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Sucharit Bhakdi - One of the best experts on this subject based on the ideXlab platform.

  • differential rOle Of p38 mitOgen activated prOtein kinase fOr cellular recOvery frOm attack by pOre fOrming s aureus α tOxin Or StreptOlysin O
    Biochemical and Biophysical Research Communications, 2006
    Co-Authors: Matthias Husmann, Katrin Dersch, Wiesia Bobkiewicz, Erik Beckmann, Gunnaporn Veerachato, Sucharit Bhakdi
    Abstract:

    FOllOwing the ObservatiOn that cells are able tO recOver frOm membrane lesiOns incurred by StaphylOcOccus aureus α-tOxin and StreptOlysin O (SLO), we investigated the rOle Of p38 in this prOcess. p38 phOsphOrylatiOn Occurred in respOnse tO attack by bOth tOxins, cOmmencing within minutes after tOxin treatment and waning after several hOurs. While SLO repOrtedly activates p38 via ASK1 and ROS, we shOw that this pathway dOes nOt play a majOr rOle fOr p38 inductiOn in α-tOxin-treated cells. Strikingly divergent effects Of p38 blOckade were nOted depending On the tOxin emplOyed. In the case Of α-tOxin, inhibitiOn Of p38 within the time frame Of its activatiOn led tO disruptiOn Of the recOvery prOcess and tO cell death. In cOntrast, blOckade Of p38 in SLO permeabilized cells did nOt affect the capacity Of the cells tO replenish their ATP stOres.

  • resealing Of large transmembrane pOres prOduced by StreptOlysin O in nucleated cells is accOmpanied by nf κb activatiOn and dOwnstream events
    The FASEB Journal, 2002
    Co-Authors: Iwan Walev, Sucharit Bhakdi, Michael Hombach, Wieslawa Bobkiewicz, Dominic Fenske, Matthias Husmann
    Abstract:

    SPECIFIC AIMSWe questiOned whether transient membrane permeabilizatiOn by StreptOlysin O, which creates very large lesiOns, will trigger transcriptiOnal events in the affected cells.PRINCIPAL FINDI...

  • delivery Of prOteins intO living cells by reversible membrane permeabilizatiOn with StreptOlysin O
    Proceedings of the National Academy of Sciences of the United States of America, 2001
    Co-Authors: Iwan Walev, Angela Valeva, Sebastian Chakrit Bhakdi, Fred Hofmann, Nabil Djonder, Klaus Aktories, Sucharit Bhakdi
    Abstract:

    The pOre-fOrming tOxin StreptOlysin O (SLO) can be used tO reversibly permeabilize adherent and nOnadherent cells, allOwing delivery Of mOlecules with up tO 100 kDa mass tO the cytOsOl. Using FITC-labeled albumin, 10(5)-10(6) mOlecules were estimated tO be entrapped per cell. Repair Of tOxin lesiOns depended On Ca(2+)-calmOdulin and On intact micrOtubules, but was nOt sensitive tO actin disruptiOn Or tO inhibitiOn Of prOtein synthesis. Resealed cells were viable fOr days and retained the capacity tO endOcytOse and tO prOliferate. The active dOmains Of large clOstridial tOxins were intrOduced intO three different cell lines. The dOmains were derived frOm ClOstridium difficile B-tOxin and ClOstridium sOrdelli lethal tOxin, which glycOsylate small G-prOteins, and frOm ClOstridium bOtulinum C2 tOxin, which ADP-ribOsylates actin. After delivery with SLO, all three tOxins disrupted the actin cytOskeletOn tO cause rOunding up Of the cells. GlucOsylatiOn assays demOnstrated that G-prOteins RhO and Ras were retained in the permeabilized cells and were mOdified by the respective tOxins. InactivatiOn Of these G-prOteins resulted in reduced stimulus-dependent granule secretiOn, whereas ADP-ribOsylatiOn Of actin by the C. bOtulinum C2-tOxin resulted in enhanced secretiOn in cells. The presented methOd fOr intrOducing prOteins intO living cells shOuld find multifaceted applicatiOn in cell biOlOgy.

  • StreptOlysin O-permeabilized granulOcytes shed L-selectin cOncOmitantly with ceramide generatiOn via neutral sphingOmyelinase.
    Journal of leukocyte biology, 2000
    Co-Authors: Iwan Walev, Dennis Tappe, Erich Gulbins, Sucharit Bhakdi
    Abstract:

    Cleavage Of membrane-assOciated L-selectin regulates leukOcyte rOlling On vascular endOthelium at sites Of inflammatiOn. We repOrt that rapid and massive shedding Of L-selectin Occurs frOm granulOcytes attacked by the pOre-fOrming bacterial tOxin StreptOlysin O (SLO). Shedding was nOt induced by an SLO mutant that retained binding capacity but lacked pOre-fOrming activity. Cells permeabilized with SLO exhibited a 1.5-fOld increase in the activity Of neutral sphingOmyelinase, which was accOmpanied by increased ceramide fOrmatiOn. L-selectin cleavage was inducible by treatment Of cells with bacterial sphingOmyelinase, and alsO thrOugh exOgenOus applicatiOn Of a cell-permeable ceramide analOg. Our data identify a nOvel path tO the shedding prOcess and shOw that activatiOn Of neutral sphingOmyelinase with the generatiOn Of ceramide is an impOrtant event underlying enhanced sheddase functiOn in cells permeabilized by a pOre-fOrming tOxin.

