil, Vincent; Chantier, Thibaut; André, Patrice; Lotteau, Vincent

    2013-10-01

    Virus-host interactomes are instrumental to understand global perturbations of cellular functions induced by infection and discover new therapies. The construction of such interactomes is, however, technically challenging and time consuming. Here we describe an original method for the prediction of high-confidence interactions between viral and human proteins through a combination of structure and high-quality interactome data. Validation was performed for the NS1 protein of the influenza virus, which led to the identification of new host factors that control viral replication.

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

    Science.gov (United States)

    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...

  2. Cell penetrating peptides to dissect host-pathogen protein-protein interactions in Theileria -transformed leukocytes

    KAUST Repository

    Haidar, Malak; de Laté , Perle Latré ; Kennedy, Eileen J.; Langsley, Gordon

    2017-01-01

    One powerful application of cell penetrating peptides is the delivery into cells of molecules that function as specific competitors or inhibitors of protein-protein interactions. Ablating defined protein-protein interactions is a refined way

  3. The Host E3-Ubiquitin Ligase TRIM6 Ubiquitinates the Ebola Virus VP35 Protein and Promotes Virus Replication.

    Science.gov (United States)

    Bharaj, Preeti; Atkins, Colm; Luthra, Priya; Giraldo, Maria Isabel; Dawes, Brian E; Miorin, Lisa; Johnson, Jeffrey R; Krogan, Nevan J; Basler, Christopher F; Freiberg, Alexander N; Rajsbaum, Ricardo

    2017-09-15

    Ebola virus (EBOV), a member of the Filoviridae family, is a highly pathogenic virus that causes severe hemorrhagic fever in humans and is responsible for epidemics throughout sub-Saharan, central, and West Africa. The EBOV genome encodes VP35, an important viral protein involved in virus replication by acting as an essential cofactor of the viral polymerase as well as a potent antagonist of the host antiviral type I interferon (IFN-I) system. By using mass spectrometry analysis and coimmunoprecipitation assays, we show here that VP35 is ubiquitinated on lysine 309 (K309), a residue located on its IFN antagonist domain. We also found that VP35 interacts with TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family. We recently reported that TRIM6 promotes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, to induce efficient antiviral IFN-I-mediated responses. Consistent with this notion, VP35 also associated noncovalently with polyubiquitin chains and inhibited TRIM6-mediated IFN-I induction. Intriguingly, we also found that TRIM6 enhances EBOV polymerase activity in a minigenome assay and TRIM6 knockout cells have reduced replication of infectious EBOV, suggesting that VP35 hijacks TRIM6 to promote EBOV replication through ubiquitination. Our work provides evidence that TRIM6 is an important host cellular factor that promotes EBOV replication, and future studies will focus on whether TRIM6 could be targeted for therapeutic intervention against EBOV infection. IMPORTANCE EBOV belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans and other mammals with high mortality rates (40 to 90%). Because of its high pathogenicity and lack of licensed antivirals and vaccines, EBOV is listed as a tier 1 select-agent risk group 4 pathogen. An important mechanism for the severity of EBOV infection is its suppression of innate immune responses. The EBOV VP35

  4. Exploitation of the host cell ubiquitin machinery by microbial effector proteins.

    Science.gov (United States)

    Lin, Yi-Han; Machner, Matthias P

    2017-06-15

    Pathogenic bacteria are in a constant battle for survival with their host. In order to gain a competitive edge, they employ a variety of sophisticated strategies that allow them to modify conserved host cell processes in ways that favor bacterial survival and growth. Ubiquitylation, the covalent attachment of the small modifier ubiquitin to target proteins, is such a pathway. Ubiquitylation profoundly alters the fate of a myriad of cellular proteins by inducing changes in their stability or function, subcellular localization or interaction with other proteins. Given the importance of ubiquitylation in cell development, protein homeostasis and innate immunity, it is not surprising that this post-translational modification is exploited by a variety of effector proteins from microbial pathogens. Here, we highlight recent advances in our understanding of the many ways microbes take advantage of host ubiquitylation, along with some surprising deviations from the canonical theme. The lessons learned from the in-depth analyses of these host-pathogen interactions provide a fresh perspective on an ancient post-translational modification that we thought was well understood.This article is part of a Minifocus on Ubiquitin Regulation and Function. For further reading, please see related articles: 'Mechanisms of regulation and diversification of deubiquitylating enzyme function' by Pawel Leznicki and Yogesh Kulathu ( J. Cell Sci. 130 , 1997-2006). 'Cell scientist to watch - Mads Gyrd-Hansen' ( J. Cell Sci. 130 , 1981-1983). © 2017. Published by The Company of Biologists Ltd.

  5. Jasmonate ZIM-domain (JAZ protein regulates host and nonhost pathogen-induced cell death in tomato and Nicotiana benthamiana.

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    Yasuhiro Ishiga

    Full Text Available The nonhost-specific phytotoxin coronatine (COR produced by several pathovars of Pseudomonas syringae functions as a jasmonic acid-isoleucine (JA-Ile mimic and contributes to disease development by suppressing plant defense responses and inducing reactive oxygen species in chloroplast. It has been shown that the F-box protein CORONATINE INSENSITIVE 1 (COI1 is the receptor for COR and JA-Ile. JASMONATE ZIM DOMAIN (JAZ proteins act as negative regulators for JA signaling in Arabidopsis. However, the physiological significance of JAZ proteins in P. syringae disease development and nonhost pathogen-induced hypersensitive response (HR cell death is not completely understood. In this study, we identified JAZ genes from tomato, a host plant for P. syringae pv. tomato DC3000 (Pst DC3000, and examined their expression profiles in response to COR and pathogens. Most JAZ genes were induced by COR treatment or inoculation with COR-producing Pst DC3000, but not by the COR-defective mutant DB29. Tomato SlJAZ2, SlJAZ6 and SlJAZ7 interacted with SlCOI1 in a COR-dependent manner. Using virus-induced gene silencing (VIGS, we demonstrated that SlJAZ2, SlJAZ6 and SlJAZ7 have no effect on COR-induced chlorosis in tomato and Nicotiana benthamiana. However, SlJAZ2-, SlJAZ6- and SlJAZ7-silenced tomato plants showed enhanced disease-associated cell death to Pst DC3000. Furthermore, we found delayed HR cell death in response to the nonhost pathogen Pst T1 or a pathogen-associated molecular pattern (PAMP, INF1, in SlJAZ2- and SlJAZ6-silenced N. benthamiana. These results suggest that tomato JAZ proteins regulate the progression of cell death during host and nonhost interactions.

  6. Identification of Proteins Bound to Dengue Viral RNA In Vivo Reveals New Host Proteins Important for Virus Replication

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    Stacia L. Phillips

    2016-01-01

    Full Text Available Dengue virus is the most prevalent cause of arthropod-borne infection worldwide. Due to the limited coding capacity of the viral genome and the complexity of the viral life cycle, host cell proteins play essential roles throughout the course of viral infection. Host RNA-binding proteins mediate various aspects of virus replication through their physical interactions with viral RNA. Here we describe a technique designed to identify such interactions in the context of infected cells using UV cross-linking followed by antisense-mediated affinity purification and mass spectrometry. Using this approach, we identified interactions, several of them novel, between host proteins and dengue viral RNA in infected Huh7 cells. Most of these interactions were subsequently validated using RNA immunoprecipitation. Using small interfering RNA (siRNA-mediated gene silencing, we showed that more than half of these host proteins are likely involved in regulating virus replication, demonstrating the utility of this method in identifying biologically relevant interactions that may not be identified using traditional in vitro approaches.

  7. On the involvement of host proteins in Cowpea mosaic virus intercellular spread

    NARCIS (Netherlands)

    Hollander, den P.W.

    2014-01-01

    Abstract of thesis Paulus den Hollander entitled “On the involvement of host proteins in Cowpea mosaic virus intercellular spread”.

    Defence: 18th of November 13.30 h

    Abstract

    Intercellular spread of Cowpea mosaic virus (CPMV) occurs via movement

  8. Heterologously expressed Staphylococcus aureus fibronectin-binding proteins are sufficient for invasion of host cells

    NARCIS (Netherlands)

    Sinha, B; Francois, P; Que, Y A; Hussain, M; Heilmann, C; Moreillon, P; Lew, D; Krause, K H; Peters, Georg; Herrmann, M

    2000-01-01

    Staphylococcus aureus invasion of mammalian cells, including epithelial, endothelial, and fibroblastic cells, critically depends on fibronectin bridging between S. aureus fibronectin-binding proteins (FnBPs) and the host fibronectin receptor integrin alpha(5)beta(1) (B. Sinha et al., Cell.

  9. Identification of host cell proteins which interact with herpes simplex virus type 1 tegument protein pUL37.

    Science.gov (United States)

    Kelly, Barbara J; Diefenbach, Eve; Fraefel, Cornel; Diefenbach, Russell J

    2012-01-20

    The herpes simplex virus type 1 (HSV-1) structural tegument protein pUL37, which is conserved across the Herpesviridae family, is known to be essential for secondary envelopment during the egress of viral particles. To shed light on additional roles of pUL37 during viral replication a yeast two-hybrid screen of a human brain cDNA library was undertaken. This screen identified ten host cell proteins as potential pUL37 interactors. One of the interactors, serine threonine kinase TAOK3, was subsequently confirmed to interact with pUL37 using an in vitro pulldown assay. Such host cell/pUL37 interactions provide further insights into the multifunctional role of this herpesviral tegument protein. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. Bacillus anthracis Overcomes an Amino Acid Auxotrophy by Cleaving Host Serum Proteins

    Science.gov (United States)

    Terwilliger, Austen; Swick, Michelle C.; Pflughoeft, Kathryn J.; Pomerantsev, Andrei; Lyons, C. Rick; Koehler, Theresa M.

    2015-01-01

    ABSTRACT Bacteria sustain an infection by acquiring nutrients from the host to support replication. The host sequesters these nutrients as a growth-restricting strategy, a concept termed “nutritional immunity.” Historically, the study of nutritional immunity has centered on iron uptake because many bacteria target hemoglobin, an abundant circulating protein, as an iron source. Left unresolved are the mechanisms that bacteria use to attain other nutrients from host sources, including amino acids. We employed a novel medium designed to mimic the chemical composition of human serum, and we show here that Bacillus anthracis, the causative agent of anthrax disease, proteolyzes human hemoglobin to liberate essential amino acids which enhance its growth. This property can be traced to the actions of InhA1, a secreted metalloprotease, and extends to at least three other serum proteins, including serum albumin. The results suggest that we must also consider proteolysis of key host proteins to be a way for bacterial pathogens to attain essential nutrients, and we provide an experimental framework to determine the host and bacterial factors involved in this process. IMPORTANCE The mechanisms by which bacterial pathogens acquire nutrients during infection are poorly understood. Here we used a novel defined medium that approximates the chemical composition of human blood serum, blood serum mimic (BSM), to better model the nutritional environment that pathogens encounter during bacteremia. Removing essential amino acids from BSM revealed that two of the most abundant proteins in blood—hemoglobin and serum albumin—can satiate the amino acid requirement for Bacillus anthracis, the causative agent of anthrax. We further demonstrate that hemoglobin is proteolyzed by the secreted protease InhA1. These studies highlight that common blood proteins can be a nutrient source for bacteria. They also challenge the historical view that hemoglobin is solely an iron source for

  11. Tailored protein encapsulation into a DNA host using geometrically organized supramolecular interactions

    Science.gov (United States)

    Sprengel, Andreas; Lill, Pascal; Stegemann, Pierre; Bravo-Rodriguez, Kenny; Schöneweiß, Elisa-C.; Merdanovic, Melisa; Gudnason, Daniel; Aznauryan, Mikayel; Gamrad, Lisa; Barcikowski, Stephan; Sanchez-Garcia, Elsa; Birkedal, Victoria; Gatsogiannis, Christos; Ehrmann, Michael; Saccà, Barbara

    2017-02-01

    The self-organizational properties of DNA have been used to realize synthetic hosts for protein encapsulation. However, current strategies of DNA-protein conjugation still limit true emulation of natural host-guest systems, whose formation relies on non-covalent bonds between geometrically matching interfaces. Here we report one of the largest DNA-protein complexes of semisynthetic origin held in place exclusively by spatially defined supramolecular interactions. Our approach is based on the decoration of the inner surface of a DNA origami hollow structure with multiple ligands converging to their corresponding binding sites on the protein surface with programmable symmetry and range-of-action. Our results demonstrate specific host-guest recognition in a 1:1 stoichiometry and selectivity for the guest whose size guarantees sufficient molecular diffusion preserving short intermolecular distances. DNA nanocontainers can be thus rationally designed to trap single guest molecules in their native form, mimicking natural strategies of molecular recognition and anticipating a new method of protein caging.

  12. Infectious Bursal Disease Virus-Host Interactions: Multifunctional Viral Proteins that Perform Multiple and Differing Jobs

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    Yao Qin

    2017-01-01

    Full Text Available Infectious bursal disease (IBD is an acute, highly contagious and immunosuppressive poultry disease caused by IBD virus (IBDV. The consequent immunosuppression increases susceptibility to other infectious diseases and the risk of subsequent vaccination failure as well. Since the genome of IBDV is relatively small, it has a limited number of proteins inhibiting the cellular antiviral responses and acting as destroyers to the host defense system. Thus, these virulence factors must be multifunctional in order to complete the viral replication cycle in a host cell. Insights into the roles of these viral proteins along with their multiple cellular targets in different pathways will give rise to a rational design for safer and effective vaccines. Here we summarize the recent findings that focus on the virus–cell interactions during IBDV infection at the protein level.

  13. Hepatitis C Virus Frameshift/Alternate Reading Frame Protein Suppresses Interferon Responses Mediated by Pattern Recognition Receptor Retinoic-Acid-Inducible Gene-I.

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    Seung Bum Park

    Full Text Available Hepatitis C virus (HCV actively evades host interferon (IFN responses but the mechanisms of how it does so are not completely understood. In this study, we present evidence for an HCV factor that contributes to the suppression of retinoic-acid-inducible gene-I (RIG-I-mediated IFN induction. Expression of frameshift/alternate reading frame protein (F/ARFP from HCV -2/+1 frame in Huh7 hepatoma cells suppressed type I IFN responses stimulated by HCV RNA pathogen-associated molecular pattern (PAMP and poly(IC. The suppression occurred independently of other HCV factors; and activation of interferon stimulated genes, TNFα, IFN-λ1, and IFN-λ2/3 was likewise suppressed by HCV F/ARFP. Point mutations in the full-length HCV sequence (JFH1 genotype 2a strain were made to introduce premature termination codons in the -2/+1 reading frame coding for F/ARFP while preserving the original reading frame, which enhanced IFNα and IFNβ induction by HCV. The potentiation of IFN response by the F/ARFP mutations was diminished in Huh7.5 cells, which already have a defective RIG-I, and by decreasing RIG-I expression in Huh7 cells. Furthermore, adding F/ARFP back via trans-complementation suppressed IFN induction in the F/ARFP mutant. The F/ARFP mutants, on the other hand, were not resistant to exogenous IFNα. Finally, HCV-infected human liver samples showed significant F/ARFP antibody reactivity, compared to HCV-uninfected control livers. Therefore, HCV F/ARFP likely cooperates with other viral factors to suppress type I and III IFN induction occurring through the RIG-I signaling pathway. This study identifies a novel mechanism of pattern recognition receptor modulation by HCV and suggests a biological function of the HCV alternate reading frame in the modulation of host innate immunity.

  14. Purification of infectious human herpesvirus 6A virions and association of host cell proteins

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    Garoff Henrik

    2007-10-01

    Full Text Available Abstract Background Viruses that are incorporating host cell proteins might trigger autoimmune diseases. It is therefore of interest to identify possible host proteins associated with viruses, especially for enveloped viruses that have been suggested to play a role in autoimmune diseases, like human herpesvirus 6A (HHV-6A in multiple sclerosis (MS. Results We have established a method for rapid and morphology preserving purification of HHV-6A virions, which in combination with parallel analyses with background control material released from mock-infected cells facilitates qualitative and quantitative investigations of the protein content of HHV-6A virions. In our iodixanol gradient purified preparation, we detected high levels of viral DNA by real-time PCR and viral proteins by metabolic labelling, silver staining and western blots. In contrast, the background level of cellular contamination was low in the purified samples as demonstrated by the silver staining and metabolic labelling analyses. Western blot analyses showed that the cellular complement protein CD46, the receptor for HHV-6A, is associated with the purified and infectious virions. Also, the cellular proteins clathrin, ezrin and Tsg101 are associated with intact HHV-6A virions. Conclusion Cellular proteins are associated with HHV-6A virions. The relevance of the association in disease and especially in autoimmunity will be further investigated.

  15. Antiviral RNA silencing suppression activity of Tomato spotted wilt virus NSs protein.

    Science.gov (United States)

    Ocampo Ocampo, T; Gabriel Peralta, S M; Bacheller, N; Uiterwaal, S; Knapp, A; Hennen, A; Ochoa-Martinez, D L; Garcia-Ruiz, H

    2016-06-17

    In addition to regulating gene expression, RNA silencing is an essential antiviral defense system in plants. Triggered by double-stranded RNA, silencing results in degradation or translational repression of target transcripts. Viruses are inducers and targets of RNA silencing. To condition susceptibility, most plant viruses encode silencing suppressors that interfere with this process, such as the Tomato spotted wilt virus (TSWV) NSs protein. The mechanism by which NSs suppresses RNA silencing and its role in viral infection and movement remain to be determined. We cloned NSs from the Hawaii isolate of TSWV and using two independent assays show for the first time that this protein restored pathogenicity and supported the formation of local infection foci by suppressor-deficient Turnip mosaic virus and Turnip crinkle virus. Demonstrating the suppression of RNA silencing directed against heterologous viruses establishes the foundation to determine the means used by NSs to block this antiviral process.

  16. Host cell subversion by Toxoplasma GRA16, an exported dense granule protein that targets the host cell nucleus and alters gene expression.

    Science.gov (United States)

    Bougdour, Alexandre; Durandau, Eric; Brenier-Pinchart, Marie-Pierre; Ortet, Philippe; Barakat, Mohamed; Kieffer, Sylvie; Curt-Varesano, Aurélie; Curt-Bertini, Rose-Laurence; Bastien, Olivier; Coute, Yohann; Pelloux, Hervé; Hakimi, Mohamed-Ali

    2013-04-17

    After invading host cells, Toxoplasma gondii multiplies within a parasitophorous vacuole (PV) that is maintained by parasite proteins secreted from organelles called dense granules. Most dense granule proteins remain within the PV, and few are known to access the host cell cytosol. We identify GRA16 as a dense granule protein that is exported through the PV membrane and reaches the host cell nucleus, where it positively modulates genes involved in cell-cycle progression and the p53 tumor suppressor pathway. GRA16 binds two host enzymes, the deubiquitinase HAUSP and PP2A phosphatase, which exert several functions, including regulation of p53 and the cell cycle. GRA16 alters p53 levels in a HAUSP-dependent manner and induces nuclear translocation of the PP2A holoenzyme. Additionally, certain GRA16-deficient strains exhibit attenuated virulence, indicating the importance of these host alterations in pathogenesis. Therefore, GRA16 represents a potentially emerging subfamily of exported dense granule proteins that modulate host function. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. Pathogenic Leptospires Modulate Protein Expression and Post-translational Modifications in Response to Mammalian Host Signals.

    Science.gov (United States)

    Nally, Jarlath E; Grassmann, Andre A; Planchon, Sébastien; Sergeant, Kjell; Renaut, Jenny; Seshu, Janakiram; McBride, Alan J; Caimano, Melissa J

    2017-01-01

    Pathogenic species of Leptospira cause leptospirosis, a bacterial zoonotic disease with a global distribution affecting over one million people annually. Reservoir hosts of leptospirosis, including rodents, dogs, and cattle, exhibit little to no signs of disease but shed large numbers of organisms in their urine. Transmission occurs when mucosal surfaces or abraded skin come into contact with infected urine or urine-contaminated water or soil. Whilst little is known about how Leptospira adapt to and persist within a reservoir host, in vitro studies suggest that leptospires alter their transcriptomic and proteomic profiles in response to environmental signals encountered during mammalian infection. We applied the dialysis membrane chamber (DMC) peritoneal implant model to compare the whole cell proteome of in vivo derived leptospires with that of leptospires cultivated in vitro at 30°C and 37°C by 2-dimensional difference in-gel electrophoresis (2-D DIGE). Of 1,735 protein spots aligned across 9 2-D DIGE gels, 202 protein spots were differentially expressed ( p 1.25 or expressed proteins were excised for identification by mass spectrometry. Data are available via ProteomeXchange with identifier PXD006995. The greatest differences were detected when DMC-cultivated leptospires were compared with IV30- or IV37-cultivated leptospires, including the increased expression of multiple isoforms of Loa22, a known virulence factor. Unexpectedly, 20 protein isoforms of LipL32 and 7 isoforms of LipL41 were uniformly identified by DIGE as differentially expressed, suggesting that unique post-translational modifications (PTMs) are operative in response to mammalian host conditions. To test this hypothesis, a rat model of persistent renal colonization was used to isolate leptospires directly from the urine of experimentally infected rats. Comparison of urinary derived leptospires to IV30 leptospires by 2-D immunoblotting confirmed that modification of proteins with

  18. Pathogenic Leptospires Modulate Protein Expression and Post-translational Modifications in Response to Mammalian Host Signals

    Directory of Open Access Journals (Sweden)

    Jarlath E. Nally

    2017-08-01

    Full Text Available Pathogenic species of Leptospira cause leptospirosis, a bacterial zoonotic disease with a global distribution affecting over one million people annually. Reservoir hosts of leptospirosis, including rodents, dogs, and cattle, exhibit little to no signs of disease but shed large numbers of organisms in their urine. Transmission occurs when mucosal surfaces or abraded skin come into contact with infected urine or urine-contaminated water or soil. Whilst little is known about how Leptospira adapt to and persist within a reservoir host, in vitro studies suggest that leptospires alter their transcriptomic and proteomic profiles in response to environmental signals encountered during mammalian infection. We applied the dialysis membrane chamber (DMC peritoneal implant model to compare the whole cell proteome of in vivo derived leptospires with that of leptospires cultivated in vitro at 30°C and 37°C by 2-dimensional difference in-gel electrophoresis (2-D DIGE. Of 1,735 protein spots aligned across 9 2-D DIGE gels, 202 protein spots were differentially expressed (p < 0.05, fold change >1.25 or < −1.25 across all three conditions. Differentially expressed proteins were excised for identification by mass spectrometry. Data are available via ProteomeXchange with identifier PXD006995. The greatest differences were detected when DMC-cultivated leptospires were compared with IV30- or IV37-cultivated leptospires, including the increased expression of multiple isoforms of Loa22, a known virulence factor. Unexpectedly, 20 protein isoforms of LipL32 and 7 isoforms of LipL41 were uniformly identified by DIGE as differentially expressed, suggesting that unique post-translational modifications (PTMs are operative in response to mammalian host conditions. To test this hypothesis, a rat model of persistent renal colonization was used to isolate leptospires directly from the urine of experimentally infected rats. Comparison of urinary derived leptospires to IV30

  19. Questiomycin A stimulates sorafenib-induced cell death via suppression of glucose-regulated protein 78.

    Science.gov (United States)

    Machihara, Kayo; Tanaka, Hidenori; Hayashi, Yoshihiro; Murakami, Ichiro; Namba, Takushi

    2017-10-07

    Hepatocellular carcinoma (HCC) is one of the most difficult cancers to treat owing to the lack of effective chemotherapeutic methods. Sorafenib, the first-line and only available treatment for HCC, extends patient overall survival by several months, with a response rate below 10%. Thus, the identification of an agent that enhances the anticancer effect of sorafenib is critical for the development of therapeutic options for HCC. Endoplasmic reticulum (ER) stress response is one of the methods of sorafenib-induced cell death. Here we report that questiomycin A suppresses expression of GRP78, a cell-protective ER chaperone protein. Analysis of the molecular mechanisms of questiomycin A revealed that this compound stimulated GRP78 protein degradation in an ER stress response-independent manner. Cotreatment with sorafenib and questiomycin A suppressed GRP78 protein expression, which is essential for the stimulation of sorafenib-induced cell death. Moreover, our in vivo study demonstrated that the coadministration of sorafenib and questiomycin A suppressed tumor formation in HCC-induced xenograft models. These results suggest that cotreatment with sorafenib and questiomycin A is a novel therapeutic strategy for HCC by enhancing sorafenib-dependent ER stress-induced cell death, and downregulation of GRP78 is a new target for the stimulation of the therapeutic effects of sorafenib in HCC. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Genome-wide RNAi screen identifies novel host proteins required for alphavirus entry.

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    Yaw Shin Ooi

    Full Text Available The enveloped alphaviruses include important and emerging human pathogens such as Chikungunya virus and Eastern equine encephalitis virus. Alphaviruses enter cells by clathrin-mediated endocytosis, and exit by budding from the plasma membrane. While there has been considerable progress in defining the structure and function of the viral proteins, relatively little is known about the host factors involved in alphavirus infection. We used a genome-wide siRNA screen to identify host factors that promote or inhibit alphavirus infection in human cells. Fuzzy homologue (FUZ, a protein with reported roles in planar cell polarity and cilia biogenesis, was required for the clathrin-dependent internalization of both alphaviruses and the classical endocytic ligand transferrin. The tetraspanin membrane protein TSPAN9 was critical for the efficient fusion of low pH-triggered virus with the endosome membrane. FUZ and TSPAN9 were broadly required for infection by the alphaviruses Sindbis virus, Semliki Forest virus, and Chikungunya virus, but were not required by the structurally-related flavivirus Dengue virus. Our results highlight the unanticipated functions of FUZ and TSPAN9 in distinct steps of alphavirus entry and suggest novel host proteins that may serve as targets for antiviral therapy.

  1. Mechanisms of Host-Pathogen Protein Complex Formation and Bacterial Immune Evasion of Streptococcus suis Protein Fhb.

    Science.gov (United States)

    Li, Xueqin; Liu, Peng; Gan, Shuzhen; Zhang, Chunmao; Zheng, Yuling; Jiang, Yongqiang; Yuan, Yuan

    2016-08-12

    Streptococcus suis serotype 2 (S. suis 2)-induced sepsis and meningitis are often accompanied by bacteremia. The evasion of polymorphonuclear leukocyte-mediated phagocytic clearance is central to the establishment of bacteremia caused by S. suis 2 and is facilitated by the ability of factor H (FH)-binding protein (Fhb) to bind FH on the bacterial surface, thereby impeding alternative pathway complement activation and phagocytic clearance. Here, C3b/C3d was found to bind to Fhb, along with FH, forming a large immune complex. The formation of this immune complex was mediated by domain II of Fhb via electrostatic and hydrophobic interactions, which, to our knowledge, is a new type of interaction. Interestingly, Fhb was found to be associated with the cell envelope and also present in the culture supernatant, where secreted Fhb inhibited complement activation via interactions with domain II, thereby enhancing antiphagocytic clearance by polymorphonuclear leukocytes. Thus, Fhb is a multifunctional bacterial protein, which binds host complement component C3 as well as FH and interferes with innate immune recognition in a secret protein manner. S. suis 2 therefore appears to have developed a new strategy to combat host innate immunity and enhance survival in host blood. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Mechanisms of Host-Pathogen Protein Complex Formation and Bacterial Immune Evasion of Streptococcus suis Protein Fhb*

    Science.gov (United States)

    Li, Xueqin; Liu, Peng; Gan, Shuzhen; Zhang, Chunmao; Zheng, Yuling; Jiang, Yongqiang; Yuan, Yuan

    2016-01-01

    Streptococcus suis serotype 2 (S. suis 2)-induced sepsis and meningitis are often accompanied by bacteremia. The evasion of polymorphonuclear leukocyte-mediated phagocytic clearance is central to the establishment of bacteremia caused by S. suis 2 and is facilitated by the ability of factor H (FH)-binding protein (Fhb) to bind FH on the bacterial surface, thereby impeding alternative pathway complement activation and phagocytic clearance. Here, C3b/C3d was found to bind to Fhb, along with FH, forming a large immune complex. The formation of this immune complex was mediated by domain II of Fhb via electrostatic and hydrophobic interactions, which, to our knowledge, is a new type of interaction. Interestingly, Fhb was found to be associated with the cell envelope and also present in the culture supernatant, where secreted Fhb inhibited complement activation via interactions with domain II, thereby enhancing antiphagocytic clearance by polymorphonuclear leukocytes. Thus, Fhb is a multifunctional bacterial protein, which binds host complement component C3 as well as FH and interferes with innate immune recognition in a secret protein manner. S. suis 2 therefore appears to have developed a new strategy to combat host innate immunity and enhance survival in host blood. PMID:27342778

  3. Aspirin augments the expression of Adenomatous Polyposis Coli protein by suppression of IKKβ

    International Nuclear Information System (INIS)

    Ashida, Noboru; Kishihata, Masako; Tien, Dat Nguyen; Kamei, Kaeko; Kimura, Takeshi; Yokode, Masayuki

    2014-01-01

    Highlights: • Clinical studies revealed aspirin inhibits cancer, but the mechanism is not known. • Adenomatous Polyposis Coli (APC) is a well-known tumor-suppressing gene. • We found aspirin up-regulates the protein of APC. • Aspirin suppressed the expression of IKKβ, an essential kinase in NFκB activation. • The deletion of IKKβ significantly increases the expression of APC protein. - Abstract: Aspirin has been widely used as analgesic, antipyretic and anti-inflammatory medicine for long. In addition to these traditional effects, clinical studies suggest that aspirin can protect against cancer, but its mechanism has not been explored. To unveil it, we identified the proteins up- or down-regulated after incubation with aspirin by using proteomics analysis with Nano-flow LC/MALDI-TOF system. Interestingly, the analysis identified the protein of Adenomatous Polyposis Coli (APC) as one of the most up-regulated protein. APC regulates cell proliferation or angiogenesis, and is widely known as a tumor-suppressing gene which can cause colorectal cancer when it is mutated. Western blots confirmed this result, and real-time PCR indicated it is transcriptionally regulated. We further tried to elucidate the molecular mechanism with focusing on IKKβ. IKKβ is the essential kinase in activation of nuclear factor-kappa B (NF-κB), major transcriptional factors that regulate genes responsible for inflammation or immune response. Previous reports indicated that aspirin specifically inhibits IKKβ activity, and constitutively active form of IKKβ accelerates APC loss. We found that aspirin suppressed the expression of IKKβ, and the deletion of IKKβ by siRNA increases the expression of APC in HEK294 cells. Finally, we observed similar effects of aspirin in human umbilical vein endothelial cells. Taken together, these results reveal that aspirin up-regulates the expression of APC via the suppression of IKKβ. This can be a mechanism how aspirin prevents cancer at

  4. Aspirin augments the expression of Adenomatous Polyposis Coli protein by suppression of IKKβ

    Energy Technology Data Exchange (ETDEWEB)

    Ashida, Noboru, E-mail: nashida@kuhp.kyoto-u.ac.jp [Department of Clinical Innovative Medicine, Institute for Advancement of Clinical and Translational Science, Faculty of Medicine, Kyoto University, Kyoto (Japan); Kishihata, Masako [Department of Clinical Innovative Medicine, Institute for Advancement of Clinical and Translational Science, Faculty of Medicine, Kyoto University, Kyoto (Japan); Tien, Dat Nguyen [Department of Clinical Innovative Medicine, Institute for Advancement of Clinical and Translational Science, Faculty of Medicine, Kyoto University, Kyoto (Japan); Department of Biomolecular Engineering, Kyoto Institute of Technology, Kyoto (Japan); Kamei, Kaeko [Department of Biomolecular Engineering, Kyoto Institute of Technology, Kyoto (Japan); Kimura, Takeshi [Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto (Japan); Yokode, Masayuki [Department of Clinical Innovative Medicine, Institute for Advancement of Clinical and Translational Science, Faculty of Medicine, Kyoto University, Kyoto (Japan)

    2014-04-04

    Highlights: • Clinical studies revealed aspirin inhibits cancer, but the mechanism is not known. • Adenomatous Polyposis Coli (APC) is a well-known tumor-suppressing gene. • We found aspirin up-regulates the protein of APC. • Aspirin suppressed the expression of IKKβ, an essential kinase in NFκB activation. • The deletion of IKKβ significantly increases the expression of APC protein. - Abstract: Aspirin has been widely used as analgesic, antipyretic and anti-inflammatory medicine for long. In addition to these traditional effects, clinical studies suggest that aspirin can protect against cancer, but its mechanism has not been explored. To unveil it, we identified the proteins up- or down-regulated after incubation with aspirin by using proteomics analysis with Nano-flow LC/MALDI-TOF system. Interestingly, the analysis identified the protein of Adenomatous Polyposis Coli (APC) as one of the most up-regulated protein. APC regulates cell proliferation or angiogenesis, and is widely known as a tumor-suppressing gene which can cause colorectal cancer when it is mutated. Western blots confirmed this result, and real-time PCR indicated it is transcriptionally regulated. We further tried to elucidate the molecular mechanism with focusing on IKKβ. IKKβ is the essential kinase in activation of nuclear factor-kappa B (NF-κB), major transcriptional factors that regulate genes responsible for inflammation or immune response. Previous reports indicated that aspirin specifically inhibits IKKβ activity, and constitutively active form of IKKβ accelerates APC loss. We found that aspirin suppressed the expression of IKKβ, and the deletion of IKKβ by siRNA increases the expression of APC in HEK294 cells. Finally, we observed similar effects of aspirin in human umbilical vein endothelial cells. Taken together, these results reveal that aspirin up-regulates the expression of APC via the suppression of IKKβ. This can be a mechanism how aspirin prevents cancer at

  5. Mining Host-Pathogen Protein Interactions to Characterize Burkholderia mallei Infectivity Mechanisms

    Science.gov (United States)

    2015-03-04

    the cytoskeleton, in lysosomes , and in the nuclear lumen. These results were consistent with the experimentally observed pathogen interference with...RESEARCH ARTICLE Mining Host- Pathogen Protein Interactions to Characterize Burkholderia mallei Infectivity Mechanisms Vesna Memišević1, Nela...Bacteriology Division, U.S. Army Medical Research Institute of Infectious Diseases , Fort Detrick, Maryland, United States of America * jaques.reifman.civ

  6. Stealth proteins: in silico identification of a novel protein family rendering bacterial pathogens invisible to host immune defense.

    Directory of Open Access Journals (Sweden)

    Peter Sperisen

    2005-11-01

    Full Text Available There are a variety of bacterial defense strategies to survive in a hostile environment. Generation of extracellular polysaccharides has proved to be a simple but effective strategy against the host's innate immune system. A comparative genomics approach led us to identify a new protein family termed Stealth, most likely involved in the synthesis of extracellular polysaccharides. This protein family is characterized by a series of domains conserved across phylogeny from bacteria to eukaryotes. In bacteria, Stealth (previously characterized as SacB, XcbA, or WefC is encoded by subsets of strains mainly colonizing multicellular organisms, with evidence for a protective effect against the host innate immune defense. More specifically, integrating all the available information about Stealth proteins in bacteria, we propose that Stealth is a D-hexose-1-phosphoryl transferase involved in the synthesis of polysaccharides. In the animal kingdom, Stealth is strongly conserved across evolution from social amoebas to simple and complex multicellular organisms, such as Dictyostelium discoideum, hydra, and human. Based on the occurrence of Stealth in most Eukaryotes and a subset of Prokaryotes together with its potential role in extracellular polysaccharide synthesis, we propose that metazoan Stealth functions to regulate the innate immune system. Moreover, there is good reason to speculate that the acquisition and spread of Stealth could be responsible for future epidemic outbreaks of infectious diseases caused by a large variety of eubacterial pathogens. Our in silico identification of a homologous protein in the human host will help to elucidate the causes of Stealth-dependent virulence. At a more basic level, the characterization of the molecular and cellular function of Stealth proteins may shed light on fundamental mechanisms of innate immune defense against microbial invasion.

  7. Stealth Proteins: In Silico Identification of a Novel Protein Family Rendering Bacterial Pathogens Invisible to Host Immune Defense.

    Directory of Open Access Journals (Sweden)

    2005-11-01

    Full Text Available There are a variety of bacterial defense strategies to survive in a hostile environment. Generation of extracellular polysaccharides has proved to be a simple but effective strategy against the host's innate immune system. A comparative genomics approach led us to identify a new protein family termed Stealth, most likely involved in the synthesis of extracellular polysaccharides. This protein family is characterized by a series of domains conserved across phylogeny from bacteria to eukaryotes. In bacteria, Stealth (previously characterized as SacB, XcbA, or WefC is encoded by subsets of strains mainly colonizing multicellular organisms, with evidence for a protective effect against the host innate immune defense. More specifically, integrating all the available information about Stealth proteins in bacteria, we propose that Stealth is a D-hexose-1-phosphoryl transferase involved in the synthesis of polysaccharides. In the animal kingdom, Stealth is strongly conserved across evolution from social amoebas to simple and complex multicellular organisms, such as Dictyostelium discoideum, hydra, and human. Based on the occurrence of Stealth in most Eukaryotes and a subset of Prokaryotes together with its potential role in extracellular polysaccharide synthesis, we propose that metazoan Stealth functions to regulate the innate immune system. Moreover, there is good reason to speculate that the acquisition and spread of Stealth could be responsible for future epidemic outbreaks of infectious diseases caused by a large variety of eubacterial pathogens. Our in silico identification of a homologous protein in the human host will help to elucidate the causes of Stealth-dependent virulence. At a more basic level, the characterization of the molecular and cellular function of Stealth proteins may shed light on fundamental mechanisms of innate immune defense against microbial invasion.

  8. Cell viability and protein turnover in nongrowing Bacillus megaterium at sporulation suppressing temperature.

    Science.gov (United States)

    Kucerová, H; Strnadová, M; Ludvík, J; Chaloupka, J

    1999-01-01

    In Bacillus megaterium, a temperature that suppresses sporulation (43 degrees C) only slightly exceeds both the optimum growth temperature and the temperature still permitting sporulation (40-41 degrees C). Here we show that, when cells grown at 35 degrees C and transferred to a sporulation medium, were subjected to shifts between 35 degrees C and the sporulation suppressing temperature (SST, 43 degrees C), their development and proteolytic activities were deeply affected. During the reversible sporulation phase that took place at 35 degrees C for 2-3 h (T2-T3), the cells developed forespores and their protein turnover was characterized by degradation of short-lived proteins and proteins made accessible to the proteolytic attack because of starvation. During the following irreversible sporulation phase refractile heat-resistant spores appeared at T4-T5. Protein turnover rate increased again after T2 and up to T8 60-70% prelabelled proteins were degraded. The SST suppressed sporulation at its beginning; at T3 no asymmetric septa were observed and the amount of heat-resistant spores at T8 was by 4-5 orders lower than at 35 degrees C. However, the cells remained viable and were able to sporulate when transferred to a lower temperature. Protein degradation was increased up to T3 but then its velocity sharply dropped and the amount of degraded protein at T8 corresponded to slightly more than one-half of that found at 35 degrees C. The cytoplasmic proteolytic activity was enhanced but the activity in the membrane fraction was decreased. When a temperature shift to SST was applied at the beginning of the irreversible sporulation phase (T2.5), the sporulation process was impaired. A portion of forespores lyzed, the others were able to complete their development but most spores were not heat-resistant and their coats showed defects. Protein degradation increased again because an effective proteolytic system was developed during the reversible sporulation phase but the

  9. Viroids: how to infect a host and cause disease without encoding proteins.

    Science.gov (United States)

    Navarro, Beatriz; Gisel, Andreas; Rodio, Maria-Elena; Delgado, Sonia; Flores, Ricardo; Di Serio, Francesco

    2012-07-01

    Despite being composed by a single-stranded, circular, non-protein-coding RNA of just 246-401 nucleotides (nt), viroids can incite in their host plants symptoms similar to those caused by DNA and RNA viruses, which have genomes at least 20-fold bigger and encode proteins. On the other hand, certain non-protein-coding plant satellite RNAs display structural similarities with viroids but for replication and transmission they need to parasitize specific helper viruses (modifying concomitantly the symptoms they induce). While phenotypic alterations accompanying infection by viruses may partly result from expressing the proteins they code for, how the non-protein-coding viroids (and satellite RNAs) cause disease remains a conundrum. Initial ideas on viroid pathogenesis focused on a direct interaction of the genomic RNA with host proteins resulting in their malfunction. With the advent of RNA silencing, it was alternatively proposed that symptoms could be produced by viroid-derived small RNAs (vd-sRNAs) -generated by the host defensive machinery- targeting specific host mRNA or DNA sequences for post-transcriptional or transcriptional gene silencing, respectively, a hypothesis that could also explain pathogenesis of non-protein-coding satellite RNAs. Evidence sustaining this view has been circumstantial, but recent data provide support for it in two cases: i) the yellow symptoms associated with a specific satellite RNA result from a 22-nt small RNA (derived from the 24-nt fragment of the satellite genome harboring the pathogenic determinant), which is complementary to a segment of the mRNA of the chlorophyll biosynthetic gene CHLI and targets it for cleavage by the RNA silencing machinery, and ii) two 21-nt vd-sRNAS containing the pathogenic determinant of the albino phenotype induced by a chloroplast-replicating viroid target for cleavage the mRNA coding for the chloroplastic heat-shock protein 90 via RNA silencing too. This evidence, which is compelling for the

  10. Predicting Zoonotic Risk of Influenza A Viruses from Host Tropism Protein Signature Using Random Forest.

    Science.gov (United States)

    Eng, Christine L P; Tong, Joo Chuan; Tan, Tin Wee

    2017-05-25

    Influenza A viruses remain a significant health problem, especially when a novel subtype emerges from the avian population to cause severe outbreaks in humans. Zoonotic viruses arise from the animal population as a result of mutations and reassortments, giving rise to novel strains with the capability to evade the host species barrier and cause human infections. Despite progress in understanding interspecies transmission of influenza viruses, we are no closer to predicting zoonotic strains that can lead to an outbreak. We have previously discovered distinct host tropism protein signatures of avian, human and zoonotic influenza strains obtained from host tropism predictions on individual protein sequences. Here, we apply machine learning approaches on the signatures to build a computational model capable of predicting zoonotic strains. The zoonotic strain prediction model can classify avian, human or zoonotic strains with high accuracy, as well as providing an estimated zoonotic risk. This would therefore allow us to quickly determine if an influenza virus strain has the potential to be zoonotic using only protein sequences. The swift identification of potential zoonotic strains in the animal population using the zoonotic strain prediction model could provide us with an early indication of an imminent influenza outbreak.

  11. Predicting Zoonotic Risk of Influenza A Viruses from Host Tropism Protein Signature Using Random Forest

    Directory of Open Access Journals (Sweden)

    Christine L. P. Eng

    2017-05-01

    Full Text Available Influenza A viruses remain a significant health problem, especially when a novel subtype emerges from the avian population to cause severe outbreaks in humans. Zoonotic viruses arise from the animal population as a result of mutations and reassortments, giving rise to novel strains with the capability to evade the host species barrier and cause human infections. Despite progress in understanding interspecies transmission of influenza viruses, we are no closer to predicting zoonotic strains that can lead to an outbreak. We have previously discovered distinct host tropism protein signatures of avian, human and zoonotic influenza strains obtained from host tropism predictions on individual protein sequences. Here, we apply machine learning approaches on the signatures to build a computational model capable of predicting zoonotic strains. The zoonotic strain prediction model can classify avian, human or zoonotic strains with high accuracy, as well as providing an estimated zoonotic risk. This would therefore allow us to quickly determine if an influenza virus strain has the potential to be zoonotic using only protein sequences. The swift identification of potential zoonotic strains in the animal population using the zoonotic strain prediction model could provide us with an early indication of an imminent influenza outbreak.

  12. HpARI Protein Secreted by a Helminth Parasite Suppresses Interleukin-33.

    Science.gov (United States)

    Osbourn, Megan; Soares, Dinesh C; Vacca, Francesco; Cohen, E Suzanne; Scott, Ian C; Gregory, William F; Smyth, Danielle J; Toivakka, Matilda; Kemter, Andrea M; le Bihan, Thierry; Wear, Martin; Hoving, Dennis; Filbey, Kara J; Hewitson, James P; Henderson, Holly; Gonzàlez-Cìscar, Andrea; Errington, Claire; Vermeren, Sonja; Astier, Anne L; Wallace, William A; Schwarze, Jürgen; Ivens, Alasdair C; Maizels, Rick M; McSorley, Henry J

    2017-10-17

    Infection by helminth parasites is associated with amelioration of allergic reactivity, but mechanistic insights into this association are lacking. Products secreted by the mouse parasite Heligmosomoides polygyrus suppress type 2 (allergic) immune responses through interference in the interleukin-33 (IL-33) pathway. Here, we identified H. polygyrus Alarmin Release Inhibitor (HpARI), an IL-33-suppressive 26-kDa protein, containing three predicted complement control protein (CCP) modules. In vivo, recombinant HpARI abrogated IL-33, group 2 innate lymphoid cell (ILC2) and eosinophilic responses to Alternaria allergen administration, and diminished eosinophilic responses to Nippostrongylus brasiliensis, increasing parasite burden. HpARI bound directly to both mouse and human IL-33 (in the cytokine's activated state) and also to nuclear DNA via its N-terminal CCP module pair (CCP1/2), tethering active IL-33 within necrotic cells, preventing its release, and forestalling initiation of type 2 allergic responses. Thus, HpARI employs a novel molecular strategy to suppress type 2 immunity in both infection and allergy. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  13. Expression of Early Immune-Response Genes in Lepidopteran Host are Suppressed by Venom From an Endoparasitoid, Pteromalus puparum

    Science.gov (United States)

    The relationships between parasitoids and their insect hosts have attracted attention at two levels. First, the basic biology of host-parasitoid interactions is of fundamental interest. Second, parasitoids have tremendous potential as biological control agents in sustainable agriculture programs. Pt...

  14. Effect of vitamin D status on chick kidney proteins: detection of a 45-kilodalton mitochondrial protein suppressed by vitamin D

    International Nuclear Information System (INIS)

    Kain, S.R.; Kamrath, K.S.; Henry, H.L.

    1988-01-01

    Two-dimensional polyacrylamide gel electrophoresis along with L-[ 35 S]methionine radiolabeling studies were used to examine the effect of chronic vitamin D status on the composition and relative abundance of chick kidney proteins. Comparison of silver-stained gels revealed no extensive differences in either the electrophoretic mobility or the amounts of kidney proteins present in the mitochondrial fraction from vitamin D-replete and vitamin D-deficient chicks. A similar result was obtained in studies with L-[ 35 S]methionine-labeled proteins. Vitamin D deficiency specifically elevated levels of a 45-kilodalton mitochondrial protein (pI 5.0 to 5.5) by approximately 5- to 12-fold relative to amounts present in vitamin D-replete tissue. This protein could not be detected in postmitochondrial supernatant fractions and was only faintly visible in crude kidney homogenates. The specificity of the observed suppression of this 45-kilodalton protein by vitamin D suggests that it may play an important role in renal functions influenced by the vitamin D endocrine system

  15. Interactions between the Hepatitis C Virus Nonstructural 2 Protein and Host Adaptor Proteins 1 and 4 Orchestrate Virus Release

    Directory of Open Access Journals (Sweden)

    Fei Xiao

    2018-03-01

    Full Text Available Hepatitis C virus (HCV spreads via secreted cell-free particles or direct cell-to-cell transmission. Yet, virus-host determinants governing differential intracellular trafficking of cell-free- and cell-to-cell-transmitted virus remain unknown. The host adaptor proteins (APs AP-1A, AP-1B, and AP-4 traffic in post-Golgi compartments, and the latter two are implicated in basolateral sorting. We reported that AP-1A mediates HCV trafficking during release, whereas the endocytic adaptor AP-2 mediates entry and assembly. We demonstrated that the host kinases AAK1 and GAK regulate HCV infection by controlling these clathrin-associated APs. Here, we sought to define the roles of AP-4, a clathrin-independent adaptor; AP-1A; and AP-1B in HCV infection. We screened for interactions between HCV proteins and the μ subunits of AP-1A, AP-1B, and AP-4 by mammalian cell-based protein fragment complementation assays. The nonstructural 2 (NS2 protein emerged as an interactor of these adaptors in this screening and by coimmunoprecipitations in HCV-infected cells. Two previously unrecognized dileucine-based motifs in the NS2 C terminus mediated AP binding and HCV release. Infectivity and coculture assays demonstrated that while all three adaptors mediate HCV release and cell-free spread, AP-1B and AP-4, but not AP-1A, mediate cell-to-cell spread. Live-cell imaging revealed HCV cotrafficking with AP-1A, AP-1B, and AP-4 and that AP-4 mediates HCV trafficking in a post-Golgi compartment. Lastly, HCV cell-to-cell spread was regulated by AAK1 and GAK and thus susceptible to treatment with AAK1 and GAK inhibitors. These data provide a mechanistic understanding of HCV trafficking in distinct release pathways and reveal a requirement for APs in cell-to-cell viral spread.

  16. Bacteria modulate the CD8+ T cell epitope repertoire of host cytosol-exposed proteins to manipulate the host immune response.

    Directory of Open Access Journals (Sweden)

    Yaakov Maman

    2011-10-01

    Full Text Available The main adaptive immune response to bacteria is mediated by B cells and CD4+ T-cells. However, some bacterial proteins reach the cytosol of host cells and are exposed to the host CD8+ T-cells response. Both gram-negative and gram-positive bacteria can translocate proteins to the cytosol through type III and IV secretion and ESX-1 systems, respectively. The translocated proteins are often essential for the bacterium survival. Once injected, these proteins can be degraded and presented on MHC-I molecules to CD8+ T-cells. The CD8+ T-cells, in turn, can induce cell death and destroy the bacteria's habitat. In viruses, escape mutations arise to avoid this detection. The accumulation of escape mutations in bacteria has never been systematically studied. We show for the first time that such mutations are systematically present in most bacteria tested. We combine multiple bioinformatic algorithms to compute CD8+ T-cell epitope libraries of bacteria with secretion systems that translocate proteins to the host cytosol. In all bacteria tested, proteins not translocated to the cytosol show no escape mutations in their CD8+ T-cell epitopes. However, proteins translocated to the cytosol show clear escape mutations and have low epitope densities for most tested HLA alleles. The low epitope densities suggest that bacteria, like viruses, are evolutionarily selected to ensure their survival in the presence of CD8+ T-cells. In contrast with most other translocated proteins examined, Pseudomonas aeruginosa's ExoU, which ultimately induces host cell death, was found to have high epitope density. This finding suggests a novel mechanism for the manipulation of CD8+ T-cells by pathogens. The ExoU effector may have evolved to maintain high epitope density enabling it to efficiently induce CD8+ T-cell mediated cell death. These results were tested using multiple epitope prediction algorithms, and were found to be consistent for most proteins tested.

  17. The Ebola virus VP35 protein binds viral immunostimulatory and host RNAs identified through deep sequencing.

    Directory of Open Access Journals (Sweden)

    Kari A Dilley

    Full Text Available Ebola virus and Marburg virus are members of the Filovirdae family and causative agents of hemorrhagic fever with high fatality rates in humans. Filovirus virulence is partially attributed to the VP35 protein, a well-characterized inhibitor of the RIG-I-like receptor pathway that triggers the antiviral interferon (IFN response. Prior work demonstrates the ability of VP35 to block potent RIG-I activators, such as Sendai virus (SeV, and this IFN-antagonist activity is directly correlated with its ability to bind RNA. Several structural studies demonstrate that VP35 binds short synthetic dsRNAs; yet, there are no data that identify viral immunostimulatory RNAs (isRNA or host RNAs bound to VP35 in cells. Utilizing a SeV infection model, we demonstrate that both viral isRNA and host RNAs are bound to Ebola and Marburg VP35s in cells. By deep sequencing the purified VP35-bound RNA, we identified the SeV copy-back defective interfering (DI RNA, previously identified as a robust RIG-I activator, as the isRNA bound by multiple filovirus VP35 proteins, including the VP35 protein from the West African outbreak strain (Makona EBOV. Moreover, RNAs isolated from a VP35 RNA-binding mutant were not immunostimulatory and did not include the SeV DI RNA. Strikingly, an analysis of host RNAs bound by wild-type, but not mutant, VP35 revealed that select host RNAs are preferentially bound by VP35 in cell culture. Taken together, these data support a model in which VP35 sequesters isRNA in virus-infected cells to avert RIG-I like receptor (RLR activation.

