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Infection and Immunity, October 2001, p. 6532-6536, Vol. 69, No. 10
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.10.6532-6536.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Purification of Anthrax Edema Factor from
Escherichia coli and Identification of Residues Required
for Binding to Anthrax Protective Antigen
Praveen
Kumar,
Nidhi
Ahuja, and
Rakesh
Bhatnagar*
Centre for Biotechnology, Jawaharlal Nehru
University, New Delhi 110067, India
Received 25 April 2001/Returned for modification 11 June
2001/Accepted 9 July 2001
 |
ABSTRACT |
The structural gene for anthrax edema factor (EF) was
expressed in Escherichia coli under the control of a
powerful T5 promoter to yield the 89-kDa recombinant protein that
reacted with anti-EF antibodies. Recombinant EF was purified to
homogeneity by a two-step procedure involving metal chelate affinity
chromatography and cation-exchange chromatography. From 1 liter of
culture, 2.5 mg of biologically active EF was easily purified. This is
the first report of purification of anthrax EF from E.
coli. EF purified from E. coli was
biologically and functionally as active as its Bacillus
anthracis counterpart. The recombinant protein could compete with lethal factor for binding to protective antigen. Sequence
analysis revealed a stretch of seven amino acids, Val Tyr Tyr Glu Ile
Gly Lys, present both in EF (residues 136 to 142) and lethal factor
(residues 147 to 153). To investigate the role of these seven residues
in binding to protective antigen, the residues were individually
mutated to alanine in EF. Mutations in residues Tyr137, Tyr138, Ile140,
and Lys142 of EF specifically blocked its interaction with anthrax
protective antigen. The adenylate cyclase activity of the mutants
remained unaffected. The results suggested that residues Tyr137,
Tyr138, Ile140, and Lys142 are required for binding of EF to anthrax
protective antigen, which facilitates its entry into susceptible cells.
| |
TEXT |
Bacillus
anthracis, the causative agent of anthrax, is a highly
pathogenic bacterium. It primarily affects animals. Humans acquire the
disease via infected animals or contaminated animal products. Two major
virulence factors of this bacterial pathogen are antiphagocytic
poly-D-glutamic acid capsule (8) and
the three-component protein exotoxin called the anthrax toxin complex (17). The three proteins that form the complex are
protective antigen (PA; 83 kDa; 735 amino acids), edema factor (EF; 89 kDa; 767 amino acids), and lethal factor (LF; 90 kDa; 776 amino acids). Individually, all three proteins are nontoxic. However, a combination of PA and LF, called the lethal toxin, causes death in experimental animals (24) and lysis of mouse peritoneal macrophages and
macrophage-like cell lines (1, 3, 6, 7), whereas a
combination of PA and EF, known as the edema toxin, induces an increase
in intracellular cyclic AMP (cAMP) levels in eukaryotic cells
(16) and elicits skin edema after subcutaneous injection
(25).
Anthrax toxin fits the A-B model of classification of toxins, according
to which the A (activity) moiety and the B (binding) moiety reside on
different proteins that interact during intoxication of cells. Anthrax
toxin utilizes a single B moiety (PA) to deliver one of the alternative
A moieties (EF or LF) into the cell cytosol (17). PA binds
to cell surface receptors and is cleaved by a cell surface protease
such as furin (15). Proteolysis releases an N-terminal
20-kDa fragment, PA20, from the cell surface and exposes a high-affinity binding site on the 63-kDa fragment,
PA63, still bound to the receptor.
PA63 then binds to EF or LF, and the entire
complex undergoes receptor-mediated endocytosis (6). Acidification of the endosome results in insertion of
PA63 into the endosomal membrane and
translocation of EF and LF into the cytosol, where they exert their
toxic effects. It appears that ion-conductive channels, formed upon
oligomerization of PA63 fragments, facilitate the
translocation process (19).
EF is a bacterial adenylate cyclase which, upon activation by its
eukaryotic cofactor, calmodulin, causes a rapid increase in the
intracellular cAMP levels of host cells (16). Anthrax edema toxin can differentially regulate lipopolysaccharide-induced production of tumor necrosis factor alpha and interleukin-6 by increasing intracellular cAMP in monocytes (12). The
disruption of the cytokine network may contribute to clinical symptoms
of anthrax, such as edema formation.
