From the Department of Bacteriology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390, Japan
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ABSTRACT |
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Cytotoxin of Pseudomonas aeruginosa
is a cytolytic toxin that forms a pore on the target membrane by
oligomerizing into a pentamer. This toxin is produced as an inactive
precursor (proCTX) and is converted to an active form by proteolytic
cleavage at the C terminus. We purified proCTX to apparent homogeneity
and characterized it in a comparison with the active toxin. ProCTX bound to the erythrocyte membrane but did not form an oligomer on the
membrane, hence the lack of hemolytic activity in proCTX. Circular
dichroic experiments showed that active and proCTX have similar
-sheet dominant structures. Intrinsic fluorescence analysis indicated that a molecule-buried tryptophan residue(s) of proCTX was
exposed to the surface of the molecule as a result of conversion to the
active form. In analytical gel filtration, chemical cross-linking, and
analytical ultracentrifugation experiments, dimer to monomer conversion
occurred with proteolytic activation.
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INTRODUCTION |
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Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, causes severe infections in compromised and immunosuppressed hosts. The bacterium produces various toxic substances that contribute to the virulence of this opportunistic pathogen (1, 2). Some strains of P. aeruginosa produce a cytolytic toxin, cytotoxin (CTX), which is cytotoxic to a wide range of eukaryotic cells (3, 4). The gene encoding the toxin (ctx) is carried by bacteriophages, and lysogenization of the phages converts CTX non-producing P. aeruginosa strains to CTX producers (5, 6). The acquisition of the ctx gene increases the virulence of the bacterium (7).
CTX has been considered a channel-forming toxin, generating a pore of 2 nm in diameter (8, 9). Although morphological identification has yet to be made, we recently found that CTX forms an oligomer on the target membrane (10). This formation correlated with the cytolytic activity; thus, CTX may form a pore by oligomerization on the membrane, as is the case for several cytolytic channel-forming toxins (11, 12). Because the molecular size of the oligomer was estimated to be 145 kDa, using SDS-PAGE1 (10), it likely consists of a pentamerized CTX.
CTX produced by P. aeruginosa is inactive and requires proteolytic action for activation. Analyses of the ctx gene and an active toxin purified from the trypsin-treated crude extract of P. aeruginosa (PACTX) showed that CTX is produced as a precursor (procytotoxin (proCTX)) of 286 amino acids and is activated by proteolytic cleavage at the C terminus (13). The cleavage occurs at the carboxyl side of Arg266 (13). Among the channel-forming toxins, aerolysin from Aeromonas spp. (12) and the alpha toxin of Clostridium septicum (14, 15) also require proteolytic cleavages at the C-terminal regions for conversion from inactive precursors to active forms. In the case of proaerolysin, the C-terminal region was seen to mask hydrophobic patches of the toxin molecule and to inhibit oligomerization of the toxin on the membrane (12, 16). Concerning proCTX, nothing is known of the function of the C-terminal sequence or what changes occur on the CTX molecule by the C-terminal cleavage.
Using a method developed for the purification of the recombinant CTX, we have now purified proCTX for the first time. Purified proCTX bound to the erythrocyte membrane but did not form the oligomer on the membrane. In circular dichroic experiments, significant changes in the secondary structure were not observed between the active CTX and proCTX. However, a striking difference was found in the oligomeric states of the proteins. We describe here a novel activation mechanism of the pore-forming toxin, involving a dimer-to-monomer conversion.
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EXPERIMENTAL PROCEDURES |
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Materials-- Trypsin (Type XIII: L-1-tosylamido-2-phenylethylchloromethyl ketone-treated), bovine serum albumin, and apomyoglobin were from Sigma. The molecular size standards for SDS-PAGE were obtained from Bio-Rad and New England Biolabs. All columns used for protein purification were purchased from Pharmacia Biotech.
Site-directed Mutagenesis and Overproduction of the Toxins in
Escherichia coli--
A codon for threonine at position 267 of the
ctx gene was replaced with a premature stop codon by
changing ACA to TAA. The mutagenesis was performed by the gapped duplex
DNA method of Kramer and Frits (17) using a Mutan-G system (Takara).
