Cathepsin B-mediated Autophagy Flux Facilitates the Anthrax Toxin Receptor 2-mediated Delivery of Anthrax Lethal Factor into the Cytoplasm

Sung Ouk Kim,Paul Saftig,Shengcai Lin,Boram Ham,Soon-Duck Ha,Jeremy Mogridge

doi:10.1074/jbc.m109.065813

Sung Ouk Kim, Paul Saftig + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m109.065813

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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.

Highlights

Delivery of lethal factor (LF) into the cytoplasm involves the formation of the heptameric PA63 pore, which becomes an SDS-resistant complex in acidified endosomes [28]
Cathepsin B Activity Is Required for Autophagic Flux—Because previous results suggested that autophagy was required for an efficient delivery of LF into the cytoplasm but CA074 enhanced LC3-II formation, we investigated whether CTSB was involved in the process of autophagy and, if so, in which step of mitochondria or other organelles, in lethal toxin (LeTx)-treated RAW264.7 autophagy process using DQTM Red BSA (DQ-BSA)
The autophagy inhibitor, 3-MA, which at least partially prevented LC3-II formation by LeTx in either ANTHR1 or ANTXR2 knockdown cells, was able to prevent MEK1 cleavage in ANTXR1 but not ANTXR2 knockdown cells (Fig. 6C). These results collectively suggest that ANTXR2 but not ANTXR1 delivery of LF into the cytoplasm was mediated through a CTSB- and autophagy-dependent pathway

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—RAW264.7 murine macrophages, HEK293 cells, and the lysosome-associated membrane protein 1 (LAMP-1)/LAMP-2 double-deficient or wild type mouse embryonic fibroblasts cell lines were cultured in DMEM medium containing 10% heated-inactivated fetal bovine serum (Sigma), 10 mM MEM nonessential amino acids solution, 100 units/ml penicillin G sodium, 100 ␮g/ml streptomycin sulfate, and 1 mM sodium pyruvate. The human monocytic cell line THP-1 was cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (Sigma), 10 mM MEM nonessential amino acids solution, 100 units/ml penicillin G sodium, 100 ␮g/ml streptomycin sulfate, and 1 mM sodium pyruvate. GFP-LC3 transiently expressing-RAW264.7 cells were incubated in RPMI media containing DQ-BSA (10 ␮g/ml) for 30 min and washed twice with PBS. The human monocytic cell line THP-1 was incubated in RPMI media containing DQ-BSA (10 ␮g/ml) for 15 min at 37 °C in 5% CO2. The fluorescent degradation products of DQ-BSA in lysosomes were imaged using a Bio-Rad Radiance 2000 two-photon confocal microscope and LaserSharp 2000 software

RESULTS

The total amounts of intracellular

The fusion of late endosomes and lysosomes requires the presence of

DISCUSSION