  • StreptOlysin O inhibitiOn Of the cOnfOrmatiOnal change during membrane binding Of the mOnOmer prevents OligOmerizatiOn and pOre fOrmatiOn
    Biochemistry, 1999
    Co-Authors: El Moeiz Abdel Ghani, Michael A Kehoe, Sucharit Bhakdi, Silvia Weis, Ivan Walev, Michael Palmer
    Abstract:

    StreptOlysin O is a fOur-dOmain prOtein tOxin that permeabilizes animal cell membranes. The tOxin first binds as a mOnOmer tO membrane chOlesterOl and subsequently assembles intO OligOmeric transmembrane pOres. Binding is mediated by a C-terminally lOcated tryptOphan-rich mOtif. In a previOus study, cOnfOrmatiOnal effects Of membrane binding were characterized by intrOducing single mutant cysteine residues that were then thiOl-specifically derivatized with the envirOnmentally sensitive fluOrOphOr acrylOdan. Membrane binding Of the labeled prOteins was accOmpanied by spectral shifts Of the prObe fluOrescence, suggesting that the tOxin mOlecule had undergOne a cOnfOrmatiOnal change. Here we prOvide evidence that this change cOrrespOnds tO an allOsteric transitiOn Of the tOxin mOnOmer that is required fOr the subsequent OligOmerizatiOn and pOre fOrmatiOn. The cOnfOrmatiOnal change is reversible with reversal Of binding, and it is related tO temperature in a fashiOn that clOsely parallels the temperature-depe...

Michael A Kehoe - One of the best experts on this subject based on the ideXlab platform.

  • cOmbined cOntributiOns Of StreptOlysin O and StreptOlysin s tO virulence Of serOtype m5 streptOcOccus pyOgenes strain manfredO
    Infection and Immunity, 2003
    Co-Authors: Michael C Fontaine, Jeong Jin Lee, Michael A Kehoe
    Abstract:

    StreptOlysin O (SLO) and StreptOlysin S (SLS) are pOtent cytOlytic tOxins prOduced by almOst all clinical isOlates Of grOup A streptOcOcci (GAS). Allele-replacement mutagenesis was used tO cOnstruct nOnpOlar (in-frame) deletiOn mutatiOns in the slO and sagB genes Of the serOtype M5 GAS strain ManfredO, prOducing isOgenic single and dOuble SLO- and SLS-defective mutants. In cOntrast tO recent repOrts On SLS-defective insertiOn mutants (I. Biswas, P. GermOn, K. McDade, and J. ScOtt, Infect. Immun. 69:7029-7038, 2001; Z. Li, D. Sledjeski, B. Kreikemeyer, A.POdbielski, and M. BOyle, J. BacteriOl. 181:6019-6027, 1999), nOne Of the mutants described here had nOtable pleiOtrOpic effects On the expressiOn Of Other virulence factOrs examined. COmparisOn Of isOgenic parent and mutant strains in variOus virulence mOdels revealed nO differences in their abilities tO multiply in human blOOd Or in their 50% lethal dOses (LD50s) upOn intraperitOneal infectiOn Of BALB/c mice. A single lOg unit difference in the LD50s Of the parent and SLS-defective mutant strains was Observed upOn infectiOn by the subcutaneOus (s.c.) rOute. COmparisOns Over a range Of infective dOses shOwed that bOth SLO and SLS cOntributed tO the early stages Of infectiOn and tO the inductiOn Of necrOtic lesiOns in the murine s.c. mOdel. Individually, each tOxin made an incremental cOntributiOn tO virulence that was nOt apparent at higher infective dOses, althOugh the absence Of bOth tOxins reduced virulence Over the entire dOse range examined. Interestingly, in sOme cases, the cOntributiOn Of SLO tO virulence was clear Only frOm an analysis Of the dOuble-mutant strain, highlighting the value Of nOt cOnfining virulence studies tO mutant strains defective in the expressiOn Of Only single virulence factOrs.

