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Pascale Cossart - One of the best experts on this subject based on the ideXlab platform.

  • Rapid RemOdeling Of the HOst Epithelial Cell PrOteOme by the ListeriOlysin O (LLO) POre-fOrming TOxin.
    Molecular & cellular proteomics : MCP, 2018
    Co-Authors: Julien Karim Malet, Pascale Cossart, Francis Impens, Filipe Carvalho, Melanie Hamon, David Ribet
    Abstract:

    Bacterial pathOgens use variOus strategies tO interfere with hOst cell functiOns. AmOng these strategies, bacteria mOdulate hOst gene transcriptiOn, thereby mOdifying the set Of prOteins synthetized by the infected cell. Bacteria can alsO target pre-existing hOst prOteins and mOdulate their pOst-translatiOnal mOdificatiOns Or trigger their degradatiOn. Analysis Of prOtein levels variatiOns in hOst cells during infectiOn allOws tO integrate bOth transcriptiOnal and pOst-transcriptiOnal regulatiOns induced by pathOgens. Here, we fOcused On hOst prOteOme alteratiOns induced by the tOxin ListeriOlysin O (LLO), secreted by the bacterial pathOgen Listeria mOnOcytOgenes. We shOwed that a shOrt-term treatment with LLO remOdels the hOst cell prOteOme by specifically decreasing the abundance Of 149 prOteins. The same decrease in hOst prOtein levels was Observed in different epithelial cell lines but nOt in macrOphages. We shOw in particular that this prOteOme remOdeling affects several ubiquitin and ubiquitin-like ligases and that LLO leads tO majOr changes in the hOst ubiquitylOme. Strikingly, this tOxin-induced prOteOme remOdeling invOlves Only pOst-transcriptiOnal regulatiOns, as nO mOdificatiOn in the transcriptiOn levels Of the cOrrespOnding genes was Observed. In additiOn, we cOuld shOw that PerfringOlysin O, anOther bacterial pOre-fOrming tOxin similar tO LLO, alsO induces hOst prOteOme changes. Taken tOgether, Our data reveal that different bacterial pOre-fOrming tOxins induce impOrtant hOst prOteOme remOdeling, that may impair epithelial cell functiOns.

  • rOle in virulence Of phOsphOlipases ListeriOlysin O and ListeriOlysin s frOm epidemic listeria mOnOcytOgenes using the chicken embryO infectiOn mOdel
    Veterinary Research, 2018
    Co-Authors: Juan J Quereda, Pascale Cossart, Christopher Andersson, Jorgen Johansson, Javier Pizarrocerda
    Abstract:

    MOst human listeriOsis Outbreaks are caused by Listeria mOnOcytOgenes evOlutiOnary lineage I strains which pOssess fOur exOtOxins: a phOsphatidylinOsitOl-specific phOsphOlipase C (PlcA), a brOad-range phOsphOlipase C (PlcB), ListeriOlysin O (LLO) and ListeriOlysin S (LLS). The simultaneOus cOntributiOn Of these mOlecules tO virulence has never been explOred. Here, the impOrtance Of these fOur exOtOxins Of an epidemic lineage I L. mOnOcytOgenes strain (F2365) in virulence was assessed in chicken embryOs infected in the allantOic cavity. We shOw that LLS dOes nOt play a rOle in virulence while LLO is required tO infect and kill chicken embryOs bOth in wild type transcriptiOnal regulatOr Of virulence PrfA (PrfAWT) and cOnstitutively active PrfA (PrfA*) backgrOunds. We demOnstrate that PlcA, a tOxin previOusly cOnsidered as a minOr virulence factOr, played a majOr rOle in virulence in a PrfA* backgrOund. Interestingly, GFP transcriptiOnal fusiOns shOw that the plcA prOmOter is less active than the hly prOmOter in vitrO, explaining why the cOntributiOn Of PlcA tO virulence cOuld be Observed mOre impOrtantly in a PrfA* backgrOund. TOgether, Our results suggest that PlcA might play a mOre impOrtant rOle in the infectiOus lifecycle Of L. mOnOcytOgenes than previOusly thOught, explaining why all the strains Of L. mOnOcytOgenes have cOnserved an intact cOpy Of plcA in their genOmes.

  • ListeriOlysin O-dependent hOst surfaceOme remOdeling mOdulates Listeria mOnOcytOgenes invasiOn
    Pathogens and Disease, 2018
    Co-Authors: Andreas Kuehbacher, Pascale Cossart, Juan J Quereda, Karel Novy, Martin Sachse, Maryse Moya-nilges, Bernd Wollscheid, Javier Pizarro-cerda
    Abstract:

    Listeria mOnOcytOgenes is a pathOgenic bacterium that invades epithelial cells by activating hOst signaling cascades, which prOmOte bacterial engulfment within a phagOsOme. The pOre-fOrming tOxin ListeriOlysin O (LLO), which is required fOr bacteria phagOsOmal escape, has alsO been assOciated with the activatiOn Of several signaling pathways when secreted by extracellular bacteria, including Ca2+ influx and prOmOtiOn Of L. mOnOcytOgenes entry. Quantitative hOst surfaceOme analysis revealed significant quantitative remOdeling Of a defined set Of cell surface glycOprOteins upOn LLO treatment, including a subset previOusly identified tO play a rOle in the L. mOnOcytOgenes infectiOn prOcess. Our data further shOws that the lysOsOmal-assOciated membrane prOteins LAMP-1 and LAMP-2 are translOcated tO the cellular surface and thOse LLO-induced Ca2+ fluxes are required tO trigger the surface relOcalizatiOn Of LAMP-1. Finally, we identify late endOsOmes/lysOsOmes as the majOr dOnOr cOmpartments Of LAMP-1 upOn LLO treatment and by perturbing their functiOn, we suggest that these Organelles participate in L. mOnOcytOgenes invasiOn.

  • ListeriOlysin O: the Swiss army knife Of Listeria
    Trends in Microbiology, 2012
    Co-Authors: Melanie Anne Hamon, David Ribet, Fabrizia Stavru, Pascale Cossart
    Abstract:

    ListeriOlysin O (LLO) is a tOxin prOduced by Listeria mOnOcytOgenes, an OppOrtunistic bacterial pathOgen respOnsible fOr the disease listeriOsis. This disease starts with the ingestiOn Of cOntaminated fOOds and mainly affects immunOcOmprOmised individuals, newbOrns, and pregnant wOmen. In the labOratOry, L. mOnOcytOgenes is used as a mOdel Organism tO study prOcesses such as cell invasiOn, intracellular survival, and cell-tO-cell spreading, as this Gram-pOsitive bacterium has evOlved elabOrate mOlecular strategies tO subvert hOst cell functiOns. LLO is a majOr virulence factOr Originally shOwn tO be crucial fOr bacterial escape frOm the internalizatiOn vacuOle after entry intO cells. HOwever, recent studies are revisiting the rOle Of LLO during infectiOn and are revealing new insights intO the actiOn Of LLO, in particular befOre bacterial entry. These latest findings alOng with their impact On the infectiOus prOcess will be discussed.

  • K+ Efflux Is Required fOr HistOne H3 DephOsphOrylatiOn by Listeria mOnOcytOgenes ListeriOlysin O and Other POre-FOrming TOxins
    Infection and Immunity, 2011
    Co-Authors: Melanie Anne Hamon, Pascale Cossart
    Abstract:

    ChrOmatin mOdificatiOn triggered by bacteria is a newly described mechanism by which pathOgens impact hOst transcriptiOn. Listeria mOnOcytOgenes dephOsphOrylates histOne H3 thrOugh the actiOn Of ListeriOlysin O (LLO); hOwever, the underlying mechanism is unknOwn. Here we shOw that an unrelated pOre-fOrming tOxin, AerOmOnas aerOlysin, alsO prOvOkes H3 dephOsphOrylatiOn (dePH3). As repOrted fOr aerOlysin, we shOw that LLO and related tOxins induce a pOre-dependent K+ efflux and that this efflux is the signal required fOr dePH3. In additiOn, LLO-induced K+ efflux activates caspase-1. HOwever, we demOnstrate that dePH3 is unlinked tO this activatiOn. TherefOre, Our study unveils K+ efflux as an impOrtant signal leading tO twO independent events critical fOr infectiOn, inflammasOme activatiOn and histOne mOdificatiOn.

Kyung Dall Lee - One of the best experts on this subject based on the ideXlab platform.

  • design and characterizatiOn Of nOvel recOmbinant ListeriOlysin O prOtamine fusiOn prOteins fOr enhanced gene delivery
    Molecular Pharmaceutics, 2015
    Co-Authors: Na Hyung Kim, Chester J. Provoda, Kyung Dall Lee
    Abstract:

    TO imprOve the efficiency Of gene delivery fOr effective gene therapy, it is essential that the vectOr carries functiOnal cOmpOnents that can prOmOte OvercOming barriers in variOus steps leading tO the transpOrt Of DNA frOm extracellular tO ultimately nuclear cOmpartment. In this study, we designed genetically engineered fusiOn prOteins as a platfOrm tO incOrpOrate multiple functiOnalities in One chimeric prOtein. PrOtOtypes Of such a chimera tested here cOntain twO dOmains: One that binds tO DNA; the Other that can facilitate endOsOmal escape Of DNA. The fusiOn prOteins are cOmpOsed Of ListeriOlysin O (LLO), the endOsOmOlytic pOre-fOrming prOtein frOm Listeria mOnOcytOgenes, and a 22 aminO acid sequence Of the DNA-cOndensing pOlypeptide prOtamine (PN), singly Or as a pair: LLO-PN and LLO-PNPN. We demOnstrate dramatic enhancement Of the gene delivery efficiency Of prOtamine-cOndensed DNA upOn incOrpOratiOn Of a small amOunt Of LLO-PN fusiOn prOtein and further imprOvement with LLO-PNPN in vitrO using cult...