  18. The Ebola virus VP35 protein binds viral immunostimulatory and host RNAs identified through deep sequencing.

    Science.gov (United States)

    Dilley, Kari A; Voorhies, Alexander A; Luthra, Priya; Puri, Vinita; Stockwell, Timothy B; Lorenzi, Hernan; Basler, Christopher F; Shabman, Reed S

    2017-01-01

    Ebola virus and Marburg virus are members of the Filovirdae family and causative agents of hemorrhagic fever with high fatality rates in humans. Filovirus virulence is partially attributed to the VP35 protein, a well-characterized inhibitor of the RIG-I-like receptor pathway that triggers the antiviral interferon (IFN) response. Prior work demonstrates the ability of VP35 to block potent RIG-I activators, such as Sendai virus (SeV), and this IFN-antagonist activity is directly correlated with its ability to bind RNA. Several structural studies demonstrate that VP35 binds short synthetic dsRNAs; yet, there are no data that identify viral immunostimulatory RNAs (isRNA) or host RNAs bound to VP35 in cells. Utilizing a SeV infection model, we demonstrate that both viral isRNA and host RNAs are bound to Ebola and Marburg VP35s in cells. By deep sequencing the purified VP35-bound RNA, we identified the SeV copy-back defective interfering (DI) RNA, previously identified as a robust RIG-I activator, as the isRNA bound by multiple filovirus VP35 proteins, including the VP35 protein from the West African outbreak strain (Makona EBOV). Moreover, RNAs isolated from a VP35 RNA-binding mutant were not immunostimulatory and did not include the SeV DI RNA. Strikingly, an analysis of host RNAs bound by wild-type, but not mutant, VP35 revealed that select host RNAs are preferentially bound by VP35 in cell culture. Taken together, these data support a model in which VP35 sequesters isRNA in virus-infected cells to avert RIG-I like receptor (RLR) activation.

  19. Differentially Regulated Host Proteins Associated with Chronic Rhinosinusitis Are Correlated with the Sinonasal Microbiome

    Directory of Open Access Journals (Sweden)

    Kristi Biswas

    2017-12-01

    Full Text Available The chronic inflammatory nature of chronic rhinosinusitis (CRS makes it a morbid condition for individuals with the disease and one whose pathogenesis is poorly understood. To date, proteomic approaches have been applied successfully in a handful of CRS studies. In this study we use a multifaceted approach, including proteomics (iTRAQ labeling and microbiome (bacterial 16S rRNA gene sequencing analyses of middle meatus swabs, as well as immune cell analysis of the underlying tissue, to investigate the host-microbe interaction in individuals with CRS (n = 10 and healthy controls (n = 9. Of the total 606 proteins identified in this study, seven were significantly (p < 0.05 more abundant and 104 were significantly lower in the CRS cohort compared with healthy controls. The majority of detected proteins (82% of proteins identified were not significantly correlated with disease status. Elevated levels of blood and immune cell proteins in the CRS cohort, together with significantly higher numbers of B-cells and macrophages in the underlying tissue, confirmed the inflammatory status of CRS individuals. Protein PRRC2C and Ras-related protein (RAB14 (two of the seven elevated proteins showed the biggest fold difference between the healthy and CRS groups. Validation of the elevated levels of these two proteins in CRS samples was provided by immunohistochemistry. Members of the bacterial community in the two study cohorts were not associated with PRRC2C, however members of the genus Moraxella did correlate with RAB14 (p < 0.0001, rho = −0.95, which is a protein involved in the development of basement membrane. In addition, significant correlations between certain members of the CRS bacterial community and 33 lower abundant proteins in the CRS cohort were identified. Members of the genera Streptococcus, Haemophilus and Veillonella were strongly correlated with CRS and were significantly associated with a number of proteins with varying functions. The

  20. Herpes simplex virus types 1 and 2 induce shutoff of host protein synthesis by different mechanisms in Friend erythroleukemia cells

    International Nuclear Information System (INIS)

    Hill, T.M.; Sinden, R.R.; Sadler, J.R.

    1983-01-01

    Herpes simplex virus type 1 (HSV-1) and HSV-2 disrupt host protein synthesis after viral infection. We have treated both viral types with agents which prevent transcription of the viral genome and used these treated viruses to infect induced Friend erythroleukemia cells. By measuring the changes in globin synthesis after infection, we have determined whether expression of the viral genome precedes the shutoff of host protein synthesis or whether the inhibitor molecule enters the cells as part of the virion. HSV-2-induced shutoff of host protein synthesis was insensitive to the effects of shortwave (254-nm) UV light and actinomycin D. Both of the treatments inhibited HSV-1-induced host protein shutoff. Likewise, treatment of HSV-1 with the cross-linking agent 4,5',8-trimethylpsoralen and longwave (360-nm) UV light prevented HSV-1 from inhibiting cellular protein synthesis. Treatment of HSV-2 with 4,5',8-trimethylpsoralen did not affect the ability of the virus to interfere with host protein synthesis, except at the highest doses of longwave UV light. It was determined that the highest longwave UV dosage damaged the HSV-2 virion as well as cross-linking the viral DNA. The results suggest that HSV-2 uses a virion-associated component to inhibit host protein synthesis and that HSV-1 requires the expression of the viral genome to cause cellular protein synthesis shutoff

  1. Cell penetrating peptides to dissect host-pathogen protein-protein interactions in Theileria -transformed leukocytes

    KAUST Repository

    Haidar, Malak

    2017-09-08

    One powerful application of cell penetrating peptides is the delivery into cells of molecules that function as specific competitors or inhibitors of protein-protein interactions. Ablating defined protein-protein interactions is a refined way to explore their contribution to a particular cellular phenotype in a given disease context. Cell-penetrating peptides can be synthetically constrained through various chemical modifications that stabilize a given structural fold with the potential to improve competitive binding to specific targets. Theileria-transformed leukocytes display high PKA activity, but PKAis an enzyme that plays key roles in multiple cellular processes; consequently genetic ablation of kinase activity gives rise to a myriad of confounding phenotypes. By contrast, ablation of a specific kinase-substrate interaction has the potential to give more refined information and we illustrate this here by describing how surgically ablating PKA interactions with BAD gives precise information on the type of glycolysis performed by Theileria-transformed leukocytes. In addition, we provide two other examples of how ablating specific protein-protein interactions in Theileria-infected leukocytes leads to precise phenotypes and argue that constrained penetrating peptides have great therapeutic potential to combat infectious diseases in general.

  2. Activated protein synthesis and suppressed protein breakdown signaling in skeletal muscle of critically ill patients.

    Directory of Open Access Journals (Sweden)

    Jakob G Jespersen

    Full Text Available BACKGROUND: Skeletal muscle mass is controlled by myostatin and Akt-dependent signaling on mammalian target of rapamycin (mTOR, glycogen synthase kinase 3β (GSK3β and forkhead box O (FoxO pathways, but it is unknown how these pathways are regulated in critically ill human muscle. To describe factors involved in muscle mass regulation, we investigated the phosphorylation and expression of key factors in these protein synthesis and breakdown signaling pathways in thigh skeletal muscle of critically ill intensive care unit (ICU patients compared with healthy controls. METHODOLOGY/PRINCIPAL FINDINGS: ICU patients were systemically inflamed, moderately hyperglycemic, received insulin therapy, and showed a tendency to lower plasma branched chain amino acids compared with controls. Using Western blotting we measured Akt, GSK3β, mTOR, ribosomal protein S6 kinase (S6k, eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1, and muscle ring finger protein 1 (MuRF1; and by RT-PCR we determined mRNA expression of, among others, insulin-like growth factor 1 (IGF-1, FoxO 1, 3 and 4, atrogin1, MuRF1, interleukin-6 (IL-6, tumor necrosis factor α (TNF-α and myostatin. Unexpectedly, in critically ill ICU patients Akt-mTOR-S6k signaling was substantially higher compared with controls. FoxO1 mRNA was higher in patients, whereas FoxO3, atrogin1 and myostatin mRNAs and MuRF1 protein were lower compared with controls. A moderate correlation (r2=0.36, p<0.05 between insulin infusion dose and phosphorylated Akt was demonstrated. CONCLUSIONS/SIGNIFICANCE: We present for the first time muscle protein turnover signaling in critically ill ICU patients, and we show signaling pathway activity towards a stimulation of muscle protein synthesis and a somewhat inhibited proteolysis.

  3. Protein O-fucosylation in Plasmodium falciparum ensures efficient infection of mosquito and vertebrate hosts.

    Science.gov (United States)

    Lopaticki, Sash; Yang, Annie S P; John, Alan; Scott, Nichollas E; Lingford, James P; O'Neill, Matthew T; Erickson, Sara M; McKenzie, Nicole C; Jennison, Charlie; Whitehead, Lachlan W; Douglas, Donna N; Kneteman, Norman M; Goddard-Borger, Ethan D; Boddey, Justin A

    2017-09-15

    O-glycosylation of the Plasmodium sporozoite surface proteins CSP and TRAP was recently identified, but the role of this modification in the parasite life cycle and its relevance to vaccine design remain unclear. Here, we identify the Plasmodium protein O-fucosyltransferase (POFUT2) responsible for O-glycosylating CSP and TRAP. Genetic disruption of POFUT2 in Plasmodium falciparum results in ookinetes that are attenuated for colonizing the mosquito midgut, an essential step in malaria transmission. Some POFUT2-deficient parasites mature into salivary gland sporozoites although they are impaired for gliding motility, cell traversal, hepatocyte invasion, and production of exoerythrocytic forms in humanized chimeric liver mice. These defects can be attributed to destabilization and incorrect trafficking of proteins bearing thrombospondin repeats (TSRs). Therefore, POFUT2 plays a similar role in malaria parasites to that in metazoans: it ensures the trafficking of Plasmodium TSR proteins as part of a non-canonical glycosylation-dependent endoplasmic reticulum protein quality control mechanism.The role of O-glycosylation in the malaria life cycle is largely unknown. Here, the authors identify a Plasmodium protein O-fucosyltransferase and show that it is important for normal trafficking of a subset of surface proteins, particularly CSP and TRAP, and efficient infection of mosquito and vertebrate hosts.

  4. Hepatitis B virus X protein suppresses caveolin-1 expression in hepatocellular carcinoma by regulating DNA methylation

    International Nuclear Information System (INIS)

    Yan, Jun; Lu, Qian; Dong, Jiahong; Li, Xiaowu; Ma, Kuansheng; Cai, Lei

    2012-01-01

    To understand the molecular mechanisms of caveolin-1 downregulation by hepatitis B virus X protein (HBx). The DNA methylation status of the caveolin-1 promoter was examined by nested methylation-specific PCR of 33 hepatitis B virus (HBV)-infected hepatocellular carcinoma (HCC) samples. The SMMC-7721 hepatoma cell line was transfected with a recombinant HBx adenoviral vector, and the effects of HBx protein on caveolin-1 expression and promoter methylation were examined and confirmed by sequencing. A reporter gene containing the caveolin-1 promoter region was constructed, and the effects of HBx on the transcriptional activity of the promoter were also studied. Methylation of the caveolin-1 promoter was detected in 84.8% (28/33) of HBV-infected HCC samples. Expression of caveolin-1 was significantly downregulated (P = 0.022), and multiple CpG sites in the promoter region of caveolin-1 were methylated in SMMC-7721 cells after HBx transfection. Transfected HBx significantly suppressed caveolin-1 promoter activity (P = 0.001). HBx protein induces methylation of the caveolin-1 promoter region and suppresses its expression

  5. Suppression of cotton leaf curl disease symptoms in Gossypium hirsutum through over expression of host-encoded miRNAs.

    Science.gov (United States)

    Akmal, Mohd; Baig, Mirza S; Khan, Jawaid A

    2017-12-10

    in any of the healthy looking transgenic lines. In this study for the first time, efficacy of the host (G. arboreum)-encoded miRNAs against CLCuD symptoms was experimentally demonstrated through overexpression of miR398 and miR2950 in G. hirsutum var. HS6 plants. Computational prediction of miRNAs targeting virus genome and their subsequent implication in translational inhibition or cleavage based suppression of viral mRNA via overexpression could help in generating virus resistant plants. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Tick-Host Range Adaptation: Changes in Protein Profiles in Unfed Adult Ixodes scapularis and Amblyomma americanum Saliva Stimulated to Feed on Different Hosts

    Directory of Open Access Journals (Sweden)

    Lucas Tirloni

    2017-12-01

    Full Text Available Understanding the molecular basis of how ticks adapt to feed on different animal hosts is central to understanding tick and tick-borne disease (TBD epidemiology. There is evidence that ticks differentially express specific sets of genes when stimulated to start feeding. This study was initiated to investigate if ticks such as Ixodes scapularis and Amblyomma americanum that are adapted to feed on multiple hosts utilized the same sets of proteins to prepare for feeding. We exposed I. scapularis and A. americanum to feeding stimuli of different hosts (rabbit, human, and dog by keeping unfed adult ticks enclosed in a perforated microfuge in close contact with host skin, but not allowing ticks to attach on host. Our data suggest that ticks of the same species differentially express tick saliva proteins (TSPs when stimulated to start feeding on different hosts. SDS-PAGE and silver staining analysis revealed unique electrophoretic profiles in saliva of I. scapularis and A. americanum that were stimulated to feed on different hosts: rabbit, human, and dog. LC-MS/MS sequencing and pairwise analysis demonstrated that I. scapularis and A. americanum ticks expressed unique protein profiles in their saliva when stimulated to start feeding on different hosts: rabbit, dog, or human. Specifically, our data revealed TSPs that were unique to each treatment and those that were shared between treatments. Overall, we identified a total of 276 and 340 non-redundant I. scapularis and A. americanum TSPs, which we have classified into 28 functional classes including: secreted conserved proteins (unknown functions, proteinase inhibitors, lipocalins, extracellular matrix/cell adhesion, heme/iron metabolism, signal transduction and immunity-related proteins being the most predominant in saliva of unfed ticks. With exception of research on vaccines against Rhipicephalus microplus, which its natural host, cattle, research on vaccine against other ticks relies feeding ticks

  7. Cocoa Procyanidins Suppress Transformation by Inhibiting Mitogen-activated Protein Kinase Kinase*S⃞

    Science.gov (United States)

    Kang, Nam Joo; Lee, Ki Won; Lee, Dong Eun; Rogozin, Evgeny A.; Bode, Ann M.; Lee, Hyong Joo; Dong, Zigang

    2008-01-01

    Cocoa was shown to inhibit chemically induced carcinogenesis in animals and exert antioxidant activity in humans. However, the molecular mechanisms of the chemopreventive potential of cocoa and its active ingredient(s) remain unknown. Here we report that cocoa procyanidins inhibit neoplastic cell transformation by suppressing the kinase activity of mitogen-activated protein kinase kinase (MEK). A cocoa procyanidin fraction (CPF) and procyanidin B2 at 5 μg/ml and 40 μm, respectively, inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation of JB6 P+ mouse epidermal (JB6 P+) cells by 47 and 93%, respectively. The TPA-induced promoter activity and expression of cyclooxygenase-2, which is involved in tumor promotion and inflammation, were dose-dependently inhibited by CPF or procyanidin B2. The activation of activator protein-1 and nuclear factor-κB induced by TPA was also attenuated by CPF or procyanidin B2. The TPA-induced phosphorylation of MEK, extracellular signal-regulated kinase, and p90 ribosomal s6 kinase was suppressed by CPF or procyanidin B2. In vitro and ex vivo kinase assay data demonstrated that CPF or procyanidin B2 inhibited the kinase activity of MEK1 and directly bound with MEK1. CPF or procyanidin B2 suppressed JB6 P+ cell transformation induced by epidermal growth factor or H-Ras, both of which are known to be involved in MEK/ERK signal activation. In contrast, theobromine (up to 80 μm) had no effect on TPA-induced transformation, cyclooxygenase-2 expression, the transactivation of activator protein-1 or nuclear factor-κB, or MEK. Notably, procyanidin B2 exerted stronger inhibitory effects compared with PD098059 (a well known pharmacological inhibitor of MEK) on MEK1 activity and neoplastic cell transformation. PMID:18519570

  8. Bacillus anthracis secretome time course under host-simulated conditions and identification of immunogenic proteins

    Directory of Open Access Journals (Sweden)

    Whittington Jessica

    2007-07-01

    Full Text Available Abstract Background The secretion time course of Bacillus anthracis strain RA3R (pXO1+/pXO2- during early, mid, and late log phase were investigated under conditions that simulate those encountered in the host. All of the identified proteins were analyzed by different software algorithms to characterize their predicted mode of secretion and cellular localization. In addition, immunogenic proteins were identified using sera from humans with cutaneous anthrax. Results A total of 275 extracellular proteins were identified by a combination of LC MS/MS and MALDI-TOF MS. All of the identified proteins were analyzed by SignalP, SecretomeP, PSORT, LipoP, TMHMM, and PROSITE to characterize their predicted mode of secretion, cellular localization, and protein domains. Fifty-three proteins were predicted by SignalP to harbor the cleavable N-terminal signal peptides and were therefore secreted via the classical Sec pathway. Twenty-three proteins were predicted by SecretomeP for secretion by the alternative Sec pathway characterized by the lack of typical export signal. In contrast to SignalP and SecretomeP predictions, PSORT predicted 171 extracellular proteins, 7 cell wall-associated proteins, and 6 cytoplasmic proteins. Moreover, 51 proteins were predicted by LipoP to contain putative Sec signal peptides (38 have SpI sites, lipoprotein signal peptides (13 have SpII sites, and N-terminal membrane helices (9 have transmembrane helices. The TMHMM algorithm predicted 25 membrane-associated proteins with one to ten transmembrane helices. Immunogenic proteins were also identified using sera from patients who have recovered from anthrax. The charge variants (83 and 63 kDa of protective antigen (PA were the most immunodominant secreted antigens, followed by charge variants of enolase and transketolase. Conclusion This is the first description of the time course of protein secretion for the pathogen Bacillus anthracis. Time course studies of protein secretion and

  9. Exploring new biological functions of amyloids: bacteria cell agglutination mediated by host protein aggregation.

    Directory of Open Access Journals (Sweden)

    Marc Torrent

    Full Text Available Antimicrobial proteins and peptides (AMPs are important effectors of the innate immune system that play a vital role in the prevention of infections. Recent advances have highlighted the similarity between AMPs and amyloid proteins. Using the Eosinophil Cationic Protein as a model, we have rationalized the structure-activity relationships between amyloid aggregation and antimicrobial activity. Our results show how protein aggregation can induce bacteria agglutination and cell death. Using confocal and total internal reflection fluorescence microscopy we have tracked the formation in situ of protein amyloid-like aggregates at the bacteria surface and on membrane models. In both cases, fibrillar aggregates able to bind to amyloid diagnostic dyes were detected. Additionally, a single point mutation (Ile13 to Ala can suppress the protein amyloid behavior, abolishing the agglutinating activity and impairing the antimicrobial action. The mutant is also defective in triggering both leakage and lipid vesicle aggregation. We conclude that ECP aggregation at the bacterial surface is essential for its cytotoxicity. Hence, we propose here a new prospective biological function for amyloid-like aggregates with potential biological relevance.

  10. Conflict RNA modification, host-parasite co-evolution, and the origins of DNA and DNA-binding proteins1.

    Science.gov (United States)

    McLaughlin, Paul J; Keegan, Liam P

    2014-08-01

    Nearly 150 different enzymatically modified forms of the four canonical residues in RNA have been identified. For instance, enzymes of the ADAR (adenosine deaminase acting on RNA) family convert adenosine residues into inosine in cellular dsRNAs. Recent findings show that DNA endonuclease V enzymes have undergone an evolutionary transition from cleaving 3' to deoxyinosine in DNA and ssDNA to cleaving 3' to inosine in dsRNA and ssRNA in humans. Recent work on dsRNA-binding domains of ADARs and other proteins also shows that a degree of sequence specificity is achieved by direct readout in the minor groove. However, the level of sequence specificity observed is much less than that of DNA major groove-binding helix-turn-helix proteins. We suggest that the evolution of DNA-binding proteins following the RNA to DNA genome transition represents the major advantage that DNA genomes have over RNA genomes. We propose that a hypothetical RNA modification, a RRAR (ribose reductase acting on genomic dsRNA) produced the first stretches of DNA in RNA genomes. We discuss why this is the most satisfactory explanation for the origin of DNA. The evolution of this RNA modification and later steps to DNA genomes are likely to have been driven by cellular genome co-evolution with viruses and intragenomic parasites. RNA modifications continue to be involved in host-virus conflicts; in vertebrates, edited cellular dsRNAs with inosine-uracil base pairs appear to be recognized as self RNA and to suppress activation of innate immune sensors that detect viral dsRNA.

  11. Vaccinia Virus Immunomodulator A46: A Lipid and Protein-Binding Scaffold for Sequestering Host TIR-Domain Proteins.

    Directory of Open Access Journals (Sweden)

    Sofiya Fedosyuk

    2016-12-01

    Full Text Available Vaccinia virus interferes with early events of the activation pathway of the transcriptional factor NF-kB by binding to numerous host TIR-domain containing adaptor proteins. We have previously determined the X-ray structure of the A46 C-terminal domain; however, the structure and function of the A46 N-terminal domain and its relationship to the C-terminal domain have remained unclear. Here, we biophysically characterize residues 1-83 of the N-terminal domain of A46 and present the X-ray structure at 1.55 Å. Crystallographic phases were obtained by a recently developed ab initio method entitled ARCIMBOLDO_BORGES that employs tertiary structure libraries extracted from the Protein Data Bank; data analysis revealed an all β-sheet structure. This is the first such structure solved by this method which should be applicable to any protein composed entirely of β-sheets. The A46(1-83 structure itself is a β-sandwich containing a co-purified molecule of myristic acid inside a hydrophobic pocket and represents a previously unknown lipid-binding fold. Mass spectrometry analysis confirmed the presence of long-chain fatty acids in both N-terminal and full-length A46; mutation of the hydrophobic pocket reduced the lipid content. Using a combination of high resolution X-ray structures of the N- and C-terminal domains and SAXS analysis of full-length protein A46(1-240, we present here a structural model of A46 in a tetrameric assembly. Integrating affinity measurements and structural data, we propose how A46 simultaneously interferes with several TIR-domain containing proteins to inhibit NF-κB activation and postulate that A46 employs a bipartite binding arrangement to sequester the host immune adaptors TRAM and MyD88.

  12. Genistein suppresses adhesion-induced protein tyrosine phosphorylation and invasion of B16-BL6 melanoma cells.

    Science.gov (United States)

    Yan, C; Han, R

    1998-07-03

    Protein tyrosine phosphorylation occurs as one of the earlier events in cancer cell-extracellular matrix (ECM) interaction. With immunoblot analysis and immunofluorescence microscopy, genistein was found to suppress the tyrosine phosphorylation of proteins located at the cell periphery, including a 125 kDa protein, when B16-BL6 melanoma cells attached to and interacted with ECM. When accompanied by the suppression of adhesion-induced protein tyrosine phosphorylation, the invasive potential of B16-BL6 cells through reconstituted basement membrane was decreased significantly. However, neither adhesive capability nor cell growth was significantly affected by genistein. Therefore, the interruption of cancer cell-ECM interaction by suppression of protein tyrosine phosphorylation may contribute to invasion prevention of genistein.

  13. Host Immunization with Recombinant Proteins to Screen Antigens for Tick Control.

    Science.gov (United States)

    Galay, Remil Linggatong; Miyata, Takeshi; Umemiya-Shirafuji, Rika; Mochizuki, Masami; Fujisaki, Kozo; Tanaka, Tetsuya

    2016-01-01

    Ticks (Parasitiformes: Ixodida) are known for their obligate blood feeding habit and their role in transmitting pathogens to various vertebrate hosts. Tick control using chemical acaricides is extensively used particularly in livestock management, but several disadvantages arise from resistance development of many tick species, and concerns on animal product and environmental contamination. Vaccination offers better protection and more cost-effective alternative to application of chemical acaricides, addressing their disadvantages. However, an ideal anti-tick vaccine targeting multiple tick species and all the tick stages is still wanting. Here, we describe the procedures involved in the evaluation of a vaccine candidate antigen against ticks at the laboratory level, from the preparation of recombinant proteins, administration to the rabbit host and monitoring of antibody titer, to tick infestation challenge and determination of the effects of immunization to ticks.

  14. Implication of haematophagous arthropod salivary proteins in host-vector interactions.

    Science.gov (United States)

    Fontaine, Albin; Diouf, Ibrahima; Bakkali, Nawal; Missé, Dorothée; Pagès, Frédéric; Fusai, Thierry; Rogier, Christophe; Almeras, Lionel

    2011-09-28

    The saliva of haematophagous arthropods contains an array of anti-haemostatic, anti-inflammatory and immunomodulatory molecules that contribute to the success of the blood meal. The saliva of haematophagous arthropods is also involved in the transmission and the establishment of pathogens in the host and in allergic responses. This survey provides a comprehensive overview of the pharmacological activity and immunogenic properties of the main salivary proteins characterised in various haematophagous arthropod species. The potential biological and epidemiological applications of these immunogenic salivary molecules will be discussed with an emphasis on their use as biomarkers of exposure to haematophagous arthropod bites or vaccine candidates that are liable to improve host protection against vector-borne diseases.

  15. Relationship between recombinant protein expression and host metabolome as determined by two-dimensional NMR spectroscopy.

    Directory of Open Access Journals (Sweden)

    Young Kee Chae

    Full Text Available Escherichia coli has been the most widely used host to produce large amounts of heterologous proteins. However, given an input plasmid DNA, E. coli may produce soluble protein, produce only inclusion bodies, or yield little or no protein at all. Many efforts have been made to surmount these problems, but most of them have involved time-consuming and labor-intensive trial-and-error. We hypothesized that different metabolomic fingerprints might be associated with different protein production outcomes. If so, then it might be possible to change the expression pattern by manipulating the metabolite environment. As a first step in testing this hypothesis, we probed a subset of the intracellular metabolites by partially labeling it with 13C-glucose. We tested 71 genes and identified 17 metabolites by employing the two-dimensional NMR spectroscopy. The statistical analysis showed that there existed the metabolite compositions favoring protein production. We hope that this work would help devise a systematic and predictive approach to the recombinant protein production.

  16. Mutation of a Conserved Nuclear Export Sequence in Chikungunya Virus Capsid Protein Disrupts Host Cell Nuclear Import.

    Science.gov (United States)

    Jacobs, Susan C; Taylor, Adam; Herrero, Lara J; Mahalingam, Suresh; Fazakerley, John K

    2017-10-20

    Transmitted by mosquitoes; chikungunya virus (CHIKV) is responsible for frequent outbreaks of arthritic disease in humans. CHIKV is an arthritogenic alphavirus of the Togaviridae family. Capsid protein, a structural protein encoded by the CHIKV RNA genome, is able to translocate to the host cell nucleus. In encephalitic alphaviruses nuclear translocation induces host cell shut off; however, the role of capsid protein nuclear localisation in arthritogenic alphaviruses remains unclear. Using replicon systems, we investigated a nuclear export sequence (NES) in the N-terminal region of capsid protein; analogous to that found in encephalitic alphavirus capsid but uncharacterised in CHIKV. The chromosomal maintenance 1 (CRM1) export adaptor protein mediated CHIKV capsid protein export from the nucleus and a region within the N-terminal part of CHIKV capsid protein was required for active nuclear targeting. In contrast to encephalitic alphaviruses, CHIKV capsid protein did not inhibit host nuclear import; however, mutating the NES of capsid protein (∆NES) blocked host protein access to the nucleus. Interactions between capsid protein and the nucleus warrant further investigation.

  17. Nonstructural 5A Protein of Hepatitis C Virus Interferes with Toll-Like Receptor Signaling and Suppresses the Interferon Response in Mouse Liver.

    Directory of Open Access Journals (Sweden)

    Takeya Tsutsumi

    Full Text Available The hepatitis C virus nonstructural protein NS5A is involved in resistance to the host immune response, as well as the viral lifecycle such as replication and maturation. Here, we established transgenic mice expressing NS5A protein in the liver and examined innate immune responses against lipopolysaccharide (LPS in vivo. Intrahepatic gene expression levels of cytokines such as interleukin-6, tumor necrosis factor-α, and interferon-γ were significantly suppressed after LPS injection in the transgenic mouse liver. Induction of the C-C motif chemokine ligand 2, 4, and 5 was also suppressed. Phosphorylation of the signal transducer and activator of transcription 3, which is activated by cytokines, was also reduced, and expression levels of interferon-stimulated genes, 2'-5' oligoadenylate synthase, interferon-inducible double-stranded RNA-activated protein kinase, and myxovirus resistance 1 were similarly suppressed. Since LPS binds to toll-like receptor 4 and stimulates the downstream pathway leading to induction of these genes, we examined the extracellular signal-regulated kinase and IκB-α. The phosphorylation levels of these molecules were reduced in transgenic mouse liver, indicating that the pathway upstream of the molecules was disrupted by NS5A. Further analyses revealed that the interaction between interleukin-1 receptor-associated kinase-1 and tumor necrosis factor receptor associated factor-6 was dispersed in transgenic mice, suggesting that NS5A may interfere with this interaction via myeloid differentiation primary response gene 88, which was shown to interact with NS5A. Since the gut microbiota, a source of LPS, is known to be associated with pathological conditions in liver diseases, our results suggest the involvement of NS5A in the pathogenesis of HCV infected-liver via the suppression of innate immunity.

  18. Blood protein turnover in parasitized ruminants. The influence of host nutrition

    International Nuclear Information System (INIS)

    Dargie, J.D.

    1981-01-01

    Ruminants infected with helminth or protozoal parasites generally become anaemic and hypoalbuminaemic, as well as losing their appetite. Since feed intake plays an important part in determining blood protein levels, it is necessary, when attempting to determine the mechanisms by which parasites cause anaemia and hypoalbuminaemia, to differentiate between the effects of feed intake per se and the specific effects of the parasite on blood protein turnover. This can be done by a variety of radioisotope techniques using infected and pair-fed control animals. Additionally, animals on a poor plane of nutrition suffer more from parasitism than those which are well fed. To understand the reason for this, it is necessary to determine whether diet influences susceptibility to parasite establishment or survival, and/or susceptibility to the metabolic consequences of parasitism. Described here is the current state of knowledge on the interaction between host nutrition and susceptibility to parasitic infection and parasitic disease processes, with particular reference to anaemia and hypoalbuminaemia. It is concluded that there is little evidence that nutrition has a significant bearing on resistance or susceptibility to infection, but that it does not have a profound influence on the ability of animals to withstand the pathogenic effects of parasites. The reasons for this are discussed in detail, but the principal benefit of a good plane of nutrition is that it enables the synthetic machinery of the host to keep pace with the concurrent parasite-induced hypercatabolism of blood proteins. (author)

  19. Knockdown of Pokemon protein expression inhibits hepatocellular carcinoma cell proliferation by suppression of AKT activity.

    Science.gov (United States)

    Zhu, Xiaosan; Dai, Yichen; Chen, Zhangxin; Xie, Junpei; Zeng, Wei; Lin, Yuanyuan

    2013-01-01

    Overexpression of Pokemon, which is an erythroid myeloid ontogenic factor protein, occurs in different cancers, including hepatocellular carcinoma (HCC). Pokemon is also reported to have an oncogenic activity in various human cancers. This study investigated the effect of Pokemon knockdown on the regulation of HCC growth. POK shRNA suppressed the expression of Pokemon protein in HepG2 cells compared to the negative control vector-transfected HCC cells. Pokemon knockdown also reduced HCC cell viability and enhanced cisplatin-induced apoptosis in HCC cells. AKT activation and the expression of various cell cycle-related genes were inhibited following Pokemon knockdown. These data demonstrate that Pokemon may play a role in HCC progression, suggesting that inhibition of Pokemon expression using Pokemon shRNA should be further evaluated as a novel target for the control of HCC.

  20. Corruption of host seven-transmembrane proteins by pathogenic microbes: a common theme in animals and plants?

    Science.gov (United States)

    Panstruga, Ralph; Schulze-Lefert, Paul

    2003-04-01

    Human diseases like AIDS, malaria, and pneumonia are caused by pathogens that corrupt host chemokine G-protein coupled receptors for molecular docking. Comparatively, little is known about plant host factors that are required for pathogenesis and that may serve as receptors for the entry of pathogenic microbes. Here, we review potential analogies between human chemokine receptors and the plant seven-transmembrane MLO protein, a candidate serving a dual role as docking molecule and defence modulator for the phytopathogenic powdery mildew fungus.

  1. hnRNP A2/B1 interacts with influenza A viral protein NS1 and inhibits virus replication potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nuclear export

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yimeng; Zhou, Jianhong; Du, Yuchun, E-mail: ydu@uark.edu

    2014-01-20

    The NS1 protein of influenza viruses is a major virulence factor and exerts its function through interacting with viral/cellular RNAs and proteins. In this study, we identified heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) as an interacting partner of NS1 proteins by a proteomic method. Knockdown of hnRNP A2/B1 by small interfering RNA (siRNA) resulted in higher levels of NS vRNA, NS1 mRNA, and NS1 protein in the virus-infected cells. In addition, we demonstrated that hnRNP A2/B1 proteins are associated with NS1 and NS2 mRNAs and that knockdown of hnRNP A2/B1 promotes transport of NS1 mRNA from the nucleus to the cytoplasm in the infected cells. Lastly, we showed that knockdown of hnRNP A2/B1 leads to enhanced virus replication. Our results suggest that hnRNP A2/B1 plays an inhibitory role in the replication of influenza A virus in host cells potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nucleocytoplasmic translocation. - Highlights: • Cellular protein hnRNP A2/B1 interacts with influenza viral protein NS1. • hnRNP A2/B1 suppresses the levels of NS1 protein, vRNA and mRNA in infected cells. • hnRNP A2/B1 protein is associated with NS1 and NS2 mRNAs. • hnRNP A2/B1 inhibits the nuclear export of NS1 mRNAs. • hnRNP A2/B1 inhibits influenza virus replication.

  2. Arctigenin suppresses unfolded protein response and sensitizes glucose deprivation-mediated cytotoxicity of cancer cells.

    Science.gov (United States)

    Sun, Shengrong; Wang, Xiong; Wang, Changhua; Nawaz, Ahmed; Wei, Wen; Li, Juanjuan; Wang, Lijun; Yu, De-Hua

    2011-01-01

    The involvement of unfolded protein response (UPR) activation in tumor survival and resistance to chemotherapies suggests a new anticancer strategy targeting UPR pathway. Arctigenin, a natural product, has been recently identified for its antitumor activity with selective toxicity against cancer cells under glucose starvation with unknown mechanism. Here we found that arctigenin specifically blocks the transcriptional induction of two potential anticancer targets, namely glucose-regulated protein-78 (GRP78) and its analog GRP94, under glucose deprivation, but not by tunicamycin. The activation of other UPR pathways, e.g., XBP-1 and ATF4, by glucose deprivation was also suppressed by arctigenin. A further transgene experiment showed that ectopic expression of GRP78 at least partially rescued arctigenin/glucose starvation-mediated cell growth inhibition, suggesting the causal role of UPR suppression in arctigenin-mediated cytotoxicity under glucose starvation. These observations bring a new insight into the mechanism of action of arctigenin and may lead to the design of new anticancer therapeutics. © Georg Thieme Verlag KG Stuttgart · New York.

  3. Anaplasma phagocytophilum MSP4 and HSP70 Proteins Are Involved in Interactions with Host Cells during Pathogen Infection

    Directory of Open Access Journals (Sweden)

    Marinela Contreras

    2017-07-01

    Full Text Available Anaplasma phagocytophilum transmembrane and surface proteins play a role during infection and multiplication in host neutrophils and tick vector cells. Recently, A. phagocytophilum Major surface protein 4 (MSP4 and Heat shock protein 70 (HSP70 were shown to be localized on the bacterial membrane, with a possible role during pathogen infection in ticks. In this study, we hypothesized that A. phagocytophilum MSP4 and HSP70 have similar functions in tick-pathogen and host-pathogen interactions. To address this hypothesis, herein we characterized the role of these bacterial proteins in interaction and infection of vertebrate host cells. The results showed that A. phagocytophilum MSP4 and HSP70 are involved in host-pathogen interactions, with a role for HSP70 during pathogen infection. The analysis of the potential protective capacity of MSP4 and MSP4-HSP70 antigens in immunized sheep showed that MSP4-HSP70 was only partially protective against pathogen infection. This limited protection may be associated with several factors, including the recognition of non-protective epitopes by IgG in immunized lambs. Nevertheless, these antigens may be combined with other candidate protective antigens for the development of vaccines for the control of human and animal granulocytic anaplasmosis. Focusing on the characterization of host protective immune mechanisms and protein-protein interactions at the host-pathogen interface may lead to the discovery and design of new effective protective antigens.

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

    KAUST Repository

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

    2015-01-01

    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

  5. Activated protein synthesis and suppressed protein breakdown signaling in skeletal muscle of critically ill patients

    DEFF Research Database (Denmark)

    Jespersen, Jakob G; Nedergaard, Anders; Reitelseder, Søren

    2011-01-01

    Skeletal muscle mass is controlled by myostatin and Akt-dependent signaling on mammalian target of rapamycin (mTOR), glycogen synthase kinase 3β (GSK3β) and forkhead box O (FoxO) pathways, but it is unknown how these pathways are regulated in critically ill human muscle. To describe factors invol...... involved in muscle mass regulation, we investigated the phosphorylation and expression of key factors in these protein synthesis and breakdown signaling pathways in thigh skeletal muscle of critically ill intensive care unit (ICU) patients compared with healthy controls....

  6. Activated protein synthesis and suppressed protein breakdown signaling in skeletal muscle of critically ill patients

    DEFF Research Database (Denmark)

    Jespersen, Jakob G; Nedergaard, Anders; Reitelseder, Søren

    2011-01-01

    Skeletal muscle mass is controlled by myostatin and Akt-dependent signaling on mammalian target of rapamycin (mTOR), glycogen synthase kinase 3ß (GSK3ß) and forkhead box O (FoxO) pathways, but it is unknown how these pathways are regulated in critically ill human muscle. To describe factors invol...... involved in muscle mass regulation, we investigated the phosphorylation and expression of key factors in these protein synthesis and breakdown signaling pathways in thigh skeletal muscle of critically ill intensive care unit (ICU) patients compared with healthy controls....

  7. Evaluation of two novel leptospiral proteins for their interaction with human host components.

    Science.gov (United States)

    Silva, Lucas P; Fernandes, Luis G V; Vieira, Monica L; de Souza, Gisele O; Heinemann, Marcos B; Vasconcellos, Silvio A; Romero, Eliete C; Nascimento, Ana L T O

    2016-07-01

    Pathogenic species of the genus Leptospira are the etiological agents of leptospirosis, the most widespread zoonosis. Mechanisms involved in leptospiral pathogenesis are not well understood. By data mining the genome sequences of Leptospira interrogans we have identified two proteins predicted to be surface exposed, LIC10821 and LIC10064. Immunofluorescence and proteinase K assays confirmed that the proteins are exposed. Reactivity of the recombinant proteins with human sera has shown that rLIC10821, but not rLIC10064, is recognized by antibodies in confirmed leptospirosis serum samples, suggesting its expression during infection. The rLIC10821 was able to bind laminin, in a dose-dependent fashion, and was called Lsa37 (leptospiral surface adhesin of 37 kDa). Studies with human plasma components demonstrated that rLIC10821 interacts with plasminogen (PLG) and fibrinogen (Fg). The binding of Lsa37 with PLG generates plasmin when PLG activator was added. Fibrin clotting reduction was observed in a thrombin-catalyzed reaction, when Fg was incubated with Lsa37, suggesting that this protein may interfere in the coagulation cascade during the disease. Although LIC10064 protein is more abundant than the corresponding Lsa37, binding activity with all the components tested was not detected. Thus, Lsa37 is a novel versatile adhesin that may mediate Leptospira-host interactions. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. Cellular protein receptors of maculosin, a host specific phytotoxin of spotted knapweed (Centaurea maculosa L.).

    Science.gov (United States)

    Park, S H; Strobel, G A

    1994-01-05

    Maculosin (the diketopiperazine, cyclo (L-Pro-L-Tyr)) is a host specific phytotoxin produced by Alternaria alternata on spotted knapweed (Centaurea maculosa L.). Receptors for this phytotoxin have been isolated from spotted knapweed. Knapweed leaves possess most of the maculosin-binding activity in the cytosolic fraction. However, activity was also observed in the whole membrane fraction of the leaf. The binding component of the cytosolic fraction was identified as a protein(s) because of its heat-lability and sensitivity to proteases. A 16-fold purification of a toxin-binding protein was carried out by ammonium sulfate fractionation, and Sephadex G-200, and maculosin-affinity column chromatography. The affinity column was prepared with epoxy activated Sepharose 6B to which the phenolic group of maculosin was attached. The receptor was estimated to contain more than one binding protein by native and SDS-PAGE. At least one of the maculosin-binding proteins was identified as ribulose-1,5-biphosphate carboxylase (RuBPcase).

  9. Suppression of cell division by pKi-67 antisense-RNA and recombinant protein.

    Science.gov (United States)

    Duchrow, M; Schmidt, M H; Zingler, M; Anemüller, S; Bruch, H P; Broll, R

    2001-01-01

    The human antigen defined by the monoclonal antibody Ki-67 (pKi-67) is a human nuclear protein strongly associated with cell proliferation and found in all tissues studied. It is widely used as a marker of proliferating cells, yet its function is unknown. To investigate its function we suppressed pKi-67 expression by antisense RNA and overexpressed a partial structure of pKi-67 in HeLa cells. A BrdU-incorporation assay showed a significant decrease in DNA synthesis after antisense inhibition. Cell cycle analysis indicated a higher proportion of cells in G1 phase and a lower proportion of cells in S phase while the number of G(2)/M phase cells remained constant. Overexpression of a recombinant protein encoding three of the repetitive elements from exon 13 of pKi-67 had a similar effect to that obtained by antisense inhibition. The similarity of the effect of expressing 'Ki-67 repeats' and pKi-67 antisense RNA could be explained by a negative effect on the folding of the endogenous protein in the endoplasmatic reticulum. Furthermore excessive self-association of pKi-67 via the repeat structure could inhibit its nuclear transport, preventing it from getting to its presumptive site of action. We conclude that the Ki-67 protein has an important role in the regulation of the cell cycle, which is mediated in part by its repetitive elements. Copyright 2001 S. Karger AG, Basel

  10. Adjustment of host cells for accommodation of symbiotic bacteria: vacuole defunctionalization, HOPS suppression, and TIP1g retargeting in Medicago

    NARCIS (Netherlands)

    Gavrin, A.Y.; Kaiser, B.N.; Geiger, D.; Tyerman, S.D.; Wen, Z.; Bisseling, T.; Fedorova, E.E.

    2014-01-01

    In legume–rhizobia symbioses, the bacteria in infected cells are enclosed in a plant membrane, forming organelle-like compartments called symbiosomes. Symbiosomes remain as individual units and avoid fusion with lytic vacuoles of host cells. We observed changes in the vacuole volume of infected

  11. Phage Lambda P Protein: Trans-Activation, Inhibition Phenotypes and their Suppression

    Science.gov (United States)

    Hayes, Sidney; Erker, Craig; Horbay, Monique A.; Marciniuk, Kristen; Wang, Wen; Hayes, Connie

    2013-01-01

    The initiation of bacteriophage λ replication depends upon interactions between the oriλ DNA site, phage proteins O and P, and E. coli host replication proteins. P exhibits a high affinity for DnaB, the major replicative helicase for unwinding double stranded DNA. The concept of P-lethality relates to the hypothesis that P can sequester DnaB and in turn prevent cellular replication initiation from oriC. Alternatively, it was suggested that P-lethality does not involve an interaction between P and DnaB, but is targeted to DnaA. P-lethality is assessed by examining host cells for transformation by ColE1-type plasmids that can express P, and the absence of transformants is attributed to a lethal effect of P expression. The plasmid we employed enabled conditional expression of P, where under permissive conditions, cells were efficiently transformed. We observed that ColE1 replication and plasmid establishment upon transformation is extremely sensitive to P, and distinguish this effect from P-lethality directed to cells. We show that alleles of dnaB protect the variant cells from P expression. P-dependent cellular filamentation arose in ΔrecA or lexA[Ind-] cells, defective for SOS induction. Replication propagation and restart could represent additional targets for P interference of E. coli replication, beyond the oriC-dependent initiation step. PMID:23389467

  12. Albugo-imposed changes to tryptophan-derived antimicrobial metabolite biosynthesis may contribute to suppression of non-host resistance to Phytophthora infestans in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Prince, David C.; Rallapalli, Ghanasyam; Xu, Deyang

    2017-01-01

    -derived antimicrobial compounds enables P. infestans colonization of Arabidopsis, although to a lesser extent than Albugo-infected tissue. A. laibachii also suppresses a subset of genes regulated by salicylic acid; however, salicylic acid plays only a minor role in non-host resistance to P. infestans. CONCLUSIONS......: Albugo sp. alter tryptophan-derived metabolites and suppress elements of the responses to salicylic acid in Arabidopsis. Albugo sp. imposed alterations in tryptophan-derived metabolites may play a role in Arabidopsis non-host resistance to P. infestans. Understanding the basis of non-host resistance...

  13. An in silico pipeline to filter the Toxoplasma gondii proteome for proteins that could traffic to the host cell nucleus and influence host cell epigenetic regulation.

    Science.gov (United States)

    Syn, Genevieve; Blackwell, Jenefer M; Jamieson, Sarra E; Francis, Richard W

    2018-01-01

    Toxoplasma gondii uses epigenetic mechanisms to regulate both endogenous and host cell gene expression. To identify genes with putative epigenetic functions, we developed an in silico pipeline to interrogate the T. gondii proteome of 8313 proteins. Step 1 employs PredictNLS and NucPred to identify genes predicted to target eukaryotic nuclei. Step 2 uses GOLink to identify proteins of epigenetic function based on Gene Ontology terms. This resulted in 611 putative nuclear localised proteins with predicted epigenetic functions. Step 3 filtered for secretory proteins using SignalP, SecretomeP, and experimental data. This identified 57 of the 611 putative epigenetic proteins as likely to be secreted. The pipeline is freely available online, uses open access tools and software with user-friendly Perl scripts to automate and manage the results, and is readily adaptable to undertake any such in silico search for genes contributing to particular functions.

  14. Genome, secretome and glucose transport highlight unique features of the protein production host Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Mattanovich Diethard

    2009-06-01

    Full Text Available Abstract Background Pichia pastoris is widely used as a production platform for heterologous proteins and model organism for organelle proliferation. Without a published genome sequence available, strain and process development relied mainly on analogies to other, well studied yeasts like Saccharomyces cerevisiae. Results To investigate specific features of growth and protein secretion, we have sequenced the 9.4 Mb genome of the type strain DSMZ 70382 and analyzed the secretome and the sugar transporters. The computationally predicted secretome consists of 88 ORFs. When grown on glucose, only 20 proteins were actually secreted at detectable levels. These data highlight one major feature of P. pastoris, namely the low contamination of heterologous proteins with host cell protein, when applying glucose based expression systems. Putative sugar transporters were identified and compared to those of related yeast species. The genome comprises 2 homologs to S. cerevisiae low affinity transporters and 2 to high affinity transporters of other Crabtree negative yeasts. Contrary to other yeasts, P. pastoris possesses 4 H+/glycerol transporters. Conclusion This work highlights significant advantages of using the P. pastoris system with glucose based expression and fermentation strategies. As only few proteins and no proteases are actually secreted on glucose, it becomes evident that cell lysis is the relevant cause of proteolytic degradation of secreted proteins. The endowment with hexose transporters, dominantly of the high affinity type, limits glucose uptake rates and thus overflow metabolism as observed in S. cerevisiae. The presence of 4 genes for glycerol transporters explains the high specific growth rates on this substrate and underlines the suitability of a glycerol/glucose based fermentation strategy. Furthermore, we present an open access web based genome browser http://www.pichiagenome.org.

  15. Host cell proteins in biologics development: Identification, quantitation and risk assessment.

    Science.gov (United States)

    Wang, Xing; Hunter, Alan K; Mozier, Ned M

    2009-06-15

    Host cell proteins (HCPs) are those produced or encoded by the organisms and unrelated to the intended recombinant product. Some are necessary for growth, survival, and normal cellular processing whereas others may be non-essential, simply carried along as baggage. Like the recombinant product, HCPs may also be modified by the host with a number of post-translational modifications. Regardless of the utility, or lack thereof, HCPs are undesirable in the final drug substance. Though commonly present in small quantities (parts per million expressed as nanograms per milligrams of the intended recombinant protein) much effort and cost is expended by industry to remove them. The purpose of this review is to summarize what is of relevance in regards to the biology, the impact of genomics and proteomics on HCP evaluation, the regulatory expectations, analytical approaches, and various methodologies to remove HCPs with bioprocessing. Historical data, bioinformatics approaches and industrial case study examples are provided. Finally, a proposal for a risk assessment tool is provided which brings these facets together and proposes a means for manufacturers to classify and organize a control strategy leading to meaningful product specifications. 2009 Wiley Periodicals, Inc.

  16. Host iron binding proteins acting as niche indicators for Neisseria meningitidis.

    Directory of Open Access Journals (Sweden)

    Philip W Jordan

    Full Text Available Neisseria meningitidis requires iron, and in the absence of iron alters its gene expression to increase iron acquisition and to make the best use of the iron it has. During different stages of colonization and infection available iron sources differ, particularly the host iron-binding proteins haemoglobin, transferrin, and lactoferrin. This study compared the transcriptional responses of N. meningitidis, when grown in the presence of these iron donors and ferric iron, using microarrays.Specific transcriptional responses to the different iron sources were observed, including genes that are not part of the response to iron restriction. Comparisons between growth on haemoglobin and either transferrin or lactoferrin identified changes in 124 and 114 genes, respectively, and 33 genes differed between growth on transferrin or lactoferrin. Comparison of gene expression from growth on haemoglobin or ferric iron showed that transcription is also affected by the entry of either haem or ferric iron into the cytoplasm. This is consistent with a model in which N. meningitidis uses the relative availability of host iron donor proteins as niche indicators.Growth in the presence of haemoglobin is associated with a response likely to be adaptive to survival within the bloodstream, which is supported by serum killing assays that indicate growth on haemoglobin significantly increases survival, and the response to lactoferrin is associated with increased expression of epithelial cell adhesins and oxidative stress response molecules. The transferrin receptor is the most highly transcribed receptor and has the fewest genes specifically induced in its presence, suggesting this is the favoured iron source for the bacterium. Most strikingly, the responses to haemoglobin, which is associated with unrestricted growth, indicates a low iron transcriptional profile, associated with an aggressive phenotype that may be adaptive to access host iron sources but which may also

  17. Multi-faceted proteomic characterization of host protein complement of Rift Valley fever virus virions and identification of specific heat shock proteins, including HSP90, as important viral host factors.

    Science.gov (United States)

    Nuss, Jonathan E; Kehn-Hall, Kylene; Benedict, Ashwini; Costantino, Julie; Ward, Michael; Peyser, Brian D; Retterer, Cary J; Tressler, Lyal E; Wanner, Laura M; McGovern, Hugh F; Zaidi, Anum; Anthony, Scott M; Kota, Krishna P; Bavari, Sina; Hakami, Ramin M

    2014-01-01

    Rift Valley fever is a potentially fatal disease of humans and domestic animals caused by Rift Valley fever virus (RVFV). Infection with RVFV in ruminants can cause near 100% abortion rates and recent outbreaks in naïve human populations have suggested case fatality rates of greater than thirty percent. To elucidate the roles that host proteins play during RVFV infection, proteomic analysis of RVFV virions was conducted using complementary analytical approaches, followed by functional validation studies of select identified host factors. Coupling the more traditional Gel LC/MS/MS approach (SDS PAGE followed by liquid chromatography tandem mass spectrometry) with an alternative technique that preserves protein complexes allowed the protein complement of these viral particles to be thoroughly examined. In addition to viral proteins present within the virions and virion-associated host proteins, multiple macromolecular complexes were identified. Bioinformatic analysis showed that host chaperones were among over-represented protein families associated with virions, and functional experiments using siRNA gene silencing and small molecule inhibitors identified several of these heat shock proteins, including heat shock protein 90 (HSP90), as important viral host factors. Further analysis indicated that HSP inhibition effects occur during the replication/transcription phase of the virus life cycle, leading to significant lowering of viral titers without compromising the functional capacity of released virions. Overall, these studies provide much needed further insight into interactions between RVFV and host cells, increasing our understanding of the infection process and suggesting novel strategies for anti-viral development. In particular, considering that several HSP90 inhibitors have been advancing through clinical trials for cancer treatment, these results also highlight the exciting potential of repurposing HSP90 inhibitors to treat RVF.

  18. Multi-faceted proteomic characterization of host protein complement of Rift Valley fever virus virions and identification of specific heat shock proteins, including HSP90, as important viral host factors.

    Directory of Open Access Journals (Sweden)

    Jonathan E Nuss

    Full Text Available Rift Valley fever is a potentially fatal disease of humans and domestic animals caused by Rift Valley fever virus (RVFV. Infection with RVFV in ruminants can cause near 100% abortion rates and recent outbreaks in naïve human populations have suggested case fatality rates of greater than thirty percent. To elucidate the roles that host proteins play during RVFV infection, proteomic analysis of RVFV virions was conducted using complementary analytical approaches, followed by functional validation studies of select identified host factors. Coupling the more traditional Gel LC/MS/MS approach (SDS PAGE followed by liquid chromatography tandem mass spectrometry with an alternative technique that preserves protein complexes allowed the protein complement of these viral particles to be thoroughly examined. In addition to viral proteins present within the virions and virion-associated host proteins, multiple macromolecular complexes were identified. Bioinformatic analysis showed that host chaperones were among over-represented protein families associated with virions, and functional experiments using siRNA gene silencing and small molecule inhibitors identified several of these heat shock proteins, including heat shock protein 90 (HSP90, as important viral host factors. Further analysis indicated that HSP inhibition effects occur during the replication/transcription phase of the virus life cycle, leading to significant lowering of viral titers without compromising the functional capacity of released virions. Overall, these studies provide much needed further insight into interactions between RVFV and host cells, increasing our understanding of the infection process and suggesting novel strategies for anti-viral development. In particular, considering that several HSP90 inhibitors have been advancing through clinical trials for cancer treatment, these results also highlight the exciting potential of repurposing HSP90 inhibitors to treat RVF.