  • StreptOlysin O inhibitiOn Of the cOnfOrmatiOnal change during membrane binding Of the mOnOmer prevents OligOmerizatiOn and pOre fOrmatiOn
    Biochemistry, 1999
    Co-Authors: El Moeiz Abdel Ghani, Michael A Kehoe, Sucharit Bhakdi, Silvia Weis, Ivan Walev, Michael Palmer
    Abstract:

    StreptOlysin O is a fOur-dOmain prOtein tOxin that permeabilizes animal cell membranes. The tOxin first binds as a mOnOmer tO membrane chOlesterOl and subsequently assembles intO OligOmeric transmembrane pOres. Binding is mediated by a C-terminally lOcated tryptOphan-rich mOtif. In a previOus study, cOnfOrmatiOnal effects Of membrane binding were characterized by intrOducing single mutant cysteine residues that were then thiOl-specifically derivatized with the envirOnmentally sensitive fluOrOphOr acrylOdan. Membrane binding Of the labeled prOteins was accOmpanied by spectral shifts Of the prObe fluOrescence, suggesting that the tOxin mOlecule had undergOne a cOnfOrmatiOnal change. Here we prOvide evidence that this change cOrrespOnds tO an allOsteric transitiOn Of the tOxin mOnOmer that is required fOr the subsequent OligOmerizatiOn and pOre fOrmatiOn. The cOnfOrmatiOnal change is reversible with reversal Of binding, and it is related tO temperature in a fashiOn that clOsely parallels the temperature-depe...

  • StreptOlysin O a prOpOsed mOdel Of allOsteric interactiOn between a pOre fOrming prOtein and its target lipid bilayer
    Biochemistry, 1998
    Co-Authors: Michael Palmer, Angela Valeva, Michael A Kehoe, Peter Saweljew, Ivan Vulicevic, Sucharit Bhakdi
    Abstract:

    StreptOlysin O, a pOlypeptide Of 571 aminO acids, belOngs tO the family Of thiOl-activated tOxins that permeabilize animal cell membranes. The prOtein binds as a mOnOmer tO membrane chOlesterOl. Binding invOlves a cOnserved regiOn clOse tO the C-terminus and triggers subsequent pOlymerizatiOn intO large arc- and ring-shaped structures surrOunding pOres Of up tO 30 nm. Besides the C-terminus, a distantly lOcated regiOn spanning residues 213−305 is invOlved in OligOmerizatiOn and in membrane insertiOn. Here, we searched fOr cOnfOrmatiOnal effects Of mOnOmer binding tO the latter functiOnally impOrtant regiOn. TO this end, single cysteine substitutiOn mutants were prOduced and derivatized with the pOlarity-sensitive fluOrOphOre acrylOdan. FluOrimetric measurements revealed that binding Of the mOnOmer tO membranes is accOmpanied by distinct envirOnmental changes at aminO acid residues 218, 248, 266, and 277. COnspicuOusly, the envirOnment Of residues 218 and 266 became mOre hydrOphilic, suggesting mOvement Of...

  • membrane penetrating dOmain Of StreptOlysin O identified by cysteine scanning mutagenesis
    Journal of Biological Chemistry, 1996
    Co-Authors: Michael Palmer, Angela Valeva, Michael A Kehoe, Peter Saweljew, Ivan Vulicevic, Sucharit Bhakdi
    Abstract:

    StreptOlysin O (SLO), a pOlypeptide Of 571 aminO acids, belOngs tO a family Of highly hOmOlOgOus tOxins that bind tO cell membranes cOntaining chOlesterOl and then pOlymerize tO fOrm large transmembrane pOres. A cOnserved regiOn clOse tO the C terminus cOntains the single cysteine residue Of SLO and has been implicated in membrane binding, which has been the Only clear assignment Of functiOn tO a part Of the sequence. We have used a cysteine-less active mutant Of SLO tO intrOduce single cysteine residues at 19 pOsitiOns distributed thrOughOut the sequence. The cysteines were derivatized with the pOlarity-sensitive fluOrOphOre acrylOdan, and the fluOrescence emissiOn Of the label was examined at the different stages Of SLO pOre assembly. With several mutants, OligOmerizatiOn On membranes was accOmpanied by emissiOn blue-shifts, indicating mOvement Of the label intO a mOre hydrOphObic envirOnment. These effects were essentially cOnfined tO the range Of aminO acids 213-305. With OligOmeric mutants L274C, S286C, and S305C, additiOnal envirOnmental alteratiOns were induced when different nOndenaturing detergents were used tO dislOdge the membrane lipids frOm the OligOmers. The cOrrespOnding aminO acid residues thus insert intO the lipid bilayer during pOre fOrmatiOn. COnversely, the spectra Of OligOmeric mutants A213C and T245C were nOt affected by detergents. DevOid Of cOntact with the lipid bilayer, these aminO acid residues prObably participate in the interactiOn Of SLO mOlecules within the OligOmer.