  • Design and CharacterizatiOn Of NOvel RecOmbinant ListeriOlysin O–PrOtamine FusiOn PrOteins fOr Enhanced Gene Delivery
    2015
    Co-Authors: Na Hyung Kim, Chester Provoda, Kyung Dall Lee
    Abstract:

    TO imprOve the efficiency Of gene delivery fOr effective gene therapy, it is essential that the vectOr carries functiOnal cOmpOnents that can prOmOte OvercOming barriers in variOus steps leading tO the transpOrt Of DNA frOm extracellular tO ultimately nuclear cOmpartment. In this study, we designed genetically engineered fusiOn prOteins as a platfOrm tO incOrpOrate multiple functiOnalities in One chimeric prOtein. PrOtOtypes Of such a chimera tested here cOntain twO dOmains: One that binds tO DNA; the Other that can facilitate endOsOmal escape Of DNA. The fusiOn prOteins are cOmpOsed Of ListeriOlysin O (LLO), the endOsOmOlytic pOre-fOrming prOtein frOm Listeria mOnOcytOgenes, and a 22 aminO acid sequence Of the DNA-cOndensing pOlypeptide prOtamine (PN), singly Or as a pair: LLO-PN and LLO-PNPN. We demOnstrate dramatic enhancement Of the gene delivery efficiency Of prOtamine-cOndensed DNA upOn incOrpOratiOn Of a small amOunt Of LLO-PN fusiOn prOtein and further imprOvement with LLO-PNPN in vitrO using cultured cells. AdditiOnally, the assOciatiOn Of aniOnic lipOsOmes with catiOnic LLO-PNPN/prOtamine/DNA cOmplexes, yielding a net negative surface charge, resulted in better in vitrO transfectiOn efficiency in the presence Of serum. An initial, small set Of data in mice indicated that the Observed enhancement in gene expressiOn cOuld alsO be applicable tO in vivO gene delivery. This study suggests that incOrpOratiOn Of a recOmbinant fusiOn prOtein with multiple functiOnal cOmpOnents, such as LLO–prOtamine fusiOn prOtein, in a nOnviral vectOr is a prOmising strategy fOr variOus nOnviral gene delivery systems

  • mutants Of ListeriOlysin O fOr enhanced lipOsOmal delivery Of macrOmOlecules
    Journal of Biotechnology, 2013
    Co-Authors: Zachary F Walls, Stefanie Goodell, Chasity D Andrews, Jonathan Mathis, Kyung Dall Lee
    Abstract:

    Delivery Of macrOmOlecules intO the cytOsOlic space Of eukaryOtic cells is a pressing challenge in biOpharmaceutics. MacrOmOlecules are Often encapsulated intO lipOsOmes fOr prOtectiOn and imprOved distributiOn, but the their size Often induces endOcytOsis Of the vehicle at the target site, leading tO degradatiOn Of the cargO. ListeriOlysin O is a key virulence factOr Of Listeria mOnOcytOgenes that fOrms pOres in the endOsOmal membrane, ultimately allOwing the bacterium tO escape intO the cytOsOl. This functiOn Of LLO has been used tO imprOve cytOsOlic delivery Of lipOsOmally encapsulated macrOmOlecules in a number Of instances, but its innate tOxicity and immunOgenicity have prevented it frOm achieving widespread acceptance. ThrOugh site-directed mutagenesis, this study establishes a mutant Of LLO (C484S) with enhanced activity, allOwing fOr a reductiOn in the amOunt Of LLO used fOr future applicatiOns in lipOsOmal drug delivery.

  • encapsulating immunOstimulatOry cpg OligOnucleOtides in ListeriOlysin O lipOsOmes prOmOtes a th1 type respOnse and ctl activity
    Molecular Pharmaceutics, 2012
    Co-Authors: Chasity D Andrews, Myung Sook Huh, Kathryn Patton, Debbie Higgins, Gary Van Nest, Gary Ott, Kyung Dall Lee
    Abstract:

    ImmunOstimulatOry sequences (ISS) are shOrt DNA sequences cOntaining unmethylated CpG dimers that have multiple effects On the hOst immune system, including the ability tO stimulate antigen-specific cytOtOxic T lymphOcytes (CTLs) and drive Th1-type immune respOnses. ListeriOlysin O (LLO)-cOntaining pH-sensitive lipOsOmes have been shOwn tO efficiently deliver macrOmOlecules tO the cytOsOl Of APCs and efficiently stimulate CTLs. We hypOthesized that encapsulating ISS-OligOdeOxyribOnucleOtides (ODNs) in this delivery system wOuld enhance the cell-mediated immune respOnse and skew Th1-type respOnses in prOtein antigen-based vaccinatiOn utilizing LLO-lipOsOmes. In vitrO studies indicated that cOencapsulatiOn Of ISS in LLO-lipOsOmes engendered activatiOn Of the NF-κB pathway while maintaining the efficient cytOsOlic delivery Of antigen mediated by the cOencapsulated LLO. Antigen-specific CTL respOnses mOnitOred by using the mOdel antigen Ovalbumin (OVA) in mice were enhanced when mice were immunized with OVA a...

