A Dominant Negative Mutant of Bacillus anthracis Protective Antigen Inhibits Anthrax Toxin Action in Vivo*

Yogendra SinghDagger, Hemant Khanna§, Arun P. Chopra, and Varsha Mehra§

From the Centre for Biochemical Technology, Mall Road, Delhi-110007, India

Received for publication, November 9, 2000, and in revised form, March 6, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

PA63, a proteolytically activated 63-kDa form of anthrax protective antigen (PA), forms heptameric oligomers and has the ability to bind and translocate the catalytic moieties, lethal factor (LF), and edema factor (EF) into the cytosol of mammalian cells. Acidic pH triggers oligomerization and membrane insertion by PA63. A disordered amphipathic loop in domain II of PA (2beta 2-2beta 3 loop) is involved in membrane insertion by PA63. Because conditions required for membrane insertion coincide with those for oligomerization of PA63 in mammalian cells, residues constituting the 2beta 2-2beta 3 loop were replaced with the residues of the amphipathic membrane-inserting loop of its homologue iota-b toxin secreted by Clostridium perfringens. It was hypothesized that such a molecule might assemble into hetero-heptameric structures with wild-type PA ultimately leading to the inhibition of cellular intoxication. The mutation blocked the ability of PA to mediate membrane insertion and translocation of LF into the cytosol but had no effect on proteolytic activation, oligomerization, or binding LF. Moreover, an equimolar mixture of purified mutant PA (PA-I) and wild-type PA showed complete inhibition of toxin activity both in vitro on J774A.1 cells and in vivo in Fischer 344 rats thereby exhibiting a dominant negative effect. In addition, PA-I inhibited the channel-forming ability of wild-type PA on the plasma membrane of CHO-K1 cells thereby indicating protein-protein interactions between the two proteins resulting in the formation of mixed oligomers with defective functional activity. Our findings provide a basis for understanding the mechanism of translocation and exploring the possibility of the use of this PA molecule as a therapeutic agent against anthrax toxin action in vivo.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Bacillus anthracis, the etiologic agent of anthrax, is a potential agent of bioterrorism (1). The toxic action has been attributed to anthrax toxin produced by the bacterium. The anthrax toxin can be resolved into three distinct protein components: protective antigen (PA),1 lethal factor (LF), and edema factor (EF). The combination of EF and PA (an edema toxin) produces skin edema, whereas LF and PA (a lethal toxin) are lethal to animals (2). The three proteins are individually non-toxic (2). Whereas EF is a calcium- and calmodulin-dependent adenylate cyclase that acts by increasing the intracellular cAMP levels in eukaryotic cells (3), LF is a Zn2+-dependent metalloprotease (4) that leads to an increase in IL-1 and TNF-alpha production by susceptible cells (5) and cleaves several mitogen-activated protein kinase kinases (MKK 1, 2 and 3) (6-8).

According to the current model of anthrax toxin action, PA binds to an as yet unknown cell surface receptor and gets proteolytically activated by cell surface protease furin to PA63. This allows oligomerization and binding of LF/EF. The toxin complex is internalized by receptor-mediated endocytosis and is exposed to acidic pH inside the endosome. This change in pH triggers both membrane insertion by PA63 and translocation of LF/EF into the cytosol (recently reviewed in 9).

Membrane insertion and channel formation are brought about by a large 2beta 2-2beta 3 loop (amino acid residues 302-325) in the domain II of PA (10). The loop shows a conserved pattern of alternating hydrophilic and hydrophobic amino acid residues similar to that observed in Clostridium perfringens iota-b toxin and Staphylococcus aureus alpha -hemolysin (11). PA also has been shown to possess a high degree of homology with the iota-b toxin secreted by C. perfringens (12).

