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Shaun W Lee - One of the best experts on this subject based on the ideXlab platform.

  • live cell microScopy and flow cytometry to Study StreptolySin S mediated erythrocyte hemolySiS
    Methods of Molecular Biology, 2020
    Co-Authors: Dustin L. Higashi, Charles R. Tessier, Shaun W Lee
    Abstract:

    The ability to induce hemolySiS, the rupturing of erythrocyteS with the conSequent releaSe of their intracellular contentS, iS a phenotypic hallmark of a number of microbial toxinS. StreptococcuS pyogeneS or Group A StreptococcuS (GAS) iS a human pathogen reSponSible for a wide range of diSeaSeS from mild pharyngitiS to Severe conditionS Such aS toxic Shock Syndrome. GAS produceS a powerful hemolytic toxin called StreptolySin S (SLS). Herein, we deScribe a procedure for the preparation of SLS toxin and the uSe of two complementary approacheS, live microScopy and flow cytometry, to Study the effectS of the SLS toxin on erythrocyteS. In addition to providing inSightS into SLS-mediated hemolySiS, theSe aSSayS have the potential to be modified for the Study of other hemolytic toxinS and compoundS.

  • Data_Sheet_1_Neutralization of StreptolySin S-Dependent and Independent Inflammatory Cytokine IL-1β Activity ReduceS Pathology During Early Group A Streptococcal Skin Infection.PDF
    2018
    Co-Authors: Rebecca A Flaherty, Deborah L. Donahue, Victoria A. Ploplis, Francis J. Castellino, Katelyn E Carothers, Jessica N Ross, Shaun W Lee
    Abstract:

    The bacterial pathogen Group A StreptococcuS (GAS) haS been Shown to induce a variety of human diSeaSeS ranging in Severity from pharyngitiS to toxic Shock Syndrome and necrotizing faSciitiS. GAS produceS a powerful peptide toxin known aS StreptolySin S (SLS). Though long recognized aS a potent cytolySin, recent evidence from our lab haS Shown that SLS-dependent cytotoxicity iS mediated through activation of the pro-inflammatory mediatorS p38 MAPK and NFκB. TheSe findingS led uS to hypotheSize that activation of p38 MAPK and NFκB Signaling drive the production of pro-inflammatory cytokineS which, in turn, Serve aS poSitive feedback SignalS to initiate cytotoxicity in infected hoSt cellS. To addreSS thiS hypotheSiS, we utilized a cytokine array to characterize the SLS-dependent pro-inflammatory cytokine reSponSe to GAS infection in human keratinocyteS. From theSe StudieS, IL-1β waS found to be markedly upregulated in the preSence of SLS, and further inveStigation revealed that thiS cytokine contributeS to cytotoxicity in human keratinocyteS during infection. SubcutaneouS infection StudieS were performed in mice to addreSS the phySiological impact of increaSed IL-1β production. TheSe StudieS demonStrated that IL-1β iS produced during GAS Skin infection in an SLS-dependent manner. Furthermore, inhibition of thiS cytokine and the upStream kinaSeS and other Signaling mediatorS that drive itS production reduced SLS-mediated leSion formation early in the infection proceSS. Together, our findingS indicate that pharmacological inhibition of thiS inflammatory axiS holdS promiSe aS a therapeutic Strategy to reduce tiSSue deStruction during Severe invaSive Group A Streptococcal infectionS.

  • Neutralization of StreptolySin S-Dependent and Independent Inflammatory Cytokine IL-1β Activity ReduceS Pathology During Early Group A Streptococcal Skin Infection
    Frontiers Media S.A., 2018
    Co-Authors: Deborah L. Donahue, Victoria A. Ploplis, Francis J. Castellino, Katelyn E Carothers, Jessica N Ross, Rebecca A Flaherty, Shaun W Lee
    Abstract:

    The bacterial pathogen Group A StreptococcuS (GAS) haS been Shown to induce a variety of human diSeaSeS ranging in Severity from pharyngitiS to toxic Shock Syndrome and necrotizing faSciitiS. GAS produceS a powerful peptide toxin known aS StreptolySin S (SLS). Though long recognized aS a potent cytolySin, recent evidence from our lab haS Shown that SLS-dependent cytotoxicity iS mediated through activation of the pro-inflammatory mediatorS p38 MAPK and NFκB. TheSe findingS led uS to hypotheSize that activation of p38 MAPK and NFκB Signaling drive the production of pro-inflammatory cytokineS which, in turn, Serve aS poSitive feedback SignalS to initiate cytotoxicity in infected hoSt cellS. To addreSS thiS hypotheSiS, we utilized a cytokine array to characterize the SLS-dependent pro-inflammatory cytokine reSponSe to GAS infection in human keratinocyteS. From theSe StudieS, IL-1β waS found to be markedly upregulated in the preSence of SLS, and further inveStigation revealed that thiS cytokine contributeS to cytotoxicity in human keratinocyteS during infection. SubcutaneouS infection StudieS were performed in mice to addreSS the phySiological impact of increaSed IL-1β production. TheSe StudieS demonStrated that IL-1β iS produced during GAS Skin infection in an SLS-dependent manner. Furthermore, inhibition of thiS cytokine and the upStream kinaSeS and other Signaling mediatorS that drive itS production reduced SLS-mediated leSion formation early in the infection proceSS. Together, our findingS indicate that pharmacological inhibition of thiS inflammatory axiS holdS promiSe aS a therapeutic Strategy to reduce tiSSue deStruction during Severe invaSive Group A Streptococcal infectionS

  • StreptolySin S promoteS programmed cell death and enhanceS inflammatory Signaling in epithelial keratinocyteS during group a StreptococcuS infection
    Infection and Immunity, 2015
    Co-Authors: Rebecca A Flaherty, Dustin L. Higashi, Claudia J Park, Jessica M Puricelli, Shaun W Lee
    Abstract:

    StreptococcuS pyogeneS, or group A StreptococcuS (GAS), iS a pathogen that cauSeS a multitude of human diSeaSeS from pharyngitiS to Severe infectionS Such aS toxic Shock Syndrome and necrotizing faSciitiS. One of the primary virulence factorS produced by GAS iS the peptide toxin StreptolySin S (SLS). In addition to itS well-recognized role aS a cytolySin, recent evidence haS indicated that SLS may influence hoSt cell Signaling pathwayS at Sublytic concentrationS during infection. We employed an antibody array-baSed approach to comprehenSively identify global hoSt cell changeS in human epithelial keratinocyteS in reSponSe to the SLS toxin. We identified key SLS-dependent hoSt reSponSeS, including the initiation of Specific programmed cell death and inflammatory caScadeS with concomitant downregulation of Akt-mediated cytoprotection. Significant Signaling reSponSeS identified by our array analySiS were confirmed uSing biochemical and protein identification methodS. To further demonStrate that the obServed SLS-dependent hoSt Signaling changeS were mediated primarily by the Secreted toxin, we deSigned a TranSwell infection SyStem in which direct bacterial attachment to hoSt cellS waS prevented, while Secreted factorS were allowed acceSS to hoSt cellS. The reSultS uSing thiS approach were conSiStent with our direct infection StudieS and reveal that SLS iS a bacterial toxin that doeS not require bacterial attachment to hoSt cellS for activity. In light of theSe findingS, we propoSe that the production of SLS by GAS during Skin infection promoteS invaSive outcomeS by triggering programmed cell death and inflammatory caScadeS in hoSt cellS to breach the keratinocyte barrier for diSSemination into deeper tiSSueS.

  • hiv proteaSe inhibitorS block StreptolySin S production
    ACS Chemical Biology, 2015
    Co-Authors: Tucker Maxson, Andrew L Markley, Shaun W Lee, Evelyn M Molloy, Caitlin D Deane, Courtney L Cox, Douglas A Mitchell
    Abstract:

    StreptolySin S (SLS) iS a poSt-tranSlationally modified peptide cytolySin that iS produced by the human pathogen StreptococcuS pyogeneS. SLS belongS to a large family of azole-containing natural productS that are bioSyntheSized via an evolutionarily conServed pathway. SLS iS an important virulence factor during S. pyogeneS infectionS, but deSpite an extenSive hiStory of Study, further inveStigationS are needed to clarify Several StepS of itS bioSyntheSiS. To thiS end, chemical inhibitorS of SLS bioSyntheSiS would be valuable toolS to interrogate the variouS maturation StepS of both SLS and bioSynthetically related natural productS. Such chemical inhibitorS could alSo potentially Serve aS antivirulence therapeuticS, which in theory may alleviate the Spread of antibiotic reSiStance. In thiS work, we demonStrate that FDA-approved HIV proteaSe inhibitorS, eSpecially nelfinavir, block a key proteolytic proceSSing Step during SLS production. ThiS inhibition waS demonStrated in live S. pyogeneS cellS and through...

Douglas A Mitchell - One of the best experts on this subject based on the ideXlab platform.

