Staphylococcus aureus Panton-Valentine leukocidin directly targets mitochondria and induces Bax-independent apoptosis of human neutrophils

Abstract

Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by Staphylococcus aureus that has recently been associated with necrotizing pneumonia. In the present study, we report that in vitro, PVL induces polymorphonuclear cell death by necrosis or by apoptosis, depending on the PVL concentration. PVL-induced apoptosis was associated with a rapid disruption of mitochondrial homeostasis and activation of caspase-9 and caspase-3, suggesting that PVL-induced apoptosis is preferentially mediated by the mitochondrial pathway. Polymorphonuclear cell exposure to PVL leads to mitochondrial localization of the toxin, whereas Bax, 1 of the 2 essential proapoptotic members of the Bcl-2 family, was still localized in the cytosol. Addition of PVL to isolated mitochondria induced the release of the apoptogenic proteins cytochrome c and Smac/DIABLO. Therefore, we suggest that PVL, which belongs to the pore-forming toxin family, could act at the mitochondrion level by creating pores in the mitochondrial outer membrane. Furthermore, LukS-PV, 1 of the 2 components of PVL, was detected in lung sections of patients with necrotizing pneumonia together with DNA fragmentation, suggesting that PVL induces apoptosis in vivo and thereby is directly involved in the pathophysiology of necrotizing pneumonia.

Figure 1

PMN apoptosis and necrosis as a function of the rPVL concentration. PMNs were incubated with medium for 6 hours, with 5 nM rPVL for 6 hours, or with 200 nM rPVL for 1 hour. PMNs were then cytospun on glass coverslips and observed after either May-Grünwald/Giemsa staining (A) or Hoechst 33342 staining (B) for nuclear morphology or prepared for TEM (C and D). Scale bars: 20 μm.

Figure 2

PMN apoptosis is induced by native as well as rPVL and culture supernatant of the S. aureus isogenic strains RN6390 expressing PVL. (A) PMNs were incubated with rLukF-PV, rLukS-PV, rPVL, or nPVL at various concentrations. Cell death was measured by surface binding of annexin V after 6 hours when treated with 5 nM rLukF-PV, 5 nM rLukS-PV, 5 nM rPVL, or 0.5 nPVL and after 1 hour when treated with 200 nM rPVL or 5 nM nPVL. (B) Culture supernatants of the S. aureus isogenic strains RN6390 (luk-PV -); RN6390 lysogenized with ΦSLT phage carrying luk-PV; or a deleted luk-PV (Δluk-PV) mutant of S. aureus RN6390 lysogenized with ΦSLT phage or rPVL were separated on 12% SDS-PAGE followed by Western blotting with anti–LukS-PV 1C12 mAb. PMNs were treated with a 1:2,000 dilution of culture supernatant of the S. aureus isogenic strains RN6390 (luk-PV -); RN6390 lysogenized with ΦSLT phage carrying luk-PV; or a deleted luk-PV (Δluk-PV) mutant of S. aureus RN6390 lysogenized with ΦSLT phage. PMN apoptosis was monitored by measuring the decrease in Δψm after 5 minutes and the surface binding of annexin V after 6 hours. Data are from 1 of 3 independent experiments that gave similar results.

Figure 3

Kinetics of plasma membrane and mitochondrial alterations during rPVL-induced PMN apoptosis. PMNs were incubated with medium or with 5 nM rPVL and studied for annexin V binding (A), Δψm (B), mitochondrial membrane alterations (staining with MitoTracker GreenFM) (C), or ROS production (D). Results in A, B, and D are mean ± SD of 3 data points. Data are from 1 of 3 independent experiments that gave similar results. (C) PMNs were observed using a laser scanning confocal microscope. Scale bars: 10 μm.

Figure 4

Effect of the caspase inhibitor zVAD-fmk on rPVL-induced PMN apoptosis. PMNs were incubated with zVAD-fmk (100 μM) for 1 hour, then with medium or 5 nM rPVL. (A) PMN morphology was observed after 6 hours using TEM. Scale bars: 20 μm. (B) DNA fragmentation was analyzed at 6 hours using the F7-26 mAb; the number indicated for each histogram represents the percentage of cells with fragmented DNA. (C) The Δψm decrease after zVAD-fmk treatment was observed after 15 minutes of PMN exposure to medium or 5 nM rPVL. Results are mean ± SD of 3 data points. Data are from 1 of 3 independent experiments that gave similar results.

Figure 5

rPVL-induced PMN apoptosis uses caspases involved in the mitochondrial pathway. (A) Three-day-activated PBLs were incubated with medium or 5 μM STS for 6 hours and PMNs were incubated with medium or with 5 nM rPVL, and, at the indicated times, total protein extracts were prepared and proteins were separated on 12% SDS-PAGE followed by Western blotting with anti–caspase-3, -8 or -9 Ab. Data are from 1 of 2 independent experiments that gave similar results. The cross represents a nonspecific band. (B) PMNs were incubated with medium or with 5 nM rPVL in the presence or absence of zVAD-fmk (100 μM), zIETD-fmk (100 μM), or zLEHD-fmk (100 μM). Apoptosis was measured as surface binding of annexin V. Results are mean ± SD of 3 data points. Data are from 1 of 3 independent experiments that gave similar results. *P ≤ 0.01; **P ≤ 0.001.

Figure 6

rPVL is localized in mitochondria during rPVL-induced PMN apoptosis. (A) PMNs were treated for 5 minutes with or without 5 nM biotinylated rPVL and were then stained with streptavidin-FITC before (without permeabilization) or after (permeabilization) treatment with Cytofix/Cytoperm. (B) PMNs were incubated with medium or with 5 nM PVL and, at the indicated times, cytosolic and mitochondrial protein extracts were prepared and proteins were separated on 12% SDS-PAGE followed by Western blotting with anti-MnSOD, anti–β-actin, anti–LukS-PV, or anti-Bax Ab. Anti-MnSOD Ab served as a marker of mitochondrial contamination of the cytosolic fractions (Cyto), while anti–β-actin Ab served as a marker of cytosolic contamination of mitochondrial fractions (Mito). Data are from 1 of 2 independent experiments that gave similar results. (C) PMNs were treated for 5 minutes with or without 5 nM biotinylated rPVL, prepared for TEM, and stained with gold-conjugated streptavidin. Arrows indicate gold-positive cells. Scale bars: 500 nm.

Figure 7

rPVL induces the release of the apoptogenic proteins cytochrome c and Smac/DIABLO from isolated mitochondria. Mitochondria from rat liver were treated with increasing concentrations of rPVL (A), rLukS (B), or Bax α (C) for 1 hour at 30°C. Mitochondrial proteins were then separated by SDS-PAGE and analyzed by Western blotting using antibodies directed against cytochrome c, Smac/DIABLO, and cytochrome oxydase subunit IV (COx IV). Data are from 1 of 2 independent experiments that gave similar results.

Figure 8

Detection of LukS-PV and of DNA-fragmented cells in lung sections of patients who died from necrotizing pneumonia. (A) Sections were stained with anti–LukS-PV 1C12 mAb. Arrows indicate LukS-PV–positive cells. (B) Serial sections were stained by the TUNEL method to detect DNA fragmentation.