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Address correspondence to F. Gisou van der Goot, Dept. of Microbiology and Molecular Medicine, University of Geneva, 1 rue Michel Servet, Geneva, Switzerland 1211. Tel.: 41-22-379-5652. Fax: 41-22-379-5702. email: gisou.vandergoot{at}medecine.unige.ch
Abstract
The protective antigen (PA) of anthrax toxin binds to a cell surface receptor, undergoes heptamerization, and binds the enzymatic subunits, the lethal factor (LF) and the edema factor (EF). The resulting complex is then endocytosed. Via mechanisms that depend on the vacuolar ATPase and require membrane insertion of PA, LF and EF are ultimately delivered to the cytoplasm where their targets reside. Here, we show that membrane insertion of PA already occurs in early endosomes, possibly only in the multivesicular regions, but that subsequent delivery of LF to the cytoplasm occurs preferentially later in the endocytic pathway and relies on the dynamics of internal vesicles of multivesicular late endosomes.
Key Words: diphtheria toxin; LBPA; ALIX; MAPK; multivesicular; COP
Introduction
Anthrax toxin, one of the two main virulence factors produced by Bacillus anthracis, is an A-B type toxin, where the B subunit, called the protective antigen (PA), is involved in cell binding and the A subunits, of which there are two, lethal factor (LF) and edema factor (EF), bare the enzymatic toxic activities (Collier and Young, 2003). LF, a metalloprotease that targets MAPK kinases (MAPKKs), is responsible for lethality of the toxin (Collier and Young, 2003). EF, a CaM-dependent adenylate cyclase that elevates intracellular levels of cAMP (Collier and Young, 2003), is responsible for edema observed in anthrax patients.
PA (83 kD) binds to one of the two identified anthrax toxin receptors, ANTXR1 and ANTXR2 (Collier and Young, 2003), and is then processed at the NH2 terminus by the endoprotease furin, leaving a 63-kD form bound to the receptor. PA63 subsequently heptamerizes giving rise to a complex (PAheptamer) that is able to bind up to three molecules of LF and/or EF (Collier and Young, 2003). Heptamerization is accompanied by a spatial redistribution of the receptor from the glycerophospholipid region of the plasma membrane to specialized microdomains, so-called lipid rafts (Abrami et al., 2003). This redistribution triggers endocytosis of the PAheptamerEF/LF complex (Abrami et al., 2003). Upon encounter of a sufficiently acidic milieu, PAheptamer undergoes a conformational change that leads to membrane insertion, which allows translocation of LF/EF across the endosomal membrane and delivery to the cytoplasm (Collier and Young, 2003). It is not clear at which stations of the endocytic pathway membrane insertion of PAheptamer, translocation of the enzymatic units, and their release into the cytoplasm occur. Here, we show that membrane insertion of PAheptamer can be uncoupled from cytoplasmic delivery of LF, each occurring at different stages of the endocytic pathway.
Results and discussion
We have previously shown that upon heptamerization, PAheptamer is internalized, transported to early endosomes, and then rapidly degraded (Abrami et al., 2003) indicating efficient transport to lysosomes and exclusion from the recycling pathway. Here, we investigated whether PAheptamer undergoes pH-induced membrane insertion in early or in late endosomes. Early and late endosomes were isolated from toxin-treated BHK cells using a well-established subcellular fractionation protocol (Aniento et al., 1993; Gruenberg, 2001). The SDS-resistant PAheptamer, which only forms after the pH-dependent conformational change, was highly enriched in early endosomes (Fig. 1 A), co-fractionating with the small GTPase rab5 (Gruenberg, 2001), indicating that membrane insertion already occurred in early endosomes. In contrast, little SDS-resistant PAheptamer was detected in late endosomes containing rab7, presumably because degradation is extremely rapid (Abrami et al., 2003). Interestingly, LF was abundant in early endosomes and clearly detectable in late endosomes (Fig. 1 A).