  • TOMM D ML Tree
    2016
    Co-Authors: Evelyn M Molloy, Tucker Maxson, Courtney L Cox, Nicole A Ethridge, Gabriele Margos, Volker Fingerle, Sherwood R. Casajens, Douglas A Mitchell
    Abstract:

    A maximum-likelihood tree of a repreSentative Sample of TOMMS baSed on the D protein from each cluSter. CladeS are color-coded baSed on the predicted claSS of TOMM natural product. NHLP, nitrile hydrataSe leader peptide; NHLP-Burk, NHLP from Burkholderia; PZN, plantazolicin; Balh, uncharacterized TOMM from BacilluS Sp. Al Hakam; McB, microcin B from Gammaproteobacteria. SLS, StreptolySin S; Bor TOMM, putative SLS-like cytolySin from BbSl

  • identification of the minimal cytolytic unit for StreptolySin S and an expanSion of the toxin family
    BMC Microbiology, 2015
    Co-Authors: Evelyn M Molloy, Tucker Maxson, Sherwood R Casjens, Nicole A Ethridge, Gabriele Margos, Volker Fingerle, Douglas A Mitchell
    Abstract:

    Background StreptolySin S (SLS) iS a cytolytic virulence factor produced by the human pathogen StreptococcuS pyogeneS and other StreptococcuS SpecieS. Related “SLS-like” toxinS have been characterized in Select StrainS of CloStridium and LiSteria, with homologouS cluSterS bioinformatically identified in a variety of other SpecieS. SLS iS a member of the thiazole/oxazole-modified microcin (TOMM) family of natural productS. The Structure of SLS haS yet to be deciphered and many queStionS remain regarding itS Structure-activity relationShipS.

  • identification of the minimal cytolytic unit for StreptolySin S and an expanSion of the toxin family
    BMC Microbiology, 2015
    Co-Authors: Evelyn M Molloy, Tucker Maxson, Courtney L Cox, Sherwood R Casjens, Nicole A Ethridge, Gabriele Margos, Volker Fingerle, Douglas A Mitchell
    Abstract:

    StreptolySin S (SLS) iS a cytolytic virulence factor produced by the human pathogen StreptococcuS pyogeneS and other StreptococcuS SpecieS. Related “SLS-like” toxinS have been characterized in Select StrainS of CloStridium and LiSteria, with homologouS cluSterS bioinformatically identified in a variety of other SpecieS. SLS iS a member of the thiazole/oxazole-modified microcin (TOMM) family of natural productS. The Structure of SLS haS yet to be deciphered and many queStionS remain regarding itS Structure-activity relationShipS. In thiS work, we aSSeSSed the hemolytic activity of a SerieS of C-terminally truncated SLS peptideS expreSSed in SLS-deficient S. pyogeneS. Our data indicate that while the N-terminal poly-heterocyclizable (NPH) region of SLS SubStantially contributeS to itS bioactivity, the variable C-terminal region of the toxin iS largely diSpenSable. Through genome mining we identified additional SLS-like cluSterS in diverSe FirmicuteS, SpirochaeteS and Actinobacteria. Among the Spirochaete cluSterS, naturally truncated SLS-like precurSorS were found in the genomeS of three Lyme diSeaSe-cauSing Borrelia burgdorferi SenSu lato (BbSl) StrainS. Although unable to reStore hemolySiS in SLS-deficient S. pyogeneS, a BbSl SLS-like precurSor peptide waS converted to a cytolySin uSing purified SLS bioSynthetic enzymeS. A PCR-baSed Screen demonStrated that SLS-like cluSterS are SubStantially more prevalent in BbSl than inferred from publicly available genome SequenceS. The mutageneSiS data deScribed herein indicate that the minimal cytolytic unit of SLS encompaSSeS the NPH region of the core peptide. IntereStingly, thiS region iS found in all characterized TOMM cytolySinS, aS well aS the novel putative TOMM cytolySinS we diScovered. We propoSe that thiS conServed region repreSentS the defining feature of the SLS-like TOMM family. We demonStrate the cytolytic potential of a BbSl SLS-like precurSor peptide, which haS a core region of Similar length to the SLS minimal cytolytic unit, when modified with purified SLS bioSynthetic enzymeS. AS Such, we Speculate that Some Borrelia have the potential to produce a TOMM cytolySin, although the biological Significance of thiS finding remainS to be determined. In addition to providing new inSight into the Structure-activity relationShipS of SLS, thiS Study greatly expandS the cytolySin group of TOMMS.

