Go to the content. | Move to the navigation | Go to the site search | Go to the menu | Contacts | Accessibility

| Create Account

Dal Molin, Federica (2006) Studi sul meccanismo di azione cellulare delle tossine prodotte da Bacillus anthracis. [Ph.D. thesis]

Full text disponibile come:

PDF Document

Abstract (english)

The major determinants of anthrax pathogenesis are anthrax toxins. They are composed of three proteins, PA, LF and EF. PA binds to specific cellular receptors and the complex PA+LF+EF is endocytosed. Once inside the cell, the toxins follow the endocytic route until pH-dependent translocation of LF and EF occurs. LF is a metal-dependent protease that cleaves MEKs, whereas EF is a calmodulin-dependent adenylate cyclase. Informations about the site of EF release into the citosol are lacking. Biochemical analysis of intracellular cAMP increase in cultured cells reveals that EF activity begins after 30 minutes from intoxication and cAMP levels are still high after 4 hours. These results can provide two kinds of informations. The first one suggests that a long travel through the endocytic pathway may occur before EF can reach the cytosol, in accordance with translocation from late endosomes. The second one suggests a severe impairment of cellular signalling, since for several hours the cell is unable to overcome the massive production of cAMP.
To investigate the site of EF translocation we performed FRET imaging of cAMP dynamics induced by the toxin. Mapping of narrow regions inside intoxicated cells and FRET efficiency measurements revealed a preferential site of cAMP increase, corresponding to perinuclear regions. This result is in agreement with the hypothesis of translocation from late endosomes.
Following another approach, we fused LF and EF to green and red fluorescent proteins. Those chimerae provide a tool for tracking the route of the toxins inside the cell. Simultaneous visualization of ETx and LTx could clarify the mechanisms of their synergistic activity. Moreover, colocalization with cellular markers could define the pattern of toxicity and the advantages of translocating closely to the nucleus.
Finally, we performed RT-PCR to test the presence of both anthrax receptors, TEM8 and CMG2, on the same cells that were used in our experiments. CMG2 has the highest affinity for PA and requires a low pH to allow dissociation and PA pore formation. Thus, the presence of CMG2 validates the hypothesis of EF translocation from late endosomes. The clarification of the precise intracellular localization of anthrax toxins is compulsory to understand their final effect, since microdomains with a broad variety and concentration of signal molecules are present in a cell.

Abstract (italian)

Le tossine di antrace hanno un ruolo centrale nella patogenesi dell’infezione. Sono composte da tre proteine, PA, LF e EF. PA si lega a specifici recettori cellulari e il complesso PA+LF+EF è endocitato. Le tossine dentro la cellula iniziano a percorrere la via endocitica fino a che LF e EF traslocano nel citosol con un meccanismo pH-dipendente. LF è una metalloproteasi che taglia le MEKs e EF è un’adenilato ciclasi calmodulina-dipendente.
Ci sono poche informazioni riguardo il sito di rilascio di EF nel citoplasma. La misura dei livelli di ciclico AMP intracellulare rivela che l’attività di EF inizia dopo 30 minuti dall’intossicazione e dopo 4 ore i livelli sono ancora alti. Queste osservazioni portano due diverse considerazioni. La prima è che la tossina debba compiere un lungo precorso lungo la via di endocitosi prima di raggiungere il citosol. Infatti, i tempi di inizio dell’attività sono compatibili con una traslocazione dai late endosomes. La seconda considerazione è che per diverse ore dall’intossicazione la cellula non riesce a fronteggiare il massiccio aumento di cAMP indotto da ETx e resta a lungo sotto l’effetto di una condizione di disregolazione.
Per indagare il percorso intracellulare e il sito di traslocazione della tossina è stata usata la tecnica della FRET, che permette di analizzare le dinamiche dell’aumento di cAMP indotto da EF. La mappatura di piccole zone all’interno della cellula ha rivelato che il cAMP aumenta prima nelle regioni perinucleari. Questo è in accordo con l’ipotesi della traslocazione dai late endosomes.
Un altro approccio è stato quello di fondere LF e EF a green e red fluorescent proteins. Queste chimere sono uno strumento per tracciare il cammino delle tossine dentro la cellula. Inoltre, la visualizzazione simultanea di ETx e LTx può chiarire le basi della loro azione sinergica. In futuro si potranno fare delle colocalizzazioni con markers cellulari per chiarire i siti di interazione con i componenti dell’ospite.