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

    Science.gov (United States)

    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...

  20. Budesonide suppresses pulmonary antibacterial host defense by down-regulating cathelicidin-related antimicrobial peptide in allergic inflammation mice and in lung epithelial cells

    Directory of Open Access Journals (Sweden)

    Wang Peng

    2013-02-01

    Full Text Available Abstract Background Glucocorticoids are widely regarded as the most effective treatment for asthma. However, the direct impact of glucocorticoids on the innate immune system and antibacterial host defense during asthma remain unclear. Understanding the mechanisms underlying this process is critical to the clinical application of glucocorticoids for asthma therapy. After sensitization and challenge with ovalbumin (OVA, BALB/c mice were treated with inhaled budesonide and infected with Pseudomonas aeruginosa (P. aeruginosa. The number of viable bacteria in enflamed lungs was evaluated, and levels of interleukin-4 (IL-4 and interferon-γ (IFN-γ in serum were measured. A lung epithelial cell line was pretreated with budesonide. Levels of cathelicidin-related antimicrobial peptide (CRAMP were measured by immunohistochemistry and western blot analysis. Intracellular bacteria were observed in lung epithelial cells. Results Inhaled budesonide enhanced lung infection in allergic mice exposed to P. aeruginosa and increased the number of viable bacteria in lung tissue. Higher levels of IL-4 and lower levels of IFN-γ were observed in the serum. Budesonide decreased the expression of CRAMP, increased the number of internalized P. aeruginosa in OVA-challenged mice and in lung epithelial cell lines. These data indicate that inhaled budesonide can suppress pulmonary antibacterial host defense by down-regulating CRAMP in allergic inflammation mice and in cells in vitro. Conclusions Inhaled budesonide suppressed pulmonary antibacterial host defense in an asthmatic mouse model and in lung epithelium cells in vitro. This effect was dependent on the down-regulation of CRAMP.

  1. Effect of lactoferrin protein on red blood cells and macrophages: mechanism of parasite–host interaction

    Directory of Open Access Journals (Sweden)

    An

    2015-07-01

    Full Text Available Namrata Anand,1 Rupinder K Kanwar,2 Mohan Lal Dubey,1 R K Vahishta,3 Rakesh Sehgal,1,* Anita K Verma,4 Jagat R Kanwar2,*1Department of Medical Parasitology, Postgraduate Institute of Medical Education and Research, Chandigarh, India; 2Nanomedicine Laboratory of Immunology and Molecular Biomedical Research, School of Medicine, Molecular and Medical Research Strategic Research Centre, Faculty of Health, Deakin University, Geelong, VIC, Australia; 3Department of Histopathology, Postgraduate Institute of Medical Education and Research, Chandigarh, 4Nanobiotech Laboratory, Department of Zoology, Kirorimal College, University of Delhi, Delhi, India*These authors contributed equally to this workBackground: Lactoferrin is a natural multifunctional protein known to have antitumor, antimicrobial, and anti-inflammatory activity. Apart from its antimicrobial effects, lactoferrin is known to boost the immune response by enhancing antioxidants. Lactoferrin exists in various forms depending on its iron saturation. The present study was done to observe the effect of lactoferrin, isolated from bovine and buffalo colostrum, on red blood cells (RBCs and macrophages (human monocytic cell line-derived macrophages THP1 cells.Methods: Lactoferrin obtained from both species and in different iron saturation forms were used in the present study, and treatment of host cells were given with different forms of lactoferrin at different concentrations. These treated host cells were used for various studies, including morphometric analysis, viability by MTT assay, survivin gene expression, production of reactive oxygen species, phagocytic properties, invasion assay, and Toll-like receptor-4, Toll-like receptor-9, and MDR1 expression, to investigate the interaction between lactoferrin and host cells and the possible mechanism of action with regard to parasitic infections.Results: The mechanism of interaction between host cells and lactoferrin have shown various aspects of gene

  2. Proteomic Analysis of Rhizoctonia solani Identifies Infection-specific, Redox Associated Proteins and Insight into Adaptation to Different Plant Hosts*

    Science.gov (United States)

    Anderson, Jonathan P.; Hane, James K.; Stoll, Thomas; Pain, Nicholas; Hastie, Marcus L.; Kaur, Parwinder; Hoogland, Christine; Gorman, Jeffrey J.; Singh, Karam B.

    2016-01-01

    Rhizoctonia solani is an important root infecting pathogen of a range of food staples worldwide including wheat, rice, maize, soybean, potato and others. Conventional resistance breeding strategies are hindered by the absence of tractable genetic resistance in any crop host. Understanding the biology and pathogenicity mechanisms of this fungus is important for addressing these disease issues, however, little is known about how R. solani causes disease. This study capitalizes on recent genomic studies by applying mass spectrometry based proteomics to identify soluble, membrane-bound and culture filtrate proteins produced under wheat infection and vegetative growth conditions. Many of the proteins found in the culture filtrate had predicted functions relating to modification of the plant cell wall, a major activity required for pathogenesis on the plant host, including a number found only under infection conditions. Other infection related proteins included a high proportion of proteins with redox associated functions and many novel proteins without functional classification. The majority of infection only proteins tested were confirmed to show transcript up-regulation during infection including a thaumatin which increased susceptibility to R. solani when expressed in Nicotiana benthamiana. In addition, analysis of expression during infection of different plant hosts highlighted how the infection strategy of this broad host range pathogen can be adapted to the particular host being encountered. Data are available via ProteomeXchange with identifier PXD002806. PMID:26811357

  3. Mistletoe infection alters the transpiration flow path and suppresses water regulation of host trees during extreme events

    Science.gov (United States)

    Griebel, A.; Maier, C.; Barton, C. V.; Metzen, D.; Renchon, A.; Boer, M. M.; Pendall, E.

    2017-12-01

    Mistletoe is a globally distributed group of parasitic plants that infiltrates the vascular tissue of its host trees to acquire water, carbon and nutrients, making it a leading agent of biotic disturbance. Many mistletoes occur in water-limited ecosystems, thus mistletoe infection in combination with increased climatic stress may exacerbate water stress and potentially accelerate mortality rates of infected trees during extreme events. This is an emerging problem in Australia, as mistletoe distribution is increasing and clear links between mistletoe infection and mortality have been established. However, direct observations about how mistletoes alter host physiological processes during extreme events are rare, which impedes our understanding of mechanisms underlying increased tree mortality rates. We addressed this gap by continuously monitoring stem and branch sap flow and a range of leaf traits of infected and uninfected trees of two co-occurring eucalypt species during a severe heatwave in south-eastern Australia. We demonstrate that mistletoes' leaf water potentials were maintained 30% lower than hosts' to redirect the trees' transpiration flow path towards mistletoe leaves. Eucalypt leaves reduced water loss through stomatal regulation when atmospheric dryness exceeded 2 kPa, but the magnitude of stomatal regulation in non-infected eucalypts differed by species (between 40-80%). Remarkably, when infected, sap flow rates of stems and branches of both eucalypt species remained unregulated even under extreme atmospheric dryness (>8 kPa). Our observations indicate that excessive water use of mistletoes likely increases xylem cavitation rates in hosts during prolonged droughts and supports that hydraulic failure contributes to increased mortality of infected trees. Hence, in order to accurately model the contribution of biotic disturbances to tree mortality under a changing climate, it will be crucial to increase our process-based understanding of the interaction

  4. Quantitative Proteomic Analysis of Mosquito C6/36 Cells Reveals Host Proteins Involved in Zika Virus Infection.

    Science.gov (United States)

    Xin, Qi-Lin; Deng, Cheng-Lin; Chen, Xi; Wang, Jun; Wang, Shao-Bo; Wang, Wei; Deng, Fei; Zhang, Bo; Xiao, Gengfu; Zhang, Lei-Ke

    2017-06-15

    Zika virus (ZIKV) is an emerging arbovirus belonging to the genus Flavivirus of the family Flaviviridae During replication processes, flavivirus manipulates host cell systems to facilitate its replication, while the host cells activate antiviral responses. Identification of host proteins involved in the flavivirus replication process may lead to the discovery of antiviral targets. The mosquitoes Aedes aegypti and Aedes albopictus are epidemiologically important vectors for ZIKV, and effective restrictions of ZIKV replication in mosquitoes will be vital in controlling the spread of virus. In this study, an iTRAQ-based quantitative proteomic analysis of ZIKV-infected Aedes albopictus C6/36 cells was performed to investigate host proteins involved in the ZIKV infection process. A total of 3,544 host proteins were quantified, with 200 being differentially regulated, among which CHCHD2 can be upregulated by ZIKV infection in both mosquito C6/36 and human HeLa cells. Our further study indicated that CHCHD2 can promote ZIKV replication and inhibit beta interferon (IFN-β) production in HeLa cells, suggesting that ZIKV infection may upregulate CHCHD2 to inhibit IFN-I production and thus promote virus replication. Bioinformatics analysis of regulated host proteins highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the 20S proteasome, bortezomib, can inhibit ZIKV infection in vivo Our study illustrated how host cells respond to ZIKV infection and also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients. IMPORTANCE ZIKV infection poses great threats to human health, and there is no FDA-approved drug available for the treatment of ZIKV infection. During replication, ZIKV manipulates host cell systems to facilitate its replication, while host cells activate

  5. Effects of minimal exposures to atmospheric pressure plasma on the activity of Salmonella Typhimurium: Deactivation of bacterial motility and suppression of host-cell invasion.

    Science.gov (United States)

    Park, Jin-Sung; Kim, Kijung; Han, Je-Hyun; Gweon, Bomi; Ko, Ung Hyun; Yoo, Suk Jae; Choe, Wonho; Shin, Jennifer H

    2016-09-01

    Atmospheric pressure plasma (APP) has been shown effective in sterilization by reducing the number of viable microbes during surface cleaning, food processing, or human tissue treatment. For safe conduct, the majority of previous research focused on complete abolition of microbes, which may require severe treatments. Our aim is to investigate the minimal treatment conditions necessary for effective inactivation of bacteria in such a manner that the APP treated bacteria would not be able to harm the host cells. For this, we ought to identify the objective criteria to make the bacteria dysfunctional. We choose the motile properties and the host-cell invasion capability as two measures to quantify the pathogenic state of bacteria. In this paper, we investigated how the APP treatment in a minimal dosage affects the activity of Salmonella Typhimurium. At 100 W and 15 kHz for 20 s, the APP treatment effectively suppressed active "run and tumble" type motility and induced formation of abnormally long structures. With 20 s exposure, the bacterial cells failed to cause pyroptosis in the host cells with >90% survival after 12 h of co-incubation. Our results suggest novel measures to evaluate the functional pathogenic state for identifying safe APP treatment conditions. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Serum-dependent expression of promyelocytic leukemia protein suppresses propagation of influenza virus

    International Nuclear Information System (INIS)

    Iki, Shigeo; Yokota, Shin-ichi; Okabayashi, Tamaki; Yokosawa, Noriko; Nagata, Kyosuke; Fujii, Nobuhiro

    2005-01-01

    The rate of propagation of influenza virus in human adenocarcinoma Caco-2 cells was found to negatively correlate with the concentration of fetal bovine serum (FBS) in the culture medium. Virus replicated more rapidly at lower FBS concentrations (0 or 2%) than at higher concentrations (10 or 20%) during an early stage of infection. Basal and interferon (IFN)-induced levels of typical IFN-inducible anti-viral proteins, such as 2',5'-oligoadenylate synthetase, dsRNA-activated protein kinase and MxA, were unaffected by variation in FBS concentrations. But promyelocytic leukemia protein (PML) was expressed in a serum-dependent manner. In particular, the 65 to 70 kDa isoform of PML was markedly upregulated following the addition of serum. In contrast, other isoforms were induced by IFN treatment, and weakly induced by FBS concentrations. Immunofluorescence microscopy indicated that PML was mainly formed nuclear bodies in Caco-2 cells at various FBS concentrations, and the levels of the PML-nuclear bodies were upregulated by FBS. Overexpression of PML isoform consisting of 560 or 633 amino acid residues by transfection of expression plasmid results in significantly delayed viral replication rate in Caco-2 cells. On the other hand, downregulation of PML expression by RNAi enhanced viral replication. These results indicate that PML isoforms which are expressed in a serum-dependent manner suppress the propagation of influenza virus at an early stage of infection

  7. Full automation and validation of a flexible ELISA platform for host cell protein and protein A impurity detection in biopharmaceuticals.

    Science.gov (United States)

    Rey, Guillaume; Wendeler, Markus W

    2012-11-01

    Monitoring host cell protein (HCP) and protein A impurities is important to ensure successful development of recombinant antibody drugs. Here, we report the full automation and validation of an ELISA platform on a robotic system that allows the detection of Chinese hamster ovary (CHO) HCPs and residual protein A of in-process control samples and final drug substance. The ELISA setup is designed to serve three main goals: high sample throughput, high quality of results, and sample handling flexibility. The processing of analysis requests, determination of optimal sample dilutions, and calculation of impurity content is performed automatically by a spreadsheet. Up to 48 samples in three unspiked and spiked dilutions each are processed within 24 h. The dilution of each sample is individually prepared based on the drug concentration and the expected impurity content. Adaptable dilution protocols allow the analysis of sample dilutions ranging from 1:2 to 1:2×10(7). The validity of results is assessed by automatic testing for dilutional linearity and spike recovery for each sample. This automated impurity ELISA facilitates multi-project process development, is easily adaptable to other impurity ELISA formats, and increases analytical capacity by combining flexible sample handling with high data quality. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Host and bacterial proteins that repress recruitment of LC3 to Shigella early during infection.

    Directory of Open Access Journals (Sweden)

    Leigh A Baxt

    Full Text Available Shigella spp. are intracytosolic gram-negative pathogens that cause disease by invasion and spread through the colonic mucosa, utilizing host cytoskeletal components to form propulsive actin tails. We have previously identified the host factor Toca-1 as being recruited to intracellular S. flexneri and being required for efficient bacterial actin tail formation. We show that at early times during infection (40 min., the type three-secreted effector protein IcsB recruits Toca-1 to intracellular bacteria and that recruitment of Toca-1 is associated with repression of recruitment of LC3, as well as with repression of recruitment of the autophagy marker NDP52, around these intracellular bacteria. LC3 is best characterized as a marker of autophagosomes, but also marks phagosomal membranes in the process LC3-associated phagocytosis. IcsB has previously been demonstrated to be required for S. flexneri evasion of autophagy at late times during infection (4-6 hr by inhibiting binding of the autophagy protein Atg5 to the Shigella surface protein IcsA (VirG. Our results suggest that IcsB and Toca-1 modulation of LC3 recruitment restricts LC3-associated phagocytosis and/or LC3 recruitment to vacuolar membrane remnants. Together with published results, our findings suggest that IcsB inhibits innate immune responses in two distinct ways, first, by inhibiting LC3-associated phagocytosis and/or LC3 recruitment to vacuolar membrane remnants early during infection, and second, by inhibiting autophagy late during infection.

  9. Subtype-specific suppression of Shiga toxin 2 released from Escherichia coli upon exposure to protein synthesis inhibitors

    DEFF Research Database (Denmark)

    Pedersen, Malene Gantzhorn; Hansen, Claus; Riise, Erik

    2008-01-01

    Shiga toxins (Stx) are important virulence factors in the pathogenesis of severe disease including hemolytic-uremic syndrome, caused by Stx-producing Escherichia coli (STEC). STEC strains increase the release of Stx in vitro following the addition of fluoroquinolones, whereas protein synthesis...... inhibitors previously have been reported to suppress the release of Stx. The amount of Stx released from wild-type STEC strains incubated with protein synthesis inhibitors was examined by a Vero cell cytotoxicity assay. The amounts released were compared to the Stx type (Stx1 or Stx2) and additionally...... to the individual subtypes and toxin variants of Stx2. In general, Stx2 release was suppressed significantly upon exposure to protein synthesis inhibitors at MICs, which was not observed in the case of Stx1. Also, the average amount of different Stx2 toxin variants released was suppressed to various levels ranging...

  10. LIM-domain protein AJUBA suppresses malignant mesothelioma cell proliferation via Hippo signaling cascade.

    Science.gov (United States)

    Tanaka, I; Osada, H; Fujii, M; Fukatsu, A; Hida, T; Horio, Y; Kondo, Y; Sato, A; Hasegawa, Y; Tsujimura, T; Sekido, Y

    2015-01-02

    Malignant mesothelioma (MM) is one of the most aggressive neoplasms usually associated with asbestos exposure and is highly refractory to current therapeutic modalities. MMs show frequent activation of a transcriptional coactivator Yes-associated protein (YAP), which is attributed to the neurofibromatosis type 2 (NF2)-Hippo pathway dysfunction, leading to deregulated cell proliferation and acquisition of a malignant phenotype. However, the whole mechanism of disordered YAP activation in MMs has not yet been well clarified. In the present study, we investigated various components of the NF2-Hippo pathway, and eventually found that MM cells frequently showed downregulation of LIM-domain protein AJUBA, a binding partner of large tumor suppressor type 2 (LATS2), which is one of the last-step kinases of the NF2-Hippo pathway. Although loss of AJUBA expression was independent of the alteration status of other Hippo pathway components, MM cell lines with AJUBA inactivation showed a more dephosphorylated (activated) level of YAP. Immunohistochemical analysis showed frequent downregulation of AJUBA in primary MMs, which was associated with YAP constitutive activation. We found that AJUBA transduction into MM cells significantly suppressed promoter activities of YAP-target genes, and the suppression of YAP activity by AJUBA was remarkably canceled by knockdown of LATS2. In connection with these results, transduction of AJUBA-expressing lentivirus significantly inhibited the proliferation and anchorage-independent growth of the MM cells that harbored ordinary LATS family expression. Taken together, our findings indicate that AJUBA negatively regulates YAP activity through the LATS family, and inactivation of AJUBA is a novel key mechanism in MM cell proliferation.

  11. Virus-producing cells determine the host protein profiles of HIV-1 virion cores

    Science.gov (United States)

    2012-01-01

    incorporation of some RNA binding (RHA and HELIC2) and DNA binding proteins (MCM5 and Ku80) in the viral cores from T cells was higher than in the cores from both mMΦ and mMN and did not correlate with the abundance of these proteins in virus producing cells. Conclusions Profiles of host proteins packaged in the cores of HIV-1 virions depend on the type of virus producing cell. The pool of proteins present in the cores of all virions is likely to contain factors important for viral functions. Incorporation ratio of certain RNA- and DNA-binding proteins suggests their more efficient, non-random packaging into virions in T cells than in mMΦ and mMN. PMID:22889230

  12. ABA suppresses Botrytis cinerea elicited NO production in tomato to influence H2O2 generation and increase host susceptibility

    Directory of Open Access Journals (Sweden)

    Anushen eSivakumaran

    2016-05-01

    Full Text Available Abscisic acid (ABA production has emerged a susceptibility factor in plant-pathogen interactions. This work examined the interaction of ABA with NO in tomato following challenge with the ABA-synthesising pathogen, Botrytis cinerea. Trace gas detection using a quantum cascade laser detected NO production within minutes of challenge with B. cinerea whilst photoacoustic laser detection detected ethylene production – an established mediator of defence against this pathogen - occurring after 6 h. Application of the NO generation inhibitor N-Nitro-L-arginine methyl ester (L-NAME suppressed both NO and ethylene production and resistance against B. cinerea. The tomato mutant sitiens fails to accumulate ABA (abscisic acid, shows increased resistance to B. cinerea and we noted exhibited elevated NO and ethylene production. Exogenous application of L-NAME or ABA reduced NO production in sitiens and reduced resistance to B. cinerea. Increased resistance to B. cinerea in sitiens have previously been linked to increased reactive oxygen species (ROS generation but this was reduced in both L-NAME and ABA treated sitiens. Taken together, our data suggests that ABA can decreases resistance to B. cinerea via reduction of NO production which also suppresses both ROS and ethylene production.

  13. Interaction of CSFV E2 protein with swine host factors as detected by yeast two-hybrid system.

    Directory of Open Access Journals (Sweden)

    Douglas P Gladue

    Full Text Available E2 is one of the envelope glycoproteins of pestiviruses, including classical swine fever virus (CSFV and bovine viral diarrhea virus (BVDV. E2 is involved in several critical functions, including virus entry into target cells, induction of a protective immune response and virulence in swine. However, there is no information regarding any host binding partners for the E2 proteins. Here, we utilized the yeast two-hybrid system and identified fifty-seven host proteins as positive binding partners which bound E2 from both CSFV and BVDV with the exception of two proteins that were found to be positive for binding only to CSFV E2. Alanine scanning of CSFV E2 demonstrated that the binding sites for these cellular proteins on E2 are likely non-linear binding sites. The possible roles of the identified host proteins are discussed as the results presented here will be important for future studies to elucidate mechanisms of host protein-virus interactions during pestivirus infection. However, due to the limitations of the yeast two hybrid system, the proteins identified is not exhaustive and each interaction identified needs to be confirmed by independent experimental approaches in the context of virus-infected cells before any definitive conclusion can be drawn on relevance for the virus life cycle.

  14. Identification of RNA Binding Proteins Associated with Dengue Virus RNA in Infected Cells Reveals Temporally Distinct Host Factor Requirements.

    Directory of Open Access Journals (Sweden)

    Olga V Viktorovskaya

    2016-08-01

    Full Text Available There are currently no vaccines or antivirals available for dengue virus infection, which can cause dengue hemorrhagic fever and death. A better understanding of the host pathogen interaction is required to develop effective therapies to treat DENV. In particular, very little is known about how cellular RNA binding proteins interact with viral RNAs. RNAs within cells are not naked; rather they are coated with proteins that affect localization, stability, translation and (for viruses replication.Seventy-nine novel RNA binding proteins for dengue virus (DENV were identified by cross-linking proteins to dengue viral RNA during a live infection in human cells. These cellular proteins were specific and distinct from those previously identified for poliovirus, suggesting a specialized role for these factors in DENV amplification. Knockdown of these proteins demonstrated their function as viral host factors, with evidence for some factors acting early, while others late in infection. Their requirement by DENV for efficient amplification is likely specific, since protein knockdown did not impair the cell fitness for viral amplification of an unrelated virus. The protein abundances of these host factors were not significantly altered during DENV infection, suggesting their interaction with DENV RNA was due to specific recruitment mechanisms. However, at the global proteome level, DENV altered the abundances of proteins in particular classes, including transporter proteins, which were down regulated, and proteins in the ubiquitin proteasome pathway, which were up regulated.The method for identification of host factors described here is robust and broadly applicable to all RNA viruses, providing an avenue to determine the conserved or distinct mechanisms through which diverse viruses manage the viral RNA within cells. This study significantly increases the number of cellular factors known to interact with DENV and reveals how DENV modulates and usurps

  15. Host ESCRT proteins are required for bromovirus RNA replication compartment assembly and function.

    Directory of Open Access Journals (Sweden)

    Arturo Diaz

    2015-03-01

    Full Text Available Positive-strand RNA viruses genome replication invariably is associated with vesicles or other rearranged cellular membranes. Brome mosaic virus (BMV RNA replication occurs on perinuclear endoplasmic reticulum (ER membranes in ~70 nm vesicular invaginations (spherules. BMV RNA replication vesicles show multiple parallels with membrane-enveloped, budding retrovirus virions, whose envelopment and release depend on the host ESCRT (endosomal sorting complexes required for transport membrane-remodeling machinery. We now find that deleting components of the ESCRT pathway results in at least two distinct BMV phenotypes. One group of genes regulate RNA replication and the frequency of viral replication complex formation, but had no effect on spherule size, while a second group of genes regulate RNA replication in a way or ways independent of spherule formation. In particular, deleting SNF7 inhibits BMV RNA replication > 25-fold and abolishes detectable BMV spherule formation, even though the BMV RNA replication proteins accumulate and localize normally on perinuclear ER membranes. Moreover, BMV ESCRT recruitment and spherule assembly depend on different sets of protein-protein interactions from those used by multivesicular body vesicles, HIV-1 virion budding, or tomato bushy stunt virus (TBSV spherule formation. These and other data demonstrate that BMV requires cellular ESCRT components for proper formation and function of its vesicular RNA replication compartments. The results highlight growing but diverse interactions of ESCRT factors with many viruses and viral processes, and potential value of the ESCRT pathway as a target for broad-spectrum antiviral resistance.

  16. Friends or Foes? Host defense (antimicrobial) peptides and proteins in human skin diseases.

    Science.gov (United States)

    Niyonsaba, François; Kiatsurayanon, Chanisa; Chieosilapatham, Panjit; Ogawa, Hideoki

    2017-11-01

    Host defense peptides/proteins (HDPs), also known as antimicrobial peptides/proteins (AMPs), are key molecules in the cutaneous innate immune system. AMPs/HDPs historically exhibit broad-spectrum killing activity against bacteria, enveloped viruses, fungi and several parasites. Recently, AMPs/HDPs were shown to have important biological functions, including inducing cell proliferation, migration and differentiation; regulating inflammatory responses; controlling the production of various cytokines/chemokines; promoting wound healing; and improving skin barrier function. Despite the fact that AMPs/HDPs protect our body, several studies have hypothesized that these molecules actively contribute to the pathogenesis of various skin diseases. For example, AMPs/HDPs play crucial roles in the pathological processes of psoriasis, atopic dermatitis, rosacea, acne vulgaris, systemic lupus erythematosus and systemic sclerosis. Thus, AMPs/HDPs may be a double-edged sword, promoting cutaneous immunity while simultaneously initiating the pathogenesis of some skin disorders. This review will describe the most common skin-derived AMPs/HDPs (defensins, cathelicidins, S100 proteins, ribonucleases and dermcidin) and discuss the biology and both the positive and negative aspects of these AMPs/HDPs in skin inflammatory/infectious diseases. Understanding the regulation, functions and mechanisms of AMPs/HDPs may offer new therapeutic opportunities in the treatment of various skin disorders. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. Multiple Functional Domains and Complexes of the Two Nonstructural Proteins of Human Respiratory Syncytial Virus Contribute to Interferon Suppression and Cellular Location▿

    Science.gov (United States)

    Swedan, Samer; Andrews, Joel; Majumdar, Tanmay; Musiyenko, Alla; Barik, Sailen

    2011-01-01

    Human respiratory syncytial virus (RSV), a major cause of severe respiratory diseases, efficiently suppresses cellular innate immunity, represented by type I interferon (IFN), using its two unique nonstructural proteins, NS1 and NS2. In a search for their mechanism, NS1 was previously shown to decrease levels of TRAF3 and IKKε, whereas NS2 interacted with RIG-I and decreased TRAF3 and STAT2. Here, we report on the interaction, cellular localization, and functional domains of these two proteins. We show that recombinant NS1 and NS2, expressed in lung epithelial A549 cells, can form homo- as well as heteromers. Interestingly, when expressed alone, substantial amounts of NS1 and NS2 localized to the nuclei and to the mitochondria, respectively. However, when coexpressed with NS2, as in RSV infection, NS1 could be detected in the mitochondria as well, suggesting that the NS1-NS2 heteromer localizes to the mitochondria. The C-terminal tetrapeptide sequence, DLNP, common to both NS1 and NS2, was required for some functions, but not all, whereas only the NS1 N-terminal region was important for IKKε reduction. Finally, NS1 and NS2 both interacted specifically with host microtubule-associated protein 1B (MAP1B). The contribution of MAP1B in NS1 function was not tested, but in NS2 it was essential for STAT2 destruction, suggesting a role of the novel DLNP motif in protein-protein interaction and IFN suppression. PMID:21795342

  18. Zebrafish Mnx proteins specify one motoneuron subtype and suppress acquisition of interneuron characteristics

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    Seredick Steve D

    2012-11-01

    Full Text Available Abstract Background Precise matching between motoneuron subtypes and the muscles they innervate is a prerequisite for normal behavior. Motoneuron subtype identity is specified by the combination of transcription factors expressed by the cell during its differentiation. Here we investigate the roles of Mnx family transcription factors in specifying the subtypes of individually identified zebrafish primary motoneurons. Results Zebrafish has three Mnx family members. We show that each of them has a distinct and temporally dynamic expression pattern in each primary motoneuron subtype. We also show that two Mnx family members are expressed in identified VeLD interneurons derived from the same progenitor domain that generates primary motoneurons. Surprisingly, we found that Mnx proteins appear unnecessary for differentiation of VeLD interneurons or the CaP motoneuron subtype. Mnx proteins are, however, required for differentiation of the MiP motoneuron subtype. We previously showed that MiPs require two temporally-distinct phases of Islet1 expression for normal development. Here we show that in the absence of Mnx proteins, the later phase of Islet1 expression is initiated but not sustained, and MiPs become hybrids that co-express morphological and molecular features of motoneurons and V2a interneurons. Unexpectedly, these hybrid MiPs often extend CaP-like axons, and some MiPs appear to be entirely transformed to a CaP morphology. Conclusions Our results suggest that Mnx proteins promote MiP subtype identity by suppressing both interneuron development and CaP axon pathfinding. This is, to our knowledge, the first report of transcription factors that act to distinguish CaP and MiP subtype identities. Our results also suggest that MiP motoneurons are more similar to V2 interneurons than are CaP motoneurons.

  19. The Role of NLR-related Protein 3 Inflammasome in Host Defense and Inflammatory Diseases

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    Chul-Su Yang

    2012-03-01

    Full Text Available Among a number of innate receptors, the nucleotide-binding domain leucine-rich repeat containing (NLR nucleotide oligomerization domain (NOD-like receptor families are involved in the recognition of cytosolic pathogen- or danger-associated molecules. Activation of these specific sets of receptors leads to the assembly of a multiprotein complex, the inflammasome, leading to the activation of caspase-1 and maturation of the cytokines interleukin (IL-1β, IL-18, and IL-33. Among NLRs, NLR-related protein 3 (NLRP3 is one of the best-characterized receptors that activates the inflammasome. There is no doubt that NLRP3 inflammasome activation is important for host defense and effective pathogen clearance against fungal, bacterial, and viral infection. In addition, mounting evidence indicates that the NLRP3 inflammasome plays a role in a variety of inflammatory diseases, including gout, atherosclerosis, and type II diabetes, as well as under conditions of cellular stress or injury. Here, we review recent advances in our understanding of the role of the NLRP3 inflammasome in host defense and various inflammatory diseases.

  20. Mucin-like protein, a saliva component involved in brown planthopper virulence and host adaptation.

    Science.gov (United States)

    Huang, Hai-Jian; Liu, Cheng-Wen; Xu, Hai-Jun; Bao, Yan-Yuan; Zhang, Chuan-Xi

    2017-04-01

    The rice brown planthopper (BPH), Nilaparvata lugens, can rapidly adapt to new resistant rice varieties within several generations, rendering its management burdensome. However, the molecular mechanism underlying its adaptability remains unclear. In this study, we investigated the potential role of mucin-like protein (NlMul) in N. lugens virulence and adaptation to host resistance. NlMul is an important glycoprotein that constitutes both gelling and watery saliva, and specifically expressed in the salivary glands at all developmental stages except the egg period. Knocking down the expression of NlMul resulted in the secretion of short and single-branched salivary sheaths. NlMul might help BPH deal with plant resistance, and altered gene expression was observed when BPHs were transferred from a susceptible rice variety to a resistant one. The NlMul-deficient BPHs showed disordered developmental duration and a portion of these insects reared on resistant rice exhibited lethal effects. Our results uncover a saliva-mediated interaction between insect and host plant, and provide useful information in rice breeding and planthopper management. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Pharmacological Inhibition of Host Heme Oxygenase-1 Suppresses Mycobacterium tuberculosis Infection In Vivo by a Mechanism Dependent on T Lymphocytes

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    Diego L. Costa

    2016-10-01

    Full Text Available Heme oxygenase-1 (HO-1 is a stress response antioxidant enzyme which catalyzes the degradation of heme released during inflammation. HO-1 expression is upregulated in both experimental and human Mycobacterium tuberculosis infection, and in patients it is a biomarker of active disease. Whether the enzyme plays a protective versus pathogenic role in tuberculosis has been the subject of debate. To address this controversy, we administered tin protoporphyrin IX (SnPPIX, a well-characterized HO-1 enzymatic inhibitor, to mice during acute M. tuberculosis infection. These SnPPIX-treated animals displayed a substantial reduction in pulmonary bacterial loads comparable to that achieved following conventional antibiotic therapy. Moreover, when administered adjunctively with antimycobacterial drugs, the HO-1 inhibitor markedly enhanced and accelerated pathogen clearance. Interestingly, both the pulmonary induction of HO-1 expression and the efficacy of SnPPIX treatment in reducing bacterial burden were dependent on the presence of host T lymphocytes. Although M. tuberculosis expresses its own heme-degrading enzyme, SnPPIX failed to inhibit its enzymatic activity or significantly restrict bacterial growth in liquid culture. Together, the above findings reveal mammalian HO-1 as a potential target for host-directed monotherapy and adjunctive therapy of tuberculosis and identify the immune response as a critical regulator of this function.

  2. HIV-1 uncoating: connection to nuclear entry and regulation by host proteins

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    Ambrose, Zandrea, E-mail: zaa4@pitt.edu [Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15261 (United States); Aiken, Christopher [Department of Pathology, Microbiology and Immunology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States)

    2014-04-15

    The RNA genome of human immunodeficiency virus type 1 (HIV-1) is enclosed by a capsid shell that dissociates within the cell in a multistep process known as uncoating, which influences completion of reverse transcription of the viral genome. Double-stranded viral DNA is imported into the nucleus for integration into the host genome, a hallmark of retroviral infection. Reverse transcription, nuclear entry, and integration are coordinated by a capsid uncoating process that is regulated by cellular proteins. Although uncoating is not well understood, recent studies have revealed insights into the process, particularly with respect to nuclear import pathways and protection of the viral genome from DNA sensors. Understanding uncoating will be valuable toward developing novel antiretroviral therapies for HIV-infected individuals.

  3. Suppression of interleukin-6-induced C-reactive protein expression by FXR agonists

    International Nuclear Information System (INIS)

    Zhang Songwen; Liu Qiangyuan; Wang Juan; Harnish, Douglas C.

    2009-01-01

    C-reactive protein (CRP), a human acute-phase protein, is a risk factor for future cardiovascular events and exerts direct pro-inflammatory and pro-atherogenic properties. The farnesoid X receptor (FXR), a member of the nuclear hormone receptor superfamily, plays an essential role in the regulation of enterohepatic circulation and lipid homeostasis. In this study, we report that two synthetic FXR agonists, WAY-362450 and GW4064, suppressed interleukin-6-induced CRP expression in human Hep3B hepatoma cells. Knockdown of FXR by short interfering RNA attenuated the inhibitory effect of the FXR agonists and also increased the ability of interleukin-6 to induce CRP production. Furthermore, treatment of wild type C57BL/6 mice with the FXR agonist, WAY-362450, attenuated lipopolysaccharide-induced serum amyloid P component and serum amyloid A3 mRNA levels in the liver, whereas no effect was observed in FXR knockout mice. These data provide new evidence for direct anti-inflammatory properties of FXR.

  4. The Y-Box Binding Protein 1 Suppresses Alzheimer's Disease Progression in Two Animal Models.

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    N V Bobkova

    Full Text Available The Y-box binding protein 1 (YB-1 is a member of the family of DNA- and RNA binding proteins. It is involved in a wide variety of DNA/RNA-dependent events including cell proliferation and differentiation, stress response, and malignant cell transformation. Previously, YB-1 was detected in neurons of the neocortex and hippocampus, but its precise role in the brain remains undefined. Here we show that subchronic intranasal injections of recombinant YB-1, as well as its fragment YB-11-219, suppress impairment of spatial memory in olfactory bulbectomized (OBX mice with Alzheimer's type degeneration and improve learning in transgenic 5XFAD mice used as a model of cerebral amyloidosis. YB-1-treated OBX and 5XFAD mice showed a decreased level of brain β-amyloid. In OBX animals, an improved morphological state of neurons was revealed in the neocortex and hippocampus; in 5XFAD mice, a delay in amyloid plaque progression was observed. Intranasally administered YB-1 penetrated into the brain and could enter neurons. In vitro co-incubation of YB-1 with monomeric β-amyloid (1-42 inhibited formation of β-amyloid fibrils, as confirmed by electron microscopy. This suggests that YB-1 interaction with β-amyloid prevents formation of filaments that are responsible for neurotoxicity and neuronal death. Our data are the first evidence for a potential therapeutic benefit of YB-1 for treatment of Alzheimer's disease.

  5. Extracellular vesicle-derived protein from Bifidobacterium longum alleviates food allergy through mast cell suppression.

    Science.gov (United States)

    Kim, Jung-Hwan; Jeun, Eun-Ji; Hong, Chun-Pyo; Kim, Seong-Hoon; Jang, Min Seong; Lee, Eun-Jung; Moon, Sook Jin; Yun, Chang Ho; Im, Sin-Hyeog; Jeong, Seok-Geun; Park, Beom-Young; Kim, Kyong-Tai; Seoh, Ju-Young; Kim, Yoon-Keun; Oh, Sung-Jong; Ham, Jun-Sang; Yang, Bo-Gie; Jang, Myoung Ho

    2016-02-01

    The incidence of food allergies has increased dramatically during the last decade. Recently, probiotics have been studied for the prevention and treatment of allergic disease. We examined whether Bifidobacterium longum KACC 91563 and Enterococcus faecalis KACC 91532 have the capacity to suppress food allergies. B longum KACC 91563 and E faecalis KACC 91532 were administered to BALB/c wild-type mice, in which food allergy was induced by using ovalbumin and alum. Food allergy symptoms and various immune responses were assessed. B longum KACC 91563, but not E faecalis KACC 91532, alleviated food allergy symptoms. Extracellular vesicles of B longum KACC 91563 bound specifically to mast cells and induced apoptosis without affecting T-cell immune responses. Furthermore, injection of family 5 extracellular solute-binding protein, a main component of extracellular vesicles, into mice markedly reduced the occurrence of diarrhea in a mouse food allergy model. B longum KACC 91563 induces apoptosis of mast cells specifically and alleviates food allergy symptoms. Accordingly, B longum KACC 91563 and family 5 extracellular solute-binding protein exhibit potential as therapeutic approaches for food allergies. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  6. Cell-Permeable Parkin Proteins Suppress Parkinson Disease-Associated Phenotypes in Cultured Cells and Animals

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    Duong, Tam; Kim, Jaetaek; Ruley, H. Earl; Jo, Daewoong

    2014-01-01

    Parkinson’s disease (PD) is a neurodegenerative disorder of complex etiology characterized by the selective loss of dopaminergic neurons, particularly in the substantia nigra. Parkin, a tightly regulated E3 ubiquitin ligase, promotes the survival of dopaminergic neurons in both PD and Parkinsonian syndromes induced by acute exposures to neurotoxic agents. The present study assessed the potential of cell-permeable parkin (CP-Parkin) as a neuroprotective agent. Cellular uptake and tissue penetration of recombinant, enzymatically active parkin was markedly enhanced by the addition of a hydrophobic macromolecule transduction domain (MTD). The resulting CP-Parkin proteins (HPM13 and PM10) suppressed dopaminergic neuronal toxicity in cells and mice exposed to 6-hydroxydopamine (6-OHDH) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). These included enhanced survival and dopamine expression in cultured CATH.a and SH-SY5Y neuronal cells; and protection against MPTP-induced damage in mice, notably preservation of tyrosine hydroxylase-positive cells with enhanced dopamine expression in the striatum and midbrain, and preservation of gross motor function. These results demonstrate that CP-Parkin proteins can compensate for intrinsic limitations in the parkin response and provide a therapeutic strategy to augment parkin activity in vivo. PMID:25019626

  7. Interaction Between 2 Nutraceutical Treatments and Host Immune Status in the Pediatric Critical Illness Stress-Induced Immune Suppression Comparative Effectiveness Trial.

    Science.gov (United States)

    Carcillo, Joseph A; Dean, J Michael; Holubkov, Richard; Berger, John; Meert, Kathleen L; Anand, Kanwaljeet J S; Zimmerman, Jerry J; Newth, Christopher J L; Harrison, Rick; Burr, Jeri; Willson, Douglas F; Nicholson, Carol; Bell, Michael J; Berg, Robert A; Shanley, Thomas P; Heidemann, Sabrina M; Dalton, Heidi; Jenkins, Tammara L; Doctor, Allan; Webster, Angie; Tamburro, Robert F

    2017-11-01

    The pediatric Critical Illness Stress-induced Immune Suppression (CRISIS) trial compared the effectiveness of 2 nutraceutical supplementation strategies and found no difference in the development of nosocomial infection and sepsis in the overall population. We performed an exploratory post hoc analysis of interaction between nutraceutical treatments and host immune status related to the development of nosocomial infection/sepsis. Children from the CRISIS trial were analyzed according to 3 admission immune status categories marked by decreasing immune competence: immune competent without lymphopenia, immune competent with lymphopenia, and previously immunocompromised. The comparative effectiveness of the 2 treatments was analyzed for interaction with immune status category. There were 134 immune-competent children without lymphopenia, 79 previously immune-competent children with lymphopenia, and 27 immunocompromised children who received 1 of the 2 treatments. A significant interaction was found between treatment arms and immune status on the time to development of nosocomial infection and sepsis ( P patient characteristic.

  8. Hijacked then lost in translation: the plight of the recombinant host cell in membrane protein structural biology projects.

    Science.gov (United States)

    Bill, Roslyn M; von der Haar, Tobias

    2015-06-01

    Membrane protein structural biology is critically dependent upon the supply of high-quality protein. Over the last few years, the value of crystallising biochemically characterised, recombinant targets that incorporate stabilising mutations has been established. Nonetheless, obtaining sufficient yields of many recombinant membrane proteins is still a major challenge. Solutions are now emerging based on an improved understanding of recombinant host cells; as a 'cell factory' each cell is tasked with managing limited resources to simultaneously balance its own growth demands with those imposed by an expression plasmid. This review examines emerging insights into the role of translation and protein folding in defining high-yielding recombinant membrane protein production in a range of host cells. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  9. A diverse host thrombospondin-type-1 repeat protein repertoire promotes symbiont colonization during establishment of cnidarian-dinoflagellate symbiosis.

    Science.gov (United States)

    Neubauer, Emilie-Fleur; Poole, Angela Z; Neubauer, Philipp; Detournay, Olivier; Tan, Kenneth; Davy, Simon K; Weis, Virginia M

    2017-05-08

    The mutualistic endosymbiosis between cnidarians and dinoflagellates is mediated by complex inter-partner signaling events, where the host cnidarian innate immune system plays a crucial role in recognition and regulation of symbionts. To date, little is known about the diversity of thrombospondin-type-1 repeat (TSR) domain proteins in basal metazoans or their potential role in regulation of cnidarian-dinoflagellate mutualisms. We reveal a large and diverse repertoire of TSR proteins in seven anthozoan species, and show that in the model sea anemone Aiptasia pallida the TSR domain promotes colonization of the host by the symbiotic dinoflagellate Symbiodinium minutum . Blocking TSR domains led to decreased colonization success, while adding exogenous TSRs resulted in a 'super colonization'. Furthermore, gene expression of TSR proteins was highest at early time-points during symbiosis establishment. Our work characterizes the diversity of cnidarian TSR proteins and provides evidence that these proteins play an important role in the establishment of cnidarian-dinoflagellate symbiosis.

  10. Inter-species protein trafficking endows dodder (Cuscuta pentagona) with a host-specific herbicide-tolerant trait.

    Science.gov (United States)

    Jiang, Linjian; Qu, Feng; Li, Zhaohu; Doohan, Douglas

    2013-06-01

    · Besides photosynthates, dodder (Cuscuta spp.) acquires phloem-mobile proteins from host; however, whether this could mediate inter-species phenotype transfer was not demonstrated. Specifically, we test whether phosphinothricin acetyl transferase (PAT) that confers host plant glufosinate herbicide tolerance traffics and functions inter-specifically. · Dodder tendrils excised from hosts can grow in vitro for weeks or resume in vivo by parasitizing new hosts. The level of PAT in in vivo and in vitro dodder tendrils was quantified by enzyme-linked immunosorbent assay. The glufosinate sensitivity was examined by dipping the distal end of in vivo and in vitro tendrils, growing on or excised from LibertyLink (LL; PAT-transgenic and glufosinate tolerant) and conventional (CN; glufosinate sensitive) soybean hosts, into glufosinate solutions for 5 s. After in vitro tendrils excised from LL hosts reparasitized new CN and LL hosts, the PAT level and the glufosinate sensitivity were also examined. · When growing on LL host, dodder tolerated glufosinate and contained PAT at a level of 0.3% of that encountered in LL soybean leaf. After PAT was largely degraded in dodders, they became glufosinate sensitive. PAT mRNA was not detected by reverse transcription PCR in dodders. · In conclusion, the results indicated that PAT inter-species trafficking confers dodder glufosinate tolerance. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

  11. Symptomless endophytic fungi suppress endogenous levels of salicylic acid and interact with the jasmonate-dependent indirect defense traits of their host, lima bean (Phaseolus lunatus).

    Science.gov (United States)

    Navarro-Meléndez, Ariana L; Heil, Martin

    2014-07-01

    Symptomless ‘type II’ fungal endophytes colonize their plant host horizontally and exert diverse effects on its resistance phenotype. Here, we used wild Lima bean (Phaseolus lunatus) plants that were experimentally colonized with one of three strains of natural endophytes (Bartalinia pondoensis, Fusarium sp., or Cochliobolus lunatus) to investigate the effects of fungal colonization on the endogenous levels of salicylic acid (SA) and jasmonic acid (JA) and on two JA-dependent indirect defense traits. Colonization with Fusarium sp. enhanced JA levels in intact leaves, whereas B. pondoensis suppressed the induction of endogenous JA in mechanically damaged leaves. Endogenous SA levels in intact leaves were significantly decreased by all strains and B. pondoensis and Fusarium sp. decreased SA levels after mechanical damage. Colonization with Fusarium sp. or C. lunatus enhanced the number of detectable volatile organic compounds (VOCs) emitted from intact leaves, and all three strains enhanced the relative amount of several VOCs emitted from intact leaves as well as the number of detectable VOCs emitted from slightly damaged leaves. All three strains completely suppressed the induced secretion of extrafloral nectar (EFN) after the exogenous application of JA. Symptomless endophytes interact in complex and strain-specific ways with the endogenous levels of SA and JA and with the defense traits that are controlled by these hormones. These interactions can occur both upstream and downstream of the defense hormones.

  12. Quercetin suppresses hypoxia-induced accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) through inhibiting protein synthesis.

    Science.gov (United States)

    Lee, Dae-Hee; Lee, Yong J

    2008-10-01

    Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells and induce the accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) in normoxia. In this study, under hypoxic conditions (1% O(2)), we examined the effect of quercetin on the intracellular level of HIF-1alpha and extracellular level of vascular endothelial growth factor (VEGF) in a variety of human cancer cell lines. Surprisingly, we observed that quercetin suppressed the HIF-1alpha accumulation during hypoxia in human prostate cancer LNCaP, colon cancer CX-1, and breast cancer SkBr3 cells. Quercetin treatment also significantly reduced hypoxia-induced secretion of VEGF. Suppression of HIF-1alpha accumulation during treatment with quercetin in hypoxia was not prevented by treatment with 26S proteasome inhibitor MG132 or PI3K inhibitor LY294002. Interestingly, hypoxia (1% O(2)) in the presence of 100 microM quercetin inhibited protein synthesis by 94% during incubation for 8 h. Significant quercetin concentration-dependent inhibition of protein synthesis and suppression of HIF-1alpha accumulation were observed under hypoxic conditions. Treatment with 100 microM cycloheximide, a protein synthesis inhibitor, replicated the effect of quercetin by inhibiting HIF-1alpha accumulation during hypoxia. These results suggest that suppression of HIF-1alpha accumulation during treatment with quercetin under hypoxic conditions is due to inhibition of protein synthesis. (c) 2008 Wiley-Liss, Inc.

  13. Recognition of HIV-1 peptides by host CTL is related to HIV-1 similarity to human proteins.

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    Morgane Rolland

    Full Text Available BACKGROUND: While human immunodeficiency virus type 1 (HIV-1-specific cytotoxic T lymphocytes preferentially target specific regions of the viral proteome, HIV-1 features that contribute to immune recognition are not well understood. One hypothesis is that similarities between HIV and human proteins influence the host immune response, i.e., resemblance between viral and host peptides could preclude reactivity against certain HIV epitopes. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the extent of similarity between HIV-1 and the human proteome. Proteins from the HIV-1 B consensus sequence from 2001 were dissected into overlapping k-mers, which were then probed against a non-redundant database of the human proteome in order to identify segments of high similarity. We tested the relationship between HIV-1 similarity to host encoded peptides and immune recognition in HIV-infected individuals, and found that HIV immunogenicity could be partially modulated by the sequence similarity to the host proteome. ELISpot responses to peptides spanning the entire viral proteome evaluated in 314 individuals showed a trend indicating an inverse relationship between the similarity to the host proteome and the frequency of recognition. In addition, analysis of responses by a group of 30 HIV-infected individuals against 944 overlapping peptides representing a broad range of individual HIV-1B Nef variants, affirmed that the degree of similarity to the host was significantly lower for peptides with reactive epitopes than for those that were not recognized. CONCLUSIONS/SIGNIFICANCE: Our results suggest that antigenic motifs that are scarcely represented in human proteins might represent more immunogenic CTL targets not selected against in the host. This observation could provide guidance in the design of more effective HIV immunogens, as sequences devoid of host-like features might afford superior immune reactivity.

  14. Human Adenovirus Core Protein V Is Targeted by the Host SUMOylation Machinery To Limit Essential Viral Functions.

    Science.gov (United States)

    Freudenberger, Nora; Meyer, Tina; Groitl, Peter; Dobner, Thomas; Schreiner, Sabrina

    2018-02-15

    Human adenoviruses (HAdV) are nonenveloped viruses containing a linear, double-stranded DNA genome surrounded by an icosahedral capsid. To allow proper viral replication, the genome is imported through the nuclear pore complex associated with viral core proteins. Until now, the role of these incoming virion proteins during the early phase of infection was poorly understood. The core protein V is speculated to bridge the core and the surrounding capsid. It binds the genome in a sequence-independent manner and localizes in the nucleus of infected cells, accumulating at nucleoli. Here, we show that protein V contains conserved SUMO conjugation motifs (SCMs). Mutation of these consensus motifs resulted in reduced SUMOylation of the protein; thus, protein V represents a novel target of the host SUMOylation machinery. To understand the role of protein V SUMO posttranslational modification during productive HAdV infection, we generated a replication-competent HAdV with SCM mutations within the protein V coding sequence. Phenotypic analyses revealed that these SCM mutations are beneficial for adenoviral replication. Blocking protein V SUMOylation at specific sites shifts the onset of viral DNA replication to earlier time points during infection and promotes viral gene expression. Simultaneously, the altered kinetics within the viral life cycle are accompanied by more efficient proteasomal degradation of host determinants and increased virus progeny production than that observed during wild-type infection. Taken together, our studies show that protein V SUMOylation reduces virus growth; hence, protein V SUMOylation represents an important novel aspect of the host antiviral strategy to limit virus replication and thereby points to potential intervention strategies. IMPORTANCE Many decades of research have revealed that HAdV structural proteins promote viral entry and mainly physical stability of the viral genome in the capsid. Our work over the last years showed that this

  15. A novel Meloidogyne graminicola effector, MgGPP, is secreted into host cells and undergoes glycosylation in concert with proteolysis to suppress plant defenses and promote parasitism.

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    Jiansong Chen

    2017-04-01

    Full Text Available Plant pathogen effectors can recruit the host post-translational machinery to mediate their post-translational modification (PTM and regulate their activity to facilitate parasitism, but few studies have focused on this phenomenon in the field of plant-parasitic nematodes. In this study, we show that the plant-parasitic nematode Meloidogyne graminicola has evolved a novel effector, MgGPP, that is exclusively expressed within the nematode subventral esophageal gland cells and up-regulated in the early parasitic stage of M. graminicola. The effector MgGPP plays a role in nematode parasitism. Transgenic rice lines expressing MgGPP become significantly more susceptible to M. graminicola infection than wild-type control plants, and conversely, in planta, the silencing of MgGPP through RNAi technology substantially increases the resistance of rice to M. graminicola. Significantly, we show that MgGPP is secreted into host plants and targeted to the ER, where the N-glycosylation and C-terminal proteolysis of MgGPP occur. C-terminal proteolysis promotes MgGPP to leave the ER, after which it is transported to the nucleus. In addition, N-glycosylation of MgGPP is required for suppressing the host response. The research data provide an intriguing example of in planta glycosylation in concert with proteolysis of a pathogen effector, which depict a novel mechanism by which parasitic nematodes could subjugate plant immunity and promote parasitism and may present a promising target for developing new strategies against nematode infections.

