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Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds
18:25

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Published on: August 25, 2013

Cytolysis by Ca-permeable transmembrane channels. Pore formation causes extensive DNA degradation and cell lysis

A Hameed1, K J Olsen, M K Lee

  • 1Department of Microbiology and Immunology, University of Miami, School of Medicine, Florida 33101.

The Journal of Experimental Medicine
|March 1, 1989
PubMed

Insights

Staphylococcal alpha-toxin and CTL perforin cause cell lysis and DNA damage. This DNA degradation requires intracellular calcium and lysosomes, similar to cytotoxic T lymphocyte (CTL) and NK cell activity.

Area of Science:

  • Immunology
  • Cell Biology

Background:

  • Cytotoxic T lymphocytes (CTLs) and NK cells induce target cell lysis and DNA damage.
  • Membrane pore-forming toxins share mechanistic similarities with immune cell-mediated cytotoxicity.

Purpose of the Study:

  • To investigate the role of purified pore-forming toxins, staphylococcal alpha-toxin and CTL perforin, in target cell lysis and nuclear damage.
  • To elucidate the mechanisms underlying toxin-induced DNA degradation.

Main Methods:

  • Measuring 51Cr release for cell lysis.
  • Assessing DNA degradation and 125IUdR release for nuclear damage.
  • Utilizing calcium chelators (Quin 2, EGTA) and lysosomotropic agents (NH4Cl, chloroquine, monensin) to investigate pathway requirements.

Main Results:

  • Both alpha-toxin and CTL perforin induced dose-dependent cell lysis and DNA release.
  • DNA degradation was dependent on intracellular calcium and sensitive to lysosomotropic agents.
  • Recombinant tumor necrosis factor (rTNF) enhanced alpha-toxin-mediated DNA degradation.

Conclusions:

  • Pore-forming toxins can independently mediate DNA degradation.
  • Toxin-induced DNA degradation requires active target cell participation, involving intracellular calcium and lysosomes.
  • These findings highlight similarities between pore-former-induced cytolysis and immune cell-mediated cytotoxicity (CTL and NK cells).

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