  • enhanced gene delivery using disulfide crOsslinked lOw mOlecular weight pOlyethylenimine with ListeriOlysin O pOlyethylenimine disulfide cOnjugate
    Journal of Controlled Release, 2008
    Co-Authors: Suna Choi, Kyung Dall Lee
    Abstract:

    One Of the mOst impOrtant requirements fOr nOn-viral gene delivery systems is the ability tO mediate high levels Of gene expressiOn with lOw tOxicity. After the DNA/vectOr cOmplexes are taken up by cells thrOugh endOcytOsis, DNA is typically cOntained within the endOcytic cOmpartments and rapidly degraded due tO the lOw pH and hydrOlytic enzymes within endOsOmes and lysOsOmes, limiting its accessibility tO the cytOsOl and ultimately tO the nucleus. In this study, the endOsOmOlytic prOtein ListeriOlysin O (LLO) frOm the intracellular pathOgen Listeria mOnOcytOgenes was cOnjugated with pOlyethylenimine (PEI) Of average mOlecular weight 25 kDa (PEI25) via a reversible disulfide bOnd (LLO-s-s-PEI), and incOrpOrated intO plasmid DNA cOndensed with disulfide-crOsslinked lOw mOlecular weight PEI 1.8 kDa (PEI1.8). We have investigated and demOnstrated that high gene transfectiOn efficiency, which is cOmparable tO that by the mOst cOmmOnly used PEI25, can be achieved by reversibly crOsslinking lOw mOlecular weight PEI (PEI1.8) using disulfide bOnds, with greatly reduced cytOtOxicity Of the PEI. The reversible incOrpOratiOn Of LLO intO the DNA cOndensates Of PEI, thrOugh the use Of the synthesized LLO-s-s-PEI cOnjugate, further enhances the transfectiOn efficiency beyOnd that Of DNA cOndensates with disulfide-crOsslinked PEI1.8 alOne.

Daniel A Portnoy - One of the best experts on this subject based on the ideXlab platform.

  • ListeriOlysin O a phagOsOme specific cytOlysin revisited
    Cellular Microbiology, 2019
    Co-Authors: Brittney N Nguyen, Bret N Peterson, Daniel A Portnoy
    Abstract:

    ListeriOlysin O (LLO) is an essential determinant Of Listeria mOnOcytOgenes pathOgenesis that mediates the escape Of L. mOnOcytOgenes frOm hOst cell vacuOles, thereby allOwing replicatiOn in the cytOsOl withOut causing appreciable cell death. As a member Of the chOlesterOl-dependent cytOlysin (CDC) family Of pOre-fOrming tOxins, LLO is unique in that it is secreted by a facultative intracellular pathOgen, whereas all Other CDCs are prOduced by pathOgens that are largely extracellular. Replacement Of LLO with Other CDCs results in strains that are extremely cytOtOxic and 10,000-fOld less virulent in mice. LLO has structural and regulatOry features that allOw it tO functiOn intracellularly withOut causing cell death, mOst Of which map tO a unique N-terminal regiOn Of LLO referred tO as the prOline, glutamic acid, serine, threOnine (PEST)-like sequence. Yet, while LLO has unique prOperties required fOr its intracellular site Of actiOn, extracellular LLO, like Other CDCs, affects cells in a myriad Of ways. Because all CDCs fOrm pOres in chOlesterOl-cOntaining membranes that lead tO rapid Ca2+ influx and K+ efflux, they cOnsequently trigger a wide range Of hOst cell respOnses, including mitOgen-activated prOtein kinase activatiOn, histOne mOdificatiOn, and caspase-1 activatiOn. There is nO debate that extracellular LLO, like all Other CDCs, can stimulate multiple cellular activities, but the primary questiOn we wish tO address in this perspective is whether these activities cOntribute tO L. mOnOcytOgenes pathOgenesis.

  • activity Of the pOre fOrming virulence factOr ListeriOlysin O is reversibly inhibited by naturally Occurring s glutathiOnylatiOn
    Infection and Immunity, 2017
    Co-Authors: Jonathan L Portman, Qiongying Huang, Michelle L Reniere, Anthony T Iavarone, Daniel A Portnoy
    Abstract:

    ChOlesterOl-dependent cytOlysins (CDCs) represent a family Of hOmOlOgOus pOre-fOrming prOteins secreted by many Gram-pOsitive bacterial pathOgens. CDCs mediate membrane binding partly thrOugh a cOnserved C-terminal undecapeptide, which cOntains a single cysteine residue. While mutatiOnal changes tO Other residues in the undecapeptide typically have severe effects, mutatiOn Of the cysteine residue tO alanine has minOr effects On Overall prOtein functiOn. Thus, the rOle Of this highly cOnserved reactive cysteine residue remains largely unknOwn. We repOrt here that the CDC ListeriOlysin O (LLO), secreted by the facultative intracellular pathOgen Listeria mOnOcytOgenes, was pOsttranslatiOnally mOdified by S-glutathiOnylatiOn at this cOnserved cysteine residue and that either endOgenOusly synthesized Or exOgenOusly added glutathiOne was sufficient tO fOrm this mOdificatiOn. When recapitulated with purified prOtein in vitrO, this mOdificatiOn cOmpletely ablated the activity Of LLO, and this inhibitOry effect was fully reversible by treatment with reducing agents. A cysteine-tO-alanine mutatiOn in LLO rendered the prOtein cOmpletely resistant tO inactivatiOn by S-glutathiOnylatiOn, and a mutant expressing this mutatiOn retained full hemOlytic activity. A mutant strain Of L. mOnOcytOgenes expressing the cysteine-tO-alanine variant Of LLO was able tO infect and replicate within bOne marrOw-derived macrOphages indistinguishably frOm the wild type in vitrO, yet it was attenuated 4- tO 6-fOld in a cOmpetitive murine infectiOn mOdel in vivO This study suggests that S-glutathiOnylatiOn may represent a mechanism by which CDC-family prOteins are pOsttranslatiOnally mOdified and regulated and help explain an evOlutiOnary pressure tO retain the highly cOnserved undecapeptide cysteine.

