©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Effect of Anthrax Toxins Lethal Factor on Ion Channels Formed by the Protective Antigen (*)

(Received for publication, April 25, 1995; and in revised form, June 1, 1995)

Jianmin Zhao Jill C. Milne R. John Collier (§)

From the Department of Microbiology and Molecular Genetics and Shipley Institute of Medicine, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Protective antigen (PA), a component of anthrax toxin, mediates translocation of the toxin's lethal and edema factors (LF and EF, respectively) to the cytoplasm, via a pathway involving their release from an acidic intracellular compartment. PA, a 63-kDa proteolytic fragment of PA, can be induced to form ion-conductive channels in the plasma membrane of mammalian cells by acidification of the medium. These channels are believed to be comprised of dodecyl sulfate-resistant oligomers (heptameric rings) of PA seen by electron microscopy of the purified protein. Here we report that the PA-mediated efflux of Rb from preloaded CHO-K1 cells under acidic conditions is strongly inhibited (geq70%) by LF or LF(N), a PA-binding fragment of LF. Control proteins caused no inhibition. Evidence is presented that the inhibition involves partial blockage of ion conductance by the PA channel. Also, oligomer formation is slowed somewhat by LF at pH values near the pH threshold of channel formation (pH 5.3), suggesting that channel formation may also be retarded under these conditions. The relevance of these results to the location of the LF-binding site on PA and the mechanism of LF and EF translocation is discussed.


INTRODUCTION

Many toxic proteins are multifunctional enzymes that are able to enter cells and covalently modify intracellular substrates. After binding to a cell-surface receptor, these toxins generally undergo receptor-mediated endocytosis and are transported to an appropriate intracellular compartment, where their enzymic moieties are transferred across the membrane and into the cytosol. Such toxins are usually composed of two structurally distinct moieties, termed A and B, with B serving to bind the toxin to receptors and facilitate transfer of the enzymically active A moiety to cytoplasm(1, 2, 3) . The precise mechanism by which the B moiety promotes membrane traversal by A is yet to be established in detail for any toxin.

Bacillus anthracis, the causative agent of anthrax, secretes three proteins: edema factor (EF, (^1)89 kDa), lethal factor (LF, 90 kDa), and protective antigen (PA, 83 kDa), which have been collectively termed anthrax toxin. EF and LF serve as alternative A moieties, which act in combination with PA, the B moiety, to generate different toxic effects on cells(4) . EF is a calcium/calmodulin-dependent adenylate cyclase, whose entry into the cytosol elevates the level of cAMP(5) , causing various effects, depending on the nature of the cell. In contrast, LF only affects macrophages, causing them to produce high levels of interleukin 1 and tumor necrosis factor alpha(6) , which in turn induce shock and death of the host. High concentrations of LF+PA result in cytolysis of macrophages(7) . Although the biochemical activity of LF has not yet been elucidated, analysis of its primary structure suggests that the protein may be a metalloprotease(8) .

According to our current concept of anthrax toxin action(4) , the entry of EF or LF into cells proceeds as follows. PA binds to a specific cell surface receptor(9) , and its NH(2)-terminal 20-kDa domain (PA) is removed by a cellular protease, implicated as furin(10) , leaving the COOH-terminal 63-kDa domain (PA) bound to the receptor(11) . Removal of PA exposes a site on PA to which either EF or LF can bind(4) . The complex of receptor-bound PAbulletEF or PAbulletLF then undergoes receptor-mediated endocytosis and is delivered to an acidic membrane-bound compartment, presumably the endosome. There, PA facilitates translocation of EF and LF across the endosomal membrane into the cytosol(12, 13) .

PA is capable of forming ion-conductive channels in artificial bilayers and cellular membranes(14, 15, 16) , and in both systems proteolytic activation of full-length PA and exposure of the PA fragment to acidic pH are required for membrane insertion and channel formation. The relevance of proteolytic activation and exposure to acidic pH has been demonstrated in vivo: mutation or deletion of the protease cleavage site in PA abolishes EF and LF binding(10, 17) , and lysosomotropic agents protect cells from the PA-mediated toxic effect of EF and LF(12, 13) . Trypsin-activated (``nicked'') PA, consisting of PA complexed with the complementary fragment, PA, is capable of forming well-defined, cation-selective channels when added to planar lipid bilayer systems(14) . Also, cells preloaded with Rb release this isotope when exposed to PA or nicked PA followed by a low pH pulse(16) .