Translocation of LF or EF to the cytosol is believed to occur through a channel formed by insertion of heptameric PA63 into the membrane (11). The formation of ion-conductive channels by PA63 has been demonstrated in both artificial lipid membranes (13) and in CHO-K1 cells (14). Acidic pH triggers oligomerization, membrane insertion by PA63, and translocation of LF into the cytosol of mammalian cells (10, 15, 16). In this paper, we show that a mutant PA protein, in which amino acid residues comprising the 2beta 2-2beta 3 loop of PA (PA-I) were substituted with the residues of the amphipathic loop of the homologous iota-b toxin, is defective in its ability to insert into the membrane and completely inhibits the lethal effect of the wild-type toxin at equimolar concentrations.

    EXPERIMENTAL PROCEDURES
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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REFERENCES

Materials-- Biochemicals and reagents were purchased from Sigma. Bacterial culture media were purchased from Difco Laboratories. The enzymes and chemicals for DNA manipulations were obtained from New England BioLabs. [3H]Leucine and [35S]methionine were obtained from Amersham Pharmacia Biotech.

Cell Culture-- The Chinese Hamster Ovary cell line (CHO-K1) and J774A.1 macrophage cell line were maintained in Dulbecco's modified Eagle's medium supplemented with 10% calf serum and 50 µg/ml gentamicin sulfate (Life Technologies Inc.) at 37 °C in a CO2 incubator.

Plasmid Construction and Mutagenesis-- Mutations in the PA gene were constructed in a previously described plasmid pYS5 (17). A non-mutagenic oligonucleotide primer corresponding to nucleotides 2169-2200 and spanning the unique HindIII site was used for polymerase chain reaction with a mutagenic primer corresponding to nucleotides 2785-2860 encompassing the unique PstI site and containing the desired mutations at nucleotides 2792-2851 (nucleotide numbering is according to Ref. 18). The amplified polymerase chain reaction product was digested with PstI and HindIII and purified on a 1% low melting point agarose gel. The plasmid pYS5 was digested with the same enzymes, purified on agarose gel, and ligated to the mutant fragment. The DNA sequence of the mutant PA gene was verified by DNA sequencing of at least 200 base pairs spanning the mutated region.

Expression and Purification of PA-- The plasmid carrying the desired sequence was transformed into E. coli dam dcm strain SCS110. Unmethylated plasmid DNA was purified and used to transform B. anthracis BH441. Wild-type and mutant PA proteins were purified from the cell supernatants of B. anthracis according to the method described earlier (16). Proteins were assayed using the Bio-Rad protein assay kit (Bio-Rad) according to the manufacturer's instructions.

Cytotoxicity Assay-- To study the cytotoxicity, varying concentrations of PA and its mutant protein were added to J774A.1 cells together with LF (1 µg/ml) and incubated for 3 h at 37 °C. At the end of the experiment, cell viability was determined using MTT assay (19).

Protein Synthesis Inhibition Assay-- PA and its mutant protein were added to CHO-K1 cells in combination of LF fusion protein (LF-(1-254)·TR·PE-(398-613)) (16) and incubated at 37 °C for 3 h. At the end of 3 h, [3H]leucine (1 µCi/ml) was added in each well and incubated for another 1 h at 37 °C. The incorporation of [3H]leucine during protein synthesis was determined as described previously (16).

Ion-conducting Channel Formation-- Ability of the mutant PA protein to form ion conductive channels in the plasma membrane of mammalian cells was studied according to the method described earlier (16). CHO-K1 cells, preloaded with 86Rb+ (1 µCi/ml, PerkinElmer Life Sciences), were incubated with medium containing 2 µg/ml nicked wild-type and mutant PA proteins for 2 h at 4 °C. The cells were then washed two times with cold phosphate-buffered saline to remove unbound PA and treated with isotonic buffer (20 mM MES-gluconate, 145 mM NaCl, pH 5.0 or 7.0) for 30 min at 37 °C. At the end of the incubation period, an aliquot of medium was removed, and released 86Rb+ was measured.

Inhibition of the channel-forming ability of wild-type PA by mutant PA protein was assayed by adding trypsin-nicked wild-type and mutant PA proteins mixed in equimolar concentrations to CHO-K1 cells preloaded with 86Rb+. Release of the radiolabel was determined as described above.