  • staphylOcOccal alpha tOxin StreptOlysin O and escherichia cOli hemOlysin prOtOtypes Of pOre fOrming bacterial cytOlysins
    Archives of Microbiology, 1996
    Co-Authors: Sucharit Bhakdi, Angela Valeva, Michael A Kehoe, Iwan Walev, Hagan Bayley, Barbara Walker, U Weller, Michael Palmer
    Abstract:

    StaphylOcOccal alpha-tOxin, StreptOlysin-O, and Escherichia cOli hemOlysin are well-studied prOtOtypes Of pOre-fOrming bacterial cytOtOxins. Each is prOduced as a water-sOluble single-chain pOlypeptide that inserts intO target membranes tO fOrm aqueOus transmembrane pOres. This review will cOmpare prOperties Of the three tOxin prOtOtypes, highlighting the similarities and alsO the differences in their structure, mOde Of binding, mechanism Of pOre fOrmatiOn, and the respOnses they elicit in target cells. POre-fOrming tOxins represent the mOst pOtent and versatile weapOns with which invading micrObes damage the hOst macrOOrganism.

Michael Palmer - One of the best experts on this subject based on the ideXlab platform.

  • differential interactiOn Of the twO chOlesterOl dependent membrane damaging tOxins StreptOlysin O and vibriO chOlerae cytOlysin with enantiOmeric chOlesterOl
    FEBS Letters, 2003
    Co-Authors: Alexander Zitzer, Emily J Westover, Douglas F Covey, Michael Palmer
    Abstract:

    Membrane chOlesterOl is essential tO the activity Of at least twO structurally unrelated families Of bacterial pOre-fOrming tOxins, represented by StreptOlysin O (SLO) and VibriO chOlerae cytOlysin (VCC), respectively. Here, we repOrt that SLO and VCC differ sharply in their interactiOn with lipOsOme membranes cOntaining enantiOmeric chOlesterOl (ent-chOlesterOl). VCC had very lOw activity with ent-chOlesterOl, which is in line with a stereOspecific mOde Of interactiOn Of this tOxin with chOlesterOl. In cOntrast, SLO was Only slightly less active with ent-chOlesterOl than with chOlesterOl, suggesting a rather limited degree Of structural specificity in the tOxin-chOlesterOl interactiOn.

  • StreptOlysin O the c terminal tryptOphan rich dOmain carries functiOnal sites fOr bOth membrane binding and self interactiOn but nOt fOr stable OligOmerizatiOn
    Biochimica et Biophysica Acta, 2001
    Co-Authors: Silvia Weis, Michael Palmer
    Abstract:

    StreptOlysin O belOngs tO the class Of thiOl-activated tOxins, which are single chain, fOur-dOmain prOteins that bind tO membranes cOntaining chOlesterOl and then assemble tO fOrm large OligOmeric pOres. Membrane binding invOlves a cOnserved tryptOphan-rich sequence mOtif lOcated within the C-terminally lOcated dOmain 4. In cOntrast, sites invOlved in OligOmerizatiOn and pOre fOrmatiOn have been assigned tO dOmains 1 and 3, respectively. We here examined the functiOnal prOperties Of dOmain 4, which was recOmbinantly expressed with an N-terminal histidine tag fOr purificatiOn and an additiOnal cysteine residue fOr cOvalent labeling. The fluOrescently labeled fragment readily bOund tO membranes, but it did nOt fOrm OligOmers nOr lyse cell membranes. MOreOver, the labeled fragment did nOt detectably becOme incOrpOrated intO hybrid OligOmers when cOmbined with lytically active full-length tOxin. HOwever, when present in large excess Over the active tOxin, the dOmain 4 fragment effected reductiOn Of hemOlytic activity and Of functiOnal pOre size, which indicates interference with OligOmerizatiOn Of the lytically active species. Our findings suppOrt the nOtiOn that dOmain 4 Of the StreptOlysin O mOlecule may fOld autOnOmOusly, is essential fOr membrane binding and is capable nOt Of irreversible but Of reversible assOciatiOn with the entire tOxin mOlecule.

  • StreptOlysin O inhibitiOn Of the cOnfOrmatiOnal change during membrane binding Of the mOnOmer prevents OligOmerizatiOn and pOre fOrmatiOn
    Biochemistry, 1999
    Co-Authors: El Moeiz Abdel Ghani, Michael A Kehoe, Sucharit Bhakdi, Silvia Weis, Ivan Walev, Michael Palmer
    Abstract:

    StreptOlysin O is a fOur-dOmain prOtein tOxin that permeabilizes animal cell membranes. The tOxin first binds as a mOnOmer tO membrane chOlesterOl and subsequently assembles intO OligOmeric transmembrane pOres. Binding is mediated by a C-terminally lOcated tryptOphan-rich mOtif. In a previOus study, cOnfOrmatiOnal effects Of membrane binding were characterized by intrOducing single mutant cysteine residues that were then thiOl-specifically derivatized with the envirOnmentally sensitive fluOrOphOr acrylOdan. Membrane binding Of the labeled prOteins was accOmpanied by spectral shifts Of the prObe fluOrescence, suggesting that the tOxin mOlecule had undergOne a cOnfOrmatiOnal change. Here we prOvide evidence that this change cOrrespOnds tO an allOsteric transitiOn Of the tOxin mOnOmer that is required fOr the subsequent OligOmerizatiOn and pOre fOrmatiOn. The cOnfOrmatiOnal change is reversible with reversal Of binding, and it is related tO temperature in a fashiOn that clOsely parallels the temperature-depe...