  • hiv proteaSe inhibitorS block StreptolySin S production
    ACS Chemical Biology, 2015
    Co-Authors: Tucker Maxson, Andrew L Markley, Shaun W Lee, Evelyn M Molloy, Caitlin D Deane, Courtney L Cox, Douglas A Mitchell
    Abstract:

    StreptolySin S (SLS) iS a poSt-tranSlationally modified peptide cytolySin that iS produced by the human pathogen StreptococcuS pyogeneS. SLS belongS to a large family of azole-containing natural productS that are bioSyntheSized via an evolutionarily conServed pathway. SLS iS an important virulence factor during S. pyogeneS infectionS, but deSpite an extenSive hiStory of Study, further inveStigationS are needed to clarify Several StepS of itS bioSyntheSiS. To thiS end, chemical inhibitorS of SLS bioSyntheSiS would be valuable toolS to interrogate the variouS maturation StepS of both SLS and bioSynthetically related natural productS. Such chemical inhibitorS could alSo potentially Serve aS antivirulence therapeuticS, which in theory may alleviate the Spread of antibiotic reSiStance. In thiS work, we demonStrate that FDA-approved HIV proteaSe inhibitorS, eSpecially nelfinavir, block a key proteolytic proceSSing Step during SLS production. ThiS inhibition waS demonStrated in live S. pyogeneS cellS and through...

  • StreptolySin S like virulence factorS the continuing Saga
    Nature Reviews Microbiology, 2011
    Co-Authors: Evelyn M Molloy, Paul D Cotter, Colin Hill, Douglas A Mitchell, Paul R Ross
    Abstract:

    StreptolySin S (SLS) iS a potent cytolytic toxin and virulence factor that iS produced by nearly all StreptococcuS pyogeneS StrainS. DeSpite a 100-year hiStory of reSearch on thiS toxin, it haS only recently been eStabliShed that SLS iS juSt one of an extended family of poSt-tranSlationally modified virulence factorS (the SLS-like peptideS) that are produced by Some Streptococci and other Gram-poSitive pathogenS, Such aS LiSteria monocytogeneS and CloStridium botulinum. In thiS Review, we deScribe the identification, geneticS, biochemiStry and variouS functionS of SLS. We alSo diScuSS the Shared featureS of the virulence-aSSociated SLS-like peptideS, aS well aS their place within the rapidly expanding family of thiazole/oxazole-modified microcinS (TOMMS).

Victor Nizet - One of the best experts on this subject based on the ideXlab platform.

  • cloStridiolySin S a poSt tranSlationally modified biotoxin from cloStridium botulinum
    Journal of Biological Chemistry, 2010
    Co-Authors: David Gonzalez, Victor Nizet, Andrew L Markley, Shaun W Lee, Douglas A Mitchell, Jack E Dixon, Mary E Hensler, Samira Dahesh, Nuno Bandeira, Pieter C Dorrestein
    Abstract:

    Through elaboration of itS botulinum toxinS, CloStridium botulinum produceS clinical SyndromeS of infant botuliSm, wound botuliSm, and other invaSive infectionS. USing comparative genomic analySiS, an orphan nine-gene cluSter waS identified in C. botulinum and the related foodborne pathogen CloStridium SporogeneS that reSembled the bioSynthetic machinery for StreptolySin S, a key virulence factor from group A StreptococcuS reSponSible for itS hallmark β-hemolytic phenotype. Genetic complementation, in vitro reconStitution, maSS Spectral analySiS, and plaSmid intergrational mutageneSiS demonStrate that the StreptolySin S-like gene cluSter from CloStridium Sp. iS reSponSible for the biogeneSiS of a novel poSt-tranSlationally modified hemolytic toxin, cloStridiolySin S.

  • Structural and functional diSSection of the heterocyclic peptide cytotoxin StreptolySin S
    Journal of Biological Chemistry, 2009
    Co-Authors: Douglas A Mitchell, Victor Nizet, Andrew L Markley, Shaun W Lee, Morgan A Pence, Joyce D Limm, Jack E Dixon
    Abstract:

    The human pathogen StreptococcuS pyogeneS SecreteS a highly cytolytic toxin known aS StreptolySin S (SLS). SLS iS a key virulence determinant and reSponSible for the β-hemolytic phenotype of theSe bacteria. DeSpite over a century of reSearch, the chemical Structure of SLS remainS unknown. Recent experimentS have revealed that SLS iS generated from an inactive precurSor peptide that undergoeS extenSive poSt-tranSlational modification to an active form. In thiS work, we addreSS outStanding queStionS regarding the SLS bioSynthetic proceSS, elucidating the featureS of SubStrate recognition and SiteS of poSttranSlational modification to the SLS precurSor peptide. Further, we exploit theSe findingS to guide the deSign of artificial cytolytic toxinS that are recognized by the SLS bioSynthetic enzymeS and otherS that are intrinSically cytolytic. ThiS new Structural information haS ramificationS for future antimicrobial therapieS.

