Membrane Bound Monomer of Staphylococcal α-Hemolysin Induces Caspase Activation and Apoptotic Cell Death despite Initiation of Membrane Repair Pathway

BACKGROUND: Wild type Staphylococcal alpha-hemolysin (alpha-HL) assembly on target mammalian cells usually results in necrotic form of cell death; however, caspase activation also occurs. The pathways of caspase activation due to binding/partial assembly by alpha-HL are unknown till date. RESULTS: Cells treated with H35N (a mutant of alpha-HL that remains as membrane bound monomer), have been shown to accumulate hypodiploid nuclei, activate caspases and induce intrinsic mitochondrial apoptotic pathway. We have earlier shown that the binding and assembly of alpha-HL requires functional form of Caveolin-1 which is an integral part of caveolae. In this report, we show that the caveolae of mammalian cells, which undergo a continuous cycle of 'kiss and run' dynamics with the plasma membrane, have become immobile upon the binding of the monomer. The cells treated with H35N were unable to recover despite activation of membrane repair mechanism involving caspase-1 dependent activation of sterol regulatory element binding protein-1. CONCLUSIONS: This is for the first time we show the range of cellular changes and responses that take place immediately after the binding of the monomeric form of staphylococcal alpha-hemolysin

Molecular modeling.

<p><b>A</b>) Energy minimized structure of <b>4. B</b>) Surface diagram of PKCαC1B (blue) docked with <b>4</b> (magenta). <b>C</b>) Overlaid structures of αC1B (blue) and εC1B (magenta). Structure of <b>4</b> was minimized using Chem3D pro 12.0.2. Molecular docking was done using sybyl 8.0. The protein structures were visualized using UCSF chimera 1.6.1.</p

Effect of 4 on the membrane translocation of PKCα and PKCε.

<p>Upper panels, Western blot analysis of the cytosolic (C) and the membrane (M) fractions of (<b>A</b>) PKCα and (<b>B</b>) PKCε after the cells were treated with varying concentration of <b>4</b> for 1 h. Lower panel, bar graph depicts the densitometry analysis of the upper panel immunoblots (Mean± SE, n = 3). Control refers to the sample with no addition of compounds.</p

Effect of 1–5 on CHO-K1 cell viability.

<p>The graph shows the percentage of viable cell after treatment with <b>1–5</b> at three different concentrations for 48 h. The cell viability was measured by MTT assay. Mean and standard deviation (SD) were obtained from three experiments done in triplicate.</p

Effect of 1, 2, 3, and 5 on the membrane translocation of PKCα and PKCε.

<p>Upper panels, Western blot analysis of the cytosolic (C) and the membrane (M) fractions of (<b>A</b>) PKCα and (<b>B</b>) PKCε after the cells were treated with 100 µM of <b>2, 3</b> or <b>5</b> for 1 h. Lower panel, bar graph of densitometry analysis of the upper panel immunoblots (Mean± SE, n = 3). Control refers to the sample with no addition of compounds.</p

Effect of solvent polarity on the absorption and fluorescence properties of 2 (5–10 µM).

<p>A), Normalized absorption and B) normalized florescence emission spectra of <b>2</b> in a) water, b) ethanol, c) acetonitrile and d) hexane.</p

Effect of 1 on TPA-induced membrane translocation of PKCα and PKCε.

<p>Upper panels, Western blot analysis of the cytosolic (C) and the membrane (M) fraction of (<b>A</b>) PKCα and (<b>B</b>) PKCε. Lower panel, bar graph of densitometry analysis of upper panel immunoblots (Mean ± SE, *P<0.05, n = 3). Cells were treated with 100 µM of <b>1</b> in the presence and absence of 100 nM TPA for 1 h. Control refers to the sample with no addition of compounds.</p

Effect of 1 and 4 on the membrane translocation of PKCα.

<p>Western blot analysis of the cytosolic (C) and the membrane (M) fractions of PKCα after the cells were treated with varying concentration of <b>1</b> and <b>4</b> for 24 h. Control refers to the sample with no addition of compounds.</p

Effect of 1 on the membrane translocation of PKCα and PKCε.

<p>Upper panels, Western blot analysis of the cytosolic (C) and the membrane (M) fractions of (<b>A</b>) PKCα and (<b>B</b>) PKCε after the cells were treated with varying concentration <b>1</b> for 1 h. Lower panel, bar graph of densitometry analysis of the upper panel immunoblots (Mean± SE, n = 3). Control refers to the sample with no addition of compounds.</p

Effect of 2–5 on TPA-induced membrane translocation of PKCα and PKCε.

<p>Upper panels, Western blot analysis of the cytosolic (C) and membrane (M) fraction of (<b>A</b>) PKCα and (<b>B</b>) PKCε. Lower panel, bar graph of densitometry analysis of upper panel immunoblots (Mean ± SE, n = 3). Cells were treated with 100 µM of <b>2–5</b> and 100 nM of TPA for 1 h. Control refers to the sample with no addition of compounds.</p