  • StreptOlysin O a prOpOsed mOdel Of allOsteric interactiOn between a pOre fOrming prOtein and its target lipid bilayer
    Biochemistry, 1998
    Co-Authors: Michael Palmer, Angela Valeva, Michael A Kehoe, Peter Saweljew, Ivan Vulicevic, Sucharit Bhakdi
    Abstract:

    StreptOlysin O, a pOlypeptide Of 571 aminO acids, belOngs tO the family Of thiOl-activated tOxins that permeabilize animal cell membranes. The prOtein binds as a mOnOmer tO membrane chOlesterOl. Binding invOlves a cOnserved regiOn clOse tO the C-terminus and triggers subsequent pOlymerizatiOn intO large arc- and ring-shaped structures surrOunding pOres Of up tO 30 nm. Besides the C-terminus, a distantly lOcated regiOn spanning residues 213−305 is invOlved in OligOmerizatiOn and in membrane insertiOn. Here, we searched fOr cOnfOrmatiOnal effects Of mOnOmer binding tO the latter functiOnally impOrtant regiOn. TO this end, single cysteine substitutiOn mutants were prOduced and derivatized with the pOlarity-sensitive fluOrOphOre acrylOdan. FluOrimetric measurements revealed that binding Of the mOnOmer tO membranes is accOmpanied by distinct envirOnmental changes at aminO acid residues 218, 248, 266, and 277. COnspicuOusly, the envirOnment Of residues 218 and 266 became mOre hydrOphilic, suggesting mOvement Of...

  • chOlesterOl StreptOlysin O interactiOn an em study Of wild type and mutant StreptOlysin O
    Journal of Structural Biology, 1998
    Co-Authors: Robin J Harris, Sucharit Bhakdi, Marc Adrian, Michael Palmer
    Abstract:

    Abstract We present transmissiOn electrOn micrOscOpical data frOm negatively stained specimens Of chOlesterOl fOllOwing interactiOn with the thiOl-activated bacterial tOxin StreptOlysin O (SLO) (wild-type and a number Of cysteine substitutiOn mutants), with and withOut chemical mOdificatiOn Of the cysteine residues. TwO experimental systems were used, One with an aqueOus suspensiOn Of chOlesterOl micrOcrystals and the Other with immObilized thin planar chOlesterOl crystals attached tO a carbOn film. In bOth systems the wild-type SLO and twO cytOlytically active mutants, Cys 530 → Ala (C530A) and Ser 101 → Cys (S101C), readily generated the characteristic SLO arc- and ring-like OligOmers On the surface Of chOlesterOl micrOcrystals and immObilized planar chOlesterOl crystals. An underlying array Of bOund tOxin can sOmetimes be detected. In the presence Of high cOncentratiOns Of SLO mOnOmer, extensive sheet-like netwOrks Of linked OligOmers extend frOm the micrOcrystals. The SLO mutant Thr250 → Cys (T250C), which alsO pOssesses a relatively high cytOlytic activity, has been fOund tO create ring-like tOxin OligOmers sOmewhat mOre slOwly than wild-type SLO, but the linear mOnOmOlecular layer array Of chOlesterOl-bOund tOxin is mOre readily detected. With mutant Asn402 → Cys (N402C), which has ≈10% cytOlytic activity cOmpared tO wild-type SLO, the fOrmatiOn Of ring-like OligOmers is markedly reduced, with incOmplete arcs and the parallel arrays predOminating. Chemical mOdificatiOn Of the functiOnal cysteine grOups Of SLO mutants T250C and N402C cOmpletely inhibits the fOrmatiOn Of tOxin OligOmers, but dOes nOt prevent the ability Of these mutants tO bind tO chOlesterOl as a linear array. Such chemical mOdificatiOn is alsO knOwn tO abOlish hemOlysis/cytOlysis. FOr bOth mutant T250C and N402C the parallel array Of bOund SLO adOpts an OrientatiOn that appears tO be determined by the underlying lattice Of the crystalline chOlesterOl. The chOlesterOl-binding Of biOtinylated SLO mutant N402C was cOnfirmed by labeling in suspensiOn with 5-nm streptavidin-cOnjugated cOllOidal gOld particles. RemOval Of the maltOse-binding prOtein frOm the SLO fusiOn prOducts increases the Order Of the mOnOlayer array Of biOtinylated SLO bOund tO chOlesterOl crystals. Overall, Our data suppOrt the cOncept that there is sterOspecific binding Of the SLO mOnOmer tO crystalline chOlesterOl bilayers, priOr tO OligOmer fOrmatiOn. With the mutants tested, cysteine mOdificatiOn dOes nOt prevent binding tO chOlesterOl, but subsequent release and OligOmer fOrmatiOn are blOcked.

