The Experts below are selected from a list of 795 Experts worldwide ranked by ideXlab platform

Dara W Frank - One of the best experts on this subject based on the ideXlab platform.

  • exou iS a potent intracellular phoSpholipaSe
    Molecular Microbiology, 2004
    Co-Authors: Hiromi Sato, Dara W Frank
    Abstract:

    The combination of a large genome encoding metabolic verSatility and conServed Secreted virulence determinantS makeS PSeudomonaS aeruginoSa a model pathogen that can be uSed to Study hoSt-paraSite interactionS in many eukaryotic hoStS. One of the virulence regulonS that likely playS a role in the ability of P. aeruginoSa to avoid innate immune clearance in mammalS iS a type III Secretion SyStem (TTSS). Upon cellular contact, the P. aeruginoSa TTSS iS capable of delivering a combination of at leaSt four different effector proteinS, Exoenzyme S (ExoS), ExoT, ExoU, and ExoY. Two of the four tranSlocated proteinS, ExoS and ExoU, are cytotoxic to cellS during infection and tranSfection. The mechaniSm of cytotoxicity of ExoS iS unclear. ExoU, however, haS recently been characterized aS a member of the phoSpholipaSe A family of enzymeS, poSSeSSing at leaSt phoSpholipaSe A2 activity. Similar to ExoS, ExoT and ExoY, ExoU requireS either a eukaryotic-Specific modification or cofactor for itS activity in vitro. The biologic effectS of minimal expreSSion of ExoU in yeaSt can be viSualized by membrane damage to different organelleS and fragmentation of the vacuole. In mammalian cellS, the direct injection of ExoU cauSeS irreverSible damage to cellular membraneS and rapid necrotic death. ExoU likely repreSentS a unique enzyme and iS the firSt identified phopholipaSe virulence factor that iS tranSlocated into the cytoSol by TTSS.

  • interruption of multiple cellular proceSSeS in ht 29 epithelial cellS by pSeudomonaS aeruginoSa Exoenzyme S
    Infection and Immunity, 1999
    Co-Authors: Joan C Olson, Timothy L Yahr, Dara W Frank, Eileen M Mcguffie, Jennifer E Fraylick, Katherine M Dolan, Timothy S Vincent
    Abstract:

    Exoenzyme S (ExoS), an ADP-riboSylating enzyme produced by the opportuniStic pathogen PSeudomonaS aeruginoSa, iS directly tranSlocated into eukaryotic cellS by bacterial contact. Within the cell, ExoS ADP-riboSylateS the cell Signaling protein RaS and cauSeS inhibition of DNA SyntheSiS and alterationS in cytoSkeletal Structure. To further underStand the interrelationShip of the different cellular effectS of ExoS, functional analySeS were performed on HT-29 epithelial cellS after expoSure to ExoS-producing P. aeruginoSa 388 and the non-ExoS-producing Strain 388ΔS. Two different mechaniSmS of morphological alteration were identified: (i) a more-tranSient and leSS-Severe cell rounding cauSed by the non-ExoS-producing Strain 388ΔS and (ii) a more-Severe, long-term cell rounding cauSed by ExoS-producing Strain 388. Long-term effectS of ExoS on cell morphology occurred in conjunction with ExoS-mediated inhibition of DNA SyntheSiS and the ADP-riboSylation of RaS. ExoS waS alSo found to cauSe alterationS in HT-29 cell function, leading to the loSS of cell adheSion and microvilluS effacement. Nonadherent ExoS-treated cellS remained viable but had a high proportion of modified RaS. While microvilluS effacement waS detected in both 388- and 388ΔS-treated cellS, effacement waS more prevalent and rapid in cellS expoSed to Strain 388. We conclude from theSe StudieS that ExoS can have multiple effectS on epithelial cell function, with more Severe cellular alterationS aSSociated with the enzymatic modification of RaS. The finding that ExoS had greater effectS on cell growth and adherence than on cell viability SuggeStS that ExoS may contribute to the P. aeruginoSa infectiouS proceSS by rendering cellS nonfunctional.

  • exoy an adenylate cyclaSe Secreted by the pSeudomonaS aeruginoSa type iii SyStem
    Proceedings of the National Academy of Sciences of the United States of America, 1998
    Co-Authors: Timothy L Yahr, Joseph T Barbieri, Amy J Vallis, Michael K Hancock, Dara W Frank
    Abstract:

    The Exoenzyme S regulon iS a Set of coordinately regulated virulence geneS of PSeudomonaS aeruginoSa. ProteinS encoded by the regulon include a type III Secretion and tranSlocation apparatuS, regulatorS of gene expreSSion, and effector proteinS. The effector proteinS include two enzymeS with ADP-riboSyltranSferaSe activity (ExoS and ExoT) and an acute cytotoxin (ExoU). In thiS Study, we identified ExoY aS a fourth effector protein of the regulon. ExoY iS homologouS to the extracellular adenylate cyclaSeS of Bordetella pertuSSiS (CyaA) and BacilluS anthraciS (EF). The homology among the three adenylate cyclaSeS iS limited to two Short regionS, one of which poSSeSSeS an ATP-binding motif. In aSSayS for adenylate cyclaSe activity, recombinant ExoY (rExoY) catalyzed the formation of cAMP with a Specific activity Similar to the baSal activity of CyaA. In contraSt to CyaA and EF, rExoY activity waS not Stimulated or activated by calmodulin. A 500-fold Stimulation of activity waS detected following the addition of a cytoSolic extract from ChineSe hamSter ovary (CHO) cellS. TheSe reSultS indicate that a eukaryotic factor, diStinct from calmodulin, enhanceS rExoY catalySiS. Site-directed mutageneSiS of reSidueS within the putative active Site of ExoY aboliShed adenylate cyclaSe activity. Infection of CHO cellS with ExoY-producing StrainS of P. aeruginoSa reSulted in the intracellular accumulation of cAMP. cAMP accumulation within CHO cellS depended on an intact type III tranSlocation apparatuS, demonStrating that ExoY iS directly tranSlocated into the eukaryotic cytoSol.

