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Activity requirements of epidermolytic toxin from Staphylococcus aureus studied by an in vitro assay
ToxtconVol. 28, No. 6, pp. 675-683, 1990. Printed in Great Britain.
0041--0101/90 $3.00+.00 PergamonPresspie
ACTIVITY REQUIREMENTS OF EPIDERMOLYTIC TOXIN FROM S T A P H Y L O C O C C U S AUREUS STUDIED BY AN IN VITRO ASSAY THOMAS P . SMITH a n d CHRISTOPHER J. BAILEY Department o f Biochemistry, Trinity College, Dublin, Ireland
(Accepted for publication 7 November 1989)
T. P. SMITH and C. J. BAILEY. Activity requirements of epidermolytic toxin from Staphylococcus aureus studied by an in vitro assay. Toxicon 28, 675-683, 1990.--The activity of epidermolytic toxin from Staphylococcus aureus was studied in vitro using discs of neonatal mouse skin. By assessing the loss of skin integrity as a function of toxin dose and time, it was possible to put the assay on a semi-quantitative basis. Epidermolysis occurred without any change in rate from pH 3.8 to 8.7, and at an increasing rate in the temperature range of 0-37°C. Activity was observed even at the lowest temperature. More than 30 inhibitors of energy metabolism, central metabolic pathways, receptor binding or proteolysis, individually failed to prevent epidermolysis and it is suggested that intoxication cannot be dependent on receptor-mediated endocytosis. Five metal-ion chelators inhibited epidermolysis, due to an effect on the tissue rather than on the toxin. Using X-ray fluorescence and atomic absorption spectroscopy, it was shown that epidermolytic toxins do not contain any essential metal ions. Some transition metals, but not Ca 2÷ or Mg 2+, prevented the chelator-dependent inhibition of epidermolysis.
INTRODUCTION
THE EPIDERMOLYTIC toxins (ET) of Staphylococcus aureus are responsible for the neonatal condition of staphylococcal scalded skin syndrome. Two forms of the toxin, known conveniently as ETA and ETB (reviewed by FREER and ARBUTt-rNOTT, 1983), are related proteins (LEE et al., 1987) with indistinguishable biological activity. In the usual end-point assay system (MELISH et al., 1970) the minimal dose is identical for the two toxins (JOHNSON et al., 1979). The toxin acts in vivo to release intercellular adhesion within the epidermis at the level of the stratum granulosum (LILLmRII~E et al., 1972). It is not known how this happens, although an intracellular target protein, profilaggrin (SMrm and BAILEY, 1986; SMmt et al., 1987) has been detected, and it has not been possible to associate metabolic changes with epidermolysis. This means that the only toxicity assay is the effect on the skin, usually as observed in vivo. It is also possible to observe the effect of the toxin in vitro using simple organ culture (or maintenance) systems (M¢CALLUM, 1972; ELIAS et al., 1974; NISmOKA et al., 1981) which allow the possibility of testing for the activity requirements of epidermolytic toxin. Thus ELIAS et al. (1977) demonstrated that intoxication was insensitive to the presence of inhibitors of respiration or of protein synthesis. NmHIOKA et al. (1981) showed that proteinase inhibitors were similarly without effect, but they were able to demonstrate an 675
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T. P. SMITH and C. J. BAILEY
inhibitory activity associated with the mitochondrial pellet, from mouse epidermis. The only claim of an exogenous interference with the process of epidermolysis is that EDTA will remove a necessary metal-ion from ETA, although ETB is not a metallo-toxin (KoNDO et al., 1976; SAKURAI and KONDO, 1978). In this paper we report a systematic search, using the in vitro assay, for inhibitors of ET.
MATERIALS AND METHODS Purification o f E T Samples of ETA and ETB were purified from standard strains by isoelectric focusing and chromatography on Sephadex G-50 S in 0.01 M NH4HCO3 (DE AZAVEDO et al., 1988). On a larger scale, ETA was purified from culture filtrates of an overproducing strain of S. aureus (O'TOOLE, P. W., Ph.D thesis, University of Dublin, 1987) by (NH4)2SO4 fractionation and Sephadex G-75 S chromatography in 0.01 M NH,HCO3 (LOCKHART, B. P., unpublished). All the preparations used were homogeneous by polyacrylamide gel electrophoresis (LAEMMLI, 1970). The toxin was stored as a lyophilized powder and assayed in solution by spectrophotometry at 280 nm (BA1LEY et al., 1982).
lnhibitors Amiloride, bumetanide and furosemide were a kind gift from Dr M. J. RYAN, University College Dublin; phytohemagglutinin and pokeweed mitogen solutions were from Gibco Ltd., Paisley, U.K.; ~:macroglobulin was from Boehringer Mannheim, Lewes, U.K.; Chelex-100 was from Bio-Rad Laboratories, Watford, U.K.; all other inhibitors were from Sigma Chem. Co., Poole, U.K.
