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Anthrax toxin triggers endocytosis of its receptor via a lipid raft-mediated clathrin-dependent process.

Abrami L, Liu S, Cosson P, Leppla SH, van der Goot FG - J. Cell Biol. (2003)

Bottom Line: The protective antigen (PA) of the anthrax toxin binds to a cell surface receptor and thereby allows lethal factor (LF) to be taken up and exert its toxic effect in the cytoplasm.Here, we report that clustering of the anthrax toxin receptor (ATR) with heptameric PA or with an antibody sandwich causes its association to specialized cholesterol and glycosphingolipid-rich microdomains of the plasma membrane (lipid rafts).We find that although endocytosis of ATR is slow, clustering it into rafts either via PA heptamerization or using an antibody sandwich is necessary and sufficient to trigger efficient internalization and allow delivery of LF to the cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Microbiology, University of Geneva, 1211 Geneva 4, Switzerland.

ABSTRACT
The protective antigen (PA) of the anthrax toxin binds to a cell surface receptor and thereby allows lethal factor (LF) to be taken up and exert its toxic effect in the cytoplasm. Here, we report that clustering of the anthrax toxin receptor (ATR) with heptameric PA or with an antibody sandwich causes its association to specialized cholesterol and glycosphingolipid-rich microdomains of the plasma membrane (lipid rafts). We find that although endocytosis of ATR is slow, clustering it into rafts either via PA heptamerization or using an antibody sandwich is necessary and sufficient to trigger efficient internalization and allow delivery of LF to the cytoplasm. Importantly, altering raft integrity using drugs prevented LF delivery and cleavage of cytosolic MAPK kinases, suggesting that lipid rafts could be therapeutic targets for drugs against anthrax. Moreover, we show that internalization of PA is dynamin and Eps15 dependent, indicating that the clathrin-dependent pathway is the major route of anthrax toxin entry into the cell. The present work illustrates that although the physiological role of the ATR is unknown, its trafficking properties, i.e., slow endocytosis as a monomer and rapid clathrin-mediated uptake on clustering, make it an ideal anthrax toxin receptor.

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Related in: MedlinePlus

Proteolytic processing of PA triggers partitioning of the anthrax toxin into lipid rafts. (A) Wild-type CHO cells were incubated for 1 h at 4°C with 500 ng/ml of either a mixture of native and trypsin-nicked PA83 (PA63), or PASNKE. DRMs were prepared and analyzed by Western blotting against PA and caveolin-1. The load (L) corresponds to 1/10 of the total material on the gradient. (B) Wild-type, sphingomyelin-deficient (CHO SPB), and recomplemented (CHO SPB(lcb1)) CHO cells were treated or not with β-MCD or filipin, then incubated with 500 ng/ml PA83 for 1 h at 4°C followed by 30 min at 37°C. DRMs were prepared and fractions were probed for the presence of PA, caveolin-1, and flotillin-1 by Western blotting. (C) ATR-deficient CHO cells were stably transfected with human ATR having an HA-tag at the COOH terminus. DRMs were prepared and fractions were probed using a biotinylated anti-HA antibody. The two upper bands are endogenous biotinylated CHO cell proteins recognized by the streptavidin-HRP, even in cells not expressing ATR-HA. (D) CHO cells were incubated with 500 ng/ml of either trypsin-nicked PA83 or of PASNKE for 1 h at 4°C followed by 30 min at 37°C, washed at 4°C, and further incubated with 1 μg/ml LF for 1 h at 4°C before preparation of DRMs. Fractions were analyzed by Western blot with anti-LF pAb.
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fig1: Proteolytic processing of PA triggers partitioning of the anthrax toxin into lipid rafts. (A) Wild-type CHO cells were incubated for 1 h at 4°C with 500 ng/ml of either a mixture of native and trypsin-nicked PA83 (PA63), or PASNKE. DRMs were prepared and analyzed by Western blotting against PA and caveolin-1. The load (L) corresponds to 1/10 of the total material on the gradient. (B) Wild-type, sphingomyelin-deficient (CHO SPB), and recomplemented (CHO SPB(lcb1)) CHO cells were treated or not with β-MCD or filipin, then incubated with 500 ng/ml PA83 for 1 h at 4°C followed by 30 min at 37°C. DRMs were prepared and fractions were probed for the presence of PA, caveolin-1, and flotillin-1 by Western blotting. (C) ATR-deficient CHO cells were stably transfected with human ATR having an HA-tag at the COOH terminus. DRMs were prepared and fractions were probed using a biotinylated anti-HA antibody. The two upper bands are endogenous biotinylated CHO cell proteins recognized by the streptavidin-HRP, even in cells not expressing ATR-HA. (D) CHO cells were incubated with 500 ng/ml of either trypsin-nicked PA83 or of PASNKE for 1 h at 4°C followed by 30 min at 37°C, washed at 4°C, and further incubated with 1 μg/ml LF for 1 h at 4°C before preparation of DRMs. Fractions were analyzed by Western blot with anti-LF pAb.