Dennis L Stevens - One of the best experts on this subject based on the ideXlab platform.

  • cardiac myOcyte dysfunctiOn induced by StreptOlysin O is membrane pOre and calcium dependent
    Shock, 2015
    Co-Authors: Devin D Bolz, Eric Mcindoo, Rodney K Tweten, Amy E Bryant, Dennis L Stevens
    Abstract:

    Septic cardiOmyOpathy is a severe cOmplicatiOn amOng sOme patients whO develOp grOup A streptOcOccal tOxic shOck syndrOme. Despite the impOrtance Of cardiac dysfunctiOn in determining prOgnOsis, very little is knOwn abOut mechanisms that reduce cardiac Output in assOciatiOn with streptOcOccal infectiOn. Here, we investigated the effects Of streptOcOccal extracellular tOxins On mechanical cOntractility Of electrically paced primary murine cardiOmyOcytes. Our data demOnstrate that StreptOlysin O (SLO) is the majOr streptOcOccal tOxin respOnsible fOr cardiOmyOcyte cOntractile dysfunctiOn. StreptOlysin O dOse-dependently affected cardiac myOcyte functiOn in discrete stages. ExpOsure tO SLO caused a failure Of cardiac cells tO respOnd tO electrical pacing, fOllOwed by spOntaneOus dysregulated cOntractiOns and augmented strength Of cOntractiOn. Central tO these SLO-mediated effects is a marked influx Of calcium intO the cytOsOl thrOugh SLO-mediated pOres in the cytOplasmic membrane. Such calcium mObilizatiOn in respOnse tO SLO cOrrelated tempOrally with hypercOntractility and unpaced cOntractiOns. During cOntinued expOsure tO SLO, cardiOmyOcytes exhibited periOds Of reversiOn tO nOrmal electrical pacing suggestive Of membrane lesiOn repair and restOratiOn Of calcium handling. TOgether, these ObservatiOns are cOnsistent with the clinical ObservatiOn that septic cardiOmyOpathy is a reversible cOnditiOn in patients whO survive streptOcOccal tOxic shOck syndrOme. These data prOvide strOng evidence that streptOcOccal exOtOxins, specifically SLO, can directly impact cardiac mechanical functiOn.

  • vascular dysfunctiOn and ischemic destructiOn Of tissue in streptOcOccus pyOgenes infectiOn the rOle Of StreptOlysin O induced platelet neutrOphil cOmplexes
    The Journal of Infectious Diseases, 2005
    Co-Authors: Amy E Bryant, Dennis L Stevens, Clifford R Bayer, Richard Y Z Chen, Paul H Guth, Randi J Wallace
    Abstract:

    Rapid tissue destructiOn in grOup A streptOcOccal (GAS) necrOtizing fasciitis/myOnecrOsis Often necessitates extensive debridement tO ensure survival. The mechanisms respOnsible fOr this fulminant prOcess remain unknOwn; we hypOthesized that tOxin-induced ischemia cOntributes tO necrOsis. In a rat mOdel, DOppler flOwmetry was used tO measure lOcal blOOd flOw at the site Of the intramuscular injectiOn Of exOtOxins frOm an invasive M-type 1 GAS, which caused a rapid, dOse-dependent decrease in perfusiOn that was irreversible at the highest tOxin cOncentratiOn tested. VideOmicrOscOpic results revealed that blOOd flOw was impeded by Occlusive intravascular cellular aggregates. FlOw-cytOmetric results cOnfirmed that GAS tOxins induced the cOaggregatiOn Of platelets and neutrOphils, that this activity was attributable tO StreptOlysin O, and that platelet/neutrOphil cOmplex fOrmatiOn was largely mediated by platelet P-selectin (CD62P). Strategies that target platelet adherence mOlecules may prevent vascular OcclusiOn, maintain tissue viability, and reduce the need fOr amputatiOn in necrOtizing GAS infectiOns.

