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

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Abstract (inglese)

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 (italiano)

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.

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Tipo di EPrint:Tesi di dottorato
Relatore:Montecucco, Cesare
Correlatore:Tonello, Fiorella
Dottorato (corsi e scuole):Dottorato > Scuole di dottorato > Bioscienze
Data di deposito della tesi:31 Dicembre 2005
Anno di Pubblicazione:26 Marzo 2006
Parole chiave (italiano / inglese):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 > Dipartimento di Scienze Biomediche Sperimentali
Codice ID:119
Depositato il:24 Set 2009 09:30
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