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.—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.
Highlights
According to the MTT data, the LD50 was approximately 100 hemolytic units (HU) after 1 hour; stromal cells were treated with a sublytic concentration of 50 HU PLO for up to 20 minutes, using LPS as a positive control to induce phosphorylation of MAPK [23] (Supplemental Fig. 1A)
To further confirm that the effect on the MAPK pathway was due to the effect of PLO, cells were treated with the dsPLO, which is able to bind to cell membranes but does not form oligomers [22]
Because T. pyogenes commonly causes endometritis [9], primary bovine endometrial stromal cells were used to determine cellular response to PLO, because these stromal cells are highly sensitive to PLO [15]
Summary
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. 10 mM 3-hydroxynaphthalene-2-carboxylic acid-(3,4dihydroxybenzylidene)-hydrazide (Dynasore), a dynamin guanosine 59-triphosphatase inhibitor, protected stromal cells against PLO-induced cytolysis as determined by 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (85 6 17% versus 50 6 9% cell viability), measuring extracellular ATP, and kinetic assays. This was a generalized mechanism because Dynasore protected HeLa cells against streptolysin O. 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.
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