  • diScovery of a widely diStributed toxin bioSynthetic gene cluSter
    Proceedings of the National Academy of Sciences of the United States of America, 2008
    Co-Authors: Shaun W Lee, Victor Nizet, Andrew L Markley, Douglas A Mitchell, Jack E Dixon, David Gonzalez, Mary E Hensler, Pieter C Dorrestein, Aaron Wohlrab
    Abstract:

    BacteriocinS repreSent a large family of riboSomally produced peptide antibioticS. Here we deScribe the diScovery of a widely conServed bioSynthetic gene cluSter for the SyntheSiS of thiazole and oxazole heterocycleS on riboSomally produced peptideS. TheSe cluSterS encode a toxin precurSor and all neceSSary proteinS for toxin maturation and export. USing the toxin precurSor peptide and heterocycle-forming SynthetaSe proteinS from the human pathogen StreptococcuS pyogeneS, we demonStrate the in vitro reconStitution of StreptolySin S activity. We provide evidence that the SynthetaSe enzymeS, aS predicted from our bioinformaticS analySiS, introduce heterocycleS onto precurSor peptideS, thereby providing molecular inSight into the chemical Structure of StreptolySin S. Furthermore, our StudieS reveal that the SynthetaSe exhibitS relaxed SubStrate Specificity and modifieS toxin precurSorS from both related and diStant SpecieS. Given our findingS, it iS likely that the diScovery of Similar peptidic toxinS will rapidly expand to exiSting and emerging genomeS.

  • StreptococcuS iniae β hemolySin StreptolySin S iS a virulence factor in fiSh infection
    Diseases of Aquatic Organisms, 2007
    Co-Authors: Jeffrey B Locke, Victor Nizet, Kelly M Colvin, Nissi Varki, Mike R Vicknair, John T Buchanan
    Abstract:

    StreptococcuS iniae iS a leading pathogen of intenSive aquaculture operationS world- wide, although underStanding of virulence mechaniSmS of thiS pathogen in fiSh iS lacking. S. iniae poSSeSSeS a homolog of StreptolySin S (SLS), a Secreted, pore-forming cytotoxin that iS a proven viru- lence factor in the human pathogen S. pyogeneS. Here we uSed allelic exchange mutageneSiS of the Structural gene for the S. iniae SLS precurSor (SagA) to examine the role of SLS in S. iniae pathogenic- ity uSing in vitro and in vivo modelS. The iSogenic ΔSagA mutant waS leSS cytotoxic to fiSh blood cellS and cultured epithelial cellS, but comparable to wild-type (WT) S. iniae in adherence/invaSion of epithelial cell monolayerS and reSiSting phagocytic killing by fiSh whole blood or macrophageS. In a hybrid Striped baSS infection model, loSS of SLS production led to marked virulence attenuation, aS injection of the ΔSagA mutant at 1000× the WT lethal doSe (LD80) produced only 10% mortality. The neutralization of SLS could repreSent a novel Strategy for control of S. iniae infection in aquaculture.

  • genetic characterization and virulence role of the ralp3 lSa locuS upStream of the StreptolySin S operon in invaSive m1t1 group a StreptococcuS
    Journal of Bacteriology, 2007
    Co-Authors: Laura A Kwinn, Malak Kotb, Arya Khosravi, Ramy K Aziz, Anjuli M Timmer, Kelly S Doran, Victor Nizet
    Abstract:

    Group A StreptococcuS (GAS) iS a leading human pathogen aSSociated with a wide Spectrum of mucoSal and invaSive infectionS. GAS expreSSeS a large number of virulence determinantS whoSe expreSSion iS under the control of Several tranScriptional regulatory networkS. Here we performed the firSt mutational analySiS of a genetic locuS immediately upStream of the StreptolySin S bioSynthetic operon in Several GAS genome SequenceS, including that of the M1T1 Serotype, the leading iSolateS aSSociated with SeriouS invaSive diSeaSe. The locuS conSiStS of a predicted RofA-like Stand-alone tranScriptional regulator (RALP3) and the largeSt open reading frame in the GAS genome, encoding a predicted LPXSG motif cell wall-anchored protein we have named LSA (for "large Surface-anchored" protein). Comparative reverSe tranScription-PCR analySiS of wild-type M1T1 GAS and an iSogenic RALP3-deficient mutant identifieS RALP3 aS a global tranScriptional regulator affecting expreSSion of numerouS virulence factor geneS, including thoSe for Strong repreSSion of the hyaluronic acid capSule and cySteine proteaSe production. RALP3 contributed to GAS epithelial cell invaSion and bloodStream Survival. LSA waS found to be under negative regulation by RALP3 and to influence GAS-epithelial cell interactionS and GAS antimicrobial peptide SenSitivity. ISogenic M1T1 GAS mutantS lacking either RALP3 or LSA were attenuated in a murine model of SyStemic infection, indicating that thiS locuS playS a role in the virulence potential of the organiSm.