The mutagenized ctx gene was inserted into a broad host
range expression plasmid, pMMB22 (18), to construct pMMB(C20), using
the same strategy as described for pMMB(CTX) for the overproduction of
procytotoxin (13).
Purification Procedure-- All purification procedures were performed at 4 °C, except that fast protein liquid chromatography procedures (Pharmacia Biotech Inc.) were carried out at room temperature.
For purification ofMeasurement of Cytotoxic Activity and Oligomer Formation-- Cytotoxic activities of the toxins were determined by means of a hemolytic activity assay (10). Oligomer formation of toxins on the rat erythrocyte membrane was detected by immunoblotting as described previously (10). Activation of proCTX was done by incubation with trypsin at 37 °C for 60 min at 1:20 trypsin per proCTX (w/w) ratio. The reaction was quenched by adding phenylmethylsulfonyl fluoride (final concentration, 1 mM).
Preparation of Antisera and Affinity Purification of
Antibody--
Rabbit anti-C20 serum was prepared as described (4).
For the specific detection for proCTX, a 9-mer peptide corresponding to
the C-terminal sequence of proCTX (LETRVRSAE) with a cysteine residue
at the N terminus was synthesized by the Fmoc
(9-fluorenylmethoxycarbonyl) strategy using a peptide synthesizer
(Applied Biosystems Model 431A). The peptide (0.1 mg) was cross-linked
to 0.1 mg of bovine serum albumin with
m-maleimidobenzoyl-N-hydrosuccinimide ester and
used to immunize a rabbit.
Immunoadsorption of Toxins with Immobilized Antibody--
The
affinity purified antibody was immobilized on Affi-Gel 10 (Bio-Rad) in
coupling buffer (0.1 M HEPES, pH 8.0). Toxins (0.5 µg)
were incubated with 10 µl of the antibody-coupled gel (1.1 µg of
IgG/µl gel) in 40 µl of binding buffer (coupling buffer with 0.1 M NaCl) for 1 h at 37 °C. After unbound materials
were separated by brief centrifugation, pellets were washed five times with the binding buffer (200 µl). Bound materials were eluted with
SDS loading buffer, and the bound and unbound fractions were analyzed
by SDS-PAGE. For immunostaining, rabbit anti-C20 serum and
peroxidase-conjugated anti-rabbit IgG antibody specific to the Fc
region (Promega) were used.
Measurement of Circular Dichroism (CD) spectra-- CD spectra of toxins were measured at 25 °C in a Jasco J-600 spectrometer with a quartz cell of 0.2 mm path length. The protein concentration was 150 µg/ml in 20 mM sodium phosphate buffer, pH 7.2. Contents were calculated using the reference described by Yang et al. (19). Estimates of the secondary structure were made using a SSE-338 program (Jasco).
Measurement of Tryptophan Fluorescence-- Fluorescence measurements were made using a Hitachi fluorometer F3000 with a quartz cuvette. The excitation wavelength was 290 nm, and the range of emission wavelength was from 300 to 400 nm. The protein concentration was adjusted to 0.6 µM in Buffer B.
Mass Spectrometry--
Samples for spectrometry were prepared
according to Nakanishi et al. (20). Briefly, 33 pmol of
toxins in 10 µl of Buffer A was incubated with an equal volume of
anti C20 serum for 3 h at room temperature, the samples were
centrifuged, and the pellets were washed once with 0.6 ml of 0.9% NaCl
and twice with 0.6 ml distilled water and lyophilized. Pellets were
dissolved in 3.3 µl of distilled water and mixed with an equal volume
of saturated sinapinic acid matrix solution in 33% (v/v)
acetonitrile/distilled water. Mass measurements were made on a
matrix-assisted laser desorption ionization time-of-flight mass
spectrometry (Voyager ELITE XL, PerSeptive Biosystems) according to the
manufacturer's instructions. Bovine serum albumin and apomyoglobin
were used as standards.
