Tyrosine 331 and phenylalanine 334 in Clostridium perfringens α-toxin are essential for cytotoxic activity

AbstractDifferences in the biological properties of the Clostridium perfringens phospholipase C (α-toxin) and the C. bifermentans phospholipase C (Cbp) have been attributed to differences in their carboxy-terminal domains. Three residues in the carboxy-terminal domain of α-toxin, which have been proposed to play a role in membrane recognition (D269, Y331 and F334), are not conserved in Cbp (Y, L and I respectively). We have characterised D269Y, Y331L and F334I variant forms of α-toxin. Variant D269Y had reduced phospholipase C activity towards aggregated egg yolk phospholipid but increased haemolytic and cytotoxic activity. Variants Y331L and F334I showed a reduction in phospholipase C, haemolytic and cytotoxic activities indicating that these substitutions contribute to the reduced haemolytic and cytotoxic activity of Cbp

Clostridium perfringens phospholipase C induced ROS production and cytotoxicity require PKC, MEK1 and NFκB activation.

Clostridium perfringens phospholipase C (CpPLC), also called α-toxin, is the most toxic extracellular enzyme produced by this bacteria and is essential for virulence in gas gangrene. At lytic concentrations, CpPLC causes membrane disruption, whereas at sublytic concentrations this toxin causes oxidative stress and activates the MEK/ERK pathway, which contributes to its cytotoxic and myotoxic effects. In the present work, the role of PKC, ERK 1/2 and NFκB signalling pathways in ROS generation induced by CpPLC and their contribution to CpPLC-induced cytotoxicity was evaluated. The results demonstrate that CpPLC induces ROS production through PKC, MEK/ERK and NFκB pathways, the latter being activated by the MEK/ERK signalling cascade. Inhibition of either of these signalling pathways prevents CpPLC's cytotoxic effect. In addition, it was demonstrated that NFκB inhibition leads to a significant reduction in the myotoxicity induced by intramuscular injection of CpPLC in mice. Understanding the role of these signalling pathways could lead towards developing rational therapeutic strategies aimed to reduce cell death during a clostridialmyonecrosis

Proposed model describing the mechanism of action of CpPLC leading towards cytotoxicity on ganglioside-deficient cells Don Q.

<p>The present study suggests a model where CpPLC activates PKC, MEK/ERK-1/2, and NFκB pathways. MEK/ERK pathway activates NFκB. Inhibition of both ERK-NFκB and PKC pathways inhibit ROS production and cytotoxicity by CpPLC in Don Q cells. (*) Results previously published <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086475#pone.0086475-MonturiolGross1" target="_blank">[16]</a>. Abbreviations: N-acetyl-L-cysteine(NAC); manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrinpenta- chloride (MnTMPyP); glutathione monoethyl ester (GSH-MEE).</p

PKC activation is required for the cytotoxic effect of CpPLC.

<p>Don Q cells (A) or GM95 cells (B) were treated overnight with GF109203x (GF, 20 µM (A), 10 µM (B)); Safingol (Saf, 10 µM (A), 30 µM (B)); Hispidin (His, 2 µM); P205 (66 µg/ml (A), 100 µg/ml (B) P222 (40 µg/ml); P219 (66 µg/ml); P223 (66 µg/ml) and Rottlerin (Rott, 7,2 µM) or MEM (control) before exposure to CpPLC. Cell viability was determined 18 h later using the neutral red assay. Results are expressed as the percentage of neutral red incorporated by the remaining cells, in comparison with the neutral red incorporated by control cells incubated with each treatment, but not exposed to the toxin. The results represent the average of two-four independent experiments with three replicate samples. (** p<0.001, * p<0.01). (C) Don Q cells were treated without (0 minutes) or with CpPLC for 10, 20, 30 or 60 minutes at 37°C. PMA was used as positive control for PKC activation. Cells were collected and PKC activation was measured using Promega'sPepTag® Non-Radioactive Protein Kinase Assays, according to manufacturer instructions. Results are representative of three independent experiments. (D) Don Q cells were treated without (0 minutes) or with CpPLC (3 ng/ml) for 5, 15, 30 or 60 minutes. Cytosol and membrane fractions were separated as described in the Materials and Methods section, and immunoblotted against PKCβII. After stripping of the blot, actin or clathrin were also detected. Densitometric analysis was performed using Image J software. Results are representative of three independent experiments.</p

NF-κB activation is required for the cytotoxic and myotoxic effects of CpPLC.