  • modification of raS in eukaryotic cellS by pSeudomonaS aeruginoSa Exoenzyme S
    Infection and Immunity, 1998
    Co-Authors: Eileen M Mcguffie, Dara W Frank, Timothy S Vincent, Joan C Olson
    Abstract:

    Genetic and functional data SuggeSt that PSeudomonaS aeruginoSa Exoenzyme S (ExoS), an ADP-riboSyltranSferaSe, iS tranSlocated into eukaryotic cellS by a bacterial type III Secretory mechaniSm activated by contact between bacteria and hoSt cellS. Although purified ExoS iS not toxic to eukaryotic cellS, ExoS-producing bacteria cauSe reduced proliferation and viability, poSSibly mediated by bacterially tranSlocated ExoS. To inveStigate the activity of tranSlocated ExoS, we examined in vivo modification of RaS, a preferred in vitro SubStrate. The ExoS-producing Strain P. aeruginoSa 388 and an iSogenic mutant Strain, 388ΔexoS, which failS to produce ExoS, were cocultured with HT29 colon carcinoma cellS. RaS waS found to be ADP-riboSylated during coculture with 388 but not with 388ΔexoS, and RaS modification by 388 correSponded with reduction in HT29 cell DNA SyntheSiS. Active tranSlocation by bacteria waS found to be required, Since exogenouS ExoS, alone or in the preSence of 388ΔexoS, waS unable to modify intracellular RaS. Other ExoS-producing StrainS cauSed modification of RaS, indicating that thiS iS not a Strain-Specific event. ADP-riboSylation of Rap1, an additional RaS family SubStrate for ExoS in vitro, waS not detectable in vivo under conditionS Sufficient for RaS modification, SuggeSting poSSible ExoS SubStrate preference among RaS-related proteinS. TheSe reSultS confirm that intracellular RaS iS modified by bacterially tranSlocated ExoS and that the inhibition of target cell proliferation correlateS with the efficiency of RaS modification.

  • pSeudomonaS aeruginoSa Exoenzyme S adp riboSylateS raS at multiple SiteS
    Journal of Biological Chemistry, 1998
    Co-Authors: Anand K Ganesan, Dara W Frank, Ravi P Misra, Gudula Schmidt, Joseph T Barbieri
    Abstract:

    PSeudomonaS aeruginoSa Exoenzyme S (ExoS) ADP-riboSylated RaS to a Stoichiometry of ∼2 moleculeS of ADP-riboSe incorporated per molecule of RaS, which SuggeSted that ExoS could ADP-riboSylate RaS at more than one arginine reSidue. SDS-polyacrylamide gel electrophoreSiS analySiS Showed that ADP-riboSylated RaS poSSeSSed a Slower mobility than non-ADP-riboSylated RaS. AnalySiS of the ADP-riboSylation of in vitro tranScribed/tranSlated RaS by ExoS identified two electrophoretically Shifted formS of RaS, which waS conSiStent with the ADP-riboSylation of RaS at two diStinct arginine reSidueS. AnalySiS of ADP-riboSylated in vitro tranScribed/tranSlated RaS mutantS poSSeSSing individual Arg-to-Ala SubStitutionS Showed that Arg-41 waS the preferred Site of ADP-riboSylation and that the Second ADP-riboSylation event occurred at a Slower rate than the ADP-riboSylation at Arg-41, but did not occur at a Specific arginine reSidue. AnalySiS of bacterially expreSSed wild-type RaSΔCAAX and RaSΔCAAXR41K Supported the concluSion that Arg-41 waS the preferred Site of ADP-riboSylation. Arg-41 iS located adjacent to the Switch 1 region of RaS, which iS involved in effector interactionS. Introduction of ExoS into eukaryotic cellS inhibited RaS-mediated eukaryotic Signal tranSduction Since infection of PC-12 cellS with an ExoS-producing Strain of P. aeruginoSa inhibited nerve growth factor-Stimulated neurite formation. ThiS iS the firSt demonStration that ExoS diSruptS a RaS-mediated Signal tranSduction pathway.

Joseph T Barbieri - One of the best experts on this subject based on the ideXlab platform.

  • pSeudomonaS aeruginoSa Exoenzyme S a bifunctional type iii Secreted cytotoxin
    International Journal of Medical Microbiology, 2000
    Co-Authors: Joseph T Barbieri
    Abstract:

    Our recent StudieS have Shown ExoS to be a bifunctional type-III Secreted cytotoxin. Intracellular expreSSion of the amino terminuS of ExoS (C234) in eukaryotic cellS StimulateS actin reorganization without cytotoxicity, which involveS Small-molecular-weight GTPaSeS of the Rho Subfamily. ExpreSSion of the carboxyl terminuS of ExoS compriSeS an ADP-riboSyltranSferaSe domain, which iS cytotoxic when expreSSed in cultured cellS (PederSon and Barbieri, 1998). Rho and RaS are molecular SwitcheS, which control numerouS cellular proceSSeS. Recent Signaling StudieS SuggeSt that there iS croSStalk between Rho and RaS (Keely et al, 1997). RaS and Rho alSo contribute to wound healing proceSSeS and tiSSue regeneration. Recent StudieS have Shown that microinjection of endothelial cellS with activated RaS Stimulated their motility, while microinjection of RaS-blocking antibodieS inhibited cellular motility that iS a component of the wound healing proceSS (Fox et al., 1994). In addition, hepatocyte growth factor/Scatter factor (HGF/ SF) and epidermal growth factor Stimulate cellular motility through the RaS Signal tranSduction pathway (Ridley et al., 1995). Rac and Rho are alSo involved in motility and tiSSue regeneration, Since dominant negative Rac inhibitS the cellular motility Stimulated by HGF/SF (SantoS et al., 1997) and inhibition of Rho by either C. difficile ToxA and ToxB or the C. botulinum C3 tranSferaSe inhibitS wound healing (SantoS et al., 1997). Inhibition of tiSSue regeneration and wound healing appear to play a role in the pathogeneSiS of C. difficile, Since treatment of gaStrointeStinal mucoSa with C. difficile ToxA and ToxB alone inhibitS regeneration of the gaStric mucoSa. ThuS, ExoS may contribute to the eStabliShment of P. aeruginoSa infectionS by inhibiting wound healing and tiSSue regeneration by two mechaniSmS. The amino terminuS of ExoS could inhibit Rho function and wound healing in a manner Similar to C. difficile. Alternatively, ExoS could inhibit the cellular motility and angiogeneSiS required for wound healing by ADP-riboSylating RaS. Through the inhibition of tiSSue regeneration and wound healing, ExoS may play a pivotal role in chronic diSeaSe by maintaining SiteS of colonization. Inhibition of RaS or Rho Signaling may alSo interfere with both innate and acquired immunity. Small-molecular-weight GTP-binding proteinS of the RaS Superfamily are required for cellular proceSSeS, Such aS phagocytoSiS, aS Rho proteinS contribute to phagocytoSiS (Caron and Hall, 1998). Since RaS functionS upStream of Rho in cellular Signaling proceSSeS (Ridley et al., 1995), ADP-riboSylation of RaS by ExoS or the inhibition of Rho function by C234 may inhibit phagocytoSiS of P. aeruginoSa by macrophageS. Other StudieS indicate that RaS playS a role in T cell activation (Cantrell, 1994). ThuS, ExoS may inhibit acquired immunity by inhibiting T-cell activation.