In Vitro Translocation Assay and Electron Microscopy-- An assay for measuring PA-mediated translocation of a labeled ligand into cells has been previously described (20). Briefly, CHO-K1 cells were chilled to 4 °C and then incubated with 2 µg/ml of trypsin-nicked wild-type PA or mutant PA proteins for 2 h. The cells were washed and incubated with in vitro transcribed and translated LFn (LF 1-254 amino acids) labeled with [35S]methionine for 1 h. After another washing step, the cells were either lysed with 100 µl of lysis buffer (20 mM sodium phosphate, pH 7.4, 10 mM EDTA, 1% Triton X-100) directly or incubated with isotonic buffer (20 mM MES-gluconate, 145 mM NaCl, pH 5.0 or 7.0) at 37 °C for 5 min, treated with Pronase E and then lysed. Proteins that translocated to the interior of the cells during the low pH pulse were protected from Pronase treatment. Cell-associated radioactivity was then measured in the samples. Percent translocation was calculated as: counts protected from Pronase/counts bound to cells × 100. Purified 63-kDa fragments of PA and mutant PA proteins (40 µg/ml) were adsorbed to a thin carbon film and negatively stained with 1% uranyl formate as described earlier (21).

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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Prior work showed that proteolytic cleavage of PA at the sequence 164RKKR167 in solution or on the surface of mammalian cells results in the removal of the N-terminal 20-kDa fragment (PA20) that leads to heptamer formation (11). The heptamer has been assumed to insert into membranes at acidic pH (15). Acidic pH inside the endosome leads to insertion of PA63 into the membrane by forming a beta -barrel composed of an amphipathic 2beta 2-2beta 3 loop consisting of an alternating arrangement of hydrophilic and hydrophobic amino acids (11).

Because acidic pH is necessary for both oligomerization and membrane insertion by PA63 (15), we investigated the functional significance of the correlation between the conditions required for both of the events to occur. Mutant PA protein (PA-I) was produced in which residues constituting the 2beta 2-2beta 3 loop were replaced with the corresponding residues of iota-b toxin, a closely related toxin secreted by C. perfringens (12), so that the alternating arrangement of hydrophilic and hydrophobic amino acids was retained. PA and PA-I were purified from the culture supernatant of B. anthracis (Fig. 1A). PA-I was tested by immunoblot analysis for reactivity against anti-PA polyclonal antibodies (Fig. 1B). The results suggested that PA-I was purified to more than 90% homogeneity and was reactive to anti-PA rabbit polyclonal antibodies like wild-type PA. The typical yield of the proteins was 10 mg/l. PA-I did not aggregate in solution and behaved similar to wild-type PA.


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Fig. 1.   Purification of PA. A, PA and PA-I were purified from the cell supernatants of B. anthracis and analyzed on 10% SDS-PAGE. Lane 1, molecular weight marker (kDa); Lane 2, wild-type PA; Lane 3, PA-I. B, the samples of PA and PA-I were analyzed on 10% SDS-PAGE, transferred to nitrocellulose paper, and probed with anti-PA polyclonal antibody. Lane 1, PA; Lane 2, PA-I.

Purified PA-I was tested for its ability to lyse J774A.1 macrophage cells in combination with LF. Whereas wild-type PA lysed 50% of the cells at a concentration of 0.04 µg/ml in 3 h (Fig. 2A), PA-I was completely non-toxic to J774A.1 cells at the highest concentration tested (100 µg/ml) (Fig. 2A). The data suggest that PA-I is inactive in exhibiting a lethal effect to macrophage cells as compared with wild-type PA. A more sensitive assay to measure the toxic activity of PA is to study the PA-dependent inhibition of protein synthesis in combination with LF-(1-254)·TR·PE-(398-613) (16). The fusion protein is comprised of the N-terminal 254 amino acids of LF (LF-(1-254); LFn) fused to the ADP-ribosylating domain of Pseudomonas aeruginosa exotoxin (16). Cytotoxicity in this assay is measured by the inhibition of protein synthesis catalyzed by Pseudomonas exotoxin and resulting from the ADP-ribosylation of elongation factor 2 (22). Whereas wild-type PA (0.1 µg/ml) showed 90% inhibition in protein synthesis when administered in combination with LF-(1-254)·TR·PE-(398-613) (1 ng/ml) to CHO-K1 cells as measured by the percent incorporation of [3H]leucine, no inhibition in protein synthesis was detected with PA-I when used even at a concentration of 100 µg/ml (Fig. 2B). The marked inhibition of the biological activity of the mutant PA protein confirmed the functional significance of 2beta 2-2beta 3 loop of PA for the biological activity of anthrax toxin.