<p>Don Q cells (<b>A</b>) or GM95 cells (<b>B</b>) were preincubated overnight with helenalin (1 µg/ml (A), 0.5 µg/ml (B)), BAY 11-7085 (BAY, 5 µg/ml (A), 0.66 µg/ml (B)), CAPE (10 µM (A), 4 µM (B)), β-Lactone (3 µM (A), 5 µM (B)) or MEM (Control) before exposure to CpPLC(<b>C</b>) GM95 cells were exposed to NF-κB SN50 (P600, 133 µg/ml), IKK-NBD (P607, 133 µg/ml) or MEM (Control) overnight before exposure to CpPLC. (A, B, C) Cell viability was determined 18 h later using the neutral red assay. Results are expressed as the percentage of neutral red incorporated by the remaining cells in comparison with the neutral red incorporated by control cells incubated with each treatment, but not exposed to the toxin. The results represent the average of two-four independent experiments with three replicate samples (mean±SE, ** p<0.001). (<b>D</b>) Groups of 10CD-1 mice were challenged intramuscularly with 1.1 µg of CpPLC, and after 3 hours, creatin kinase (CK) activity was measured in plasma. One hour before and one hour after toxin injection, mice received intraperitonealy 100 µl of PBS (Control), BAY 117085 (BAY, 20 µg) or Helenalin (3 µg). Results are means±SE of 2–3 independent experiments. (**p≤0,001).</p

activates NFκB in Don Q cells.

<p>(A) Don Q cells were treated without (control) or with CpPLC at different times (3, 5 or 7 hours), and separation of nuclear fraction was performed. Equal amount of protein in the nuclear fraction was electrophoresed and transfered. NFκB p50 was detected, followed by stripping and actin detection (with remaining of p50 just above actin band). Results represent three independent experiments (mean ± SE) (**p<0,01 vs control) (B) Don Q cells were treated overnight with Safingol (Saf, 10 µM), P222 (40 µg/ml), PD98059 (PD, 187 µM), Tiron (TIR, 2 mg/ml), or BAY117085 (BAY, 0,004 µg/µl), and then treated with or without CpPLC (Control) for 7 hours. Cells were lysed and immunoblotted against phospho-IκB. After stripping of the blot, actin was also detected. Densitometric analysis was determined using Image J software. Results represent three independent experiments (mean ± SE) (*p<0,05).</p

ERK1/2 becomes activated independently of PKC and ROS on Don Q cells exposed to CpPLC.

<p>(A) Don Q cells were treated overnight with GF109203x (GF, 20 µM), 2-Aminoethoxydiphenyl borate (2APB, 70 µM) PD98059 (PD, 187 µM), Tiron (TIR, 2 mg/ml) or Helenalin (Hel, 1 µg/ml), previously to CpPLC (PLC) exposure (PC: positive control, NC: negative control, C: control of cells without CpPLC). Cells were collected and PKC activation was measured using Promega'sPepTag® Non-Radioactive Protein Kinase Assays, according to manufacturer instructions. Results are representative of two independent experiments. (B) Don Q cells were treated overnight with GF109203x (GF, 20 µM), PD98059 (PD, 187 µM), Tiron (TIR, 2 mg/ml) or BAY117085 (BAY, 0,004 µg/µl) or PMA (100 nM) as positive control (Y value = 1451±1095), then cells were lysed and immunoblot-ted against phospho-ERK. After stripping of the blot, total ERK1/2 was also evaluated. Densitometric analysis was determined using Image J software. Results are representative of five independent experiments (mean±SD) (*p<0,001 vs PLC).</p

MEK1 and NFκB are required for CpPLC's ROS production in Don Q cells.

<p>Don Q cells were treated overnight with GF109203x (GF, 20 µM), P222 (40 µg/ml), Safingol (15 µM), PD98059 (PD, 187 µM), Tiron (2 mg/ml), BAY117085 (BAY, 0,004 µg/µl) or P607 (66 µg/ml) prior to a 7 hour exposure to CpPLC. Cells were then loaded with DCFDA (5 µM) for 30 min, washed with PBS, tripsinized and propidium iodine stained. A FACSCalibur flow cytometer (Becton Dickinson) was immediately used to collect data for 5×10³ cells, and analysis was performed using CELLQUEST (Becton and Dickinson) to determine intensity and percentage of DCF fluorescent cells (FL1-H) among living cells. Results are representative of 3–4 independent experiments (mean ± SE) (**p<0,001; *p<0,05).</p