Recently, we found that purified PA forms high molecular weight, dodecyl sulfate-resistant oligomers, which appear predominantly as heptameric rings by electron microscopy(18) . Similar or identical oligomers are generated in a time-dependent manner in cells incubated with PA, and oligomer formation in vivo appears to require exposure of PA to acidic pH, presumably in an acidic intracellular compartment. Thus, inhibitors of internalization or endosome acidification block conversion of cell-associated PA to high molecular mass species, and acidification of the medium induces oligomer formation either in the presence or absence of these inhibitors. The conditions required for PA oligomerization therefore correlate with those required for channel formation and translocation of EF and LF across cellular membranes, suggesting that the oligomer may play a role in both activities.

If the PA oligomer functions in translocation of EF and LF, as hypothesized, its interaction with these proteins might affect either the formation of PA ion channels or the conductance properties of the channels once formed. Here we report that LF inhibits the PA-induced permeabilization of the plasma membranes of CHO-K1 cells under acidic pH conditions and present data relevant to the mechanism of the inhibition.


EXPERIMENTAL PROCEDURES

Toxin Purification

PA and LF were purified from the Sterne strain of B. anthracis, according to the method of Leppla (19) , with modifications, as described previously(14) . The LF(N) fragment, comprising the amino-terminal 255 residues of LF, was cloned from the toxin plasmid (pXO1) of the Sterne strain (20) of B. anthracis by polymerase chain reaction, with appropriate restriction sites introduced by the 3` (NdeI) and 5` (BamHI) polymerase chain reaction primers. The polymerase chain reaction product was then cloned into an Escherichia coli expression vector, pET15b (Novagen, Inc, Madison, WI), replacing the NdeI-BamHI fragment. The recombinant protein was produced with an amino-terminal hexa-histidine tag, allowing protein to be purified by Ni-chelate affinity chromatography. Briefly, cultures were grown in Luria broth-ampicillin (100 µg/ml) to an OD of 0.6-1.0, and protein expression was induced by addition of isopropyl-1-thio-beta-D-galactopyranoside (1 mM). Cell lysates were prepared by sonication, cleared by centrifigation, and loaded onto a Ni-charged column(21) . The column was washed, and the protein was eluted with imidazole according to the manufacturer's protocol. The eluted protein was further purified by anion-exchange chromatography (Mono-Q column on the fast proteim liquid chromatography system, Pharmacia), yielding 1-2 mg of purified protein/liter of culture. The purity of all proteins used in the experiments was greater than 95%, as judged by SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining. Protein concentrations were determined using the Bradford reagent (Pierce). Nicked PA was produced by treatment of PA (0.3 mg/ml = 3.6 mM) with trypsin (1 µg/ml) for 45 min at 37 °C, and the reaction was stopped by adding soybean trypsin inhibitor to 10 µg/ml.

Cell Culture

The Chinese hamster ovary cell line (CHO-K1), from the American Type Culture Collection, was grown in Ham's F-12 medium supplemented with 10% calf serum, 500 units/ml penicillin G, and 500 units/ml streptomycin sulfate (Life Technologies, Inc.).

Rb Efflux Measurements

CHO-K1 cells were plated at a density of 10^6 cells/well in 6-well culture plates or 2 10^5 cells/well in 24-well culture plates, depending on the experiment. Approximately 10-12 h after plating, fresh medium containing Rb (1 µCi/ml, NEN DuPont) was added, and the cells were incubated for an additional 9-10 h. PA binding was carried out by incubation of cells with fresh medium containing 6 nM nicked PA for 2 h at 4 °C. The cells were then washed two times with cold Dulbecco's phophate-buffered saline (PBS) (Sigma) to remove unbound PA and treated with isotonic buffers (20 mM MES-Tris, 145 mM NaCl, pH 4.0-7.0) to initiate the Rb release assay. After 30 min at 4 °C, the medium was removed, and released Rb was measured using a counter (LKB-Wallac Clinigamma). Finally, SDS (1%) was added to the cells to determine cell-retained Rb. For the kinetic study of Rb release, the cells were pulsed with pH 5.0 buffer, and an aliquot of medium was removed at intervals for isotopic assay. All experiments were performed in duplicate.