In vitro assay Whole thickness mouse skin (Q strain, 1-3 days old) was scraped with a blunt scalpel to remove subcutaneous fat. Circular discs (5 mm 2) were cut using a leather punch and stored briefly on ice until required. Assays were started by addition of two skin punches to 200 #1 of phosphate-buffered saline (PBS), pH 7.4, containing a given amount of ET (usually 10 pg of ETA). After incubation, usually at 37°C, for a given time the effect of the toxin was assessed by attempting removal of the superficial layers (corneal and granular) of the epidermis using fine forceps. When the effect of pH was tested, the phosphate content of the medium was increased and NaC1 reduced to retain isotonicity, and the amount of tissue added to the medium was halved, in order to avoid gross pH changes in solutions not at neutral pH. The effect of temperature was assessed after separate preincubation of skin and toxin for 15 min at the appropriate temperature. To examine the effect of exogenous substances, two sorts of experiments were carried out, each in duplicate. All substances were tested by a procedure in which skin plus test compound were pre-incubated at 37°C for 30 min before addition of ET (10 #g). The assay was then allowed to proceed for 30 min and the tissue discs were examined. In addition, some compounds were tested after preincubation of ET plus compound. The scoring systems used was as follows: ( + +), the upper epidermis separated easily from the underlying tissue of the disc; (+), separation was achieved with difficulty; ( + / - ) , separation could only be achieved at the edges of the disc; ( - ) no separation possible. Skin samples were tested once and then discarded. A system of positive and negative controls was incorporated with each assay set. The positive control contained 10/~g of ET and was tested under standard conditions. Negative controls (no toxin added) were used to test that assay conditions or exogenous additives did not compromise the integrity of the tissue over the period of assay. In all of the reported exogenous substance tests, the negative controls scored ( - ) after the 30 min assay period.
Metal ion analysis ET samples (0.2-2.5 mg/ml) in 0.01 M NH4HCO3 were analysed for zinc, copper, cobalt and magnesium using Perkin Elmer 372 and International Laboratories 257 atomic absorption spectrophotometers. Qualitative analysis of protein samples (approx. 50 mg) as a dry powder was carried out on a Link Systems Meca 10--44 automatic X-ray fluorescence spectrometer, equipped with a silver target.
Light microscopy Skin was fixed overnight at 20°C in a 4% (v/v) solution of formaldehyde in 75 mM sodium phosphate, pH 7.4, dehydrated in ethanol and embedded in paraffin wax. Transverse sections (5/tin) were cut with a rotary microtome, stained with haematoxylin and eosin, and examined in a Zeiss microscope.
Activity of Epidermolytic Toxin
677
RESULTS
Properties of the in vitro assay system The response of the skin punch assay to varying doses of ETA is summarized in Table 1. As the dose decreased the time required for epidermolysis increased. The speed of the response can vary with the age of the animal tissue but the test was always sensitive to epidermolytic toxin at the levels shown in Table 1. In more than 10 tests in the range of toxin dosage 0.5-10 #g, it was always observed that a two-fold difference in toxin quantity produced an observable difference in speed of response. On the basis of the nonparametric sign test (SNEDECOR and COO-mAN, 1967), this corresponds to greater than 99% confidence that the two-fold difference can be measured. The effect at 0.1 #g of toxin was also reproduced so that there is greater than 95% confidence that this amount could be detected in 2 hr. The minimum effective dose at 2 hr was not estimated more exactly. For comparative purposes, assays were carried out under standard conditions, in PBS at 37°C with 10 #g of ET in sample and control. An indistinguishable positive effect, ( + + ) at 30 min, corresponds to at least 5 #g (P = 0.01) of ET activity in the sample. A negative effect was followed to a time of at least 1 hr and corresponds to less than 0.1 #g of ET activity (P = 0.05) in the sample. ETB (10 #g) was indistinguishably positive, when compared with an equal quantity of ETA, in the test. ETA was active over a broad temperature range (0-37°C), even at the lowest temperature (Table 2). A 10°C drop in temperature approximately doubled the TABLE 1. In vitro DOSE--RESPONSE OF SKIN TO EP1DERMOLYTIC TOXIN A
Assay results (min)
Toxin ~g/tube)
15
30 .
60
0.0
.
0.1
--
--
.
+/--
.
0.5
--
--
+
1.0
-
+/-
+
5.0 lO.O
+
+ ++
++ ++
90
120
+
++ ++ ++ ++ ++
.
++ ++ ++ ++
T h e scoring system indicates: ( - ) , no separation possible; ( + / - ) , separation only achieved at the edge of the disc; (+), complete separation achieved with difficulty; ( + + ) , upper epidermis readily peeled away from the underlying tissue.