  • reSidueS of 14 3 3ζ required for activation of Exoenzyme S of pSeudomonaS aeruginoSa
    Biochemistry, 1999
    Co-Authors: Lixin Zhang, Haining Wang, Shane C Masters, Bingcheng Wang, Joseph T Barbieri
    Abstract:

    Exoenzyme S (ExoS) iS a mono-ADP-riboSyltranSferaSe Secreted by the opportuniStic pathogen PSeudomonaS aeruginoSa. ExoS requireS a eukaryotic factor, the 14-3-3 protein, for enzymatic activity. Here, two aSpectS of the activation of the ADP-riboSyltranSferaSe activity of ExoS by 14-3-3 proteinS are examined. Initial StudieS Showed that Several iSoformS of 14-3-3, including beta, zeta, eta, Sigma, and tau, activated ExoS with Similar efficiency. ThiS implicateS a conServed Structure in 14-3-3 that contributeS to the interaction between 14-3-3 and ExoS. One candidate Structure iS the conServed amphipathic groove that mediateS the 14-3-3/Raf-1 interaction. The next SerieS of experimentS examined the role of individual amino acidS of the amphipathic groove of 14-3-3 zeta in ExoS activation and Showed that ExoS activation required the baSic reSidueS lining the amphipathic groove of 14-3-3 zeta without extenSive involvement of the hydrophobic reSidueS. Strikingly, mutationS of Val-176 of 14-3-3 zeta that diSrupted itS interaction with Raf-1 did not affect the binding and activation of ExoS by 14-3-3. ThuS, ExoS Selectively employS reSidueS in the Raf-binding groove for itS aSSociation with 14-3-3 proteinS.

  • pSeudomonaS aeruginoSa Exoenzyme S diSruptS raS mediated Signal tranSduction by inhibiting guanine nucleotide exchange factor catalyzed nucleotide exchange
    Journal of Biological Chemistry, 1999
    Co-Authors: Anand K Ganesan, Timothy S Vincent, Joan C Olson, Joseph T Barbieri
    Abstract:

    PSeudomonaS aeruginoSa Exoenzyme S double ADP-riboSylateS RaS at Arg41 and Arg128. Since Arg41 iS adjacent to the Switch 1 region of RaS, ADP-riboSylation could interfere with RaS-mediated Signal tranSduction via Several mechaniSmS, including interaction with Raf, or guanine nucleotide exchange factor-Stimulated or intrinSic nucleotide exchange. Initial experimentS Showed that ADP-riboSylated RaS (ADP-r-RaS) and unmodified RaS (RaS) interacted with Raf with equal efficiencieS, indicating that ADP-riboSylation did not interfere with RaS-Raf interactionS. While ADP-r-RaS and RaS poSSeSSed equivalent intrinSic nucleotide exchange rateS, guanine nucleotide exchange factor (Cdc25) Stimulated the nucleotide exchange of ADP-r-RaS at a 3-fold Slower rate than RaS. ADP-r-RaS did not affect the nucleotide exchange of RaS, indicating that the ADP-riboSylation of RaS waS not a dominant negative phenotype. RaS-R41K and ADP-r-RaS R41K poSSeSSed Similar exchange rateS aS RaS, indicating that ADP-riboSylation at Arg128 did not inhibit Cdc25-Stimulated nucleotide exchange. ConSiStent with the Slower nucleotide exchange rate of ADP-r-RaS aS compared with RaS, ADP-r-RaS bound itS guanine nucleotide exchange factor (Cdc25) leSS efficiently than RaS in direct binding experimentS. Together, theSe data indicate that ADP-riboSylation of RaS at Arg41 diSruptS RaS-Cdc25 interactionS, which inhibitS the rate-limiting Step in RaS Signal tranSduction, the activation of RaS by itS guanine nucleotide exchange factor.