The percentage of released Rb was calculated as follows: % Rb release = (released Rb)/(total Rb [released + retained]) 100. Spontaneous release was determined in the absence of PA and was subtracted from the reported values. Spontaneous release of Rb was approximately 20% for cells treated with pH 5.0 buffer for 30 min.

Detection of PA Oligomerization in Cells by Immunoblotting

CHO-K1 cells were seeded at a density of 2 10^5 cells/well in 24-well culture plates. After 24 h the plate was placed on ice, and binding of nicked PA (6 nM) was carried out as described above at 4 °C for 2 h. Unbound PA was removed by washing cells with cold PBS three times. The cells were treated with isotonic buffers at different pH values (0.4 ml/well). For analysis of the effect of LF on PA oligomerization, LF (0.1 µM) was included in these buffers. After 20 min the buffer was removed, and stop solution (PBS plus 1% Triton X-100 and 1% SDS) was added. The samples were then loaded on a 7.5% SDS-polyacrylamide gel, and after electrophoresis the proteins were transferred to a nitrocellulose membrane. The membrane was incubated with rabbit anti-PA antibody in TBS (10 mM Tris-HCl, 150 mM NaCl, pH 7.5) containing 5% milk. After washing three time with TBST (TBS plus 1% Triton X-100) a secondary antibody (horseradish peroxidase-conjugated anti-rabbit IgG from Sigma) was added in TBS containing 5% milk. PA was visualized using the DuPont Renaissance Kit according to the manufacturer's protocol. The relative amounts of PA monomer and oligomer were calculated from the intensity of protein bands after densitometric scanning (LKB Ultroscan).

Na Influx Measurements

CHO-K1 cells (2.5 10^5 cell/well) were plated in 24-well culture plates 24 h prior to the start of an experiment. Nicked PA (6 nM) was incubated with cells for 2 h at 4 °C, and cells were washed with cold PBS. The cells were treated with low pH (5.0) medium for 30 min at 4 °C to induce ion channel formation, and LF (0.2 µM) was then added into the low pH medium for an additional 30 min at 4 °C. An equal volume of the same low pH medium containing 2 µCi/ml Na (NEN Dupont) was added at time 0, the incubation was continued at 4 °C, and at intervals the medium was removed from selected samples and the cells were washed four times with cold PBS. The cell-associated radioactivity was determined after dissolving the cells in 1% SDS. Spontaneous influx of Na determined in the absence of PA has been subtracted from the values reported. In a parallel experiment cell-associated protein was determined at intervals with Bradford reagent. All experiments were performed in duplicate.


RESULTS

Permeabilization of the plasma membrane of CHO-K1 cells by PA was monitored by measuring Rb efflux. Briefly, CHO-K1 cells preloaded with Rb were incubated with trypsin-activated PA [nicked PA (PA + PA); 6 nM] for 2 h at 4 °C, unbound toxin was removed by washing, and medium buffered at low pH (pH 5.0) was added. Release of Rb from the cells was then quantified at intervals by assaying aliquots of the medium. Upon addition of pH 5.0 isotonic buffer, Rb efflux began immediately, continued linearly for the first 10 min, and was complete by 15-20 min, at which point 70-80% of total cell-associated Rb had been released (Fig. 1). Inclusion of LF (0.1 µM) in the acidic pulse buffer caused a rapid and strong inhibition (5-fold) of the rate of Rb release (Fig. 1). Rb release continued at an approximately constant rate over the 30-min period of the experiment in the presence of LF. A more gradual decline in the rate of Rb release was seen when LF was added 1, 2, or 5 min after the low pH buffer. LF(N) (residues 1-255), the PA-binding domain of LF, had a similar inhibitory effect, whereas bovine serum albumin or lysozyme caused no inhibition (Table 1). Tetrahexylammonium bromide (HeNBr) also inhibited Rb release, consistent with earlier reports (14, 16) .