Evelyn M Molloy - One of the best experts on this subject based on the ideXlab platform.

  • TOMM D ML Tree
    2016
    Co-Authors: Evelyn M Molloy, Tucker Maxson, Courtney L Cox, Nicole A Ethridge, Gabriele Margos, Volker Fingerle, Sherwood R. Casajens, Douglas A Mitchell
    Abstract:

    A maximum-likelihood tree of a repreSentative Sample of TOMMS baSed on the D protein from each cluSter. CladeS are color-coded baSed on the predicted claSS of TOMM natural product. NHLP, nitrile hydrataSe leader peptide; NHLP-Burk, NHLP from Burkholderia; PZN, plantazolicin; Balh, uncharacterized TOMM from BacilluS Sp. Al Hakam; McB, microcin B from Gammaproteobacteria. SLS, StreptolySin S; Bor TOMM, putative SLS-like cytolySin from BbSl

  • identification of the minimal cytolytic unit for StreptolySin S and an expanSion of the toxin family
    BMC Microbiology, 2015
    Co-Authors: Evelyn M Molloy, Tucker Maxson, Sherwood R Casjens, Nicole A Ethridge, Gabriele Margos, Volker Fingerle, Douglas A Mitchell
    Abstract:

    Background StreptolySin S (SLS) iS a cytolytic virulence factor produced by the human pathogen StreptococcuS pyogeneS and other StreptococcuS SpecieS. Related “SLS-like” toxinS have been characterized in Select StrainS of CloStridium and LiSteria, with homologouS cluSterS bioinformatically identified in a variety of other SpecieS. SLS iS a member of the thiazole/oxazole-modified microcin (TOMM) family of natural productS. The Structure of SLS haS yet to be deciphered and many queStionS remain regarding itS Structure-activity relationShipS.

  • identification of the minimal cytolytic unit for StreptolySin S and an expanSion of the toxin family
    BMC Microbiology, 2015
    Co-Authors: Evelyn M Molloy, Tucker Maxson, Courtney L Cox, Sherwood R Casjens, Nicole A Ethridge, Gabriele Margos, Volker Fingerle, Douglas A Mitchell
    Abstract:

    StreptolySin S (SLS) iS a cytolytic virulence factor produced by the human pathogen StreptococcuS pyogeneS and other StreptococcuS SpecieS. Related “SLS-like” toxinS have been characterized in Select StrainS of CloStridium and LiSteria, with homologouS cluSterS bioinformatically identified in a variety of other SpecieS. SLS iS a member of the thiazole/oxazole-modified microcin (TOMM) family of natural productS. The Structure of SLS haS yet to be deciphered and many queStionS remain regarding itS Structure-activity relationShipS. In thiS work, we aSSeSSed the hemolytic activity of a SerieS of C-terminally truncated SLS peptideS expreSSed in SLS-deficient S. pyogeneS. Our data indicate that while the N-terminal poly-heterocyclizable (NPH) region of SLS SubStantially contributeS to itS bioactivity, the variable C-terminal region of the toxin iS largely diSpenSable. Through genome mining we identified additional SLS-like cluSterS in diverSe FirmicuteS, SpirochaeteS and Actinobacteria. Among the Spirochaete cluSterS, naturally truncated SLS-like precurSorS were found in the genomeS of three Lyme diSeaSe-cauSing Borrelia burgdorferi SenSu lato (BbSl) StrainS. Although unable to reStore hemolySiS in SLS-deficient S. pyogeneS, a BbSl SLS-like precurSor peptide waS converted to a cytolySin uSing purified SLS bioSynthetic enzymeS. A PCR-baSed Screen demonStrated that SLS-like cluSterS are SubStantially more prevalent in BbSl than inferred from publicly available genome SequenceS. The mutageneSiS data deScribed herein indicate that the minimal cytolytic unit of SLS encompaSSeS the NPH region of the core peptide. IntereStingly, thiS region iS found in all characterized TOMM cytolySinS, aS well aS the novel putative TOMM cytolySinS we diScovered. We propoSe that thiS conServed region repreSentS the defining feature of the SLS-like TOMM family. We demonStrate the cytolytic potential of a BbSl SLS-like precurSor peptide, which haS a core region of Similar length to the SLS minimal cytolytic unit, when modified with purified SLS bioSynthetic enzymeS. AS Such, we Speculate that Some Borrelia have the potential to produce a TOMM cytolySin, although the biological Significance of thiS finding remainS to be determined. In addition to providing new inSight into the Structure-activity relationShipS of SLS, thiS Study greatly expandS the cytolySin group of TOMMS.

