title: Clostridium difficile binary toxin CDT induces clustering of the lipolysis-stimulated lipoprotein receptor into lipid rafts.
abstract: Clostridium difficile is the leading cause of antibiotics-associated diarrhea and pseudomembranous colitis. Hypervirulent C. difficile strains produce the binary actin-ADP-ribosylating toxin CDT (C. difficile transferase), in addition to the Rho-glucosylating toxins A and B. We recently identified the lipolysis-stimulated lipoprotein receptor (LSR) as the host receptor that mediates uptake of CDT into target cells. Here we investigated in H1-HeLa cells, which ectopically express LSR, the influence of CDT on the plasma membrane distribution of the receptor. We found by fluorescence microscopy that the binding component of CDT (CDTb) induces clustering of LSR into subcompartments of the plasma membrane. Detergent extraction of cells treated with CDTb, followed by sucrose gradient fractionation, uncovered accumulation of LSR in detergent-resistant membranes (DRMs) that contained typical marker proteins of lipid rafts. Membrane cholesterol depletion with methyl-β-cyclodextrin inhibited the association of LSR with DRMs upon addition of CDTb. The receptor-binding domain of CDTb also triggered LSR clustering into DRMs. CDTb-triggered clustering of LSR into DRMs could be confirmed in Caco-2 cells. Our data suggest that CDT forces its receptor to cluster into lipid rafts and that oligomerization of the B component might enhance but is not essential for this process.
  
  
  OBJECTIVE
  C. difficile binary toxin CDT is a member of the iota-like, actin ADP-ribosylating toxin family. The mechanism that mediates endocytic uptake of these toxins still remains elusive. Previous studies highlighted the importance of lipid rafts for oligomerization of the binding component of these toxins and for cell entry. Recently, the host cell receptor for this toxin family, namely, the lipolysis-stimulated lipoprotein receptor (LSR), has been identified. Our study now demonstrates that the binding component of CDT (CDTb) induces clustering of LSR into lipid rafts. Importantly, LSR clustering is efficiently induced also by the receptor-binding domain of CDTb, suggesting that oligomerization of the B component of CDT is not the main trigger of this process. The current work extends our knowledge on the cooperative play between iota-like toxins and their receptor.
authors: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany.
authors: Panagiotis
authors: P
authors: Papatheodorou
authors: Daniel
authors: D
authors: Hornuss
authors: Thilo
authors: T
authors: Nölke
authors: Sarah
authors: S
authors: Hemmasi
authors: Jan
authors: J
authors: Castonguay
authors: Monica
authors: M
authors: Picchianti
authors: Klaus
authors: K
authors: Aktories
mesh_terms: D036002:ADP Ribose Transferases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D018938:Caco-2 Cells; D002462:Cell Membrane / Q000737:chemistry* / Q000187:drug effects*; D016360:Clostridioides difficile / Q000378:metabolism*; D004252:DNA Mutational Analysis; D006367:HeLa Cells; D006801:Humans; D021962:Membrane Microdomains / Q000737:chemistry* / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D017434:Protein Structure, Tertiary; D018110:Receptors, Lipoprotein / Q000032:analysis*
chemical_list: D001426:Bacterial Proteins; D018110:Receptors, Lipoprotein; D036002:ADP Ribose Transferases; C410164:actin-specific ADP-ribosyltransferase, Clostridium
references: Infect Immun. 2006 Dec;74(12):6571-80
references: 17030583
references: Mol Cell Biol. 2005 Jul;25(13):5752-62
references: 15964828
references: J Biol Chem. 2000 Oct 13;275(41):32363-70
references: 10921915
references: Curr Opin Lipidol. 1998 Jun;9(3):221-4
references: 9645504
references: Infect Immun. 2012 Oct;80(10):3410-6
references: 22825447
references: PLoS Pathog. 2009 Oct;5(10):e1000626
references: 19834554
references: Toxicon. 2012 Sep 15;60(4):572-81
references: 22543189
references: J Biol Chem. 1997 Jul 11;272(28):17744-8
references: 9211926
references: J Biol Chem. 2004 Oct 22;279(43):45175-84
references: 15302864
references: Infect Immun. 2002 Apr;70(4):1909-14
references: 11895954
references: Infect Immun. 2004 Jun;72(6):3267-75
references: 15155629
references: J Biol Chem. 2011 Aug 19;286(33):29356-29365
references: 21705797
references: Microbiol Mol Biol Rev. 2004 Sep;68(3):373-402, table of contents
references: 15353562
references: Infect Immun. 2001 Apr;69(4):2435-41
references: 11254604
references: Infect Immun. 2010 Apr;78(4):1468-74
references: 20145093
references: Cell Microbiol. 2011 Jan;13(1):154-70
references: 20846184
references: Infect Immun. 2001 May;69(5):2980-7
references: 11292715
references: Nature. 1986 Jul 24-30;322(6077):390-2
references: 3736664
references: J Biol Chem. 2000 Jun 23;275(25):18704-11
references: 10749859
references: Infect Immun. 2012 Apr;80(4):1418-23
references: 22252869
references: Proc Natl Acad Sci U S A. 2011 Sep 27;108(39):16422-7
references: 21930894
references: Cell. 1992 Feb 7;68(3):533-44
references: 1531449
references: Nat Rev Microbiol. 2009 Jul;7(7):526-36
references: 19528959
references: J Cell Biol. 2003 Feb 3;160(3):321-8
references: 12551953
references: Mol Cell. 2001 Mar;7(3):661-71
references: 11463390
references: J Cell Sci. 2011 Feb 15;124(Pt 4):548-55
references: 21245199
references: Infect Immun. 2000 Jun;68(6):3475-84
references: 10816501
references: J Proteome Res. 2005 Mar-Apr;4(2):523-31
references: 15822930
references: Infect Immun. 2011 Oct;79(10):3913-21
references: 21768281
references: Eur J Biochem. 2004 Aug;271(15):3103-14
references: 15265030
references: PLoS One. 2012;7(12):e51356
references: 23236484
references: N Engl J Med. 2005 Dec 8;353(23):2433-41
references: 16322603
references: Nat Rev Microbiol. 2011 Jun 16;9(7):487-98
references: 21677684
references: Infect Immun. 2004 Apr;72(4):2186-93
references: 15039342
references: Biochem J. 2002 Nov 1;367(Pt 3):801-8
references: 12175336
references: J Biol Chem. 1999 May 7;274(19):13390-8
references: 10224102
references: J Biol Chem. 2000 Feb 11;275(6):4417-21
references: 10660613