TABLE 2. TEMPERATURE-DEPENDENCE OF /~ vitro EPIDERMOLY$1S BY E T A
Assay results* (hr)
Temperature (°C)
1
2
o
-
7 15 20 27 37
-+/++ Each
3
4
-
-
+/-
+/++ ++
+/+ ++ ++ ++
+ ++ ++ ++ ++
5
6
+
++ ++ ++ ++ ++ ++
++ ++ ++ ++ ++
assay contained 10 #g of ETA. Negative controls scored ( - ) at all times up
to 6 hr. *See T a b l e 1
for explanation.
678
T.P. SMITH and C. J. BAILEY
time required for complete epidermolysis. The effect of pH was studied at 37°C in the presence of 10/tg ETA. In the pH range 3.8-8.7 (final pH value), epidermolysis occurred exactly as at pH 7.4. Beyond this range, the skin samples began to break up in the absence of toxin, probably because of splitting at the dermal-epidermal junction.
Effects of exogenous substances A large number of substances were tested for their ability to perturb the course of epidermolysis. The respiratory inhibitors fluoride, arsenate, iodoacetate, azide, cyanide and 2,4 dinitrophenol (1 mM of each) were preincubated with the tissue for 30 rain. None of these inhibitors blocked epidermolysis or caused any apparent change in its rate, under the standard conditions. Inhibitors of microtubule assembly (colchicine, l mM), ion transport (ouabain, 1 #M; amiloride, 0.1 mM; bumetanide, 0.1 mM; furosemide, 1 mM) and lysosomal systems (ammonium chloride, 10 mM; chloroquine, 10 mM) were ineffective under standard conditions. To test whether the toxin required a carbohydratedependent uptake system, the lectins conconavalin A (10/~M), pokewood mitogen (1:20 dilution) and phytohaemagglutinin (1:20 dilution) and the sugars D-glucose, N-acetyl-Dglucosamine, D-galactose, N-acetyl-D-galactosamine, D-mannose, D-fructose, D-sorbitol (all at 100mM) of D-glucosamine, D-galactosamine, N-acetylneuraminic acid (all at 50 mM) were each incorporated into the assay system without any effect on the progress of epidermolysis. Inhibitors of the four major classes of proteolytic enzymes were tested. Leupeptin (1 mM); aprotinin (0.5mg/ml); soyabean trypsin inhibitor (0.5mg/ml); phenylmethane sulphonyl fluoride (1 mM); pepstatin (0.1 mM); phosphoramidon (0.1 mg/ml) and ~2macroglobulin (0.5 mg/ml) had no effect on epidermolysis under standard conditions. But in the presence of EDTA (10 mM), a negative response was observed at 30 min incubation under standard conditions. As the two other metalloprotease inhibitors, phosphoramidon and ~2-macroglobulin, had no effect, it seemed possible that EDTA might inhibit epidermolysis by removal of an essential metal ion which is not necessarily associated with a protease.
Effect of chelators EDTA at 10 mM blocked epidermolysis by ETA after pre-incubation of skin plus chelator or pre-incubation of toxin plus chelator. EDTA (10 mM) also inhibited ETB after pre-incubation of skin plus chelator. However the result is complicated by the observation that EDTA alone caused skin-splitting after 2 hr in the assay system. It was only possible to assay for inhibition by EDTA because the action of the toxin is apparent after 30 min. A quantitative distinction between the skin-splitting activities was made by histological examination. Epidermolytic toxin split the skin at the granular layer (Fig. la). Toxin plus EDTA produced a split at the dermal-epidermal boundary (Fig. l b) and it was shown that EDTA alone gave the same cleavage. To confirm that chelation can prevent intoxication, we searched for chelators which did not cause epidermolysis. The results are shown in Table 3 together with representative data for the metal-ion affinities. EGTA (10 mM) prevented epidermolysis by ETA after pre-incubation of chelator with skin or toxin (Fig. lc), and by ETB, after pre-incubation of chelator with skin, and by itself caused no morphological or histological effect on the epidermis. Three other chelators, 1,10-phenanthroline, citrate and oxalate prevented epidermolysis after pre-incubation with skin, EGTA, oxalate and citrate blocked epiderm-
FIG. 1. HISTOLOGYOF NEONATALMOUSESKIN. Tissue sections were processed for microscopy after incubation in phosphate-buffered saline with: (a) ETA (50#g/ml) after 30min; (b) ETA (50pg/ml)+EDTA (10mM), after 2hr; (c) ETA (50 #g/ml) + EGTA (10 mM), after 2 hr. The positions of the stratum corneum (SC); stratum granulosum (SG) and dermis (D) are indicated. In (a) the tissue has split intra-epidermally, just below the SG. In (b) the split is at the junction of the dermis and epidermis, no splitting was observed after 30 min. In (c) no split occurred. The scale bar indicates 10 #m.
