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Departments of
*
Microbiology and
Biochemistry, University of Minnesota Medical School, Minneapolis, MN 55455; and
Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, Moscow, ID 83843
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Later in that decade, it was shown that ETA possessed both edematous activity (4) and mitogenic activity toward T lymphocytes (5). It was hypothesized that these properties may account for the epidermolytic activity and edema and skin rash seen in SSSS. It is noteworthy that several other superantigens have been described that belong to a different family of toxins, namely, the pyrogenic toxin superantigen (PTSAg) family (which includes toxic shock syndrome toxin-1, the staphylococcal enterotoxins, and the scarlet fever toxins of streptococci) that also have edematous and T cell proliferative activity that result in skin rash and peeling (6).
Studies in the late 1980s and early 1990s involving the sequencing of the ETA gene (eta) (7) led many researchers to believe that ETA may be a member of the large family of serine protease enzymes. Several studies demonstrated that ETA had esterase activity (which is an intrinsic property of serine proteases; Refs. 8, 9, 10). The solution of the three-dimensional structure of the molecule in 1997 definitely established that ETA had structural elements similar to serine proteases (11, 12). These structural similarities were most notable in the active site region known as the catalytic triad, which consists of three residues (His72, Asp120, and Ser195 in ETA) that comprise the functional active site in a serine protease enzyme. Ser195 and one mutant of Ser195 (S195C, in which Ser195 was mutated to Cys) have been studied in detail by several groups already (8, 10, 13). These findings contributed to the idea that ETA causes SSSS by acting directly or indirectly as an epidermal site-specific serine protease.
More recently, ETA has been demonstrated to have T lymphocyte mitogenic activity and a specific Vß stimulation profile (14), which has further defined the status of ETA as a superantigen.
This work is a study of the superantigenic, epidermolytic, and esterolytic activities of wild-type and mutant forms of ETA to determine the contributions of these properties to SSSS. Several amino acids in ETA were targeted for mutagenesis for a number of reasons, such as their location in the active site structure (His72, Asp120, Ser195), maintenance of active site structure (Pro192, Ser211), presence in uniquely oriented regions such as the D-loop, which is comprised of residues 162 through 169 (Pro162, Phe163, Asp164), and potential role in endowing ETA with substrate specificity (Lys213). Other charged residues on exterior surfaces (R87G) that may be important in binding to various ligands, and conserved mutations (I62V) were made by random mutagenesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ETA gene was first inserted into the plasmid vector pCE104
(ampr, ermr) resulting in
pCE117 (11). Most mutants of plasmid pCE117
(ETA+, ampr,
ermr) were constructed by use of a variation of a
whole-plasmid PCR technique based on the procedure described in the
Quik-Change site-directed mutagenesis protocol (Stratagene, La Jolla,
CA). The mutants made and primers used are listed in Table I
. The positions of the residues targeted
for mutagenesis are shown on the ribbon diagram of the
three-dimensional structure of ETA in Fig. 1. Individual residues with more than one
mutation (e.g., K213A, K213E, and K213T) employed the use of mutant
primers with degenerate codons at the position of the mutation. Two
mutants were constructed via random mutagenesis (i.e., were created by
using limiting amounts of a nonmutagenized primer in a standard PCR
reaction) reaction (15, 16). Sequencing of mutant DNA was
performed by a modification of the dideoxynucleotide chain termination
method for dsDNA (Sequenase T7 DNA polymerase version 2.0 kit; United
States Biochemicals, Cleveland, OH). Randomly mutagenized ETA was
sequenced along the entire gene to insure no aberrant second-site
mutations had occurred. Mutants made by the Quik-Change method were
sequenced across the region of the desired mutation to insure the
change had been made.
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Synthesized mutant DNA was transformed into DH5
Escherichia coli made competent with 50 mM
CaCl2 (pH 6.4) for the purposes of sequencing and
plasmid propagation. For toxin production, DNA was transformed into
S. aureus strain RN4220 via protoplast transformation
(17). Positive clones were tested for ETA expression in a
double immunodiffusion assay (18) by use of a rabbit
polyclonal antisera raised against wild-type ETA.