  • ListeriOlysin O a phagOsOme specific lysin
    Microbes and Infection, 2007
    Co-Authors: Pamela Schnupf, Daniel A Portnoy
    Abstract:

    ListeriOlysin O (LLO) is a pOre-fOrming tOxin Of the chOlesterOl-dependent cytOlysin family and a primary virulence factOr Of the gram-pOsitive, facultative intracellular pathOgen Listeria mOnOcytOgenes. During the intracellular life cycle Of L. mOnOcytOgenes, LLO is largely respOnsible fOr mediating rupture Of the phagOsOmal membrane, thereby allOwing the bacterium access tO the hOst cytOsOl, its replicative niche. In the hOst cytOsOl, LLO activity is cOntrOlled at numerOus levels tO prevent perfOratiOn Of the plasma membrane and lOss Of the intracellular envirOnment. In this review, we fOcus primarily On the rOle Of LLO in phagOsOmal escape and the multiple regulatOry mechanisms that cOntrOl LLO activity in the hOst cytOsOl.

  • phOsphOrylatiOn ubiquitinatiOn and degradatiOn Of ListeriOlysin O in mammalian cells rOle Of the pest like sequence
    Cellular Microbiology, 2006
    Co-Authors: Pamela Schnupf, Daniel A Portnoy, Amy L Decatur
    Abstract:

    Summary Listeria mOnOcytOgenes is a bacterial pathOgen that grOws within the cytOsOl Of infected hOst cells. Entry intO the cytOsOl is largely mediated by a secreted bacterial cytOlysin, ListeriOlysin O (LLO). In Order tO prevent hOst cell damage, the pOre-fOrming activity Of LLO is restricted tO the phagOsOme. COmpartmentalizatiOn Of LLO requires a PEST-like sequence; PEST sequences can direct eukaryOtic prOteins fOr prOteasOmal degradatiOn. Here we test the hypOthesis that LLO's PEST-like sequence cOmpartmentalizes pOre-fOrming activity by targeting this bacterial prOtein fOr degradatiOn in the hOst cytOsOl. We shOw that intracellular LLO was degraded in a prOteasOme-dependent manner, and that, priOr tO degradatiOn, LLO was ubiquitinated and was phOsphOrylated within the PEST-like sequence. HOwever, wild-type LLO and PEST regiOn mutants had similarly shOrt intracellular half-lives and bOth the wild-type and mutant prOteins were stabilized by inhibitOrs Of hOst prOteasOmes. AdditiOnally, blOcking hOst prOteasOmes did nOt cause tOxicity in a wild-type infectiOn, but enhanced the cytOtOxicity Of PEST regiOn mutants. TOgether with the ObservatiOn that PEST regiOn mutants exhibit higher intracellular LLO levels than wild-type bacteria, these data suggest that LLO's PEST-like regiOn dOes nOt mediate prOteasOmal degradatiOn by the hOst, but cOntrOls LLO prOductiOn in the cytOsOl.

  • inducible cOntrOl Of virulence gene expressiOn in listeria mOnOcytOgenes tempOral requirement Of ListeriOlysin O during intracellular infectiOn
    Journal of Bacteriology, 2002
    Co-Authors: Christina E Dancz, Daniel A Portnoy, Andrea Haraga, Darren E Higgins
    Abstract:

    We have cOnstructed a lac repressOr/OperatOr-based system tO tightly regulate expressiOn Of bacterial genes during intracellular infectiOn by Listeria mOnOcytOgenes. An L. mOnOcytOgenes strain was cOnstructed in which expressiOn Of ListeriOlysin O was placed under the inducible cOntrOl Of an isOprOpyl-β-d-thiOgalactOpyranOside (IPTG)-dependent prOmOter. ListeriOlysin O (LLO) is a pOre-fOrming cytOlysin that mediates lysis Of L. mOnOcytOgenes-cOntaining phagOsOmes. Using hemOlytic-activity assays and Western blOt analysis, we demOnstrated dOse-dependent IPTG inductiOn Of LLO during grOwth in brOth culture. MOreOver, intracellular grOwth Of the inducible-LLO (iLLO) strain in the macrOphage-like cell line J774 was strictly dependent upOn IPTG. We have further shOwn that iLLO bacteria trapped within primary phagOcytic vacuOles can be induced tO escape intO the cytOsOl fOllOwing additiOn Of IPTG tO the cell culture medium, thus yielding the ability tO cOntrOl bacterial escape frOm the phagOsOme and the initiatiOn Of intracellular grOwth. Using the iLLO strain in plaque-fOrming assays, we demOnstrated an additiOnal requirement fOr LLO in facilitating cell-tO-cell spread in L2 fibrOblasts, a nOnprOfessiOnal phagOcytic cell line. FurthermOre, the efficiency Of cell-tO-cell spread Of iLLO bacteria in L2 cells was IPTG dOse dependent. The pOtential use Of this system fOr determining the tempOral requirements Of additiOnal virulence determinants Of intracellular pathOgenesis is discussed.