680
T. P. S M I T H and C. J. BAILEY TABLE 3. EFFECT OF CHELATORSON it/ vitro EPIDERMOLYS1S
Chelator (mM) E D T A (10) E G T A (I0) 1, 10-Phenanthroline (10) 2,2'-Dipyridyl (I0) Diethyldithiocarbamate (10) Chelex-100 (250 mg/ml) Citrate (50) Oxalate (50) Malonate (50) Succinate (50)
Stability constant (log KApp) Ca 2+ Zn 2÷ (stoichiometry) 7.7 7.5 0.5 0.5 (Mg 2÷) n.a. n.a. 3.6 3.0 1.5 1.2
13.5 (1) 9.4 (I) 17 (3) 13.5 (3) 11.5 (2) n.a. 5.0 (1) 8.2 (3) 4.5 (2) 1.8 (1)
Assay result* 30 min + + + + + + + + + +
The log KApp values are apparent values recalculated at pH 7.4 from the data o f DAWSON et al. (1986). The ratio o f complexed to free metal ion, in the presence of excess chelator, m a y be calculated from the formula: log [Complexed/Free] = log KApp+ (Stoichiometry)X log [Ligand]. The stoichiometry o f the complexes is the molar ratio of chelator to metal ion in the dominant complex at excess chelator. The stoichiometry of all Ca 2+ complexes is 1, but varies as indicated for Zn 2÷ complexes. n.a. not available. *See Table 1 for additional information.
olysis for up to 2hr. 1,10-Phenanthroline caused a rate change so that, although a negative result was obtained at 30 min (Table 3), epidermolysis was apparent ( + / - ) at 60 min and complete (+ +) at 90 min. This chelator on its own did not produce any skinsplitting reaction at times up to 90 min. Three more chelators with high affinities for metal ions (Table 3), 2,2'-dipyridyl, diethyldithiocarbamate and Chelex-100 did not inhibit the toxin. Of these, the resin is a solid which cannot be in intimate contact with the inner layers of the skin and it may be that the skin site is also inaccessible to the other two inactive chelators. Since two of the inhibitors are carboxylic acids, others of that class were tested. Succinate and malonate are without substantial metal-ion binding ability (Table 3) and did not inhibit the toxin. On the basis of the wide variety of different chemical substances which inhibit epidermolysis by ETA, it seems reasonable to conclude that the metal-chelating ability is the cause of inhibition. The test protocol does not indicate whether the required metal ion is sequestered from the skin or the toxin or both. Therefore experiments were carried out in which skin or toxin samples were separated from the chelators after pre-incubation but before assay. Skin punches were incubated in 5 mM EGTA in PBS (200/~1) for 30 min at 37°C, removed, washed in PBS (20 ml) for 1 min and blotted before the test. In the assay system these samples remained entirely unresponsive to 10 #g of ETA for up to 2 hr. To test for the existence of essential metal ions in the toxin, samples of ET were assayed and analysed before and after EDTA treatment. Samples of ETA and ETB were each incubated with 10 mM EDTA and after 2 hr incubation the toxins were separated from the chelator by Sephadex chromatography. They were found to be as active as untreated toxin: epidermolysis (+ +) occurred after 30 min incubation with 10/ag of either ETA or ETB. Samples of ET were also assayed for the presence of metal ions. Two samples of ETA, isolated by Sephadex chromatography without EDTA pre-treatment, gave 0.3 and 0.7 gram-Atom (gA) per mole protein of copper plus 0.7(2) gA per mole of zinc, together with traces of magnesium; similar samples of ETB contained 0.2 and 0.4 gA per mole of copper and 0,1 and 0.3gA of zinc. An ETA sample was adjusted to 10mM EDTA,
Activity of Epidermolytic Toxin
681
dialysed for three days at 2°C, then isolated by Sephadex chromatography. Zinc and copper was undeteetable in this ETA ( < 0.01 gA of each metal per mole of protein). A large quantity of freeze-dried toxin from the latter preparation was examined for the presence of metal ions using X-ray fluorescence techniques. No X-ray emissions in the region 4-40 keV (covering the range of atomic numbers for all the transition metals) in comparison with pure hen-egg lysozome, a metal-free protein, could be detected. The sensitivity of this technique is very great, although it may vary a little between elements. For zinc, control experiments showed that a level of 0.02 gA per mole was readily detectable. It is concluded that the evidence is consistent with the occurrence of a required metal ion in the skin, that EDTA does not remove essential metal ions from ETA or ETB, and that ETA does not contain any essential ions, although ETA as usually isolated may contain adventitious, non-essential metal ions, at levels approaching 1 gA per mole protein. Attempts were made to restore sensitivity to skin rendered resistant by EGTA pretreatment. After addition of 0.1 mM of any of Ca 2+, Mg 2+, Zn 2+, Co 2+, Fe 2+ (none of which inhibited even at 6 mM), no epidermolysis was evident when the skin was challenged with 10#g ETA at 37°C for 60min. However it was possible to protect the epidermolytic reaction from EGTA inhibition by cation swamping. In the experiments, 5 mM EGTA and 6 mM cation were briefly pre-incubated before the addition of ETA and skin punches. The transition metals, Mn 2÷, Cu 2+, Fe 2+, Zn 2+, and Co 2+ prevented the EGTA-dependent inhibition of epidermolysis, but Ca :+ and Mg 2+ did not. DISCUSSION