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Fig. 2.   Cytotoxicity assay with PA and mutant PA proteins. A, J774A.1 cells were cultured in 96-well plates in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and incubated with LF (1 µg/ml) in combination with varying concentrations of PA and PA-I for 3 h at 37 °C. At the end of the experiment, toxicity was determined by MTT assay as described under "Experimental Procedures." B, CHO-K1 cells were incubated with varying concentrations of PA or PA-I together with LF-(1-254)·TR·PE-(398-613) (1 ng/ml) at 37 °C for 3 h. At the end of the incubation, medium was replaced with medium containing [3H]leucine (1 µCi/ml) and the extent of protein synthesis was measured as described under "Experimental Procedures." Results are expressed as percent of [3H]leucine incorporation by viable cells in the absence of added proteins. Counts equivalent to 12,500 were considered as 100% [3H]leucine incorporation.

Several previous studies have shown that cleavage at the sequence 164RKKR167 by trypsin/furin is a prerequisite for anthrax toxin action (17) and leads to the formation of SDS-resistant oligomers by PA63 at acidic pH (15). Analysis of the mutant PA protein for sensitivity toward trypsin showed that the mutant was equally susceptible to trypsin as wild-type PA (Table I). The mutation introduced did not affect the ability of PA-I to bind LFn on cell surface (Table I) and, therefore, did not alter the ability of PA to bind to the receptor.

                              
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Table I
Characteristics of mutant PA protein

Purified PA63 forms SDS and boiling-resistant oligomers when exposed to acidic pH on mammalian cells (15). We, thus, determined whether PA-I retained the ability to form SDS-resistant oligomers in solution as well as when incubated with mammalian cells. PA-I was equally as effective in oligomerizing in solution as was wild-type PA (Table I). Electron microscopy data confirmed the formation of heptamers by PA-I as well as wild-type PA (Fig. 3, A and B). These results suggest that PA-I retained the ability to perform intermolecular interaction leading to oligomerization.


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Fig. 3.   Electron microscopy of the oligomeric 63-kDa fragments of PA and PA-I. Samples (40 µg/ml) were negatively stained with 1% uranyl formate and analyzed by electron microscopy. A, PA; B, PA-I.

We next examined the ability of PA-I to insert into the plasma membrane of CHO-K1 cells at acidic pH. Membrane insertion was tested by measuring the ability of PA-I to form ion-conductive channels in the plasma membrane of mammalian cells. PA63 forms ion-conductive selective channels in artificial membranes and plasma membrane of mammalian at acidic pH (13, 14). Earlier studies have correlated the ability of PA to insert into membranes with the extent of 86Rb+ released at acidic pH (16). Cells preloaded with 86Rb+ were incubated with trypsin-nicked PA or PA-I at 4 °C and placed into acidic medium, and release of 86Rb+ was measured. Incubation of trypsin-nicked wild-type PA (2 µg/ml) induced release of 70% 86Rb+, whereas trypsin-nicked PA-I did not cause leakage of 86Rb+ (Fig. 4). The result suggests that PA-I is unable to form ion-conductive channels and that integrity of the 2beta 2-2beta 3 loop is essential for proper membrane insertion by PA63.