Mentions: We investigated whether the selective uptake of PA63, and not of PA83, was due to a change in surface distribution on conversion of PA83 to PA63. The similarity between the structure and the mode of action of PA and that of certain bacterial pore-forming toxins such as aerolysin (Abrami et al., 2000) prompted us to determine whether PA63 was associated with raftlike lipid microdomains of the plasma membrane. These domains are thought to form through lateral movement and assembly of cholesterol and glycosphingolipids. A specific subclass of rafts form flasklike invaginations at the plasma membrane and are then called caveolae (Simons and Ikonen, 1997; Brown and London, 1998). Rafts act as surface platforms in signal transduction, cholesterol homeostasis, and endocytosis (Brown and London, 1998; Simons and Toomre, 2000). Lipid rafts have also been implicated in various infectious processes (Fivaz et al., 1999), and in particular, were shown to favor heptamerization of the pore-forming toxin aerolysin (Abrami and van der Goot, 1999) via mechanisms that could well apply to PA. One biochemical characteristic of rafts is their resistance to nonionic detergents at 4°C, which allows their purification on density gradients. Native, full-size PA83 was associated with detergent-soluble domains of the plasma membrane (Fig. 1 A) in agreement with previous observations (Beauregard et al., 1999), as was a variant of PA mutated in the consensus furin cleavage site (PASNKE; Gordon et al., 1995). In contrast, PA63 (which can be obtained either in vitro by trypsin cleavage or in vivo by cell surface furin processing) was almost exclusively associated with detergent-resistant membranes (DRMs; Fig. 1 A). To confirm that the presence of PA63 in more buoyant, upper fractions of the gradient, is indeed due to its association with lipid rafts, we investigated whether the distribution of PA63 was altered by removal of plasma membrane cholesterol using β-methyl cyclodextrin (β-MCD) or sequestration of cholesterol using filipin (Simons and Toomre, 2000). As shown in Fig. 1 B, PA63 was partially (β-MCD) or completely (filipin) redistributed to the bottom of the gradient, in a manner similar to that of the normally raft-associated protein flotillin-1 (Bickel et al., 1997), but in contrast to the caveolar protein caveolin-1 (Kurzchalia and Parton, 1999). Because rafts are also rich in sphingomyelin, we analyzed whether reduction of sphingomyelin levels would affect the surface distribution of PA63. In sphingomyelin-deficient mutant CHO cells (Hanada et al., 1998), PA63 was no longer associated with DRMs (Fig. 1 B). Flotillin-1 was also partially redistributed to detergent-soluble fractions, in contrast to caveolin-1, which remained at the top of the gradient. Thus, PA63 is associated with noncaveolar cholesterol and sphingolipid-rich domains of the plasma membrane.