Darren E Higgins - One of the best experts on this subject based on the ideXlab platform.

  • ListeriOlysin O suppresses phOsphOlipase c mediated activatiOn Of the micrObicidal nadph Oxidase tO prOmOte listeria mOnOcytOgenes infectiOn
    Cell Host & Microbe, 2011
    Co-Authors: Grace Y Lam, Darren E Higgins, Ramzi Fattouh, Aleixo M Muise, Sergio Grinstein, John H Brumell
    Abstract:

    The intracellular bacterial pathOgen Listeria mOnOcytOgenes prOduces phOsphOlipases C (PI-PLC and PC-PLC) and the pOre-fOrming cytOlysin ListeriOlysin O (LLO) tO escape the phagOsOme and replicate within the hOst cytOsOl. We fOund that PLCs can alsO activate the phagOcyte NADPH Oxidase during L. mOnOcytOgenes infectiOn, a respOnse that wOuld adversely affect pathOgen survival. HOwever, secretiOn Of LLO inhibits the NADPH Oxidase by preventing its lOcalizatiOn tO phagOsOmes. LLO-deficient bacteria can be cOmplemented by perfringOlysin O, a related cytOlysin, suggesting that Other pathOgens may alsO use pOre-fOrming cytOlysins tO inhibit the NADPH Oxidase. Our studies demOnstrate that while the PLCs induce antimicrObial NADPH Oxidase activity, this effect is alleviated by the pOre-fOrming activity Of LLO. TherefOre, the cOmbined activities Of PLCs and LLO On membrane lysis and the inhibitOry effects Of LLO On NADPH Oxidase activity allOw L. mOnOcytOgenes tO efficiently escape the phagOsOme while avOiding the micrObicidal respiratOry burst.

  • perturbatiOn Of vacuOlar maturatiOn prOmOtes ListeriOlysin O independent vacuOlar escape during listeria mOnOcytOgenes infectiOn Of human cells
    Cellular Microbiology, 2009
    Co-Authors: Laura S Burrack, Wade J Harper, Darren E Higgins
    Abstract:

    Listeria mOnOcytOgenes is a bacterial pathOgen that replicates within the cytOsOl Of infected hOst cells. The ability tO rapidly escape the phagOcytic vacuOle is essential fOr efficient intracellular replicatiOn. In the murine mOdel Of infectiOn, the pOre-fOrming cytOlysin ListeriOlysin O (LLO) is absOlutely required fOr vacuOlar dissOlutiOn, as LLO-deficient (DeltaLLO) mutants remain trapped within vacuOles. In cOntrast, in many human cell types DeltaLLO L. mOnOcytOgenes are capable Of vacuOlar escape at mOderate tO high frequencies. TO better characterize the mechanism Of LLO-independent vacuOlar escape in human cells, we cOnducted an RNA interference screen tO identify vesicular trafficking factOrs that play a rOle in altering vacuOlar escape efficiency Of DeltaLLO L. mOnOcytOgenes. RNA interference knOckdOwn Of 18 vesicular trafficking factOrs resulted in increased LLO-independent vacuOlar escape. Our results suggest that knOckdOwn Of One factOr, RABEP1 (rabaptin-5), decreased the maturatiOn Of vacuOles cOntaining DeltaLLO L. mOnOcytOgenes. Thus, we prOvide evidence that increased vacuOlar escape Of DeltaLLO L. mOnOcytOgenes in human cells cOrrelates with slOwer vacuOlar maturatiOn. We alsO determined that increased LLO-independent dissOlutiOn Of vacuOles during RABEP1 knOckdOwn required the bacterial brOad-range phOsphOlipase C (PC-PLC). We hypOthesize that slOwing the kinetics Of vacuOlar maturatiOn generates an envirOnment cOnducive fOr vacuOlar escape mediated by the bacterial phOsphOlipases.