Analytical Gel Filtration Analysis-- Analytical gel filtration was performed using a Superose TM12 HR 16/60 column equilibrated with Buffer B. Two hundred µl of toxin solution (150 µg/ml) was applied to the column and then eluted at a flow rate of 0.5 ml/min. Molecular weight standards used were ovotransferrin (76,000-78,000), ovalbumin (45,000), carbonic anhydrase (30,000), and myoglobin (17,200).
Cross-linking-- Purified proteins (50 µg/ml in 50 mM triethanolamine, pH 8.5) were incubated with 2.5 mM or10 mM dimethyl suberimidate for 10 min at 37 °C. Reaction was quenched by incubation with Tris-HCl, pH 8.0 (final concentration, 100 mM), and then SDS loading buffer was added. Aliquots of the reaction mixture were boiled and analyzed by SDS-PAGE.
Analytical Ultracentrifugation-- Analytical ultracentrifugation was carried out using a Beckman Optima XL-A ultracentrifuge equipped with an optical scanning detector. Proteins in Buffer B at a concentration of 150 µg/ml were analyzed. Data were collected in a Beckman An-60Ti rotor with double sector charcoal-filled Epon cells and quartz windows.
A sedimentation velocity run was carried out at 50,000 rpm at 25 °C. Boundaries were recorded at 280 nm. Apparent sedimentation coefficients were standardized to water at 20 °C. For sedimentation equilibrium experiment, samples were brought to equilibrium at 4 °C for 20 h at 24,000 rpm. Partial specific volumes for proCTX andOther Methods--
N-terminal amino acid sequences of the
purified proteins were determined by Edman degradation using an
automated protein sequencer (Applied Biosystems Model 476A). SDS-PAGE
was performed as described by Laemmli (22). Proteins were stained with
Coomassie Brilliant Blue R-250. Immunoblotting was done as described
previously (13). Protein concentrations were assessed according to
Lowry et al. (23) for unpurified materials and by absorbance
at 280 nm for purified preparations. Absorption coefficients of C20
and proCTX (2.24 and 2.32, respectively) were determined from amino
acid compositions of the proteins.
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RESULTS |
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Purification of an Active Form of Toxin-- We earlier reported a method for purification of an active form of CTX from the crude extract of P. aeruginosa (4). The procedure involved five steps, including trypsin treatment for activation of proCTX in the crude extract. Purification procedures described by other researchers were complicated and included an activation step (24, 25). Because these methods were not applicable to the purification of CTX or its mutants expressed in E. coli, we tried to design a simple purification method for recombinant CTX.
For direct expression of an active form of CTX (
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Purification of proCTX--
We applied the method developed here
to purify proCTX because this approach needed no activation process.
ProCTX produced in E. coli was also insoluble and could be
solubilized with urea. The optimal concentration of urea for
solubilization of proCTX was 3 M. Solubilized proCTX was
purified to apparent homogeneity by the procedure used for the
purification of C20 (Fig. 1B), although the extra step of
anion exchange chromatography was necessary. From 10 liters of culture,
5.5 mg of proCTX was obtained.
ProCTX Does Not Form a 145-kDa Oligomer on the Target
Membrane--
After trypsin treatment, the purified proCTX exhibited
hemolytic activity on rat erythrocytes comparable to the activity of PACTX, as well as that of C20 (Fig. 2). Without activation, proCTX had no hemolytic activity (up to 4 µM). When erythrocytes
incubated with proCTX were analyzed by immunoblotting, proCTX at the
concentration of 0.06 µM bound to the erythrocytes.
Binding of proCTX was in a dose-dependent manner. ProCTX,
however, did not form the oligomer of 145 kDa on the erythrocyte
membrane (Fig. 3). Formation of the oligomer by proCTX was not detected
even at the higher concentration (up to 4 µM; data not
shown). Trypsin-treated proCTX formed the oligomer as seen for
C20.
These data suggested that the inactive nature of proCTX was due to the
lack of potential to form a 145 kDa oligomer on the target
membrane.