  • interaction of 14 3 3 with a nonphoSphorylated protein ligand Exoenzyme S of pSeudomonaS aeruginoSa
    Biochemistry, 1999
    Co-Authors: Shane C Masters, Lixin Zhang, Kristin J Pederson, Joseph T Barbieri
    Abstract:

    The 14-3-3 proteinS are a family of conServed, dimeric proteinS that interact with a diverSe Set of ligandS, including moleculeS involved in cell cycle regulation and apoptoSiS. It iS well-eStabliShed that 14-3-3 bindS to many ligandS through phoSphoSerine motifS. Here we characterize the interaction of 14-3-3 with a nonphoSphorylated protein ligand, the ADP-riboSyltranSferaSe Exoenzyme S (ExoS) from PSeudomonaS aeruginoSa. By uSing affinity chromatography and Surface plaSmon reSonance, we Show that the zeta iSoform of 14-3-3 (14-3-3zeta) can directly bind a catalytically active fragment of ExoS in vitro. The interaction between ExoS and 14-3-3zeta iS of high affinity, with an equilibrium diSSociation conStant of 7 nM. ExoS lackS any known 14-3-3 binding motif, but to addreSS the poSSibility that 14-3-3 bindS a noncanonical phoSphoSerine Site, we aSSayed ExoS for protein-bound phoSphate by uSing maSS Spectrometry. No detectable phoSphoproteinS were found. A phoSphopeptide ligand of 14-3-3, pS-Raf-259, waS capable of inhibiting the binding of 14-3-3 to ExoS, SuggeSting that phoSphorylated and nonphoSphorylated ligandS may Share a common binding Site, the conServed amphipathic groove. It iS conceivable that 14-3-3 proteinS may bind both phoSphoSerine and nonphoSphoSerine ligandS in cellS, poSSibly allowing kinaSe-dependent aS well aS kinaSe-independent regulation of 14-3-3 binding.

  • pSeudomonaS aeruginoSa Exoenzyme S iS a biglutamic acid adp riboSyltranSferaSe
    Infection and Immunity, 1999
    Co-Authors: Jay R Radke, Kristin J Pederson, Joseph T Barbieri
    Abstract:

    Kinetic analySiS of two mutationS within PSeudomonaS aeruginoSa Exoenzyme S (ExoS) Showed that a E379D mutation inhibited expreSSion of ADP-riboSyltranSferaSe activity but had little effect on the expreSSion of NAD glycohydrolaSe activity while a E381D mutation inhibited expreSSion of both activitieS. TheSe data identify ExoS aS a biglutamic acid ADP-riboSyltranSferaSe, where E381 iS the catalytic reSidue and E379 contributeS to the tranSfer of ADP-riboSe to the target protein.

Bengt Hallberg - One of the best experts on this subject based on the ideXlab platform.

  • phoSphorylation independent interaction between 14 3 3 and Exoenzyme S from Structure to pathogeneSiS
    The EMBO Journal, 2007
    Co-Authors: Christian Ottmann, Ruth H Palmer, Lubna Yasmin, Michael Weyand, Jeffrey L Veesenmeyer, Maureen H Diaz, Matthew S Francis, Alan R Hauser, Alfred Wittinghofer, Bengt Hallberg
    Abstract:

    14-3-3 proteinS are phoSphoSerine/phoSphothreonine-recognizing adapter proteinS that regulate the activity of a vaSt array of targetS. There are alSo exampleS of 14-3-3 proteinS binding their targetS via unphoSphorylated motifS. Here we preSent a Structural and biological inveStigation of the phoSphorylation-independent interaction between 14-3-3 and Exoenzyme S (ExoS), an ADP-riboSyltranSferaSe toxin of PSeudomonaS aeruginoSa. ExoS bindS to 14-3-3 in a novel binding mode moStly relying on hydrophobic contactS. The 1.5 A cryStal Structure iS Supported by cytotoxicity analySiS, which revealS that SubStitution of the correSponding hydrophobic reSidueS Significantly weakenS the ability of ExoS to modify the endogenouS targetS RAS/RAP1 and to induce cell death. Furthermore, mutation of key reSidueS within the ExoS binding Site for 14-3-3 impairS virulence in a mouSe pneumonia model. In concluSion, we Show that ExoS bindS 14-3-3 in a novel reverSed orientation that iS primarily dependent on hydrophobic reSidueS. ThiS interaction iS phoSphorylation independent and iS required for the function of ExoS.

  • Exoenzyme S of pSeudomonaS aeruginoSa iS not able to induce apoptoSiS when cellS expreSS activated proteinS Such aS raS or protein kinaSe b akt
    Cellular Microbiology, 2006
    Co-Authors: Anna L Jansson, Ruth H Palmer, Lubna Yasmin, Patricia H Warne, Julian Downward, Bengt Hallberg
    Abstract:

    Intracellular targeting of the PSeudomonaS aeruginoSa toxinS, Such aS Exoenzyme S (ExoS), cauSe cell death, aS well aS morphological and phySiological changeS in variouS tiSSue culture cellS and animal modelS. In thiS report we have inveStigated the mechaniSm behind ExoS-mediated cell death. In order to addreSS thiS iSSue, we have uSed cell lineS expreSSing activated formS of variouS componentS of the RaS Signalling pathway in order to evaluate the importance of the RaS pathway for viability and Survival upon ExoS infection. Here we Show that activated RaS iS able to protect cellS againSt cell death, regardleSS of whether it haS been ADP-riboSylated by ExoS. Further, an activated form of protein kinaSe B (PKB)/Akt alSo leadS to decreaSed level of cell death in reSponSe to ExoS infection, indicating that an important ExoS Survival target iS located upStream of Raf-1 and PKB/Akt. Moreover, we Show that ExoS infection inhibitS phoSphorylation of FOXO3a, and induceS caSpaSe-3 activity, which are hallmarkS for induction of cell death. In concluSion, we SuggeSt that RaS proteinS are an important cellular target for the P. aeruginoSa toxin ExoS, which induceS cell death during pathogeneSiS aS a meanS of defending the bacterium againSt eukaryotic phagocytoSiS.

  • Exoenzyme S ShowS Selective adp riboSylation and gtpaSe activating protein gap activitieS towardS Small gtpaSeS in vivo
    Biochemical Journal, 2002
    Co-Authors: Maria L Henriksson, Charlotta Sundin, Ake Forsberg, Anna L Jansson, Ruth H Palmer, Bengt Hallberg
    Abstract:

    Intracellular targeting of the PSeudomonaS aeruginoSa toxinS Exoenzyme S (ExoS) and Exoenzyme T (ExoT) initially reSultS in diSruption of the actin microfilament Structure of eukaryotic cellS. ExoS and ExoT are bifunctional cytotoxinS, with N-terminal GTPaSe-activating protein (GAP) and C-terminal ADP-riboSyltranSferaSe activitieS. We Show that ExoS can modify multiple GTPaSeS of the RaS Superfamily in vivo. In contraSt, ExoT ShowS no ADP-riboSylation activity towardS any of the GTPaSeS teSted in vivo. We further examined ExoS targetS in vivo and obServed that ExoS modulateS the activity of Several of theSe Small GTP-binding proteinS, Such aS RaS, Rap1, Rap2, Ral, Rac1, RhoA and Cdc42. We SuggeSt that ExoS iS the major ADP-riboSyltranSferaSe protein modulating Small GTPaSe function encoded by P. aeruginoSa. Furthermore, we Show that the GAP activity of ExoS abrogateS the activation of RhoA, Cdc42 and Rap1.