  16. Attachment and entry of Chlamydia have distinct requirements for host protein disulfide isomerase.

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    Stephanie Abromaitis

    2009-04-01

    Full Text Available Chlamydia is an obligate intracellular pathogen that causes a wide range of diseases in humans. Attachment and entry are key processes in infectivity and subsequent pathogenesis of Chlamydia, yet the mechanisms governing these interactions are unknown. It was recently shown that a cell line, CHO6, that is resistant to attachment, and thus infectivity, of multiple Chlamydia species has a defect in protein disulfide isomerase (PDI N-terminal signal sequence processing. Ectopic expression of PDI in CHO6 cells led to restoration of Chlamydia attachment and infectivity; however, the mechanism leading to this recovery was not ascertained. To advance our understanding of the role of PDI in Chlamydia infection, we used RNA interference to establish that cellular PDI is essential for bacterial attachment to cells, making PDI the only host protein identified as necessary for attachment of multiple species of Chlamydia. Genetic complementation and PDI-specific inhibitors were used to determine that cell surface PDI enzymatic activity is required for bacterial entry into cells, but enzymatic function was not required for bacterial attachment. We further determined that it is a PDI-mediated reduction at the cell surface that triggers bacterial uptake. While PDI is necessary for Chlamydia attachment to cells, the bacteria do not appear to utilize plasma membrane-associated PDI as a receptor, suggesting that Chlamydia binds a cell surface protein that requires structural association with PDI. Our findings demonstrate that PDI has two essential and independent roles in the process of chlamydial infectivity: it is structurally required for chlamydial attachment, and the thiol-mediated oxido-reductive function of PDI is necessary for entry.

  17. Accumulation and dissemination of prion protein in experimental sheep scrapie in the natural host

    Directory of Open Access Journals (Sweden)

    Warner Richard

    2009-02-01

    Full Text Available Abstract Background In order to study the sites of uptake and mechanisms of dissemination of scrapie prions in the natural host under controlled conditions, lambs aged 14 days and homozygous for the VRQ allele of the PrP gene were infected by the oral route. Infection occurred in all lambs with a remarkably short and highly consistent incubation period of approximately 6 months. Challenge of lambs at approximately eight months of age resulted in disease in all animals, but with more variable incubation periods averaging significantly longer than those challenged at 14 days. This model provides an excellent system in which to study the disease in the natural host by virtue of the relatively short incubation period and close resemblance to natural infection. Results Multiple sites of prion uptake were identified, of which the most important was the Peyer's patch of the distal ileum. Neuroinvasion was detected initially in the enteric nervous system prior to infection of the central nervous system. At end stage disease prion accumulation was widespread throughout the entire neuraxis, but vacuolar pathology was absent in most animals that developed disease at 6–7 months of age. Conclusion Initial spread of detectable PrP was consistent with drainage in afferent lymph to dependent lymph nodes. Subsequent accumulation of prions in lymphoid tissue not associated with the gut is consistent with haematogenous spread. In addition to macrophages and follicular dendritic cells, prion containing cells consistent with afferent lymph dendritic cells were identified and are suggested as a likely vehicle for carriage of prions from initial site of uptake to the lymphoreticular system, and as potential carriers of prion protein in blood. It is apparent that spongiform change, the characteristic lesion of scrapie and other prion diseases, is not responsible for the clinical signs in sheep, but may develop in an age dependent manner.

  18. Plasmodium falciparum Plasmodium helical interspersed subtelomeric proteins contribute to cytoadherence and anchor P. falciparum erythrocyte membrane protein 1 to the host cell cytoskeleton

    DEFF Research Database (Denmark)

    Oberli, Alexander; Zurbrügg, Laura; Rusch, Sebastian

    2016-01-01

    is anchored to the cytoskeleton, and the Plasmodium helical interspersed subtelomeric (PHIST) gene family plays a role in many host cell modifications including binding the intracellular domain of PfEMP1. Here, we show that conditional reduction of the PHIST protein PFE1605w strongly reduces adhesion...... interacts with both the intracellular segment of PfEMP1 and with cytoskeletal components. This is the first report of a PHIST protein interacting with key molecules of the cytoadherence complex and the host cytoskeleton, and this functional role seems to play an essential role in the pathology of P...

  19. Suppressing Type 2C Protein Phosphatases Alters Fruit Ripening and the Stress Response in Tomato.

    Science.gov (United States)

    Zhang, Yushu; Li, Qian; Jiang, Li; Kai, Wenbin; Liang, Bin; Wang, Juan; Du, Yangwei; Zhai, Xiawan; Wang, Jieling; Zhang, Yingqi; Sun, Yufei; Zhang, Lusheng; Leng, Ping

    2018-01-01

    Although ABA signaling has been widely studied in Arabidopsis, the roles of core ABA signaling components in fruit remain poorly understood. Herein, we characterize SlPP2C1, a group A type 2C protein phosphatase that negatively regulates ABA signaling and fruit ripening in tomato. The SlPP2C1 protein was localized in the cytoplasm close to AtAHG3/AtPP2CA. The SlPP2C1 gene was expressed in all tomato tissues throughout development, particularly in flowers and fruits, and it was up-regulated by dehydration and ABA treatment. SlPP2C1 expression in fruits was increased at 30 d after full bloom and peaked at the B + 1 stage. Suppression of SlPP2C1 expression significantly accelerated fruit ripening which was associated with higher levels of ABA signaling genes that are reported to alter the expression of fruit ripening genes involved in ethylene release and cell wall catabolism. SlPP2C1-RNAi (RNA interference) led to increased endogenous ABA accumulation and advanced release of ethylene in transgenic fruits compared with wild-type (WT) fruits. SlPP2C1-RNAi also resulted in abnormal flowers and obstructed the normal abscission of pedicels. SlPP2C1-RNAi plants were hypersensitized to ABA, and displayed delayed seed germination and primary root growth, and increased resistance to drought stress compared with WT plants. These results demonstrated that SlPP2C1 is a functional component in the ABA signaling pathway which participates in fruit ripening, ABA responses and drought tolerance. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  20. Identification and Initial Characterization of the Effectors of an Anther Smut Fungus and Potential Host Target Proteins

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    Venkata S. Kuppireddy

    2017-11-01

    Full Text Available (1 Background: Plant pathogenic fungi often display high levels of host specificity and biotrophic fungi; in particular, they must manipulate their hosts to avoid detection and to complete their obligate pathogenic lifecycles. One important strategy of such fungi is the secretion of small proteins that serve as effectors in this process. Microbotryum violaceum is a species complex whose members infect members of the Caryophyllaceae; M. lychnidis-dioicae, a parasite on Silene latifolia, is one of the best studied interactions. We are interested in identifying and characterizing effectors of the fungus and possible corresponding host targets; (2 Methods: In silico analysis of the M. lychnidis-dioicae genome and transcriptomes allowed us to predict a pool of small secreted proteins (SSPs with the hallmarks of effectors, including a lack of conserved protein family (PFAM domains and also localized regions of disorder. Putative SSPs were tested for secretion using a yeast secretion trap method. We then used yeast two-hybrid analyses for candidate-secreted effectors to probe a cDNA library from a range of growth conditions of the fungus, including infected plants; (3 Results: Roughly 50 SSPs were identified by in silico analysis. Of these, 4 were studied further and shown to be secreted, as well as examined for potential host interactors. One of the putative effectors, MVLG_01732, was found to interact with Arabidopsis thaliana calcium-dependent lipid binding protein (AtCLB and with cellulose synthase interactive protein 1 orthologues; and (4 Conclusions: The identification of a pool of putative effectors provides a resource for functional characterization of fungal proteins that mediate the delicate interaction between pathogen and host. The candidate targets of effectors, e.g., AtCLB, involved in pollen germination suggest tantalizing insights that could drive future studies.

  1. Arginine-aromatic interactions and their effects on arginine-induced solubilization of aromatic solutes and suppression of protein aggregation

    KAUST Repository

    Shah, Dhawal; Li, Jianguo; Shaikh, Abdul Rajjak; Rajagopalan, Raj

    2011-01-01

    We examine the interaction of aromatic residues of proteins with arginine, an additive commonly used to suppress protein aggregation, using experiments and molecular dynamics simulations. An aromatic-rich peptide, FFYTP (a segment of insulin), and lysozyme and insulin are used as model systems. Mass spectrometry shows that arginine increases the solubility of FFYTP by binding to the peptide, with the simulations revealing the predominant association of arginine to be with the aromatic residues. The calculations further show a positive preferential interaction coefficient, Γ XP, contrary to conventional thinking that positive Γ XP's indicate aggregation rather than suppression of aggregation. Simulations with lysozyme and insulin also show arginine's preference for aromatic residues, in addition to acidic residues. We use these observations and earlier results reported by us and others to discuss the possible implications of arginine's interactions with aromatic residues on the solubilization of aromatic moieties and proteins. Our results also highlight the fact that explanations based purely on Γ XP, which measures average affinity of an additive to a protein, could obscure or misinterpret the underlying molecular mechanisms behind additive-induced suppression of protein aggregation. © 2011 American Institute of Chemical Engineers (AIChE).

  2. Arginine-aromatic interactions and their effects on arginine-induced solubilization of aromatic solutes and suppression of protein aggregation

    KAUST Repository

    Shah, Dhawal

    2011-09-21

    We examine the interaction of aromatic residues of proteins with arginine, an additive commonly used to suppress protein aggregation, using experiments and molecular dynamics simulations. An aromatic-rich peptide, FFYTP (a segment of insulin), and lysozyme and insulin are used as model systems. Mass spectrometry shows that arginine increases the solubility of FFYTP by binding to the peptide, with the simulations revealing the predominant association of arginine to be with the aromatic residues. The calculations further show a positive preferential interaction coefficient, Γ XP, contrary to conventional thinking that positive Γ XP\\'s indicate aggregation rather than suppression of aggregation. Simulations with lysozyme and insulin also show arginine\\'s preference for aromatic residues, in addition to acidic residues. We use these observations and earlier results reported by us and others to discuss the possible implications of arginine\\'s interactions with aromatic residues on the solubilization of aromatic moieties and proteins. Our results also highlight the fact that explanations based purely on Γ XP, which measures average affinity of an additive to a protein, could obscure or misinterpret the underlying molecular mechanisms behind additive-induced suppression of protein aggregation. © 2011 American Institute of Chemical Engineers (AIChE).

  3. Improved Detection of Invasive Pulmonary Aspergillosis Arising during Leukemia Treatment Using a Panel of Host Response Proteins and Fungal Antigens.

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    Allan R Brasier

    Full Text Available Invasive pulmonary aspergillosis (IPA is an opportunistic fungal infection in patients undergoing chemotherapy for hematological malignancy, hematopoietic stem cell transplant, or other forms of immunosuppression. In this group, Aspergillus infections account for the majority of deaths due to mold pathogens. Although early detection is associated with improved outcomes, current diagnostic regimens lack sensitivity and specificity. Patients undergoing chemotherapy, stem cell transplantation and lung transplantation were enrolled in a multi-site prospective observational trial. Proven and probable IPA cases and matched controls were subjected to discovery proteomics analyses using a biofluid analysis platform, fractionating plasma into reproducible protein and peptide pools. From 556 spots identified by 2D gel electrophoresis, 66 differentially expressed post-translationally modified plasma proteins were identified in the leukemic subgroup only. This protein group was rich in complement components, acute-phase reactants and coagulation factors. Low molecular weight peptides corresponding to abundant plasma proteins were identified. A candidate marker panel of host response (9 plasma proteins, 4 peptides, fungal polysaccharides (galactomannan, and cell wall components (β-D glucan were selected by statistical filtering for patients with leukemia as a primary underlying diagnosis. Quantitative measurements were developed to qualify the differential expression of the candidate host response proteins using selective reaction monitoring mass spectrometry assays, and then applied to a separate cohort of 57 patients with leukemia. In this verification cohort, a machine learning ensemble-based algorithm, generalized pathseeker (GPS produced a greater case classification accuracy than galactomannan (GM or host proteins alone. In conclusion, Integration of host response proteins with GM improves the diagnostic detection of probable IPA in patients

  4. Olopatadine Suppresses the Migration of THP-1 Monocytes Induced by S100A12 Protein

    Directory of Open Access Journals (Sweden)

    2006-01-01

    Full Text Available Olopatadine hydrochloride (olopatadine is an antiallergic drug with histamine H 1 receptor antagonistic activity. Recently, olopatadine has been shown to bind to S100A12 which is a member of the S100 family of calcium-binding proteins, and exerts multiple proinflammatory activities including chemotaxis for monocytes and neutrophils. In this study, we examined the possibility that the interaction of olopatadine with S100A12 inhibits the proinflammatory effects of S100A12. Pretreatment of olopatadine with S100A12 reduced migration of THP-1, a monocyte cell line, induced by S100A12 alone, but did not affect recombinant human regulated upon activation, normal T cell expressed and secreted (RANTES-induced migration. Amlexanox, which also binds to S100A12, inhibited the THP-1 migration induced by S100A12. However, ketotifen, another histamine H 1 receptor antagonist, had little effect on the activity of S100A12. These results suggest that olopatadine has a new mechanism of action, that is, suppression of the function of S100A12, in addition to histamine H 1 receptor antagonistic activity.

  5. Investigation of the Relationship between Lactococcal Host Cell Wall Polysaccharide Genotype and 936 Phage Receptor Binding Protein Phylogeny

    DEFF Research Database (Denmark)

    Mahony, Jennifer; Kot, Witold Piotr; Murphy, James

    2013-01-01

    Comparative genomics of 11 lactococcal 936-type phages combined with host range analysis allowed subgrouping of these phage genomes, particularly with respect to their encoded receptor binding proteins. The so-called pellicle or cell wall polysaccharide of Lactococcus lactis, which has been...... implicated as a host receptor of (certain) 936-type phages, is specified by a large gene cluster, which, among different lactococcal strains, contains highly conserved regions as well as regions of diversity. The regions of diversity within this cluster on the genomes of lactococcal strains MG1363, SK11, IL......1403, KF147, CV56, and UC509.9 were used for the development of a multiplex PCR system to identify the pellicle genotype of lactococcal strains used in this study. The resulting comparative analysis revealed an apparent correlation between the pellicle genotype of a given host strain and the host range...

  6. Identification and characterization of the host protein DNAJC14 as a broadly active flavivirus replication modulator.

    Directory of Open Access Journals (Sweden)

    Zhigang Yi

    2011-01-01

    Full Text Available Viruses in the Flavivirus genus of the Flaviviridae family are arthropod-transmitted and contribute to staggering numbers of human infections and significant deaths annually across the globe. To identify cellular factors with antiviral activity against flaviviruses, we screened a cDNA library using an iterative approach. We identified a mammalian Hsp40 chaperone protein (DNAJC14 that when overexpressed was able to mediate protection from yellow fever virus (YFV-induced cell death. Further studies revealed that DNAJC14 inhibits YFV at the step of viral RNA replication. Since replication of bovine viral diarrhea virus (BVDV, a member of the related Pestivirus genus, is also known to be modulated by DNAJC14, we tested the effect of this host factor on diverse Flaviviridae family members. Flaviviruses, including the pathogenic Asibi strain of YFV, Kunjin, and tick-borne Langat virus, as well as a Hepacivirus, hepatitis C virus (HCV, all were inhibited by overexpression of DNAJC14. Mutagenesis showed that both the J-domain and the C-terminal domain, which mediates self-interaction, are required for anti-YFV activity. We found that DNAJC14 does not block YFV nor HCV NS2-3 cleavage, and using non-inhibitory mutants demonstrate that DNAJC14 is recruited to YFV replication complexes. Immunofluorescence analysis demonstrated that endogenous DNAJC14 rearranges during infection and is found in replication complexes identified by dsRNA staining. Interestingly, silencing of endogenous DNAJC14 results in impaired YFV replication suggesting a requirement for DNAJC14 in YFV replication complex assembly. Finally, the antiviral activity of overexpressed DNAJC14 occurs in a time- and dose-dependent manner. DNAJC14 overexpression may disrupt the proper stoichiometry resulting in inhibition, which can be overcome upon restoration of the optimal ratios due to the accumulation of viral nonstructural proteins. Our findings, together with previously published work

  7. Suppression or activation of immune responses by predicted secreted proteins of the soybean rust pathogen Phakopsora pachyrhizi

    Science.gov (United States)

    Rust fungi, such as Phakopsora pachyrhizi, are major threats to crop production. They form specialized haustoria that are intimately associated with plant cells. These haustoria have roles in acquiring nutrients and secreting effector proteins that manipulate host immune systems. Functional characte...

  8. Aspergillus flavus induced alterations in tear protein profile reveal pathogen-induced host response to fungal infection.

    Science.gov (United States)

    Kandhavelu, Jeyalakshmi; Demonte, Naveen Luke; Namperumalsamy, Venkatesh Prajna; Prajna, Lalitha; Thangavel, Chitra; Jayapal, Jeya Maheshwari; Kuppamuthu, Dharmalingam

    2017-01-30

    Aspergillus flavus and Fusarium sp. are primary causative agents of keratitis that results in corneal tissue damage leading to vision loss particularly in individuals from the tropical parts of the world. Proteins in the tear film collected from control and keratitis patients was profiled and compared. A total of 1873 proteins from control and 1400 proteins from patient tear were identified by mass spectrometry. While 847 proteins were found to be glycosylated in the patient tear, only 726 were glycosylated in control tear. And, some of the tear proteins showed alterations in their glycosylation pattern after infection. Complement system proteins, proteins specific for neutrophil extracellular traps and proteins involved in would healing were found only in the patient tear. The presence of these innate immune system proteins in the tear film of patients supports the previous data indicating the involvement of neutrophil and complement pathways in antifungal defense. High levels of wound healing proteins in keratitis patient tear implied activation of tissue repair during infection. The early appearance of the host defense proteins and wound healing response indicates that tear proteins could be used as an early marker system for monitoring the progression of pathogenesis. Identification of negative regulators of the above defense pathways in keratitis tear indicates an intricate balance of pro and anti-defense mechanisms operating in fungal infection of the eye. Tear proteins from control and mycotic keratitis patients were separated into glycoproteins and non-glycosylated proteins and then identified by mass spectrometry. Tear proteins from keratitis patients showed alteration in the glycosylation pattern indicating the alteration of glycosylation machinery due to infection. Neutrophil extracellular traps specific proteins, complement pathway proteins, as well as wound healing proteins, were found only in patient tear showing the activation of antifungal defense

  9. Plant innate immunity induced by flagellin suppresses the hypersensitive response in non-host plants elicited by Pseudomonas syringae pv. averrhoi.

    Directory of Open Access Journals (Sweden)

    Chia-Fong Wei

    Full Text Available A new pathogen, Pseudomonas syringae pv. averrhoi (Pav, which causes bacterial spot disease on carambola was identified in Taiwan in 1997. Many strains of this pathovar have been isolated from different locations and several varieties of hosts. Some of these strains, such as HL1, are nonmotile and elicit a strong hypersensitive response (HR in nonhost tobacco leaves, while other strains, such as PA5, are motile and elicit a weak HR. Based on the image from a transmission electron microscope, the results showed that HL1 is flagellum-deficient and PA5 has normal flagella. Here we cloned and analyzed the fliC gene and glycosylation island from Pav HL1 and PA5. The amino acid sequences of FliC from HL1 and PA5 are identical to P. s. pvs. tabaci (Pta, glycinea and phaseolicola and share very high similarity with other pathovars of P. syringae. In contrast to the flagellin mutant PtaΔfliC, PA5ΔfliC grows as well as wild type in the host plant, but it elicits stronger HR than wild type does in non-host plants. Furthermore, the purified Pav flagellin, but not the divergent flagellin from Agrobacterium tumefaciens, is able to impair the HR induced by PA5ΔfliC. PA5Δfgt1 possessing nonglycosylated flagella behaved as its wild type in both bacterial growth in host and HR elicitation. Flagellin was infiltrated into tobacco leaves either simultaneously with flagellum-deficient HL1 or prior to the inoculation of wild type HL1, and both treatments impaired the HR induced by HL1. Moreover, the HR elicited by PA5 and PA5ΔfliC was enhanced by the addition of cycloheximide, suggesting that the flagellin is one of the PAMPs (pathogen-associated molecular patterns contributed to induce the PAMP-triggered immunity (PTI. Taken together, the results shown in this study reveal that flagellin in Pav is capable of suppressing HR via PTI induction during an incompatible interaction.

  10. Plant Innate Immunity Induced by Flagellin Suppresses the Hypersensitive Response in Non-Host Plants Elicited by Pseudomonas syringae pv. averrhoi

    Science.gov (United States)

    Wei, Chia-Fong; Hsu, Shih-Tien; Deng, Wen-Ling; Wen, Yu-Der; Huang, Hsiou-Chen

    2012-01-01

    A new pathogen, Pseudomonas syringae pv. averrhoi (Pav), which causes bacterial spot disease on carambola was identified in Taiwan in 1997. Many strains of this pathovar have been isolated from different locations and several varieties of hosts. Some of these strains, such as HL1, are nonmotile and elicit a strong hypersensitive response (HR) in nonhost tobacco leaves, while other strains, such as PA5, are motile and elicit a weak HR. Based on the image from a transmission electron microscope, the results showed that HL1 is flagellum-deficient and PA5 has normal flagella. Here we cloned and analyzed the fliC gene and glycosylation island from Pav HL1 and PA5. The amino acid sequences of FliC from HL1 and PA5 are identical to P. s. pvs. tabaci (Pta), glycinea and phaseolicola and share very high similarity with other pathovars of P. syringae. In contrast to the flagellin mutant PtaΔfliC, PA5ΔfliC grows as well as wild type in the host plant, but it elicits stronger HR than wild type does in non-host plants. Furthermore, the purified Pav flagellin, but not the divergent flagellin from Agrobacterium tumefaciens, is able to impair the HR induced by PA5ΔfliC. PA5Δfgt1 possessing nonglycosylated flagella behaved as its wild type in both bacterial growth in host and HR elicitation. Flagellin was infiltrated into tobacco leaves either simultaneously with flagellum-deficient HL1 or prior to the inoculation of wild type HL1, and both treatments impaired the HR induced by HL1. Moreover, the HR elicited by PA5 and PA5ΔfliC was enhanced by the addition of cycloheximide, suggesting that the flagellin is one of the PAMPs (pathogen-associated molecular patterns) contributed to induce the PAMP-triggered immunity (PTI). Taken together, the results shown in this study reveal that flagellin in Pav is capable of suppressing HR via PTI induction during an incompatible interaction. PMID:22911741

  11. Induction of cell death by tospoviral protein NSs and the motif critical for cell death does not control RNA silencing suppression activity.

    Science.gov (United States)

    Singh, Ajeet; Permar, Vipin; Jain, R K; Goswami, Suneha; Kumar, Ranjeet Ranjan; Canto, Tomas; Palukaitis, Peter; Praveen, Shelly

    2017-08-01

    Groundnut bud necrosis virus induces necrotic symptoms in different hosts. Previous studies showed reactive oxygen species-mediated programmed cell death (PCD) resulted in necrotic symptoms. Transgenic expression of viral protein NSs mimics viral symptoms. Here, we showed a role for NSs in influencing oxidative burst in the cell, by analyzing H 2 O 2 accumulation, activities of antioxidant enzymes and expression levels of vacuolar processing enzymes, H 2 O 2 -responsive microRNA 319a.2 plus its possible target metacaspase-8. The role of NSs in PCD, was shown using two NSs mutants: one in the Trp/GH3 motif (a homologue of pro-apototic domain) (NSs S189R ) and the other in a non-Trp/GH3 motif (NSs L172R ). Tobacco rattle virus (TRV) expressing NSs S189R enhanced the PCD response, but not TRV-NSs L172R , while RNA silencing suppression activity was lost in TRV-NSs L172R , but not in TRV-NSs S189R . Therefore, we propose dual roles of NSs in RNA silencing suppression and induction of cell death, controlled by different motifs. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Hydroxysafflor yellow A suppress oleic acid-induced acute lung injury via protein kinase A

    International Nuclear Information System (INIS)

    Wang, Chaoyun; Huang, Qingxian; Wang, Chunhua; Zhu, Xiaoxi; Duan, Yunfeng; Yuan, Shuai; Bai, Xianyong

    2013-01-01

    Inflammation response and oxidative stress play important roles in acute lung injury (ALI). Activation of the cAMP/protein kinase A (PKA) signaling pathway may attenuate ALI by suppressing immune responses and inhibiting the generation of reactive oxygen species (ROS). Hydroxysafflor yellow A (HSYA) is a natural flavonoid compound that reduces oxidative stress and inflammatory cytokine-mediated damage. In this study, we examined whether HSYA could protect the lungs from oleic acid (OA)-induced injury, which was used to mimic ALI, and determined the role of the cAMP/PKA signaling pathway in this process. Arterial oxygen tension (PaO 2 ), carbon dioxide tension, pH, and the PaO 2 /fraction of inspired oxygen ratio in the blood were detected using a blood gas analyzer. We measured wet/dry lung weight ratio and evaluated tissue morphology. The protein and inflammatory cytokine levels in the bronchoalveolar lavage fluid and serum were determined using enzyme-linked immunoassay. The activities of superoxide dismutase, glutathione peroxidase, PKA, and nicotinamide adenine dinucleotide phosphate oxidase, and the concentrations of cAMP and malondialdehyde in the lung tissue were detected using assay kits. Bcl-2, Bax, caspase 3, and p22 phox levels in the lung tissue were analyzed using Western blotting. OA increased the inflammatory cytokine and ROS levels and caused lung dysfunction by decreasing cAMP synthesis, inhibiting PKA activity, stimulating caspase 3, and reducing the Bcl-2/Bax ratio. H-89 increased these effects. HSYA significantly increased the activities of antioxidant enzymes, inhibited the inflammatory response via cAMP/PKA pathway activation, and attenuated OA-induced lung injury. Our results show that the cAMP/PKA signaling pathway is required for the protective effect of HSYA against ALI. - Highlights: • Oleic acid (OA) cause acute lung injury (ALI) via inhibiting cAMP/PKA signal pathway. • Blocking protein kinase A (PKA) activation may enhance Cytokine

  13. DNA-hosted copper nanoclusters/graphene oxide based fluorescent biosensor for protein kinase activity detection.

    Science.gov (United States)

    Wang, Mengke; Lin, Zihan; Liu, Qing; Jiang, Shan; Liu, Hua; Su, Xingguang

    2018-07-05

    A novel fluorescent biosensor for protein kinase activity (PKA) detection was designed by applying double-strands DNA-hosted copper nanoclusters (dsDNA-CuNCs) and graphene oxide (GO). One DNA strand of the dsDNA consisted of two domains, one domain can hybridize with another complementary DNA strand to stabilize the fluorescent CuNCs and another domain was adenosine 5'-triphosphate (ATP) aptamer. ATP aptamer of the dsDNA-CuNCs would be spontaneously absorbed onto the GO surface through π-π stacking interactions. Thus GO can efficiently quench the fluorescence (FL) of dsDNA-CuNCs through fluorescence resonance energy transfer (FRET). In the present of ATP, ATP specifically combined with ATP aptamer to form ATP-ATP aptamer binding complexes, which had much less affinity to GO, resulting in the fluorescence recovery of the system. Nevertheless, in the presence of PKA, ATP could be translated into ADP and ADP could not combine with ATP aptamer resulting in the fluorescence quenching of dsDNA-CuNCs again. According to the change of the fluorescence signal, PKA activity could be successfully monitored in the range of 0.1-5.0 U mL -1 with a detection limit (LOD) of 0.039 U mL -1 . Besides, the inhibitory effect of H-89 on PKA activity was studied. The sensor was performed for PKA activity detection in cell lysates with satisfactory results. Copyright © 2018 Elsevier B.V. All rights reserved.

  14. Comprehensive Identification of Immunodominant Proteins of Brucella abortus and Brucella melitensis Using Antibodies in the Sera from Naturally Infected Hosts

    Directory of Open Access Journals (Sweden)

    Gamal Wareth

    2016-04-01

    Full Text Available Brucellosis is a debilitating zoonotic disease that affects humans and animals. The diagnosis of brucellosis is challenging, as accurate species level identification is not possible with any of the currently available serology-based diagnostic methods. The present study aimed at identifying Brucella (B. species-specific proteins from the closely related species B. abortus and B. melitensis using sera collected from naturally infected host species. Unlike earlier reported investigations with either laboratory-grown species or vaccine strains, in the present study, field strains were utilized for analysis. The label-free quantitative proteomic analysis of the naturally isolated strains of these two closely related species revealed 402 differentially expressed proteins, among which 63 and 103 proteins were found exclusively in the whole cell extracts of B. abortus and B. melitensis field strains, respectively. The sera from four different naturally infected host species, i.e., cattle, buffalo, sheep, and goat were applied to identify the immune-binding protein spots present in the whole protein extracts from the isolated B. abortus and B. melitensis field strains and resolved on two-dimensional gel electrophoresis. Comprehensive analysis revealed that 25 proteins of B. abortus and 20 proteins of B. melitensis were distinctly immunoreactive. Dihydrodipicolinate synthase, glyceraldehyde-3-phosphate dehydrogenase and lactate/malate dehydrogenase from B. abortus, amino acid ABC transporter substrate-binding protein from B. melitensis and fumarylacetoacetate hydrolase from both species were reactive with the sera of all the tested naturally infected host species. The identified proteins could be used for the design of serological assays capable of detecting pan-Brucella, B. abortus- and B. melitensis-specific antibodies.

  15. Comprehensive Identification of Immunodominant Proteins of Brucella abortus and Brucella melitensis Using Antibodies in the Sera from Naturally Infected Hosts.

    Science.gov (United States)

    Wareth, Gamal; Eravci, Murat; Weise, Christoph; Roesler, Uwe; Melzer, Falk; Sprague, Lisa D; Neubauer, Heinrich; Murugaiyan, Jayaseelan

    2016-04-30

    Brucellosis is a debilitating zoonotic disease that affects humans and animals. The diagnosis of brucellosis is challenging, as accurate species level identification is not possible with any of the currently available serology-based diagnostic methods. The present study aimed at identifying Brucella (B.) species-specific proteins from the closely related species B. abortus and B. melitensis using sera collected from naturally infected host species. Unlike earlier reported investigations with either laboratory-grown species or vaccine strains, in the present study, field strains were utilized for analysis. The label-free quantitative proteomic analysis of the naturally isolated strains of these two closely related species revealed 402 differentially expressed proteins, among which 63 and 103 proteins were found exclusively in the whole cell extracts of B. abortus and B. melitensis field strains, respectively. The sera from four different naturally infected host species, i.e., cattle, buffalo, sheep, and goat were applied to identify the immune-binding protein spots present in the whole protein extracts from the isolated B. abortus and B. melitensis field strains and resolved on two-dimensional gel electrophoresis. Comprehensive analysis revealed that 25 proteins of B. abortus and 20 proteins of B. melitensis were distinctly immunoreactive. Dihydrodipicolinate synthase, glyceraldehyde-3-phosphate dehydrogenase and lactate/malate dehydrogenase from B. abortus, amino acid ABC transporter substrate-binding protein from B. melitensis and fumarylacetoacetate hydrolase from both species were reactive with the sera of all the tested naturally infected host species. The identified proteins could be used for the design of serological assays capable of detecting pan-Brucella, B. abortus- and B. melitensis-specific antibodies.

  16. Molecular characterization of Trypanosoma cruzi SAP proteins with host-cell lysosome exocytosis-inducing activity required for parasite invasion.

    Science.gov (United States)

    Zanforlin, Tamiris; Bayer-Santos, Ethel; Cortez, Cristian; Almeida, Igor C; Yoshida, Nobuko; da Silveira, José Franco

    2013-01-01

    To invade target cells, Trypanosoma cruzi metacyclic forms engage distinct sets of surface and secreted molecules that interact with host components. Serine-, alanine-, and proline-rich proteins (SAP) comprise a multigene family constituted of molecules with a high serine, alanine and proline residue content. SAP proteins have a central domain (SAP-CD) responsible for interaction with and invasion of mammalian cells by metacyclic forms. Using a 513 bp sequence from SAP-CD in blastn analysis, we identified 39 full-length SAP genes in the genome of T. cruzi. Although most of these genes were mapped in the T. cruzi in silico chromosome TcChr41, several SAP sequences were spread out across the genome. The level of SAP transcripts was twice as high in metacyclic forms as in epimastigotes. Monoclonal (MAb-SAP) and polyclonal (anti-SAP) antibodies produced against the recombinant protein SAP-CD were used to investigate the expression and localization of SAP proteins. MAb-SAP reacted with a 55 kDa SAP protein released by epimastigotes and metacyclic forms and with distinct sets of SAP variants expressed in amastigotes and tissue culture-derived trypomastigotes (TCTs). Anti-SAP antibodies reacted with components located in the anterior region of epimastigotes and between the nucleus and the kinetoplast in metacyclic trypomastigotes. In contrast, anti-SAP recognized surface components of amastigotes and TCTs, suggesting that SAP proteins are directed to different cellular compartments. Ten SAP peptides were identified by mass spectrometry in vesicle and soluble-protein fractions obtained from parasite conditioned medium. Using overlapping sequences from SAP-CD, we identified a 54-aa peptide (SAP-CE) that was able to induce host-cell lysosome exocytosis and inhibit parasite internalization by 52%. This study provides novel information about the genomic organization, expression and cellular localization of SAP proteins and proposes a triggering role for extracellular SAP

  17. Specific chlamydial inclusion membrane proteins associate with active Src family kinases in microdomains that interact with the host microtubule network.

    Science.gov (United States)

    Mital, Jeffrey; Miller, Natalie J; Fischer, Elizabeth R; Hackstadt, Ted

    2010-09-01

    Chlamydiae are Gram-negative obligate intracellular bacteria that cause diseases with significant medical and economic impact. Chlamydia trachomatis replicates within a vacuole termed an inclusion, which is extensively modified by the insertion of a number of bacterial effector proteins known as inclusion membrane proteins (Incs). Once modified, the inclusion is trafficked in a dynein-dependent manner to the microtubule-organizing centre (MTOC), where it associates with host centrosomes. Here we describe a novel structure on the inclusion membrane comprised of both host and bacterial proteins. Members of the Src family of kinases are recruited to the chlamydial inclusion in an active form. These kinases display a distinct, localized punctate microdomain-like staining pattern on the inclusion membrane that colocalizes with four chlamydial inclusion membrane proteins (Incs) and is enriched in cholesterol. Biochemical studies show that at least two of these Incs stably interact with one another. Furthermore, host centrosomes associate with these microdomain proteins in C. trachomatis-infected cells and in uninfected cells exogenously expressing one of the chlamydial effectors. Together, the data suggest that a specific structure on the C. trachomatis inclusion membrane may be responsible for the known interactions of chlamydiae with the microtubule network and resultant effects on centrosome stability.

  18. Mutations in Encephalomyocarditis Virus 3A Protein Uncouple the Dependency of Genome Replication on Host Factors Phosphatidylinositol 4-Kinase IIIα and Oxysterol-Binding Protein

    NARCIS (Netherlands)

    Dorobantu, Cristina M|info:eu-repo/dai/nl/372622283; Albulescu, Lucian|info:eu-repo/dai/nl/369492382; Lyoo, Heyrhyoung|info:eu-repo/dai/nl/412352931; van Kampen, Mirjam; De Francesco, Raffaele; Lohmann, Volker; Harak, Christian; van der Schaar, Hilde M|info:eu-repo/dai/nl/318007568; Strating, Jeroen R P M|info:eu-repo/dai/nl/298979594; Gorbalenya, Alexander E; van Kuppeveld, Frank J M|info:eu-repo/dai/nl/156614723

    2016-01-01

    Positive-strand RNA [(+)RNA] viruses are true masters of reprogramming host lipid trafficking and synthesis to support virus genome replication. Via their membrane-associated 3A protein, picornaviruses of the genus Enterovirus (e.g., poliovirus, coxsackievirus, and rhinovirus) subvert Golgi

  19. Three genes for mitochondrial proteins suppress null-mutations in both Afg3 and Rca1 when over-expressed.

    Science.gov (United States)

    Rep, M; Nooy, J; Guélin, E; Grivell, L A

    1996-08-01

    The AFG3 gene of Saccharomyces cerevisiae encodes a mitochondrial inner membrane protein with ATP-dependent protease activity. To gain more insight into the function of this protein, multi-copy suppressors of an afg3-null mutation were isolated. Three genes were found that restored partial growth on non-fermentable carbon sources, all of which affect the biogenesis of respiratory competent mitochondria: PIM1(LON) encodes a matrix-localized ATP-dependent protease involved in the turnover of matrix proteins; OXA1(PET1402) encodes a putative mitochondrial inner membrane protein involved in the biogenesis of the respiratory chain; and MBA1 encodes a mitochondrial protein required for optimal respiratory growth. All three genes also suppressed a null mutation in a related gene, RCA1, as well as in the combination of afg3- and rca1-null.

  20. Endoglin-mediated suppression of prostate cancer invasion is regulated by activin and bone morphogenetic protein type II receptors.

    Directory of Open Access Journals (Sweden)

    Michael J Breen

    Full Text Available Mortality from prostate cancer (PCa is due to the formation of metastatic disease. Understanding how that process is regulated is therefore critical. We previously demonstrated that endoglin, a type III transforming growth factor β (TGFβ superfamily receptor, suppresses human PCa cell invasion and metastasis. Endoglin-mediated suppression of invasion was also shown by us to be dependent upon the type I TGFβ receptor, activin receptor-like kinase 2 (ALK2, and the downstream effector, Smad1. In this study we demonstrate for the first time that two type II TGFβ receptors are required for endoglin-mediated suppression of invasion: activin A receptor type IIA (ActRIIA and bone morphogenetic protein receptor type II (BMPRII. Downstream signaling through these receptors is predominantly mediated by Smad1. ActRIIA stimulates Smad1 activation in a kinase-dependent manner, and this is required for suppression of invasion. In contrast BMPRII regulates Smad1 in a biphasic manner, promoting Smad1 signaling through its kinase domain but suppressing it through its cytoplasmic tail. BMPRII's Smad1-regulatory effects are dependent upon its expression level. Further, its ability to suppress invasion is independent of either kinase function or tail domain. We demonstrate that ActRIIA and BMPRII physically interact, and that each also interacts with endoglin. The current findings demonstrate that both BMPRII and ActRIIA are necessary for endoglin-mediated suppression of human PCa cell invasion, that they have differential effects on Smad1 signaling, that they make separate contributions to regulation of invasion, and that they functionally and physically interact.

  1. Increased plasma ghrelin suppresses insulin release in wethers fed with a high-protein diet.

    Science.gov (United States)

    Takahashi, T; Sato, K; Kato, S; Yonezawa, T; Kobayashi, Y; Ohtani, Y; Ohwada, S; Aso, H; Yamaguchi, T; Roh, S G; Katoh, K

    2014-06-01

    Ghrelin is a multifunctional peptide that promotes an increase of food intake and stimulates GH secretion. Ghrelin secretion is regulated by nutritional status and nutrients. Although a high-protein (HP) diet increases plasma ghrelin secretion in mammals, the mechanisms and the roles of the elevated ghrelin concentrations due to a HP diet have not been fully established. To clarify the roles of elevated acylated ghrelin upon intake of a HP diet, we investigated the regulation of ghrelin concentrations in plasma and tissues in wethers fed with either the HP diet or the control (CNT) diet for 14 days, and examined the action of the elevated plasma ghrelin by using a ghrelin-receptor antagonist. The HP diet gradually increased the plasma acylated-ghrelin concentrations, but the CNT diet did not. Although the GH concentrations did not vary significantly across the groups, an injection of ghrelin-receptor antagonist enhanced insulin levels in circulation in the HP diet group. In the fundus region of the stomach, the ghrelin levels did not differ between the HP and CNT diet groups, whereas ghrelin O-acyltransferase mRNA levels were higher in the group fed with HP diet than those of the CNT diet group were. These results indicate that the HP diet elevated the plasma ghrelin levels by increasing its synthesis; this elevation strongly suppresses the appearance of insulin in the circulation of wethers, but it is not involved in GH secretion. Overall, our findings indicate a role of endogenous ghrelin action in secretion of insulin, which acts as a regulator after the consumption of a HP diet. © 2014 Society for Endocrinology.

  2. Brugia malayi excreted/secreted proteins at the host/parasite interface: stage- and gender-specific proteomic profiling.

    Directory of Open Access Journals (Sweden)

    Sasisekhar Bennuru

    Full Text Available Relatively little is known about the filarial proteins that interact with the human host. Although the filarial genome has recently been completed, protein profiles have been limited to only a few recombinants or purified proteins of interest. Here, we describe a large-scale proteomic analysis using microcapillary reverse-phase liquid chromatography-tandem-mass spectrometry to identify the excretory-secretory (ES products of the L3, L3 to L4 molting ES, adult male, adult female, and microfilarial stages of the filarial parasite Brugia malayi. The analysis of the ES products from adult male, adult female, microfilariae (Mf, L3, and molting L3 larvae identified 852 proteins. Annotation suggests that the functional and component distribution was very similar across each of the stages studied; however, the Mf contributed a higher proportion to the total number of identified proteins than the other stages. Of the 852 proteins identified in the ES, only 229 had previous confirmatory expressed sequence tags (ESTs in the available databases. Moreover, this analysis was able to confirm the presence of 274 "hypothetical" proteins inferred from gene prediction algorithms applied to the B. malayi (Bm genome. Not surprisingly, the majority (160/274 of these "hypothetical" proteins were predicted to be secreted by Signal IP and/or SecretomeP 2.0 analysis. Of major interest is the abundance of previously characterized immunomodulatory proteins such as ES-62 (leucyl aminopeptidase, MIF-1, SERPIN, glutathione peroxidase, and galectin in the ES of microfilariae (and Mf-containing adult females compared to the adult males. In addition, searching the ES protein spectra against the Wolbachia database resulted in the identification of 90 Wolbachia-specific proteins, most of which were metabolic enzymes that have not been shown to be immunogenic. This proteomic analysis extends our knowledge of the ES and provides insight into the host-parasite interaction.

  3. Inhibition of host protein synthesis by Sindbis virus: correlation with viral RNA replication and release of nuclear proteins to the cytoplasm.

    Science.gov (United States)

    Sanz, Miguel A; García-Moreno, Manuel; Carrasco, Luis

    2015-04-01

    Infection of mammalian cells by Sindbis virus (SINV) profoundly blocks cellular mRNA translation. Experimental evidence points to viral non-structural proteins (nsPs), in particular nsP2, as the mediator of this inhibition. However, individual expression of nsP1, nsP2, nsP3 or nsP1-4 does not block cellular protein synthesis in BHK cells. Trans-complementation of a defective SINV replicon lacking most of the coding region for nsPs by the co-expression of nsP1-4 propitiates viral RNA replication at low levels, and inhibition of cellular translation is not observed. Exit of nuclear proteins including T-cell intracellular antigen and polypyrimidine tract-binding protein is clearly detected in SINV-infected cells, but not upon the expression of nsPs, even when the defective replicon was complemented. Analysis of a SINV variant with a point mutation in nsP2, exhibiting defects in the shut-off of host protein synthesis, indicates that both viral RNA replication and the release of nuclear proteins to the cytoplasm are greatly inhibited. Furthermore, nucleoside analogues that inhibit cellular and viral RNA synthesis impede the blockade of host mRNA translation, in addition to the release of nuclear proteins. Prevention of the shut-off of host mRNA translation by nucleoside analogues is not due to the inhibition of eIF2α phosphorylation, as this prevention is also observed in PKR(-/-) mouse embryonic fibroblasts that do not phosphorylate eIF2α after SINV infection. Collectively, our observations are consistent with the concept that for the inhibition of cellular protein synthesis to occur, viral RNA replication must take place at control levels, leading to the release of nuclear proteins to the cytoplasm. © 2014 John Wiley & Sons Ltd.

  4. Host range restriction of vaccinia virus in Chinese hamster ovary cells: relationship to shutoff of protein synthesis

    International Nuclear Information System (INIS)

    Drillien, R.; Spehner, D.; Kirn, A.

    1978-01-01

    Chinese hamster ovary cells were found to be nonpermissive for vaccinia virus. Although early virus-induced events occurred in these cells (RNA and polypeptide synthesis), subsequent events appeared to be prevented by a very rapid and nonselective shutoff of protein synthesis. Within less than 2 h after infection, both host and viral protein syntheses were arrested. At low multiplicities of infection, inhibition of RNA synthesis with cordycepin resulted in failure of the virus to block protein synthesis. Moreover, infection of the cells in the presence of cycloheximide prevented the immediate onset of shutoff after reversal of cycloheximide. Inactivation of virus particles by uv irradiation also impaired the capacity of the virus to inhibit protein synthesis. These results suggested that an early vaccinia virus-coded product was implicated in the shutoff of protein synthesis. Either the nonpermissive Chinese hamster ovary cells were more sensitive to this inhibition than permissive cells, or a regulatory control of the vaccinia shutoff function was defective

  5. MvaT Family Proteins Encoded on IncP-7 Plasmid pCAR1 and the Host Chromosome Regulate the Host Transcriptome Cooperatively but Differently.

    Science.gov (United States)

    Yun, Choong-Soo; Takahashi, Yurika; Shintani, Masaki; Takeda, Toshiharu; Suzuki-Minakuchi, Chiho; Okada, Kazunori; Yamane, Hisakazu; Nojiri, Hideaki

    2016-02-01

    MvaT proteins are members of the H-NS family of proteins in pseudomonads. The IncP-7 conjugative plasmid pCAR1 carries an mvaT-homologous gene, pmr. In Pseudomonas putida KT2440 bearing pCAR1, pmr and the chromosomally carried homologous genes, turA and turB, are transcribed at high levels, and Pmr interacts with TurA and TurB in vitro. In the present study, we clarified how the three MvaT proteins regulate the transcriptome of P. putida KT2440(pCAR1). Analyses performed by a modified chromatin immunoprecipitation assay with microarray technology (ChIP-chip) suggested that the binding regions of Pmr, TurA, and TurB in the P. putida KT2440(pCAR1) genome are almost identical; nevertheless, transcriptomic analyses using mutants with deletions of the genes encoding the MvaT proteins during the log and early stationary growth phases clearly suggested that their regulons were different. Indeed, significant regulon dissimilarity was found between Pmr and the other two proteins. Transcription of a larger number of genes was affected by Pmr deletion during early stationary phase than during log phase, suggesting that Pmr ameliorates the effects of pCAR1 on host fitness more effectively during the early stationary phase. Alternatively, the similarity of the TurA and TurB regulons implied that they might play complementary roles as global transcriptional regulators in response to plasmid carriage. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  6. Myostatin inhibits eEF2K-eEF2 by regulating AMPK to suppress protein synthesis.

    Science.gov (United States)

    Deng, Zhao; Luo, Pei; Lai, Wen; Song, Tongxing; Peng, Jian; Wei, Hong-Kui

    2017-12-09

    Growth of skeletal muscle is dependent on the protein synthesis, and the rate of protein synthesis is mainly regulated in the stage of translation initiation and elongation. Myostatin, a member of the transforming growth factor-β (TGF-β) superfamily, is a negative regulator of protein synthesis. C2C12 myotubes was incubated with 0, 0.01, 0.1, 1, 2, 3 μg/mL myostatin recombinant protein, and then we detected the rates of protein synthesis by the method of SUnSET. We found that high concentrations of myostatin (2 and 3 μg/mL) inhibited protein synthesis by blocking mTOR and eEF2K-eEF2 pathway, while low concentration of myostatin (0.01, 0.1 and 1 μg/mL) regulated eEF2K-eEF2 pathway activity to block protein synthesis without affected mTOR pathway, and myostatin inhibited eEF2K-eEF2 pathway through regulating AMPK pathway to suppress protein synthesis. It provided a new mechanism for myostatin regulating protein synthesis and treating muscle atrophy. Copyright © 2017. Published by Elsevier Inc.

  7. Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

    Science.gov (United States)

    Kuss, Sharon K.; Mata, Miguel A.; Zhang, Liang; Fontoura, Beatriz M. A.

    2013-01-01

    Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA) that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses. PMID:23872491

  8. Protein interaction networks at the host-microbe interface in Diaphorina citri, the insect vector of the citrus greening pathogen.

    Science.gov (United States)

    Ramsey, J S; Chavez, J D; Johnson, R; Hosseinzadeh, S; Mahoney, J E; Mohr, J P; Robison, F; Zhong, X; Hall, D G; MacCoss, M; Bruce, J; Cilia, M

    2017-02-01

    The Asian citrus psyllid ( Diaphorina citri) is the insect vector responsible for the worldwide spread of ' Candidatus Liberibacter asiaticus' (CLas), the bacterial pathogen associated with citrus greening disease. Developmental changes in the insect vector impact pathogen transmission, such that D. citri transmission of CLas is more efficient when bacteria are acquired by nymphs when compared with adults. We hypothesize that expression changes in the D. citri immune system and commensal microbiota occur during development and regulate vector competency. In support of this hypothesis, more proteins, with greater fold changes, were differentially expressed in response to CLas in adults when compared with nymphs, including insect proteins involved in bacterial adhesion and immunity. Compared with nymphs, adult insects had a higher titre of CLas and the bacterial endosymbionts Wolbachia, Profftella and Carsonella. All Wolbachia and Profftella proteins differentially expressed between nymphs and adults are upregulated in adults, while most differentially expressed Carsonella proteins are upregulated in nymphs. Discovery of protein interaction networks has broad applicability to the study of host-microbe relationships. Using protein interaction reporter technology, a D. citri haemocyanin protein highly upregulated in response to CLas was found to physically interact with the CLas coenzyme A (CoA) biosynthesis enzyme phosphopantothenoylcysteine synthetase/decarboxylase. CLas pantothenate kinase, which catalyses the rate-limiting step of CoA biosynthesis, was found to interact with a D. citri myosin protein. Two Carsonella enzymes involved in histidine and tryptophan biosynthesis were found to physically interact with D. citri proteins. These co-evolved protein interaction networks at the host-microbe interface are highly specific targets for controlling the insect vector responsible for the spread of citrus greening.

  9. Suppression of adhesion-induced protein tyrosine phosphorylation decreases invasive and metastatic potentials of B16-BL6 melanoma cells by protein tyrosine kinase inhibitor genistein.

    Science.gov (United States)

    Yan, C; Han, R

    1997-01-01

    Protein tyrosine kinase (PTK) appears to be involved in the activation of signaling during cell attachment to and spreading on extracellular matrix (ECM) in the metastatic cascade. To verify the assumption that PTK inhibitors might impair ECM signaling and prevent cancer metastasis, the highly metastatic B16-BL6 mouse melanoma cells were exposed to the PTK inhibitor genistein for 3 days. The ability of the cells to invade through reconstituted basement membrane (Matrigel) and to establish experimental pulmonary metastatic foci in C57BL/6 mice decreased after genistein exposure. The genistein-treated cells were also prevented from attaching to Matrigel and spread extremely poorly on the ECM substratum. Immunoblot analysis showed that tyrosine phosphorylation of a 125-kD protein in response to cell spreading on Matrigel was suppressed in the genistein-treated cells. Adhesion-induced protein tyrosine phosphorylation represents the earlier and specific event in the activation of ECM signaling, so this result implied ECM signaling was impaired in the treated cells. With immunofluorescence microscopy, the adhesion-induced tyrosine phosphorylated proteins were located at the pericytoplasms of well-spread cells, but not at the periphery of poorly spread genistein-treated cells. Therefore, this paper suggests that genistein might impair ECM signaling and subsequently prevent cancer cells from spreading well and invading or establishing metastasis through the suppression of adhesion-induced protein tyrosine phosphorylation. PTKs and adhesion-induced protein tyrosine phosphorylation might play a role in the control of invasion and metastasis.

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

    Science.gov (United States)

    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...

  11. Transcriptome analysis reveals the host response to Schmallenberg virus in bovine cells and antagonistic effects of the NSs protein.

    Science.gov (United States)

    Blomström, Anne-Lie; Gu, Quan; Barry, Gerald; Wilkie, Gavin; Skelton, Jessica K; Baird, Margaret; McFarlane, Melanie; Schnettler, Esther; Elliott, Richard M; Palmarini, Massimo; Kohl, Alain

    2015-04-19

    Schmallenberg virus (SBV) is a member of the Orthobunyavirus genus (Bunyaviridae family) causing malformations and abortions in ruminants. Although, as for other members of this family/genus, the non-structural protein NSs has been shown to be an interferon antagonist, very little is known regarding the overall inhibitory effects and targets of orthobunyavirus NSs proteins on host gene expression during infection. Therefore, using RNA-seq this study describes changes to the transcriptome of primary bovine cells following infection with Schmallenberg virus (SBV) or with a mutant lacking the non-structural protein NSs (SBVdelNSs) providing a detailed comparison of the effect of NSs expression on the host cell. The sequence reads from all samples (uninfected cells, SBV and SBVdelNSs) assembled well to the bovine host reference genome (on average 87.43% of the reads). During infection with SBVdelNSs, 649 genes were differentially expressed compared to uninfected cells (78.7% upregulated) and many of these were known antiviral and IFN-stimulated genes. On the other hand, only nine genes were differentially expressed in SBV infected cells compared to uninfected control cells, demonstrating the strong inhibitory effect of NSs on cellular gene expression. However, the majority of the genes that were expressed during SBV infection are involved in restriction of viral replication and spread indicating that SBV does not completely manage to shutdown the host antiviral response. In this study we show the effects of SBV NSs on the transcriptome of infected cells as well as the cellular response to wild type SBV. Although NSs is very efficient in shutting down genes of the host innate response, a number of possible antiviral factors were identified. Thus the data from this study can serve as a base for more detailed mechanistic studies of SBV and other orthobunyaviruses.