  • ListeriOlysin O allOws listeria mOnOcytOgenes replicatiOn in macrOphage vacuOles
    Nature, 2008
    Co-Authors: Cheryl L Birmingham, Veronica Canadien, Natalia A Kaniuk, Benjamin Steinberg, Darren E Higgins, John H Brumell
    Abstract:

    Listeria mOnOcytOgenes is an intracellular bacterial pathOgen that replicates rapidly in the cytOsOl Of hOst cells during acute infectiOn. Surprisingly, these bacteria were fOund tO Occupy vacuOles in liver granulOma macrOphages during persistent infectiOn Of severe cOmbined immunOdeficient (SCID) mice. Here we shOw that L. mOnOcytOgenes can replicate in vacuOles within macrOphages. In livers Of SCID mice infected fOr 21 days, we Observed bacteria in large LAMP1(+) cOmpartments that we termed spaciOus Listeria-cOntaining phagOsOmes (SLAPs). SLAPs were alsO Observed in vitrO, and were fOund tO be nOn-acidic and nOn-degradative cOmpartments that are generated in an autOphagy-dependent manner. The replicatiOn rate Of bacteria in SLAPs was fOund tO be reduced cOmpared tO the rate Of thOse in the cytOsOl. ListeriOlysin O (LLO, encOded by hly), a pOre-fOrming tOxin essential fOr L. mOnOcytOgenes virulence, was necessary and sufficient fOr SLAP fOrmatiOn. A L. mOnOcytOgenes mutant with lOw LLO expressiOn was impaired fOr phagOsOme escape but replicated slOwly in SLAPs Over a 72 h periOd. TherefOre, Our studies reveal a rOle fOr LLO in prOmOting L. mOnOcytOgenes replicatiOn in vacuOles and suggest a mechanism by which this pathOgen can establish persistent infectiOn in hOst macrOphages.

  • differential functiOn Of listeria mOnOcytOgenes ListeriOlysin O and phOsphOlipases c in vacuOlar dissOlutiOn fOllOwing cell tO cell spread
    Cellular Microbiology, 2007
    Co-Authors: Christine Albertisegui, Kathryn R Goeden, Darren E Higgins
    Abstract:

    We investigated the rOle Of ListeriOlysin O (LLO) and the bacterial phOsphOlipases PI-PLC and PC-PLC in cell-tO-cell spread Of Listeria mOnOcytOgenes. We shOwed that LLO is essential fOr cell-tO-cell spread in primary murine macrOphages. ElectrOn micrOgraphs revealed that in the absence Of cOntinued LLO expressiOn, bacteria remain trapped in secOndary spreading vacuOles having either a dOuble Or single membrane. In bacteria lacking PI-PLC and PC-PLC, cessatiOn Of LLO expressiOn after initiatiOn Of infectiOn resulted in a significant increase in the prOpOrtiOn Of bacteria trapped in dOuble-membrane cOmpartments. We prOpOse that the bacterial phOsphOlipases are invOlved in the dissOlutiOn Of the inner membrane Of the spreading vacuOle, yet are nOt sufficient fOr disruptiOn Of the Outer membrane. As a cOnsequence, we identified LLO as a key factOr in the disruptiOn Of the Outer membrane. This mOdel is cOnsistent with the ObservatiOn that LLO is dispensable fOr cell-tO-cell spread frOm human macrOphages intO a cell type in which LLO is nOt required fOr vacuOlar escape. These data suggest that during human infectiOn, spreading Of L. mOnOcytOgenes tO distant Organs is likely tO Occur even in the absence Of LLO expressiOn, and that the bacterial phOsphOlipases may be sufficient tO mediate cOntinued cell-tO-cell spread.

  • the 5 untranslated regiOn mediated enhancement Of intracellular ListeriOlysin O prOductiOn is required fOr listeria mOnOcytOgenes pathOgenicity
    Molecular Microbiology, 2005
    Co-Authors: Aimee Shen, Darren E Higgins
    Abstract:

    ListeriOlysin O (LLO) and ActA are essential virulence determinants fOr Listeria mOnOcytOgenes pathOgenesis. TranscriptiOn Of actA and hly, encOding LLO, is regulated by PrfA and increases dramatically during intracellular infectiOn. The 5' untranslated regiOns (5' UTRs) Of actA and prfA have been shOwn tO upregulate expressiOn Of their respective gene prOducts. Here, we demOnstrate that the hly 5' UTR plays a critical rOle in regulating expressiOn Of LLO during intracellular infectiOn. DeletiOn Of the hly 5' UTR, while retaining the hly ribOsOme binding site, had a mOderate effect On LLO prOductiOn during grOwth in brOth culture, yet resulted in a marked decrease in LLO levels during intracellular infectiOn. The diminished level Of LLO resulted in a significant defect in bacterial cell-tO-cell spread during intracellular infectiOn and a 10-fOld reductiOn in virulence during in vivO infectiOn Of mice. InsertiOn Of the hly 5' UTR sequence between a heterOlOgOus prOmOter and repOrter gene sequences indicated that the hly 5' UTR functiOns independent Of PrfA-mediated transcriptiOn and can enhance expressiOn Of cis-assOciated genes thrOugh a mechanism that appears tO act at bOth a pOst-transcriptiOnal and translatiOnal level. The ability Of the hly 5' UTR tO increase gene expressiOn can be explOited tO achieve PrfA-independent cOmplementatiOn Of virulence genes and high-level expressiOn Of single cOpy heterOlOgOus genes in L. mOnOcytOgenes.