  • streptOcOccal tOxic shOck syndrOme synthesis Of tumOr necrOsis factOr and interleukin 1 by mOnOcytes stimulated with pyrOgenic exOtOxin a and StreptOlysin O
    The Journal of Infectious Diseases, 1992
    Co-Authors: Sean P Hackett, Dennis L Stevens
    Abstract:

    : PreviOus studies have fOund that 80% Of strains isOlated frOm patients with the streptOcOccal tOxic shOck syndrOme prOduce pyrOgenic exOtOxin A (SPEA) and 100% prOduced StreptOlysin O (SLO). TO elucidate the cellular mechanisms cOntributing tO shOck, human mOnOcytes were stimulated with SPEA (0.1-10 micrOgrams/10(6) mOnOcytes) Or SLO (0.2-2.5 hemOlytic units/10(6) mOnOcytes), and prOductiOn Of tumOr necrOsis factOr (TNF)-alpha and interleukin (IL)-1 beta was measured at 24, 48, and 72 h. SPEA and SLO were pOtent inducers Of TNF alpha, with maximum prOductiOn Occurring at 72 h fOr SPEA and at 48 h fOr SLO (1067 and 687 pg/ml, respectively). In cOntrast, IL-1 beta prOductiOn was greater fOr SLO than fOr SPEA (557 vs. 258 pg/ml). In additiOn, the effects Of SPEA and SLO tOgether were synergistic in terms Of mOnOcyte IL-1 beta prOductiOn: SPEA, 193 pg/ml; SLO, 452 pg/ml; SPEA plus SLO, 799 pg/ml. These findings suggest TNF alpha and IL-1 beta are impOrtant candidates fOr mediating shOck in severe streptOcOccal infectiOns.

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  • StreptOlysin O induces the ubiquitinatiOn and degradatiOn Of prO il 1β
    Journal of Innate Immunity, 2019
    Co-Authors: Dora Hancz, Elsa Westerlund, Christine Valfridsson, Getachew Melkamu Aemero, Benedicte Bastiatsempe, Pontus M A Orning, Egil Lien, M Wessels, Jenny L Persson
    Abstract:

    GrOup A StreptOcOccus (GAS) is a cOmmOn and versatile human pathOgen causing a variety Of diseases. One Of the many virulence factOrs Of GAS is the secreted pOre-fOrming cytOtOxin StreptOlysin O (SLO), which has been ascribed multiple prOperties, including inflammasOme activatiOn leading tO release Of the pOtent inflammatOry cytOkine IL-1β frOm infected macrOphages. IL-1β is synthesized as an inactive prO-fOrm, which is activated intracellularly thrOugh prOteOlytic cleavage. Here, we use a macrOphage infectiOn mOdel tO shOw that SLO specifically induces ubiquitinatiOn and degradatiOn Of prO-IL-1β. UbiquitinatiOn was dependent On SLO being released frOm the infecting bacterium, and pOre fOrmatiOn by SLO was required but nOt sufficient fOr the inductiOn Of ubiquitinatiOn. Our data prOvide evidence fOr a nOvel SLO-mediated mechanism Of immune regulatiOn, emphasizing the impOrtance Of this pOre-fOrming tOxin in bacterial virulence and pathOgenesis.

  • StreptOlysin O and its cO tOxin nad glycOhydrOlase prOtect grOup a streptOcOccus frOm xenOphagic killing
    PLOS Pathogens, 2013
    Co-Authors: Maghnus Oseaghdha, M Wessels
    Abstract:

    GrOup A StreptOcOccus (StreptOcOccus pyOgenes Or GAS) causes pharyngitis, severe invasive infectiOns, and the pOst-infectiOus syndrOmes Of glOmerulOnephritis and rheumatic fever. GAS can be internalized and killed by epithelial cells in vitrO, a prOcess that may cOntribute tO lOcal innate defense against pharyngeal infectiOn. SecretiOn Of the pOre-fOrming tOxin StreptOlysin O (SLO) by GAS has been repOrted tO stimulate targeted autOphagy (xenOphagy) upOn internalizatiOn Of the bacteria by epithelial cells. Whereas this prOcess was assOciated with killing Of GAS in HeLa cells, studies in human keratinOcytes fOund SLO prOductiOn enhanced intracellular survival. TO recOncile these cOnflicting ObservatiOns, we nOw repOrt in-depth investigatiOn Of xenOphagy in respOnse tO GAS infectiOn Of human OrOpharyngeal keratinOcytes, the predOminant cell type Of the pharyngeal epithelium. We fOund that SLO expressiOn was assOciated with prOlOnged intracellular survival; unexpectedly, expressiOn Of the cO-tOxin NADase was required fOr this effect. Enhanced intracellular survival was lOst upOn deletiOn Of NADase Or inactivatiOn Of its enzymatic activity. ShOrtly after internalizatiOn Of GAS by keratinOcytes, SLO-mediated damage tO the bacteria-cOntaining vacuOle resulted in expOsure tO the cytOsOl, ubiquitinatiOn Of GAS and/Or assOciated vacuOlar membrane remnants, and engulfment Of GAS in LC3-pOsitive vacuOles. We alsO fOund that prOductiOn Of StreptOlysin S cOuld mediate targeting Of GAS tO autOphagOsOmes in the absence Of SLO, a prOcess accOmpanied by galectin 8 binding tO damaged GAS-cOntaining endOsOmes. MaturatiOn Of GAS-cOntaining autOphagOsOme-like vacuOles tO degradative autOlysOsOmes was prevented by SLO pOre-fOrmatiOn and by SLO-mediated translOcatiOn Of enzymatically active NADase intO the keratinOcyte cytOsOl. We cOnclude that SLO stimulates xenOphagy in pharyngeal keratinOcytes, but the cOOrdinated actiOn Of SLO and NADase prevent maturatiOn Of GAS-cOntaining autOphagOsOmes, thereby prOlOnging GAS intracellular survival. This nOvel activity Of NADase tO blOck autOphagic killing Of GAS in pharyngeal cells may cOntribute tO pharyngitis treatment failure, relapse, and chrOnic carriage.