The genes for PA, LF, and EF (called pagA, lef,
and cya, respectively) reside on a 185-kb plasmid, pXO1, of
B. anthracis. Strains lacking pXO1 do not produce toxin and
are essentially avirulent (13). All three genes have been
cloned and sequenced (4, 5, 21, 26). Efforts have been
made to express and purify PA and LF from other expression hosts, such
as Bacillus subtilis (14) and Escherichia
coli. Large amounts of biologically active PA and LF can now be
obtained from E. coli with relative ease (10,
11). Unfortunately, attempts have not been made to overexpress
and purify full-length EF from other expression hosts. Therefore,
culture supernatant of B. anthracis remains by and large the
major source for the purification of EF (18, 20).
Purification of EF from B. anthracis cultures requires P-3
containment facilities. Moreover, B. anthracis cultures give poor yields of EF that are often contaminated with other proteins. This
has discouraged researchers from undertaking studies of EF (23,
27).
The aim of the present study was to develop a rapid and efficient
system for purification of EF. EF was expressed in E. coli as a six-histidine-tagged protein and was purified to
homogeneity using a two-step procedure involving metal chelate affinity
chromatography and cation-exchange chromatography. Further, we have
used this system for cloning, expression, and purification of mutant
proteins of anthrax EF to investigate the role of a stretch of seven
amino acids (9), Val Tyr Tyr Glu Ile Gly Lys, that is
present both in EF (residues 136 to 142) and LF (residues 147 to 153).
Plasmid construction.
The full-length structural gene for EF
was amplified by PCR using pXO1 as a template and primers that added
BamHI and KpnI sites to the 5' and 3' ends of the
PCR product, respectively. The sequences of the forward and reverse
primers were 5' GAT GGC GCG GAT CCA TGA ATG AAC ATT ACA CTG AGA G 3'
and 5' GAT GGC GGG GTA CCT TAT TTT TCA TCA ATA ATT TTT TGG 3',
respectively. The amplified PCR product was digested with the
restriction enzymes BamHI and KpnI and then
ligated to the BamHI- and KpnI-digested plasmid
pQE30 (Qiagen) to generate the construct pPN-EF. Cloning of the gene
was verified by restriction analysis and sequencing (22).
The construct pPN-EF has a six-histidine coding sequence at the 5' end
of the gene. The expression of the EF gene is under the control of a
powerful T5 promoter. There are two lac operator sequences,
which in combination with the lac repressor protein ensure
tight regulation of gene expression.
Expression and purification of EF.
For high-level expression
of the gene, pPN-EF was transformed into E. coli
SG13009(pREP4) competent cells. Cells bearing pPN-EF were grown
at 37°C and 250 rpm in Luria broth containing 100 µg of
ampicillin and 25 µg of kanamycin per ml. When the
A600 reached 0.8, isopropyl-1-thio-
-D-galactopyranoside (IPTG)
was added to a final concentration of 0.5 mM. After 4 h of
induction, the cells were harvested by centrifugation. The sodium
dodecyl sulfate-polyacrylamide gel electrophoresis profile of these
cells showed EF migrating at 89 kDa. The protein reacted
with rabbit polyclonal anti-EF antibodies on immunoblots. The
periplasm, cytosol, and inclusion bodies were checked for the
presence of EF, which was found to be mainly localized in the cytosol.
For the purification of EF from the cytosol of SG13009 cells, all
procedures were performed at 4°C. The pellet obtained from 1 liter of
culture was resuspended in 30 ml of sonication buffer (50 mM potassium
phosphate, pH 8.0, 300 mM NaCl, 2 mM -mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride). Lysozyme (1 mg/ml) was added, and the cell suspension was incubated on ice for 30 min. The
cells were disrupted by sonication at 4°C. Cell debris was removed by
centrifugation, and the supernatant was mixed with 5 ml of 50%
Ni-nitrilotriacetic acid resin previously equilibrated with sonication
buffer. The matrix was washed with the sonication buffer containing 20 mM imidazole until the A280 of the
flowthrough was less than 0.01. The protein was then eluted with 300 mM
imidazole chloride in elution buffer (20 mM potassium phosphate, pH
7.0, 50 mM NaCl, 1% glycerol, 2 mM -mercaptoethanol, 1 mM
EDTA, and 1 mM phenylmethylsulfonyl fluoride). Affinity chromatography
resulted in 456-fold purification of EF (calculated by dividing the
specific activity of the protein eluting from the column by that
obtained for the total cell lysate).