Selection of mutants
Active site residues. The catalytic triad of a serine protease enzyme consists of a conserved structural motif (19) consisting of a histidine residue, an aspartic acid residue, and a serine residue. His72, Asp120, and Ser195 of ETA comprise what is sequentially and structurally believed to be the putative active site of ETA (11, 12). In serine protease hydrolysis of peptide (and ester) bonds, the Ser195 residue initiates a nucleophilic attack on the substrate, whereas His72 accepts a proton from Ser195 and Asp120 orients His72 to accept the proton from Ser195.
Active site structure maintenance residues. Crystallographic analyses have shown that several residues in ETA are important for the maintenance of the conformation of the putative active site (11, 12, 19). The carbonyl oxygen of Pro192 occupies the oxyanion hole in serine protease enzymes. Ser211 is also believed to stabilize the active site by hydrogen bonding to and ensuring the proper orientation of Asp120. Ser211 is also thought to stabilize the charge that builds up on the Asp-His pair during catalysis. Although Asp164 is also believed to be important in maintaining the structure of the active site, it will hereafter be described with the other D-loop residues.
D-loop residues.
Although most serine protease enzymes have a relatively conserved
structure, ETA has several unique structural characteristics and
motifs, such as the amphipathic N-terminal -helix and the
orientation of what is known as the D-loop.
Pro162, Phe163, and
Asp164 are located in this D-loop region and are
believed to contribute to the unique orientation of the D-loop.
Asp164 is involved in the stabilization of the
uniquely oriented bond between Pro192 and
Gly193 by hydrogen bonding to the main chain
nitrogen of Gly193. Asp164
is also located in and probably involved in the unique orientation of
the D-loop of ETA. Two mutants of D164 were made,
one with an alanine substitution (to analyze the change while
maintaining main-chain structure and rigidity), the other with a
glycine substitution (to allow main-chain flexibility and thusly, more
flexibility in the D-loop).
Substrate specificity mutants. Lys213 is believed to endow ETA with specificity for a cleavage site that follows glutamic acid (and possibly, aspartic acid) in its putative substrate. The positively charged lysine residue is thought to stabilize a negatively charged side chain in a substrate (i.e., aspartic acid and glutamic acid) (11, 12). It also appears to be integral in the biological activity of ETA. For this reason, several different mutations of Lys213 were used, namely, K213A, K213E, and K213T.
N-terminal -helix residues.
Glu12 is located in the highly charged N-terminal
-helix. An N-terminal -helix deletion mutant was also made, where
residues 5–22 of ETA, which comprise the actual -helical portion of
the N terminus, were deleted. The first four amino acids remained in
the mutant to ensure proper synthesis and secretion.
Other mutants. Two mutants were made via random mutagenesis and were also used in our studies. I62V was used to study the effects of a small-scale amino acid substitution in a putative "unimportant," interior area of the protein. R87G was also made and is a mutation in a charged residue on the exterior surface of the first of the two ß-barrels near the active site.
Toxin production and purification
Wild-type and mutant toxins were produced in RN4220 S. aureus grown to stationary phase in 1.2 liters of a pyrogen-free, dialyzable beef-heart medium with 5 µg/ml erythromycin at 37°C and the proteins precipitated in four volumes of ethanol for 48 h (20). Precipitates were resuspended in pyrogen-free water and cleared by centrifugation. Supernatants were dialyzed against deionized water for 2 days (changing water each day), and ETA and mutants forms of ETA were purified via flatbed isoelectric focusing (which involves separating proteins based on their isoelectric points) in pH gradients of 3.5–10, and then 6–8. Toxin-positive fractions were collected and dialyzed against deionized water for 4 days (changing water each day). Purified toxin was quantified via a double immunodiffusion dilution assay and lyophilized until needed (18). On average, 2–10 mg toxin or mutants were obtained per liter of medium. Multiple preparations of toxin were used to insure reproducibility among toxin preparations. Toxin purity was assessed by reverse-phase HPLC (14) and SDS PAGE (21). For reverse-phase HPLC, toxins were loaded onto a Hewlett Packard HP1090 apparatus (Waldbronn, Germany) equipped with a 15-cm C18 column (VYDAC, Hesperia, CA) in 0.1% trifluoroacetic acid (TFA) and eluted in a gradient of 0–60% acetonitrile in 0.1% TFA at a flow rate of 1.0 ml/min. ETA and mutants were pure as tested by these techniques.
