title: Cholesterol-dependent actin remodeling via RhoA and Rac1 activation by the Streptococcus pneumoniae toxin pneumolysin.
abstract: The Streptococcus pneumoniae toxin pneumolysin belongs to the group of cholesterol-dependent cytolysins. It produces rapid cell lysis at higher concentrations or apoptosis at lower concentrations. In cell membranes, it forms prepores and pores. Here, we show that sublytic concentrations of pneumolysin produce rapid activation of Rho and Rac GTPases and formation of actin stress fibers, filopodia, and lamellipodia. That Rac1-specific and Rho-associated kinase (ROCK)-specific inhibitors reverted the formation of lamellipodia and stress fibers, respectively, identifies RhoA and Rac1 as key toxin effectors. Live imaging excluded macropore formation (as judged by membrane impermeability toward calcein) but indicated very early membrane depolarization [as judged by bis-(1,3-dibutylbarbituric acid)trimethine oxanol staining], indicative of formation of micropores with ion channel properties. That Rac1-dependent lamellipodia formation was reverted by the voltage-gated calcium channel inhibitor SKF96365 and by toxin exposure in calcium-free medium suggests a role for calcium influx via endogenous calcium channels in the Rac1 activation. Cellular cholesterol depletion by methyl-beta-cyclodextrin or incubation of the toxin with cholesterol before cell treatment eliminated its membrane binding and the subsequent GTPase activation. Thus, that our experiments show small GTPase activation by a cholesterol-dependent cytolysin suggests a membrane cholesterol-dependent activation mechanism.
authors.affiliation: Cell Biophysics Group, European Neuroscience Institute Göttingen, Waldweg 33, 37073 Göttingen, Germany. ailiev@gwdg.de
authors.forename: Asparouh I
authors.initials: AI
authors.lastname: Iliev
authors.forename: Jasmin Roya
authors.initials: JR
authors.lastname: Djannatian
authors.forename: Roland
authors.initials: R
authors.lastname: Nau
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Mitchell
authors.forename: Fred S
authors.initials: FS
authors.lastname: Wouters
mesh_terms: D000199:Actins / Q000378:metabolism*; D001426:Bacterial Proteins / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002784:Cholesterol / Q000494:pharmacology*; D022162:Cytoplasmic Vesicles / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006801:Humans; D010641:Phenotype; D011554:Pseudopodia / Q000187:drug effects; D013301:Streptolysins / Q000494:pharmacology*; D022502:Stress Fibers / Q000187:drug effects; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: D000199:Actins; D001426:Bacterial Proteins; D020662:Guanine Nucleotide Exchange Factors; D013301:Streptolysins; C032290:plY protein, Streptococcus pneumoniae; D002784:Cholesterol; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein; D002118:Calcium