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Therefore, we analyzed PA-treated cells by electron microscopy. Specific labeling was initially observed on the PM (Fig. 1 E) but, after warming the cells to 37°C, labeling was increasingly found on the intraluminal vesicles of a subset of multivesicular endosomes (Fig. 1, GJ, arrowheads). Interestingly, at intermediate times, a consistent observation was labeling in the region of the coat patch (Fig. 1 F; not depicted) implicated in sorting of receptors into multivesicular endosomes (Sachse et al., 2002).
To further address whether cytoplasmic release of LF required delivery to late endosomes, we inhibited microtubule-dependent transport using the depolymerizing agent nocodazole. LF-dependent MEK1 cleavage was delayed, without affecting the formation of SDS-resistant PAheptamer (Fig. 2 A, note that degradation was inhibited as expected because access to late endosomes and lysosomes is impaired). To rule out the possibility that this delay was somehow linked to the presence of some MEK1 on late endosomes (Wunderlich et al., 2001), we also followed LF-induced cleavage of another MAPKK, MKK3, which is involved in the p38 MAPK signaling cascade, different from the MEK1-dependent ERK pathway. As for MEK1, MKK3 cleavage was delayed in nocodazole-treated cells (Fig. 2 B). Interestingly, the kinetics of cleavage of MKK3 were slower than those of MEK1 (Fig. 2 C).
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Because the above observations indicated that LF delivery preferentially occurs from late endosomes, we decided to affect this organelle by overexpressing a dominant-negative mutant of rab7 (N125I), a small GTPase known to be involved in late endosome function and dynamics (Gruenberg, 2001). Although formation (Fig. 2 E) and degradation (not depicted) of SDS-resistant PAheptamer occurred normally, cleavage of MEK1 was again delayed (Fig. 2 E) confirming the involvement of late endosomes in cytoplasmic delivery of LF.
Altogether, the above experiments support the following sequence of events: the LFPAheptamer complex is internalized; in early endosomes, PAheptamer undergoes membrane insertion and mediates translocation of LF, in vitro studies indeed indicate that channel formation by PA is sufficient to allow translocation of LF. At that stage however, LF remains associated with early endosomes and microtubule-dependent transport to late endosomes is required for efficient delivery to the cytoplasm where LF can reach MAPKKs. The question arises why translocated LF can reach the cytoplasm from late endosomes but not from early endosomes. One possibility is that PAheptamer preferentially inserts into the membrane of intraluminal vesicles as suggested by the electron microscopy images (Fig. 1, GJ), which would lead to translocation of LF into the lumen of these vesicles.
Sorting into and formation of intraluminal vesicles occurs in early endosomes and seems to be, at that stage, a one-way street (Katzmann et al., 2002; Gruenberg and Stenmark, 2004). Once these intraluminal vesicles have reached late endosomes, some apparently acquire the ability to undergo regulated back fusion with the limiting membrane. The membrane of intraluminal vesicles indeed not only contains proteins destined to be degraded but also proteins in transit to other destinations in the cell (Kobayashi et al., 2000; Chow et al., 2002), which must get back to the limiting membrane from which budding of outgoing vesicles occurs (Gruenberg, 2001; Murk et al., 2003). To test whether this localized ability of back fusion of intraluminal vesicles could be used by LF to reach the cytoplasm, we affected one of the abundant and important components of intraluminal vesicles, the unconventional lipid lysobisphosphatidic acid (LBPA; Gruenberg, 2001). This lipid is unique to late endosomes and it was shown that feeding cells with a monoclonal antibody against LBPA, 6c4, impairs sorting of proteins and lipids leading to a traffic jam in the compartment (Kobayashi et al., 1999). We found that incubating cells with the 6c4 antibody did not affect the kinetics of formation of SDS-resistant PAheptamer as expected, but significantly delayed cleavage of MEK1 by LF (Fig. 3 A).
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To rule out that the effects of 6c4 and ALIX siRNA were somehow related to the metalloprotease activity of LF rather than the trafficking of the toxin, we used a hybrid (called fusion protein 59; FP59) in which the metalloprotease domain of LF had been replaced by the ADP-ribosyltransferase domain of Pseudomonas exotoxin A, which modifies elongation factor 2 (EF-2). Because this modification leads to a change in charge of EF-2 it can be monitored on native gels (Liu and Leppla, 2003b). Incubation of cells with nocodazole (not depicted), anti-LBPA antibody 6c4, as well as the knockdown of ALIX, delayed the kinetics of ADP-ribosylation of EF-2 by FP59 (Fig. 3, D and E).
