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Projekt Druckansicht

Modification of actin by bacterial protein toxins and effectors

Fachliche Zuordnung Zellbiologie
Förderung Förderung von 2003 bis 2011
Projektkennung Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 5407435
 
Erstellungsjahr 2011

Zusammenfassung der Projektergebnisse

This project focused on bacterial virulence factors which mono-ADP-ribosylate actin and thereby directly attack the cytoskeleton of eukaryotic cells. The binary C2 toxin from Clostridium botulinum has a transport component (C2IIa) that forms a complex with and delivers the separate enzyme component (C2I) into the cytosol while Salmonella enterica SpvB is directly delivered from intracellular located bacteria into the cytosol. We characterized the mode of action of the catalytic domain of SpvB (C/SpvB) in vitro and in intact cells in direct comparison to C2 toxin. During the first period, we characterized C/SpcB in vitro and identified arginine-177 as the modification-site for SpvB in the actin homologue Act(88F) from Drosophila. As for C2I and related binary clostridial actin-ADP-ribosylating toxins, G-actin was the preferred substrate for SpvB but in contrast to the binary toxins, SpvB had no significant NAD glycohydrolase activity. During the second period, we investigated the mode of action of C/SpvB versus C2I in intact cells and the cellular responses following toxin-catalyzed ADP-ribosylation of actin. Since SpvB is not taken up into cells, we constructed, expressed and purified a recombinant fusion toxin (C2IN-C/SpvB) to deliver C/SpvB into the cytosol via the C2IIa transporter of C2 toxin. As C2I, C/SpvB ADP-ribosylated G-actin at arginine-177 in living cells, leading to depolymerization of F-actin and thereby to cell rounding. ADP-ribosylation did not trigger degradation of actin in the mammalian cells (HeLa, Vero, CaCo-2) used in this study. The destruction of actin filaments did not emanate from a defined region of the cell but filaments likely started to depolymerize from the cytoplasmic regions surrounding the endosomes, from which C2I and C/SpvB entered the cytosol, as investigated in cells expressing EGFP-actin. No further substrate besides actin was found in cells, both C/SpvB and C2I exclusively ADP-ribosylated actin. Thus, the overall mode of action of C/SpvB and C2I in intact cells was quite similar. However, in contrast to C2 toxin or related binary actin ADP-ribosylating toxins, which induced caspase activation and apoptosis after about 24 h. The cytopathic effect of C2IN-C/SpvB was transient due to its degradation in the host cell cytosol. Most C2IN-C/SpvB-treated round cells resumed a flat and significantly enlarged morphology, rebuilt F-actin structures after several hours and the amount of non-ADP-ribosylated actin was increasing over time in such cells. The cells failed to divide, resulting in bi- and multinuclear cells, which stayed viable for a few days. In the SpvB protein, 7 proline residues link the N-terminal domain to C/SpvB and in C2IN-C/SpvB, the presence of a poly-proline region between C2IN and C/SpvB was essential for the translocation of C2IN-C/SpvB across cell membranes into the cytosol. This result led to the observation that host cell peptidyl prolyl "cis/trans" isomerases such as cyclophilins facilitate the intracellular translocation of actin ADP-ribosylating toxins through trans-membrane pores.

Projektbezogene Publikationen (Auswahl)

 
 

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