  12. The Importance of the KR-Rich Region of the Coat Protein of Ourmia melon virus for Host Specificity, Tissue Tropism, and Interference With Antiviral Defense.

    Science.gov (United States)

    Rossi, Marika; Vallino, Marta; Abbà, Simona; Ciuffo, Marina; Balestrini, Raffaella; Genre, Andrea; Turina, Massimo

    2015-01-01

    The N-terminal region of the Ourmia melon virus (OuMV) coat protein (CP) contains a short lysine/arginine-rich (KR) region. By alanine scanning mutagenesis, we showed that the KR region influences pathogenicity and virulence of OuMV without altering viral particle assembly. A mutant, called OuMV6710, with three basic residue substitutions in the KR region, was impaired in the ability to maintain the initial systemic infection in Nicotiana benthamiana and to infect both cucumber and melon plants systemically. The integrity of this protein region was also crucial for encapsidation of viral genomic RNA; in fact, certain mutations within the KR region partially compromised the RNA encapsidation efficiency of the CP. In Arabidopsis thaliana Col-0, OuMV6710 was impaired in particle accumulation; however, this phenotype was abolished in dcl2/dcl4 and dcl2/dcl3/dcl4 Arabidopsis mutants defective for antiviral silencing. Moreover, in contrast to CPwt, in situ immunolocalization experiments indicated that CP6710 accumulates efficiently in the spongy mesophyll tissue of infected N. benthamiana and A. thaliana leaves but only occasionally infects palisade tissues. These results provided strong evidence of a crucial role for OuMV CP during viral infection and highlighted the relevance of the KR region in determining tissue tropism, host range, pathogenicity, and RNA affinity, which may be all correlated with a possible CP silencing-suppression activity.

  13. Suppression of NYVAC Infection in HeLa Cells Requires RNase L but Is Independent of Protein Kinase R Activity

    Science.gov (United States)

    Fernández-Escobar, Mercedes; Nájera, José Luis; Baldanta, Sara; Rodriguez, Dolores; Way, Michael; Esteban, Mariano

    2015-01-01

    Protein kinase R (PKR) and RNase L are host cell components that function to contain viral spread after infections. In this study, we analyzed the role of both proteins in the abortive infection of human HeLa cells with the poxvirus strain NYVAC, for which an inhibition of viral A27L and B5R gene expression is described. Specifically, the translation of these viral genes is independent of PKR activation, but their expression is dependent on the RNase L activity. PMID:26656695

  14. Surface-layer protein A (SlpA is a major contributor to host-cell adherence of Clostridium difficile.

    Directory of Open Access Journals (Sweden)

    Michelle M Merrigan

    Full Text Available Clostridium difficile is a leading cause of antibiotic-associated diarrhea, and a significant etiologic agent of healthcare-associated infections. The mechanisms of attachment and host colonization of C. difficile are not well defined. We hypothesize that non-toxin bacterial factors, especially those facilitating the interaction of C. difficile with the host gut, contribute to the initiation of C. difficile infection. In this work, we optimized a completely anaerobic, quantitative, epithelial-cell adherence assay for vegetative C. difficile cells, determined adherence proficiency under multiple conditions, and investigated C. difficile surface protein variation via immunological and DNA sequencing approaches focused on Surface-Layer Protein A (SlpA. In total, thirty-six epidemic-associated and non-epidemic associated C. difficile clinical isolates were tested in this study, and displayed intra- and inter-clade differences in attachment that were unrelated to toxin production. SlpA was a major contributor to bacterial adherence, and individual subunits of the protein (varying in sequence between strains mediated host-cell attachment to different extents. Pre-treatment of host cells with crude or purified SlpA subunits, or incubation of vegetative bacteria with anti-SlpA antisera significantly reduced C. difficile attachment. SlpA-mediated adherence-interference correlated with the attachment efficiency of the strain from which the protein was derived, with maximal blockage observed when SlpA was derived from highly adherent strains. In addition, SlpA-containing preparations from a non-toxigenic strain effectively blocked adherence of a phylogenetically distant, epidemic-associated strain, and vice-versa. Taken together, these results suggest that SlpA plays a major role in C. difficile infection, and that it may represent an attractive target for interventions aimed at abrogating gut colonization by this pathogen.

  15. NSs protein of severe fever with thrombocytopenia syndrome virus suppresses interferon production through different mechanism than Rift Valley fever virus.

    Science.gov (United States)

    Zhang, S; Zheng, B; Wang, T; Li, A; Wan, J; Qu, J; Li, C H; Li, D; Liang, M

    Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly identified Phlebovirus that causes severe fever with thrombocytopenia syndrome. Our study demonstrated that SFTSV NSs functioned as IFN antagonist mainly by suppressing TBK1/IKKε-IRF3 signaling pathway. NSs interacted with and relocalized TANK-binding kinase 1 (TBK1) into NSs-induced cytoplasmic structures and this interaction could effectively inhibit downstream phosphorylation and dimerization of interferon regulatory factor 3 (IRF3), resulting in the suppression of antiviral signaling and IFN induction. Functional sites of SFTSV NSs binding with TBK1 were then studied and results showed that NSs had lost their IFN-inhibiting activity after deleting the 25 amino acids in N-terminal. Furthermore, the mechanism of Rift Valley fever virus (RVFV) NSs blocking IFN-β response were also investigated. Preliminary results showed that RVFV NSs proteins could neither interact nor co-localize with TBK1 in cytoplasm, but suppressed its expression levels, phosphorylation and dimerization of IRF3 in the subsequent steps, resulting in inhibition of the IFN-β production. Altogether, our data demonstrated the probable mechanism used by SFTSV to inhibit IFN responses which was different from RVFV and pointed toward a novel mechanism for RVFV suppressing IFN responses.

  16. Comparative and functional genomics of Legionella identified eukaryotic like proteins as key players in host-pathogen interactions

    Directory of Open Access Journals (Sweden)

    Laura eGomez-Valero

    2011-10-01

    Full Text Available Although best known for its ability to cause severe pneumonia in people whose immune defenses are weakened, Legionella pneumophila and Legionella longbeachae are two species of a large genus of bacteria that are ubiquitous in nature, where they parasitize protozoa. Adaptation to the host environment and exploitation of host cell functions are critical for the success of these intracellular pathogens. The establishment and publication of the complete genome sequences of L. pneumophila and L. longbeachae isolates paved the way for major breakthroughs in understanding the biology of these organisms. In this review we present the knowledge gained from the analyses and comparison of the complete genome sequences of different L. pneumophila and L. longbeachae strains. Emphasis is given on putative virulence and Legionella life cycle related functions, such as the identification of an extended array of eukaryotic-like proteins, many of which have been shown to modulate host cell functions to the pathogen's advantage. Surprisingly, many of the eukaryotic domain proteins identified in L. pneumophila as well as many substrates of the Dot/Icm type IV secretion system essential for intracellular replication are different between these two species, although they cause the same disease. Finally, evolutionary aspects regarding the eukaryotic like proteins in Legionella are discussed.

  17. Host protein Snapin interacts with human cytomegalovirus pUL130 ...

    Indian Academy of Sciences (India)

    2016-04-07

    Apr 7, 2016 ... The interplay between the host and Human cytomegalovirus (HCMV) plays a pivotal role in the outcome of an infection. ... ed from infected cells but is incorporated into the virion envelope in a ..... Fields virology 5th ed.

  18. Smart protein biogate as a mediator to regulate competitive host-guest interaction for sensitive ratiometric electrochemical assay of prion

    Science.gov (United States)

    Yu, Peng; Zhang, Xiaohua; Zhou, Jiawan; Xiong, Erhu; Li, Xiaoyu; Chen, Jinhua

    2015-11-01

    A novel competitive host-guest strategy regulated by protein biogate was developed for sensitive and selective analysis of prion protein. The methylene blue (MB)-tagged prion aptamer (MB-Apt) was introduced to the multiwalled carbon nanotubes-β-cyclodextrins (MWCNTs-β-CD) composites-modified glassy carbon (GC) electrode through the host-guest interaction between β-CD and MB. In the absence of prion, MB-Apt could be displaced by ferrocenecarboxylic acid (FCA) due to its stronger binding affinity to β-CD, resulting in a large oxidation peak of FCA. However, in the presence of prion, the specific prion-aptamer interaction drove the formation of protein biogate to seal the cavity of β-CD, which hindered the guest displacement of MB by FCA and resulted in the oxidation peak current of MB (IMB) increased and that of FCA (IFCA) decreased. The developed aptasensor showed good response towards the target (prion protein) with a low detection limit of 160 fM. By changing the specific aptamers, this strategy could be easily extended to detect other proteins, showing promising potential for extensive applications in bioanalysis.

  19. Antibody-mediated suppression of grafted lymphoma. III. Evaluation of the role of thymic function, non-thymus-derived lymphocytes, macrophages, platelets, and polymorphonuclear leukocytes in syngeneic and allogeneic hosts

    International Nuclear Information System (INIS)

    Shin, H.S.; Hayden, M.; Langley, S.; Kaliss, N.; Smith, M.R.

    1975-01-01

    Syngeneic or allogeneic mice pretreated with sublethal whole-body irradiation were rendered incapable of suppressing the growth of grafted tumor cells sensitized with alloantibody. The growth of sensitized tumor cells was suppressed when they were mixed with donor effector cells from mice syngeneic or allogeneic to the recipients and then were inoculated in irradiated recipients. Three donor-host combinations were used to study the suppression of the murine lymphoma 6C3HED indigenous to C3H mice. These were C3H donor cells in C3H recipients, C57BL/6 donor cells in C3H recipients, or C57BL/6 donor cells in C57BL/6 recipients. In all three combinations, macrophages obtained from an inflammatory exudate, exudate lymphocytes not bearing theta antigen, and platelets were, in descending order of effectiveness, consistently active in restoring antibody-mediated suppression of tumor growth in irradiated hosts. Prior irradiation of the transferred lymphocytes somewhat diminished their effectiveness. Freeze-thawed or heat-killed macrophages (but not freeze-thawed platelets or lymphocytes) were effective in restoration. Peripheral blood mononuclear leukocytes and splenic lymphoid cells were not active in the recipients syngeneic to the donor cells but were active in recipients allogeneic to the donor cells. Polymorphonuclear leukocytes isolated from peripheral blood or an inflammatory exudate were not active. Intact thymic function seems unimportant since antibody-mediated suppression took place as effectively in thymectomized mice as in normal controls. (U.S.)

  20. The E-Id Protein Axis Specifies Adaptive Lymphoid Cell Identity and Suppresses Thymic Innate Lymphoid Cell Development.

    Science.gov (United States)

    Miyazaki, Masaki; Miyazaki, Kazuko; Chen, Kenian; Jin, Yi; Turner, Jacob; Moore, Amanda J; Saito, Rintaro; Yoshida, Kenichi; Ogawa, Seishi; Rodewald, Hans-Reimer; Lin, Yin C; Kawamoto, Hiroshi; Murre, Cornelis

    2017-05-16

    Innate and adaptive lymphoid development is orchestrated by the activities of E proteins and their antagonist Id proteins, but how these factors regulate early T cell progenitor (ETP) and innate lymphoid cell (ILC) development remains unclear. Using multiple genetic strategies, we demonstrated that E proteins E2A and HEB acted in synergy in the thymus to establish T cell identity and to suppress the aberrant development of ILCs, including ILC2s and lymphoid-tissue-inducer-like cells. E2A and HEB orchestrated T cell fate and suppressed the ILC transcription signature by activating the expression of genes associated with Notch receptors, T cell receptor (TCR) assembly, and TCR-mediated signaling. E2A and HEB acted in ETPs to establish and maintain a T-cell-lineage-specific enhancer repertoire, including regulatory elements associated with the Notch1, Rag1, and Rag2 loci. On the basis of these and previous observations, we propose that the E-Id protein axis specifies innate and adaptive lymphoid cell fate. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Novel Burkholderia mallei Virulence Factors Linked to Specific Host-Pathogen Protein Interactions

    Science.gov (United States)

    2013-06-23

    equine hosts. Thus, the genes retained in B. mallei share a high sequence similarity to genes common to B. pseudomallei (3), and many virulence...oppor- tunistic infections in mammalian hosts. Even for the equine - adapted and, thus, more genetically constrained, B. mallei pathogen, we cannot...BioDrugs: Clin. Immunotherapeut., Biopharmaceut. Gene Therapy 17, 413–424 88. Anderson, D. M., and Frank, D. W. (2012) Five mechanisms of manipula

  2. Plant RNA binding proteins for control of RNA virus infection

    Directory of Open Access Journals (Sweden)

    Sung Un eHuh

    2013-12-01

    Full Text Available Plant RNA viruses have effective strategies to infect host plants through either direct or indirect interactions with various host proteins, thus suppressing the host immune system. When plant RNA viruses enter host cells exposed RNAs of viruses are recognized by the host immune system through processes such as siRNA-dependent silencing. Interestingly, some host RNA binding proteins have been involved in the inhibition of RNA virus replication, movement, and translation through RNA-specific binding. Host plants intensively use RNA binding proteins for defense against viral infections in nature. In this mini review, we will summarize the function of some host RNA binding proteins which act in a sequence-specific binding manner to the infecting virus RNA. It is important to understand how plants effectively suppresses RNA virus infections via RNA binding proteins, and this defense system can be potentially developed as a synthetic virus defense strategy for use in crop engineering.

  3. The secreted antifungal protein thionin 2.4 in Arabidopsis thaliana suppresses the toxicity of a fungal fruit body lectin from Fusarium graminearum.

    Directory of Open Access Journals (Sweden)

    Tomoya Asano

    Full Text Available Plants possess active defense systems and can protect themselves from pathogenic invasion by secretion of a variety of small antimicrobial or antifungal proteins such as thionins. The antibacterial and antifungal properties of thionins are derived from their ability to induce open pore formation on cell membranes of phytopathogens, resulting in release of potassium and calcium ions from the cell. Wheat thionin also accumulates in the cell walls of Fusarium-inoculated plants, suggesting that it may have a role in blocking pathogen infection at the plant cell walls. Here we developed an anti-thionin 2.4 (Thi2.4 antibody and used it to show that Thi2.4 is localized in the cell walls of Arabidopsis and cell membranes of F. graminearum, when flowers are inoculated with F. graminearum. The Thi2.4 protein had an antifungal effect on F. graminearum. Next, we purified the Thi2.4 protein, conjugated it with glutathione-S-transferase (GST and coupled the proteins to an NHS-activated column. Total protein from F. graminearum was applied to GST-Thi2.4 or Thi2.4-binding columns, and the fungal fruit body lectin (FFBL of F. graminearum was identified as a Thi2.4-interacting protein. This interaction was confirmed by a yeast two-hybrid analysis. To investigate the biological function of FFBL, we infiltrated the lectin into Arabidopsis leaves and observed that it induced cell death in the leaves. Application of FFBL at the same time as inoculation with F. graminearum significantly enhanced the virulence of the pathogen. By contrast, FFBL-induced host cell death was effectively suppressed in transgenic plants that overexpressed Thi2.4. We found that a 15 kD Thi2.4 protein was specifically expressed in flowers and flower buds and suggest that it acts not only as an antifungal peptide, but also as a suppressor of the FFBL toxicity. Secreted thionin proteins are involved in this dual defense mechanism against pathogen invasion at the plant-pathogen interface.

  4. A versatile selection system for folding competent proteins using genetic complementation in a eukaryotic host

    DEFF Research Database (Denmark)

    Lyngsø, C.; Kjaerulff, S.; Muller, S.

    2010-01-01

    in vivo selection system for folded proteins. It is based on genetic complementation of the Schizosaccharomyces pombe growth marker gene invertase fused C-terminally to a protein library. The fusion proteins are directed to the secretion system, utilizing the ability of the eukaryotic protein quality...

  5. Suppression of phospholipid biosynthesis by cerulenin in the condensed Single-Protein-Production (cSPP) system

    International Nuclear Information System (INIS)

    Mao, Lili; Inoue, Koichi; Tao, Yisong; Montelione, Gaetano T.; McDermott, Ann E.; Inouye, Masayori

    2011-01-01

    Using the single-protein-production (SPP) system, a protein of interest can be exclusively produced in high yield from its ACA-less gene in Escherichia coli expressing MazF, an ACA-specific mRNA interferase. It is thus feasible to study a membrane protein by solid-state NMR (SSNMR) directly in natural membrane fractions. In developing isotope-enrichment methods, we observed that 13 C was also incorporated into phospholipids, generating spurious signals in SSNMR spectra. Notable, with the SPP system a protein can be produced in total absence of cell growth caused by antibiotics. Here, we demonstrate that cerulenin, an inhibitor of phospholipid biosynthesis, can suppress isotope incorporation in the lipids without affecting membrane protein yield in the SPP system. SSNMR analysis of ATP synthase subunit c, an E. coli inner membrane protein, produced by the SPP method using cerulenin revealed that 13 C resonance signals from phospholipid were markedly reduced, while signals for the isotope-enriched protein were clearly present.

  6. The interactomes of influenza virus NS1 and NS2 proteins identify new host factors and provide insights for ADAR1 playing a supportive role in virus replication.

    Science.gov (United States)

    de Chassey, Benoît; Aublin-Gex, Anne; Ruggieri, Alessia; Meyniel-Schicklin, Laurène; Pradezynski, Fabrine; Davoust, Nathalie; Chantier, Thibault; Tafforeau, Lionel; Mangeot, Philippe-Emmanuel; Ciancia, Claire; Perrin-Cocon, Laure; Bartenschlager, Ralf; André, Patrice; Lotteau, Vincent

    2013-01-01

    Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1) appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.

  7. The interactomes of influenza virus NS1 and NS2 proteins identify new host factors and provide insights for ADAR1 playing a supportive role in virus replication.

    Directory of Open Access Journals (Sweden)

    Benoît de Chassey

    Full Text Available Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1 appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.

  8. Plasma Membrane-Located Purine Nucleotide Transport Proteins Are Key Components for Host Exploitation by Microsporidian Intracellular Parasites

    Science.gov (United States)

    Heinz, Eva; Hacker, Christian; Dean, Paul; Mifsud, John; Goldberg, Alina V.; Williams, Tom A.; Nakjang, Sirintra; Gregory, Alison; Hirt, Robert P.; Lucocq, John M.; Kunji, Edmund R. S.; Embley, T. Martin

    2014-01-01

    Microsporidia are obligate intracellular parasites of most animal groups including humans, but despite their significant economic and medical importance there are major gaps in our understanding of how they exploit infected host cells. We have investigated the evolution, cellular locations and substrate specificities of a family of nucleotide transport (NTT) proteins from Trachipleistophora hominis, a microsporidian isolated from an HIV/AIDS patient. Transport proteins are critical to microsporidian success because they compensate for the dramatic loss of metabolic pathways that is a hallmark of the group. Our data demonstrate that the use of plasma membrane-located nucleotide transport proteins (NTT) is a key strategy adopted by microsporidians to exploit host cells. Acquisition of an ancestral transporter gene at the base of the microsporidian radiation was followed by lineage-specific events of gene duplication, which in the case of T. hominis has generated four paralogous NTT transporters. All four T. hominis NTT proteins are located predominantly to the plasma membrane of replicating intracellular cells where they can mediate transport at the host-parasite interface. In contrast to published data for Encephalitozoon cuniculi, we found no evidence for the location for any of the T. hominis NTT transporters to its minimal mitochondria (mitosomes), consistent with lineage-specific differences in transporter and mitosome evolution. All of the T. hominis NTTs transported radiolabelled purine nucleotides (ATP, ADP, GTP and GDP) when expressed in Escherichia coli, but did not transport radiolabelled pyrimidine nucleotides. Genome analysis suggests that imported purine nucleotides could be used by T. hominis to make all of the critical purine-based building-blocks for DNA and RNA biosynthesis during parasite intracellular replication, as well as providing essential energy for parasite cellular metabolism and protein synthesis. PMID:25474405

  9. Plasma membrane-located purine nucleotide transport proteins are key components for host exploitation by microsporidian intracellular parasites.

    Directory of Open Access Journals (Sweden)

    Eva Heinz

    2014-12-01

    Full Text Available Microsporidia are obligate intracellular parasites of most animal groups including humans, but despite their significant economic and medical importance there are major gaps in our understanding of how they exploit infected host cells. We have investigated the evolution, cellular locations and substrate specificities of a family of nucleotide transport (NTT proteins from Trachipleistophora hominis, a microsporidian isolated from an HIV/AIDS patient. Transport proteins are critical to microsporidian success because they compensate for the dramatic loss of metabolic pathways that is a hallmark of the group. Our data demonstrate that the use of plasma membrane-located nucleotide transport proteins (NTT is a key strategy adopted by microsporidians to exploit host cells. Acquisition of an ancestral transporter gene at the base of the microsporidian radiation was followed by lineage-specific events of gene duplication, which in the case of T. hominis has generated four paralogous NTT transporters. All four T. hominis NTT proteins are located predominantly to the plasma membrane of replicating intracellular cells where they can mediate transport at the host-parasite interface. In contrast to published data for Encephalitozoon cuniculi, we found no evidence for the location for any of the T. hominis NTT transporters to its minimal mitochondria (mitosomes, consistent with lineage-specific differences in transporter and mitosome evolution. All of the T. hominis NTTs transported radiolabelled purine nucleotides (ATP, ADP, GTP and GDP when expressed in Escherichia coli, but did not transport radiolabelled pyrimidine nucleotides. Genome analysis suggests that imported purine nucleotides could be used by T. hominis to make all of the critical purine-based building-blocks for DNA and RNA biosynthesis during parasite intracellular replication, as well as providing essential energy for parasite cellular metabolism and protein synthesis.

  10. Suppressive effect of AMP-activated protein kinase on the epithelial-mesenchymal transition in retinal pigment epithelial cells.

    Directory of Open Access Journals (Sweden)

    Ryo Matoba

    Full Text Available The epithelial-mesenchymal transition (EMT in retinal pigment epithelial (RPE cells plays a central role in the development of proliferative vitreoretinopathy (PVR. The purpose of this study was to investigate the effect of AMP-activated protein kinase (AMPK, a key regulator of energy homeostasis, on the EMT in RPE cells. In this study, EMT-associated formation of cellular aggregates was induced by co-stimulation of cultured ARPE-19 cells with tumor necrosis factor (TNF-α (10 ng/ml and transforming growth factor (TGF-β2 (5 ng/ml. 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR, a potent activator of AMPK, significantly suppressed TNF-α and TGF-β2-induced cellular aggregate formation (p < 0.01. Dipyridamole almost completely reversed the suppressive effect of AICAR, whereas 5'-amino-5'-deoxyadenosine restored aggregate formation by approximately 50%. AICAR suppressed the downregulation of E-cadherin and the upregulation of fibronectin and α-smooth muscle actin by TNF-α and TGF-β2. The levels of matrix metalloproteinase (MMP-2, MMP-9, interleukin-6, and vascular endothelial growth factor were significantly decreased by AICAR. Activation of the mitogen-activated protein kinase and mammalian target of rapamycin pathways, but not the Smad pathway, was inhibited by AICAR. These findings indicate that AICAR suppresses the EMT in RPE cells at least partially via activation of AMPK. AMPK is a potential target molecule for the prevention and treatment of PVR, so AICAR may be a promising candidate for PVR therapy.

  11. Analysis of protein targets in pathogen-host interaction in infectious diseases: a case study on Plasmodium falciparum and Homo sapiens interaction network.

    Science.gov (United States)

    Saha, Sovan; Sengupta, Kaustav; Chatterjee, Piyali; Basu, Subhadip; Nasipuri, Mita

    2017-09-23

    Infection and disease progression is the outcome of protein interactions between pathogen and host. Pathogen, the role player of Infection, is becoming a severe threat to life as because of its adaptability toward drugs and evolutionary dynamism in nature. Identifying protein targets by analyzing protein interactions between host and pathogen is the key point. Proteins with higher degree and possessing some topologically significant graph theoretical measures are found to be drug targets. On the other hand, exceptional nodes may be involved in infection mechanism because of some pathway process and biologically unknown factors. In this article, we attempt to investigate characteristics of host-pathogen protein interactions by presenting a comprehensive review of computational approaches applied on different infectious diseases. As an illustration, we have analyzed a case study on infectious disease malaria, with its causative agent Plasmodium falciparum acting as 'Bait' and host, Homo sapiens/human acting as 'Prey'. In this pathogen-host interaction network based on some interconnectivity and centrality properties, proteins are viewed as central, peripheral, hub and non-hub nodes and their significance on infection process. Besides, it is observed that because of sparseness of the pathogen and host interaction network, there may be some topologically unimportant but biologically significant proteins, which can also act as Bait/Prey. So, functional similarity or gene ontology mapping can help us in this case to identify these proteins. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  12. Nuclear assortment of eIF4E coincides with shut-off of host protein synthesis upon poliovirus infection.

    Science.gov (United States)

    Sukarieh, R; Sonenberg, N; Pelletier, J

    2010-05-01

    Eukaryotic initiation factor (eIF) 4E is a subunit of the cap-binding protein complex, eIF4F, which recognizes the cap structure of cellular mRNAs to facilitate translation initiation. eIF4E is assembled into the eIF4F complex via its interaction with eIF4G, an event that is under Akt/mTOR regulation. The eIF4E-eIF4G interaction is regulated by the eIF4E binding partners, eIF4E-binding proteins and eIF4E-transporter. Cleavage of eIF4G occurs upon poliovirus infection and is responsible for the shut-off of host-cell protein synthesis observed early in infection. Here, we document that relocalization of eIF4E to the nucleus occurs concomitantly with cleavage of eIF4G upon poliovirus infection. This event is not dependent upon virus replication, but is dependent on eIF4G cleavage. We postulate that eIF4E nuclear relocalization may contribute to the shut-off of host protein synthesis that is a hallmark of poliovirus infection by perturbing the circular status of actively translating mRNAs.

  13. A reverse-phase protein microarray-based screen identifies host signaling dynamics upon Burkholderia spp. infection

    Directory of Open Access Journals (Sweden)

    Chih-Yuan eChiang

    2015-07-01

    Full Text Available Burkholderia is a diverse genus of Gram-negative bacteria that cause high mortality rate in humans and cattle. The lack of effective therapeutic treatments poses serious public health threats. Insights toward host-Burkholderia spp. interaction are critical in understanding the pathogenesis of the infection as well as identifying therapeutic targets for drug development. Reverse-phase protein microarray (RPMA technology was previously proven to characterize novel biomarkers and molecular signatures associated with infectious diseases and cancers. In the present study, this technology was utilized to interrogate changes in host protein expression and post-translational phosphorylation events in macrophages infected with a collection of geographically diverse strains of Burkholderia spp. The expression or phosphorylation state of 25 proteins was altered during Burkholderia spp. infections and of which eight proteins were selected for further validation by immunoblotting. Kinetic expression patterns of phosphorylated AMPK-α1, Src, and GSK3β suggested the importance of their roles in regulating Burkholderia spp. mediated innate immune responses. Modulating inflammatory responses by perturbing AMPK-α1, Src, and GSK3β activities may provide novel therapeutic targets for future treatments.

  14. Natural proteasome inhibitor celastrol suppresses androgen-independent prostate cancer progression by modulating apoptotic proteins and NF-kappaB.

    Directory of Open Access Journals (Sweden)

    Yao Dai

    Full Text Available Celastrol is a natural proteasome inhibitor that exhibits promising anti-tumor effects in human malignancies, especially the androgen-independent prostate cancer (AIPC with constitutive NF-κB activation. Celastrol induces apoptosis by means of proteasome inhibition and suppresses prostate tumor growth. However, the detailed mechanism of action remains elusive. In the current study, we aim to test the hypothesis that celastrol suppresses AIPC progression via inhibiting the constitutive NF-κB activity as well as modulating the Bcl-2 family proteins.We examined the efficacy of celastrol both in vitro and in vivo, and evaluated the role of NF-κB in celastrol-mediated AIPC regression. We found that celastrol inhibited cell proliferation in all three AIPC cell lines (PC-3, DU145 and CL1, with IC₅₀ in the range of 1-2 µM. Celastrol also suppressed cell migration and invasion. Celastrol significantly induced apoptosis as evidenced by increased sub-G1 population, caspase activation and PARP cleavage. Moreover, celastrol promoted cleavage of the anti-apoptotic protein Mcl-1 and activated the pro-apoptotic protein Noxa. In addition, celastrol rapidly blocked cytosolic IκBα degradation and nuclear translocation of RelA. Likewise, celastrol inhibited the expression of multiple NF-κB target genes that are involved in proliferation, invasion and anti-apoptosis. Celastrol suppressed AIPC tumor progression by inhibiting proliferation, increasing apoptosis and decreasing angiogenesis, in PC-3 xenograft model in nude mouse. Furthermore, increased cellular IκBα and inhibited expression of various NF-κB target genes were observed in tumor tissues.Our data suggest that, via targeting the proteasome, celastrol suppresses proliferation, invasion and angiogenesis by inducing the apoptotic machinery and attenuating constitutive NF-κB activity in AIPC both in vitro and in vivo. Celastrol as an active ingredient of traditional herbal medicine could thus be

  15. ASH1L Suppresses Matrix Metalloproteinase through Mitogen-activated Protein Kinase Signaling Pathway in Pulpitis.

    Science.gov (United States)

    Bei, Yin; Tianqian, Hui; Fanyuan, Yu; Haiyun, Luo; Xueyang, Liao; Jing, Yang; Chenglin, Wang; Ling, Ye

    2017-02-01

    Pulpitis is an inflammation of dental pulp produced by a response to external stimuli. The response entails substantial cellular and molecular activities. Both genetic and epigenetic regulators contribute to the occurrence of pulpitis. However, the epigenetic mechanisms are still poorly understood. In this research, we studied the role of the absent, small, or homeotic-like (ASH1L) gene in the process of pulpitis. Human dental pulp cells (HDPCs) were stimulated with proinflammatory cytokine tumor necrosis factor alpha (TNF-α). Gene expression profiling was performed to assess the occurrence of epigenetic regulators. Pulp tissue from rat experimental pulpitis was subjected to immunofluorescence to detect the occurrence of ASH1L and trimethylation of lysine 4 histone 3 (H3K4me3). The presence of ASH1L in HDPCs that had been generated by TNF-α stimulation was analyzed by Western blot procedures and cellular immunofluorescence. Once detected, ASH1L was silenced through the use of specific small interfering RNA. The effects of ASH1L on the occurrence and operation of matrix metalloproteinases (MMPs) were then tested by analysis of quantitative polymerase chain reactions, Western blotting, and zymography. Chromatin immunoprecipitation was performed to detect whether ASH1L and H3K4me3 were present in the promoter regions of MMPs. We then used Western blot procedures to examine the nuclear factor kappa B and the mitogen-activated protein kinase (MAPK) responses to the silencing of ASH1L. We also examined the specific pathway involved in ASH1L regulation of the MMPs. After stimulating HDPCs with TNF-α, ASH1L emerged as 1 of the most strongly induced epigenetic mediators. We found that TNF-α treatment induced the expression of ASH1L through the nuclear factor kappa B and MAPK signal pathways. ASH1L was found in both the nucleus and the cytoplasm. TNF-α treatment was particularly active in inducing the accumulation of ASH1L in cellular cytoplasm. As is also consistent

  16. Cysteine-rich buccal gland protein suppressed the proliferation, migration and invasion of hela cells through akt pathway.

    Science.gov (United States)

    Han, Jianmei; Liu, Yu; Jiang, Qi; Xiao, Rong

    2017-11-01

    Cysteine-rich buccal gland protein (CRBGP) as a member of cysteine-rich secretory proteins (CRISPs) superfamily was isolated from the buccal glands of Lampetra japonica, the blood suckers in the marine. Previous studies showed CRBGP could suppress angiogenesis probably due to its ion channel blocking activity. Whether CRBGP could also affect the activity of tumor cells has not been reported yet. In this study, CRBGP suppressed the proliferation of Hela cells with an IC 50 of 6.7 μM by inducing apoptosis. Both microscopic observation and Western blot indicated that CRBGP was able to induce the nuclei shrinking, downregulate the protein level of BCL2 and caspase 3 as well as upregulate the level of BAX in Hela cells, suggested that CRBGP might induce apoptosis of Hela cells in a mitochondrial-dependent pathway. Furthermore, CRBGP could disturb F-actin organization, which would finally cause the Hela cells to lose their shape and to lessen their abilities on adhesion, migration and invasion. Finally, CRBGP was shown to reduce the phosphorylation level of Akt, which indicated that CRBGP might inhibit the proliferation and metastasis of Hela cells through Akt pathway. CRBGP, as a voltage-gated sodium channel blocker, also possesses the anti-tumor abilities which provided information on the effects and action manner of the other CRISPs. © 2017 IUBMB Life, 69(11):856-866, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

  17. Plasminogen Binding Proteins and Plasmin Generation on the Surface of Leptospira spp.: The Contribution to the Bacteria-Host Interactions

    Directory of Open Access Journals (Sweden)

    Monica L. Vieira

    2012-01-01

    Full Text Available Leptospirosis is considered a neglected infectious disease of human and veterinary concern. Although extensive investigations on host-pathogen interactions have been pursued by several research groups, mechanisms of infection, invasion and persistence of pathogenic Leptospira spp. remain to be elucidated. We have reported the ability of leptospires to bind human plasminogen (PLG and to generate enzimatically active plasmin (PLA on the bacteria surface. PLA-coated Leptospira can degrade immobilized ECM molecules, an activity with implications in host tissue penetration. Moreover, we have identified and characterized several proteins that may act as PLG-binding receptors, each of them competent to generate active plasmin. The PLA activity associated to the outer surface of Leptospira could hamper the host immune attack by conferring the bacteria some benefit during infection. The PLA-coated leptospires obstruct complement C3b and IgG depositions on the bacterial surface, most probably through degradation. The decrease of leptospiral opsonization might be an important aspect of the immune evasion strategy. We believe that the presence of PLA on the leptospiral surface may (i facilitate host tissue penetration, (ii help the bacteria to evade the immune system and, as a consequence, (iii permit Leptospira to reach secondary sites of infection.

  18. Bst1 is required for Candida albicans infecting host via facilitating cell wall anchorage of Glycosylphosphatidyl inositol anchored proteins

    Science.gov (United States)

    Liu, Wei; Zou, Zui; Huang, Xin; Shen, Hui; He, Li Juan; Chen, Si Min; Li, Li Ping; Yan, Lan; Zhang, Shi Qun; Zhang, Jun Dong; Xu, Zheng; Xu, Guo Tong; An, Mao Mao; Jiang, Yuan Ying

    2016-01-01

    Glycosylphosphatidyl inositol anchored proteins (GPI-APs) on fungal cell wall are essential for invasive infections. While the function of inositol deacylation of GPI-APs in mammalian cells has been previously characterized the impact of inositol deacylation in fungi and implications to host infection remains largely unexplored. Herein we describe our identification of BST1, an inositol deacylase of GPI-Aps in Candida albicans, was critical for GPI-APs cell wall attachment and host infection. BST1-deficient C. albicans (bst1Δ/Δ) was associated with severely impaired cell wall anchorage of GPI-APs and subsequen unmasked β-(1,3)-glucan. Consistent with the aberrant cell wall structures, bst1Δ/Δ strain did not display an invasive ability and could be recognized more efficiently by host immune systems. Moreover, BST1 null mutants or those expressing Bst1 variants did not display inositol deacylation activity and exhibited severely attenuated virulence and reduced organic colonization in a murine systemic candidiasis model. Thus, Bst1 can facilitate cell wall anchorage of GPI-APs in C. albicans by inositol deacylation, and is critical for host invasion and immune escape. PMID:27708385

  19. CD4+ Primary T Cells Expressing HCV-Core Protein Upregulate Foxp3 and IL-10, Suppressing CD4 and CD8 T Cells

    Science.gov (United States)

    Aguado, Enrique; Garcia-Cozar, Francisco

    2014-01-01

    Adaptive T cell responses are critical for controlling HCV infection. While there is clinical evidence of a relevant role for regulatory T cells in chronic HCV-infected patients, based on their increased number and function; mechanisms underlying such a phenomena are still poorly understood. Accumulating evidence suggests that proteins from Hepatitis C virus can suppress host immune responses. We and others have shown that HCV is present in CD4+ lymphocytes from chronically infected patients and that HCV-core protein induces a state of unresponsiveness in the CD4+ tumor cell line Jurkat. Here we show that CD4+ primary T cells lentivirally transduced with HCV-core, not only acquire an anergic phenotype but also inhibit IL-2 production and proliferation of bystander CD4+ or CD8+ T cells in response to anti-CD3 plus anti-CD28 stimulation. Core-transduced CD4+ T cells show a phenotype characterized by an increased basal secretion of the regulatory cytokine IL-10, a decreased IFN-γ production upon stimulation, as well as expression of regulatory T cell markers, CTLA-4, and Foxp3. A significant induction of CD4+CD25+CD127lowPD-1highTIM-3high regulatory T cells with an exhausted phenotype was also observed. Moreover, CCR7 expression decreased in HCV-core expressing CD4+ T cells explaining their sequestration in inflamed tissues such as the infected liver. This work provides a new perspective on de novo generation of regulatory CD4+ T cells in the periphery, induced by the expression of a single viral protein. PMID:24465502

  20. CD4+ primary T cells expressing HCV-core protein upregulate Foxp3 and IL-10, suppressing CD4 and CD8 T cells.

    Directory of Open Access Journals (Sweden)

    Cecilia Fernandez-Ponce

    Full Text Available Adaptive T cell responses are critical for controlling HCV infection. While there is clinical evidence of a relevant role for regulatory T cells in chronic HCV-infected patients, based on their increased number and function; mechanisms underlying such a phenomena are still poorly understood. Accumulating evidence suggests that proteins from Hepatitis C virus can suppress host immune responses. We and others have shown that HCV is present in CD4+ lymphocytes from chronically infected patients and that HCV-core protein induces a state of unresponsiveness in the CD4+ tumor cell line Jurkat. Here we show that CD4+ primary T cells lentivirally transduced with HCV-core, not only acquire an anergic phenotype but also inhibit IL-2 production and proliferation of bystander CD4+ or CD8+ T cells in response to anti-CD3 plus anti-CD28 stimulation. Core-transduced CD4+ T cells show a phenotype characterized by an increased basal secretion of the regulatory cytokine IL-10, a decreased IFN-γ production upon stimulation, as well as expression of regulatory T cell markers, CTLA-4, and Foxp3. A significant induction of CD4+CD25+CD127(lowPD-1(highTIM-3(high regulatory T cells with an exhausted phenotype was also observed. Moreover, CCR7 expression decreased in HCV-core expressing CD4+ T cells explaining their sequestration in inflamed tissues such as the infected liver. This work provides a new perspective on de novo generation of regulatory CD4+ T cells in the periphery, induced by the expression of a single viral protein.

  1. Hydrophilic crosslinked-polymeric surface capable of effective suppression of protein adsorption

    Energy Technology Data Exchange (ETDEWEB)

    Kamon, Yuri; Inoue, Naoko; Mihara, Erika; Kitayama, Yukiya; Ooya, Tooru; Takeuchi, Toshifumi, E-mail: takeuchi@gold.kobe-u.ac.jp

    2016-08-15

    Highlights: • Three hydrophilic crosslinked polymers were examined for protein adsorption. • All polymers showed low nonspecific adsorption of negatively charged proteins. • Poly(MMPC) showed the lowest adsorption for positively charged proteins. • Poly(MMPC) is able to reduce nonspecific adsorption of a wide range of proteins. - Abstract: We investigated the nonspecific adsorption of proteins towards three hydrophilic crosslinked-polymeric thin layers prepared by surface-initiated atom transfer radical polymerization using N,N′-methylenebisacrylamide, 2-(methacryloyloxy)ethyl-[N-(2-methacryloyloxy)ethyl]phosphorylcholine (MMPC), or 6,6′-diacryloyl-trehalose crosslinkers. Protein binding experiments were performed by surface plasmon resonance with six proteins of different pI values including α-lactalbumin, bovine serum albumin (BSA), myoglobin, ribonuclease A, cytochrome C, and lysozyme in buffer solution at pH 7.4. All of the obtained crosslinked-polymeric thin layers showed low nonspecific adsorption of negatively charged proteins at pH 7.4 such as α-lactalbumin, BSA, and myoglobin. Nonspecific adsorption of positively charged proteins including ribonuclease A, cytochrome C, and lysozyme was the lowest for poly(MMPC). These results suggest poly(MMPC) can effectively reduce nonspecific adsorption of a wide range of proteins that are negatively or positively charged at pH 7.4. MMPC is a promising crosslinker for a wide range of polymeric materials requiring low nonspecific protein binding.

  2. Host transcription factor Speckled 110 kDa (Sp110), a nuclear body protein, is hijacked by hepatitis B virus protein X for viral persistence.

    Science.gov (United States)

    Sengupta, Isha; Das, Dipanwita; Singh, Shivaram Prasad; Chakravarty, Runu; Das, Chandrima

    2017-12-15

    Promyelocytic leukemia nuclear bodies (PML-NB) are sub-nuclear organelles that are the hub of numerous proteins. DNA/RNA viruses often hijack the cellular factors resident in PML-NBs to promote their proliferation in host cells. Hepatitis B virus (HBV), belonging to Hepadnaviridae family, remains undetected in early infection as it does not induce the innate immune response and is known to be the cause of several hepatic diseases leading to cirrhosis and hepatocellular carcinoma. The association of PML-NB proteins and HBV is being addressed in a number of recent studies. Here, we report that the PML-NB protein Speckled 110 kDa (Sp110) is SUMO1-modified and undergoes a deSUMOylation-driven release from the PML-NB in the presence of HBV. Intriguingly, Sp110 knockdown significantly reduced viral DNA load in the culture supernatant by activation of the type I interferon-response pathway. Furthermore, we found that Sp110 differentially regulates several direct target genes of hepatitis B virus protein X (HBx), a viral co-factor. Subsequently, we identified Sp110 as a novel interactor of HBx and found this association to be essential for the exit of Sp110 from the PML-NB during HBV infection and HBx recruitment on the promoter of these genes. HBx, in turn, modulates the recruitment of its associated transcription cofactors p300/HDAC1 to these co-regulated genes, thereby altering the host gene expression program in favor of viral persistence. Thus, we report a mechanism by which HBV can evade host immune response by hijacking the PML-NB protein Sp110, and therefore, we propose it to be a novel target for antiviral therapy. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Pathogenic leptospires modulate protein expression and post-translational modifications in response to mammalian host signals

    Science.gov (United States)

    Pathogenic species of Leptospira cause leptospirosis, a bacterial zoonotic disease with a global distribution affecting over one million people annually. Reservoir hosts of leptospirosis, including rodents, dogs and cattle, exhibit little to no signs of disease but shed large numbers of organisms in...

  4. Radix isatidis Polysaccharides Inhibit Influenza a Virus and Influenza A Virus-Induced Inflammation via Suppression of Host TLR3 Signaling In Vitro

    Directory of Open Access Journals (Sweden)

    Zhengtu Li

    2017-01-01

    Full Text Available Influenza remains one of the major epidemic diseases worldwide, and rapid virus replication and collateral lung tissue damage caused by excessive pro-inflammatory host immune cell responses lead to high mortality rates. Thus, novel therapeutic agents that control influenza A virus (IAV propagation and attenuate excessive pro-inflammatory responses are needed. Polysaccharide extract from Radix isatidis, a traditional Chinese herbal medicine, exerted potent anti-IAV activity against human seasonal influenza viruses (H1N1 and H3N2 and avian influenza viruses (H6N2 and H9N2 in vitro. The polysaccharides also significantly reduced the expression of pro-inflammatory cytokines (IL-6 and chemokines (IP-10, MIG, and CCL-5 stimulated by A/PR/8/34 (H1N1 at a range of doses (7.5 mg/mL, 15 mg/mL, and 30 mg/mL; however, they were only effective against progeny virus at a high dose. Similar activity was detected against inflammation induced by avian influenza virus H9N2. The polysaccharides strongly inhibited the protein expression of TLR-3 induced by PR8, suggesting that they impair the upregulation of pro-inflammatory factors induced by IAV by inhibiting activation of the TLR-3 signaling pathway. The polysaccharide extract from Radix isatidis root therefore has the potential to be used as an adjunct to antiviral therapy for the treatment of IAV infection.

  5. The endothelial protein C receptor and activated protein C play a limited role in host defense during experimental tuberculosis

    NARCIS (Netherlands)

    Kager, Liesbeth M.; Roelofs, Joris J. T. H.; de Vos, Alex F.; Wieland, Catharina W.; Schouten, Marcel; Meijers, Joost C. M.; Isermann, Berend; van't Veer, Cornelis; Esmon, Charles T.; van der Poll, Tom

    2013-01-01

    The protein C (PC) system is an important regulator of both coagulation and inflammation. Activated PC (APC), together with its receptor the endothelial protein C receptor (EPCR), has anticoagulant and anti-inflammatory properties. During tuberculosis (TB), a devastating chronic pulmonary disease

  6. Theobromine suppresses adipogenesis through enhancement of CCAAT-enhancer-binding protein β degradation by adenosine receptor A1.

    Science.gov (United States)

    Mitani, Takakazu; Watanabe, Shun; Yoshioka, Yasukiyo; Katayama, Shigeru; Nakamura, Soichiro; Ashida, Hitoshi

    2017-12-01

    Theobromine, a methylxanthine derived from cacao beans, reportedly has various health-promoting properties but molecular mechanism by which effects of theobromine on adipocyte differentiation and adipogenesis remains unclear. In this study, we aimed to clarify the molecular mechanisms of the anti-adipogenic effect of theobromine in vitro and in vivo. ICR mice (4week-old) were administered with theobromine (0.1g/kg) for 7days. Theobromine administration attenuated gains in body and epididymal adipose tissue weights in mice and suppressed expression of adipogenic-associated genes in mouse adipose tissue. In 3T3-L1 preadipocytes, theobromine caused degradation of C/EBPβ protein by the ubiquitin-proteasome pathway. Pull down assay showed that theobromine selectively interacts with adenosine receptor A1 (AR1), and AR1 knockdown inhibited theobromine-induced C/EBPβ degradation. Theobromine increased sumoylation of C/EBPβ at Lys133. Expression of the small ubiquitin-like modifier (SUMO)-specific protease 2 (SENP2) gene, coding for a desumoylation enzyme, was suppressed by theobromine. In vivo knockdown studies showed that AR1 knockdown in mice attenuated the anti-adipogenic effects of theobromine in younger mice. Theobromine suppresses adipocyte differentiation and induced C/EBPβ degradation by increasing its sumoylation. Furthermore, the inhibition of AR1 signaling is important for theobromine-induced C/EBPβ degradation. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Simultaneous Hypoxia and Low Extracellular pH Suppress Overall Metabolic Rate and Protein Synthesis In Vitro.

    Directory of Open Access Journals (Sweden)

    Brita Singers Sørensen

    Full Text Available The tumor microenvironment is characterized by regions of hypoxia and acidosis which are linked to poor prognosis. This occurs due to an aberrant vasculature as well as high rates of glycolysis and lactate production in tumor cells even in the presence of oxygen (the Warburg effect, which weakens the spatial linkage between hypoxia and acidosis.Five different human squamous cell carcinoma cell lines (SiHa, FaDuDD, UTSCC5, UTSCC14 and UTSCC15 were treated with hypoxia, acidosis (pH 6.3, or a combination, and gene expression analyzed using microarray. SiHa and FaDuDD were chosen for further characterization of cell energetics and protein synthesis. Total cellular ATP turnover and relative glycolytic dependency was determined by simultaneous measurements of oxygen consumption and lactate synthesis rates and total protein synthesis was determined by autoradiographic quantification of the incorporation of 35S-labelled methionine and cysteine into protein.Microarray analysis allowed differentiation between genes induced at low oxygen only at normal extracellular pH (pHe, genes induced at low oxygen at both normal and low pHe, and genes induced at low pHe independent of oxygen concentration. Several genes were found to be upregulated by acidosis independent of oxygenation. Acidosis resulted in a more wide-scale change in gene expression profiles than hypoxia including upregulation of genes involved in the translation process, for example Eukaryotic translation initiation factor 4A, isoform 2 (EIF4A2, and Ribosomal protein L37 (RPL37. Acidosis suppressed overall ATP turnover and protein synthesis by 50%. Protein synthesis, but not total ATP production, was also suppressed under hypoxic conditions. A dramatic decrease in ATP turnover (SiHa and protein synthesis (both cell lines was observed when hypoxia and low pHe were combined.We demonstrate here that the influence of hypoxia and acidosis causes different responses, both in gene expression and in de

  8. Simultaneous Hypoxia and Low Extracellular pH Suppress Overall Metabolic Rate and Protein Synthesis In Vitro.

    Science.gov (United States)

    Sørensen, Brita Singers; Busk, Morten; Overgaard, Jens; Horsman, Michael R; Alsner, Jan

    2015-01-01

    The tumor microenvironment is characterized by regions of hypoxia and acidosis which are linked to poor prognosis. This occurs due to an aberrant vasculature as well as high rates of glycolysis and lactate production in tumor cells even in the presence of oxygen (the Warburg effect), which weakens the spatial linkage between hypoxia and acidosis. Five different human squamous cell carcinoma cell lines (SiHa, FaDuDD, UTSCC5, UTSCC14 and UTSCC15) were treated with hypoxia, acidosis (pH 6.3), or a combination, and gene expression analyzed using microarray. SiHa and FaDuDD were chosen for further characterization of cell energetics and protein synthesis. Total cellular ATP turnover and relative glycolytic dependency was determined by simultaneous measurements of oxygen consumption and lactate synthesis rates and total protein synthesis was determined by autoradiographic quantification of the incorporation of 35S-labelled methionine and cysteine into protein. Microarray analysis allowed differentiation between genes induced at low oxygen only at normal extracellular pH (pHe), genes induced at low oxygen at both normal and low pHe, and genes induced at low pHe independent of oxygen concentration. Several genes were found to be upregulated by acidosis independent of oxygenation. Acidosis resulted in a more wide-scale change in gene expression profiles than hypoxia including upregulation of genes involved in the translation process, for example Eukaryotic translation initiation factor 4A, isoform 2 (EIF4A2), and Ribosomal protein L37 (RPL37). Acidosis suppressed overall ATP turnover and protein synthesis by 50%. Protein synthesis, but not total ATP production, was also suppressed under hypoxic conditions. A dramatic decrease in ATP turnover (SiHa) and protein synthesis (both cell lines) was observed when hypoxia and low pHe were combined. We demonstrate here that the influence of hypoxia and acidosis causes different responses, both in gene expression and in de novo

  9. Evaluation of a Bead-Free Coimmunoprecipitation Technique for Identification of Virus-Host Protein Interactions Using High-Resolution Mass Spectrometry.

    Science.gov (United States)

    DeBlasio, Stacy L; Bereman, Michael S; Mahoney, Jaclyn; Thannhauser, Theodore W; Gray, Stewart M; MacCoss, Michael J; Cilia Heck, Michelle

    2017-09-01

    Protein interactions between virus and host are essential for viral propagation and movement, as viruses lack most of the proteins required to thrive on their own. Precision methods aimed at disrupting virus-host interactions represent new approaches to disease management but require in-depth knowledge of the identity and binding specificity of host proteins within these interaction networks. Protein coimmunoprecipitation (co-IP) coupled with mass spectrometry (MS) provides a high-throughput way to characterize virus-host interactomes in a single experiment. Common co-IP methods use antibodies immobilized on agarose or magnetic beads to isolate virus-host complexes in solutions of host tissue homogenate. Although these workflows are well established, they can be fairly laborious and expensive. Therefore, we evaluated the feasibility of using antibody-coated microtiter plates coupled with MS analysis as an easy, less expensive way to identify host proteins that interact with Potato leafroll virus (PLRV), an insect-borne RNA virus that infects potatoes. With the use of the bead-free platform, we were able to detect 36 plant and 1 nonstructural viral protein significantly coimmunoprecipitating with PLRV. Two of these proteins, a 14-3-3 signal transduction protein and malate dehydrogenase 2 (mMDH2), were detected as having a weakened or lost association with a structural mutant of the virus, demonstrating that the bead-free method is sensitive enough to detect quantitative differences that can be used to pin-point domains of interaction. Collectively, our analysis shows that the bead-free platform is a low-cost alternative that can be used by core facilities and other investigators to identify plant and viral proteins interacting with virions and/or the viral structural proteins.

  10. GLYCINE-RICH RNA-BINDING PROTEIN1 interacts with RECEPTOR-LIKE CYTOPLASMIC PROTEIN KINASE1 and suppresses cell death and defense responses in pepper (Capsicum annuum).