Anthrax toxin triggers endocytosis of its receptor via a lipid raft-mediated clathrin-dependent process.

Abrami L, Liu S, Cosson P, Leppla SH, van der Goot FG - J. Cell Biol. (2003)

Proteolytic processing of PA triggers partitioning of the anthrax toxin into lipid rafts. (A) Wild-type CHO cells were incubated for 1 h at 4°C with 500 ng/ml of either a mixture of native and trypsin-nicked PA83 (PA63), or PASNKE. DRMs were prepared and analyzed by Western blotting against PA and caveolin-1. The load (L) corresponds to 1/10 of the total material on the gradient. (B) Wild-type, sphingomyelin-deficient (CHO SPB), and recomplemented (CHO SPB(lcb1)) CHO cells were treated or not with β-MCD or filipin, then incubated with 500 ng/ml PA83 for 1 h at 4°C followed by 30 min at 37°C. DRMs were prepared and fractions were probed for the presence of PA, caveolin-1, and flotillin-1 by Western blotting. (C) ATR-deficient CHO cells were stably transfected with human ATR having an HA-tag at the COOH terminus. DRMs were prepared and fractions were probed using a biotinylated anti-HA antibody. The two upper bands are endogenous biotinylated CHO cell proteins recognized by the streptavidin-HRP, even in cells not expressing ATR-HA. (D) CHO cells were incubated with 500 ng/ml of either trypsin-nicked PA83 or of PASNKE for 1 h at 4°C followed by 30 min at 37°C, washed at 4°C, and further incubated with 1 μg/ml LF for 1 h at 4°C before preparation of DRMs. Fractions were analyzed by Western blot with anti-LF pAb.
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Related In: Results  -  Collection

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fig1: Proteolytic processing of PA triggers partitioning of the anthrax toxin into lipid rafts. (A) Wild-type CHO cells were incubated for 1 h at 4°C with 500 ng/ml of either a mixture of native and trypsin-nicked PA83 (PA63), or PASNKE. DRMs were prepared and analyzed by Western blotting against PA and caveolin-1. The load (L) corresponds to 1/10 of the total material on the gradient. (B) Wild-type, sphingomyelin-deficient (CHO SPB), and recomplemented (CHO SPB(lcb1)) CHO cells were treated or not with β-MCD or filipin, then incubated with 500 ng/ml PA83 for 1 h at 4°C followed by 30 min at 37°C. DRMs were prepared and fractions were probed for the presence of PA, caveolin-1, and flotillin-1 by Western blotting. (C) ATR-deficient CHO cells were stably transfected with human ATR having an HA-tag at the COOH terminus. DRMs were prepared and fractions were probed using a biotinylated anti-HA antibody. The two upper bands are endogenous biotinylated CHO cell proteins recognized by the streptavidin-HRP, even in cells not expressing ATR-HA. (D) CHO cells were incubated with 500 ng/ml of either trypsin-nicked PA83 or of PASNKE for 1 h at 4°C followed by 30 min at 37°C, washed at 4°C, and further incubated with 1 μg/ml LF for 1 h at 4°C before preparation of DRMs. Fractions were analyzed by Western blot with anti-LF pAb.
Mentions: We investigated whether the selective uptake of PA63, and not of PA83, was due to a change in surface distribution on conversion of PA83 to PA63. The similarity between the structure and the mode of action of PA and that of certain bacterial pore-forming toxins such as aerolysin (Abrami et al., 2000) prompted us to determine whether PA63 was associated with raftlike lipid microdomains of the plasma membrane. These domains are thought to form through lateral movement and assembly of cholesterol and glycosphingolipids. A specific subclass of rafts form flasklike invaginations at the plasma membrane and are then called caveolae (Simons and Ikonen, 1997; Brown and London, 1998). Rafts act as surface platforms in signal transduction, cholesterol homeostasis, and endocytosis (Brown and London, 1998; Simons and Toomre, 2000). Lipid rafts have also been implicated in various infectious processes (Fivaz et al., 1999), and in particular, were shown to favor heptamerization of the pore-forming toxin aerolysin (Abrami and van der Goot, 1999) via mechanisms that could well apply to PA. One biochemical characteristic of rafts is their resistance to nonionic detergents at 4°C, which allows their purification on density gradients. Native, full-size PA83 was associated with detergent-soluble domains of the plasma membrane (Fig. 1 A) in agreement with previous observations (Beauregard et al., 1999), as was a variant of PA mutated in the consensus furin cleavage site (PASNKE; Gordon et al., 1995). In contrast, PA63 (which can be obtained either in vitro by trypsin cleavage or in vivo by cell surface furin processing) was almost exclusively associated with detergent-resistant membranes (DRMs; Fig. 1 A). To confirm that the presence of PA63 in more buoyant, upper fractions of the gradient, is indeed due to its association with lipid rafts, we investigated whether the distribution of PA63 was altered by removal of plasma membrane cholesterol using β-methyl cyclodextrin (β-MCD) or sequestration of cholesterol using filipin (Simons and Toomre, 2000). As shown in Fig. 1 B, PA63 was partially (β-MCD) or completely (filipin) redistributed to the bottom of the gradient, in a manner similar to that of the normally raft-associated protein flotillin-1 (Bickel et al., 1997), but in contrast to the caveolar protein caveolin-1 (Kurzchalia and Parton, 1999). Because rafts are also rich in sphingomyelin, we analyzed whether reduction of sphingomyelin levels would affect the surface distribution of PA63. In sphingomyelin-deficient mutant CHO cells (Hanada et al., 1998), PA63 was no longer associated with DRMs (Fig. 1 B). Flotillin-1 was also partially redistributed to detergent-soluble fractions, in contrast to caveolin-1, which remained at the top of the gradient. Thus, PA63 is associated with noncaveolar cholesterol and sphingolipid-rich domains of the plasma membrane.

