title: Protective role of the dynamin inhibitor Dynasore against the cholesterol-dependent cytolysin of Trueperella pyogenes.
abstract: The virulence of many Gram-positive bacteria depends on cholesterol-dependent cytolysins (CDCs), which form pores in eukaryotic cell plasma membranes. Pyolysin (PLO) from Trueperella pyogenes provided a unique opportunity to explore cellular responses to CDCs because it does not require thiol activation. Sublytic concentrations of PLO stimulated phosphorylation of MAPK ERK and p38 in primary stromal cells, and induced autophagy as determined by protein light-chain 3B cleavage. Although, inhibitors of MAPK or autophagy did not affect PLO-induced cytolysis. However, 10 μM 3-hydroxynaphthalene-2-carboxylic acid-(3,4-dihydroxybenzylidene)-hydrazide (Dynasore), a dynamin guanosine 5'-triphosphatase inhibitor, protected stromal cells against PLO-induced cytolysis as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (85 ± 17% versus 50 ± 9% cell viability), measuring extracellular ATP, and kinetic assays. This was a generalized mechanism because Dynasore also protected HeLa cells against streptolysin O. Furthermore, the effect was reversible, with stromal cell sensitivity to PLO restored within 30 minutes of Dynasore removal. The protective effect of Dynasore was not conferred by dynamin inhibition, induction of ERK phosphorylation, or Dynasore binding to PLO. Rather, Dynasore reduced cellular cholesterol and disrupted plasma membrane lipid rafts, similar to positive control methyl-β-cyclodextrin. Dynasore is a tractable tool to explore the complexity of cholesterol homeostasis in eukaryotic cells and to develop strategies to counter CDCs.
authors.affiliation: Institute of Life Science, College of Medicine, Swansea University, Swansea, United Kingdom.
authors.forename: Giulio
authors.initials: G
authors.lastname: Preta
authors.affiliation: Institute of Life Science, College of Medicine, Swansea University, Swansea, United Kingdom.
authors.forename: Virginia
authors.initials: V
authors.lastname: Lotti
authors.affiliation: Institute of Life Science, College of Medicine, Swansea University, Swansea, United Kingdom.
authors.forename: James G
authors.initials: JG
authors.lastname: Cronin
authors.affiliation: Institute of Life Science, College of Medicine, Swansea University, Swansea, United Kingdom i.m.sheldon@swansea.ac.uk.
authors.forename: I Martin
authors.initials: IM
authors.lastname: Sheldon
mesh_terms: D000191:Actinomycetaceae / Q000472:pathogenicity*; D000818:Animals; D001343:Autophagy / Q000187:drug effects; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000633:toxicity; D002417:Cattle; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D003603:Cytotoxins / Q000037:antagonists & inhibitors* / Q000633:toxicity*; D034281:Dynamins / Q000037:antagonists & inhibitors*; D004717:Endometrium / Q000187:drug effects / Q000378:metabolism / Q000382:microbiology; D005260:Female; D006367:HeLa Cells; D006801:Humans; D006835:Hydrazones / Q000494:pharmacology*; D020935:MAP Kinase Signaling System / Q000187:drug effects; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism; D008954:Models, Biological; D013301:Streptolysins / Q000037:antagonists & inhibitors / Q000633:toxicity; D017154:Stromal Cells / Q000187:drug effects / Q000378:metabolism / Q000382:microbiology
chemical_list: D001426:Bacterial Proteins; D003603:Cytotoxins; D006835:Hydrazones; C511794:N'-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthahydrazide; D013301:Streptolysins; C025964:streptolysin O; D002784:Cholesterol; D034281:Dynamins
keywords: bacteria; host-pathogen interaction; lipid rafts; mammalian cell survival