    Science.gov (United States)

    Kim, Dae Sung; Kim, Nak Hyun; Hwang, Byung Kook

    2015-01-01

    Plants use a variety of innate immune regulators to trigger cell death and defense responses against pathogen attack. We identified pepper (Capsicum annuum) GLYCINE-RICH RNA-BINDING PROTEIN1 (CaGRP1) as a RECEPTOR-LIKE CYTOPLASMIC PROTEIN KINASE1 (CaPIK1)-interacting partner, based on bimolecular fluorescence complementation and coimmunoprecipitation analyses as well as gene silencing and transient expression analysis. CaGRP1 contains an N-terminal RNA recognition motif and a glycine-rich region at the C-terminus. The CaGRP1 protein had DNA- and RNA-binding activity in vitro. CaGRP1 interacted with CaPIK1 in planta. CaGRP1 and CaGRP1-CaPIK1 complexes were localized to the nucleus in plant cells. CaPIK1 phosphorylated CaGRP1 in vitro and in planta. Transient coexpression of CaGRP1 with CaPIK1 suppressed the CaPIK1-triggered cell death response, accompanied by a reduced CaPIK1-triggered reactive oxygen species (ROS) burst. The RNA recognition motif region of CaGRP1 was responsible for the nuclear localization of CaGRP1 as well as the suppression of the CaPIK1-triggered cell death response. CaGRP1 silencing in pepper conferred enhanced resistance to Xanthomonas campestris pv vesicatoria (Xcv) infection; however, CaPIK1-silenced plants were more susceptible to Xcv. CaGRP1 interacts with CaPIK1 and negatively regulates CaPIK1-triggered cell death and defense responses by suppressing ROS accumulation. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

  11. Helicobacter pylori neutrophil-activating protein: A potential Treg modulator suppressing allergic asthma?

    Directory of Open Access Journals (Sweden)

    Anjna eSehrawat

    2015-06-01

    Full Text Available The ultimate aim of immunology is to kill the pathogen without being harmful to the host. But what if eliminating the pathogen in itself is discomforting for the host? One such emerging case is of Helicobacter pylori. Modern medicine, infantile vaccination and ultra-hygienic conditions have led to progressive disappearance of H. pylori in different parts of the world. However, the adversities caused by H. pylori’s absence are much larger than those caused by its presence. Asthma is rising as an epidemic in last few decades and several reports suggest an inverse-relationship between H. pylori’s persistence and early-life onset asthma. Regulatory T cells play an important role in both the cases. This is further supported by experiments on mouse-models. Hence, need of the hour is to discern the relationship between H. pylori and its host and eliminating its negative impacts without disturbing our indigenous microbiota. To resolve whether H. pylori is a pathogen or an amphibiont is another important side. This review explores the biological basis of H. pylori-induced priming of immune system offering resistance to childhood-onset asthma. HP-NAP-Tregs interaction has been predicted using molecular docking and dynamic simulation.

  12. Prediction of Chlamydia pneumoniae protein localization in host mitochondria and cytoplasm and possible involvements in lung cancer etiology: a computational approach

    Directory of Open Access Journals (Sweden)

    Aws Alshamsan

    2017-12-01

    Full Text Available Collecting evidence suggests that the intercellular infection of Chlamydia pneumoniae in lungs contributes to the etiology of lung cancer. Many proteins of Chlamydia pneumoniae outmanoeuvre the various system of the host. The infection may regulate various factors, which can influence the growth of lung cancer in affected persons. In this in-silico study, we predict potential targeting of Chlamydia pneumoniae proteins in mitochondrial and cytoplasmic comportments of host cell and their possible involvement in growth and development of lung cancer. Various cellular activities are controlled in mitochondria and cytoplasm, where the localization of Chlamydia pneumoniae proteins may alter the normal functioning of host cells. The rationale of this study is to find out and explain the connection between Chlamydia pneumoniae infection and lung cancer. A sum of 183 and 513 proteins were predicted to target in mitochondria and cytoplasm of host cell out of total 1112 proteins of Chlamydia pneumoniae. In particular, many targeted proteins may interfere with normal growth behaviour of host cells, thereby altering the decision of program cell death. Present article provides a potential connection of Chlamydia pneumoniae protein targeting and proposed that various targeted proteins may play crucial role in lung cancer etiology through diverse mechanisms.

  13. Pokemon siRNA Delivery Mediated by RGD-Modified HBV Core Protein Suppressed the Growth of Hepatocellular Carcinoma.

    Science.gov (United States)

    Kong, Jing; Liu, Xiaoping; Jia, Jianbo; Wu, Jinsheng; Wu, Ning; Chen, Jun; Fang, Fang

    2015-10-01

    Hepatocellular carcinoma (HCC) is a deadly human malignant tumor that is among the most common cancers in the world, especially in Asia. Hepatitis B virus (HBV) infection has been well established as a high risk factor for hepatic malignance. Studies have shown that Pokemon is a master oncogene for HCC growth, suggesting it as an ideal therapeutic target. However, efficient delivery system is still lacking for Pokemon targeting treatment. In this study, we used core proteins of HBV, which is modified with RGD peptides, to construct a biomimetic vector for the delivery of Pokemon siRNAs (namely, RGD-HBc-Pokemon siRNA). Quantitative PCR and Western blot assays revealed that RGD-HBc-Pokemon siRNA possessed the highest efficiency of Pokemon suppression in HCC cells. In vitro experiments further indicated that RGD-HBc-Pokemon-siRNA exerted a higher tumor suppressor activity on HCC cell lines, evidenced by reduced proliferation and attenuated invasiveness, than Pokemon-siRNA or RGD-HBc alone. Finally, animal studies demonstrated that RGD-HBc-Pokemon siRNA suppressed the growth of HCC xenografts in mice by a greater extent than Pokemon-siRNA or RGD-HBc alone. Based on the above results, Pokemon siRNA delivery mediated by RGD-modified HBV core protein was shown to be an effective strategy of HCC gene therapy.

  14. Rif1 acts through Protein Phosphatase 1 but independent of replication timing to suppress telomere extension in budding yeast.

    Science.gov (United States)

    Kedziora, Sylwia; Gali, Vamsi K; Wilson, Rosemary H C; Clark, Kate R M; Nieduszynski, Conrad A; Hiraga, Shin-Ichiro; Donaldson, Anne D

    2018-05-04

    The Rif1 protein negatively regulates telomeric TG repeat length in the budding yeast Saccharomyces cerevisiae, but how it prevents telomere over-extension is unknown. Rif1 was recently shown to control DNA replication by acting as a Protein Phosphatase 1 (PP1)-targeting subunit. Therefore, we investigated whether Rif1 controls telomere length by targeting PP1 activity. We find that a Rif1 mutant defective for PP1 interaction causes a long-telomere phenotype, similar to that of rif1Δ cells. Tethering PP1 at a specific telomere partially substitutes for Rif1 in limiting TG repeat length, confirming the importance of PP1 in telomere length control. Ablating Rif1-PP1 interaction is known to cause precocious activation of telomere-proximal replication origins and aberrantly early telomere replication. However, we find that Rif1 still limits telomere length even if late replication is forced through deletion of nearby replication origins, indicating that Rif1 can control telomere length independent of replication timing. Moreover we find that, even at a de novo telomere created after DNA synthesis during a mitotic block, Rif1-PP1 interaction is required to suppress telomere lengthening and prevent inappropriate recruitment of Tel1 kinase. Overall, our results show that Rif1 controls telomere length by recruiting PP1 to directly suppress telomerase-mediated TG repeat lengthening.

  15. Predicting Zoonotic Risk of Influenza A Viruses from Host Tropism Protein Signature Using Random Forest

    OpenAIRE

    Christine L. P. Eng; Joo Chuan Tong; Tin Wee Tan

    2017-01-01

    Influenza A viruses remain a significant health problem, especially when a novel subtype emerges from the avian population to cause severe outbreaks in humans. Zoonotic viruses arise from the animal population as a result of mutations and reassortments, giving rise to novel strains with the capability to evade the host species barrier and cause human infections. Despite progress in understanding interspecies transmission of influenza viruses, we are no closer to predicting zoonotic strains th...

  16. Lactococcus lactis as expression host for the biosynthetic incorporation of tryptophan analogues into recombinant proteins

    NARCIS (Netherlands)

    El Khattabi, Mohamed; van Roosmalen, Maarten L.; Jager, Dennis; Metselaar, Heidi; Permentier, Hjalmar; Leenhouts, Kees; Broos, Jaap

    2008-01-01

    Incorporation of Trp (tryptophan) analogues into a protein may facilitate its structural analysis by spectroscopic techniques. Development of a biological system for the biosynthetic incorporation of such analogues into proteins is of considerable importance. The Gram-negative Escherichia coli is

  17. A Gene Family Coding for Salivary Proteins (SHOT) of the Polyphagous Spider Mite Tetranychus urticae Exhibits Fast Host-Dependent Transcriptional Plasticity.

    Science.gov (United States)

    Jonckheere, Wim; Dermauw, Wannes; Khalighi, Mousaalreza; Pavlidi, Nena; Reubens, Wim; Baggerman, Geert; Tirry, Luc; Menschaert, Gerben; Kant, Merijn R; Vanholme, Bartel; Van Leeuwen, Thomas

    2018-01-01

    The salivary protein repertoire released by the herbivorous pest Tetranychus urticae is assumed to hold keys to its success on diverse crops. We report on a spider mite-specific protein family that is expanded in T. urticae. The encoding genes have an expression pattern restricted to the anterior podocephalic glands, while peptide fragments were found in the T. urticae secretome, supporting the salivary nature of these proteins. As peptide fragments were identified in a host-dependent manner, we designated this family as the SHOT (secreted host-responsive protein of Tetranychidae) family. The proteins were divided in three groups based on sequence similarity. Unlike TuSHOT3 genes, TuSHOT1 and TuSHOT2 genes were highly expressed when feeding on a subset of family Fabaceae, while expression was depleted on other hosts. TuSHOT1 and TuSHOT2 expression was induced within 24 h after certain host transfers, pointing toward transcriptional plasticity rather than selection as the cause. Transfer from an 'inducer' to a 'noninducer' plant was associated with slow yet strong downregulation of TuSHOT1 and TuSHOT2, occurring over generations rather than hours. This asymmetric on and off regulation points toward host-specific effects of SHOT proteins, which is further supported by the diversity of SHOT genes identified in Tetranychidae with a distinct host repertoire.

  18. Protein-free transfection of CHO host cells with an IgG-fusion protein: selection and characterization of stable high producers and comparison to conventionally transfected clones.

    Science.gov (United States)

    Lattenmayer, Christine; Loeschel, Martina; Schriebl, Kornelia; Steinfellner, Willibald; Sterovsky, Thomas; Trummer, Evelyn; Vorauer-Uhl, Karola; Müller, Dethardt; Katinger, Hermann; Kunert, Renate

    2007-04-15

    In order to improve the current techniques of cell cultivation in the absence of serum, we have developed a protein-free transfection protocol for CHO cells, based on the Nucleofector technology. After starting with a heterogeneous pool of primary transfectants which express the fusion protein EpoFc, we isolated single clones and compared them with parallel clones generated by lipofection in serum-dependent cultivation. Our intensive characterization program was based on determination of specific productivity (q(p)) and analysis of genetic parameters. In two nucleofection experiments, transfection with 5 microg of DNA resulted in best productivities of the primary cell pools. After subcloning, the q(p) could be raised up to 27 pg x cells(-1) x day(-1). While the serum-dependent transfectants exhibited specific productivities up to 57 pg x cells(-1) x day(-1) in serum-dependent cultivation, a significant decrease that resulted in the range of q(p) of the protein-free transfectants was observed after switching to protein-free conditions. Investigation of genetic parameters revealed higher mRNA levels and gene copy numbers (GCN) for the protein-free adapted serum-dependent transfectants. Therefore, we assume that problems during protein-free adaptation (PFA) lead to a less efficient translation machinery after serum deprivation. We describe the generation of stable-producing recombinant CHO clones by protein-free transfection of a protein-free adapted host cell line, which reduces the risk of adverse clonal changes after PFA. The main advantage of this approach is the earlier predictability of clone behavior, which makes the generation of production clones by protein-free transfection, a viable and highly efficient strategy for recombinant cell line development. (c) 2006 Wiley Periodicals, Inc.

  19. Genome-Wide Comparison of Magnaporthe Species Reveals a Host-Specific Pattern of Secretory Proteins and Transposable Elements.

    Directory of Open Access Journals (Sweden)

    Meghana Deepak Shirke

    Full Text Available Blast disease caused by the Magnaporthe species is a major factor affecting the productivity of rice, wheat and millets. This study was aimed at generating genomic information for rice and non-rice Magnaporthe isolates to understand the extent of genetic variation. We have sequenced the whole genome of the Magnaporthe isolates, infecting rice (leaf and neck, finger millet (leaf and neck, foxtail millet (leaf and buffel grass (leaf. Rice and finger millet isolates infecting both leaf and neck tissues were sequenced, since the damage and yield loss caused due to neck blast is much higher as compared to leaf blast. The genome-wide comparison was carried out to study the variability in gene content, candidate effectors, repeat element distribution, genes involved in carbohydrate metabolism and SNPs. The analysis of repeat element footprints revealed some genes such as naringenin, 2-oxoglutarate 3-dioxygenase being targeted by Pot2 and Occan, in isolates from different host species. Some repeat insertions were host-specific while other insertions were randomly shared between isolates. The distributions of repeat elements, secretory proteins, CAZymes and SNPs showed significant variation across host-specific lineages of Magnaporthe indicating an independent genome evolution orchestrated by multiple genomic factors.

  20. Immune recognition of salivary proteins from the cattle tick Rhipicephalus microplus differs according to the genotype of the bovine host.

    Science.gov (United States)

    Garcia, Gustavo Rocha; Maruyama, Sandra Regina; Nelson, Kristina T; Ribeiro, José Marcos Chaves; Gardinassi, Luiz Gustavo; Maia, Antonio Augusto Mendes; Ferreira, Beatriz Rossetti; Kooyman, Frans N J; de Miranda Santos, Isabel K F

    2017-03-14

    -specific antibodies were not directly correlated with infestation phenotypes. However, in spite of receiving apparently lower amounts of tick saliva, tick-resistant bovines recognized more tick salivary proteins. These reactive salivary proteins are putatively involved in several functions of parasitism and blood-feeding. Our results indicate that neutralization by host antibodies of tick salivary proteins involved in parasitism is essential to control tick infestations.

  1. Soilborne wheat mosaic virus (SBWMV 19K protein belongs to a class of cysteine rich proteins that suppress RNA silencing

    Directory of Open Access Journals (Sweden)

    Howard Amanda

    2005-03-01

    Full Text Available Abstract Amino acid sequence analyses indicate that the Soilborne wheat mosaic virus (SBWMV 19K protein is a cysteine-rich protein (CRP and shares sequence homology with CRPs derived from furo-, hordei-, peclu- and tobraviruses. Since the hordei- and pecluvirus CRPs were shown to be pathogenesis factors and/or suppressors of RNA silencing, experiments were conducted to determine if the SBWMV 19K CRP has similar activities. The SBWMV 19K CRP was introduced into the Potato virus X (PVX viral vector and inoculated to tobacco plants. The SBWMV 19K CRP aggravated PVX-induced symptoms and restored green fluorescent protein (GFP expression to GFP silenced tissues. These observations indicate that the SBWMV 19K CRP is a pathogenicity determinant and a suppressor of RNA silencing.

  2. Is Peripheral Benzodiazepine Receptor (PBR) Gene Expression Involved in Breast Cancer Suppression by Dietary Soybean Protein?

    National Research Council Canada - National Science Library

    Das, Salil

    2006-01-01

    .... It has been established that women in Asian countries consume more soy protein than women in the United States and that the incidence of breast cancer in women in Asian countries is generally lower...

  3. Is Peripheral Benzodiazepine Receptor (PBR) Gene Expression Involved in Breast Cancer Suppression by Dietary Soybean Protein

    National Research Council Canada - National Science Library

    Das, Salil

    2004-01-01

    ...% casein and those of groups 3 and 4 received same diet containing 20% soybean protein. Animals of groups 2 and 4 received DMBA in sesame oil by gavage (15 mg per animal). Control animals (groups 1 and 3...

  4. Suppression of allergic reactions in ovalbumin-sensitized mice by yam storage proteins dioscorins.

    Science.gov (United States)

    Hsu, Yu-Jhen; Weng, Ching-Feng; Lin, Kuo-Wei; Lin, Kuo-Chih

    2013-11-27

    To study the biomedical functions of dioscorins isolated from various species of Dioscorea , we investigated their antiallergic potential using an OVA-induced allergy mouse model. All the dioscorins suppressed allergic reactions by decreasing the serum IgE and histamine levels. The serum IFN-γ and IgG2a levels increased in all the dioscorin-treated mice. The spleen cells from the dioscorin-treated mice also exhibited an up-regulation of IFN-γ secretion in response to ConA stimulation. Although dioscorins did not affect the IgG1 levels, the IL-5 levels decreased to basal levels in mice treated with dioscorins of D. alata or D. japonica and in most of the lymphoid cells of the dioscorin-treated mice in response to ConA stimulation. The decrease of IgE and histamine levels was concomitant with an increase in IFN-γ and IgG2a levels and with a decrease in IL-5 levels, suggesting that dioscorins suppressed the OVA-induced allergic reactions, possibly through modulating an imbalanced Th1/Th2 immune response.

  5. A new black Aspergillus species, A. vadensis, is a promising host for homologous and heterologous protein production

    DEFF Research Database (Denmark)

    de Vries, R.P.; Burgers, K.; van de Vondervoort, P.J.I

    2004-01-01

    A new species of the group of black aspergilli, Aspergillus vadensis, was analyzed for its potential as a host for homologous and heterologous protein production. Unlike the other black aspergilli, this strain does not acidify the culture medium when nitrate is the nitrogen source and only produces...... very low levels of extracellular proteases, mainly serine metalloproteases. The stability of A. tubingensis feruloyl esterase A (FaeA) was compared upon production in wild-type A. vadensis, A. tubingensis, and an A. niger strain in which the three main protease-encoding genes were disrupted....... The production of FaeA in A. vadensis resulted in larger amounts of intact protein than production in A. tubingensis and was similar to production in an A. niger protease disruptant, confirming in vivo the low proteolytic activity of A. vadensis. The protoplast formation and transformation efficiencies of A...

  6. The Human Cytomegalovirus Major Immediate-Early Proteins as Antagonists of Intrinsic and Innate Antiviral Host Responses

    Directory of Open Access Journals (Sweden)

    Michael Nevels

    2009-11-01

    Full Text Available The major immediate-early (IE gene of human cytomegalovirus (CMV is believed to have a decisive role in acute infection and its activity is an important indicator of viral reactivation from latency. Although a variety of gene products are expressed from this region, the 72-kDa IE1 and the 86-kDa IE2 nuclear phosphoproteins are the most abundant and important. Both proteins have long been recognized as promiscuous transcriptional regulators. More recently, a critical role of the IE1 and IE2 proteins in counteracting nonadaptive host cell defense mechanisms has been revealed. In this review we will briefly summarize the available literature on IE1- and IE2-dependent mechanisms contributing to CMV evasion from intrinsic and innate immune responses.

  7. GSK3 is required for rapalogs to induce degradation of some oncogenic proteins and to suppress cancer cell growth.

    Science.gov (United States)

    Koo, Junghui; Wang, Xuerong; Owonikoko, Taofeek K; Ramalingam, Suresh S; Khuri, Fadlo R; Sun, Shi-Yong

    2015-04-20

    The single-agent activity of rapalogs (rapamycin and its analogues) in most tumor types has been modest at best. The underlying mechanisms are largely unclear. In this report, we have uncovered a critical role of GSK3 in regulating degradation of some oncogenic proteins induced by rapalogs and cell sensitivity to rapalogs. The basal level of GSK3 activity was positively correlated with cell sensitivity of lung cancer cell lines to rapalogs. GSK3 inhibition antagonized rapamycin's growth inhibitory effects both in vitro and in vivo, while enforced activation of GSK3β sensitized cells to rapamycin. GSK3 inhibition rescued rapamcyin-induced reduction of several oncogenic proteins such as cyclin D1, Mcl-1 and c-Myc, without interfering with the ability of rapamycin to suppress mTORC1 signaling and cap binding. Interestingly, rapamycin induces proteasomal degradation of these oncogenic proteins, as evidenced by their decreased stabilities induced by rapamcyin and rescue of their reduction by proteasomal inhibition. Moreover, acute or short-time rapamycin treatment dissociated not only raptor, but also rictor from mTOR in several tested cell lines, suggesting inhibition of both mTORC1 and mTORC2. Thus, induction of GSK3-dependent degradation of these oncogenic proteins is likely secondary to mTORC2 inhibition; this effect should be critical for rapamycin to exert its anticancer activity.

  8. Systems Biology Analysis of Temporal In vivo Brucella melitensis and Bovine Transcriptomes Predicts host:Pathogen Protein–Protein Interactions

    Directory of Open Access Journals (Sweden)

    Carlos A. Rossetti

    2017-07-01

    Full Text Available To date, fewer than 200 gene-products have been identified as Brucella virulence factors, and most were characterized individually without considering how they are temporally and coordinately expressed or secreted during the infection process. Here, we describe and analyze the in vivo temporal transcriptional profile of Brucella melitensis during the initial 4 h interaction with cattle. Pathway analysis revealed an activation of the “Two component system” providing evidence that the in vivo Brucella sense and actively regulate their metabolism through the transition to an intracellular lifestyle. Contrarily, other Brucella pathways involved in virulence such as “ABC transporters” and “T4SS system” were repressed suggesting a silencing strategy to avoid stimulation of the host innate immune response very early in the infection process. Also, three flagellum-encoded loci (BMEII0150-0168, BMEII1080-1089, and BMEII1105-1114, the “flagellar assembly” pathway and the cell components “bacterial-type flagellum hook” and “bacterial-type flagellum” were repressed in the tissue-associated B. melitensis, while RopE1 sigma factor, a flagellar repressor, was activated throughout the experiment. These results support the idea that Brucella employ a stealthy strategy at the onset of the infection of susceptible hosts. Further, through systems-level in silico host:pathogen protein–protein interactions simulation and correlation of pathogen gene expression with the host gene perturbations, we identified unanticipated interactions such as VirB11::MAPK8IP1; BtaE::NFKBIA, and 22 kDa OMP precursor::BAD and MAP2K3. These findings are suggestive of new virulence factors and mechanisms responsible for Brucella evasion of the host's protective immune response and the capability to maintain a dormant state. The predicted protein–protein interactions and the points of disruption provide novel insights that will stimulate advanced hypothesis

  9. Using structural knowledge in the protein data bank to inform the search for potential host-microbe protein interactions in sequence space: application to Mycobacterium tuberculosis.

    Science.gov (United States)

    Mahajan, Gaurang; Mande, Shekhar C

    2017-04-04

    A comprehensive map of the human-M. tuberculosis (MTB) protein interactome would help fill the gaps in our understanding of the disease, and computational prediction can aid and complement experimental studies towards this end. Several sequence-based in silico approaches tap the existing data on experimentally validated protein-protein interactions (PPIs); these PPIs serve as templates from which novel interactions between pathogen and host are inferred. Such comparative approaches typically make use of local sequence alignment, which, in the absence of structural details about the interfaces mediating the template interactions, could lead to incorrect inferences, particularly when multi-domain proteins are involved. We propose leveraging the domain-domain interaction (DDI) information in PDB complexes to score and prioritize candidate PPIs between host and pathogen proteomes based on targeted sequence-level comparisons. Our method picks out a small set of human-MTB protein pairs as candidates for physical interactions, and the use of functional meta-data suggests that some of them could contribute to the in vivo molecular cross-talk between pathogen and host that regulates the course of the infection. Further, we present numerical data for Pfam domain families that highlights interaction specificity on the domain level. Not every instance of a pair of domains, for which interaction evidence has been found in a few instances (i.e. structures), is likely to functionally interact. Our sorting approach scores candidates according to how "distant" they are in sequence space from known examples of DDIs (templates). Thus, it provides a natural way to deal with the heterogeneity in domain-level interactions. Our method represents a more informed application of local alignment to the sequence-based search for potential human-microbial interactions that uses available PPI data as a prior. Our approach is somewhat limited in its sensitivity by the restricted size and

  10. Signal transduction of Helicobacter pylori during interaction with host cell protein receptors of epithelial and immune cells

    Science.gov (United States)

    Pachathundikandi, Suneesh Kumar; Tegtmeyer, Nicole; Backert, Steffen

    2013-01-01

    Helicobacter pylori infections can induce pathologies ranging from chronic gastritis, peptic ulceration to gastric cancer. Bacterial isolates harbor numerous well-known adhesins, vacuolating cytotoxin VacA, protease HtrA, urease, peptidoglycan, and type IV secretion systems (T4SS). It appears that H. pylori targets more than 40 known host protein receptors on epithelial or immune cells. A series of T4SS components such as CagL, CagI, CagY, and CagA can bind to the integrin α5β1 receptor. Other targeted membrane-based receptors include the integrins αvβ3, αvβ5, and β2 (CD18), RPTP-α/β, GP130, E-cadherin, fibronectin, laminin, CD46, CD74, ICAM1/LFA1, T-cell receptor, Toll-like receptors, and receptor tyrosine kinases EGFR, ErbB2, ErbB3, and c-Met. In addition, H. pylori is able to activate the intracellular receptors NOD1, NOD2, and NLRP3 with important roles in innate immunity. Here we review the interplay of various bacterial factors with host protein receptors. The contribution of these interactions to signal transduction and pathogenesis is discussed. PMID:24280762

  11. 6-Hydroxydopamine Inhibits the Hepatitis C Virus through Alkylation of Host and Viral Proteins and the Induction of Oxidative Stress.

    Science.gov (United States)

    Lafreniere, Matthew A; Powdrill, Megan H; Singaravelu, Ragunath; Pezacki, John Paul

    2016-11-11

    Many viruses, including the hepatitis C virus (HCV), are dependent on the host RNA silencing pathway for replication. In this study, we screened small molecule probes, previously reported to disrupt loading of the RNA-induced silencing complex (RISC), including 6-hydroxydopamine (6-OHDA), suramin (SUR), and aurintricarboxylic acid (ATA), to examine their effects on viral replication. We found that 6-OHDA inhibited HCV replication; however, 6-OHDA was a less potent inhibitor of RISC than either SUR or ATA. By generating a novel chemical probe (6-OHDA-yne), we determined that 6-OHDA covalently modifies host and virus proteins. Moreover, 6-OHDA was shown to be an alkylating agent that is capable of generating adducts with a number of enzymes involved in the oxidative stress response. Furthermore, modification of viral enzymes with 6-OHDA and 6-OHDA-yne was found to inhibit their enzymatic activity. Our findings suggest that 6-OHDA is a probe for oxidative stress as well as protein alkylation, and these properties together contribute to the antiviral effects of this compound.

  12. Sequence Variation in Rhoptry Neck Protein 10 Gene among Toxoplasma gondii Isolates from Different Hosts and Geographical Locations.

    Science.gov (United States)

    Zhao, Yu; Zhou, Donghui; Chen, Jia; Sun, Xiaolin

    2017-01-01

    Toxoplasma gondii, as a eukaryotic parasite of the phylum Apicomplexa, can infect almost all the warm-blooded animals and humans, causing toxoplasmosis. Rhoptry neck proteins (RONs) play a key role in the invasion process of T. gondii and are potential vaccine candidate molecules against toxoplasmosis. The present study examined sequence variation in the rhoptry neck protein 10 (TgRON10) gene among 10 T. gondii isolates from different hosts and geographical locations from Lanzhou province during 2014, and compared with the corresponding sequences of strains ME49 and VEG obtained from the ToxoDB database, using polymerase chain reaction (PCR) amplification, sequence analysis, and phylogenetic reconstruction by Bayesian inference (BI) and maximum parsimony (MP). Analysis of all the 12 TgRON10 genomic and cDNA sequences revealed 7 exons and 6 introns in the TgRON10 gDNA. The complete genomic sequence of the TgRON10 gene ranged from 4759 bp to 4763 bp, and sequence variation was 0-0.6% among the 12 T. gondii isolates, indicating a low sequence variation in TgRON10 gene. Phylogenetic analysis of TgRON10 sequences showed that the cluster of the 12 T. gondii isolates was not completely consistent with their respective genotypes. TgRON10 gene is not a suitable genetic marker for the differentiation of T. gondii isolates from different hosts and geographical locations, but may represent a potential vaccine candidate against toxoplasmosis, worth further studies.

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

    Science.gov (United States)

    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...

  14. Tissue-Specific Ablation of Prkar1a Causes Schwannomas by Suppressing Neurofibromatosis Protein Production

    Directory of Open Access Journals (Sweden)

    Georgette N. Jones

    2008-11-01

    Full Text Available Signaling events leading to Schwann cell tumor initiation have been extensively characterized in the context of neurofibromatosis (NF. Similar tumors are also observed in patients with the endocrine neoplasia syndrome Carney complex, which results from inactivating mutations in PRKAR1A. Loss of PRKAR1A causes enhanced protein kinase A activity, although the pathways leading to tumorigenesis are not well characterized. Tissue-specific ablation of Prkar1a in neural crest precursor cells (TEC3KO mice causes schwannomas with nearly 80% penetrance by 10 months. These heterogeneous neoplasms were clinically characterized as genetically engineered mouse schwannomas, grades II and III. At the molecular level, analysis of the tumors revealed almost complete loss of both NF proteins, despite the fact that transcript levels were increased, implying posttranscriptional regulation. Although Erk and Akt signaling are typically enhanced in NF-associated tumors, we observed no activation of either of these pathways in TEC3KO tumors. Furthermore, the small G proteins Ras, Rac1, and RhoA are all known to be involved with NF signaling. In TEC3KO tumors, all three molecules showed modest increases in total protein, but only Rac1 showed significant activation. These data suggest that dysregulated protein kinase A activation causes tumorigenesis through pathways that overlap but are distinct from those described in NF tumorigenesis.

  15. Immunization with a recombinant vaccinia virus that encodes nonstructural proteins of the hepatitis C virus suppresses viral protein levels in mouse liver.

    Science.gov (United States)

    Sekiguchi, Satoshi; Kimura, Kiminori; Chiyo, Tomoko; Ohtsuki, Takahiro; Tobita, Yoshimi; Tokunaga, Yuko; Yasui, Fumihiko; Tsukiyama-Kohara, Kyoko; Wakita, Takaji; Tanaka, Toshiyuki; Miyasaka, Masayuki; Mizuno, Kyosuke; Hayashi, Yukiko; Hishima, Tsunekazu; Matsushima, Kouji; Kohara, Michinori

    2012-01-01

    Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV), is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV) that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid-polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25), which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29((+/-))/MxCre((+/-)) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) TNF-α and (interleukin) IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine.

  16. Immunization with a recombinant vaccinia virus that encodes nonstructural proteins of the hepatitis C virus suppresses viral protein levels in mouse liver.

    Directory of Open Access Journals (Sweden)

    Satoshi Sekiguchi

    Full Text Available Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV, is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid-polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis, liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25, which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29((+/-/MxCre((+/- mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor TNF-α and (interleukin IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine.

  17. Evaluation of spectral libraries and sample preparation for DIA-LC-MS analysis of host cell proteins: A case study of a bacterially expressed recombinant biopharmaceutical protein.

    Science.gov (United States)

    Heissel, Søren; Bunkenborg, Jakob; Kristiansen, Max Per; Holmbjerg, Anne Fich; Grimstrup, Marie; Mørtz, Ejvind; Kofoed, Thomas; Højrup, Peter

    2018-07-01

    Recombinantly expressed biopharmaceutical proteins often undergo a series of purification steps with the aim of removing contaminating material. Depending on the application of the protein, there are various requirements for the degree of purity, but host cell proteins (HCPs) will in general remain in small amounts. LC-MS has emerged as an orthogonal technique, capable of providing detailed information regarding the individual proteins. The aim of this case study was to characterize the HCPs associated with a biopharmaceutical protein, provided by Statens Serum Institut (DK), which is used in the field of tuberculosis and has not previously been studied by LC-MS. The developed method and acquired experiences served to develop a generalized strategy for HCP-characterization in our laboratory. We evaluated the use of different spectral libraries, recorded in data-dependent mode for obtaining the highest HCP coverage, combined with SWATH-based absolute quantification. The accuracy of two label-free absolute quantification strategies was evaluated using stable isotope peptides. Two different sample preparation workflows were evaluated for optimal HCP yield. . The label-free strategy produced accurate quantification across several orders of magnitude, and the calculated purity was found to be in agreement with previously obtained ELISA data. Copyright © 2018 Elsevier Inc. All rights reserved.

  18. Two-step interrogation then recognition of DNA binding site by Integration Host Factor: an architectural DNA-bending protein.

    Science.gov (United States)

    Velmurugu, Yogambigai; Vivas, Paula; Connolly, Mitchell; Kuznetsov, Serguei V; Rice, Phoebe A; Ansari, Anjum

    2018-02-28

    The dynamics and mechanism of how site-specific DNA-bending proteins initially interrogate potential binding sites prior to recognition have remained elusive for most systems. Here we present these dynamics for Integration Host factor (IHF), a nucleoid-associated architectural protein, using a μs-resolved T-jump approach. Our studies show two distinct DNA-bending steps during site recognition by IHF. While the faster (∼100 μs) step is unaffected by changes in DNA or protein sequence that alter affinity by >100-fold, the slower (1-10 ms) step is accelerated ∼5-fold when mismatches are introduced at DNA sites that are sharply kinked in the specific complex. The amplitudes of the fast phase increase when the specific complex is destabilized and decrease with increasing [salt], which increases specificity. Taken together, these results indicate that the fast phase is non-specific DNA bending while the slow phase, which responds only to changes in DNA flexibility at the kink sites, is specific DNA kinking during site recognition. Notably, the timescales for the fast phase overlap with one-dimensional diffusion times measured for several proteins on DNA, suggesting that these dynamics reflect partial DNA bending during interrogation of potential binding sites by IHF as it scans DNA.

  19. The contribution of different prion protein types and host polymorphisms to clinicopathological variations in Creutzfeldt-Jakob disease.

    Science.gov (United States)

    Head, Mark W; Ironside, James W

    2012-07-01

    Creutzfeldt-Jakob disease is a fatal neurodegenerative disease that primarily affects the central nervous system. In this respect, it can be considered alongside the more frequently occurring neurodegenerative diseases, such as Alzheimer's disease. Creutzfeldt-Jakob disease is perhaps the paradigmatic protein misfolding disorder, so comparisons between the mechanisms involved in Creutzfeldt-Jakob disease and other neurodegenerative diseases associated with protein misfolding (such as the tauopathies and synucleinopathies) may also be informative. Like many of these diseases, Creutzfeldt-Jakob disease occurs sporadically or can, more rarely, be associated with mutations. However, Creutzfeldt-Jakob disease can also be acquired and is experimentally transmissible. These properties have had profound public health implications and made the disease of interest to virologists, in addition to those interested in protein misfolding disorders and neurodegeneration. The possible causes for the pronounced phenotypic variation among different forms of Creutzfeldt-Jakob disease are beginning to become understood, and these appear to depend in large measure on the genetics of the host (specifically the sequence of the prion protein gene, PRNP) and the epigenetic aspects of the agent (thought to be a misfolded and aggregated form of the PRNP gene product, termed a prion). This review will examine whether this model in its present form has sufficient complexity and subtlety to account for the clinicopathological variation evident in Creutzfeldt-Jakob disease and will outline the ways in which a more complete and informative molecular definition of human prions are currently being sought. Copyright © 2012 John Wiley & Sons, Ltd.

  20. Brucella TIR-like protein TcpB/Btp1 specifically targets the host adaptor protein MAL/TIRAP to promote infection.

    Science.gov (United States)

    Li, Wenna; Ke, Yuehua; Wang, Yufei; Yang, Mingjuan; Gao, Junguang; Zhan, Shaoxia; Xinying, Du; Huang, Liuyu; Li, Wenfeng; Chen, Zeliang; Li, Juan

    2016-08-26

    Brucella spp. are known to avoid host immune recognition and weaken the immune response to infection. Brucella like accomplish this by employing two clever strategies, called the stealth strategy and hijacking strategy. The TIR domain-containing protein (TcpB/Btp1) of Brucella melitensis is thought to be involved in inhibiting host NF-κB activation by binding to adaptors downstream of Toll-like receptors. However, of the five TIR domain-containing adaptors conserved in mammals, whether MyD88 or MAL, even other three adaptors, are specifically targeted by TcpB has not been identified. Here, we confirmed the effect of TcpB on B.melitensis virulence in mice and found that TcpB selectively targets MAL. By using siRNA against MAL, we found that TcpB from B.melitensis is involved in intracellular survival and that MAL affects intracellular replication of B.melitensis. Our results confirm that TcpB specifically targets MAL/TIRAP to disrupt downstream signaling pathways and promote intra-host survival of Brucella spp. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Evidence for suppression of immunity as a driver for genomic introgressions and host range expansion in races of Albugo candida, a generalist parasite

    DEFF Research Database (Denmark)

    McMullan, Mark; Gardiner, Anastasia; Bailey, Kate

    2015-01-01

    How generalist parasites with wide host ranges can evolve is a central question in parasite evolution. Albugo candida is an obligate biotrophic parasite that consists of many physiological races that each specialize on distinct Brassicaceae host species. By analyzing genome sequence assemblies...... by normally non-infecting races. This facilitates introgression and the exchange of effector repertoires, and may enable the evolution of novel races that can undergo clonal population expansion on new hosts. We discuss recent studies on hybridization in other eukaryotes such as yeast, Heliconius butterflies...

  2. Agrobacterium T-DNA-encoded protein Atu6002 interferes with the host auxin response

    Science.gov (United States)

    Lacroix, Benoît; Gizatullina, Diana I.; Babst, Benjamin A.; Gifford, Andrew N.; Citovsky, Vitaly

    2013-01-01

    Summary Several genes in the Agrobacterium tumefaciens transferred (T) DNA encode proteins that are involved in developmental alterations leading to the formation of tumors in infected plants. We investigated the role of the protein encoded by the Atu6002 gene, the function of which is completely unknown. The Atu6002 expression occurs in Agrobacterium-induced tumors, and is also activated upon activation of plant cell division by growth hormones. Within the expressing plant cells, the Atu6002 protein is targeted to the plasma membrane. Interestingly, constitutive ectopic expression of Atu6002 in transgenic tobacco plants lead to a severe developmental phenotype characterized by stunted growth, shorter internodes, lanceolate leaves, increased branching, and modified flower morphology. These Atu6002-expressing plants also displayed impaired response to auxin. However, auxin cellular uptake and polar transport were not significantly inhibited in these plants, suggesting that Atu6002 interferes with auxin perception or signaling pathways. PMID:24128370

  3. ZNF383, a novel KRAB-containing zinc finger protein, suppresses MAPK signaling pathway

    International Nuclear Information System (INIS)

    Cao Lei; Wang Zhi; Zhu Chuanbing; Zhao Yulian; Yuan Wuzhou; Li Jing; Wang Yuequn; Ying Zhaochu; Li Yongqing; Yu Weishi; Wu Xiushan; Liu Mingyao

    2005-01-01

    Mitogen-activated protein kinases (MAPKs) are major components of pathways controlling embryogenesis, cell differentiation, cell proliferation, and cell death. One of the most explored functions of MAPK signaling is the regulation of gene expression by direct or indirect phosphorylation and subsequent activation of transcription factors. In this article, we isolated a novel KRAB-related zinc finger gene named ZNF383 from an early embryo heart cDNA library. The cDNA of ZNF383 is 2220 bp, encoding a protein of 475 amino acids. The protein is conserved in evolution across different species. Northern blot analysis indicates that a 2.2 kb transcript specific for ZNF383 is detected in most of the examined human adult and embryonic tissues with a higher level in skeletal muscle. In COS-7 cells, ZNF383 protein is localized to nucleus and cytoplasm. ZNF383 is a transcription repressor when fused to Gal-4 DNA-binding domain and cotransfected with VP-16. Deletion analysis indicates that the KRAB box of ZNF383 is responsible for the transcriptional repressor activity. Overexpression of ZNF383 in cells inhibits the transcriptional activities of AP-1 and SRE, suggesting that ZNF383 may act as a negative regulator in MAPK-mediated signaling pathways

  4. The host antimicrobial peptide Bac71-35 binds to bacterial ribosomal proteins and inhibits protein synthesis.

    Science.gov (United States)

    Mardirossian, Mario; Grzela, Renata; Giglione, Carmela; Meinnel, Thierry; Gennaro, Renato; Mergaert, Peter; Scocchi, Marco

    2014-12-18

    Antimicrobial peptides (AMPs) are molecules from innate immunity with high potential as novel anti-infective agents. Most of them inactivate bacteria through pore formation or membrane barrier disruption, but others cross the membrane without damages and act inside the cells, affecting vital processes. However, little is known about their intracellular bacterial targets. Here we report that Bac71-35, a proline-rich AMP belonging to the cathelicidin family, can reach high concentrations (up to 340 μM) inside the E. coli cytoplasm. The peptide specifically and completely inhibits in vitro translation in the micromolar concentration range. Experiments of incorporation of radioactive precursors in macromolecules with E. coli cells confirmed that Bac71-35 affects specifically protein synthesis. Ribosome coprecipitation and crosslinking assays showed that the peptide interacts with ribosomes, binding to a limited subset of ribosomal proteins. Overall, these results indicate that the killing mechanism of Bac71-35 is based on a specific block of protein synthesis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Schiff Base Metal Derivatives Enhance the Expression of HSP70 and Suppress BAX Proteins in Prevention of Acute Gastric Lesion

    Directory of Open Access Journals (Sweden)

    Shahram Golbabapour

    2013-01-01

    Full Text Available Schiff base complexes have appeared to be promising in the treatment of different diseases and disorders and have drawn a lot of attention to their biological activities. This study was conducted to evaluate the regulatory effect of Schiff base metal derivatives on the expression of heat shock proteins (HSP 70 and BAX in protection against acute haemorrhagic gastric ulcer in rats. Rats were assigned to 6 groups of 6 rats: the normal control (Tween 20 5% v/v, 5 mL/kg, the positive control (Tween 20 5% v/v, 5 mL/kg, and four Schiff base derivative groups named Schiff_1, Schiff_2, Schiff_3, and Schiff_4 (25 mg/kg. After 1 h, all of the groups received ethanol 95% (5 mL/kg but the normal control received Tween 20 (Tween 20 5% v/v, 5 mL/kg. The animals were euthanized after 60 min and the stomachs were dissected for histology (H&E, immunohistochemistry, and western blot analysis against HSP70 and BAX proteins. The results showed that the Schiff base metal derivatives enhanced the expression of HSP70 and suppressed the expression of BAX proteins during their gastroprotection against ethanol-induced gastric lesion in rats.

  6. Schiff base metal derivatives enhance the expression of HSP70 and suppress BAX proteins in prevention of acute gastric lesion.

    Science.gov (United States)

    Golbabapour, Shahram; Gwaram, Nura Suleiman; Al-Obaidi, Mazen M Jamil; Soleimani, A F; Ali, Hapipah Mohd; Abdul Majid, Nazia

    2013-01-01

    Schiff base complexes have appeared to be promising in the treatment of different diseases and disorders and have drawn a lot of attention to their biological activities. This study was conducted to evaluate the regulatory effect of Schiff base metal derivatives on the expression of heat shock proteins (HSP) 70 and BAX in protection against acute haemorrhagic gastric ulcer in rats. Rats were assigned to 6 groups of 6 rats: the normal control (Tween 20 5% v/v, 5 mL/kg), the positive control (Tween 20 5% v/v, 5 mL/kg), and four Schiff base derivative groups named Schiff_1, Schiff_2, Schiff_3, and Schiff_4 (25 mg/kg). After 1 h, all of the groups received ethanol 95% (5 mL/kg) but the normal control received Tween 20 (Tween 20 5% v/v, 5 mL/kg). The animals were euthanized after 60 min and the stomachs were dissected for histology (H&E), immunohistochemistry, and western blot analysis against HSP70 and BAX proteins. The results showed that the Schiff base metal derivatives enhanced the expression of HSP70 and suppressed the expression of BAX proteins during their gastroprotection against ethanol-induced gastric lesion in rats.

  7. Assessment of protein solution versus crystal structure determination using spin- diffusion-suppressed NOE and heteronuclear relaxation data

    International Nuclear Information System (INIS)

    LeMaster, David M.

    1997-01-01

    A spin-diffusion-suppressed NOE buildup series has been measured for E. coli thioredoxin.The extensive 13C and 15N relaxation data previously reported for this protein allow for direct interpretation of dynamical contributions to the 1H-1H cross-relaxation rates for a large proportion of the NOE cross peaks. Estimates of the average accuracy for these derived NOE distances are bounded by 4% and 10%, based on a comparison to the corresponding X-ray distances. An independent fluctuation model is proposed for prediction of the dynamical corrections to 1H-1H cross-relaxation rates, based solely on experimental structural and heteronuclear relaxation data. This analysis is aided by the demonstration that heteronuclear order parameters greater than 0.6 depend only on the variance of the H-X bond orientation,independent of the motional model in either one- or two-dimensional diffusion (i.e., 1- S2 = 3/4 sin2 2 θσ). The combination of spin-diffusion-suppressed NOE data and analysis of dynamical corrections to 1H-1H cross-relaxation rates based on heteronuclear relaxation data has allowed for a detailed interpretation of various discrepancies between the reported solution and crystal structures

  8. Suppression of complement regulatory protein C1 inhibitor in vascular endothelial activation by inhibiting vascular cell adhesion molecule-1 action

    International Nuclear Information System (INIS)

    Zhang, Haimou; Qin, Gangjian; Liang, Gang; Li, Jinan; Chiu, Isaac; Barrington, Robert A.; Liu, Dongxu

    2007-01-01

    Increased expression of adhesion molecules by activated endothelium is a critical feature of vascular inflammation associated with the several diseases such as endotoxin shock and sepsis/septic shock. Our data demonstrated complement regulatory protein C1 inhibitor (C1INH) prevents endothelial cell injury. We hypothesized that C1INH has the ability of an anti-endothelial activation associated with suppression of expression of adhesion molecule(s). C1INH blocked leukocyte adhesion to endothelial cell monolayer in both static assay and flow conditions. In inflammatory condition, C1INH reduced vascular cell adhesion molecule (VCAM-1) expression associated with its cytoplasmic mRNA destabilization and nuclear transcription level. Studies exploring the underlying mechanism of C1INH-mediated suppression in VCAM-1 expression were related to reduction of NF-κB activation and nuclear translocation in an IκBα-dependent manner. The inhibitory effects were associated with reduction of inhibitor IκB kinase activity and stabilization of the NF-κB inhibitor IκB. These findings indicate a novel role for C1INH in inhibition of vascular endothelial activation. These observations could provide the basis for new therapeutic application of C1INH to target inflammatory processes in different pathologic situations

  9. [Suppressive Effects of Extract of Cedar Wood on Heat-induced Expression of Cellular Heat Shock Protein].

    Science.gov (United States)

    Miyakoshi, Junji; Matsubara, Eri; Narita, Eijiro; Koyama, Shin; Shimizu, Yoko; Kawai, Shuichi

    2018-01-01

     In recent years, highly antimicrobial properties of cedar heartwood essential oil against the wood-rotting fungi and pathogenic fungi have been reported in several papers. Antimicrobial properties against oral bacteria by hinokitiol contained in Thujopsis have been also extensively studied. The relation of naturally derived components and human immune system has been studied in some previous papers. In the present study, we focused on Japanese cedar, which has the widest artificial afforestation site in the country among various tree species. Extract oil was obtained from mixture of sapwood and heartwood of about 40-year cedar grown in Oguni, Kumamoto, Japan. We examined the influence of extract components from Japanese cedar woods on the expression of heat shock protein 70 (Hsp70) during heating, and on the micronucleus formation induced by the treatment of bleomycin as a DNA damaging agent. Cell lines used in this study were human fetal glial cells (SVGp12) and human glioma cells (MO54). Remarkable suppression of the Hsp70 expression induced by heating at 43°C was detected by the treatment of cedar extract in both SVGp12 and MO54 cells. We also found that cedar extract had an inhibitory tendency to reduce the micronucleus formation induced by bleomycin. From these results, the extract components from Japanese cedar woods would have an inhibitory effect of the stress response as a suppression of the heat-induced Hsp70 expression, and might have a reductive effect on carcinogenicity.

  10. Suppressive effects of a novel compound on interphotoreceptor retinoid-binding protein-induced experimental autoimmune uveoretinitis in rats

    Directory of Open Access Journals (Sweden)

    Jun-ichi Sakai

    1999-01-01

    Full Text Available The immunosuppressive effect of ethyl O-(N-(pcarboxyphenyl-carbamoyl-mycophenolate(CAM was examined in interphotoreceptor retinoid-binding protein (IRBP-induced experimental autoimmune uveoretinitis (EAU in rats. Lewis rats immunized with bovine IRBP were treated with various oral doses of CAM postimmunization. The degree of inflammation was assessed clinically each day and histologically on day 14 or day 20. Production of various cytokines and IRBP-specific antibody, as well as IRBP-specific proliferation response, was assessed. Complete inhibition of EAU in rats, both by clinical and histologic criteria, was achieved with 50 mg/kg CAM when administered daily for 14 days following IRBP immunization. Partial inhibition was observed at lesser doses of CAM. This CAM-mediated response was accompanied by diminished production of cytokines interleukin-2, interferon-γ and tumor necrosis factor-α, as well as a reduction in IRBP-specific antibody production. Furthermore, administration of CAM either in the induction phase only (days 0–7 or in the effector phase only (days 9 or 11 to day 20 was also capable of suppressing EAU, as assessed histopathologically on day 20. We conclude that CAM is effective in suppressing EAU in rats and its mechanism of action appears to involve modulation of T cell function.

  11. Alteration of a recombinant protein N-glycan structure in silkworms by partial suppression of N-acetylglucosaminidase gene expression.

    Science.gov (United States)

    Kato, Tatsuya; Kikuta, Kotaro; Kanematsu, Ayumi; Kondo, Sachiko; Yagi, Hirokazu; Kato, Koichi; Park, Enoch Y

    2017-09-01

    To synthesize complex type N-glycans in silkworms, shRNAs against the fused lobe from Bombyx mori (BmFDL), which codes N-acetylglucosaminidase (GlcNAcase) in the Golgi, was expressed by recombinant B. mori nucleopolyhedrovirus (BmNPV) in silkworm larvae. Expression was under the control of the actin promoter of B. mori or the U6-2 and i.e.-2 promoters from Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV). The reduction of specific GlcNAcase activity was observed in Bm5 cells and silkworm larvae using the U6-2 promoter. In silkworm larvae, the partial suppression of BmFDL gene expression was observed. When shRNA against BmFDL was expressed under the control of U6-2 promoter, the Man 3 GlcNAc(Fuc)GlcNAc structure appeared in a main N-glycans of recombinant human IgG. These results suggested that the control of BmFDL expression by its shRNA in silkworms caused the modification of its N-glycan synthetic pathway, which may lead to the alteration of N-glycans in the expressed recombinant proteins. Suppression of BmFDL gene expression by shRNA is not sufficient to synthesize complex N-glycans in silkworm larvae but can modify the N-glycan synthetic pathway.

  12. Receptor for activated protein kinase C 1 suppresses gastric tumor progression through nuclear factor-kB pathway.

    Science.gov (United States)

    Yong-Zheng, X; Wan-Li, M; Ji-Ming, M; Xue-Qun, R

    2015-12-01

    Nuclear factor-kB (NF-kB) activity is crucial for survival and proliferation of many kinds of malignancies, including gastric cancer (GC). The receptor for activated protein kinase C 1 (RACK1) is known to regulate tumor development, whereas the underlined mechanism has not been described clearly. We analyzed expression of RACK1 in paired human GC samples by both real-time polymerase chain reaction (PCR) and western blot. Effects of RACK inhibition with small interfering RNA or its overexpression in cultured GC cell lines were evaluated in cell viabilities. NF-kB signaling was investigated using luciferase reporter assay and real-time PCR. RACK1 was significantly decreased in GC samples. Knockdown of RACK elevated GC cell viabilities, whereas overexpression of RACK1 suppressed tumorigenesis of GC cells. Importantly, NF-kB signaling was enhanced after RACK1 expression was inhibited, suggesting the negative regulation of the pro-oncogenic NF-kB activity by RACK1 might contribute to its tumor suppressor role in GC cells. Our results support that RACK1 suppresses gastric tumor progression through the NF-kB signaling pathway.

  13. Chitooligosaccharides suppress the level of protein expression and acetylcholinesterase activity induced by Abeta25-35 in PC12 cells.

    Science.gov (United States)

    Lee, Sang-Hoon; Park, Jin-Sook; Kim, Se-Kwon; Ahn, Chang-Bum; Je, Jae-Young

    2009-02-01

    Clinical applications of acetylcholinesterase (AChE) inhibitors are widespread in Alzheimer's sufferers in order to activate central cholinergic system and alleviate cognitive deficits by inhibiting the hydrolysis of acetylcholine. In this study, six kinds of chitooligosaccharides (COSs) with different molecular weight and degree of deacetylation were examined for their inhibitory effects against AChE. The 90-COSs exhibited potent AChE inhibitory activities compared to 50-COSs, while 90-MMWCOS (1000-5000 Da) in the 90-COSs showed the highest activity. Cell culture experiment revealed that 90-MMWCOS suppressed the level of AChE protein expression and AChE activity induced by Abeta(25-35) in PC12 cell lines.