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Fig. 4.   Ion channel formation by PA and PA-I. CHO-K1 cells preloaded with 86Rb+ were incubated with trypsin-nicked PA or PA-I at 4 °C for 2 h and subjected to acidic pH shock. The leakage of 86Rb+ into the medium was determined as described under "Experimental Procedures." Results are expressed as percent of 86Rb+ associated with cells in the absence of added proteins.

To test the ability of PA-I to translocate LF into the cytosol of mammalian cells, we employed the previously described in vitro translocation assay (20) that uses the in vitro transcribed and translated LFn labeled with [35S]methionine. We measured the ability of PA-I to translocate radiolabeled LFn across the plasma membrane of CHO-K1 cells upon treatment with low pH buffer. As shown in Fig. 5, whereas wild-type PA translocated 45% of the bound LFn at pH 5.0, PA-I showed no translocation of LFn. Insignificant translocation of LFn was observed with wild-type PA at pH 7.0 consistent with the earlier reports that showed that acidic pH is a prerequisite for the translocation event to occur (20). The results suggest that the mutant PA protein is inactive in translocation. The results confirm the previous propositions that membrane insertion by PA63 is a prerequisite for the translocation of LF into the cytosol but do not, in any way, suggest that LF passes through the lumen of the channel formed by the 2beta 2-2beta 3 loop.


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Fig. 5.   In vitro translocation assay. CHO-K1 cells were chilled in ice for 15 min and incubated with trypsin-nicked PA or PA-I for 2 h at 4 °C. After washing with phosphate-buffered saline, the cells were further incubated for 1 h with 35S-labeled LFn at 4 °C. After another washing step, the pH of the medium was either maintained at 7.0 or lowered to 5.0. The cells were either lysed directly or treated with Pronase E to remove surface bound proteins. Cell-associated radioactivity was then counted. Results are expressed as percent 35S-labeled LFn translocated of the total bound to cells.

We next investigated whether mixing of the mutant PA protein and wild-type PA at varying ratios resulted in alterations in the cytotoxic activity of the wild-type toxin. When the mutant and wild-type PA were present at equimolar concentrations, complete inhibition in protein synthesis of CHO-K1 cells was observed (Fig. 6). A significant inhibition could be detected when the ratio of PA-I to PA was 1:4. These data suggest that the PA-I inhibits wild-type PA-mediated cellular intoxication.


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Fig. 6.   Inhibition of toxic activity of PA in combination with LF-(1-254)·TR·PE-(398-613). CHO-K1 cells were incubated with PA-I or PA-D mixed with varying concentrations of wild-type PA at 37 °C for 3 h in combination with LF-(1-254)·TR·PE-(398-613). At the end of 3 h, cells were incubated with medium containing [3H]leucine (1 µCi/ml) for 1 h at 37 °C. At the end of the experiment, the amount of [3H]leucine incorporation was measured. Results are expressed as percent of [3H]leucine incorporated by viable cells in the absence of added proteins.

It has been reported previously that a mutant form of PA, in which the furin cleavage site 164RKKR167 was deleted (PA-D), blocked the anthrax toxin effect in vitro and in vivo (17). Because this molecule cannot be cleaved with trypsin or furin, does not form oligomers, and can only inhibit the action of wild-type PA by competing for cellular receptor, this molecule was employed as a control in our experiments to investigate whether inhibition in the cytotoxic activity was due to the competition for binding to the receptor. PA-D failed to inhibit the cytotoxic effect when mixed with wild-type PA in all the ratios tested (Fig. 6). It has been shown previously that PA-D, rather than wild-type PA, inhibits the lethal toxin activity when present at a 10-fold excess concentration (17). Typically, a substantial excess of mutant protein is required to inhibit the binding of an active ligand to cell surface receptors, and PA-I more likely inhibits the action of anthrax toxin by interacting with wild-type PA to form an inactive hetero-heptameric complex and thus, is a more potent inhibitor of anthrax toxin action. This model is consistent with the ability of purified PA-I to inhibit wild-type PA-mediated cytotoxic activity when the ratio of PA-I to PA is 1:4. Purification of active homoheptamers of PA and PA-I to homogeneity was not successful due to the presence of lower order oligomers as well.

