Cathepsin B-mediated autophagy flux facilitates the anthrax toxin receptor 2-mediated delivery of anthrax lethal factor into the cytoplasm

Abstract

Anthrax lethal toxin (LeTx) is a virulence factor secreted by Bacillus anthracis and has direct cytotoxic effects on most cells once released into the cytoplasm. The cytoplasmic delivery of the proteolytically active component of LeTx, lethal factor (LF), is carried out by the transporter component, protective antigen, which interacts with either of two known surface receptors known as anthrax toxin receptor (ANTXR) 1 and 2. We found that the cytoplasmic delivery of LF by ANTXR2 was mediated by cathepsin B (CTSB) and required lysosomal fusion with LeTx-containing endosomes. Also, binding of protective antigen to ANXTR1 or -2 triggered autophagy, which facilitated the cytoplasmic delivery of ANTXR2-associated LF. We found that whereas cells treated with the membrane-permeable CTSB inhibitor CA074-Me- or CTSB-deficient cells had no defect in fusion of LC3-containing autophagic vacuoles with lysosomes, autophagic flux was significantly delayed. These results suggested that the ANTXR2-mediated cytoplasmic delivery of LF was enhanced by CTSB-dependent autophagic flux.

FIGURE 1.

The cathepsin B inhibitor CA074 prevents cell death induced by anthrax lethal toxin in RAW 264.7 macrophages. A, RAW264.7 murine macrophages were treated with different kinds of cathepsin inhibitors (cathepsin G inhibitor (CTSGi; 100 μm), cathepsin K inhibitor (CTSKi;100 μm), cathepsin B inhibitor (CA074-Me; 100 μm), and proteasome inhibitor (MG132; 10 μm) for 1 h and then treated with LeTx (250 ng/ml LF and 500 ng/ml PA) in the presence of the inhibitors for 5 h. MTT was added 2 h before the end of the experiment for cell death determination. Data are expressed as the mean ± S.D. (n = 3). B, RAW264.7 cells were pretreated with different concentrations of CA074-Me and then treated with LeTx (250 ng/ml LF and 500 ng/ml PA) for 5 h. Cell death assays were performed as above in A. Data are expressed as the mean ± S.D. (n = 4).

FIGURE 2.

Inhibition of cathepsin B blocks MEK1 cleavage induced by anthrax lethal toxin through delaying LeTx release into the cytoplasm. A, RAW264.7 cells were pretreated with CA074-Me (100 μm) or MG132 (10 μm) for 1 h and treated with LeTx (250 ng/ml LF and 500 ng/ml PA) for 2 h. MEK1 cleavage was analyzed by Western blots. Immunoblotting for p38 was used as a loading control. NT, N terminus. B, bone marrow-derived macrophages from C57BL/6 (Ctsb^+/+) or (Ctsb^−/−) mice were treated with LeTx (250 ng/ml LF and 500 ng/ml PA) for the indicated times, and MEK1 cleavage was analyzed by Western blots. The immunoblot for p38 was used as a loading control. C, C57 bone marrow-derived immortalized macrophage cells were incubated in the presence or absence of CA074-Me (100 μm) for 1 h and treated with LeTx (250 ng/ml LF and 500 ng/ml PA) for 1 h. LeTx-treated cells were washed twice with PBS and incubated for another 1 h or 2 h with fresh media. Cell homogenates were heated at 96 °C for 5 min and separated using SDS-PAGE. PA-heptamer stability was analyzed by immunoblot. D, RAW264.7 macrophages were pretreated with CA074-Me (100 μm) for 1 h and treated with LeTx (250 ng/ml LF and 500 ng/ml PA) for 1 h. Macrophages were washed twice with PBS and harvested after another 1 h of incubation at 37°C. Macrophage homogenates were prepared in 1× extraction buffer as described under “Experimental Procedures,” and the gradient was centrifuged for 10 h at 35,000 rpm in the SW41 rotor (Beckman). Gradient fractions were collected and analyzed by Western blots. E, bone marrow-derived macrophages from C57BL/6 (Ctsb^+/+) or (Ctsb^−/−) mice were treated with LeTx (250 ng/ml LF and 500 ng/ml PA) for 90 min, and macrophages were washed twice with PBS. Half of the cells were harvested, and intracellular total endosomal compartments were prepared as under “Experimental Procedures.” The remaining cells were further incubated at 37°C for 90 min, and intracellular total endosomal compartments were prepared as above. PA-heptamer stabilities were analyzed by immunoblots (top panel). Bone marrow-derived macrophages from C57BL/6 (Ctsb^+/+) or (Ctsb^−/−) mice were treated with LeTx (250 ng/ml LF and 500 ng/ml PA) for 90 min. The cytosolic and endosomal compartments were prepared as above, and LF was analyzed by Western blot. Immunoblots for Rab7 and p38 were used as loading controls (bottom panel).