The in vitro assay utilized in this report provided a rapid and sensitive end-point assay system. The convenient limit of 0.1 pg at 0.5 #g/ml (2 x 10-SM) could readily be lowered by extending the assay period and by accepting detection at a low level of confidence. In the in vitro assay the lowest confirmed limit is 0.1 #g at 2-4 #g/ml. (WUEPPERet al., 1975; NISHIOKA et al., 1981). The main advantage of the in vitro assay is not an increase in sensitivity but the ability to vary the assay conditions and to test the effects of exogenous additives. By scoring the ease of epidermolysis as a function of dose and incubation time, it is possible to put the assay on a semi-quantitative basis that makes it convenient to search for inhibitory effects. The insensitivity of epidermolysis to a range of variations and supplementations extends the earlier reports (ELIAS et al., 1977; NISHIOKAet al., 1981) and has important implications for the possible mechanism of epidermolytic toxin. For a large proportion of protein toxins, intoxication requires uptake into eukaryotic cells via receptor-mediated endocytosis (MIDDLEBROOKand KOHN, 1981). Such uptake systems involve complex membrane transport events and are expected to be energydependent, temperature-sensitive, pH-dependent and also susceptible to inhibition by lysosomatropic agents and high concentrations of analogues of receptor molecules (typically carbohydrate). The individual properties of the toxins vary so that not all systems are susceptible to all the factors listed. For example, ricin and abrin show a measurable uptake at 5°C, there is a high temperature-dependence, but no critical temperature, of uptake. Lysosomatropic substances do not prevent inhibition at neutral pH; but inhibitors of glycolysis and respiration completely protect cells (reviewed by OLSNESand S^NDVIG, 1983). In the case of diptheria toxin (PAPPENI-mIMER, 1977) intoxication is inhibited at low temperatures, by ammonium chloride and by fluoride but not by other inhibitors of energy metabolism or by uncoupling agents.
682
T.P. SMITH and C. J. BAILEY
The uniformly-negative effects of so m a n y inhibitors of receptor-mediated endocytosis, suggest that epidermolytic toxin does not enter cells by such a mechanism. The strength of this conclusion is limited only by the nature of the skin test assay. It may be that epidermolysis is an effect which occurs at saturation levels of toxin where the effects of inhibition could be less obvious. Positive effects were observed with a group of chelators suggesting that chelation is the unifying property of this group of inhibitors. One of these, E D T A , has been reported to inhibit ETA by removal of a metal ion which could be replaced by Zn 2+, Co 2+, Mg 2÷ and Mn 2+ (KONDO et al., 1976; SAKURAI and KONDO, 1978). In samples of the toxin purified from culture medium, it was reported that copper was present in the native toxin at stoichiometric amounts. Although our experiments confirm that purified ETA routinely contains copper (and zinc) in amounts close to a stoichiometric ratio, they also show that the metal-ions can be removed by E D T A , after which the toxin is active and metal-free. The interpretation of the original experiments of KONDO and collaborators depends critically on the assumption that dialysis has reduced the E D T A concentration from 200 mM to less than 2.5 m M (DIMONO et al., 1976) in order that the chelator does not interfere with the assay. The results in the present paper are consistent with the theory that ETA and ETB have the same binding properties and mechanism, as is expected for two proteins showing a high degree of amino acid sequence homology (O'ToOLE and FOSTER, 1987; LEE et al., 1987). Our observation that E D T A splits the skin at the dermal-epidermal junction is in agreement with previous observations (SCALETTA and MACCALLUM, 1972; DIMOND et al., 1976). Histochemical evidence has been produced to show that the EDTA-separated epidermis may be subsequently split by ET (ELIAS et al., 1976), but there is no gross morphological evidence to support this claim. The major result of the present work is that epidermolysis requires a metal ion which is provided by the tissue, not the toxin. The identity of the metal cannot be established exactly, but the experiments on protective effects imply that the cation must have a higher affinity for E G T A than do Ca 2+ and Mg2÷; that is, a transition metal rather than an alkaline earth metal. Since metal ions are implicated in m a n y biochemical mechanisms it remains uncertain whether the ion is necessary for transport to the putative intracellular target which is profilaggrin (SMITH and BAILEY, 1986; SMITH et aL, 1987) or for some other event. It is, however, known (LOCKHART, unpublished) that E D T A does not interfere with the binding of ET to profilaggrin on western blots. Acknowledgements--We thank DAVIDDOFF, TERRYWILLIAMSand ALBERTMcQUAID for assistance with the metal ion analyses and PAT HARTIGANfor advice on histology. The research was supported by grants from the Irish Medical Research Council.