Lymphocyte mitogenicity assay
Splenocytes from American Dutch-belted rabbits were isolated and resuspended in RPMI 1640 medium (supplemented with penicillin-streptomycin and 2% FCS) and dispensed into 96-well microtiter plates at a concentration of 2 x 105 cells/well in 200-µl volumes (22). Varying dilutions of ETA (10 µg, 1.0 µg, 0.1 µg, 10 ng, 1.0 ng, and 0.1 ng) were added to each well (in quadruplicate) and incubated for up to 8 days at 37°C in 7% CO2. Cells were then pulsed for 18 h with [3H]thymidine (1.0 µCi). DNA was harvested onto fiberglass filters, and cpm due to [3H]thymidine incorporation into the DNA of proliferating cells were measured in a standard scintillation counter. Background cpm were measured by taking counts on cells not treated with toxin. Potential contaminating factors that may have been contributed by the plasmid vector for ETA (pCE104) or cell line used for toxin purification (RN4220) were taken into account by growing RN4220 transformed with pCE104 and purifying culture fluids in the same manner as described for wild-type ETA. These purified fractions (which came from the same fractions from the isoelectric focusing (IEF) plates as did wild-type ETA) were used as a negative control in mitogenicity assays. The highest cpm for ETA were seen on day 6 at a concentration of 1.0 µg/well. ETA at 0.1 µg/well also caused significant but lower mitogenic activity that peaked on day 6. All subsequent assays of mitogenicity of wild-type and mutant ETAs were therefore harvested after 6 days, and dose response curves to several different concentrations of these same proteins (10, 1.0, and 0.1 µg, and 10, 1.0, and 0.1ng) were performed. Data shown for mitogenicity assays are representative of several different experiments, graphs are representative of at least 2 different wild-type or mutant toxin preparations, and each assay was done with splenocytes from different rabbits.
Epidermolytic assay
A standard Nikolsky test for assaying the epidermolytic capacity of each toxin was performed (1). Varying concentrations of each toxin were resuspended in PBS (PBS, 0.15 M NaCl, 5.0 mM NaPO4, pH 7.1) in 50-µl amounts and injected subcutaneously into 1- to 3-day-old BALB/c mice(Harlan Sprague Dawley, Indianapolis, IN). If given an epidermolytically active toxin, exfoliation of epidermal layers of skin occurs after 1–3 h when the skin is gently stroked. Each toxin was first assayed at 10 µg/mouse, and two to four mice were used per concentration of toxin. If active, lower doses of toxin were used until the minimal dose at which epidermolysis occurred was reached. Likewise, if inactive, the concentration of toxin was raised.
Protease assay
An azocasein substrate (Sigma, St. Louis, MO) was used in the following colorimetric assay. A proteolytically active protein will liberate azo-dye-labeled amino acids from the substrate, resulting in a color change that can be detected spectrophotometrically. One hundred microliters of 50 µg/ml toxin (in sterile, pyrogen-free water) was added to 350 µl of a protease assay buffer (0.1 M NaPO4, 0.01 mM EDTA, 0.01 mM DTT, pH 7.6). Azocasein (50 µl of a 2% w/v solution) was then added, and the mixture was incubated at 37°C for varying time intervals. The remaining intact protein was precipitated with 200 µl of 10% TCA, incubated overnight at 4°C, and removed via centrifugation. The remaining supernatant was assayed for liberated azo-labeled amino acids at 450 nm in a standard spectrophotometer.
Esterase assay
N-t-Boc-L-glutamic acid
-phenyl ester (Sigma) was diluted to 500 mM in 1–4 dioxane and used
as a substrate (13). Five microliters of 500 mM substrate
was added to 3.0 µg toxin suspended in 0.5 ml of 50 mM HEPES buffer
(pH 7.4). Catalytic reactions were performed in 20 mM Tris/phosphate
buffer (pH 7.8). Readings were taken at 60 s at 270 nm with a
molar absorption coefficient of 1.5
mM-1cm-1. Data were
fitted to a standard Henderson-Hasselbach equation using a least
square-fitting program (Kaleidagraph, Version 3.0) to generate values
for Km, Vmax and
Kcat.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Active site mutants. Mutants H72A, D120A, and
S195C retained significant mitogenic activity (Fig. 2), and the peak activity
(
105 cpm) and concentration at which
lymphocyte mitogenicity was lost (0.1 ng/well) were similar and
comparable to wild-type ETA. In contrast, esterase (Table II) and epidermolytic (Table III) activity were completely abolished.