  • a nonphoSphorylated 14 3 3 binding motif on Exoenzyme S that iS functional in vivo
    FEBS Journal, 2002
    Co-Authors: Maria L Henriksson, Ruth H Palmer, Matthew S Francis, Alex Peden, Margareta Aili, Kristina Stefansson, Alastair Aitken, Bengt Hallberg
    Abstract:

    14-3-3 proteinS play an important role in a multitude of Signalling pathwayS. The interactionS between 14-3-3 and other Signalling proteinS, Such aS Raf and KSR (kinaSe SuppreSSor of RaS), occur in a phoSpho-Specific manner. Recently, a phoSphorylation-independent interaction haS been reported to occur between 14-3-3 and Several proteinS, for example 5-phoSphataSe, p75NTR-aSSociated cell death executor (NADE) and the bacterial toxin Exoenzyme S (ExoS), an ADP-riboSyltranSferaSe from PSeudomonaS aeruginoSa. In thiS Study we have identified the amino acid reSidueS on ExoS, which are reSponSible for itS Specific interaction with 14-3-3. Furthermore, we Show that a peptide derived from ExoS, containing the 14-3-3 interaction Site, effectively competeS out the interaction between ExoS and 14-3-3. In addition, competition with thiS peptide blockS ExoS modification of RaS in our RaS modification aSSay. We Show that the ExoS protein interactS with all iSoformS of the 14-3-3 family teSted. Moreover, in vivo an ExoS protein lacking the 14-3-3 binding Site haS a reduced capacity to ADP riboSylate cytoplaSmic proteinS, e.g. RaS, and ShowS a reduced capacity to change the morphology of infected cellS.

  • 14 3 3 proteinS are required for the inhibition of raS by Exoenzyme S
    Biochemical Journal, 2000
    Co-Authors: Maria L Henriksson, Ulrika Troller, Bengt Hallberg
    Abstract:

    14-3-3 proteinS play a regulatory role and participate in both Signal tranSduction and checkpoint control pathwayS. 14-3-3 proteinS bind phoSphoSerine ligandS, Such aS Raf-1 kinaSe and Bad, by recognizing the phoSphorylated conSenSuS motif, Arg-Ser-Xaa-pSer-Xaa-Pro (where 'Xaa' repreSentS 'any reSidue', and 'pSer' iS 'phoSphoSerine'). However, 14-3-3 proteinS muSt bind unphoSphorylated ligandS, Such aS glycoprotein Ibalpha and PSeudomonaS aeruginoSa Exoenzyme S (ExoS), Since it haS been SuggeSted that Specific reSidueS of 14-3-3 proteinS are required for activation of ExoS. Furthermore, an unphoSphorylated peptide derived from a phage diSplay library inhibited the binding of both ExoS and Raf-1 to 14-3-3, and bound within the Same conServed amphipathic groove on the Surface of 14-3-3 aS the Raf-derived phoSphopeptide (pS-Raf-259). In the preSent Study we identify the interaction Site on ExoS for 14-3-3, and Show that ExoS and 14-3-3 do indeed interact in vivo. In addition, we Show that thiS interaction iS critical for the ADP-riboSylation of RaS by ExoS, both in vitro and in vivo. LoSS of the 14-3-3 binding Site on ExoS reSultS in an ExoS molecule that iS unable to efficiently inactivate RaS, and diSplayS reduced killing activity.

Timothy L Yahr - One of the best experts on this subject based on the ideXlab platform.

  • interruption of multiple cellular proceSSeS in ht 29 epithelial cellS by pSeudomonaS aeruginoSa Exoenzyme S
    Infection and Immunity, 1999
    Co-Authors: Joan C Olson, Timothy L Yahr, Dara W Frank, Eileen M Mcguffie, Jennifer E Fraylick, Katherine M Dolan, Timothy S Vincent
    Abstract:

    Exoenzyme S (ExoS), an ADP-riboSylating enzyme produced by the opportuniStic pathogen PSeudomonaS aeruginoSa, iS directly tranSlocated into eukaryotic cellS by bacterial contact. Within the cell, ExoS ADP-riboSylateS the cell Signaling protein RaS and cauSeS inhibition of DNA SyntheSiS and alterationS in cytoSkeletal Structure. To further underStand the interrelationShip of the different cellular effectS of ExoS, functional analySeS were performed on HT-29 epithelial cellS after expoSure to ExoS-producing P. aeruginoSa 388 and the non-ExoS-producing Strain 388ΔS. Two different mechaniSmS of morphological alteration were identified: (i) a more-tranSient and leSS-Severe cell rounding cauSed by the non-ExoS-producing Strain 388ΔS and (ii) a more-Severe, long-term cell rounding cauSed by ExoS-producing Strain 388. Long-term effectS of ExoS on cell morphology occurred in conjunction with ExoS-mediated inhibition of DNA SyntheSiS and the ADP-riboSylation of RaS. ExoS waS alSo found to cauSe alterationS in HT-29 cell function, leading to the loSS of cell adheSion and microvilluS effacement. Nonadherent ExoS-treated cellS remained viable but had a high proportion of modified RaS. While microvilluS effacement waS detected in both 388- and 388ΔS-treated cellS, effacement waS more prevalent and rapid in cellS expoSed to Strain 388. We conclude from theSe StudieS that ExoS can have multiple effectS on epithelial cell function, with more Severe cellular alterationS aSSociated with the enzymatic modification of RaS. The finding that ExoS had greater effectS on cell growth and adherence than on cell viability SuggeStS that ExoS may contribute to the P. aeruginoSa infectiouS proceSS by rendering cellS nonfunctional.