REFERENCES BAILEY,C. J., MARTIN,S. R. and BAYLEY,P. M. (1982) A circular dichroism study of epidermolytictoxins A and
B from Staphylococcus aureus. Biochem. J. 203, 775-778. DAWSON,R. M. C., ELLIOT,D. C., ELLIOTT,W. H. and HONES,K. M. (1986) Stability constants for metal ion complexes. In: Data for Biochemical Research (3rd edn), pp 399-415. Oxford: Clarendon Press. DEAZAVEDO, J., BAILEY, C. J. and ARBUTBNOTT,J. P. (1988) Purification of epidermolytic toxins from Staphylococcus aureus. Methods Enzymol. 165, 32-37. DIMOND,R. L., ERICKSON,K. L. and WUEPPER,K. D. (1976) The role of divalent cations in epidermolysis. Brit. J. DermatoL 95, 25-34. ELIAS,P. M., FRITSCn,P., TAPPE1NER,G., MITTERMAYER,H. and WOLFE,K. (1974) Experimental staphylococcal toxic epidermal necrolysis (TEN) in adult humans and mice. J. Lab. Clin. Med. 84, 414~23.
Activity of Epidermolytic Toxin
683
ELIAS, P. M., FRITSCH,P. and MITTERMAYER,H. (1976) Staphylococcal toxic epidermal necrolysis: species and tissue susceptibility and resistance. J. Invest. Dermatol. 66, 80-89. ELIAS, P. M., FRITSCH, P. and EPSTE,N, E. H. (1977) Staphylococcal scalded skin syndrome: clinical features, pathogenesis, and recent microbiological and biochemical developments. Arch. Dermatol. 113, 207-219. FREER, J. H. and ARnUTHNOTI',J. P. (1983) Toxins of Staphylococcus aureus Pharmacol. Therapeut. 19, 55-106. JOHNSON,A. D., SPERO,L., CADES,J. S. and DE CIcco, B. T. (1979) Purification and characterization of different types of exfoliative toxin from Staphylococcus aureus. Infect. lmmun. 24, 679-684. KONDO, I., SAKURAI, S. and SAg1, Y. (1976) Staphylococcal exfoliatin A and B. In: Staphylococci and Staphylococcal Diseases, pp. 489-498 (JEI.ZASZEWlCZ,J., Ed.) Stuttgart: Gustav Fischer. LAEMMLI,U. K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage 1"4. Nature 227, 680-685. LEE, C. Y., SCHMIDT, J. J., JOHtqSON-WIh~GAR, A. D., SPERO, L. and IANOOt.O, J. J. (1987) Sequence determination and comparison of the exfoliative toxin A and B from genes from Staphylococcus aureus. J. Bacteriol. 169, 3904-3909. LILLmRIOGE, C. B., MELISH, M. E. and GLASC,OW, L. A. (1972) Site of action of exfoliative toxin in the staphylococcal scalded skin syndrome. Pediatrics 50, 728-738. MCCALLUM,H. M. (1972) Action of staphylococcal epidermolytic toxin on mouse skin in organ culture. Br. J. Dermatol. 86 (Suppl 8), 40-41. MELlSH, M. E., GLASGOW, L. A. and TuR~a~R, M. D. (1970) Staphylococcal scalded skin syndrome (SSSS). Experimental model and isolation of a new exfoliative toxin (ET). Pediatr. Res. 4, 378-379. MIDDLEnROOK, J. L. and KOHN, L. D. (1981) Receptor-mediated binding and internalisation of toxins and hormones. New York, London: Academic Press. NISH1OKA,K., KATAYAMA,I. and SANO,S. (I 981) Possible binding of epidermolytic toxin to a subcellular fraction of the epidermis. J. Dermatol. 8, 7-12. OLSh'ES, S. and SANDWG, K. (1983) Entry of toxic proteins into cells In: Receptor-mediated endocytosis, pp. 187-236 (CuATRECASAS,P. and Ro'm, T., Eds). London: Chapman and Hall. O'TOOLE, P. W. and FOSTER,T. J. (1987) Nucleotide sequence of the epidermolytic toxin serotype A gene of Staphylococcus aureus. J. Bacteriol. 169, 3910-3915. PAPPE~a-IEIMER,A. W. (1977) Diphtheria Toxin. Ann. Rev. Biochem. 46, 69-94. SAgURAI,S. and KONDO, I. (1978) Characterisation of staphylococcal exfoliatin A as a metallotoxin with special reference to determination of the contained metal by radioactivation analysis. Jpn. J. Med. Sci. Biol. 31, 208-211. SCALETTA,L. S. and MACCULLUM,D. K. (1972) A fine structural study of divalent cation-mediated epithelial union with connective tissue in human oral mucosa. Amer. J. Anat. 133, 431-454. SMm~, T. P. and BAILEY,C. J. (1986) Epidermolytic toxin from Staphylococcus aureus binds to filaggrins. FEBS Left. 194, 309-312. SMITH, T. P., JOHN, D. A. and BAILEY,C. J. (1987) The binding of epidermolytic toxin from Staphylococcus aureus to mouse epidermal tissue. Histochem. J. 19, 137-149. SNEDECOR, G. W. and COCHRAtq,W. G. (1967) Statistical Methods, 6th edn, pp. 125-128. Ames: Iowa State University Press. WUEPPER, K. D.. DIMOND,R. L. and KNUTSON,D. D. (1975) Studies on the mechanism of epidermal injury by a staphylococcal epidermolytic toxin. J. Invest. Dermatol. 65, 191-200.