For further purification of EF, the fractions containing EF were pooled
and dialyzed against binding buffer (20 mM potassium
phosphate, pH 7.0, 1% glycerol, 2 mM
-mercaptoethanol, and 1
mM EDTA) to remove
imidazole and NaCl. The dialyzed sample was
loaded onto an SP-Sepharose
(Sigma) cation-exchange column previously
equilibrated with binding
buffer. The protein was eluted with
a linear gradient of 0 to 500 mM
NaCl in the same buffer. EF eluted
from the exchanger at approximately
200 mM NaCl concentration
as a homogeneous preparation. This was
evident from the single
band obtained for EF on both native and
denaturing gels. Purified
EF was dialyzed against 10 mM HEPES buffer
containing 50 mM NaCl
and was stored in aliquots at
70°C.
One liter of culture yielded 2.5 mg of purified EF with a specific
activity of 230 µmol/min/mg. It was found that the two-step
purification procedure resulted in a total of 1,045-fold purification
compared to the cell
lysate.
Having purified recombinant EF to homogeneity, further experiments were
done to examine whether the biological activity of
the recombinant
protein was comparable to that of native protein
purified from
B. anthracis. In order to cause toxicity in susceptible
cells, EF
must first interact with PA, which facilitates its transport
to the
cell cytosol. The binding of
E. coli-purified EF to PA
was demonstrated both in solution and on cultured cells. Next,
to
evaluate the biological activity of recombinant EF in cultured
cells,
the cAMP response generated by the protein in CHO cells
was studied.
Competition assays with LF were also done to establish
that the
recombinant protein was biologically and functionally
active.
In vitro binding of EF to PA.
To study the binding of EF to PA
in solution, PA was cleaved with trypsin, and the nicked PA was
incubated with EF (1 µg/ml) for 15 min in 20 mM Tris, pH 9.0, containing 2 mg of CHAPS
{3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}/ml. Analysis of these samples on a nondenaturing 5 to 10% gradient gel demonstrated that EF purified from E. coli could
bind to trypsin-digested PA and retard its mobility.
Binding of EF to receptor-bound PA.
To study the
interaction between E. coli-purified EF and
receptor-bound PA, J774A.1 macrophage-like cells plated in 12-well plates were cooled and incubated with PA (1 µg/ml) and radioiodinated EF (1 µg/ml) at 4°C, as described in detail previously for LF (3). PA, when added to the cells, binds to its receptors
on the cell surface. Nicking by cell surface proteases leads to the exposure of binding sites to which EF rapidly binds. Unbound protein was removed by washing the cells with cold Hanks balanced salt solution. The cells were then solubilized in 0.5 ml of 0.1 N NaOH, and
radioactive counts associated with the cells were measured to
determine the amount of EF that bound to receptor-bound PA. E. coli-purified EF (specific binding, 12,759 ± 180) and B. anthracis-purified EF (specific binding,
12,467 ± 131) were found to be equally capable of binding
to receptor-bound PA.
Biological activity of purified EF.
To evaluate the biological
activity and the potency of E. coli-purified EF,
the cAMP response generated by the protein in CHO cells was
studied. Initial experiments showed that EF, like other agents
that increase intracellular cAMP, induces characteristic morphological
changes in CHO cells. Treatment of the cells with purified EF
(even at concentrations as low as 10 ng/ml) along with 1 µg of PA/ml
caused elongation of the cells and a 20-fold increase in the
intracellular cAMP level (measured with a Biotrak cAMP EIA kit
[Amersham Pharmacia]).
In fact, cAMP levels in cell cytosol rise proportionally in response to
increasing doses of EF (with PA held constant). However,
beyond a
saturating dose of EF (1 µg/ml), no further increase
in cAMP was
observed. PA or EF alone can neither increase cAMP
nor induce
morphological changes in CHO cells. EF purified from
E. coli and
B. anthracis was found to be equally potent in
generating
cAMP response in cultured cells (data not
shown).
Competition with LF.
EF or LF binds with high affinity to the
site exposed on domain 1 of PA upon cleavage with proteases, such as
trypsin and furin. It is expected that the binding of the alternate
activity moiety to this site would make the site unavailable for the
other moiety. This competition between LF and E. coli-purified EF for the binding site of PA was demonstrated on
both J774A.1 and CHO cells.