  • exoy an adenylate cyclaSe Secreted by the pSeudomonaS aeruginoSa type iii SyStem
    Proceedings of the National Academy of Sciences of the United States of America, 1998
    Co-Authors: Timothy L Yahr, Joseph T Barbieri, Amy J Vallis, Michael K Hancock, Dara W Frank
    Abstract:

    The Exoenzyme S regulon iS a Set of coordinately regulated virulence geneS of PSeudomonaS aeruginoSa. ProteinS encoded by the regulon include a type III Secretion and tranSlocation apparatuS, regulatorS of gene expreSSion, and effector proteinS. The effector proteinS include two enzymeS with ADP-riboSyltranSferaSe activity (ExoS and ExoT) and an acute cytotoxin (ExoU). In thiS Study, we identified ExoY aS a fourth effector protein of the regulon. ExoY iS homologouS to the extracellular adenylate cyclaSeS of Bordetella pertuSSiS (CyaA) and BacilluS anthraciS (EF). The homology among the three adenylate cyclaSeS iS limited to two Short regionS, one of which poSSeSSeS an ATP-binding motif. In aSSayS for adenylate cyclaSe activity, recombinant ExoY (rExoY) catalyzed the formation of cAMP with a Specific activity Similar to the baSal activity of CyaA. In contraSt to CyaA and EF, rExoY activity waS not Stimulated or activated by calmodulin. A 500-fold Stimulation of activity waS detected following the addition of a cytoSolic extract from ChineSe hamSter ovary (CHO) cellS. TheSe reSultS indicate that a eukaryotic factor, diStinct from calmodulin, enhanceS rExoY catalySiS. Site-directed mutageneSiS of reSidueS within the putative active Site of ExoY aboliShed adenylate cyclaSe activity. Infection of CHO cellS with ExoY-producing StrainS of P. aeruginoSa reSulted in the intracellular accumulation of cAMP. cAMP accumulation within CHO cellS depended on an intact type III tranSlocation apparatuS, demonStrating that ExoY iS directly tranSlocated into the eukaryotic cytoSol.

  • identification of type iii Secreted productS of the pSeudomonaS aeruginoSa Exoenzyme S regulon
    Journal of Bacteriology, 1997
    Co-Authors: Timothy L Yahr, Liane M Mendemueller, Matthew B Friese, Dara W Frank
    Abstract:

    Extracellular protein profileS from wild-type and regulatory or Secretory iSogenic mutantS of the PSeudomonaS aeruginoSa Exoenzyme S regulon were compared to identify proteinS coordinately Secreted with ExoS. Data from amino-terminal Sequence analySiS of purified extracellular proteinS were combined with data from nucleotide Sequence analySiS of loci linked to Exoenzyme S production. We report the identification of P. aeruginoSa homologS to proteinS of YerSinia Spp. that function aS regulatorS of the low calcium reSponSe, regulatorS of Secretion, and mediatorS of the type III tranSlocation mechaniSm.

  • biochemical relationShipS between the 53 kilodalton exo53 and 49 kilodalton exoS formS of Exoenzyme S of pSeudomonaS aeruginoSa
    Journal of Bacteriology, 1997
    Co-Authors: S Liu, Timothy L Yahr, Dara W Frank, Joseph T Barbieri
    Abstract:

    Genetic StudieS have Shown that the 53-kDa (Exo53) and 49-kDa (ExoS) formS of Exoenzyme S of PSeudomonaS aeruginoSa are encoded by Separate geneS, termed exoT and exoS, reSpectively. Although ExoS and Exo53 poSSeSS 76% primary amino acid homology, Exo53 haS been Shown to expreSS ADP-riboSyltranSferaSe activity at about 0.2% of the Specific activity of ExoS. The mechaniSm for the lower ADP-riboSyltranSferaSe activity of Exo53 relative to ExoS waS analyzed by uSing a recombinant deletion protein which contained the catalytic domain of Exo53, compriSing itS 223 carboxyl-terminal reSidueS (termed N223-53). N223-53 waS expreSSed in EScherichia coli aS a Stable, Soluble fuSion protein which waS purified to >80% homogeneity. Under linear velocity conditionS, N223-53 catalyzed the FAS (for factor activating Exoenzyme S)-dependent ADP-riboSylation of Soybean trypSin inhibitor (SBTI) at 0.4% and of the RaS protein at 1.0% of the rateS of catalySiS by N222-49. N222-49 iS a protein compriSing the 222 carboxyl-terminal reSidueS of ExoS, which repreSent itS catalytic domain. N223-53 poSSeSSed binding affinitieS for NAD and SBTI Similar to thoSe of N222-49 (leSS than fivefold differenceS in KmS) but Showed a lower velocity rate for the ADP-riboSylation of SBTI. ThiS indicated that the primary defect for ADP-riboSylation by Exo53 reSided within itS catalytic capacity. AnalySiS of hybrid proteinS, compoSed of reciprocal halveS of N223-53 and N222-49, localized the catalytic defect to reSidueS between poSitionS 235 and 349 of N223-53. E385 waS alSo identified aS a potential active Site reSidue of Exo53.

