Clostridium difficile toxin CDT hijacks microtubule organization and reroutes vesicle traffic to increase pathogen adherence Carsten Schwan a,1 , Anna S. Kruppke a,1 , Thilo Nölke a , Lucas Schumacher b , Friedrich Koch-Nolte b , Mikhail Kudryashev c , Henning Stahlberg c , and Klaus Aktories a,d,2 a Institute of Experimental and Clinical Pharmacology and Toxicology, Albert-Ludwigs-University of Freiburg, 79104 Freiburg, Germany; b Universitätsklinikum Hamburg Eppendorf, Institute of Immunology, 20246 Hamburg, Germany; c Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, CH-4058 Basel, Switzerland; and d Centre for Biological Signalling Studies (BIOSS), Albert-Ludwigs-University of Freiburg, 79104 Freiburg, Germany Edited by Rino Rappuoli, Novartis Vaccines and Diagnostics Srl, Siena, Italy, and approved December 30, 2013 (received for review June 18, 2013) Clostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis by the actions of Rho-glucosylating toxins A and B. Recently identified hypervirulent strains, which are associated with increased morbidity and mortality, addition- ally produce the actin-ADP–ribosylating toxin C. difficile transfer- ase (CDT). CDT depolymerizes actin, causes formation of microtubule- based protrusions, and increases pathogen adherence. Here we show that CDT-induced protrusions allow vesicle traffic and contain endo- plasmic reticulum tubules, connected to microtubules via the calcium sensor Stim1. The toxin reroutes Rab11-positive vesicles containing fibronectin, which is involved in bacterial adherence, from basolateral to the apical membrane sides in a microtubule- and Stim1-dependent manner. The data yield a model of C. difficile adherence regulated by actin depolymerization, microtubule restructuring, subsequent Stim1-dependent Ca 2+ signaling, vesicle rerouting, and secretion of ECM proteins to increase bacterial adherence. T he anaerobe and spore-forming bacterium Clostridium difficile is the causative pathogen of pseudomembranous colitis and antibiotic-associated diarrhea. During recent years, morbidity and mortality of C. difficile infections (CDIs) increased and emerged as a major health threat in developed countries (1). Main pathogenic factors of C. difficile are the Rho-inactivating toxins A and B (2, 3). Recently, so-called hypervirulent strains (e.g., NAP1/027) have been associated with severe courses of CDIs (4). These strains have deletions in a regulatory gene of the pathogenicity locus, are resistant towards fluoroquinolons, and produce the binary toxin C. difficile transferase (CDT). CDT ADP ribosylates actin at arginine-177, thereby inhibiting actin poly- merization (5–7). CDT-induced depolymerization of F-actin induces formation of long microtubule-based protrusions, which form a meshwork on the surface of intestinal host cells (8). Clostridia are embedded in this microtubule-based meshwork, resulting in increased host cell adherence. Here, we report that toxin-induced protrusions contain vesi- cles and tubular ER structures, which are functionally linked to microtubules and to the cell membrane via Stim1. The data in- dicate that CDT alters the secretory machinery of host cells and reroutes ECM proteins like fibronectin from basolateral to the apical surface of host cells. This redistribution increases adherence and colonization of clostridia in a microtubule-dependent manner. Results CDT-Induced Protrusions Contain ER Membranes. Cryo-electron to- mography of human coloncarcinoma (Caco-2) cells revealed that CDT-induced protrusions contained intracellular membranes, resembling membrane vesicles and membrane tubules (Fig. 1 A and B). The membranes were frequently found along the pro- trusion stem close to the plasma membrane or at the protrusion tip, where protrusions often form a bulbous head. Because microtubules interact with the ER (9), we studied ER dynamics using the ER localization signal peptide of calreticulin as a GFP fusion protein (Fig. 1C). In line with the electron tomography data, CDT-induced protrusion formation was accompanied by translocation of ER into the protrusions. About 60% of the protrusions contained ER structures. The ER tubules revealed dynamic movements similar to microtubule-based protrusions (Movie S1). Notably, protruding ER tubules remained in contact with the entire cellular ER network and were apparently not physically separated (Fig. 1C). Formation of protrusions was also observed in other intestinal epithelial cell lines (e.g., T84 and HCT116 cells; Fig. S1A). Moreover, CDT-induced redistribution of the ER did not induce ER stress (Fig. S1 B and C). Stim1 Is Involved in CDT-Induced ER Translocation. Next we studied Stim1, which is involved in the interaction of the ER with microtubules via the tip attachment complex (TAC) (9). Mi- croscopic time lapse studies revealed that Stim1 comets moved constantly into the protrusions (Movie S2). Transfection of shRNA targeting Stim1 (Fig. 1D) in Caco-2 cells reduced the number of ER tubules in protrusions from ∼60% to ∼30%. Control shRNA had no effect on ER in protrusions. In line with these findings, Stim1, lacking the EB1 binding site, reduced the number of ER tubules in protrusions from 60% to 45%, whereas the overexpression of WT Stim1 increased the number of pro- trusions containing ER from 60% to >75%. Similar results were obtained in HeLa cells stably expressing the CDT receptor lipolysis-stimulated lipoprotein receptor (LSR; Fig. S2 A and B). Significance Hypervirulent strains of Clostridium difficile frequently pro- duce the actin-ADP–ribosylating toxin Clostridium difficile transferase (CDT), which increases bacteria adherence by for- mation of microtubule-based protrusions. Here we report that CDT-induced protrusions contain trafficking vesicles and en- doplasmic reticulum, connected to microtubules via the calcium sensor Stim1. CDT increases calcium signaling and reroutes fibronectin-containing vesicles from the basolateral to the apical side of intestinal epithelial cells, where protrusions are formed. Released fibronectin enhances adherence of bacteria. The data reassess the role of the actin cytoskeleton in bacterial adherence and infection. Author contributions: C.S., A.S.K., F.K.-N., M.K., H.S., and K.A. designed research; C.S., A.S.K., T.N., L.S., and M.K. performed research; L.S., F.K.-N., M.K., and H.S. contributed new re- agents/analytic tools; C.S., A.S.K., F.K.-N., and K.A. analyzed data; and C.S., A.S.K., and K.A. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1 C.S. and A.S.K. contributed equally to this work. 2 To whom correspondence should be addressed. E-mail: [email protected] freiburg.de. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1311589111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1311589111 PNAS | February 11, 2014 | vol. 111 | no. 6 | 2313–2318 MICROBIOLOGY Downloaded from https://www.pnas.org by 14.250.91.15 on August 13, 2023 from IP address 14.250.91.15.
Welcome message from author
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Related Documents