FIGURE 3.

Late endosome and lysosome fusion is required for release of LeTx into the cytoplasm. A, inhibition of microtubule activity blocked cell death and MEK1 degradation induced by LeTx. RAW264.7 macrophages were incubated in the absence or presence of the microtubule inhibitor vinblastine (0–250 ng/ml) for 1 h and treated with LeTx (125 ng/ml LF and 250 ng/ml PA) for 5 h. Cell death was measured using a MTT assay (top panel). Data are expressed as the mean ± S.D. (n = 3). RAW264.7 macrophages were pretreated with the vinblastine (250 ng/ml) for 1 h and treated with LeTx (125 ng/ml LF and 250 ng/ml PA) for 2 h. MEK1 degradation was analyzed by Western blots (bottom panel). NT, N terminus. B, Lamp1/2 deficiency in MEF cell lines caused delayed MEK1 degradation induced by LeTx. Mouse embryo fibroblast wild type (Lamp1/2^+/+) or Lamp1/2 double-deficient (Lamp1/2^−/−) cell lines were treated with different doses of LeTx for 2 h, and MEK1 degradation were analyzed by immunoblotting (top panel). Lamp1/2^+/+ or Lamp1/2^−/− MEF cell lines were treated with LeTx (250 ng/ml LF and 500 ng/ml PA) for the indicated times, and MEK1 degradation was analyzed by Western blot (bottom panel). C, HEK293 cells were transfected with siRNA against VAMP7 or control siRNA. A portion of the transfected cells was harvested at 42 h post-transfection for confirmation of protein knockdown, and the remaining cells were treated with LeTx (250 ng/ml LF and 500 ng/ml PA) for 2 h. MEK1 degradation was analyzed by Western blot. D, inhibition of cathepsin B activity caused enhanced colocalization with cathepsin B-containing vesicles and PA or LF. HEK293 cells were pretreated with or without CA074-Me (100 μm) for 1 h and then treated with LeTx (250 ng/ml LF and 500 ng/ml PA) for 1 h. Cells were washed twice with media and further incubated at 37°C for 1 h. After blocking with 10% goat serum, cells were immunostained with anti-CTSB, anti-PA or anti-LF antibodies, and visualized using a confocal microscope; scale bar, 5 μm.

FIGURE 4.

Induction of autophagy is required for effective trafficking of LeTx. A, LeTx induced the formation of intracellular vacuoles in macrophages. RAW264.7 macrophages were incubated in absence or presence of LeTx (250 ng/ml LF and 500 ng/ml PA) at 37 °C for 1 h and washed twice with ice-cold PBS. Cells were then fixed with 2.5% glutaraldehyde in 0.1 m sodium cacodylate buffer, and grids with specimen were prepared as described under “Experimental Procedures.” Micrographs were taken with a transmission electron microscope. Scale bars show 2 μm in length for the lower magnification and 0.2 μm for higher magnification. B, LeTx induced LC3-II formation in a PA-dependent manner. RAW264.7 macrophages were treated with PA and LF (PA + LF, 500 ng/ml PA and 250 ng/ml LF), PA and inactive LF (PA + mLF, 500 ng/ml PA and 250 ng/ml LF), PA only (500 ng/ml PA), or LF only (250 ng/ml PA) for 2 h. Induction of autophagy was analyzed by immunoblotting against LC3-II. An immunoblot for p38 MAPK was used as a loading control. C, RAW264.7 cells were pretreated with CA074-Me (50 or 100 μm) or the microtubule inhibitor vinblastine (0.25 μm) and treated with LeTx (125 ng/ml LF and 250 ng/ml PA) for 2 h. LC3-II accumulation was analyzed by Western blots. D, N-terminal GFP-conjugated LC3 was transiently transfected in RAW264.7 macrophages and treated with LeTx (250 ng/ml LF and 500 ng/ml PA) for 1 h at 16 h post-transfection. Cell were washed twice with normal media and further incubated at 37°C for 30 min. Cells were then fixed and immunostained with anti-PA or anti-LF as described under “Experimental Procedures.” GFP-conjugated LC3 and PA (top panel) or LF (bottom panel) were visualized using a Zeiss LSM510 META confocal microscope; scale bar, 5 μm. E, THP-1 cells were incubated with serum or without serum for 4 h and treated with LeTx (125 ng/ml LF and 250 ng/ml PA) for the indicated times. MEK1 degradation or LC3-II formation was analyzed by Western blots. Immunoblot for actin was used as a loading control. NT, N terminus. F, THP-1 cells were treated with different doses of 3-MA for 1 h and exposed by LeTx for 2 h. PA-heptamer and MEK1 degradation, phospho-ERK (pERK), and LC3-II formation were analyzed by Western blots.