To exclude that the various treatment had a gross effect on the endocytic pathway, we repeated the studies using a different toxin, namely diphtheria toxin (DT), known to translocate into the cytoplasm at the level of early endosomes (Papini et al., 1993; Lemichez et al., 1997). DT is also an A-B toxin, where the A subunit is, as Pseudomonas exotoxin A, an ADP-ribosyltransferase that modifies EF-2. We first tested ldlF cells at 40°C and found that they were insensitive to DT (Fig. 4 A) because ADP-ribosylation of EF-2 did not occur (Fig. 4 B). However, in contrast to what was observed for anthrax lethal toxin and PA+FP59, treatment with nocodazole had no effect on the kinetics of substrate modification by DT (Fig. 4 C), nor did incubation with the anti-LBPA antibody 6c4 (Fig. 4 D) or RNAi against ALIX (Fig. 4 E).
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Materials and methods
Cells and reagents
Wild-type PA, PASNKE, LF, FP59, a recombinant fusion toxin consisting of LF amino acids 1254, fused to the ADP-ribosylation domain of Pseudomonas exotoxin A, and DT were produced as described previously (Liu and Leppla, 2003a, b) and the corresponding polyclonal antibodies used (Abrami et al., 2003). Antibodies against the COOH terminus of MEK1 were purchased from Santa Cruz Biotechnology, Inc.; the NH2 terminus of MEK1 was purchased from Upstate Biotechnology; the NH2 terminus of MKK3, EF-2, and rab5 was purchased from Santa Cruz Biotechnology Inc.; and rab7, -COP, and LBPA were gifts from J. Gruenberg (University of Geneva). Antibodies against ALIX were a gift from R. Sadoul (University of Grenoble, Grenoble, France). Nocodazole was purchased from Sigma-Aldrich. HeLa, CHO, and mutant ldlF cells (provided by M. Krieger; Massachusetts Institute of Technology, Cambridge, MA), and RAW 264 cells were maintained as described previously (Abrami et al., 1998, 2003). The dominant-negative Rab7 N125I mutant and siRNA against ALIX (Matsuo et al., 2004) were gifts from P. Boquet (Institut National de la Santé et de la Recherche Médicale and University of Nice, Nice, France) and J. Gruenberg, respectively.
Biochemical methods
Early and late endosomes were isolated using sucrose density gradients (Kobayashi et al., 1999). For SDS-PAGE analysis, samples were boiled for 5 min. The various subunits and forms of the anthrax toxin and ADP-ribosylation of EF-2 were detected by Western blotting of SDS-PAGE and native on 420% acrylamide gradient gels (Abrami et al., 2003; Liu and Leppla, 2003b). Transient transfection experiments in HeLa cells were performed 48 h (1 µg cDNA/9.6 cm2 plate) or 72 h (200 pmoles siRNA/9.6 cm2 plate) using Fugene (Roche Diagnostics Corporation) and oligofectamine (Invitrogen) transfection reagents, respectively.
Immunofluorescence
CHO cells were incubated with 500 ng/ml PASNKE, submitted to an antibody sandwich and incubated at 37°C, submitted to an acid wash to remove remaining surface-bound PASNKE and fixed with 3% PFA (Abrami et al., 2003). Images were acquired using a 100x lens on an Axiophot (Carl Zeiss MicroImaging, Inc.), equipped with a cooled camera (Hamamatsu) using the Openlab acquisition software.
Acknowledgments
We thank M. Krieger, P. Boquet, J. Gruenberg, and R. Sadoul for reagents; D. Hsu for making toxins; and J. Gruenberg, I. Le Blanc, and M. Moayeri for critical reading of the manuscript.
This work was supported the Swiss National Science Foundation, the EMBO Young Investigator Program, and the National Institutes of Health (AI053270-01).
Submitted: 9 December 2003
Accepted: 30 June 2004
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