CHO cells were incubated with edema toxin in the presence and absence
of LF. The cAMP response of CHO cells to edema toxin
was found to
decrease when the dose of LF was increased (Table
1). A 10-fold excess of LF over
recombinant EF effectively reduces
intracellular cAMP to basal levels.
Intoxication of J774A.1 cells by lethal toxin was similarly affected by
the presence of recombinant EF. Complete cell death
occurs within
3 h when J774A.1 cells are incubated with lethal
toxin (1 µg
[each] of PA and LF/ml). However, in the presence
of EF, J774A.1
cells are protected against the cytotoxicity of
the lethal toxin (Table
2). Complete protection is achieved when
the concentration of EF is 10 times that of LF.
Role of residues 136-Val Tyr Tyr Glu Ile Gly Lys-142.
To
explain the competition between EF and LF for the binding site on PA,
the sequences of the two proteins were compared. The N-terminal 300 amino acids of EF and LF have significant homology and are considered
to be the domains that bind to PA63. The most notable feature of this region of homology is a stretch of seven amino
acids, Val Tyr Tyr Glu Ile Gly Lys, which is present in EF (residues
136 to 142) as well as LF (residues 147 to 153). To investigate the
roles of these amino acids in binding to PA63, each of the seven amino acids was individually replaced with Ala. Stratagene's Quikchange site-directed mutagenesis kit was used for
introducing mutations. pPN-EF was used as the template for PCR with
primers that contained the desired mutation. Seven mutant constructs
were made. Sequencing of the mutant constructs confirmed that
only specific mutations were introduced (V136A, Y137A, Y138A, E139A, I140A, G141A, and K142A). The constructs were then transformed into E. coli SG13009(pREP4) competent cells for
expression of the mutant protein. The EF mutants were purified to
homogeneity as detailed before for the wild-type EF.
To evaluate the biological activities of the mutant proteins, the cAMP
responses generated by the mutants in CHO cells were
studied.
Mutants Y137A, Y138A, I140A, and K142A (when added along
with PA)
failed to elicit an elongation response in CHO cells,
unlike wild-type
EF or the other three mutants, V136A, E139A,
and G141A. Quantitative
estimation of the intracellular cAMP of
the treated cells revealed that
the abilities of the mutant proteins
Y137A, Y138A, I140A, and K142A to
increase intracellular cAMP
levels were impaired (Fig.
1). The cAMP response elicited by the
mutant E139A in CHO cells was also slightly reduced in comparison
to
that generated by the wild-type protein.
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FIG. 1.
cAMP response of CHO cells to EF mutants. Cells plated
in 96-well tissue culture plates were treated with PA (1 µg/ml) and
the indicated concentrations of EF mutant or wild-type EF
(EFWT). After 2 h, the intracellular cAMP of the
treated cells was determined. The protein contents of the wells
averaged 7 µg. Untreated cells or cells treated with just one toxin
component had an average of 30 pmol of cAMP/mg of CHO cell protein. All
values are representative of three different experiments done in
triplicate, with a mean standard error of less than 5%.
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Next, the enzymatic activities of the mutant proteins were
determined. It was observed that the adenylate cyclase activities
of all of the mutant proteins were comparable to that of the wild-type
EF. This suggested that the impaired cAMP response generated in
CHO
cells by the mutants Y137A, Y138A, I140A, and K142A was not
due to
reduced adenylate cyclase activity but was due to a defect
in their
ability to enter the
cells.
To gain entry into susceptible cells, EF binds to receptor-bound PA. To
study the interaction of EF mutant proteins with PA,
the mutant
proteins were allowed to compete with radiolabeled
wild-type EF for
binding to receptor-bound PA. The results from
the competition assay
showed that cold wild-type EF, as well as
the mutants V136A, E139A, and
G141A, was able to compete with
radiolabeled wild-type EF and inhibit
its binding to receptor-bound
PA. However, the mutants Y137A, Y138A,
I140A, and K142A could
not compete with the radiolabeled
wild-type protein in binding
to receptor-bound PA (Fig.
2). This suggested that the mutants
Y137A, Y138A, I140A, and K142A were defective in the ability to
bind
PA.
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FIG. 2.
Competitive binding of EF mutants to receptor-bound PA.