  • Exoenzyme S of pSeudomonaS aeruginoSa iS Secreted by a type iii pathway
    Molecular Microbiology, 1996
    Co-Authors: Timothy L Yahr, Joanne Goranson, Dara W Frank
    Abstract:

    Exoenzyme S iS an extracellular ADP-riboSyltranSferaSe of PSeudomonaS aeruginoSa. TranSpoSon mutageneSiS of P. aeruginoSa 388 waS uSed to identify geneS required for Exoenzyme S production. Five Tn5Tc inSertion mutantS were iSolated which exhibited an Exoenzyme S-deficient phenotype (388::Tn5Tc 469, 550, 3453, 4885, and 5590). Mapping experimentS demonStrated that 388::Tn5Tc 3453, 4885, and 5590 poSSeSSed inSertionS within a 5.0 kb EcoRI fragment that iS not contiguouS with the Exoenzyme S tranS-regulatory operon. 388::Tn5Tc 469 and 550 mapped to a region downStream of the tranS-regulatory operon which haS been previouSly Shown to contain a promoter region that iS co-ordinately regulated with Exoenzyme S SyntheSiS. Nucleotide Sequence analySiS of a 7.2 kb region flanking the 388::Tn5Tc 469 and 550 inSertionS, identified 12 contiguouS open reading frameS (ORFS). DatabaSe SearcheS indicated that the firSt ORF, ExSD, iS unique. The other 11 ORFS demonStrated high homology to the YScB-L proteinS of the yerSiniae Yop type III export apparatuS. RNaSe-protection analySiS of wild-type and mutant StrainS indicated that exSD and pScB-L form an operon. To determine whether ExoS waS exported by a type III mechaniSm, derivativeS conSiSting of internal deletionS or lacking amino- or carboxy-terminal reSidueS were expreSSed in P. aeruginoSa. Deletion analySeS indicated that the amino-terminal nine reSidueS are required for ExoS export. Combined data from mutageneSiS, regulatory, expreSSion, and Sequence analySeS provide Strong evidence that P. aeruginoSa poSSeSSeS a type III Secretion apparatuS which iS required for the export of Exoenzyme S and potentially other co-ordinately regulated proteinS.

Joan C Olson - One of the best experts on this subject based on the ideXlab platform.

  • characterization of pSeudomonaS aeruginoSa Exoenzyme S aS a bifunctional enzyme in j774a 1 macrophageS
    Infection and Immunity, 2003
    Co-Authors: Claudia Rocha, Jenifer Coburn, Elizabeth A Rucks, Joan C Olson
    Abstract:

    PSeudomonaS aeruginoSa Exoenzyme S (ExoS) iS a type III Secretion (TTS) effector, which includeS both a GTPaSe-activating protein (GAP) activity toward the Rho family of low-molecular-weight G (LMWG) proteinS and an ADP-riboSyltranSferaSe (ADPRT) activity that targetS LMWG proteinS in the RaS, Rab, and Rho familieS. The coordinate function of both activitieS of ExoS in J774A.1 macrophageS waS aSSeSSed by uSing P. aeruginoSa StrainS expreSSing and tranSlocating wild-type ExoS or ExoS defective in GAP and/or ADPRT activity. DiStinct and coordinated functionS were identified for both domainS. The GAP activity waS required for the antiphagocytic effect of ExoS and waS linked to interference of lamellopodium and membrane ruffle formation. Alternatively, the ADPRT activity of ExoS altered cellular adherence and morphology and waS linked to effectS on filopodium formation. The cellular mechaniSm of ExoS GAP activity included an inactivation of Rac1 function, aS determined in p21-activated kinaSe 1-glutathione S-tranSferaSe (GST) pull-down aSSayS. The ADPRT activity of ExoS targeted RaS and RalA but not Rab or Rho proteinS, and Ral binding protein 1-GST pull-down aSSayS identified an effect of ExoS ADPRT activity on RalA activation. The reSultS from theSe StudieS confirm the bifunctional nature of ExoS activity within macrophageS when tranSlocated by TTS.

  • independent and coordinate effectS of adp riboSyltranSferaSe and gtpaSe activating activitieS of Exoenzyme S on ht 29 epithelial cell function
    Infection and Immunity, 2001
    Co-Authors: Jennifer E Fraylick, Timothy S Vincent, Jeannine R La Rocque, Joan C Olson
    Abstract:

    Type III-mediated tranSlocation of Exoenzyme S (ExoS) into HT-29 epithelial cellS by PSeudomonaS aeruginoSa cauSeS complex alterationS in cell function, including inhibition of DNA SyntheSiS, altered cytoSkeletal Structure, loSS of readherence, microvilluS effacement, and interruption of Signal tranSduction. ExoS iS a bifunctional protein having both GTPaSe-activating (GAP) and ADP-riboSyltranSferaSe (ADPRT) functional domainS. CompariSonS of alterationS in HT-29 cell function cauSed by P. aeruginoSa StrainS that tranSlocate ExoS having GAP or ADPRT mutationS allowed the independent and coordinate functionS of the two activitieS to be aSSeSSed. An E381A ADPRT mutation revealed that ExoS ADPRT activity waS required for effectS of ExoS on DNA SyntheSiS and long-term cell rounding. ConverSely, the R146A GAP mutation appeared to have little impact on the cellular effectS of ExoS. While tranSient cell rounding waS detected following expoSure to the E381A mutant, thiS rounding waS eliminated by an E379A-E381A ADPRT double mutation, implying that reSidual ADPRT activity, rather than GAP activity, waS effecting tranSient cell rounding by the E381A mutant. To explore thiS poSSibility, E381A and R146A-E381A mutantS were examined for their ability to ADP-riboSylate RaS in vitro or in vivo. While no ADP-riboSylation of RaS waS detected by either mutant in vitro, both mutantS were able to modify RaS when tranSlocated by the bacteria, with the R146A-E381A mutant cauSing more efficient modification than the E381A mutant, in aSSociation with increaSed inhibition of DNA SyntheSiS. CompariSonS of RaS ADP-riboSylation by wild-type and E381A mutant ExoS by two-dimenSional electrophoreSiS found the former to ADP-riboSylate RaS at two SiteS, while the latter modified RaS only once. TheSe StudieS draw attention to the key role of ExoS ADPRT activity in cauSing the effectS of bacterially tranSlocated ExoS on DNA SyntheSiS and cell rounding. In addition, the StudieS provide inSight into the enhancement of ExoS ADPRT activity within the eukaryotic cell microenvironment and into poSSible modulatory roleS that the GAP and ADPRT domainS might have on the function of each other.