No epidermolysis was seen at concentrations as high as 100 µg per
mouse (the highest dose tested), which is about 200 times the minimum
dose at which wild-type ETA caused epidermolysis (Table III). These
mutants were completely inactive as casein proteases (data not shown),
consistent also with the lack of casein protease activity seen for
wild-type ETA. These findings support the hypothesis that esterase, and
thus likely a specific serine protease activity (with an as yet
unidentified substrate), is required for causation of SSSS.
Furthermore, the data suggest that lymphocyte mitogenic activity is a
distinct and separable property of ETA from esterase and epidermolytic
activities.
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). S211A was
mitogenic only at 10 and 1.0 µg/well, and counts at those
concentrations were only about one half of those of wild-type ETA at
those concentrations. The S211A mutant possessed wild-type esterase
activity (in fact, S211A had slightly higher esterase activity; Table II
), but still required a higher dose of toxin (5–10 µg per mouse)
to cause epidermolysis (Table III), whereas P192A was esterolytic and
epidermolytic at wild-type concentrations. These data suggest a role
for Ser211 in binding a peptide substrate in vivo
that is neither present nor necessary for the protein to act on a
specific ester substrate, since the esterase activity remained
unaffected despite a mutation at this residue, but the dose required to
cause disease was much higher. S211A may also reorient the active site
in such a way as to affect pathogenic potential but not esterase
activity.
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). However, all of these mutants were
active as esterases (Table II) and as epidermolytic agents (Table III)
at concentrations comparable to wild type with one exception; D164A
displayed a significant increase in esterase activity ( 9-fold
higher than wild-type ETA), but had only wild-type epidermolytic
activity. D164A bound substrate better than wild type (as indicated by
Km), and D164G was intermediate
between D164A and wild type, likely because the active site of D164G
was more open than wild type but less so than for D164A. This must have
resulted from differences in the flexibility of the D-loops. In
addition, D164G had a lower Kcat
(turnover rate) than both D164A and wild type, again likely because of
differences in the flexibility of the D-loops. The above data also
demonstrate that ETA has intrinsic lymphocyte mitogenic activity in
that the D164G mutant lost activity compared with wild type, and that
mitogenicity and epidermolytic activity are separable.
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These mutants were not esterolytic (Table II), also consistent with the
lack of activity seen in active site mutants. These data agree with the
hypothesis that a mutation at Lys213 would be
rendered esterolytically inactive, since a glutamic acid ester was
used as a substrate. It was also interesting to note that these
mutants were also unable to cause epidermolysis (Table III), which may
lend support to the idea of a very specific peptide substrate in
vivo.
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-helix mutants. The mutation at
Glu12 (E12A) possessed wild-type ETA activity in
lymphocyte mitogenicity (Fig. 6),
esterase activity (Table II), and epidermolytic activity (Table III).
Interestingly, an N-terminal deletion mutant (
5–22) appeared to be
degraded as it was made in E. coli, and was either not
expressed or not transformed in S. aureus after repeated
attempts. Other studies have shown that N-terminal -helix deletion
mutants of ETA are stable and biologically active (12).
About 1.5 mg of toxin was demonstrated immediately after ethanol
precipitation of DH5 E. coli containing the deletion of
residues 5–22 in 2.4 liters of beef heart medium, and about 400 µg
remained after isoelectric focusing (IEF). After dialysis to remove
ampholytes, none of the mutant could be detected. It is thought that
the N-terminal -helix in some way might protect ETA such that it is
not subject to degradation, either from outside sources (e.g., E.
coli DH5 proteases) or via autodegradation.
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Two other mutants made via random mutagenesis were evaluated for
activity. It was shown that the conservative mutation (I62V) retained
lymphocyte mitogenic activity (Fig. 6), esterase activity (Table II),
and epidermolytic activity (Table III). This is consistent with what
one would expect to find with a fairly conservative amino acid change
in a biologically "unimportant," internal region of the molecule.
However, whereas the R87G mutant was esterolytically comparable to
wild-type ETA (Table III), the epidermolytic activity (Table II) was
slightly lower than wild-type ETA, and the lymphocyte mitogenic
activity was significantly lower than wild-type ETA (Fig. 6). Like
S211A, R87G had a higher epidermolytic dose (ED) than did
wild-type ETA.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SSSS is a disease primarily seen in neonates and is associated with production of one or both of the two exfoliative toxins (ETA and ETB). SSSS is characterized by generalized exfoliation of outer epidermal layers at the level of the desmosomes. As well, neonates often display edema and erythematous skin rash as might be expected due to cytokine release.