J774A.1 cells were incubated with 100 ng of radioiodinated wild-type EF
(EFWT)/ml along with 1 µg of PA/ml at 4°C. Various
concentrations of cold wild-type protein or the mutants were allowed to
compete with radioiodinated wild-type EF. The cells were later washed
with cold Hanks balanced salt solution and solubilized in 0.1 N NaOH,
and radioactive counts were taken.
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Similar results were obtained in vitro when the mutant proteins were
allowed to bind to PA in solution. Mutants V136A, E139A,
and G141A were
able to bind to trypsin-digested PA and retard
its mobility on native
polyacrylamide gel electrophoresis. However,
mutants Y137A,
Y138A, I140A, and K142A did not bind to trypsin-digested
PA in solution
(Fig.
3).
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FIG. 3.
Binding of EF mutants to PA in solution. Wild-type EF
(EFWT) or its mutants (1 µg) were allowed to incubate
with trypsin-nicked PA (1 µg) for 15 min, and the samples were
analyzed on a nondenaturing 5 to 10% gradient gel. The gel was stained
with silver stain.
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Thus, it was concluded that the residues Tyr137, Tyr138, Ile140, and
Lys142 are required by EF for binding to anthrax PA,
which facilitates
its entry into susceptible
cells.
To summarize, a system for expression and purification of
B. anthracis EF has been presented above. The procedure does not
involve handling of the pathogenic bacteria, since the expression
host
is
E. coli. From 1 liter of culture, 2.5 mg of
purified EF
can easily be obtained, which is a significant
improvement over
the yields (0.4 to 0.8 mg/liter) obtained
from
B. anthracis cultures.
The recombinant
protein is purified to homogeneity. Furthermore,
we have demonstrated
that EF purified from
E. coli is biologically
and
functionally comparable to its
B. anthracis counterpart. The
rapidity of the procedure (just two chromatographic steps) ensures
minimal loss of biological activity. The high reproducibility
and
simplicity of the method make it a convenient system to adopt
routinely. We hope that availability of this system will encourage
researchers to undertake the study of EF. We have used this
system
for cloning, expression, and purification of mutant
proteins of
anthrax EF (V136A, Y137A, Y138A, E139A, I140A, G141A,
and K142A).
Studies with the mutant proteins show that the residues
Tyr137,
Tyr138, Ile140, and Lys142 are required for the binding of EF
to anthrax PA. Mutations in these residues disrupt the interaction
between the two proteins, thereby affecting the entry of EF into
susceptible
cells.
EF merits study not just because it is an important virulence factor of
B. anthracis but also because it is the only toxin
known so
far which enters susceptible cells through receptor-mediated
endocytosis, where it displays intrinsic adenylate cyclase
activity.
Bordetella pertussis adenylate cyclase is another
toxin with inherent
adenylate cyclase activity, but unlike its
B. anthracis counterpart,
it enters cells through direct
penetration of the plasma membrane.
Most other toxins increase
intracellular cAMP levels by modulating
the adenylate cyclase activity
of the host
cell.
Edema toxin provides an interesting model to study how cAMP regulates
basic cellular and metabolic processes. It is through
its ability to
influence intracellular cAMP levels that edema
toxin modulates cytokine
synthesis in the infected host (
11).
Disruption of
cytokine networks impairs the host's ability to
defend against the
invading bacteria and contributes to clinical
symptoms of anthrax, such
as edema
formation.
Hitherto, biochemical investigations of the structure-function
relationship of anthrax EF were hampered by the difficulty
of purifying
protein free from the other two toxin components.
The availability of
homogeneous protein preparations will trigger
research on this subject.
Insights into the structure-function
relationship of the protein can
provide useful information. For
example, delineation of the minimal
sequence of EF required for
binding to PA and its translocation across
the heptamer can help
in developing an anthrax toxin-based protein
translocation system
to deliver heterologous proteins into the
cytoplasm of mammalian
cells. Such a system could serve as a tool by
which cellular processes
can be
modulated.
| |
ACKNOWLEDGMENTS |
This work was partly supported by a grant from DBT, Government of
India. Both Praveen Kumar and Nidhi Ahuja are financially supported by
UGC, Government of India.
We thank R. N. Saini for photographic assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Centre for
Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India.
Phone: (91) 11-6179751. Fax: (91) 11-6198234 or -6865886. E-mail:
rakbhat{at}hotmail.com.
Editor:
J. T. Barbieri
| |
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Infection and Immunity, October 2001, p. 6532-6536, Vol. 69, No. 10
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.10.6532-6536.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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