Coenzyme depletion by members of the aerolysin family of pore-forming toxins leads to diminished ATP levels and cell death

Abstract

Recent studies demonstrated that a variety of bacterial pore-forming toxins induce cell death through a process of programmed necrosis characterized by the rapid depletion of cellular ATP. However, events leading to the necrosis and depletion of ATP are not thoroughly understood. We demonstrate that ATP-depletion induced by two pore-forming toxins, the Clostridium perfringens epsilon-toxin and the Aeromonas hydrophila aerolysin toxin, is associated with decreased mitochondrial membrane potential and opening of the mitochondrial permeability transition pore. To gain further insight into the toxin-induced metabolic changes contributing to necrosis and depletion of ATP, we analyzed the biochemical profiles of 251 distinct compounds by GC/MS or LC/MS/MS following exposure of a human kidney cell line to the epsilon-toxin. As expected, numerous biochemicals were seen to increase or decrease in response to epsilon-toxin. However, the pattern of these changes was consistent with the toxin-induced disruption of major energy-producing pathways in the cell including disruptions to the beta-oxidation of lipids. In particular, treatment with epsilon-toxin led to decreased levels of key coenzymes required for energy production including carnitine, NAD (and NADH), and coenzyme A. Independent biochemical assays confirmed that epsilon-toxin and aerolysin induced the rapid decrease of these coenzymes or their synthetic precursors. Incubation of cells with NADH or carnitine-enriched medium helped protect cells from toxin-induced ATP depletion and cell death. Collectively, these results demonstrate that members of the aerolysin family of pore-forming toxins lead to decreased levels of essential coenzymes required for energy production. The resulting loss of energy substrates is expected to contribute to dissipation of the mitochondrial membrane potential, opening of the mitochondrial permeability transition pore, and ultimately cell death.

Fig. 1

Cytotoxicity. A. ACHN cells were incubated at 37°C for 24 hours with serial dilutions of purified epsilon-toxin (■, 7.8 to 500 nM) or aerolysin (□, 0.04 to 2.5 nM). Cytotoxicity was assessed by addition of CellTiter Blue reagent to detect metabolically active cells. Results were normalized to the fluorescent signal from untreated cells (100%) and cells treated with 1% Triton (0%). Results represent the mean and standard deviation of quadruplicate samples. B. ACHN cells were incubated at 37°C for 90 minutes or 24 hours with 500 nM epsilon-toxin or 2.5 nM aerolysin. Cytotoxicity was assessed by addition of CellTiter Blue reagent to detect metabolically active cells. Results were normalized to the fluorescent signal from untreated cells (100%) and cells treated with 1% Triton (0%). Results represent the mean and standard deviation of triplicate samples. C. ACHN cells were incubated at 37°C for 90 minutes with 500 nM epsilon-toxin or 2.5 nM aerolysin. Extracellular LDH was measured using the CytoTox-ONE Homogeneous Membrane Integrity Assay (Promega). Results represent the mean and standard deviation of triplicate samples. These experiments were performed at least three times with similar results.

Fig. 2

Toxin-induced ATP depletion and mitochondrial perturbation. A. ACHN cells were incubated at 37°C for 90 minutes with 500 nM epsilon-toxin (■) or 2.5 nM aerolysin (□). Cellular ATP levels were assessed using ATPLite 1 Step (Perkin Elmer). Results were normalized to the fluorescent signal from untreated cells (100%) and cells treated with 1% Triton (0%). Results represent the mean and standard deviation of triplicate samples. B. ACHN cells were preloaded with a mitochondrial membrane potential-sensitive dye at 37°C for 45 minutes. Cells then were either untreated (■) or treated with 500 nM epsilon-toxin (□), 2.5 nM aerolysin (●), or 500 nM CCCP (○) as a control. The fluorescent signal was monitored at 5 minute intervals for a period of 90 minutes after treatment. Results represent the mean and standard deviation of eight replicate samples; error bars may be too small to be visualised. C. ACHN cells were incubated with serial dilutions of cyclosporine A (62.5 to 500 nM) at 37°C for 60 minutes. Purified epsilon-toxin (■, 500 nM) or aerolysin (□, 2.5 nM) was added and cells were incubated at 37°C for 90 minutes. Cellular ATP levels were assessed using ATPLite 1 Step (Perkin Elmer). Results were normalized to the fluorescent signal from untreated cells (100%) and cells treated with 1% Triton (0%). Results represent the mean and standard deviation of triplicate samples. These experiments were performed at least three times with similar results.