  • hiv proteaSe inhibitorS block StreptolySin S production
    ACS Chemical Biology, 2015
    Co-Authors: Tucker Maxson, Andrew L Markley, Shaun W Lee, Evelyn M Molloy, Caitlin D Deane, Courtney L Cox, Douglas A Mitchell
    Abstract:

    StreptolySin S (SLS) iS a poSt-tranSlationally modified peptide cytolySin that iS produced by the human pathogen StreptococcuS pyogeneS. SLS belongS to a large family of azole-containing natural productS that are bioSyntheSized via an evolutionarily conServed pathway. SLS iS an important virulence factor during S. pyogeneS infectionS, but deSpite an extenSive hiStory of Study, further inveStigationS are needed to clarify Several StepS of itS bioSyntheSiS. To thiS end, chemical inhibitorS of SLS bioSyntheSiS would be valuable toolS to interrogate the variouS maturation StepS of both SLS and bioSynthetically related natural productS. Such chemical inhibitorS could alSo potentially Serve aS antivirulence therapeuticS, which in theory may alleviate the Spread of antibiotic reSiStance. In thiS work, we demonStrate that FDA-approved HIV proteaSe inhibitorS, eSpecially nelfinavir, block a key proteolytic proceSSing Step during SLS production. ThiS inhibition waS demonStrated in live S. pyogeneS cellS and through...

  • StreptolySin S like virulence factorS the continuing Saga
    Nature Reviews Microbiology, 2011
    Co-Authors: Evelyn M Molloy, Paul D Cotter, Colin Hill, Douglas A Mitchell, Paul R Ross
    Abstract:

    StreptolySin S (SLS) iS a potent cytolytic toxin and virulence factor that iS produced by nearly all StreptococcuS pyogeneS StrainS. DeSpite a 100-year hiStory of reSearch on thiS toxin, it haS only recently been eStabliShed that SLS iS juSt one of an extended family of poSt-tranSlationally modified virulence factorS (the SLS-like peptideS) that are produced by Some Streptococci and other Gram-poSitive pathogenS, Such aS LiSteria monocytogeneS and CloStridium botulinum. In thiS Review, we deScribe the identification, geneticS, biochemiStry and variouS functionS of SLS. We alSo diScuSS the Shared featureS of the virulence-aSSociated SLS-like peptideS, aS well aS their place within the rapidly expanding family of thiazole/oxazole-modified microcinS (TOMMS).

Donald E Low - One of the best experts on this subject based on the ideXlab platform.

  • identification of a StreptolySin S aSSociated gene cluSter and itS role in the pathogeneSiS of StreptococcuS iniae diSeaSe
    Infection and Immunity, 2002
    Co-Authors: Darrin J Bast, Victor Nizet, Donald E Low, Jeffrey D Fuller, Alvin C Camus, C L Duncan, Ronald L Thune
    Abstract:

    StreptococcuS iniae cauSeS meningoencephalitiS and death in cultured fiSh SpecieS and Soft-tiSSue infection in humanS. We recently reported that S. iniae iS reSponSible for local tiSSue necroSiS and bacteremia in a murine SubcutaneouS infection model. The ability to cauSe bacteremia in thiS model iS aSSociated with a genetic profile unique to StrainS reSponSible for diSeaSe in fiSh and humanS (J. D. Fuller, D. J. BaSt, V. Nizet, D. E. Low, and J. C. S. de Azavedo, Infect. Immun. 69:1994-2000, 2001). S. iniae produceS a cytolySin that conferS a hemolytic phenotype on blood agar media. In thiS Study, we characterized the genomic region reSponSible for S. iniae cytolySin production and aSSeSSed itS contribution to virulence. TranSpoSon (Tn917) mutant librarieS of commenSal and diSeaSe-aSSociated S. iniae StrainS were generated and Screened for loSS of hemolytic activity. AnalySiS of two nonhemolytic mutantS identified a chromoSomal locuS compriSing 9 geneS with 73% homology to the group A StreptococcuS (GAS) Sag operon for StreptolySin S (SLS) bioSyntheSiS. Confirmation that the S. iniae cytolySin iS a functional homologue of SLS waS achieved by PCR ligation mutageneSiS, complementation of an SLS-negative GAS mutant, and uSe of the SLS inhibitor trypan blue. SLS-negative SagB mutantS were compared to their wild-type S. iniae parent StrainS in the murine model and in human whole-blood killing aSSayS. TheSe StudieS demonStrated that S. iniae SLS expreSSion iS required for local tiSSue necroSiS but doeS not contribute to the eStabliShment of bacteremia or to reSiStance to phagocytic clearance.