Figure 1: Inhibition of PA-mediated Rb efflux from CHO-K1 cells by LF. CHO-K1 cells pre-loaded with Rb in 6-well culture plates were incubated with nicked PA (6 nM) for 2 h at 4 °C. Unbound PA was removed by washing with cold PBS, and low pH (5.0) isotonic buffer was added in the presence (bullet) or absence () of LF (0.1 µM). Alternatively, LF was added after exposure of cells to low pH medium for 1 (), 2 (▪), or 5 (▴) min, as indicated by arrows, to a final concentration of 0.1 µM. At the times indicated an aliquot of medium was removed to determine released Rb.





The inhibition of Rb release by LF or LF(N) was dependent both on the concentration of the inhibitory proteins and on the pH. Half-maximal inhibition was seen at 40 nM LF or 100 nM LF(N) when these proteins were included in the pH 5.0 pulse medium (Fig. 2). The inhibition by LF was maximal at or near this threshold pH and decreased as the pH was lowered toward pH 4.0 (Fig. 3). A similar dependence on pH was seen with LF(N), but the reduction in inhibitory activity with decreasing pH was less pronounced (not shown).


Figure 2: Concentration dependence of the inhibition of Rb release by LF and LF(N). CHO-K1 cells pre-loaded with Rb in 24-well culture plates were incubated with nicked PA (6 nM) at 4 °C for 2 h. Unbound PA was removed by washing with cold PBS, and low pH (5.0) buffer containing the indicated amount of LF (bullet) or LF(N) () was added. After a 30-min exposure to the acidic buffer at 4 °C, aliquots of medium were removed to measure released Rb.




Figure 3: pH dependence of the inhibition of Rb release by LF. CHO-K1 cells pre-loaded with Rb in 24-well culture plates were incubated with nicked PA (6 nM) for 2 h at 4 °C. Unbound PA was removed by washing with cold PBS, and pH (4.0-7.0) buffers were added (bullet). After 30 min the medium was removed for determination of released Rb. To determine the effect of LF on Rb release, LF (0.1 µM) () was included in the pH (4.0-5.5) buffers.



Because channel formation by nicked PA alone shows a strong pH dependence (Fig. 3)(16) , we performed an experiment to differentiate the pH dependence of the inhibition by LF from that of channel formation. Rb-loaded CHO-K1 cells to which PA had been bound were pulsed with pH 4.5 buffer for 1 min to induce ion channel formation, and after removal of the buffer, the cells were treated immediately with LF in buffers of various pH. The inhibition of Rb release by LF was maximal at pH 5.5 and declined as the pH was either raised or lowered from this value (Table 2). Control experiments (not shown) indicated that neutralization of the medium did not affect the kinetics of Rb release after channel formation by nicked PA at low pH.



The inhibition of Rb release by LF and LF(N) in the experiments reported above could be attributable to effects on channel formation, on ion conductance by the channels, or on both. To distinguish among these possibilities, we first examined the effects of LF on the formation of high molecular mass, dodecyl sulfate-resistant oligomers of PA, which are believed to represent the ring-shaped heptamers seen by electron microscopy and to be responsible for the PA channels in membranes(18) . Cells that had been incubated with nicked PA were treated with buffers of various pH for 20 min and then disrupted with dodecyl sulfate. Analysis of the extracts by SDS-polyacrylamide gel electorphoresis and Western blotting revealed oligomer in samples treated at pH 5.25 or lower (Fig. 4), which correlates with the pH dependence profile of channel formation. Inclusion of LF in the low pH buffers caused no apparent change in oligomer formation.


Figure 4: Effect of LF on PA oligomerization as a function of pH. CHO-K1 cells in 6-well culture plates were incubated in the presence of nicked PA (6 nM) at 4 °C for 2 h, and unbound PA was removed by washing with cold PBS. The cells were treated with the pH (4.0-7.0) buffers indicated at the top of the figure for 20 min (panel A, top). Alternatively, LF (0.1 µM) was included in the pH buffers (panel A, bottom). At the end of incubation, the buffer was removed, and the cells were dissolved in PBS containing 1% SDS. The cell extract was electrophoresed on a polyacrylamide gel, followed by Western blotting. The positions of the PA monomer and oligomer are indicated. The plot in B shows the percentage of oligomer formed in the presence () or absence (bullet) of LF, as determined by densitometry of the blot in A.