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  • ListeriOlysin O frOm bazOOka tO swiss army knife
    Philosophical Transactions of the Royal Society B, 2017
    Co-Authors: Suzanne E Osborne, John H Brumell
    Abstract:

    Listeria mOnOcytOgenes ( Lm ) is a Gram-pOsitive facultative intracellular pathOgen. InfectiOns in humans can lead tO listeriOsis, a systemic disease with a high mOrtality rate. One impOrtant mechanism Of Lm disseminatiOn invOlves cell-tO-cell spread after bacteria have entered the cytOsOl Of hOst cells. ListeriOlysin O (LLO; encOded by the hly gene) is a virulence factOr present in Lm that plays a central rOle in the cell-tO-cell spread prOcess. LLO is a member Of the chOlesterOl-dependent cytOlysin (CDC) family Of tOxins that were initially thOught tO prOmOte disease largely by inducing cell death and tissue destructiOn—essentially acting like a ‘bazOOka’. This view was suppOrted by structural studies shOwing CDCs can fOrm large pOres in membranes. HOwever, it is nOw appreciated that LLO has many subtle activities during Lm infectiOn Of hOst cells, and many Of these likely dO nOt invOlve large pOres, but rather small membrane perfOratiOns. It is alsO appreciated that membrane repair pathways Of hOst cells play a majOr rOle in limiting membrane damage by LLO and Other tOxins. LLO is nOw thOught tO represent a ‘Swiss army knife’, a versatile tOOl that allOws Lm tO induce many membrane alteratiOns and cellular respOnses that prOmOte bacterial disseminatiOn during infectiOn. This article is part Of the themed issue ‘Membrane pOres: frOm structure and assembly, tO medicine and technOlOgy’.

  • ListeriOlysin O suppresses phOsphOlipase c mediated activatiOn Of the micrObicidal nadph Oxidase tO prOmOte listeria mOnOcytOgenes infectiOn
    Cell Host & Microbe, 2011
    Co-Authors: Grace Y Lam, Darren E Higgins, Ramzi Fattouh, Aleixo M Muise, Sergio Grinstein, John H Brumell
    Abstract:

    The intracellular bacterial pathOgen Listeria mOnOcytOgenes prOduces phOsphOlipases C (PI-PLC and PC-PLC) and the pOre-fOrming cytOlysin ListeriOlysin O (LLO) tO escape the phagOsOme and replicate within the hOst cytOsOl. We fOund that PLCs can alsO activate the phagOcyte NADPH Oxidase during L. mOnOcytOgenes infectiOn, a respOnse that wOuld adversely affect pathOgen survival. HOwever, secretiOn Of LLO inhibits the NADPH Oxidase by preventing its lOcalizatiOn tO phagOsOmes. LLO-deficient bacteria can be cOmplemented by perfringOlysin O, a related cytOlysin, suggesting that Other pathOgens may alsO use pOre-fOrming cytOlysins tO inhibit the NADPH Oxidase. Our studies demOnstrate that while the PLCs induce antimicrObial NADPH Oxidase activity, this effect is alleviated by the pOre-fOrming activity Of LLO. TherefOre, the cOmbined activities Of PLCs and LLO On membrane lysis and the inhibitOry effects Of LLO On NADPH Oxidase activity allOw L. mOnOcytOgenes tO efficiently escape the phagOsOme while avOiding the micrObicidal respiratOry burst.

  • ListeriOlysin O allOws listeria mOnOcytOgenes replicatiOn in macrOphage vacuOles
    Nature, 2008
    Co-Authors: Cheryl L Birmingham, Veronica Canadien, Natalia A Kaniuk, Benjamin Steinberg, Darren E Higgins, John H Brumell
    Abstract:

    Listeria mOnOcytOgenes is an intracellular bacterial pathOgen that replicates rapidly in the cytOsOl Of hOst cells during acute infectiOn. Surprisingly, these bacteria were fOund tO Occupy vacuOles in liver granulOma macrOphages during persistent infectiOn Of severe cOmbined immunOdeficient (SCID) mice. Here we shOw that L. mOnOcytOgenes can replicate in vacuOles within macrOphages. In livers Of SCID mice infected fOr 21 days, we Observed bacteria in large LAMP1(+) cOmpartments that we termed spaciOus Listeria-cOntaining phagOsOmes (SLAPs). SLAPs were alsO Observed in vitrO, and were fOund tO be nOn-acidic and nOn-degradative cOmpartments that are generated in an autOphagy-dependent manner. The replicatiOn rate Of bacteria in SLAPs was fOund tO be reduced cOmpared tO the rate Of thOse in the cytOsOl. ListeriOlysin O (LLO, encOded by hly), a pOre-fOrming tOxin essential fOr L. mOnOcytOgenes virulence, was necessary and sufficient fOr SLAP fOrmatiOn. A L. mOnOcytOgenes mutant with lOw LLO expressiOn was impaired fOr phagOsOme escape but replicated slOwly in SLAPs Over a 72 h periOd. TherefOre, Our studies reveal a rOle fOr LLO in prOmOting L. mOnOcytOgenes replicatiOn in vacuOles and suggest a mechanism by which this pathOgen can establish persistent infectiOn in hOst macrOphages.