Pro-autophagic signal induction by bacterial pore-forming toxins

Abstract

Pore-forming toxins (PFT) comprise a large, structurally heterogeneous group of bacterial protein toxins. Nucleated target cells mount complex responses which allow them to survive moderate membrane damage by PFT. Autophagy has recently been implicated in responses to various PFT, but how this process is triggered is not known, and the significance of the phenomenon is not understood. Here, we show that S. aureus α-toxin, Vibrio cholerae cytolysin, streptolysin O and E. coli haemolysin activate two pathways leading to autophagy. The first pathway is triggered via AMP-activated protein kinase (AMPK). AMPK is a major energy sensor which induces autophagy by inhibiting the target of rapamycin complex 1 (TORC1) in response to a drop of the cellular ATP/AMP-ratio, as is also observed in response to membrane perforation. The second pathway is activated by the conserved eIF2α-kinase GCN2, which causes global translational arrest and promotes autophagy in response to starvation. The latter could be accounted for by impaired amino acid transport into target cells. Notably, PKR, an eIF2α-kinase which has been implicated in autophagy induction during viral infection, was also activated upon membrane perforation, and evidence was obtained that phosphorylation of eIF2α is required for the accumulation of autophagosomes in α-toxin-treated cells. Treatment with 3-methyl-adenine inhibited autophagy and disrupted the ability of cells to recover from sublethal attack by S. aureus α-toxin. We propose that PFT induce pro-autophagic signals through membrane perforation-dependent nutrient and energy depletion, and that an important function of autophagy in this context is to maintain metabolic homoeostasis.

Fig. 1

Transient ATP loss is accompanied by transient AMPK phosphorylation and dephosphorylation of S6K. HaCaT cells were left untreated (Co) or loaded with α-toxin (1 μg/ml) for 40 min on ice, washed with ice-cold PBS and incubated in medium at 37°C for indicated times, before ATP levels were measured (a) or analysis of (p−) AMPKα by Western blot was performed (b); β-actin served as loading control. c HaCaT cells were treated like in a, and phosphorylation of p70S6K was analysed by Western blot

Fig. 2

Activation of AMPK by various PFT. a HaCaT cells were incubated with 100 ng/ml VCC for indicated times, and the phosphorylation status of AMPK was analysed by Western blot. b HaCaT cells were treated with 1 μg/ml SLO in [K⁺]-n or [K⁺]—hi media at 37°C for various times and analysed as in a. c A498 cells, which lack CD14 and are insensitive to LPS, were incubated with 160 ng/ml HlyA for the indicated periods and analysed as in a

Fig. 3

a S. aureus α-toxin inhibits leucine uptake. Accumulation of tritiated l-leucine was measured in HaCaT cells that were pre-treated with α-toxin (100 ng/ml) for ten minutes. Columns indicate mean values of quadruplicate measurements; error bars indicate ± SEM. The experiment was repeated with the same result. b Activation of GCN2 by α-toxin. Cells were left untreated (Co) or treated with toxin (500 ng/ml) in [K⁺]-n or [K⁺]-hi media for various times at 37°C before analysis of (p−)GCN2 by Western blot. Cells exposed to the UV light (312 nm, 70 mJ/cm²) of a Spectroline cross-linking apparatus, and subsequently incubated for 6 min, served as a positive control (UV). c HaCaT cells were treated with mutant D152C (500 ng/ml) for various times and phosphorylated, and total GCN2 was analysed by Western blot. d, e Cells were treated with 100 ng/ml VCC (d) or 250 ng/ml SLO (e) for the indicated times and analysed like in b. f A498 cells were incubated with HlyA (160 ng/ml) for various times and analysed as in b. g HaCaT cells were treated with monensin (25 μM) or nigericin (6 μM) for the indicated periods, and subsequently analysed for p-GCN2 and GCN2 by Western blot

Fig. 4

a Activation of PKR by α-toxin. Upper panel: HaCaT cells were treated with α-toxin and phosphorylation of a double-stranded RNA-binding protein of apparent MW 75kD (referred to as PKR) was determined. Cells were incubated with the indicated agents for the times given below panels. Bottom set of panels: left PKR phosphorylation was determined as above, in cells treated with wild-type α-toxin, or mutant D152C; right same treatment as left bottom panel, but poly-C (instead of poly-I:C) beads were employed for precipitation, as a control. b α-toxin causes phosphorylation of eIF2α. HaCaT cells were loaded with α-toxin on ice (1 μg/ml), washed and incubated for the indicated times before phosphorylation of eIF2α at Ser51 was analysed by Western blot

Fig. 5

a TEM images of HaCaT cells. Left panel: untreated, size bar 1 μm; right: α-toxin for 2 h, size bar 500 nm. b Digital fluorescence microscopic image of HaCaT cells treated with AlexaFluor546-labelled α-toxin (red) for 2 h. Left image sample was fixed and stained for p62 (green), by using AlexaFluor488-coupled secondary antibody. The image of the right panel was taken from cells which were transiently transfected with EGFP-LC3, and treated with AlexaFluor546-α-tox as above. Arrows indicate colocalization of AlexaFluor546-α-tox with p62, or EGFP-LC3-positive puncta, respectively. Untreated controls exhibited diffuse distribution of p62 (not shown), and of most EGFP-LC3 (see Fig. 6a)

Fig. 6

a HaCaT cells were transfected with EGFP-LC3 and treated with 200 ng/ml wild type (α-Tox), or D152C mutant (MUT) for 4 h. (Co) denotes untreated sample and (α-Tox + 3-MA) a sample pretreated with 3-MA (10 mM) and then treated with α-toxin in the presence of 3-MA. b Cells were transiently co-transfected with EGFP-LC3 and control vector (CoV) or GADD34-expression vector, before treatment with α-toxin as in a. The box plot summarizes quantitation of data from >30 cells per treatment: the median number of puncta was 1.8-fold higher in control cells. c HaCaT cells were transfected as in a, incubated for 30 min with α-toxin-producing S. aureus (plasmid-transformed derivative of DU1090 [39], 30 CFU/cell), washed with medium and incubated for 3 h at 37°C. A representative digital fluorescence microscopic image is shown. Note juxta-nuclear accumulation of EGFP-LC3, decorating Hoechst-stained bacteria. No recruitment of EGFP-LC3 was observed when a α-toxin-non-producing parental strain was employed (not shown)

Fig. 7

a HaCaT cells were treated for 4 h with 200 ng/ml α-toxin or mutant toxin in the absence or presence of 3-MA, as indicated in the figure. The presence of EGFP-LC3-II was detected by Western blot. b HaCaT cells were treated with toxin as above, but in the presence or absence of bafilomycin (200 ng/ml). EGFP-LC3-II was analysed by Western blot. c 3-MA inhibits ATP recovery after α-toxin attack. HaCaT cells were loaded with α-toxin after pre-treatment with or w/o 3-MA (10 mM) for 1 h and subsequently incubated at 37°C in the presence (grey) or absence (black) of 3-MA. Cellular ATP was determined after 2 and 6 h. Data are mean values from four independent experiments; error bars indicate SEM, Asterisk denotes P < 0.05

Fig. 8

Hypothetical model of induction and role of autophagy after membrane perforation by PFT: membrane perforation cuts nutrient supplies by paralysing ion gradient–dependent transport systems and causes energy loss. This leads to the activation of GCN2, AMPK and possibly additional pro-autophagic signals. The autophagic response triggered by these pathways can temporarily relieve energy and nutrient shortage, and might contribute to the elimination of toxin by promoting its destruction, or leading to exocytosis of undigestable pore complexes [16]