To probe the possibility that LF might nonetheless have an effect on the initial rate of oligomerization, we performed an experiment in which the rate of oligomer formation was monitored at pH 4.6 and 5.0 in the presence and absence of LF. As shown in Fig. 5, oligomerization was essentially complete by 5 min at pH 4.6, and inclusion of LF in the acidification buffer had little or no effect. At pH 5.0, however, oligomer formation proceeded more slowly, and a slight, but reproducible, inhibition by LF was seen.


Figure 5: Kinetics of low pH-induced PA oligomerization and the effect of LF on oligomerization. CHO-K1 cells in 6-well culture plates were incubated in the presence of nicked PA (6 nM) for 2 h at 4 °C. After unbound PA was removed by washing with PBS, the cells were exposed to a low pH buffer (pH 5.0, top panel, or pH 4.6, bottom panel) for the indicated times. The reaction was stopped by removing the low pH buffer and adding PBS containing 1% SDS. The cell extract was then processed for Western blotting as described in Fig. 4. Percentage of oligomer was calculated from densitometer scans of the blot (). To determine the effect of LF on PA oligomerization, LF (0.1 µM) was added in the low pH buffer (bullet). Alternatively, LF (0.1 µM) was included in the PA-binding medium and was also present in the low pH buffer (▪).



Thus, while the inhibition of channel formation by LF might account for part of the inhibition of Rb release produced by LF, most of the inhibition appeared to be due to an effect on ion conductance of the PA channels. To support this hypothesis we performed an experiment in which PA-bound cells were first incubated with pH 5 medium for 30 min to allow channel formation to proceed to completion. After an additional 30-min incubation in the presence or absence of LF, Na was added, and its influx was measured at intervals by assaying cell-associated radioactivity. As shown in Fig. 6, a strong inhibition by LF was seen, supporting the conclusion that binding of LF to preformed PA channels significantly affected their ion conductance.


Figure 6: Effect of LF on PA-mediated influx of Na into CHO-K1 cells. CHO-K1 cells in 24-well culture plates were incubated with nicked PA (6 nM) for 2 h at 4 °C. After unbound PA was removed by washing with cold PBS, the cells were treated with low pH (5.0) buffer for 30 min, and the incubation was continued in the presence (bullet) or absence () of LF (0.2 µM) for an additional 30 min at 4 °C. Na was then added, and cell-associated radioactivity was determined at intervals as described under ``Experimental Procedures.'' Cellular protein, determined with Bradford reagent in parallel sets of samples in the absence of Na, showed no loss of cells during the course of the experiment.




DISCUSSION

The ability of LF to inhibit PA-mediated Rb release from CHO-K1 cells under acidic pH conditions strongly suggests that LF interacts directly with PA channels in the plasma membrane. (EF would presumably cause a similar inhibition, but we were unable to test it because of difficulties in obtaining sufficient quantities of the protein.) The interaction of LF with the PA channels is consistent with the known role of PA in membrane translocation of EF and LF and is supported by earlier results showing binding of LF to PAin vivo and in vitro. EF and LF have been found to bind to cells only after PA has attached and been proteolytically processed (by the endogenous protease, furin(10) ), and sedimentation analysis and native polyacrylamide gel electrophoresis have confirmed a direct interaction in vitro(4) .

The fact that LF and LF(N) showed similar inhibitory effects on PA ion channel activity ( Fig. 2and Table 1) indicates (i) that the PA-binding portion of LF is largely responsible for the observed inhibition and (ii) that the EF/LF-binding site on PA is intact and exposed after PA inserts into the plasma membrane. While the COOH-terminal portion of LF (residues 256-776) is apparently not required for the inhibition of PA ion channel activity, its presence decreases the inhibition at lower pH values, perhaps due to influences on conformation of the LF(N) moiety. Our data on the inhibition of PA channel activity by LF are consistent with the report by Finkelstein (22) that addition of EF or LF to PA channels in planar bilayers at acidic pH causes a reduction of conductance on a time scale measured in many seconds. Recently Finkelstein and co-workers have found that the ion conductance of PA is attenuated by EF and LF at the single-channel level, (^2)which is consistent with our conclusions regarding the inhibition by LF and LF(N).