There have been two major theories to explain the exfoliation seen in SSSS. First, it was hypothesized that the ETs cause epidermolysis as a consequence of direct action at the desmosomes. This is supported by our recent observations that ETs have structures similar to serine protease enzymes (11, 30). Furthermore, it has been shown that ETs have esterase activity (Ref. 13) and this study). However, protease activity has yet to be demonstrated. The second hypothesis to explain the epidermolysis seen in SSSS is that the ETs stimulate cytokine release with subsequent edema, which physically forces layers of skin apart at the desmosomes (4). In support of this hypothesis are data showing that ETs are superantigenic (14, 31) and that edema and reddening of skin occur in SSSS.
The studies presented here were conducted to evaluate the contributions of both direct activity and superantigenic activity on the causation of skin peeling in SSSS. Lymphocyte mitogenic activity remained in most mutants, irrespective of esterase or epidermolytic activity, which indicates that mitogenicity and esterase/epidermolytic activity are distinct and separable properties of ETA. However, some mutants did appear to have markedly reduced mitogenic activity (R87G, D164G, S211A, and the K213 mutants, especially K213T). Moreover, the two mutants with higher than normal (yet still epidermolytically active) epidermolytic doses, namely, R87G (1.0 µg/mouse, 2 times a normal ED) and S211A (5–10 µg/mouse, 10–20 times a normal ED) were significantly less mitogenic than wild type and most mutant forms of ETA. Collectively, these data suggest that superantigenicity is not required for the skin peeling seen in SSSS. However, superantigenicity may lead to edema and skin reddening; neither of these latter two effects were seen in the 1- to 3-h time required to cause skin peeling in mice, but are seen in SSSS. Interestingly, residues whose mutants were deficient in mitogenicity are all clustered about the active site, but are not considered to be part of the serine protease catalytic triad itself.
Most significantly, esterase and epidermolytic activity were found to correlate with one another. This lends support to the idea that some sort of serine protease activity (or at least, serine esterase activity) either directly causes or contributes to the causation of SSSS. All mutants that retained esterase activity also retained epidermolytic activity. Conversely, mutants that lost esterase activity also lost epidermolytic activity (i.e., still epidermolytically inactive at 100 µg/mouse, 500 times a normal ED). Various other serine proteases were tested for epidermolysis, such as trypsin, subtilisin, and staphylococcal V8 protease. None of these proteins were capable of causing epidermolysis, which indicates that ETA possesses special properties that endow it with its signature biological activity.
Neither wild-type ETA, nor any mutant form of ETA showed proteolytic activity for azocasein or any other peptide substrate studied to date. An ester substrate, such as used in this study, may be more accessible to the putative active site by virtue of its size and increased flexibility (ester bonds are typically more flexible than rigid, planar, peptide bonds).
The unusual properties displayed by the Asp164 mutants indicate that this may be a particularly "sensitive" residue in terms of biological activity. The fact that mitogenic activity was abolished in D164G may be due to replacing Asp164 with a residue that allows much more main-chain flexibility in an important area of the protein in terms of binding immune system cells. A D164A mutation may reorient the D-loop in such a way as to make the active site more accessible, at least to an ester substrate, without losing main chain rigidity.
In conclusion, while much is still unknown about how ETA causes SSSS, many of the structural, immunological, and biological properties of the toxin have been studied in detail, and several conclusions can be drawn from available information. Data gathered to date have shown that ETA does act as a superantigen in the absence of any potential contaminating factors. Structural analyses and biological activity (i.e., esterase activity) studies have shown that ETA is likely acting as a serine protease, possibly cleaving a protein or proteins at the stratum granulosum. The correlation between esterase activity and epidermolysis indicates that an epidermal site-specific protease activity is likely the mechanism by which ETA causes exfoliation. Superantigenicity may also play a supplementary role in the causation of SSSS such as production of rash and edema, but is not required for the progression of epidermolysis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Patrick M. Schlievert, Department of Microbiology, University of Minnesota Medical School, Box 196 Mayo, Minneapolis, MN 55455. E-mail address:
3 Abbreviations used in this paper: SSSS, staphylococcal scalded skin syndrome; ETA, exfoliative toxin A; TFA, trifluoroacetic acid; ED, epidermolytic dose.
Received for publication July 19, 1999. Accepted for publication December 3, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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