C-terminal Peptide Is Dissociated from the Active
Toxin--
Proteolytic cleavage in the C terminus is required for
activation of proCTX. It remained to be determined whether the
proteolytic cleavage removes the C-terminal peptide from the toxin
molecule or simply introduces a nick in the molecule. Using an antibody specific to the C-terminal peptide immobilized on Affi-Gel 10 beads, we
estimated the fate of the C-terminal peptide generated by trypsin
treatment. As shown in Fig.
4A, C20 and the
trypsin-treated proCTX were not adsorbed by the immobilized antibody
and recovered in the supernatants, whereas only a small portion of
proCTX remained in the supernatant fraction. Although toxins in the
pellet fractions were not clearly visible by Coomassie Blue staining
because of the presence of broad bands of the IgG light chain
co-migrating with toxins, immunostaining revealed that proCTX was
recovered in the pellet fraction, whereas trypsin-treated proCTX, as
well as
C20, was not (Fig. 4B). These observations
suggested that the C-terminal peptide left the toxin molecule once it
was generated by proteolytic cleavage. This was further support for the
notion that
C20 could serve as a model molecule for the activated
toxin.
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CD Spectra of proCTX and C20 in the Far-ultraviolet--
As a
first step to elucidate molecular events involved in the activation
process, we wanted to know whether activation of proCTX would alter the
structure. For this purpose, we carried out CD spectroscopic analysis
of proCTX and
C20. The far-UV CD spectra of both proteins, presented
in Fig. 5, were similar. ProCTX was
estimated to contain 14.3%
-helices, 52.7%
-sheet, 14.2%
-turn, and 18.8% random structure, and
C20 contained 12.5%
-helices, 54.4%
-sheet, 10.3%
-turn, and 22.9% random
structure. These findings indicated that the secondary structure of
proCTX was not affected by proteolytic removal of the C-terminal 20 amino acid residues and that CTX was a
-sheet predominant
protein.
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Tryptophan Fluorescence--
In some cases, intrinsic fluorescence
is sensitive for microenvironment transition around tryptophan
residues. Because there was no tryptophan residue in the C-terminal
region on proCTX, we compared fluorescence spectra of the active toxin
and proCTX. As shown in Fig. 6, the
maximum of the fluorescence emission spectrum of proCTX was displayed
at 337.5 nm, whereas a maximum emission wavelength of C20 spectrum
shifted to 342 nm and the intensity was decreased. The maximum
wavelengths of both proteins were not affected by shifted salt
concentration in the range of 0-1.0 M NaCl (data not
shown). Trypsin-treated proCTX without fractionation of the C-terminal
peptide also showed a red-shifted spectrum with a maximum wavelength
the same as that of
C20 (Fig. 6). Although there was a small
difference in intensity between
C20 and trypsin-treated proCTX, this
was attributed to slightly different concentrations of the proteins.
These data suggested that an environment around the tryptophan
residue(s) became hydrophilic during the activation process of
proCTX.
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Dimer to Monomer Conversion of proCTX by Proteolytic
Activation--
The active CTX was suggested to exist as a monomer in
the solution (4), and the oligomeric state of proCTX remained to be
characterized. We first did analytical gel filtration experiments. ProCTX was eluted much earlier than active toxins C20 and PACTX, and
the molecular weights of proCTX and active toxins were estimated to be
52,000 and 24,000, respectively. Furthermore, the molecular weight of
proCTX was reduced to 24,000 by trypsin treatment (data not shown).
These findings suggested that proCTX existed as a homodimer in the
solution and was converted to monomers by proteolytic activation.
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DISCUSSION |
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To purify proCTX of P. aeruginosa, we used a new
method, which is simple but includes a step of solubilizing the
aggregated CTX by urea. This may raise the possibility that the
purified proteins lost their native structures. However, the biological and physicochemical properties of the purified recombinant active CTX,
C20, were indistinguishable from those of an active toxin obtained
from P. aeruginosa. Purified proCTX was also active after trypsin treatment. Thus, the protein refolded correctly during dialysis
after being denatured by urea.