Statistiche Download - Aggiungi a RefWorks
EPrint type:Ph.D. thesis
Tutor:Montecucco, Cesare
Supervisor:Tonello, Fiorella
Ph.D. course:Dottorato > Scuole di dottorato > Bioscienze
Data di deposito della tesi:31 December 2005
Anno di Pubblicazione:26 March 2006
Key Words:Bacillus anthracis, edema toxin, lethal toxin, fluorescent proteins, cAMP
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/11 Biologia molecolare
Area 05 - Scienze biologiche > BIO/19 Microbiologia generale
Struttura di riferimento:Dipartimenti > pre 2012 - Dipartimento di Scienze Biomediche Sperimentali
Codice ID:119
Depositato il:24 Sep 2009 09:30
Simple Metadata
Full Metadata
EndNote Format


I riferimenti della bibliografia possono essere cercati con Cerca la citazione di AIRE, copiando il titolo dell'articolo (o del libro) e la rivista (se presente) nei campi appositi di "Cerca la Citazione di AIRE".
Le url contenute in alcuni riferimenti sono raggiungibili cliccando sul link alla fine della citazione (Vai!) e tramite Google (Ricerca con Google). Il risultato dipende dalla formattazione della citazione.

BIBLIOGRAFIA Cerca con Google

Abrami, L., M. Lindsay, R.G. Parton, S.H. Leppla, and F.G. van der Goot. 2004.Membrane insertion of anthrax protective antigen and cytoplasmicdelivery of lethal factor occur at different stages of the endocyticpathway. J Cell Biol. 166:645-51. Cerca con Google

Abrami, L., S. Liu, P. Cosson, S.H. Leppla, and F.G. van der Goot. 2003.Anthrax toxin triggers endocytosis of its receptor via a lipid raft-mediatedclathrin-dependent process. J Cell Biol. 160:321-8. Cerca con Google

Adone, R., P. Pasquali, G. La Rosa, C. Marianelli, M. Muscillo, A. Fasanella, M.Francia, and F. Ciuchini. 2002. Sequence analysis of the genes encodingfor the major virulence factors of Bacillus anthracis vaccine strain'Carbosap'. J Appl Microbiol. 93:117-21. Cerca con Google

Ascenzi, P., P. Visca, G. Ippolito, A. Spallarossa, M. Bolognesi, and C.Montecucco. 2002. Anthrax toxin: a tripartite lethal combination. FEBSLett. 531:384-8. Cerca con Google

Axelsson, M.A., N.G. Karlsson, D.M. Steel, J. Ouwendijk, T. Nilsson, and G.C.Hansson. 2001. Neutralization of pH in the Golgi apparatus causesredistribution of glycosyltransferases and changes in the O-glycosylationof mucins. Glycobiology. 11:633-44. Cerca con Google

Bade, S., A. Rummel, C. Reisinger, T. Karnath, G. Ahnert-Hilger, H. Bigalke,and T. Binz. 2004. Botulinum neurotoxin type D enables cytosolicdelivery of enzymatically active cargo proteins to neurones via unfoldedtranslocation intermediates. J Neurochem. 91:1461-72. Cerca con Google

Bradley, K.A., J. Mogridge, M. Mourez, R.J. Collier, and J.A. Young. 2001.Identification of the cellular receptor for anthrax toxin. Nature. 414:225-9. Cerca con Google

Brossier, F., M. Levy, A. Landier, P. Lafaye, and M. Mock. 2004. Functionalanalysis of Bacillus anthracis protective antigen by using neutralizingmonoclonal antibodies. Infect Immun. 72:6313-7. Cerca con Google