0041--0101/90 $3.00+.00 PergamonPresspie
ACTIVITY REQUIREMENTS OF EPIDERMOLYTIC TOXIN FROM S T A P H Y L O C O C C U S AUREUS STUDIED BY AN IN VITRO ASSAY THOMAS P . SMITH a n d CHRISTOPHER J. BAILEY Department o f Biochemistry, Trinity College, Dublin, Ireland
(Accepted for publication 7 November 1989)
T. P. SMITH and C. J. BAILEY. Activity requirements of epidermolytic toxin from Staphylococcus aureus studied by an in vitro assay. Toxicon 28, 675-683, 1990.--The activity of epidermolytic toxin from Staphylococcus aureus was studied in vitro using discs of neonatal mouse skin. By assessing the loss of skin integrity as a function of toxin dose and time, it was possible to put the assay on a semi-quantitative basis. Epidermolysis occurred without any change in rate from pH 3.8 to 8.7, and at an increasing rate in the temperature range of 0-37°C. Activity was observed even at the lowest temperature. More than 30 inhibitors of energy metabolism, central metabolic pathways, receptor binding or proteolysis, individually failed to prevent epidermolysis and it is suggested that intoxication cannot be dependent on receptor-mediated endocytosis. Five metal-ion chelators inhibited epidermolysis, due to an effect on the tissue rather than on the toxin. Using X-ray fluorescence and atomic absorption spectroscopy, it was shown that epidermolytic toxins do not contain any essential metal ions. Some transition metals, but not Ca 2÷ or Mg 2+, prevented the chelator-dependent inhibition of epidermolysis.
INTRODUCTION
THE EPIDERMOLYTIC toxins (ET) of Staphylococcus aureus are responsible for the neonatal condition of staphylococcal scalded skin syndrome. Two forms of the toxin, known conveniently as ETA and ETB (reviewed by FREER and ARBUTt-rNOTT, 1983), are related proteins (LEE et al., 1987) with indistinguishable biological activity. In the usual end-point assay system (MELISH et al., 1970) the minimal dose is identical for the two toxins (JOHNSON et al., 1979). The toxin acts in vivo to release intercellular adhesion within the epidermis at the level of the stratum granulosum (LILLmRII~E et al., 1972). It is not known how this happens, although an intracellular target protein, profilaggrin (SMrm and BAILEY, 1986; SMmt et al., 1987) has been detected, and it has not been possible to associate metabolic changes with epidermolysis. This means that the only toxicity assay is the effect on the skin, usually as observed in vivo. It is also possible to observe the effect of the toxin in vitro using simple organ culture (or maintenance) systems (M¢CALLUM, 1972; ELIAS et al., 1974; NISmOKA et al., 1981) which allow the possibility of testing for the activity requirements of epidermolytic toxin. Thus ELIAS et al. (1977) demonstrated that intoxication was insensitive to the presence of inhibitors of respiration or of protein synthesis. NmHIOKA et al. (1981) showed that proteinase inhibitors were similarly without effect, but they were able to demonstrate an 675
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inhibitory activity associated with the mitochondrial pellet, from mouse epidermis. The only claim of an exogenous interference with the process of epidermolysis is that EDTA will remove a necessary metal-ion from ETA, although ETB is not a metallo-toxin (KoNDO et al., 1976; SAKURAI and KONDO, 1978). In this paper we report a systematic search, using the in vitro assay, for inhibitors of ET.
MATERIALS AND METHODS Purification o f E T Samples of ETA and ETB were purified from standard strains by isoelectric focusing and chromatography on Sephadex G-50 S in 0.01 M NH4HCO3 (DE AZAVEDO et al., 1988). On a larger scale, ETA was purified from culture filtrates of an overproducing strain of S. aureus (O'TOOLE, P. W., Ph.D thesis, University of Dublin, 1987) by (NH4)2SO4 fractionation and Sephadex G-75 S chromatography in 0.01 M NH,HCO3 (LOCKHART, B. P., unpublished). All the preparations used were homogeneous by polyacrylamide gel electrophoresis (LAEMMLI, 1970). The toxin was stored as a lyophilized powder and assayed in solution by spectrophotometry at 280 nm (BA1LEY et al., 1982).
lnhibitors Amiloride, bumetanide and furosemide were a kind gift from Dr M. J. RYAN, University College Dublin; phytohemagglutinin and pokeweed mitogen solutions were from Gibco Ltd., Paisley, U.K.; ~:macroglobulin was from Boehringer Mannheim, Lewes, U.K.; Chelex-100 was from Bio-Rad Laboratories, Watford, U.K.; all other inhibitors were from Sigma Chem. Co., Poole, U.K.