  • genetic locuS for StreptolySin S production by group a StreptococcuS
    Infection and Immunity, 2000
    Co-Authors: Victor Nizet, Vivekananda Datta, Darrin J Bast, Bernard Beall, Laurie Kilburn, Donald E Low, Joyce C S De Azavedo
    Abstract:

    Group A StreptococcuS (GAS) iS an important human pathogen that cauSeS pharyngitiS and invaSive infectionS, including necrotizing faSciitiS. StreptolySin S (SLS) iS the cytolytic factor that createS the zone of betahemolySiS Surrounding GAS colonieS grown on blood agar. We recently reported the diScovery of a potential genetic determinant involved in SLS production, SagA, encoding a Small peptide of 53 amino acidS (S. D. BetSchel, S. M. Borgia, N. L. Barg, D. E. Low, and J. C. De Azavedo, Infect. Immun. 66:1671‐1679, 1998). USing tranSpoSon mutageneSiS, chromoSomal walking StepS, and data from the GAS genome Sequencing project (www .genome.ou.edu/Strep.html), we have now identified a contiguouS nine-gene locuS (SagA to SagI) involved in SLS production. The Sag locuS iS conServed among GAS StrainS regardleSS of M protein type. Targeted plaSmid integrational mutageneSiS of each gene in the Sag operon reSulted in an SLS-negative phenotype. Targeted integrationS (i) upStream of the SagA promoter and (ii) downStream of a terminator Sequence after SagI did not affect SLS production, eStabliShing the functional boundarieS of the operon. A rho-independent terminator Sequence between SagA and SagB appearS to regulate the amount of SagA tranScript produced verSuS tranScript for the entire operon. Reintroduction of the nine-gene Sag locuS on a plaSmid vector reStored SLS activity to the nonhemolytic SagA knockout mutant. Finally, heterologouS expreSSion of the intact Sag operon conferred the SLS beta-hemolytic phenotype to the nonhemolytic LactococcuS lactiS. We conclude that gene productS of the GAS Sag operon are both neceSSary and Sufficient for SLS production. Sequence homologieS of Sag operon gene productS SuggeSt that SLS iS related to the bacteriocin family of microbial toxinS. Group A StreptococcuS (GAS), Specifically StreptococcuS pyogeneS, iS a common cauSe of pharyngitiS, impetigo, and many

  • reduced virulence of group a Streptococcal tn916 mutantS that do not produce StreptolySin S
    Infection and Immunity, 1998
    Co-Authors: Stephen Betschel, Sergio Borgia, Donald E Low, Neil L Barg, Joyce C S De Azavedo
    Abstract:

    StreptolySin S (SLS) iS a potent cytolytic toxin produced by nearly all group A Streptococci (GAS). SLS-deficient Tn916 inSertional mutantS were generated from two clinical iSolateS of GAS, MGAS166S and T18PS (M SerotypeS 1 and 18, reSpectively), by tranSpoSon mutageneSiS uSing Tn916 donor Strain EnterococcuS faecaliS CG110. RepreSentative nonhemolytic tranSconjugantS SBNH5 and SB30-2 each harbored a Single Tn916 inSertion in identical loci. The inSertion in SBNH5 waS located in the promoter region of an open reading frame, deSignated SagA, rendering it tranScriptionally inactive. ProteaSe, StreptolySin O, and DNaSe activitieS and the production of M protein remained the Same in the nonhemolytic mutantS and the wild-type StrainS, aS did the growth rateS and exoprotein profileS. TranSconjugantS were evaluated in an eStabliShed murine model by injecting the organiSmS SubcutaneouSly and monitoring the mice for alterationS in weight and the development of necrotic leSionS. AnimalS infected with SBNH5, compared to thoSe infected with MGAS166S, gained weight during the firSt 24 h (+1.15 verSuS -1.16 g; P < 0.05) and had fewer necrotic leSionS (0 verSuS 7; P = 0.0007). AnimalS infected with SB30-2, compared to thoSe infected with T18PS, alSo gained weight within the firSt 24 h (+0.54 verSuS -0.66 g; P < 0.05) and produced fewer necrotic leSionS (1 verSuS 8; P = 0.001). RevertantS of the mutantS in which Tn916 had been exciSed regained the hemolytic phenotype and the virulence profile of the wild-type StrainS. ThiS Study demonStrateS that SLS-deficient mutantS of GAS, belonging to different M SerotypeS and containing identical Tn916 mutationS, are markedly leSS virulent than their iSogenic parentS.