Burns, L.L., J.M. Canaves, J.K. Pennypacker, D.K. Blumenthal, and S.S. Taylor.2003. Isoform specific differences in binding of a dual-specificity A-kinaseanchoring protein to type I and type II regulatory subunits of PKA.Biochemistry. 42:5754-63. Cerca con Google

Dell'Aica, I., M. Dona, F. Tonello, A. Piris, M. Mock, C. Montecucco, and S.Garbisa. 2004. Potent inhibitors of anthrax lethal factor from green tea.EMBO Rep. 5:418-22. Cerca con Google

Dodge-Kafka, K.L., J. Soughayer, G.C. Pare, J.J. Carlisle Michel, L.K.Langeberg, M.S. Kapiloff, and J.D. Scott. 2005. The protein kinase Aanchoring protein mAKAP coordinates two integrated cAMP effectorpathways. Nature. 437:574-8. Cerca con Google

Drum, C.L., S.Z. Yan, J. Bard, Y.Q. Shen, D. Lu, S. Soelaiman, Z. Grabarek, A.Bohm, and W.J. Tang. 2002. Structural basis for the activation of anthraxadenylyl cyclase exotoxin by calmodulin. Nature. 415:396-402. Cerca con Google

Drum, C.L., S.Z. Yan, R. Sarac, Y. Mabuchi, K. Beckingham, A. Bohm, Z.Grabarek, and W.J. Tang. 2000. An extended conformation of calmodulininduces interactions between the structural domains of adenylyl cyclasefrom Bacillus anthracis to promote catalysis. J Biol Chem. 275:36334-40. Cerca con Google

Duclos, S., R. Corsini, and M. Desjardins. 2003. Remodeling of endosomesduring lysosome biogenesis involves 'kiss and run' fusion eventsregulated by rab5. J Cell Sci. 116:907-18. Cerca con Google

Fouet, A., and M. Mock. 1996. Differential influence of the two Bacillus anthracisplasmids on regulation of virulence gene expression. Infect Immun.64:4928-32. Cerca con Google

Fuld, S., G. Borland, and S.J. Yarwood. 2005. Elevation of cyclic AMP in JurkatT-cells provokes distinct transcriptional responses through the proteinkinase A (PKA) and exchange protein activated by cyclic AMP (EPAC)pathways. Exp Cell Res. 309:161-73. Cerca con Google

Gruenberg, J. 2001. The endocytic pathway: a mosaic of domains. Nat Rev MolCell Biol. 2:721-30. Cerca con Google

Guidi-Rontani, C., M. Weber-Levy, M. Mock, and V. Cabiaux. 2000. Cerca con Google

Translocation of Bacillus anthracis lethal and oedema factors acrossendosome membranes. Cell Microbiol. 2:259-64. Cerca con Google

Guignot, J., M. Mock, and A. Fouet. 1997. AtxA activates the transcription ofgenes harbored by both Bacillus anthracis virulence plasmids. FEMSMicrobiol Lett. 147:203-7. Cerca con Google

Guo, Q., Y. Shen, N.L. Zhukovskaya, J. Florian, and W.J. Tang. 2004.Structural and kinetic analyses of the interaction of anthrax adenylylcyclase toxin with reaction products cAMP and pyrophosphate. J BiolChem. 279:29427-35. Cerca con Google

Hanks, S., S. Adams, J. Douglas, L. Arbour, D.J. Atherton, S. Balci, H. Bode,M.E. Campbell, M. Feingold, G. Keser, W. Kleijer, G. Mancini, J.A.McGrath, F. Muntoni, A. Nanda, M.D. Teare, M. Warman, F.M. Pope, A.Superti-Furga, P.A. Futreal, and N. Rahman. 2003. Mutations in the geneencoding capillary morphogenesis protein 2 cause juvenile hyaline Cerca con Google

fibromatosis and infantile systemic hyalinosis. Am J Hum Genet. 73:791-800. Cerca con Google

Hoffmaster, A.R., and T.M. Koehler. 1997. The anthrax toxin activator geneatxA is associated with CO2-enhanced non-toxin gene expression inBacillus anthracis. Infect Immun. 65:3091-9. Cerca con Google