To confirm the hypothesis that wild-type PA and PA-I might assemble to form non-functional oligomeric structures, the trypsin-nicked proteins (2 µg/ml each) were mixed together at neutral pH and incubated with CHO-K1 cells preloaded with 86Rb+ at 4 °C. After 2 h, the cells were washed to remove unbound proteins and incubated with isotonic buffer of pH 5.0 or 7.0 for 30 min at 37 °C. Whereas wild-type PA released 62% of the radiolabel from cells, equimolar mixture containing PA and PA-I showed insignificant release of 86Rb+ (Fig. 7). The results suggest that there is complete inhibition of channel-forming ability of PA by PA-I. Indeed, the capacity of PA-I to dramatically alter the channel-forming ability of wild-type PA provides evidence that these two species can interact to form dysfunctional hetero-oligomeric structures. A dominant negative mutant of VacA toxin secreted by Helicobacter pylori has recently been reported that inhibits the vacuolating activity of wild-type toxin when present at a 5-fold-less concentration than wild-type VacA (23).


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Fig. 7.   Inhibition of channel-forming activity of PA by PA-I. CHO-K1 cells, preloaded with 86Rb+, were incubated with trypsin-cleaved PA and PA-I mixed in equimolar ratios at neutral pH for 2 h at 4 °C. After washing twice with cold phosphate-buffered saline, the cells were subjected to acidic pH shock as described under "Experimental Procedures." The leakage of 86Rb+ into the medium was then determined. Results are expressed as percent of 86Rb+ associated with cells in the absence of added proteins.

Identification of such a dominant negative inhibitor might be valuable for treatment of anthrax toxin action. Animal experiments were thus initiated to test the efficacy of PA-I to act as a dominant negative inhibitor of lethal toxin action in vivo. Whereas wild-type lethal (40 µg of PA + 8 µg of LF) resulted in the death of male Fischer 344 rats in ~60 min (Table I), a 1:1 mix containing wild-type PA and PA-I (40 µg of PA + 40 µg of PA-I + 8 µg LF) protected rats, and no symptoms were evident even after 48 h. Equimolar ratio of wild-type PA and PA-D resulted in the death of rats within 70 min (Table I). Taken together, these data confirm that PA-I can act as a dominant negative and a potent inhibitor of anthrax toxin action in vivo.

Use of B. anthracis as a bioweapon has become the bane of the defense establishments in various countries (1). Keeping in view the potent activity of PA-I both in vitro and in vivo it has the potential to be used as therapeutic agent for use in neutralizing anthrax toxin action in individuals infected with B. anthracis.

    ACKNOWLEDGEMENTS

We thank Prof. S. K. Brahmachari for making this work possible. We thank Stephen H. Leppla for helpful discussions during various stages of the study. We thank Abdur Raheem for synthesizing oligonucleotide primers. We also thank Prof. R. John Collier for hosting H. K. to work in his laboratory and Bret R. Sellman and M. Mourez for their guidance. R. J. C. and co-workers have obtained results similar to those reported here but with other dominant negative mutants of PA (Sellman, B. R., Mourez, M., and Collier, R. J. (2001) Science 292, 695-697

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Center for Biochemical Technology, Mall Road, Delhi-110007, India. Tel.: 91-11-766 6156; Fax: 91-11-766 7471; E-mail: ysingh@cbt.res.in.

§ Supported by Council of Scientific and Industrial Research.

Supported by University Grants Commission, University of Delhi.

Published, JBC Papers in Press, March 16, 2001, DOI 10.1074/jbc.M010222200

    ABBREVIATIONS

The abbreviations used are: PA, protective antigen; LF, lethal factor; EF, edema factor; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PAGE, polyacrylamide gel electrophoresis; LFn, N-terminal 254 amino acids of LF.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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