When characterizing the purified proCTX, there were four major findings: (a) proCTX binds to the erythrocytes membrane but cannot form the oligomer on the membrane; (b) proteolytic action removes the C-terminal peptide from the toxin molecule; (c) activation of proCTX by the C-terminal cleavage does not induce change in the secondary structure; and (d) by proteolytic removal of the C-terminal peptide, the homodimer of proCTX is converted to monomers.
One of the crucial steps in intoxication by CTX is oligomerization on the target membrane. The oligomer formed by CTX is most likely in a pentameric form (10). From our findings, the activation process of proCTX is proposed to be as follows: proCTX existing in a dimeric form can bind to the membrane but is cytolytically inactive because it does not oligomerize into a pentamer. Once the C-terminal peptide is removed by proteolytic action, proCTX is converted into monomers, which form a pentamer on the membrane and thus are active in cytolytic events. This is in clear contrast with the activation process described for aerolysin, another pore-forming toxin that requires the C-terminal processing for activation. Aerolysin is also produced as an inactive precursor and exists as a homodimer. After activation by C-terminal cleavage, aerolysin exists as a homodimer (26). To form a heptameric oligomer on the membrane, conversion from dimer to monomer is thought to be essential for aerolysin as well. Although such an intermediate has not been detected, the conversion may be induced by a structural change upon active dimeric toxin after binding to the target membrane (12). The process of oligomerization on the membrane is one point that remains to be elucidated for pore-forming toxins.
The C terminus of proCTX contributes to stable dimer formation but the mechanism remains obscure. A synthesized 21-mer peptide corresponding to the C terminus of proCTX did not form the dimer.2 A direct interaction between the C-terminal regions may not be an event in dimer formation, or such an interaction may be stabilized by other interactions between the two proteins. Another possibility is that the C-terminal region, which contains a stretch of 10 hydrophobic amino acids followed by a sequence potentially forming a coiled-coil helix (residues 277-286), may involve an intermolecular interaction with other parts of the toxin molecule to stabilize the dimeric form of proCTX.
It also remains unclear how the change in intrinsic fluorescence of the toxin occurred by activation. The results suggest that a tryptophan residue(s) buried in the proCTX molecule was exposed to the solvent after activation; it might have been covered directly by the C-terminal peptide and appeared on the surface of the protein by removal of the peptide. Alternatively, the residue(s) might exist on the intersurface of the dimeric proCTX and be exposed to the solvent by dissociation of the toxin molecule into monomers. Identification of the tryptophan residue(s) responsible for change in intrinsic fluorescence will be informative for understanding the structural features of proCTX.
Another important finding on the structure of CTX is that the toxin
consists predominantly of the -sheet. Increasing numbers of
bacterial toxins that form a pore by oligomerization have been reported
to have structures rich in the
-sheet. This was documented most
clearly for aerolysin (16) and staphylococcal
toxin (27). The
structure of oligomerized
toxin at a resolution of 1.9 Å revealed
that a transmembrane domain of the channel is a 14-stranded
-barrel.
The
-sheet predominant feature may be essential for oligomerization
and channel formation of CTX as well. To understand the structural
roles of the
-sheet in CTX, the oligomerized toxin will have to be
isolated, and then it will be possible to examine the exact number of
CTX protomers constituting the oligomer.
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ACKNOWLEDGEMENTS |
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We thank Dr. E. Yoshihara (Tokai University) for helpful discussions; Beckman Instruments (Japan) Ltd. for analytical ultracentrifugation experiments; Drs. Y. Ogoma, T. Taketomi, and T. Tokuda for measurements of CD spectra and masses and for helpful suggestions; and M. Ohara for reading the manuscript.
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FOOTNOTES |
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* This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan.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.
To whom correspondence should be addressed. Tel.: 81-263-37-2615;
Fax: 81-263-37-2616; E-mail: omakoto{at}gipac.shinshu-u.ac.jp.
1 The abbreviations used are: CTX, cytotoxin; PACTX, P. aeruginosa CTX; proCTX, procytotoxin; PAGE, polyacrylamide gel electrophoresis; CD, circular dichroism.
2 M. Ohnishi, unpublished data.
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REFERENCES |
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