  14. A novel nuclear DnaJ protein, DNAJC8, can suppress the formation of spinocerebellar ataxia 3 polyglutamine aggregation in a J-domain independent manner

    Energy Technology Data Exchange (ETDEWEB)

    Ito, Norie [Department of Pathology, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556 (Japan); Department of Neurology, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556 (Japan); Kamiguchi, Kenjiro; Nakanishi, Katsuya; Sokolovskya, Alice; Hirohashi, Yoshihiko; Tamura, Yasuaki; Murai, Aiko; Yamamoto, Eri; Kanaseki, Takayuki; Tsukahara, Tomohide; Kochin, Vitaly [Department of Pathology, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556 (Japan); Chiba, Susumu [Department of Neurology, Clinical Brain Research Laboratory, Toyokura Memorial Hall, Sapporo Yamano-ue Hospital (Japan); Shimohama, Shun [Department of Neurology, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556 (Japan); Sato, Noriyuki [Department of Pathology, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556 (Japan); Torigoe, Toshihiko, E-mail: torigoe@sapmed.ac.jp [Department of Pathology, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556 (Japan)

    2016-06-10

    Polyglutamine (polyQ) diseases comprise neurodegenerative disorders caused by expression of expanded polyQ-containing proteins. The cytotoxicity of the expanded polyQ-containing proteins is closely associated with aggregate formation. In this study, we report that a novel J-protein, DNAJ (HSP40) Homolog, Subfamily C, Member 8 (DNAJC8), suppresses the aggregation of polyQ-containing protein in a cellular model of spinocerebellar ataxia type 3 (SCA3), which is also known as Machado-Joseph disease. Overexpression of DNAJC8 in SH-SY5Y neuroblastoma cells significantly reduced the polyQ aggregation and apoptosis, and DNAJC8 was co-localized with the polyQ aggregation in the cell nucleus. Deletion mutants of DNAJC8 revealed that the C-terminal domain of DNAJC8 was essential for the suppression of polyQ aggregation, whereas the J-domain was dispensable. Furthermore, 22-mer oligopeptide derived from C-termilal domain could suppress the polyQ aggregation. These results indicate that DNAJC8 can suppress the polyQ aggregation via a distinct mechanism independent of HSP70-based chaperone machinery and have a unique protective role against the aggregation of expanded polyQ-containing proteins such as pathogenic ataxin-3 proteins.

  15. Transcriptomic analysis of host immune and cell death responses associated with the influenza A virus PB1-F2 protein.

    Directory of Open Access Journals (Sweden)

    Ronan Le Goffic

    2011-08-01

    Full Text Available Airway inflammation plays a major role in the pathogenesis of influenza viruses and can lead to a fatal outcome. One of the challenging objectives in the field of influenza research is the identification of the molecular bases associated to the immunopathological disorders developed during infection. While its precise function in the virus cycle is still unclear, the viral protein PB1-F2 is proposed to exert a deleterious activity within the infected host. Using an engineered recombinant virus unable to express PB1-F2 and its wild-type homolog, we analyzed and compared the pathogenicity and host response developed by the two viruses in a mouse model. We confirmed that the deletion of PB1-F2 renders the virus less virulent. The global transcriptomic analyses of the infected lungs revealed a potent impact of PB1-F2 on the response developed by the host. Thus, after two days post-infection, PB1-F2 invalidation severely decreased the number of genes activated by the host. PB1-F2 expression induced an increase in the number and level of expression of activated genes linked to cell death, inflammatory response and neutrophil chemotaxis. When generating interactive gene networks specific to PB1-F2, we identified IFN-γ as a central regulator of PB1-F2-regulated genes. The enhanced cell death of airway-recruited leukocytes was evidenced using an apoptosis assay, confirming the pro-apoptotic properties of PB1-F2. Using a NF-kB luciferase adenoviral vector, we were able to quantify in vivo the implication of NF-kB in the inflammation mediated by the influenza virus infection; we found that PB1-F2 expression intensifies the NF-kB activity. Finally, we quantified the neutrophil recruitment within the airways, and showed that this type of leukocyte is more abundant during the infection of the wild-type virus. Collectively, these data demonstrate that PB1-F2 strongly influences the early host response during IAV infection and provides new insights into the

  16. RB1CC1 Protein Suppresses Type II Collagen Synthesis in Chondrocytes and Causes Dwarfism*

    Science.gov (United States)

    Nishimura, Ichiro; Chano, Tokuhiro; Kita, Hiroko; Matsusue, Yoshitaka; Okabe, Hidetoshi

    2011-01-01

    RB1-inducible coiled-coil 1 (RB1CC1) functions in various processes, such as cell growth, differentiation, senescence, apoptosis, and autophagy. The conditional transgenic mice with cartilage-specific RB1CC1 excess that were used in the present study were made for the first time by the Cre-loxP system. Cartilage-specific RB1CC1 excess caused dwarfism in mice without causing obvious abnormalities in endochondral ossification and subsequent skeletal development from embryo to adult. In vitro and in vivo analysis revealed that the dwarf phenotype in cartilaginous RB1CC1 excess was induced by reductions in the total amount of cartilage and the number of cartilaginous cells, following suppressions of type II collagen synthesis and Erk1/2 signals. In addition, we have demonstrated that two kinds of SNPs (T-547C and C-468T) in the human RB1CC1 promoter have significant influence on the self-transcriptional level. Accordingly, human genotypic variants of RB1CC1 that either stimulate or inhibit RB1CC1 transcription in vivo may cause body size variations. PMID:22049074

  17. Variation in C - reactive protein response according to host and mycobacterial characteristics in active tuberculosis

    OpenAIRE

    Brown, James; Clark, Kristina; Smith, Colette; Hopwood, Jennifer; Lynard, Oliver; Toolan, Michael; Creer, Dean; Barker, Jack; Breen, Ronan; Brown, Tim; Cropley, Ian; Lipman, Marc

    2016-01-01

    Background The C - reactive protein (CRP) response is often measured in patients with active tuberculosis (TB) yet little is known about its relationship to clinical features in TB, or whether responses differ between ethnic groups or with different Mycobacterium tuberculosis (M.tb) strain types. We report the relationship between baseline serum CRP prior to treatment and disease characteristics in a metropolitan population with TB resident in a low TB incidence region. Methods People treated...

  18. Ectopic expression of MPF2-like protein WSA206 leads to arrest in silique and seed development in heterologous host

    International Nuclear Information System (INIS)

    Khan, M.R.

    2016-01-01

    MPF2-like genes belonging to STMADS11 clade of MADS-box transcription factors are mostly involved in calyx inflation, floral reversion and fertility. However their role in fertility remained enigmatic. In this study we transformed WSA206 gene paralog - originated through genome duplication in a Solanaceous plant Withaniasomnifera - ectopically in a heterologous host Arabidopsis thaliana. Interesting phenotypes in floral organs and fruits were observed. Overexpression of WSA206 leads to arrest in silique development. The siliques were compressed and size was drastically reduced from 34mm to 3mm. Along with siliques, the seed development was also suppressed as revealed by shriveling of seeds and reduction in seed number. In extreme cases the siliques were devoid of any seeds. In cases where seeds developed, these were impaired in viability. Besides, the transgenic Arabidopsis also exhibited exorbitant growth of calyx to an extent that it resembled inflated calyx in Solanaceae. The calyx remained persistent and encapsulated the under-developed siliques containing non-viable seeds inside. Thus, fertility and sepal development are tightly coupled traits that are controlled by WSA206 paralog in heterologous system. (author)

  19. Immune recognition of Onchocerca volvulus proteins in the human host and animal models of onchocerciasis.

    Science.gov (United States)

    Manchang, T K; Ajonina-Ekoti, I; Ndjonka, D; Eisenbarth, A; Achukwi, M D; Renz, A; Brattig, N W; Liebau, E; Breloer, M

    2015-05-01

    Onchocerca volvulus is a tissue-dwelling, vector-borne nematode parasite of humans and is the causative agent of onchocerciasis or river blindness. Natural infections of BALB/c mice with Litomosoides sigmodontis and of cattle with Onchocerca ochengi were used as models to study the immune responses to O. volvulus-derived recombinant proteins (OvALT-2, OvNLT-1, Ov103 and Ov7). The humoral immune response of O. volvulus-infected humans against OvALT-2, OvNLT-1 and Ov7 revealed pronounced immunoglobulin G (IgG) titres which were, however, significantly lower than against the lysate of O. volvulus adult female worms. Sera derived from patients displaying the hyperreactive form of onchocerciasis showed a uniform trend of higher IgG reactivity both to the single proteins and the O. volvulus lysate. Sera derived from L. sigmodontis-infected mice and from calves exposed to O. ochengi transmission in a hyperendemic area also contained IgM and IgG1 specific for O. volvulus-derived recombinant proteins. These results strongly suggest that L. sigmodontis-specific and O. ochengi-specific immunoglobulins elicited during natural infection of mice and cattle cross-reacted with O. volvulus-derived recombinant antigens. Monitoring O. ochengi-infected calves over a 26-month period, provided a comprehensive kinetic of the humoral response to infection that was strictly correlated with parasite load and occurrence of microfilariae.

  20. Campylobacter jejuni acquire new host-derived CRISPR spacers when in association with bacteriophages harbouring a CRISPR-like Cas4 protein

    Directory of Open Access Journals (Sweden)

    Ian F. Connerton

    2015-01-01

    Full Text Available Campylobacter jejuni is a worldwide cause of human diarrhoeal disease. Clustered Repetitively Interspaced Palindromic Repeats (CRISPRs and associated proteins allow Bacteria and Archaea to evade bacteriophage and plasmid infection. Type II CRISPR systems are found in association with combinations of genes encoding the CRISPR-associated Cas1, Cas2, Cas4 or Csn2, and Cas9 proteins. C. jejuni possesses a minimal subtype II-C CRISPR system containing cas1, cas2, and cas9 genes whilst cas4 is notably absent. Cas4 proteins possess 5ʹ-3ʹ exonuclease activity to create recombinogenic-ends for spacer acquisition. Here we report a conserved Cas4-like protein in Campylobacter bacteriophages that creates a novel split arrangement between the bacteriophage and host that represents a new twist in the bacteriophage/host co-evolutionary arms race. The continuous association of bacteriophage and host in the carrier state life cycle of C. jejuni provided an opportunity to study spacer acquisition in this species. Remarkably all the spacer sequences observed were of host origin. We hypothesise that Campylobacter bacteriophages can use Cas4-like protein to activate spacer acquisition to use host DNA as an effective decoy to bacteriophage DNA. Bacteria that acquire self-spacers and escape phage infection must overcome CRISPR-mediated autoimmunity either by loss of the interference functions leaving them susceptible to foreign DNA incursion or tolerate changes in gene regulation.

  1. Whey protein delays gastric emptying and suppresses plasma fatty acids and their metabolites compared to casein, gluten, and fish protein

    DEFF Research Database (Denmark)

    Stanstrup, Jan; Schou, Simon S; Holmer-Jensen, Jens

    2014-01-01

    ), and cod (COD). Obese, nondiabetic subjects were included in the randomized, blinded, crossover meal study. Subjects ingested a high fat meal containing one of the four protein sources. Plasma samples were collected at five time points and metabolites analyzed using LC-Q-TOF-MS. In contrast to previous...... studies, the WI meal caused a decreased rate of gastric emptying compared to the other test meals. The WI meal also caused elevated levels of a number of amino acids, possibly stimulating insulin release leading to reduced plasma glucose. The WI meal also caused decreased levels of a number of fatty acids......, while the GLU meal caused elevated levels of a number of unidentified hydroxy fatty acids and dicarboxylic fatty acids. Also reported are a number of markers of fish intake unique to the COD meal....

  2. A viral protein promotes host SAMS1 activity and ethylene production for the benefit of virus infection.

    Science.gov (United States)

    Zhao, Shanshan; Hong, Wei; Wu, Jianguo; Wang, Yu; Ji, Shaoyi; Zhu, Shuyi; Wei, Chunhong; Zhang, Jinsong; Li, Yi

    2017-10-10

    Ethylene plays critical roles in plant development and biotic stress response, but the mechanism of ethylene in host antiviral response remains unclear. Here, we report that Rice dwarf virus (RDV) triggers ethylene production by stimulating the activity of S-adenosyl-L-methionine synthetase (SAMS), a key component of the ethylene synthesis pathway, resulting in elevated susceptibility to RDV. RDV-encoded Pns11 protein specifically interacted with OsSAMS1 to enhance its enzymatic activity, leading to higher ethylene levels in both RDV-infected and Pns11-overexpressing rice. Consistent with a counter-defense role for ethylene, Pns11-overexpressing rice, as well as those overexpressing OsSAMS1 , were substantially more susceptible to RDV infection, and a similar effect was observed in rice plants treated with an ethylene precursor. Conversely, OsSAMS1- knockout mutants, as well as an osein2 mutant defective in ethylene signaling, resisted RDV infection more robustly. Our findings uncover a novel mechanism which RDV manipulates ethylene biosynthesis in the host plants to achieve efficient infection.

  3. DNA vaccine encoding nucleocapsid and surface proteins of wild type canine distemper virus protects its natural host against distemper.

    Science.gov (United States)

    Cherpillod, P; Tipold, A; Griot-Wenk, M; Cardozo, C; Schmid, I; Fatzer, R; Schobesberger, M; Zurbriggen, R; Bruckner, L; Roch, F; Vandevelde, M; Wittek, R; Zurbriggen, A

    2000-07-01

    Canine distemper virus (CDV), a member of the genus Morbillivirus induces a highly infectious, frequently lethal disease in dogs and other carnivores. Current vaccines against canine distemper consisting of attenuated viruses have been in use for many years and have greatly reduced the incidence of distemper in the dog population. However, certain strains may not guarantee adequate protection and others can induce post vaccinal encephalitis. We tested a DNA vaccine for its ability to protect dogs, the natural host of CDV, against distemper. We constructed plasmids containing the nucleocapsid, the fusion, and the attachment protein genes of a virulent canine distemper virus strain. Mice inoculated with these plasmids developed humoral and cellular immune responses against CDV antigens. Dogs immunized with the expression plasmids developed virus-neutralizing antibodies. Significantly, vaccinated dogs were protected against challenge with virulent CDV, whereas unvaccinated animals succumbed to distemper.

  4. Low Bioavailability and High Immunogenicity of a New Brand of E. coli l-Asparaginase with Active Host Contaminating Proteins

    Directory of Open Access Journals (Sweden)

    Priscila Pini Zenatti

    2018-04-01

    Full Text Available The drug l-asparaginase is a cornerstone in the treatment of acute lymphoblastic leukemia (ALL. The native E. coli l-asparaginase used in Brazil until recently has been manufactured by Medac/Kyowa. Then a decision was taken by the Ministry of Health in 2017 to supply the National Health System with a cheaper alternative l-asparaginase manufactured by Beijing SL Pharmaceutical, called Leuginase®. As opposed to Medac, the asparaginase that has been in use in Brazil under the trade name of Aginasa®, it was not possible to find a single entry with the terms Leuginase in the Pubmed repository. The apparent lack of clinical studies and the scarcity of safety information provided to the hospitals by the drug distributor created a debate among Brazilian pediatric oncologists about issues of safety and efficacy that culminated eventually in a court decision to halt the distribution of the new drug all over the country. Boldrini Children's Center, a non-profit pediatric oncohematology hospital, has conducted its own evaluation of Leuginase®. Mass spectrometry analyses found at least 12 different contaminating host-cell proteins (HCP in Leuginase®. The presence of two HCP (beta-lactamase and malate dehydrogenase was confirmed by orthogonal methodologies. The relative number of HCP peptides ranged from 19 to 37% of the total peptides identified by mass spectrometry. In vivo studies in mice injected with Leuginase® revealed a 3 times lower plasma bioavailability and the development of higher antibody titres against l-asparaginase in comparison to Aginasa®-injected animals. The decision to buy a new drug based on its price alone is not safe. Developing countries are especially vulnerable to cheaper alternatives that lack solid quality assurance. Keywords: l-Asparaginase, Host contaminant proteins, Mass spectrometry, Bioavailability, Immunogenicity

  5. Human Immunodeficiency Virus Type 1 Nef protein modulates the lipid composition of virions and host cell membrane microdomains

    Directory of Open Access Journals (Sweden)

    Geyer Matthias

    2007-10-01

    Full Text Available Abstract Background The Nef protein of Human Immunodeficiency Viruses optimizes viral spread in the infected host by manipulating cellular transport and signal transduction machineries. Nef also boosts the infectivity of HIV particles by an unknown mechanism. Recent studies suggested a correlation between the association of Nef with lipid raft microdomains and its positive effects on virion infectivity. Furthermore, the lipidome analysis of HIV-1 particles revealed a marked enrichment of classical raft lipids and thus identified HIV-1 virions as an example for naturally occurring membrane microdomains. Since Nef modulates the protein composition and function of membrane microdomains we tested here if Nef also has the propensity to alter microdomain lipid composition. Results Quantitative mass spectrometric lipidome analysis of highly purified HIV-1 particles revealed that the presence of Nef during virus production from T lymphocytes enforced their raft character via a significant reduction of polyunsaturated phosphatidylcholine species and a specific enrichment of sphingomyelin. In contrast, Nef did not significantly affect virion levels of phosphoglycerolipids or cholesterol. The observed alterations in virion lipid composition were insufficient to mediate Nef's effect on particle infectivity and Nef augmented virion infectivity independently of whether virus entry was targeted to or excluded from membrane microdomains. However, altered lipid compositions similar to those observed in virions were also detected in detergent-resistant membrane preparations of virus producing cells. Conclusion Nef alters not only the proteome but also the lipid composition of host cell microdomains. This novel activity represents a previously unrecognized mechanism by which Nef could manipulate HIV-1 target cells to facilitate virus propagation in vivo.

  6. Sequence Variation in Rhoptry Neck Protein 10 Gene among Toxoplasma gondii Isolates from Different Hosts and Geographical Locations

    Directory of Open Access Journals (Sweden)

    Yu ZHAO

    2017-09-01

    Full Text Available Background: Toxoplasma gondii, as a eukaryotic parasite of the phylum Apicomplexa, can infect almost all the warm-blooded animals and humans, causing toxoplasmosis. Rhoptry neck proteins (RONs play a key role in the invasion process of T. gondii and are potential vaccine candidate molecules against toxoplasmosis.Methods: The present study examined sequence variation in the rhoptry neck protein 10 (TgRON10 gene among 10 T. gondii isolates from different hosts and geographical locations from Lanzhou province during 2014, and compared with the corresponding sequences of strains ME49 and VEG obtained from the ToxoDB database, using polymerase chain reaction (PCR amplification, sequence analysis, and phylogenetic reconstruction by Bayesian inference (BI and maximum parsimony (MP. Results: Analysis of all the 12 TgRON10 genomic and cDNA sequences revealed 7 exons and 6 introns in the TgRON10 gDNA. The complete genomic sequence of the TgRON10 gene ranged from 4759 bp to 4763 bp, and sequence variation was 0-0.6% among the 12 T. gondii isolates, indicating a low sequence variation in TgRON10 gene. Phylogenetic analysis of TgRON10 sequences showed that the cluster of the 12 T. gondii isolates was not completely consistent with their respective genotypes.Conclusion: TgRON10 gene is not a suitable genetic marker for the differentiation of T. gondii isolates from different hosts and geographical locations, but may represent a potential vaccine candidate against toxoplasmosis, worth further studies.

  7. Role of Pea Enation Mosaic Virus Coat Protein in the Host Plant and Aphid Vector

    Directory of Open Access Journals (Sweden)

    Juliette Doumayrou

    2016-11-01

    Full Text Available Understanding the molecular mechanisms involved in plant virus–vector interactions is essential for the development of effective control measures for aphid-vectored epidemic plant diseases. The coat proteins (CP are the main component of the viral capsids, and they are implicated in practically every stage of the viral infection cycle. Pea enation mosaic virus 1 (PEMV1, Enamovirus, Luteoviridae and Pea enation mosaic virus 2 (PEMV2, Umbravirus, Tombusviridae are two RNA viruses in an obligate symbiosis causing the pea enation mosaic disease. Sixteen mutant viruses were generated with mutations in different domains of the CP to evaluate the role of specific amino acids in viral replication, virion assembly, long-distance movement in Pisum sativum, and aphid transmission. Twelve mutant viruses were unable to assemble but were able to replicate in inoculated leaves, move long-distance, and express the CP in newly infected leaves. Four mutant viruses produced virions, but three were not transmissible by the pea aphid, Acyrthosiphon pisum. Three-dimensional modeling of the PEMV CP, combined with biological assays for virion assembly and aphid transmission, allowed for a model of the assembly of PEMV coat protein subunits.

  8. Role of Pea Enation Mosaic Virus Coat Protein in the Host Plant and Aphid Vector.

    Science.gov (United States)

    Doumayrou, Juliette; Sheber, Melissa; Bonning, Bryony C; Miller, W Allen

    2016-11-18

    Understanding the molecular mechanisms involved in plant virus-vector interactions is essential for the development of effective control measures for aphid-vectored epidemic plant diseases. The coat proteins (CP) are the main component of the viral capsids, and they are implicated in practically every stage of the viral infection cycle. Pea enation mosaic virus 1 (PEMV1, Enamovirus , Luteoviridae ) and Pea enation mosaic virus 2 (PEMV2, Umbravirus , Tombusviridae ) are two RNA viruses in an obligate symbiosis causing the pea enation mosaic disease. Sixteen mutant viruses were generated with mutations in different domains of the CP to evaluate the role of specific amino acids in viral replication, virion assembly, long-distance movement in Pisum sativum , and aphid transmission. Twelve mutant viruses were unable to assemble but were able to replicate in inoculated leaves, move long-distance, and express the CP in newly infected leaves. Four mutant viruses produced virions, but three were not transmissible by the pea aphid, Acyrthosiphon pisum . Three-dimensional modeling of the PEMV CP, combined with biological assays for virion assembly and aphid transmission, allowed for a model of the assembly of PEMV coat protein subunits.

  9. Cytoskeleton-interacting LIM-domain protein CRP1 suppresses cell proliferation and protects from stress-induced cell death

    International Nuclear Information System (INIS)

    Latonen, Leena; Jaervinen, Paeivi M.; Laiho, Marikki

    2008-01-01

    Members of the cysteine-rich protein (CRP) family are actin cytoskeleton-interacting LIM-domain proteins known to act in muscle cell differentiation. We have earlier found that CRP1, a founding member of this family, is transcriptionally induced by UV radiation in human diploid fibroblasts [M. Gentile, L. Latonen, M. Laiho, Cell cycle arrest and apoptosis provoked by UV radiation-induced DNA damage are transcriptionally highly divergent responses, Nucleic Acids Res. 31 (2003) 4779-4790]. Here we show that CRP1 is induced by growth-inhibitory signals, such as increased cellular density, and cytotoxic stress induced by UV radiation or staurosporine. We found that high levels of CRP1 correlate with differentiation-associated morphology towards the myofibroblast lineage and that expression of ectopic CRP1 suppresses cell proliferation. Following UV- and staurosporine-induced stresses, expression of CRP1 provides a survival advantage evidenced by decreased cellular death and increased cellular metabolic activity and attachment. Our studies identify that CRP1 is a novel stress response factor, and provide evidence for its growth-inhibitory and cytoprotective functions

  10. Secreted Clusterin protein inhibits osteoblast differentiation of bone marrow mesenchymal stem cells by suppressing ERK1/2 signaling pathway.

    Science.gov (United States)

    Abdallah, Basem M; Alzahrani, Abdullah M; Kassem, Moustapha

    2018-05-01

    Secreted Clusterin (sCLU, also known as Apolipoprotein J) is an anti-apoptotic glycoprotein involved in the regulation of cell proliferation, lipid transport, extracellular tissue remodeling and apoptosis. sCLU is expressed and secreted by mouse bone marrow-derived skeletal (stromal or mesenchymal) stem cells (mBMSCs), but its functional role in MSC biology is not known. In this study, we demonstrated that Clusterin mRNA expression and protein secretion in conditioned medium increased during adipocyte differentiation and decreased during osteoblast differentiation of mBMSCs. Treatment of mBMSC cultures with recombinant sCLU protein increased cell proliferation and exerted an inhibitory effect on the osteoblast differentiation while stimulated adipocyte differentiation in a dose-dependent manner. siRNA-mediated silencing of Clu expression in mBMSCs reduced adipocyte differentiation and stimulated osteoblast differentiation of mBMSCs. Furthermore, the inhibitory effect of sCLU on the osteoblast differentiation of mBMSCs was mediated by the suppression of extracellular signal-regulated kinase (ERK1/2) phosphorylation. In conclusion, we identified sCLU as a regulator of mBMSCs lineage commitment to osteoblasts versus adipocytes through a mechanism mediated by ERK1/2 signaling. Inhibiting sCLU is a possible therapeutic approach for enhancing osteoblast differentiation and consequently bone formation. Copyright © 2018 Elsevier Inc. All rights reserved.

  11. Effects of host nutrition on virulence and fitness of entomopathogenic nematodes: Lipid- and protein-based supplements in Tenebrio molitor diets

    Science.gov (United States)

    Shapiro-Ilan, David; Rojas, M. Guadalupe; Morales-Ramos, Juan A.; Lewis, Edwin E.; Tedders, W. Louis

    2008-01-01

    Entomopathogenic nematodes, Heterorhabditis indica and Steinernema riobrave, were tested for virulence and reproductive yield in Tenebrio molitor that were fed wheat bran diets with varying lipid- and protein-based supplements. Lipid supplements were based on 20% canola oil, peanut, pork or salmon, or a low lipid control (5% canola). Protein treatments consisted of basic supplement ingredients plus 0, 10, or 20% egg white; a bran-only control was also included. Some diet supplements had positive effects on nematode quality, whereas others had negative or neutral effects. All supplements with 20% lipids except canola oil caused increased T. molitor susceptibility to H. indica, whereas susceptibility to S. riobrave was not affected. Protein supplements did not affect host susceptibility, and neither lipid nor protein diet supplements affected reproductive capacity of either nematode species. Subsequently, we determined the pest control efficacy of progeny of nematodes that had been reared through T. molitor from different diets against Diaprepes abbreviatus and Otiorhynchus sulcatus. All nematode treatments reduced insect survival relative to the control (water only). Nematodes originating from T. molitor diets with the 0% or 20% protein exhibited lower efficacy versus D. abbreviatus than the intermediate level of protein (10%) or bran-only treatments. Nematodes originating from T. molitor lipid or control diets did not differ in virulence. Our research indicates that nutritional content of an insect host diet can affect host susceptibility to entomopathogenic nematodes and nematode fitness; therefore, host media could conceivably be optimized to increase in vivo nematode production efficiency. PMID:19259513

  12. Suppressing Receptor-Interacting Protein 140: a New Sight for Salidroside to Treat Cerebral Ischemia.

    Science.gov (United States)

    Chen, Tong; Ma, Zhanqiang; Zhu, Lingpeng; Jiang, Wenjiao; Wei, Tingting; Zhou, Rui; Luo, Fen; Zhang, Kai; Fu, Qiang; Ma, Chunhua; Yan, Tianhua

    2016-11-01

    The purpose of the current study was to detect the effect of salidroside (Sal) on cerebral ischemia and explore its potential mechanism. Middle cerebral artery occlusion (MCAO) was performed to investigate the effects of Sal on cerebral ischemia. The rats were randomly divided into five groups: sham group, vehicle group, clopidogrel (7.5 mg/kg) group, Sal (20 mg/kg) group, and Sal (40 mg/kg) group. SH-SY5Y cells were exposed to ischemia-reperfusion (I/R) injury to verify the protective effect of Sal in vitro. We also built the stable receptor-interacting protein 140 (RIP140)-overexpressing SH-SY5Y cells. The results showed that Sal significantly reduces brain infarct size and cerebral edema. Sal could effectively decrease the levels of interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in serum of the MCAO rats and supernatant of I/R-induced SH-SY5Y cells. Immunohistochemical and Western blot results demonstrated that Sal inhibited RIP140-mediated inflammation and apoptosis in the MCAO rats and SH-SY5Y cells. In addition, we further confirmed that RIP140/NF-κB signaling plays a crucial role by evaluating the protein expression in RIP140-overexpressing SH-SY5Y cells. Our findings suggested that Sal could be used as an effective neuroprotective agent for cerebral ischemia due to its significant effect on preventing neuronal cell injury after cerebral ischemia both in vivo and in vitro by the inhibitions of RIP140-mediated inflammation and apoptosis.

  13. The Arf GTPase-activating protein family is exploited by Salmonella enterica serovar Typhimurium to invade nonphagocytic host cells.

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    Davidson, Anthony C; Humphreys, Daniel; Brooks, Andrew B E; Hume, Peter J; Koronakis, Vassilis

    2015-02-10

    To establish intracellular infections, Salmonella bacteria trigger host cell membrane ruffling and invasion by subverting cellular Arf guanine nucleotide exchange factors (GEFs) that activate Arf1 and Arf6 GTPases by promoting GTP binding. A family of cellular Arf GTPase-activating proteins (GAPs) can downregulate Arf signaling by stimulating GTP hydrolysis, but whether they do this during infection is unknown. Here, we uncovered a remarkable role for distinct Arf GAP family members in Salmonella invasion. The Arf6 GAPs ACAP1 and ADAP1 and the Arf1 GAP ASAP1 localized at Salmonella-induced ruffles, which was not the case for the plasma membrane-localized Arf6 GAPs ARAP3 and GIT1 or the Golgi-associated Arf1 GAP1. Surprisingly, we found that loss of ACAP1, ADAP1, or ASAP1 impaired Salmonella invasion, revealing that GAPs cannot be considered mere terminators of cytoskeleton remodeling. Salmonella invasion was restored in Arf GAP-depleted cells by expressing fast-cycling Arf derivatives, demonstrating that Arf GTP/GDP cycles facilitate Salmonella invasion. Consistent with this view, both constitutively active and dominant-negative Arf derivatives that cannot undergo GTP/GDP cycles inhibited invasion. Furthermore, we demonstrated that Arf GEFs and GAPs colocalize at invading Salmonella and collaborate to drive Arf1-dependent pathogen invasion. This study revealed that Salmonella bacteria exploit a remarkable interplay between Arf GEFs and GAPs to direct cycles of Arf GTPase activation and inactivation. These cycles drive Salmonella cytoskeleton remodeling and enable intracellular infections. To initiate infections, the Salmonella bacterial pathogen remodels the mammalian actin cytoskeleton and invades host cells by subverting host Arf GEFs that activate Arf1 and Arf6 GTPases. Cellular Arf GAPs deactivate Arf GTPases and negatively regulate cell processes, but whether they target Arfs during infection is unknown. Here, we uncovered an important role for the Arf GAP

  14. Genetic analysis of ectopic growth suppression during planar growth of integuments mediated by the Arabidopsis AGC protein kinase UNICORN.

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    Enugutti, Balaji; Schneitz, Kay

    2013-01-02

    The coordination of growth within a tissue layer is of critical importance for tissue morphogenesis. For example, cells within the epidermis undergo stereotypic cell divisions that are oriented along the plane of the layer (planar growth), thereby propagating the layered epidermal structure. Little is known about the developmental control that regulates such planar growth in plants. Recent evidence suggested that the Arabidopsis AGC VIII protein kinase UNICORN (UCN) maintains planar growth by suppressing the formation of ectopic multicellular protrusions in several floral tissues including integuments. In the current model UCN controls this process during integument development by directly interacting with the ABERRANT TESTA SHAPE (ATS) protein, a member of the KANADI (KAN) family of transcription factors, thereby repressing its activity. Here we report on the further characterization of the UCN mechanism. Phenotypic analysis of flowers of ucn-1 plants impaired in floral homeotic gene activity revealed that any of the four floral whorls could produce organs carrying ucn-1 protrusions. The ectopic outgrowths of ucn integuments did not accumulate detectable signals of the auxin and cytokinin reporters DR5rev::GFP and ARR5::GUS, respectively. Furthermore, wild-type and ucn-1 seedlings showed similarly strong callus formation upon in vitro culture on callus-inducing medium. We also show that ovules of ucn-1 plants carrying the dominant ats allele sk21-D exhibited more pronounced protrusion formation. Finally ovules of ucn-1 ett-1 double mutants and ucn-1 ett-1 arf4-1 triple mutants displayed an additive phenotype. These data deepen the molecular insight into the UCN-mediated control of planar growth during integument development. The presented evidence indicates that UCN downstream signaling does not involve the control of auxin or cytokinin homeostasis. The results also reveal that UCN interacts with ATS independently of an ATS/ETT complex required for integument

  15. Surfactant protein-A suppresses eosinophil-mediated killing of Mycoplasma pneumoniae in allergic lungs.

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    Julie G Ledford

    Full Text Available Surfactant protein-A (SP-A has well-established functions in reducing bacterial and viral infections but its role in chronic lung diseases such as asthma is unclear. Mycoplasma pneumoniae (Mp frequently colonizes the airways of chronic asthmatics and is thought to contribute to exacerbations of asthma. Our lab has previously reported that during Mp infection of non-allergic airways, SP-A aides in maintaining airway homeostasis by inhibiting an overzealous TNF-alpha mediated response and, in allergic mice, SP-A regulates eosinophilic infiltration and inflammation of the airway. In the current study, we used an in vivo model with wild type (WT and SP-A(-/- allergic mice challenged with the model antigen ovalbumin (Ova that were concurrently infected with Mp (Ova+Mp to test the hypothesis that SP-A ameliorates Mp-induced stimulation of eosinophils. Thus, SP-A could protect allergic airways from injury due to release of eosinophil inflammatory products. SP-A deficient mice exhibit significant increases in inflammatory cells, mucus production and lung damage during concurrent allergic airway disease and infection (Ova+Mp as compared to the WT mice of the same treatment group. In contrast, SP-A deficient mice have significantly decreased Mp burden compared to WT mice. The eosinophil specific factor, eosinophil peroxidase (EPO, which has been implicated in pathogen killing and also in epithelial dysfunction due to oxidative damage of resident lung proteins, is enhanced in samples from allergic/infected SP-A(-/- mice as compared to WT mice. In vitro experiments using purified eosinophils and human SP-A suggest that SP-A limits the release of EPO from Mp-stimulated eosinophils thereby reducing their killing capacity. These findings are the first to demonstrate that although SP-A interferes with eosinophil-mediated biologic clearance of Mp by mediating the interaction of Mp with eosinophils, SP-A simultaneously benefits the airway by limiting inflammation

  16. JAK inhibitors suppress t(8;21) fusion protein-induced leukemia

    Science.gov (United States)

    Lo, Miao-Chia; Peterson, Luke F.; Yan, Ming; Cong, Xiuli; Hickman, Justin H.; DeKelver, Russel C.; Niewerth, Denise; Zhang, Dong-Er

    2014-01-01

    Oncogenic mutations in components of the JAK/STAT pathway, including those in cytokine receptors and JAKs, lead to increased activity of downstream signaling and are frequently found in leukemia and other hematological disorders. Thus, small-molecule inhibitors of this pathway have been the focus of targeted therapy in these hematological diseases. We previously showed that t(8;21) fusion protein AML1-ETO and its alternatively spliced variant AML1-ETO9a (AE9a) enhance the JAK/STAT pathway via down-regulation of CD45, a negative regulator of this pathway. To investigate the therapeutic potential of targeting JAK/STAT in t(8;21) leukemia, we examined the effects of a JAK2-selective inhibitor TG101209 and a JAK1/2-selective inhibitor INCB18424 on t(8;21) leukemia cells. TG101209 and INCB18424 inhibited proliferation and promoted apoptosis of these cells. Furthermore, TG101209 treatment in AE9a leukemia mice reduced tumor burden and significantly prolonged survival. TG101209 also significantly impaired the leukemia-initiating potential of AE9a leukemia cells in secondary recipient mice. These results demonstrate the potential therapeutic efficacy of JAK inhibitors in treating t(8;21) AML. PMID:23812420

  17. Topical Use of Angiopoietin-like Protein 2 RNAi-loaded Lipid Nanoparticles Suppresses Corneal Neovascularization

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    Yukako Taketani

    2016-01-01

    Full Text Available Corneal neovascularization (CNV is a sight-threatening condition that is encountered in various inflammatory settings including chemical injury. We recently confirmed that angiopoietin-like protein 2 (ANGPTL2 is a potent angiogenic and proinflammatory factor in the cornea, and we have produced a single-stranded proline-modified short hairpin anti-ANGPTL2 RNA interference molecule that is carried in a lipid nanoparticle (ANGPTL2 Li-pshRNA for topical application. In this study, we have further examined the topical delivery and anti-ANGPTL2 activity of this molecule and have found that fluorescence-labeled ANGPTL2 Li-pshRNA eye drops can penetrate all layers of the cornea and that ANGPTL2 mRNA expression was dramatically inhibited in both epithelium and stroma at 12 and 24 hours after administration. We also examined the inhibitory effect of ANGPTL2 Li-pshRNA on CNV in a mouse chemical injury model and found that the area of angiogenesis was significantly decreased in corneas treated with ANGPTL2 Li-pshRNA eye drops compared to controls. Together, these findings indicate that this modified RNA interference agent is clinically viable in a topical formulation for use against CNV.

  18. Glutathione Peroxidase-1 Suppresses the Unfolded Protein Response upon Cigarette Smoke Exposure

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    Patrick Geraghty

    2016-01-01

    Full Text Available Oxidative stress provokes endoplasmic reticulum (ER stress-induced unfolded protein response (UPR in the lungs of chronic obstructive pulmonary (COPD subjects. The antioxidant, glutathione peroxidase-1 (GPx-1, counters oxidative stress induced by cigarette smoke exposure. Here, we investigate whether GPx-1 expression deters the UPR following exposure to cigarette smoke. Expression of ER stress markers was investigated in fully differentiated normal human bronchial epithelial (NHBE cells isolated from nonsmoking, smoking, and COPD donors and redifferentiated at the air liquid interface. NHBE cells from COPD donors expressed heightened ATF4, XBP1, GRP78, GRP94, EDEM1, and CHOP compared to cells from nonsmoking donors. These changes coincided with reduced GPx-1 expression. Reintroduction of GPx-1 into NHBE cells isolated from COPD donors reduced the UPR. To determine whether the loss of GPx-1 expression has a direct impact on these ER stress markers during smoke exposure, Gpx-1−/− mice were exposed to cigarette smoke for 1 year. Loss of Gpx-1 expression enhanced cigarette smoke-induced ER stress and apoptosis. Equally, induction of ER stress with tunicamycin enhanced antioxidant expression in mouse precision-cut lung slices. Smoke inhalation also exacerbated the UPR response during respiratory syncytial virus infection. Therefore, ER stress may be an antioxidant-related pathophysiological event in COPD.

  19. Screening and identification of host proteins interacting with Theileria annulata cysteine proteinase (TaCP by yeast-two-hybrid system

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    Shuaiyang Zhao

    2017-10-01

    Full Text Available Abstract Background Theileria annulata can infect monocytes/macrophages and B lymphocytes and causes severe lymphoproliferative disease in ruminants. Meanwhile, infection by T. annulata leads to the permanent proliferation of cell population through regulating signaling pathways of host cells. Cysteine proteinases (CPs are one kind of protein hydrolase and usually play critical roles in parasite virulence, host invasion, nutrition and host immune response. However, the biological function of T. annulata CP (TaCP is still unclear. In this study, a yeast-two-hybrid assay was performed to screen host proteins interacting with TaCP, to provide information to help our understanding of the molecular mechanisms between T. annulata and host cells. Methods The cDNA from purified bovine B cells was inserted into pGADT7-SfiI vector (pGADT7-SfiI-BcDNA, Prey plasmid for constructing the yeast two-hybrid cDNA library. TaCP was cloned into the pGBKT7 vector (pGBKT7-TaCP and was considered as bait plasmid after evaluating the expression, auto-activation and toxicity tests in the yeast strain Y2HGold. The yeast two-hybrid screening was carried out via co-transforming bait and prey plasmids into yeast strain Y2HGold. Sequences of positive preys were analyzed using BLAST, Gene Ontology, UniProt and STRING. Results Two host proteins, CRBN (Bos taurus cereblon transcript variant X2 and Ppp4C (Bos indicus protein phosphatase 4 catalytic subunit were identified to interact with TaCP. The results of functional analysis showed that the two proteins were involved in many cellular processes, such as ubiquitylation regulation, microtubule organization, DNA repair, cell apoptosis and maturation of spliceosomal snRNPs. Conclusions This study is the first to screen the host proteins of bovine B cells interacting with TaCP, and 2 proteins, CRBN and Ppp4C, were identified using yeast two-hybrid technique. The results of functional analysis suggest that the two proteins are

  20. Noroviruses Co-opt the Function of Host Proteins VAPA and VAPB for Replication via a Phenylalanine-Phenylalanine-Acidic-Tract-Motif Mimic in Nonstructural Viral Protein NS1/2.

    Science.gov (United States)

    McCune, Broc T; Tang, Wei; Lu, Jia; Eaglesham, James B; Thorne, Lucy; Mayer, Anne E; Condiff, Emily; Nice, Timothy J; Goodfellow, Ian; Krezel, Andrzej M; Virgin, Herbert W

    2017-07-11

    The Norovirus genus contains important human pathogens, but the role of host pathways in norovirus replication is largely unknown. Murine noroviruses provide the opportunity to study norovirus replication in cell culture and in small animals. The human norovirus nonstructural protein NS1/2 interacts with the host protein VAMP-associated protein A (VAPA), but the significance of the NS1/2-VAPA interaction is unexplored. Here we report decreased murine norovirus replication in VAPA- and VAPB-deficient cells. We characterized the role of VAPA in detail. VAPA was required for the efficiency of a step(s) in the viral replication cycle after entry of viral RNA into the cytoplasm but before the synthesis of viral minus-sense RNA. The interaction of VAPA with viral NS1/2 proteins is conserved between murine and human noroviruses. Murine norovirus NS1/2 directly bound the major sperm protein (MSP) domain of VAPA through its NS1 domain. Mutations within NS1 that disrupted interaction with VAPA inhibited viral replication. Structural analysis revealed that the viral NS1 domain contains a mimic of the phenylalanine-phenylalanine-acidic-tract (FFAT) motif that enables host proteins to bind to the VAPA MSP domain. The NS1/2-FFAT mimic region interacted with the VAPA-MSP domain in a manner similar to that seen with bona fide host FFAT motifs. Amino acids in the FFAT mimic region of the NS1 domain that are important for viral replication are highly conserved across murine norovirus strains. Thus, VAPA interaction with a norovirus protein that functionally mimics host FFAT motifs is important for murine norovirus replication. IMPORTANCE Human noroviruses are a leading cause of gastroenteritis worldwide, but host factors involved in norovirus replication are incompletely understood. Murine noroviruses have been studied to define mechanisms of norovirus replication. Here we defined the importance of the interaction between the hitherto poorly studied NS1/2 norovirus protein and the

  1. Bactericidal/Permeability-increasing protein fold-containing family member A1 in airway host protection and respiratory disease.

    Science.gov (United States)

    Britto, Clemente J; Cohn, Lauren

    2015-05-01

    Bactericidal/permeability-increasing protein fold-containing family member A1 (BPIFA1), formerly known as SPLUNC1, is one of the most abundant proteins in respiratory secretions and has been identified with increasing frequency in studies of pulmonary disease. Its expression is largely restricted to the respiratory tract, being highly concentrated in the upper airways and proximal trachea. BPIFA1 is highly responsive to airborne pathogens, allergens, and irritants. BPIFA1 actively participates in host protection through antimicrobial, surfactant, airway surface liquid regulation, and immunomodulatory properties. Its expression is modulated in multiple lung diseases, including cystic fibrosis, chronic obstructive pulmonary disease, respiratory malignancies, and idiopathic pulmonary fibrosis. However, the role of BPIFA1 in pulmonary pathogenesis remains to be elucidated. This review highlights the versatile properties of BPIFA1 in antimicrobial protection and its roles as a sensor of environmental exposure and regulator of immune cell function. A greater understanding of the contribution of BPIFA1 to disease pathogenesis and activity may clarify if BPIFA1 is a biomarker and potential drug target in pulmonary disease.

  2. Dasatinib synergises with irinotecan to suppress hepatocellular carcinoma via inhibiting the protein synthesis of PLK1.

    Science.gov (United States)

    Xu, Li; Zhu, Yuanrun; Shao, Jinjin; Chen, Min; Yan, Hao; Li, Guanqun; Zhu, Yi; Xu, Zhifei; Yang, Bo; Luo, Peihua; He, Qiaojun

    2017-04-11

    Hepatocellular carcinoma (HCC) is one of the most common types of malignant tumour and has poor prognosis. Currently, systematic chemotherapy is the only approach to prolong survival. Thus the development of new treatment regimens is urgently needed to improve the therapeutic efficacy. Our study intended to assess the combination of dasatinib and irinotecan against HCC and made an effort to develop a potential medical choice for advanced HCC patients. We used SRB colorimetric assay and clonogenic assay to assess antitumour effect in vitro and HCC xenograft model to assess antitumour effect in vivo. We applied flow cytometry and western blotting to explore the mechanism of the combined therapy. Knockdown and overexpression of PLK1 are also applied for validation. We confirmed that dasatinib has synergistic effect with irinotecan (or SN38) on HCC both in vitro and in vivo. The effect is due to arisen apoptosis rate of HCC cells that is accompanied by mitochondria dysfunction. The enhanced antitumour efficacy of SN38 could be explained by additional inhibition of PLK1, which is triggered by dasatinib. Unlike existed PLK1 inhibitors, dasatinib does not inhibit PLK1 activity in a direct way. Instead, we found that dasatinib reduces PLK1 level by interfering with its protein synthesis progress. We validated that this kind of downregulation of PLK1 level has a key role in the synergistic effect of the two agents. Dasatinib is able to reinforce the anti-HCC efficacy of irinotecan/SN38 by downregulation of PLK1 synthesis. The combination of the two agents might be a potential medical choice for HCC therapy.

  3. Protein kinase C (PKC) isoforms in cancer, tumor promotion and tumor suppression.

    Science.gov (United States)

    Isakov, Noah

    2018-02-01

    The AGC family of serine/threonine kinases (PKA, PKG, PKC) includes more than 60 members that are critical regulators of numerous cellular functions, including cell cycle and differentiation, morphogenesis, and cell survival and death. Mutation and/or dysregulation of AGC kinases can lead to malignant cell transformation and contribute to the pathogenesis of many human diseases. Members of one subgroup of AGC kinases, the protein kinase C (PKC), have been singled out as critical players in carcinogenesis, following their identification as the intracellular receptors of phorbol esters, which exhibit tumor-promoting activities. This observation attracted the attention of researchers worldwide and led to intense investigations on the role of PKC in cell transformation and the potential use of PKC as therapeutic drug targets in cancer diseases. Studies demonstrated that many cancers had altered expression and/or mutation of specific PKC genes. However, the causal relationships between the changes in PKC gene expression and/or mutation and the direct cause of cancer remain elusive. Independent studies in normal cells demonstrated that activation of PKC is essential for the induction of cell activation and proliferation, differentiation, motility, and survival. Based on these observations and the general assumption that PKC isoforms play a positive role in cell transformation and/or cancer progression, many PKC inhibitors have entered clinical trials but the numerous attempts to target PKC in cancer has so far yielded only very limited success. More recent studies demonstrated that PKC function as tumor suppressors, and suggested that future clinical efforts should focus on restoring, rather than inhibiting, PKC activity. The present manuscript provides some historical perspectives on the tumor promoting function of PKC, reviewing some of the observations linking PKC to cancer progression, and discusses the role of PKC in the pathogenesis of cancer diseases and its

  4. Host AMPK Is a Modulator of Plasmodium Liver Infection

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    Margarida T. Grilo Ruivo

    2016-09-01

    Full Text Available Manipulation of the master regulator of energy homeostasis AMP-activated protein kinase (AMPK activity is a strategy used by many intracellular pathogens for successful replication. Infection by most pathogens leads to an activation of host AMPK activity due to the energetic demands placed on the infected cell. Here, we demonstrate that the opposite is observed in cells infected with rodent malaria parasites. Indeed, AMPK activity upon the infection of hepatic cells is suppressed and dispensable for successful infection. By contrast, an overactive AMPK is deleterious to intracellular growth and replication of different Plasmodium spp., including the human malaria parasite, P. falciparum. The negative impact of host AMPK activity on infection was further confirmed in mice under conditions that activate its function. Overall, this work establishes the role of host AMPK signaling as a suppressive pathway of Plasmodium hepatic infection and as a potential target for host-based antimalarial interventions.

  5. The host outer membrane proteins OmpA and OmpC are associated with the Shigella phage Sf6 virion

    International Nuclear Information System (INIS)

    Zhao Haiyan; Sequeira, Reuben D.; Galeva, Nadezhda A.; Tang Liang

    2011-01-01

    Assembly of dsDNA bacteriophage is a precisely programmed process. Potential roles of host cell components in phage assembly haven't been well understood. It was previously reported that two unidentified proteins were present in bacteriophage Sf6 virion (Casjens et al, 2004, J.Mol.Biol. 339, 379-394, Fig. 2A). Using tandem mass spectrometry, we have identified the two proteins as outer membrane proteins (OMPs) OmpA and OmpC from its host Shigella flexneri. The transmission electron cryo-microscopy structure of Sf6 shows significant density at specific sites at the phage capsid inner surface. This density fit well with the characteristic beta-barrel domains of OMPs, thus may be due to the two host proteins. Locations of this density suggest a role in Sf6 morphogenesis reminiscent of phage-encoded cementing proteins. These data indicate a new, OMP-related phage:host linkage, adding to previous knowledge that some lambdoid bacteriophage genomes contain OmpC-like genes that express phage-encoded porins in the lysogenic state.