Houslay, M.D., and W. Kolch. 2000. Cell-type specific integration of cross-talkbetween extracellular signal-regulated kinase and cAMP signaling. MolPharmacol. 58:659-68. Cerca con Google

Kumar, P., N. Ahuja, and R. Bhatnagar. 2002. Anthrax edema toxin requiresinflux of calcium for inducing cyclic AMP toxicity in target cells. InfectImmun. 70:4997-5007. Cerca con Google

Ladant, D., and A. Ullmann. 1999. Bordatella pertussis adenylate cyclase: atoxin with multiple talents. Trends Microbiol. 7:172-6. Cerca con Google

Le Blanc, I., P.P. Luyet, V. Pons, C. Ferguson, N. Emans, A. Petiot, N. Mayran,N. Demaurex, J. Faure, R. Sadoul, R.G. Parton, and J. Gruenberg. 2005. Cerca con Google

Endosome-to-cytosol transport of viral nucleocapsids. Nat Cell Biol.7:653-64. Cerca con Google

Leppla, S.H. 1988. Production and purification of anthrax toxin. MethodsEnzymol. 165:103-16. Cerca con Google

Llopis, J., J.M. McCaffery, A. Miyawaki, M.G. Farquhar, and R.Y. Tsien. 1998.Measurement of cytosolic, mitochondrial, and Golgi pH in single livingcells with green fluorescent proteins. Proc Natl Acad Sci U S A. 95:6803-8. Cerca con Google

Mock, M., and T. Mignot. 2003. Anthrax toxins and the host: a story of intimacy.Cell Microbiol. 5:15-23. Cerca con Google

Mongillo, M., A. Terrin, S. Evellin, V. Lissandron, and M. Zaccolo. 2005. Studyof cyclic adenosine monophosphate microdomains in cells. Methods MolBiol. 307:1-13. Cerca con Google

Mourez, M., R.S. Kane, J. Mogridge, S. Metallo, P. Deschatelets, B.R. Sellman,G.M. Whitesides, and R.J. Collier. 2001. Designing a polyvalent inhibitorof anthrax toxin. Nat Biotechnol. 19:958-61. Cerca con Google

Nassi, S., R.J. Collier, and A. Finkelstein. 2002. PA63 channel of anthrax toxin:an extended beta-barrel. Biochemistry. 41:1445-50. Cerca con Google

Paccani, S.R., F. Tonello, R. Ghittoni, M. Natale, L. Muraro, M.M. D'Elios, W.J.Tang, C. Montecucco, and C.T. Baldari. 2005. Anthrax toxins suppress Tlymphocyte activation by disrupting antigen receptor signaling. J ExpMed. 201:325-31. Cerca con Google

Pannifer, A.D., T.Y. Wong, R. Schwarzenbacher, M. Renatus, C. Petosa, J.Bienkowska, D.B. Lacy, R.J. Collier, S. Park, S.H. Leppla, P. Hanna, andR.C. Liddington. 2001. Crystal structure of the anthrax lethal factor.Nature. 414:229-33. Cerca con Google

Park, J.M., F.R. Greten, Z.W. Li, and M. Karin. 2002. Macrophage apoptosis byanthrax lethal factor through p38 MAP kinase inhibition. Science.297:2048-51. Cerca con Google

Pini, A., Y. Runci, C. Falciani, B. Lelli, J. Brunetti, S. Pileri, M. Fabbrini, L. Lozzi,C. Ricci, A. Bernini, F. Tonello, F. Dal Molin, P. Neri, N. Niccolai, and L.Bracci. 2006. Stable peptide inhibitors prevent binding of lethal andedema factors to protective antigen and neutralize anthrax toxin in vivo.Biochem J. Cerca con Google

Rainey, G.J., D.J. Wigelsworth, P.L. Ryan, H.M. Scobie, R.J. Collier, and J.A.Young. 2005. Receptor-specific requirements for anthrax toxin deliveryinto cells. Proc Natl Acad Sci U S A. 102:13278-83. Cerca con Google