Bottom Line: The protective antigen (PA) of the anthrax toxin binds to a cell surface receptor and thereby allows lethal factor (LF) to be taken up and exert its toxic effect in the cytoplasm.Here, we report that clustering of the anthrax toxin receptor (ATR) with heptameric PA or with an antibody sandwich causes its association to specialized cholesterol and glycosphingolipid-rich microdomains of the plasma membrane (lipid rafts).We find that although endocytosis of ATR is slow, clustering it into rafts either via PA heptamerization or using an antibody sandwich is necessary and sufficient to trigger efficient internalization and allow delivery of LF to the cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Microbiology, University of Geneva, 1211 Geneva 4, Switzerland.

ABSTRACT
The protective antigen (PA) of the anthrax toxin binds to a cell surface receptor and thereby allows lethal factor (LF) to be taken up and exert its toxic effect in the cytoplasm. Here, we report that clustering of the anthrax toxin receptor (ATR) with heptameric PA or with an antibody sandwich causes its association to specialized cholesterol and glycosphingolipid-rich microdomains of the plasma membrane (lipid rafts). We find that although endocytosis of ATR is slow, clustering it into rafts either via PA heptamerization or using an antibody sandwich is necessary and sufficient to trigger efficient internalization and allow delivery of LF to the cytoplasm. Importantly, altering raft integrity using drugs prevented LF delivery and cleavage of cytosolic MAPK kinases, suggesting that lipid rafts could be therapeutic targets for drugs against anthrax. Moreover, we show that internalization of PA is dynamin and Eps15 dependent, indicating that the clathrin-dependent pathway is the major route of anthrax toxin entry into the cell. The present work illustrates that although the physiological role of the ATR is unknown, its trafficking properties, i.e., slow endocytosis as a monomer and rapid clathrin-mediated uptake on clustering, make it an ideal anthrax toxin receptor.

Show MeSH
Related in: MedlinePlus