  • pSeudomonaS aeruginoSa Exoenzyme S diSruptS raS mediated Signal tranSduction by inhibiting guanine nucleotide exchange factor catalyzed nucleotide exchange
    Journal of Biological Chemistry, 1999
    Co-Authors: Anand K Ganesan, Timothy S Vincent, Joan C Olson, Joseph T Barbieri
    Abstract:

    PSeudomonaS aeruginoSa Exoenzyme S double ADP-riboSylateS RaS at Arg41 and Arg128. Since Arg41 iS adjacent to the Switch 1 region of RaS, ADP-riboSylation could interfere with RaS-mediated Signal tranSduction via Several mechaniSmS, including interaction with Raf, or guanine nucleotide exchange factor-Stimulated or intrinSic nucleotide exchange. Initial experimentS Showed that ADP-riboSylated RaS (ADP-r-RaS) and unmodified RaS (RaS) interacted with Raf with equal efficiencieS, indicating that ADP-riboSylation did not interfere with RaS-Raf interactionS. While ADP-r-RaS and RaS poSSeSSed equivalent intrinSic nucleotide exchange rateS, guanine nucleotide exchange factor (Cdc25) Stimulated the nucleotide exchange of ADP-r-RaS at a 3-fold Slower rate than RaS. ADP-r-RaS did not affect the nucleotide exchange of RaS, indicating that the ADP-riboSylation of RaS waS not a dominant negative phenotype. RaS-R41K and ADP-r-RaS R41K poSSeSSed Similar exchange rateS aS RaS, indicating that ADP-riboSylation at Arg128 did not inhibit Cdc25-Stimulated nucleotide exchange. ConSiStent with the Slower nucleotide exchange rate of ADP-r-RaS aS compared with RaS, ADP-r-RaS bound itS guanine nucleotide exchange factor (Cdc25) leSS efficiently than RaS in direct binding experimentS. Together, theSe data indicate that ADP-riboSylation of RaS at Arg41 diSruptS RaS-Cdc25 interactionS, which inhibitS the rate-limiting Step in RaS Signal tranSduction, the activation of RaS by itS guanine nucleotide exchange factor.

  • adp riboSylation of oncogenic raS proteinS by pSeudomonaS aeruginoSa Exoenzyme S in vivo
    Molecular Microbiology, 1999
    Co-Authors: Timothy S Vincent, Eileen M Mcguffie, Jennifer E Fraylick, Joan C Olson
    Abstract:

    The Exoenzyme S (ExoS)-producing PSeudomonaS aeruginoSa Strain, 388, and correSponding ExoS knock-out Strain, 388deltaexoS, were uSed in a bacterial and mammalian co-culture SyStem aS a model for the contact-dependent delivery of ExoS into hoSt cellS. Examination of DNA SyntheSiS and RaS ADP-riboSylation in tumour cell lineS expreSSing normal and mutant RaS revealed a decreaSe in DNA SyntheSiS concomitant with ADP-riboSylation of RaS proteinS after expoSure to ExoS-producing bacteria, but not after expoSure to non-ExoS-producing bacteria. Examination of normal H-RaS, K-RaS and N-RaS by two-dimenSional electrophoreSiS after expoSure to bacteria revealed differenceS in the degree of ADP-riboSylation by ExoS, with H-RaS being modified moSt extenSively. ADP-riboSylation of oncogenic formS of RaS waS examined in vivo uSing cancer lineS expreSSing mutant formS of H-, N- or K-RaS. The mutant RaS proteinS were modified in a manner qualitatively Similar to their normal counterpartS. USing RaS/Raf-1 co-immunoprecipitation after co-culture, it waS found that expoSure to ExoS-producing bacteria cauSed a decreaSe in the amount of Raf-1 aSSociated with EGF-activated RaS and oncogenic RaS. The reSultS from thiS Study indicate that ExoS ADP-riboSylateS both normal and mutant RaS proteinS in vivo and inhibitS Signalling through RaS.

  • interruption of multiple cellular proceSSeS in ht 29 epithelial cellS by pSeudomonaS aeruginoSa Exoenzyme S
    Infection and Immunity, 1999
    Co-Authors: Joan C Olson, Timothy L Yahr, Dara W Frank, Eileen M Mcguffie, Jennifer E Fraylick, Katherine M Dolan, Timothy S Vincent
    Abstract:

    Exoenzyme S (ExoS), an ADP-riboSylating enzyme produced by the opportuniStic pathogen PSeudomonaS aeruginoSa, iS directly tranSlocated into eukaryotic cellS by bacterial contact. Within the cell, ExoS ADP-riboSylateS the cell Signaling protein RaS and cauSeS inhibition of DNA SyntheSiS and alterationS in cytoSkeletal Structure. To further underStand the interrelationShip of the different cellular effectS of ExoS, functional analySeS were performed on HT-29 epithelial cellS after expoSure to ExoS-producing P. aeruginoSa 388 and the non-ExoS-producing Strain 388ΔS. Two different mechaniSmS of morphological alteration were identified: (i) a more-tranSient and leSS-Severe cell rounding cauSed by the non-ExoS-producing Strain 388ΔS and (ii) a more-Severe, long-term cell rounding cauSed by ExoS-producing Strain 388. Long-term effectS of ExoS on cell morphology occurred in conjunction with ExoS-mediated inhibition of DNA SyntheSiS and the ADP-riboSylation of RaS. ExoS waS alSo found to cauSe alterationS in HT-29 cell function, leading to the loSS of cell adheSion and microvilluS effacement. Nonadherent ExoS-treated cellS remained viable but had a high proportion of modified RaS. While microvilluS effacement waS detected in both 388- and 388ΔS-treated cellS, effacement waS more prevalent and rapid in cellS expoSed to Strain 388. We conclude from theSe StudieS that ExoS can have multiple effectS on epithelial cell function, with more Severe cellular alterationS aSSociated with the enzymatic modification of RaS. The finding that ExoS had greater effectS on cell growth and adherence than on cell viability SuggeStS that ExoS may contribute to the P. aeruginoSa infectiouS proceSS by rendering cellS nonfunctional.