Reduction of Streptolysin O (SLO) Pore-Forming Activity Enhances Inflammasome Activation
<p><b>Pore formation of streptolysin O (SLO) mutants</b>. Blebbing was induced (<b>A</b>,<b>B</b>,<b>D</b>–<b>G</b>), or not induced (<b>C</b>) in CHO cells by treatment with 25 mM paraformaldehyde and 2 mM DTT. Subsequently, no SLO (<b>A</b>), 10,000 U/mL SLO C530A (<b>B</b>,<b>D</b>,<b>E</b>) or an equivalent mass of SLO N402E (<b>F</b>) or SLO N402C (<b>G</b>,<b>H</b>) were added to the cells, and the cells were prepared for EM. Alternatively, living CHO cells were treated with 125 U/mL SLO for 5 min (<b>C</b>) and prepared for EM. Arrowheads indicate strands formed by SLO N402C. Scale bar = 1 μm (<b>A</b>,<b>B</b>) or 50 nm (<b>C</b>–<b>H</b>).</p> "> Figure 2
<p><b>SLO N402C shows reduced direct toxicity</b>. LPS-primed bone marrow-derived macrophages (BMDM)from B6 or Casp1<sup>−/−</sup> mice were treated with the indicated SLO mutants at the indicated concentrations for 5 min (<b>A</b>,<b>B</b>) or 30 min (<b>C</b>,<b>D</b>) and PI uptake analyzed by FACS. The percentage of PI<sup>high</sup>, or dead cells (<b>A</b>,<b>C</b>) or percentage of PI<sup>l</sup>°<sup>w</sup>, or transiently permeabilized cells (<b>B</b>,<b>D</b>) are shown. The graphs display the average ± SEM of 3 independent experiments. <b>*</b> indicates <span class="html-italic">p <</span> 0.05, <b>**</b> indicates <span class="html-italic">p <</span> 0.01 and <b>***</b> indicates <span class="html-italic">p <</span> 0.001 for comparisons between wild type and N402C SLO (<b>A</b>,<b>B</b>) or comparisons between B6 and Casp1<sup>−/−</sup> BMDM (<b>C</b>,<b>D</b>).</p> "> Figure 3
<p><b>SLO C530A/N402C also shows reduced toxicity.</b> LPS-primed BMDM were treated with the indicated SLO mutants at the indicated concentrations (1000 U/mL for <b>C</b>) for 5 min or 30 min in the absence (<b>A</b>–<b>C</b>) or presence (<b>C</b>) of 50 mM KCl and PI uptake analyzed by FACS. The percentage of PI<sup>high</sup>, or dead cells (<b>A</b>,<b>C</b>) or percentage of PI<sup>l</sup>°<sup>w</sup>, or transiently permeabilized cells (<b>B</b>,<b>C</b>) are shown. The graphs display the average ± SEM of 3 independent experiments. <b>*</b> indicates <span class="html-italic">p <</span> 0.05, <b>**</b> indicates <span class="html-italic">p <</span> 0.01 and <b>***</b> indicates <span class="html-italic">p <</span> 0.001.</p> "> Figure 4
<p><b>SLO N402C enhances IL-1β release</b>. (<b>A</b>–<b>E</b>) LPS-primed B6 or Casp1<sup>−/−</sup> BMDM were treated with the indicated inhibitors and concentrations of SLO mutants for 30 min, or the controls nigericin or nothing. Supernatants were harvested and assayed for IL-1β levels by ELISA. (<b>F</b>) LPS-primed BMDM were treated as indicated with controls or 2000 U/mL of each SLO mutant for 30 min. Supernatants were collected and TCA precipitated, while cells were lysed in SDS-sample buffer. Lysates and supernatants were resolved by SDS-PAGE, transferred to PVDF and probed with anti-IL-1β monoclonal antibody 3ZD and anti-actin monoclonal antibody. The graphs display the average of 4 independent experiments ± SEM, while the blot shows one representative experiment of 5. <b>*</b> indicates <span class="html-italic">p <</span> 0.05 and <b>**</b> indicates <span class="html-italic">p <</span> 0.01.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Altered Pore Formation in SLO N402C Mutant
2.2. SLO N402C Has Reduced Toxicity in Mammalian Cells
2.3. SLO C530A/N402C Has Similarly Reduced Toxicity
2.4. Increased IL-1β Production by SLO N402C
3. Discussion
4. Experimental Section
4.1. Reagents
4.2. Cell Culture
4.3. Electron Microscopy
4.4. Toxicity Assay
4.5. IL-1β Release
4.6. Statistics
5. Conclusions
Acknowledgments
Conflict of Interest
References
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Keyel, P.A.; Roth, R.; Yokoyama, W.M.; Heuser, J.E.; Salter, R.D. Reduction of Streptolysin O (SLO) Pore-Forming Activity Enhances Inflammasome Activation. Toxins 2013, 5, 1105-1118. https://doi.org/10.3390/toxins5061105
Keyel PA, Roth R, Yokoyama WM, Heuser JE, Salter RD. Reduction of Streptolysin O (SLO) Pore-Forming Activity Enhances Inflammasome Activation. Toxins. 2013; 5(6):1105-1118. https://doi.org/10.3390/toxins5061105
Chicago/Turabian StyleKeyel, Peter A., Robyn Roth, Wayne M. Yokoyama, John E. Heuser, and Russell D. Salter. 2013. "Reduction of Streptolysin O (SLO) Pore-Forming Activity Enhances Inflammasome Activation" Toxins 5, no. 6: 1105-1118. https://doi.org/10.3390/toxins5061105
APA StyleKeyel, P. A., Roth, R., Yokoyama, W. M., Heuser, J. E., & Salter, R. D. (2013). Reduction of Streptolysin O (SLO) Pore-Forming Activity Enhances Inflammasome Activation. Toxins, 5(6), 1105-1118. https://doi.org/10.3390/toxins5061105