Half-maximal inhibition of the PA ion channels in our experiments occurred at approximately 40 nM LF at pH 5.0 (Fig. 2). An apparent dissociation constant of 10 pM of LF for PA has been reported, from results obtained with a soluble competitive binding assay utilizing a monoclonal antibody against PA(4) . Competitive displacement of radiolabeled LF from macrophage-associated PA at pH 7.5 occurred at a value of 2.4 nM(23) . These results suggest that the affinity of LF for PA is strong, in the nanomolar to picomolar range, but further work will be required to obtain precise values of the affinity constant under various conditions.

We have previously established a relationship between PA oligomerization and ion channel formation in the plasma membrane of CHO-K1 cells under acidic pH conditions(18) . The conditions required for oligomerization and channel formation by PA correlate with those required for the translocation of LF and EF across cellular membranes(18) , and we infer that the PA oligomer is likely to be the structural entity that mediates both translocation and ion conductance. Although the presence of LF did not prevent PA oligomer formation in the plasma membrane, it did slow the rate of oligomerization somewhat at pH 5.0 (Fig. 5). This could be attributed to LF-bound PA undergoing self-association more slowly than free PA monomers, or to PA needing to dissociate from LF prior to oligomerization. The fact that LF had little or no effect on PA oligomerization in the plasma membrane of CHO-K1 cells at pH 4.6 and the finding that LF can efficiently inhibit PA ion channel activity at this pH suggest that PA can undergo oligomerization in cells with or without LF bound. These results also suggest that the LF-binding site on the PA molecule is accessible before, during, or after its oligomerization and insertion into the plasma membrane of CHO-K1 cells.

The role that either the oligomer or the channel plays in transfer of EF and LF across the endosomal membrane into the cytosol is not yet understood, but at least two models can be proposed. In the first, EF and LF are assumed to traverse the endosomal membrane via the PA channel. The complex of PAbulletLF or PAbulletEF may insert into the endosomal membrane, before or after oligomerization, and the channel formed by PA may then mediate transfer of unfolded LF and EF to the cytosol, where they would then refold into native form. In the second model, monomeric PA is assumed to mediate translocation. The acidic environment of the endosome may trigger insertion of PAbulletLF or PAbulletEF complexes into the endosomal membrane, resulting in transfer of LF and EF to the cytosol via the lipid phase per se or the PA-lipid interface. According to this model, oligomerization of PA and channel formation are secondary events in which, after the translocation, lateral diffusion of the membrane-inserted PA monomers results in their oligomerzation and channel formation. The ability of LF to inhibit channel activity under acidic pH conditions favors the former model.

The block in channel conductance caused by LF at acidic pH apparently results from the binding of LF to a site on PA, which partially occludes the channel and inhibits ion transport. The binding may be within the PA channel (which may correspond to the lumen of the heptameric PA rings) and thereby physically occlude the lumen, or it may be at peripheral sites and attenuate conductance via conformational alterations induced in PA. No data are yet available from crystallography or electron microscopy to localize LF or LF(N) bound to the PA oligomers. At pH 7.0 LF has only a slight inhibitory effect on the activity of the PA channel (Table 2), suggesting that LF weakly interacts with PA at neutral pH. This finding may be relevant to the mechanism by which LF is released from the channel and is able to enter the cytosol, which is near neutral pH.

The proposed models for PA membrane insertion and LF/EF translocation are likely to be directly relevant to translocation by binary toxins produced by other bacteria, and perhaps to translocation by structurally unrelated toxins(24, 25) . Diphtheria, tetanus, and botulinum toxins share with anthrax toxin the ability to insert into lipid bilayers and form ion channels under acidic conditions(26) . In planar phospholipid bilayers the B moieties of these binary toxins form voltage-dependent channels under acidic pH conditions (27, 28, 29) . Understanding of how the B moieties of the binary toxins facilitate transfer of their respective A moieties across membranes and how ion channels participate in this process, may reveal common features in the translocation process.

Addendum- After this work was completed, we obtained sufficient quantities of recombinant EF to demonstrate that this protein causes an inhibition of PA-dependent Rb release similar to that seen with LF.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant AI-22021. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 617-432-1930; Fax: 617-432-0115; collier{at}warren.med.harvard.edu.

^1
The abbreviations used are: EF, edema factor; LF, lethal factor; PA, protective antigen; MES, 4-morpholineethanesulfonic acid.

^2
A. Finkelstein and J. A. Mindell, personal communication.


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