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Protective role of the dynamin inhibitor Dynasore against the cholesterol-dependent cytolysin of Trueperella pyogenes / James, Cronin; Martin, Sheldon

The FASEB Journal, Volume: 29, Issue: 4, Pages: 1516 - 1528

Swansea University Authors: James, Cronin, Martin, Sheldon

DOI (Published version): 10.1096/fj.14-265207

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 activa...

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Published in: The FASEB Journal
Published: 2015
Online Access: http://www.fasebj.org/content/29/4/1516.full
URI: https://cronfa.swan.ac.uk/Record/cronfa19859
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spelling 2018-07-26T10:40:41.3306275 v2 19859 2015-01-05 Protective role of the dynamin inhibitor Dynasore against the cholesterol-dependent cytolysin of Trueperella pyogenes 9cfd17551c0d1f7438895121e4fbb6e8 0000-0002-0590-9462 James Cronin James Cronin true false ab0f74b794e59cc270c69e63ee1d9748 0000-0001-7902-5558 Martin Sheldon Martin Sheldon true false 2015-01-05 BMS 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.—Preta, G., Lotti, V., Cronin, J. G., and Sheldon, I. M. Protective role of the dynamin inhibitor Dynasore against the cholesterol-dependent cytolysin of Trueperella pyogenes. Journal Article The FASEB Journal 29 4 1516 1528 1 4 2015 2015-04-01 10.1096/fj.14-265207 http://www.fasebj.org/content/29/4/1516.full This work was funded by the United Kingdom Biotechnology and Biological Sciences Research Council (Grant BB/K006592/1).Giulio Preta was a postdoctoral assistant working for Sheldon, and funded by Sheldon's BBSRC grant. COLLEGE NANME Biomedical Sciences COLLEGE CODE BMS Swansea University 2018-07-26T10:40:41.3306275 2015-01-05T16:33:18.6440915 Swansea University Medical School Medicine G. Preta 1 V. Lotti 2 J. G. Cronin 3 I. M. Sheldon 4 James Cronin 0000-0002-0590-9462 5 Martin Sheldon 0000-0001-7902-5558 6 0019859-16062017082406.pdf PretaFASEB.pdf 2017-06-16T08:24:06.9600000 Output 2354274 application/pdf Version of Record true 2014-11-20T00:00:00.0000000 true eng
title Protective role of the dynamin inhibitor Dynasore against the cholesterol-dependent cytolysin of Trueperella pyogenes
spellingShingle Protective role of the dynamin inhibitor Dynasore against the cholesterol-dependent cytolysin of Trueperella pyogenes
James, Cronin
Martin, Sheldon
title_short Protective role of the dynamin inhibitor Dynasore against the cholesterol-dependent cytolysin of Trueperella pyogenes
title_full Protective role of the dynamin inhibitor Dynasore against the cholesterol-dependent cytolysin of Trueperella pyogenes
title_fullStr Protective role of the dynamin inhibitor Dynasore against the cholesterol-dependent cytolysin of Trueperella pyogenes
title_full_unstemmed Protective role of the dynamin inhibitor Dynasore against the cholesterol-dependent cytolysin of Trueperella pyogenes
title_sort Protective role of the dynamin inhibitor Dynasore against the cholesterol-dependent cytolysin of Trueperella pyogenes
author_id_str_mv 9cfd17551c0d1f7438895121e4fbb6e8
ab0f74b794e59cc270c69e63ee1d9748
author_id_fullname_str_mv 9cfd17551c0d1f7438895121e4fbb6e8_***_James, Cronin
ab0f74b794e59cc270c69e63ee1d9748_***_Martin, Sheldon
author James, Cronin
Martin, Sheldon
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url http://www.fasebj.org/content/29/4/1516.full
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description 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.—Preta, G., Lotti, V., Cronin, J. G., and Sheldon, I. M. Protective role of the dynamin inhibitor Dynasore against the cholesterol-dependent cytolysin of Trueperella pyogenes.
published_date 2015-04-01T03:36:32Z
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