  6. Chemokines, macrophage inflammatory protein-2 and stromal cell-derived factor-1{alpha}, suppress amyloid {beta}-induced neurotoxicity

    Energy Technology Data Exchange (ETDEWEB)

    Raman, Dayanidhi; Milatovic, Snjezana-Zaja [Department of Cancer Biology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States); Milatovic, Dejan [Department of Pediatrics/Pediatric Toxicology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States); Splittgerber, Ryan [Department of Cancer Biology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States); Fan, Guo-Huang [Department of Neurobiology and Neurotoxicology, Meharry Medical College, Nashville, TN 37221 (United States); Richmond, Ann, E-mail: ann.richmond@vanderbilt.edu [VA Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States)

    2011-11-15

    Alzheimer's disease (AD) is characterized by a progressive cognitive decline and accumulation of neurotoxic oligomeric peptides amyloid-{beta} (A{beta}). Although the molecular events are not entirely known, it has become evident that inflammation, environmental and other risk factors may play a causal, disruptive and/or protective role in the development of AD. The present study investigated the ability of the chemokines, macrophage inflammatory protein-2 (MIP-2) and stromal cell-derived factor-1{alpha} (SDF-1{alpha}), the respective ligands for chemokine receptors CXCR2 and CXCR4, to suppress A{beta}-induced neurotoxicity in vitro and in vivo. Pretreatment with MIP-2 or SDF-1{alpha} significantly protected neurons from A{beta}-induced dendritic regression and apoptosis in vitro through activation of Akt, ERK1/2 and maintenance of metalloproteinase ADAM17 especially with SDF-1{alpha}. Intra-cerebroventricular (ICV) injection of A{beta} led to reduction in dendritic length and spine density of pyramidal neurons in the CA1 area of the hippocampus and increased oxidative damage 24 h following the exposure. The A{beta}-induced morphometric changes of neurons and increase in biomarkers of oxidative damage, F{sub 2}-isoprostanes, were significantly inhibited by pretreatment with the chemokines MIP-2 or SDF-1{alpha}. Additionally, MIP-2 or SDF-1{alpha} was able to suppress the aberrant mislocalization of p21-activated kinase (PAK), one of the proteins involved in the maintenance of dendritic spines. Furthermore, MIP-2 also protected neurons against A{beta} neurotoxicity in CXCR2-/- mice, potentially through observed up regulation of CXCR1 mRNA. Understanding the neuroprotective potential of chemokines is crucial in defining the role for their employment during the early stages of neurodegeneration. -- Research highlights: Black-Right-Pointing-Pointer Neuroprotective ability of the chemokines MIP2 and CXCL12 against A{beta} toxicity. Black-Right-Pointing-Pointer MIP

  7. Chemokines, macrophage inflammatory protein-2 and stromal cell-derived factor-1α, suppress amyloid β-induced neurotoxicity

    International Nuclear Information System (INIS)

    Raman, Dayanidhi; Milatovic, Snjezana-Zaja; Milatovic, Dejan; Splittgerber, Ryan; Fan, Guo-Huang; Richmond, Ann

    2011-01-01

    Alzheimer's disease (AD) is characterized by a progressive cognitive decline and accumulation of neurotoxic oligomeric peptides amyloid-β (Aβ). Although the molecular events are not entirely known, it has become evident that inflammation, environmental and other risk factors may play a causal, disruptive and/or protective role in the development of AD. The present study investigated the ability of the chemokines, macrophage inflammatory protein-2 (MIP-2) and stromal cell-derived factor-1α (SDF-1α), the respective ligands for chemokine receptors CXCR2 and CXCR4, to suppress Aβ-induced neurotoxicity in vitro and in vivo. Pretreatment with MIP-2 or SDF-1α significantly protected neurons from Aβ-induced dendritic regression and apoptosis in vitro through activation of Akt, ERK1/2 and maintenance of metalloproteinase ADAM17 especially with SDF-1α. Intra-cerebroventricular (ICV) injection of Aβ led to reduction in dendritic length and spine density of pyramidal neurons in the CA1 area of the hippocampus and increased oxidative damage 24 h following the exposure. The Aβ-induced morphometric changes of neurons and increase in biomarkers of oxidative damage, F 2 -isoprostanes, were significantly inhibited by pretreatment with the chemokines MIP-2 or SDF-1α. Additionally, MIP-2 or SDF-1α was able to suppress the aberrant mislocalization of p21-activated kinase (PAK), one of the proteins involved in the maintenance of dendritic spines. Furthermore, MIP-2 also protected neurons against Aβ neurotoxicity in CXCR2−/− mice, potentially through observed up regulation of CXCR1 mRNA. Understanding the neuroprotective potential of chemokines is crucial in defining the role for their employment during the early stages of neurodegeneration. -- Research highlights: ► Neuroprotective ability of the chemokines MIP2 and CXCL12 against Aβ toxicity. ► MIP-2 or CXCL12 prevented dendritic regression and apoptosis in vitro. ► Neuroprotection through activation of Akt, ERK

  8. A putative ATP/GTP binding protein affects Leishmania mexicana growth in insect vectors and vertebrate hosts

    Science.gov (United States)

    Hlaváčová, Jana; Zimmer, Sara L.; Butenko, Anzhelika; Podešvová, Lucie; Leštinová, Tereza; Lukeš, Julius; Kostygov, Alexei; Votýpka, Jan; Volf, Petr

    2017-01-01

    Background Leishmania virulence factors responsible for the complicated epidemiology of the various leishmaniases remain mainly unidentified. This study is a characterization of a gene previously identified as upregulated in two of three overlapping datasets containing putative factors important for Leishmania’s ability to establish mammalian intracellular infection and to colonize the gut of an insect vector. Methodology/Principal findings The investigated gene encodes ATP/GTP binding motif-containing protein related to Leishmania development 1 (ALD1), a cytosolic protein that contains a cryptic ATP/GTP binding P-loop. We compared differentiation, growth rates, and infective abilities of wild-type and ALD1 null mutant cell lines of L. mexicana. Loss of ALD1 results in retarded growth kinetics but not defects in differentiation in axenic culture. Similarly, when mice and the sand fly vector were infected with the ALD1 null mutant, the primary difference in infection and colonization phenotype relative to wild type was an inability to achieve maximal host pathogenicity. While ability of the ALD1 null mutant cells to infect macrophages in vitro was not affected, replication within macrophages was clearly curtailed. Conclusions/Significance L. mexicana ALD1, encoding a protein with no assigned functional domains or motifs, was identified utilizing multiple comparative analyses with the related and often experimentally overlooked monoxenous flagellates. We found that it plays a role in Leishmania infection and colonization in vitro and in vivo. Results suggest that ALD1 functions in L. mexicana’s general metabolic network, rather than function in specific aspect of virulence as anticipated from the compared datasets. This result validates our comparative genomics approach for finding relevant factors, yet highlights the importance of quality laboratory-based analysis of genes tagged by these methods. PMID:28742133

  9. Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair

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    Gustafsson, Ann-Sofie, E-mail: ann-sofie.gustafsson@bms.uu.se; Abramenkovs, Andris; Stenerlöw, Bo

    2014-11-15

    Highlights: • We reduced the level of DNA-PKcs with siRNA and examined cells after γ-irradiation. • Low DNA-PKcs levels lead to radiosensitivity but did not affect repair of DSB. • Low DNA-PKcs levels may block progression of mitosis. • DNA-PKcs role in mitotic progression is independent of its role in DSB repair. • We suggest different mechanisms by which loss of DNA-PKcs function sensitize cells. - Abstract: Efficient and correct repair of DNA double-strand break (DSB) is critical for cell survival. Defects in the DNA repair may lead to cell death, genomic instability and development of cancer. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of the non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. In the present study, by using siRNA against DNA-PKcs in four human cell lines, we examined how low levels of DNA-PKcs affected cellular response to ionizing radiation. Decrease of DNA-PKcs levels by 80–95%, induced by siRNA treatment, lead to extreme radiosensitivity, similar to that seen in cells completely lacking DNA-PKcs and low levels of DNA-PKcs promoted cell accumulation in G2/M phase after irradiation and blocked progression of mitosis. Surprisingly, low levels of DNA-PKcs did not affect the repair capacity and the removal of 53BP1 or γ-H2AX foci and rejoining of DSB appeared normal. This was in strong contrast to cells completely lacking DNA-PKcs and cells treated with the DNA-PKcs inhibitor NU7441, in which DSB repair were severely compromised. This suggests that there are different mechanisms by which loss of DNA-PKcs functions can sensitize cells to ionizing radiation. Further, foci of phosphorylated DNA-PKcs (T2609 and S2056) co-localized with DSB and this was independent of the amount of DNA-PKcs but foci of DNA-PKcs was only seen in siRNA-treated cells. Our study emphasizes on the critical role of DNA-PKcs for maintaining survival after radiation exposure

  10. Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair

    International Nuclear Information System (INIS)

    Gustafsson, Ann-Sofie; Abramenkovs, Andris; Stenerlöw, Bo

    2014-01-01

    Highlights: • We reduced the level of DNA-PKcs with siRNA and examined cells after γ-irradiation. • Low DNA-PKcs levels lead to radiosensitivity but did not affect repair of DSB. • Low DNA-PKcs levels may block progression of mitosis. • DNA-PKcs role in mitotic progression is independent of its role in DSB repair. • We suggest different mechanisms by which loss of DNA-PKcs function sensitize cells. - Abstract: Efficient and correct repair of DNA double-strand break (DSB) is critical for cell survival. Defects in the DNA repair may lead to cell death, genomic instability and development of cancer. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of the non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. In the present study, by using siRNA against DNA-PKcs in four human cell lines, we examined how low levels of DNA-PKcs affected cellular response to ionizing radiation. Decrease of DNA-PKcs levels by 80–95%, induced by siRNA treatment, lead to extreme radiosensitivity, similar to that seen in cells completely lacking DNA-PKcs and low levels of DNA-PKcs promoted cell accumulation in G2/M phase after irradiation and blocked progression of mitosis. Surprisingly, low levels of DNA-PKcs did not affect the repair capacity and the removal of 53BP1 or γ-H2AX foci and rejoining of DSB appeared normal. This was in strong contrast to cells completely lacking DNA-PKcs and cells treated with the DNA-PKcs inhibitor NU7441, in which DSB repair were severely compromised. This suggests that there are different mechanisms by which loss of DNA-PKcs functions can sensitize cells to ionizing radiation. Further, foci of phosphorylated DNA-PKcs (T2609 and S2056) co-localized with DSB and this was independent of the amount of DNA-PKcs but foci of DNA-PKcs was only seen in siRNA-treated cells. Our study emphasizes on the critical role of DNA-PKcs for maintaining survival after radiation exposure

  11. Co-suppression of synthesis of major x-kafirin sub-class together with y-kafirin-1 and y-kafirin-2 required for substantially improved protein digestibility in transgenic sorghum

    CSIR Research Space (South Africa)

    Grootboom, AW

    2014-01-01

    Full Text Available Co-suppressing major kafirin sub-classes is fundamental to improved protein digestibility and nutritional value of sorghum. The improvement is linked to an irregularly invaginated phenotype of protein bodies....

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

    Directory of Open Access Journals (Sweden)

    Lara J. Kohler

    2016-07-01

    Full Text Available 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.

  13. Agrobacterium-delivered virulence protein VirE2 is trafficked inside host cells via a myosin XI-K-powered ER/actin network.

    Science.gov (United States)

    Yang, Qinghua; Li, Xiaoyang; Tu, Haitao; Pan, Shen Q

    2017-03-14

    Agrobacterium tumefaciens causes crown gall tumors on various plants by delivering transferred DNA (T-DNA) and virulence proteins into host plant cells. Under laboratory conditions, the bacterium is widely used as a vector to genetically modify a wide range of organisms, including plants, yeasts, fungi, and algae. Various studies suggest that T-DNA is protected inside host cells by VirE2, one of the virulence proteins. However, it is not clear how Agrobacterium -delivered factors are trafficked through the cytoplasm. In this study, we monitored the movement of Agrobacterium -delivered VirE2 inside plant cells by using a split-GFP approach in real time. Agrobacterium -delivered VirE2 trafficked via the endoplasmic reticulum (ER) and F-actin network inside plant cells. During this process, VirE2 was aggregated as filamentous structures and was present on the cytosolic side of the ER. VirE2 movement was powered by myosin XI-K. Thus, exogenously produced and delivered VirE2 protein can use the endogenous host ER/actin network for movement inside host cells. The A. tumefaciens pathogen hijacks the conserved host infrastructure for virulence trafficking. Well-conserved infrastructure may be useful for Agrobacterium to target a wide range of recipient cells and achieve a high efficiency of transformation.

  14. Alteration of protein levels during influenza virus H1N1 infection in host cells: a proteomic survey of host and virus reveals differential dynamics.

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    Susann Kummer

    Full Text Available We studied the dynamics of the proteome of influenza virus A/PR/8/34 (H1N1 infected Madin-Darby canine kidney cells up to 12 hours post infection by mass spectrometry based quantitative proteomics using the approach of stable isotope labeling by amino acids in cell culture (SILAC. We identified 1311 cell proteins and, apart from the proton channel M2, all major virus proteins. Based on their abundance two groups of virus proteins could be distinguished being in line with the function of the proteins in genesis and formation of new virions. Further, the data indicate a correlation between the amount of proteins synthesized and their previously determined copy number inside the viral particle. We employed bioinformatic approaches such as functional clustering, gene ontology, and pathway (KEGG enrichment tests to uncover co-regulated cellular protein sets, assigned the individual subsets to their biological function, and determined their interrelation within the progression of viral infection. For the first time we are able to describe dynamic changes of the cellular and, of note, the viral proteome in a time dependent manner simultaneously. Through cluster analysis, time dependent patterns of protein abundances revealed highly dynamic up- and/or down-regulation processes. Taken together our study provides strong evidence that virus infection has a major impact on the cell status at the protein level.

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

    Science.gov (United States)

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

    2017-01-01

    Fungi that cause cereal rust diseases (genus Puccinia) are important pathogens of wheat globally. Upon infection, the fungus secretes a number of effector proteins. Although a large repository of putative effectors has been predicted using bioinformatic pipelines, the lack of available high-throughput effector screening systems has limited functional studies on these proteins. In this study, we mined the available transcriptomes of Puccinia graminis and P. striiformis to look for potential effectors that suppress host hypersensitive response (HR). Twenty small (wheat, confirming its activity in a homologous system. Overall, this study provides the first evidence for the presence of effectors in Puccinia species suppressing multiple plant defense responses.

  16. The influenza fingerprints: NS1 and M1 proteins contribute to specific host cell ultrastructure signatures upon infection by different influenza A viruses

    Energy Technology Data Exchange (ETDEWEB)

    Terrier, Olivier; Moules, Vincent; Carron, Coralie; Cartet, Gaeelle [Equipe VirCell, Laboratoire de Virologie et Pathologie Humaine, VirPath EMR 4610, Universite de Lyon, Universite Claude Bernard Lyon 1, Hospices Civils de Lyon, Faculte de medecine RTH Laennec, rue Guillaume Paradin, F-69008 Lyon (France); Frobert, Emilie [Laboratoire de Virologie, Centre de Biologie et de Pathologie Est, Hospices Civils de Lyon, 59 boulevard Pinel, F-69677 Bron Cedex, Lyon (France); Yver, Matthieu; Traversier, Aurelien [Equipe VirCell, Laboratoire de Virologie et Pathologie Humaine, VirPath EMR 4610, Universite de Lyon, Universite Claude Bernard Lyon 1, Hospices Civils de Lyon, Faculte de medecine RTH Laennec, rue Guillaume Paradin, F-69008 Lyon (France); Wolff, Thorsten [Division of Influenza/Respiratory Viruses, Robert Koch Institute, Nordufer 20, D-13353 Berlin (Germany); Riteau, Beatrice [Laboratoire de Virologie et Pathologie Humaine, VirPath EMR 4610, Universite de Lyon, Universite Claude Bernard Lyon 1, Hospices Civils de Lyon, Faculte de medecine RTH Laennec, rue Guillaume Paradin, F-69008 Lyon (France); Naffakh, Nadia [Institut Pasteur, Unite de Genetique Moleculaire des Virus Respiratoires, URA CNRS 3015, EA302 Universite Paris Diderot, Paris (France); and others

    2012-10-10

    Influenza A are nuclear replicating viruses which hijack host machineries in order to achieve optimal infection. Numerous functional virus-host interactions have now been characterized, but little information has been gathered concerning their link to the virally induced remodeling of the host cellular architecture. In this study, we infected cells with several human and avian influenza viruses and we have analyzed their ultrastructural modifications by using electron and confocal microscopy. We discovered that infections lead to a major and systematic disruption of nucleoli and the formation of a large number of diverse viral structures showing specificity that depended on the subtype origin and genomic composition of viruses. We identified NS1 and M1 proteins as the main actors in the remodeling of the host ultra-structure and our results suggest that each influenza A virus strain could be associated with a specific cellular fingerprint, possibly correlated to the functional properties of their viral components.

  17. The influenza fingerprints: NS1 and M1 proteins contribute to specific host cell ultrastructure signatures upon infection by different influenza A viruses

    International Nuclear Information System (INIS)

    Terrier, Olivier; Moules, Vincent; Carron, Coralie; Cartet, Gaëlle; Frobert, Emilie; Yver, Matthieu; Traversier, Aurelien; Wolff, Thorsten; Riteau, Beatrice; Naffakh, Nadia

    2012-01-01

    Influenza A are nuclear replicating viruses which hijack host machineries in order to achieve optimal infection. Numerous functional virus–host interactions have now been characterized, but little information has been gathered concerning their link to the virally induced remodeling of the host cellular architecture. In this study, we infected cells with several human and avian influenza viruses and we have analyzed their ultrastructural modifications by using electron and confocal microscopy. We discovered that infections lead to a major and systematic disruption of nucleoli and the formation of a large number of diverse viral structures showing specificity that depended on the subtype origin and genomic composition of viruses. We identified NS1 and M1 proteins as the main actors in the remodeling of the host ultra-structure and our results suggest that each influenza A virus strain could be associated with a specific cellular fingerprint, possibly correlated to the functional properties of their viral components.

  18. Doliroside A from Dolichos falcata Klein suppressing amyloid β-protein 42 fibrillogenesis: An insight at molecular level.

    Directory of Open Access Journals (Sweden)

    Dongpu Li

    Full Text Available A bioactive chemical constituent, doliroside A, from Chinese traditional herbal medicine Dolichos falcata Klein was isolated, purified and identified by 60% ethanol extraction, thin layer chromatography (TLC, high performance liquid chromatography (HPLC and nuclear magnetic resonance (NMR spectroscopy. Molecular interaction mechanism between doliroside and amyloid β42 protein was evaluated by thioflavin T fluorescence (ThT, circular dichroism (CD, atomic force microscope (AFM, and differential scanning calorimeter (DSC from the aspects of kinetics, secondary structure, morphology, and thermodynamics, respectively. Results show that the purity of doliroside A is 99.9% by HPLC, and its chemical structure is identified by 1H- and 13C-NMR. Doliroside A is observed to be concentration-dependent inhibiting the fibrillation of Aβ42 with the IC50 value of 26.57 ± 1.6 μM. CD and DSC results imply that doliroside A can bind to the nuclei and oligomers of Aβ42 to form a stable complex and suppress Aβ42 fibrillation. AFM images show that doliroside A, after bound to the nuclei and oligomers, redirect Aβ42 into off-pathway, amorphous oligomers. These findings not only provide a full insight into the molecular interaction mechanisms between Aβ42 and doliroside A, but also facilitate the development of new native anti-AD drug of doliroside A compound.

  19. Esculetin exerts anti-proliferative effects against non-small-cell lung carcinoma by suppressing specificity protein 1 in vitro.

    Science.gov (United States)

    Lee, Ra H; Jeon, Young-Joo; Cho, Jin H; Jang, Jeong-Yun; Kong, Il-Keun; Kim, Seok-Ho; Kim, MinSeok S; Chung, Hak-Jae; Oh, Keon B; Park, Seon-Min; Shin, Jae-Cheon; Seo, Jae-Min; Ko, Sungho; Shim, Jung-Hyun; Chae, Jung-Il

    2017-01-01

    Esculetin, a coumarin derivative, is a phenolic compound isolated from Artemisia capillaris, Citrus limonia, and Euphorbia lathyris. Although it has been reported to have anti-inflammatory, anti-oxidant, and anti-proliferative activities in several human cancers, its anti-proliferative activity against non-small-cell lung carcinoma (NSCLC) and the molecular mechanisms involved have not been adequately elucidated. In this study, we used two NSCLC cell lines (NCI-H358 and NCI-H1299) to investigate the anti-proliferative activity and apoptotic effect of esculetin. Our data showed that esculetin-treated cells exhibited reduced proliferation and apoptotic cell morphologies. Intriguingly, the transcription factor specificity protein 1 (Sp1) was significantly suppressed by esculetin in a dose- and time-dependent manner. Furthermore, the levels of p27 and p21, two key regulators of the cell cycle, were up-regulated by the esculetin-mediated down-regulation of Sp1; the level of a third cell-cycle regulator, survivin, was decreased, resulting in caspase-dependent apoptosis. Therefore, we conclude that esculetin could be a potent anti-proliferative agent in patients with NSCLC.

  20. Bone morphogenetic protein-9 suppresses growth of myeloma cells by signaling through ALK2 but is inhibited by endoglin

    International Nuclear Information System (INIS)

    Olsen, O E; Wader, K F; Misund, K; Våtsveen, T K; Rø, T B; Mylin, A K; Turesson, I; Størdal, B F; Moen, S H; Standal, T; Waage, A; Sundan, A; Holien, T

    2014-01-01

    Multiple myeloma is a malignancy of plasma cells predominantly located in the bone marrow. A number of bone morphogenetic proteins (BMPs) induce apoptosis in myeloma cells in vitro, and with this study we add BMP-9 to the list. BMP-9 has been found in human serum at concentrations that inhibit cancer cell growth in vitro. We here show that the level of BMP-9 in serum was elevated in myeloma patients (median 176 pg/ml, range 8–809) compared with healthy controls (median 110 pg/ml, range 8–359). BMP-9 was also present in the bone marrow and was able to induce apoptosis in 4 out of 11 primary myeloma cell samples by signaling through ALK2. BMP-9-induced apoptosis in myeloma cells was associated with c-MYC downregulation. The effects of BMP-9 were counteracted by membrane-bound (CD105) or soluble endoglin present in the bone marrow microenvironment, suggesting a mechanism for how myeloma cells can evade the tumor suppressing activity of BMP-9 in multiple myeloma

  1. The Bacterial Effector AvrPto Targets the Regulatory Coreceptor SOBIR1 and Suppresses Defense Signaling Mediated by the Receptor-Like Protein Cf-4

    NARCIS (Netherlands)

    Wu, Jinbin; Burgh, Van Der Aranka M.; Bi, Guozhi; Zhang, Lisha; Alfano, James R.; Martin, Gregory B.; Joosten, Matthieu H.A.J.

    2018-01-01

    Receptor-like proteins (RLPs) and receptor-like kinases (RLKs) are cell-surface receptors that are essential for detecting invading pathogens and subsequent activation of plant defense responses. RLPs lack a cytoplasmic kinase domain to trigger downstream signaling leading to host resistance. The

  2. [Herbs for calming liver and suppressing yang in treatment of hyperthyroidism with hyperactive liver yang: herbal effects on lymphocyte protein expression].

    Science.gov (United States)

    Li, Xiangping; Yin, Tao; Zhong, Guangwei; Li, Wei; Luo, Yanhong; Xiang, Lingli; Liu, Zhehao

    2011-07-01

    To observe the herbal effects on hyperthyroidism patients with syndrome of hyperactivity of liver-Yang by method for calming the liver and suppressing Yang and investigate its effects on the lymphocyte protein expression. This approach may lay a foundation for the further investigation of the curative mechanisms of calming the liver and suppressing Yang treatment. A total of 48 hyperthyroidism patients with syndrome of hyperactivity of liver-Yang were randomly divided into treatment group and control group. The treatment group was treated by method for calming the liver and suppressing Yang in accordance with traditional Chinese medicine (TCM) and the control group with thiamazole tablets for three periods of treatment The therapeutic effects, the score of TCM symptom, electrocardiogram (P wave), thyroid hormones and ultrasound were observed in both groups before and after the treatment. The side effects in the treatment course were observed in both groups. The level of differential protein expression was analyzed by two-dimensional electrphoresis and matrix assisted laser desorption/ionizaton time-of-flight mass spectrometry. The treatment group has the effect on stepping down the heart rate, cutting down the P wave amplitude changes, regulating the level of thyroid hormones and decreasing the volume of thyromegaly. There are not statistically significant between the treatment group and control group. However, the treatment group has obviously better effect on regulating TCM symptom and decreasing the side reaction than the control group (Peffective between the treatment group and control group. The average spots in lymphocyte for normal people, before and after treating hyperthyroidism patients with syndrome of hyperactivity of liver-Yang were (429 +/- 31), (452 +/- 28) and (437 +/- 36) spots respectively. Eight down-regulated protein expressions and 11 up-regulated protein expressions were obtained in the hyperthyroidism patients with syndrome of hyperactivity

  3. BTB-BACK Domain Protein POB1 Suppresses Immune Cell Death by Targeting Ubiquitin E3 ligase PUB17 for Degradation.

    Directory of Open Access Journals (Sweden)

    Beatriz Orosa

    2017-01-01

    Full Text Available Hypersensitive response programmed cell death (HR-PCD is a critical feature in plant immunity required for pathogen restriction and prevention of disease development. The precise control of this process is paramount to cell survival and an effective immune response. The discovery of new components that function to suppress HR-PCD will be instrumental in understanding the regulation of this fundamental mechanism. Here we report the identification and characterisation of a BTB domain E3 ligase protein, POB1, that functions to suppress HR-PCD triggered by evolutionarily diverse pathogens. Nicotiana benthamiana and tobacco plants with reduced POB1 activity show accelerated HR-PCD whilst those with increased POB1 levels show attenuated HR-PCD. We demonstrate that POB1 dimerization and nuclear localization are vital for its function in HR-PCD suppression. Using protein-protein interaction assays, we identify the Plant U-Box E3 ligase PUB17, a well established positive regulator of plant innate immunity, as a target for POB1-mediated proteasomal degradation. Using confocal imaging and in planta immunoprecipitation assays we show that POB1 interacts with PUB17 in the nucleus and stimulates its degradation. Mutated versions of POB1 that show reduced interaction with PUB17 fail to suppress HR-PCD, indicating that POB1-mediated degradation of PUB17 U-box E3 ligase is an important step for negative regulation of specific immune pathways in plants. Our data reveals a new mechanism for BTB domain proteins in suppressing HR-PCD in plant innate immune responses.

  4. Analysis of cathepsin and furin proteolytic enzymes involved in viral fusion protein activation in cells of the bat reservoir host.

    Directory of Open Access Journals (Sweden)

    Farah El Najjar

    Full Text Available Bats of different species play a major role in the emergence and transmission of highly pathogenic viruses including Ebola virus, SARS-like coronavirus and the henipaviruses. These viruses require proteolytic activation of surface envelope glycoproteins needed for entry, and cellular cathepsins have been shown to be involved in proteolysis of glycoproteins from these distinct virus families. Very little is currently known about the available proteases in bats. To determine whether the utilization of cathepsins by bat-borne viruses is related to the nature of proteases in their natural hosts, we examined proteolytic processing of several viral fusion proteins in cells derived from two fruit bat species, Pteropus alecto and Rousettus aegyptiacus. Our work shows that fruit bat cells have homologs of cathepsin and furin proteases capable of cleaving and activating both the cathepsin-dependent Hendra virus F and the furin-dependent parainfluenza virus 5 F proteins. Sequence analysis comparing Pteropus alecto furin and cathepsin L to proteases from other mammalian species showed a high degree of conservation; however significant amino acid variation occurs at the C-terminus of Pteropus alecto furin. Further analysis of furin-like proteases from fruit bats revealed that these proteases are catalytically active and resemble other mammalian furins in their response to a potent furin inhibitor. However, kinetic analysis suggests that differences may exist in the cellular localization of furin between different species. Collectively, these results indicate that the unusual role of cathepsin proteases in the life cycle of bat-borne viruses is not due to the lack of active furin-like proteases in these natural reservoir species; however, differences may exist between furin proteases present in fruit bats compared to furins in other mammalian species, and these differences may impact protease usage for viral glycoprotein processing.

  5. Genetic analysis of ectopic growth suppression during planar growth of integuments mediated by the Arabidopsis AGC protein kinase UNICORN

    Directory of Open Access Journals (Sweden)

    Enugutti Balaji

    2013-01-01

    Full Text Available Abstract Background The coordination of growth within a tissue layer is of critical importance for tissue morphogenesis. For example, cells within the epidermis undergo stereotypic cell divisions that are oriented along the plane of the layer (planar growth, thereby propagating the layered epidermal structure. Little is known about the developmental control that regulates such planar growth in plants. Recent evidence suggested that the Arabidopsis AGC VIII protein kinase UNICORN (UCN maintains planar growth by suppressing the formation of ectopic multicellular protrusions in several floral tissues including integuments. In the current model UCN controls this process during integument development by directly interacting with the ABERRANT TESTA SHAPE (ATS protein, a member of the KANADI (KAN family of transcription factors, thereby repressing its activity. Here we report on the further characterization of the UCN mechanism. Results Phenotypic analysis of flowers of ucn-1 plants impaired in floral homeotic gene activity revealed that any of the four floral whorls could produce organs carrying ucn-1 protrusions. The ectopic outgrowths of ucn integuments did not accumulate detectable signals of the auxin and cytokinin reporters DR5rev::GFP and ARR5::GUS, respectively. Furthermore, wild-type and ucn-1 seedlings showed similarly strong callus formation upon in vitro culture on callus-inducing medium. We also show that ovules of ucn-1 plants carrying the dominant ats allele sk21-D exhibited more pronounced protrusion formation. Finally ovules of ucn-1 ett-1 double mutants and ucn-1 ett-1 arf4-1 triple mutants displayed an additive phenotype. Conclusions These data deepen the molecular insight into the UCN-mediated control of planar growth during integument development. The presented evidence indicates that UCN downstream signaling does not involve the control of auxin or cytokinin homeostasis. The results also reveal that UCN interacts with ATS

  6. Solute carrier protein family 11 member 1 (Slc11a1) activation efficiently inhibits Leishmania donovani survival in host macrophages.

    Science.gov (United States)

    Singh, Nisha; Gedda, Mallikarjuna Rao; Tiwari, Neeraj; Singh, Suya P; Bajpai, Surabhi; Singh, Rakesh K

    2017-09-01

    Visceral leishmaniasis (kala-azar), a life threatening disease caused by L. donovani , is a latent threat to more than 147 million people living in disease endemic South East Asia region of the Indian subcontinent. The therapeutic option to control leishmanial infections are very limited, and at present comprise only two drugs, an antifungal amphotericin B and an antitumor miltefosine, which are also highly vulnerable for parasitic resistance. Therefore, identification and development of alternate control measures is an exigent requirement to control leishmanial infections. In this study, we report that functionally induced expression of solute carrier protein family 11 member 1 ( Slc11a1), a transmembrane divalent cationic transporter recruited on the surface of phagolysosomes after phagocytosis of parasites, effectively inhibits Leishmania donovani growth in host macrophages. Further, the increased Slc11a1 functionality also resulted in increased production of NOx, TNF-α and IL-12 by activated macrophages. The findings of this study signify the importance of interplay between Slc11a1 expression and macrophages activation that can be effectively used to control of Leishmania growth and survival.

  7. Competitive advantage of Borrelia burgdorferi with outer surface protein BBA03 during tick-mediated infection of the mammalian host.

    Science.gov (United States)

    Bestor, Aaron; Rego, Ryan O M; Tilly, Kit; Rosa, Patricia A

    2012-10-01

    Linear plasmid lp54 is one of the most highly conserved and differentially expressed elements of the segmented genome of the Lyme disease spirochete Borrelia burgdorferi. We previously reported that deletion of a 4.1-kb region of lp54 (bba01 to bba07 [bba01-bba07]) led to a slight attenuation of tick-transmitted infection in mice following challenge with a large number of infected ticks. In the current study, we reduced the number of ticks in the challenge to more closely mimic the natural dose and found a profound defect in tick-transmitted infection of the bba01-bba07 mutant relative to wild-type B. burgdorferi. We next focused on deletion of bba03 as the most likely cause of this mutant phenotype, as previous studies have shown that expression of bba03 is increased by culture conditions that simulate tick feeding. Consistent with this hypothesis, we demonstrated increased expression of bba03 by spirochetes in fed relative to unfed ticks. We also observed that a bba03 deletion mutant, although fully competent by itself, did not efficiently infect mice when transmitted by ticks that were simultaneously coinfected with wild-type B. burgdorferi. These results suggest that BBA03 provides a competitive advantage to spirochetes carrying this protein during tick transmission to a mammalian host in the natural infectious cycle.

  8. The extracellular adherence protein (Eap) of Staphylococcus aureus inhibits wound healing by interfering with host defense and repair mechanisms.

    Science.gov (United States)

    Athanasopoulos, Athanasios N; Economopoulou, Matina; Orlova, Valeria V; Sobke, Astrid; Schneider, Darius; Weber, Holger; Augustin, Hellmut G; Eming, Sabine A; Schubert, Uwe; Linn, Thomas; Nawroth, Peter P; Hussain, Muzaffar; Hammes, Hans-Peter; Herrmann, Mathias; Preissner, Klaus T; Chavakis, Triantafyllos

    2006-04-01

    Staphylococcus aureus is a major human pathogen interfering with host-cell functions. Impaired wound healing is often observed in S aureus-infected wounds, yet, the underlying mechanisms are poorly defined. Here, we identify the extracellular adherence protein (Eap) of S aureus to be responsible for impaired wound healing. In a mouse wound-healing model wound closure was inhibited in the presence of wild-type S aureus and this effect was reversible when the wounds were incubated with an isogenic Eap-deficient strain. Isolated Eap also delayed wound closure. In the presence of Eap, recruitment of inflammatory cells to the wound site as well as neovascularization of the wound were prevented. In vitro, Eap significantly reduced intercellular adhesion molecule 1 (ICAM-1)-dependent leukocyte-endothelial interactions and diminished the consequent activation of the proinflammatory transcription factor nuclear factor kappaB (NFkappaB) in leukocytes associated with a decrease in expression of tissue factor. Moreover, Eap blocked alphav-integrin-mediated endothelial-cell migration and capillary tube formation, and neovascularization in matrigels in vivo. Collectively, the potent anti-inflammatory and antiangiogenic properties of Eap provide an underlying mechanism that may explain the impaired wound healing in S aureus-infected wounds. Eap may also serve as a lead compound for new anti-inflammatory and antiangiogenic therapies in several pathologies.

  9. Identification of amino-acid residues in the V protein of peste des petits ruminants essential for interference and suppression of STAT-mediated interferon signaling

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Xusheng, E-mail: maxushengtt@163.com [State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Lanzhou 730030 (China); Yang, Xing [State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Lanzhou 730030 (China); Nian, Xiaofeng [Institute of Pathogen Biology and Immunology, Hebei North University, Zhangjiakou 07500 (China); Zhang, Zhidong; Dou, Yongxi [State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Lanzhou 730030 (China); Zhang, Xuehu [Gansu Agricultural University, Lanzhou (China); Luo, Xuenong; Su, Junhong; Zhu, Qiyun [State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Lanzhou 730030 (China); Cai, Xuepeng, E-mail: caixp@vip.163.com [State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Lanzhou 730030 (China)

    2015-09-15

    Peste des petits ruminants virus (PPRV) causes a fatal disease in small ruminants. V protein of PPRV plays a pivotal role in interfering with host innate immunity by blocking IFNs signaling through interacting with STAT1 and STAT2. In the present study, the results demonstrated that PPRV V protein blocks IFN actions in a dose dependent manner and restrains the translocation of STAT1/2 proteins. We speculate that the translocation inhibition might be caused by the interfering of the downstream of STAT protein. Mutagenesis defines that Cys cluster and Trp motif of PPRV V protein are essential for STAT-mediated IFN signaling. These findings give a new sight for the further studies to understand the delicate mechanism of PPRV to escape the IFN signaling. - Highlights: • PPRV V protein inhibits type I IFN production and blocks its activation. • PPRV V protein negatively regulates activation of ISRE and GAS promoter. • PPRV V protein inhibits nuclear translocation of STAT protein by non-degradation. • PNT and VCT domain of PPRV V protein inhibit IFN transduction. • PPRV V protein binds with STAT protein via some conserved motifs.

  10. The intrinsically disordered structural platform of the plant defence hub protein RPM1-interacting protein 4 provides insights into its mode of action in the host-pathogen interface and evolution of the nitrate-induced domain protein family.

    Science.gov (United States)

    Sun, Xiaolin; Greenwood, David R; Templeton, Matthew D; Libich, David S; McGhie, Tony K; Xue, Bin; Yoon, Minsoo; Cui, Wei; Kirk, Christopher A; Jones, William T; Uversky, Vladimir N; Rikkerink, Erik H A

    2014-09-01

    Arabidopsis thaliana (At) RPM1-interacting protein 4 (RIN4), targeted by many defence-suppressing bacterial type III effectors and monitored by several resistance proteins, regulates plant immune responses to pathogen-associated molecular patterns and type III effectors. Little is known about the overall protein structure of AtRIN4, especially in its unbound form, and the relevance of structure to its diverse biological functions. AtRIN4 contains two nitrate-induced (NOI) domains and is a member of the NOI family. Using experimental and bioinformatic approaches, we demonstrate that the unbound AtRIN4 is intrinsically disordered under physiological conditions. The intrinsically disordered polypeptide chain of AtRIN4 is interspersed with molecular recognition features (MoRFs) and anchor-identified long-binding regions, potentially allowing it to undergo disorder-to-order transitions upon binding to partner(s). A poly-l-proline II structure, often responsible for protein recognition, is also identified in AtRIN4. By performing bioinformatics analyses on RIN4 homologues from different plant species and the NOI proteins from Arabidopsis, we infer the conservation of intrinsic disorder, MoRFs and long-binding regions of AtRIN4 in other plant species and the NOI family. Intrinsic disorder and MoRFs could provide RIN4 proteins with the binding promiscuity and plasticity required to act as hubs in a pivotal position within plant defence signalling cascades. © 2014 FEBS.

  11. Suppression of host-seeking Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae) nymphs after dual applications of plant-derived acaricides in New Jersey.

    Science.gov (United States)

    Jordan, Robert A; Dolan, Marc C; Piesman, Joseph; Schulze, Terry L

    2011-04-01

    We evaluated the ability of dual applications of natural, plant-derived acaricides to suppress nymphal Ixodes scapularis Say and Amblyomma americanum (L.) (Acari: Ixodidae) in a Lyme disease endemic area of New Jersey. An aqueous formulation of 2% nootkatone provided >90% control of I. scapularis through 7 d. Control declined to 80.9% at 14 d, and a second application was made that provided >95% control through the remaining 4 wk of the nymphal season. Nootkatone provided >90% control of A. americanum through 35 d postapplication. Applications of 2% carvacrol and EcoTrol T&O resulted in rapid knockdown of both tick species, but control declined significantly to 76.7 and 73.7%, respectively, after 14 d when a second application was made that extended control of both tick species to between 86.2 and 94.8% at 21 d. Subsequently, control declined steadily in all plots by 42 d postapplication except for I. scapularis in carvacrol-treated plots, where levels of control >90% were observed through 35 d. Of the three compounds tested, 2% nootkatone provided the most consistent results, with 96.5 and 91.9% control of I. scapularis and A. americanum through 42 and 35 d, respectively. The ability of plant-derived natural products to quickly suppress and maintain significant control of populations of these medically important ticks may represent a future alternative to the use of conventional synthetic acaricides. In addition, the demonstrated efficacy of properly-timed backpack sprayer application may enable homeowner access to these minimal-risk acaricides.

  12. Suppression of type I and type III IFN signalling by NSs protein of severe fever with thrombocytopenia syndrome virus through inhibition of STAT1 phosphorylation and activation.

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    Chaudhary, Vidyanath; Zhang, Shuo; Yuen, Kit-San; Li, Chuan; Lui, Pak-Yin; Fung, Sin-Yee; Wang, Pei-Hui; Chan, Chi-Ping; Li, Dexin; Kok, Kin-Hang; Liang, Mifang; Jin, Dong-Yan

    2015-11-01

    Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne pathogen causing significant morbidity and mortality in Asia. NSs protein of SFTSV is known to perturb type I IFN induction and signalling, but the mechanism remains to be fully understood. Here, we showed the suppression of both type I and type III IFN signalling by SFTSV NSs protein is mediated through inhibition of STAT1 phosphorylation and activation. Infection with live SFTSV or expression of NSs potently suppressed IFN-stimulated genes but not NFkB activation. NSs was capable of counteracting the activity of IFN-α1, IFN-β, IFN-λ1 and IFN-λ2. Mechanistically, NSs associated with STAT1 and STAT2, mitigated IFN-β-induced phosphorylation of STAT1 at S727, and reduced the expression and activity of STAT1 protein in IFN-β-treated cells, resulting in the inhibition of STAT1 and STAT2 recruitment to IFNstimulated promoters. Taken together, SFTSV NSs protein is an IFN antagonist that suppresses phosphorylation and activation of STAT1.

  13. Gene expression of a green fluorescent protein homolog as a host-specific biomarker of heat stress within a reef-building coral.

    Science.gov (United States)

    Smith-Keune, C; Dove, S

    2008-01-01

    Recent incidences of mass coral bleaching indicate that major reef building corals are increasingly suffering thermal stress associated with climate-related temperature increases. The development of pulse amplitude modulated (PAM) fluorometry has enabled rapid detection of the onset of thermal stress within coral algal symbionts, but sensitive biomarkers of thermal stress specific to the host coral have been slower to emerge. Differential display reverse transcription polymerase chain reaction (DDRT-PCR) was used to produce fingerprints of gene expression for the reef-building coral Acropora millepora exposed to 33 degrees C. Changes in the expression of 23 out of 399 putative genes occurred within 144 h. Down-regulation of one host-specific gene (AmA1a) occurred within just 6 h. Full-length sequencing revealed the product of this gene to be an all-protein chromatophore (green fluorescent protein [GFP]-homolog). RT-PCR revealed consistent down-regulation of this GFP-homolog for three replicate colonies within 6 h at both 32 degrees C and 33 degrees C but not at lower temperatures. Down-regulation of this host gene preceded significant decreases in the photosynthetic activity of photosystem II (dark-adapted F (v)/F (m)) of algal symbionts as measured by PAM fluorometry. Gene expression of host-specific genes such as GFP-homologs may therefore prove to be highly sensitive indicators for the onset of thermal stress within host coral cells.

  14. Plasmodium Cysteine Repeat Modular Proteins 3 and 4 are essential for malaria parasite transmission from the mosquito to the host

    Directory of Open Access Journals (Sweden)

    Mota Maria M

    2011-03-01

    Full Text Available Abstract Background The Plasmodium Cysteine Repeat Modular Proteins (PCRMP are a family of four conserved proteins of malaria parasites, that contain a number of motifs implicated in host-parasite interactions. Analysis of mutants of the rodent parasite Plasmodium berghei lacking expression of PCRMP1 or 2 showed that these proteins are essential for targeting of P. berghei sporozoites to the mosquito salivary gland and, hence, for transmission from the mosquito to the mouse. Methods In this work, the role of the remaining PCRMP family members, PCRMP3 and 4, has been investigated throughout the Plasmodium life cycle by generation and analysis of P. berghei gene deletion mutants, Δpcrmp3 and Δpcrmp4. The role of PCRMP members during the transmission and hepatic stages of the Plasmodium lifecycle has been evaluated by light- and electron microscopy and by analysis of liver stage development in HEPG2 cells in vitro and by infecting mice with mutant sporozoites. In addition, mice were immunized with live Δpcrmp3 and Δpcrmp4 sporozoites to evaluate their immunization potential as a genetically-attenuated parasite-based vaccine. Results Disruption of pcrmp3 and pcrmp4 in P. berghei revealed that they are also essential for transmission of the parasite through the mosquito vector, although acting in a distinct way to pbcrmp1 and 2. Mutants lacking expression of PCRMP3 or PCRMP4 show normal blood stage development and oocyst formation in the mosquito and develop into morphologically normal sporozoites, but these have a defect in egress from oocysts and do not enter the salivary glands. Sporozoites extracted from oocysts perform gliding motility and invade and infect hepatocytes but do not undergo further development and proliferation. Furthermore, the study shows that immunization with Δcrmp3 and Δcrmp4 sporozoites does not confer protective immunity upon subsequent challenge. Conclusions PCRMP3 and 4 play multiple roles during the Plasmodium life

  15. Virus variants with differences in the P1 protein coexist in a Plum pox virus population and display particular host-dependent pathogenicity features.

    Science.gov (United States)

    Maliogka, Varvara I; Salvador, Beatriz; Carbonell, Alberto; Sáenz, Pilar; León, David San; Oliveros, Juan Carlos; Delgadillo, Ma Otilia; García, Juan Antonio; Simón-Mateo, Carmen

    2012-10-01

    Subisolates segregated from an M-type Plum pox virus (PPV) isolate, PPV-PS, differ widely in pathogenicity despite their high degree of sequence similarity. A single amino acid substitution, K109E, in the helper component proteinase (HCPro) protein of PPV caused a significant enhancement of symptom severity in herbaceous hosts, and notably modified virus infectivity in peach seedlings. The presence of this substitution in certain subisolates that induced mild symptoms in herbaceous hosts and did not infect peach seedlings suggested the existence of uncharacterized attenuating factors in these subisolates. In this study, we show that two amino acid changes in the P1 protein are specifically associated with the mild pathogenicity exhibited by some PS subisolates. Site-directed mutagenesis studies demonstrated that both substitutions, W29R and V139E, but especially W29R, resulted in lower levels of virus accumulation and symptom severity in a woody host, Prunus persica. Furthermore, when W29R and V139E mutations were expressed concomitantly, PPV infectivity was completely abolished in this host. In contrast, the V139E substitution, but not W29R, was found to be responsible for symptom attenuation in herbaceous hosts. Deep sequencing analysis demonstrated that the W29R and V139E heterogeneities already existed in the original PPV-PS isolate before its segregation in different subisolates by local lesion cloning. These results highlight the potential complexity of potyviral populations and the relevance of the P1 protein of potyviruses in pathogenesis and viral adaptation to the host. © 2012 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2012 BSPP AND BLACKWELL PUBLISHING LTD.

  16. Diverse amino acid changes at specific positions in the N-terminal region of the coat protein allow Plum pox virus to adapt to new hosts.

    Science.gov (United States)

    Carbonell, Alberto; Maliogka, Varvara I; Pérez, José de Jesús; Salvador, Beatriz; León, David San; García, Juan Antonio; Simón-Mateo, Carmen

    2013-10-01

    Plum pox virus (PPV)-D and PPV-R are two isolates from strain D of PPV that differ in host specificity. Previous analyses of chimeras originating from PPV-R and PPV-D suggested that the N terminus of the coat protein (CP) includes host-specific pathogenicity determinants. Here, these determinants were mapped precisely by analyzing the infectivity in herbaceous and woody species of chimeras containing a fragment of the 3' region of PPV-D (including the region coding for the CP) in a PPV-R backbone. These chimeras were not infectious in Prunus persica, but systemically infected Nicotiana clevelandii and N. benthamiana when specific amino acids were modified or deleted in a short 30-amino-acid region of the N terminus of the CP. Most of these mutations did not reduce PPV fitness in Prunus spp. although others impaired systemic infection in this host. We propose a model in which the N terminus of the CP, highly relevant for virus systemic movement, is targeted by a host defense mechanism in Nicotiana spp. Mutations in this short region allow PPV to overcome the defense response in this host but can compromise the efficiency of PPV systemic movement in other hosts such as Prunus spp.

  17. The amyR-deletion strain of Aspergillus niger CICC2462 is a suitable host strain to express secreted protein with a low background.

    Science.gov (United States)

    Zhang, Hui; Wang, Shuang; Zhang, Xiang Xiang; Ji, Wei; Song, Fuping; Zhao, Yue; Li, Jie

    2016-04-28

    The filamentous fungus Aspergillus niger is widely exploited as an important expression host for industrial production. The glucoamylase high-producing strain A. niger CICC2462 has been used as a host strain for the establishment of a secretion expression system. It expresses recombinant xylanase, mannase and asparaginase at a high level, but some high secretory background proteins in these recombinant strains still remain, such as alpha-amylase and alpha-glucosidase; lead to a low-purity of fermentation products. The aim was to construct an A. niger host strain with a low background of protein secretion. The transcription factor amyR was deleted in A. niger CICC2462, and the results from enzyme activity assays and SDS-PAGE analysis showed that the glucoamylase and amylase activities of the ∆amyR strains were significantly lower than those of the wild-type strain. High-throughput RNA-sequencing and shotgun LC-MS/MS proteomic technology analysis demonstrated that the expression of amylolytic enzymes was decreased at both the transcriptional and translational levels in the ∆amyR strain. Interestingly, the ∆amyR strain growth rate better than the wild-type strain. Our findings clearly indicated that the ∆amyR strain of A. niger CICC2462 can be used as a host strain with a low background of protein secretion.

  18. Suppressive effects of mycoviral proteins encoded by Magnaporthe oryzae chrysovirus 1 strain A on conidial germination of the rice blast fungus.

    Science.gov (United States)

    Urayama, Syun-Ichi; Kimura, Yuri; Katoh, Yu; Ohta, Tomoko; Onozuka, Nobuya; Fukuhara, Toshiyuki; Arie, Tsutomu; Teraoka, Tohru; Komatsu, Ken; Moriyama, Hiromitsu

    2016-09-02

    Magnaporthe oryzae chrysovirus 1 strain A (MoCV1-A) is the causal agent of growth repression and attenuated virulence (hypovirulence) of the rice blast fungus, Magnaporthe oryzae. We previously revealed that heterologous expression of the MoCV1-A ORF4 protein resulted in cytological damage to the yeasts Saccharomyces cerevisiae and Cryptococcus neoformans. Since the ORF4 protein is one of the components of viral particles, we evaluated the inhibitory effects of the purified virus particle against the conidial germination of M. oryzae, and confirmed its suppressive effects. Recombinant MoCV1-A ORF4 protein produced in Pichia pastoris was also effective for suppression of conidial germination of M. oryzae. MoCV1-A ORF4 protein sequence showed significant similarity to 6 related mycoviral proteins; Botrysphaeria dothidea chrysovirus 1, two Fusarium graminearum viruses, Fusarium oxysporum f. sp. dianthi mycovirus 1, Penicillium janczewski chrysovirus and Agaricus bisporus virus 1 in the Chrysoviridae family. Multiple alignments of the ORF4-related protein sequences showed that their central regions (210-591 aa in MoCV1-A ORF4) are relatively conserved. Indeed, yeast transformants expressing the conserved central region of MoCV1-A ORF4 protein (325-575 aa) showed similar impaired growth phenotypes as those observed in yeasts expressing the full-length MoCV1-A ORF4 protein. These data suggest that the mycovirus itself and its encoded viral protein can be useful as anti-fungal proteins to control rice blast disease caused by M. oryzae and other pathogenic fungi. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Buddleja officinalis suppresses high glucose-induced vascular smooth muscle cell proliferation: role of mitogen-activated protein kinases, nuclear factor-kappaB and matrix metalloproteinases.

    Science.gov (United States)

    Lee, Yun Jung; Kim, Jin Sook; Kang, Dae Gill; Lee, Ho Sub

    2010-02-01

    Diabetes mellitus is a well-established risk factor for vascular diseases caused by atherosclerosis. In the development of diabetic atherogenesis, vascular smooth muscle cell proliferation is recognized as a key event. Thus, we aimed to investigate whether an ethanol extract of Buddleja officinalis (EBO) suppresses high glucose-induced proliferation in primary cultured human aortic smooth muscle cells (HASMC). [(3)H]-thymidine incorporation revealed that incubation of HASMC with a high concentration of glucose (25 mmol/L) increased cell proliferation. The expression levels of cell cycle protein were also increased by treatment with high glucose concentration. Pretreatment of HASMC with EBO significantly attenuated the increase of high glucose-induced cell proliferation as well as p38 mitogen-activated protein kinases (MAPK) and JNK phosphorylation. EBO suppressed high glucose-induced matrix metalloproteinase (MMP)-9 activity in a dose-dependent manner. In addition, EBO suppressed nuclear factor-kappaB (NF-kappaB) nuclear translocation and transcriptional activity in high glucose conditions. Taken together, the present data suggest that EBO could suppress high glucose-induced atherosclerotic processes through inhibition of p38, JNK, NF-kappaB and MMP signal pathways in HASMC.

  20. A low carbohydrate, high protein diet suppresses intratumoral androgen synthesis and slows castration-resistant prostate tumor growth in mice.

    Science.gov (United States)

    Fokidis, H Bobby; Yieng Chin, Mei; Ho, Victor W; Adomat, Hans H; Soma, Kiran K; Fazli, Ladan; Nip, Ka Mun; Cox, Michael; Krystal, Gerald; Zoubeidi, Amina; Tomlinson Guns, Emma S

    2015-06-01

    Dietary factors continue to preside as dominant influences in prostate cancer prevalence and progression-free survival following primary treatment. We investigated the influence of a low carbohydrate diet, compared to a typical Western diet, on prostate cancer (PCa) tumor growth in vivo. LNCaP xenograft tumor growth was studied in both intact and castrated mice, representing a more advanced castration resistant PCa (CRPC). No differences in LNCaP tumor progression (total tumor volume) with diet was observed for intact mice (P = 0.471) however, castrated mice on the Low Carb diet saw a statistically significant reduction in tumor growth rate compared with Western diet fed mice (P = 0.017). No correlation with serum PSA was observed. Steroid profiles, alongside serum cholesterol and cholesteryl ester levels, were significantly altered by both diet and castration. Specifically, DHT concentration with the Low Carb diet was 58% that of the CRPC-bearing mice on the Western diet. Enzymes in the steroidogenesis pathway were directly impacted and tumors isolated from intact mice on the Low Carb diet had higher AKR1C3 protein levels and lower HSD17B2 protein levels than intact mice on the Western diet (ARK1C3: P = 0.074; HSD17B2: P = 0.091, with α = 0.1). In contrast, CRPC tumors from mice on Low Carb diets had higher concentrations of both HSD17B2 (P = 0.016) and SRD5A1 (P = 0.058 with α = 0.1) enzymes. There was no correlation between tumor growth in castrated mice for Low Carb diet versus Western diet and (a) serum insulin (b) GH serum levels (c) insulin receptor (IR) or (d) IGF-1R in tumor tissue. Intact mice fed Western diet had higher serum insulin which was associated with significantly higher blood glucose and tumor tissue IR. We conclude that both diet and castration have a significant impact on the endocrinology of mice bearing LNCaP xenograft tumors. The observed effects of diet on cholesterol and steroid regulation impact tumor tissue DHT specifically and are

  1. Export of a Toxoplasma gondii rhoptry neck protein complex at the host cell membrane to form the moving junction during invasion.

    Directory of Open Access Journals (Sweden)

    Sébastien Besteiro

    2009-02-01

    Full Text Available One of the most conserved features of the invasion process in Apicomplexa parasites is the formation of a moving junction (MJ between the apex of the parasite and the host cell membrane that moves along the parasite and serves as support to propel it inside the host cell. The MJ was, up to a recent period, completely unknown at the molecular level. Recently, proteins originated from two distinct post-Golgi specialised secretory organelles, the micronemes (for AMA1 and the neck of the rhoptries (for RON2/RON4/RON5 proteins, have been shown to form a complex. AMA1 and RON4 in particular, have been localised to the MJ during invasion. Using biochemical approaches, we have identified RON8 as an additional member of the complex. We also demonstrated that all RON proteins are present at the MJ during invasion. Using metabolic labelling and immunoprecipitation, we showed that RON2 and AMA1 were able to interact in the absence of the other members. We also discovered that all MJ proteins are subjected to proteolytic maturation during trafficking to their respective organelles and that they could associate as non-mature forms in vitro. Finally, whereas AMA1 has previously been shown to be inserted into the parasite membrane upon secretion, we demonstrated, using differential permeabilization and loading of RON-specific antibodies into the host cell, that the RON complex is targeted to the host cell membrane, where RON4/5/8 remain associated with the cytoplasmic face. Globally, these results point toward a model of MJ organization where the parasite would be secreting and inserting interacting components on either side of the MJ, both at the host and at its own plasma membranes.

  2. Dengue Virus Selectively Annexes Endoplasmic Reticulum-Associated Translation Machinery as a Strategy for Co-opting Host Cell Protein Synthesis.