FIGURE 5.

Cathepsin B inhibition causes defective autophagy flux. A, RAW264.7 cells were electroporated with GFP-LC3 plasmid and incubated in DMEM containing DQ-BSA (10 μg/ml) for 30 min. Cell were then washed twice with DMEM and treated with LeTx (250 ng/ml LF and 500 ng/ml PA) for 60 min in the presence or absence of CA074-Me (100 μm). Cells were fixed, and colocalization of GFP-LC3 and red fluorescent of DQ-BSA was imaged using a Bio-Rad Radiance 2000 two-photon confocal microscope and LaserSharp 2000 software. Arrows show the colocalization of dequenched DQ-BSA vesicles with LC3-positive puncta; scale bar, 5 μm. B, THP-1 cells were incubated in RPMI media containing DQ-BSA (10 μg/ml) for 15 min at 37 °C in 5% CO₂. Cells were washed and incubated further in regular growth media for 45 min to ensure that DQ-BSA had reached the lysosomal compartment. Cells were then further incubated in regular growth media in the presence or absence of CA074-Me (50 μm), then harvested at indicated time points. Cells were analyzed using flow cytometry. C, for confocal images analysis, THP-1 cells were plated on coverslips after treatment as above in B, and cells were incubated in RPMI media with or without CA074-Me (50 μm). Cells were then fixed with 4% formaldehyde at the indicated time point. The fluorescent products of DQ-BSA were imaged using the Bio-Rad Radiance 2000 two-photon confocal microscope and LaserSharp 2000 software. Dotted lines indicate the cell margin; scale bar, 5 μm. D, C57 wild type (CTSB^+/+) or cathepsin B-deficient (CTSB^−/−) cell lines were plated on coverslips and incubated in RPMI media containing DQ-BSA (10 μg/ml) for 15 min at 37 °C in 5% CO₂. Cells were washed twice with PBS and incubated in RPMI media for the indicated times. Cells were fixed as above in C, and the fluorescent degradation products of the DQ-BSA in lysosome were imaged using Bio-Rad Radiance 2000 two-photon confocal microscope and LaserSharp 2000 software. Dotted lines indicate the cell margin.

FIGURE 6.

Cathepsin B is involved in ANTXR2-dependent LeTx delivery through autophagic flux. A, HEK293 cells were transfected with control siRNA (si-Scramble) or human (h) ANTXR1- or ANTXR2-specific siRNAs (si-ANTXR1 or si-ANTXR2), and at 42 h post-transfection, portions of the cells were harvested for reverse transcription-PCR (left panel). The remaining cells were incubated in the presence or absence of CA074-Me (100 μm), and treated with LeTx (250 ng/ml LF and 500 ng/ml PA) for 2 h. MEK1 degradation, down-regulation of phospho-ERK (pERK), and LC3-II formation were analyzed by Western blots (middle panel) and MEK1-NT immunoreactivities were analyzed using the NIH-Image program. Data are expressed as mean ± S.D. (n = 3; *, significant, p < 0.05; NS, not significant with p > 0.5). NT, N terminus. B, ANTXR1/R2-deficient CHO cells (R1⁻/R2⁻), ANTXR1-reconstituted CHO cells (R1⁺/R2⁻), or ANTXR2-reconstituted CHO cells (R1⁻/R2⁺) were treated with LeTx (500 ng/ml LF and 1000 ng/ml PA) for the indicated times. MEK1 degradation and LC3-II formation were analyzed by Western blot. Immunoblots for HA or actin were used for transfection or loading control. C, human monocytic THP-1 cells were transfected with control siRNA or siANTXRs, and after 42 h cells were treated with LeTx as above. MEK1 degradation and LC3-II formation were analyzed by Western blot (upper panel), and LC3-II immunoreactivities were analyzed using NHI-Image program (lower panel). Data are expressed as the mean ± S.D. (n = 2).

FIGURE 7.

A diagram illustrating the proposed pathway involved in the cytoplasmic delivery of LF. Unlike ANTXR1, which can deliver LF into the cytoplasm from early endosomes, ANTXR2-mediated delivery of LF requires autophagy flux, which is triggered by lysosomal fusion.