Scobie, H.M., G.J. Rainey, K.A. Bradley, and J.A. Young. 2003. Humancapillary morphogenesis protein 2 functions as an anthrax toxin receptor.Proc Natl Acad Sci U S A. 100:5170-4. Cerca con Google

Scobie, H.M., and J.A. Young. 2005. Interactions between anthrax toxinreceptors and protective antigen. Curr Opin Microbiol. 8:106-12. Cerca con Google

Shaner, N.C., R.E. Campbell, P.A. Steinbach, B.N. Giepmans, A.E. Palmer, andR.Y. Tsien. 2004. Improved monomeric red, orange and yellowfluorescent proteins derived from Discosoma sp. red fluorescent protein.Nat Biotechnol. 22:1567-72. Cerca con Google

Shen, Y., N.L. Zhukovskaya, Q. Guo, J. Florian, and W.J. Tang. 2005. Calciumindependentcalmodulin binding and two-metal-ion catalytic mechanismof anthrax edema factor. Embo J. 24:929-41. Cerca con Google

Shen, Y., N.L. Zhukovskaya, M.I. Zimmer, S. Soelaiman, P. Bergson, C.R.Wang, C.S. Gibbs, and W.J. Tang. 2004. Selective inhibition of anthraxedema factor by adefovir, a drug for chronic hepatitis B virus infection.Proc Natl Acad Sci U S A. 101:3242-7. Cerca con Google

Sirard, J.C., M. Mock, and A. Fouet. 1994. The three Bacillus anthracis toxingenes are coordinately regulated by bicarbonate and temperature. JBacteriol. 176:5188-92. Cerca con Google

Tonello, F., L. Naletto, V. Romanello, F. Dal Molin, and C. Montecucco. 2004.Tyrosine-728 and glutamic acid-735 are essential for themetalloproteolytic activity of the lethal factor of Bacillus anthracis.Biochem Biophys Res Commun. 313:496-502. Cerca con Google

Tonello, F., M. Seveso, O. Marin, M. Mock, and C. Montecucco. 2002.Screening inhibitors of anthrax lethal factor. Nature. 418:386. Cerca con Google

Vitale, G., R. Pellizzari, C. Recchi, G. Napolitani, M. Mock, and C. Montecucco.1998. Anthrax lethal factor cleaves the N-terminus of MAPKKs andinduces tyrosine/threonine phosphorylation of MAPKs in culturedmacrophages. Biochem Biophys Res Commun. 248:706-11. Cerca con Google

Wigelsworth, D.J., B.A. Krantz, K.A. Christensen, D.B. Lacy, S.J. Juris, and R.J.Collier. 2004. Binding stoichiometry and kinetics of the interaction of ahuman anthrax toxin receptor, CMG2, with protective antigen. J BiolChem. 279:23349-56. Cerca con Google

Wright, G.G., and G.L. Mandell. 1986. Anthrax toxin blocks priming ofneutrophils by lipopolysaccharide and by muramyl dipeptide. J Exp Med.164:1700-9. Cerca con Google

Yao, H., R.D. York, A. Misra-Press, D.W. Carr, and P.J. Stork. 1998. The cyclicadenosine monophosphate-dependent protein kinase (PKA) is requiredfor the sustained activation of mitogen-activated kinases and geneexpression by nerve growth factor. J Biol Chem. 273:8240-7. Cerca con Google

Zaccolo, M. 2004. Use of chimeric fluorescent proteins and fluorescenceresonance energy transfer to monitor cellular responses. Circ Res.94:866-73. Cerca con Google

Zacharias, D.A., J.D. Violin, A.C. Newton, and R.Y. Tsien. 2002. Partitioning oflipid-modified monomeric GFPs into membrane microdomains of livecells. Science. 296:913-6. Cerca con Google

Zhang, S., K. Cunningham, and R.J. Collier. 2004. Anthrax protective antigen:efficiency of translocation is independent of the number of ligands boundto the prepore. Biochemistry. 43:6339-43. Cerca con Google

Download statistics

Solo per lo Staff dell Archivio: Modifica questo record