title: Actin ADP-ribosylation at Threonine148 by Photorhabdus luminescens toxin TccC3 induces aggregation of intracellular F-actin.
abstract: Intoxication of eukaryotic cells by Photorhabdus luminescens toxin TccC3 induces cell rounding and detachment from the substratum within a few hours and compromises a number of cell functions like phagocytosis. Here, we used morphological and biochemical procedures to analyse the mechanism of TccC3 intoxication. Life imaging of TccC3-intoxicated HeLa cells transfected with AcGFP-actin shows condensation of F-actin into large aggregates. Life cell total internal reflection fluorescence (TIRF) microscopy of identically treated HeLa cells confirmed the formation of actin aggregates but also disassembly of F-actin stress fibres. Recombinant TccC3 toxin ADP-ribosylates purified skeletal and non-muscle actin at threonine148 leading to a strong propensity to polymerize and F-actin bundle formation as shown by TIRF and electron microscopy. Native gel electrophoresis shows strongly reduced binding of Thr148-ADP-ribosylated actin to the severing proteins gelsolin and its fragments G1 and G1-3, and to ADF/cofilin. Complexation of actin with these proteins inhibits its ADP-ribosylation. TIRF microscopy demonstrates rapid polymerization of Thr148-ADP-ribosylated actin to curled F-actin bundles even in the presence of thymosin β4, gelsolin or G1-3. Thr148-ADP-ribosylated F-actin cannot be depolymerized by gelsolin or G1-3 as verified by TIRF, co-sedimentation and electron microscopy and shows reduced treadmilling as indicated by a lack of stimulation of its ATPase activity after addition of cofilin-1.
authors.affiliation: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany.
authors.forename: Alexander E
authors.initials: AE
authors.lastname: Lang
authors.affiliation: Abteilung für Anatomie und Molekulare Embryologie, Ruhr-Universität Bochum, Bochum, Germany.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Qu
authors.affiliation: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Schwan
authors.affiliation: ETH Zürich, Institute for Biomechanics, University of Zürich, Balgrist Campus, Zürich, Switzerland.
authors.forename: Unai
authors.initials: U
authors.lastname: Silvan
authors.affiliation: Department of Cardiovascular Physiology, Ruhr-University Bochum, Bochum, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Unger
authors.affiliation: Focal Area Structural Biology and Biophysics, Biozentrum, University of Basel, Switzerland.
authors.forename: Cora-Ann
authors.initials: CA
authors.lastname: Schoenenberger
authors.affiliation: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany.|Centre for Biological Signalling Studies (BIOSS), University of Freiburg, Freiburg, Germany.|Freiburg Institute of Advanced Studies (FRIAS), University of Freiburg, Freiburg, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Aktories
authors.affiliation: Abteilung für Anatomie und Molekulare Embryologie, Ruhr-Universität Bochum, Bochum, Germany.
authors.forename: Hans Georg
authors.initials: HG
authors.lastname: Mannherz
mesh_terms: D000199:Actins / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D004847:Epithelial Cells / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D020637:Photorhabdus / Q000378:metabolism*; D066263:Protein Aggregation, Pathological
chemical_list: D000199:Actins; D001427:Bacterial Toxins; D000244:Adenosine Diphosphate