Fig. 3. Heat map of epsilon-toxin-induced metabolic changes

Relative levels of 251 metabolites were determined (as described in Experimental) in untreated control cells (C), cells treated with heat-inactivated epsilon toxin (HI), and cells treated with active epsilon-toxin (TT) for 30 or 60 minutes. The results are presented as the ratio of individual metabolite levels, comparing levels in toxin-treated cells to levels in either control group. For simplicity, metabolites were segregated into 7 pathways as shown. Red: indicates significant difference (ANOVA, p≤0.05) in metabolite levels between the treatment groups shown, with a mean ratio of > 1. Blue: indicates significant difference (ANOVA, p≤0.05) between the treatment groups shown, with a mean ratio of < 1. Black: mean values are not significantly different for that comparison. For additional information, see Table S1.

Fig. 4

Toxin induced decreases in pantothenate, carnitine, and NAD⁺ (and NADH). A. ACHN cells were incubated at 37°C for 90 minutes in balanced salt solution with 500 nM ε-toxin or 2.5 nM aerolysin. Culture supernatants were collected, and the cell monolayers then were washed twice in PBS and lysed using M-PER (Pierce). Pantothenate levels in the cell lysates (■, epsilon-toxin; ●, aerolysin) and culture supernatants (□, epsilon-toxin; ○, aerolysin) were assessed using a microbiological assay. The sum of pantothenate levels in the cell lysates and culture supernatants from untreated control cells were determined and the mean value was set at 100%. Results represent the mean and standard deviation of triplicate samples. The microbiological assay was performed at least three times with similar results. B. ACHN cells were incubated at 37°C for 90 minutes in balanced salt solution with 500 nM ε-toxin or 2.5 nM aerolysin. Culture supernatants were collected, and the cell monolayers then were washed twice in PBS and lysed using M-PER (Pierce). Carnitine levels in the cell lysates (■, epsilon-toxin; ●, aerolysin) and culture supernatants (□, epsilon-toxin; ○, aerolysin) were assessed using a microbiological assay. The sum of carnitine levels in the cell lysates and culture supernatants from untreated control cells were determined and the mean value was set at 100%. Results represent the mean and standard deviation of triplicate samples. The microbiological assay was performed at least three times with similar results. C. ACHN cells were incubated at 37°C for 90 minutes in balanced salt solution with 500 nM epsilon-toxin (■) or 2.5 nM aerolysin (□). Cell monolayers then were washed twice in PBS. Total NAD (NAD ⁺ plus NADH) levels in the cell lysates were assessed using an enzymatic assay. Results were normalized to untreated cells (100%). Results represent the mean and standard deviation of triplicate samples. The enzymatic assay was performed at least three times with similar results.

Fig. 5

ACHN cells were pre-incubated at 37°C for 60 minutes in balanced salt solution (filled bars) or in balanced salt solution supplemented with 5 mM NADH (open bars). Toxins, (500 nM epsilon-toxin or 2.5 nM aerolysin) then were added and the cells were incubated at 37°C for 90 minutes. Cellular ATP levels were assessed using ATPLite 1 Step (Perkin Elmer). Results were normalized to the fluorescent signal from untreated cells (100%) and cells treated with 1% Triton (0%). Results represent the mean and standard deviation of triplicate samples. B and C. ACHN cells were pre-incubated at 37°C for 60 minutes in balanced salt solution (filled bars) or in balanced salt solution supplemented with 5 mM carnitine (open bars). Toxins, (500 nM epsilon-toxin or 2.5 nM aerolysin) then were added and the cells were incubated at 37°C for 90 minutes. Cellular ATP levels were assessed using ATPLite 1 Step (Perkin Elmer) (B) and release of LDH was measured using the CytoTox-ONE Homogeneous Membrane Integrity Assay (Promega) (C). Results represent the mean and standard deviation of triplicate samples. These experiments were performed at least three times with similar results.