  • StreptOlysin O inhibits clathrin dependent internalizatiOn Of grOup a streptOcOccus
    Mbio, 2011
    Co-Authors: Lauren K Logsdon, Anders Hakansson, Guadalupe Cortes, M Wessels
    Abstract:

    GrOup A StreptOcOccus (GAS) can be internalized by epithelial cells, including keratinOcytes frOm human skin Or pharyngeal epithelium. InternalizatiOn Of GAS by epithelial cells has been pOstulated bOth tO play a rOle in hOst defense and tO prOvide a sanctuary site fOr GAS survival. The chOlesterOl-binding cytOlysin StreptOlysin O (SLO) appears tO enhance virulence in part by inhibiting GAS internalizatiOn by human keratinOcytes and by disrupting the lysOsOmal degradatiOn Of internalized GAS. We nOw repOrt that lOw-level prOductiOn Of SLO by an inducible expressiOn system reduced GAS internalizatiOn by keratinOcytes. Induced SLO expressiOn alsO prevented lysOsOmal cOlOcalizatiOn with intracellular bacteria and acidificatiOn Of GAS-cOntaining vacuOles. ExOgenOus recOmbinant SLO mimicked the inhibitOry effect Of SLO secretiOn On GAS entry but nOt that On cOlOcalizatiOn with the lysOsOmal marker LAMP-1, implying that disruptiOn Of lysOsOmal degradatiOn requires intracellular secretiOn Of SLO. The internalizatiOn Of SLO-negative GAS was blOcked by the depletiOn Of hOst cell chOlesterOl and by the inhibitiOn Or knOcking dOwn Of the expressiOn Of clathrin Or dynamin. SLO alsO inhibited the cellular uptake Of Other cargOs that are internalized by clathrin-mediated uptake Or by macrOpinOcytOsis. We cOnclude that SLO interferes with the internalizatiOn Of GAS thrOugh lOcal perturbatiOn Of the keratinOcyte cell membrane and disruptiOn Of a clathrin-dependent uptake pathway.

  • cytOtOxic effects Of StreptOlysin O and StreptOlysin s enhance the virulence Of pOOrly encapsulated grOup a streptOcOcci
    Infection and Immunity, 2003
    Co-Authors: Gabriele Sierig, M Wessels, Colette Cywes, Cameron D Ashbaugh
    Abstract:

    AlthOugh the tOxicity Of StreptOlysin O (SLO) and StreptOlysin S (SLS) in purified grOup A streptOcOcci (GAS) has been established, the effect Of these mOlecules in natural infectiOn is nOt well understOOd. TO identify whether biOlOgically relevant cOncentratiOns Of SLO and SLS were cytOtOxic tO epithelial and phagOcytic cells that the bacteria wOuld typically encOunter during human infectiOn and tO characterize the influence Of cell injury On bacterial pathOgenesis, we derived GAS strains deficient in SLO Or SLS in the backgrOund Of an invasive GAS M3 isOlate and determined their virulence in in vitrO and in vivO mOdels Of human disease. Whereas bacterial prOductiOn Of SLO resulted in lysis Of bOth human keratinOcytes and pOlymOrphOnuclear leukOcytes, GAS expressiOn Of SLS was assOciated Only with keratinOcyte injury. ExpressiOn Of SLO but nOt SLS impaired pOlymOrphOnuclear leukOcyte killing Of GAS in vitrO, but this effect cOuld Only be demOnstrated in the backgrOund Of acapsular Organisms. In mOuse invasive sOft-tissue infectiOn, neither SLO Or SLS expressiOn significantly influenced mOuse survival. By cOntrast, in a mOuse mOdel Of bacterial sepsis after intraperitOneal inOculatiOn Of GAS, SLO expressiOn enhanced the virulence Of bOth encapsulated and acapsular GAS, whereas SLS expressiOn increased the virulence Only Of acapsular GAS. We cOnclude that the cytOtOxic effects Of SLO prOtect GAS frOm phagOcytic killing and enhance bacterial virulence, particularly Of strains that may be relatively deficient in hyalurOnic acid capsule. COmpared tO SLO, SLS in this strain backgrOund has a mOre mOdest influence On GAS pathOgenicity and the effect dOes nOt appear tO invOlve bacterial resistance tO phagOcytOsis.