In vitro assay Whole thickness mouse skin (Q strain, 1-3 days old) was scraped with a blunt scalpel to remove subcutaneous fat. Circular discs (5 mm 2) were cut using a leather punch and stored briefly on ice until required. Assays were started by addition of two skin punches to 200 #1 of phosphate-buffered saline (PBS), pH 7.4, containing a given amount of ET (usually 10 pg of ETA). After incubation, usually at 37°C, for a given time the effect of the toxin was assessed by attempting removal of the superficial layers (corneal and granular) of the epidermis using fine forceps. When the effect of pH was tested, the phosphate content of the medium was increased and NaC1 reduced to retain isotonicity, and the amount of tissue added to the medium was halved, in order to avoid gross pH changes in solutions not at neutral pH. The effect of temperature was assessed after separate preincubation of skin and toxin for 15 min at the appropriate temperature. To examine the effect of exogenous substances, two sorts of experiments were carried out, each in duplicate. All substances were tested by a procedure in which skin plus test compound were pre-incubated at 37°C for 30 min before addition of ET (10 #g). The assay was then allowed to proceed for 30 min and the tissue discs were examined. In addition, some compounds were tested after preincubation of ET plus compound. The scoring systems used was as follows: ( + +), the upper epidermis separated easily from the underlying tissue of the disc; (+), separation was achieved with difficulty; ( + / - ) , separation could only be achieved at the edges of the disc; ( - ) no separation possible. Skin samples were tested once and then discarded. A system of positive and negative controls was incorporated with each assay set. The positive control contained 10/~g of ET and was tested under standard conditions. Negative controls (no toxin added) were used to test that assay conditions or exogenous additives did not compromise the integrity of the tissue over the period of assay. In all of the reported exogenous substance tests, the negative controls scored ( - ) after the 30 min assay period.
Metal ion analysis ET samples (0.2-2.5 mg/ml) in 0.01 M NH4HCO3 were analysed for zinc, copper, cobalt and magnesium using Perkin Elmer 372 and International Laboratories 257 atomic absorption spectrophotometers. Qualitative analysis of protein samples (approx. 50 mg) as a dry powder was carried out on a Link Systems Meca 10--44 automatic X-ray fluorescence spectrometer, equipped with a silver target.
Light microscopy Skin was fixed overnight at 20°C in a 4% (v/v) solution of formaldehyde in 75 mM sodium phosphate, pH 7.4, dehydrated in ethanol and embedded in paraffin wax. Transverse sections (5/tin) were cut with a rotary microtome, stained with haematoxylin and eosin, and examined in a Zeiss microscope.
Activity of Epidermolytic Toxin
677
RESULTS
Properties of the in vitro assay system The response of the skin punch assay to varying doses of ETA is summarized in Table 1. As the dose decreased the time required for epidermolysis increased. The speed of the response can vary with the age of the animal tissue but the test was always sensitive to epidermolytic toxin at the levels shown in Table 1. In more than 10 tests in the range of toxin dosage 0.5-10 #g, it was always observed that a two-fold difference in toxin quantity produced an observable difference in speed of response. On the basis of the nonparametric sign test (SNEDECOR and COO-mAN, 1967), this corresponds to greater than 99% confidence that the two-fold difference can be measured. The effect at 0.1 #g of toxin was also reproduced so that there is greater than 95% confidence that this amount could be detected in 2 hr. The minimum effective dose at 2 hr was not estimated more exactly. For comparative purposes, assays were carried out under standard conditions, in PBS at 37°C with 10 #g of ET in sample and control. An indistinguishable positive effect, ( + + ) at 30 min, corresponds to at least 5 #g (P = 0.01) of ET activity in the sample. A negative effect was followed to a time of at least 1 hr and corresponds to less than 0.1 #g of ET activity (P = 0.05) in the sample. ETB (10 #g) was indistinguishably positive, when compared with an equal quantity of ETA, in the test. ETA was active over a broad temperature range (0-37°C), even at the lowest temperature (Table 2). A 10°C drop in temperature approximately doubled the TABLE 1. In vitro DOSE--RESPONSE OF SKIN TO EP1DERMOLYTIC TOXIN A
Assay results (min)
Toxin ~g/tube)
15
30 .
60
0.0
.
0.1
--
--
.
+/--
.
0.5
--
--
+
1.0
-
+/-
+
5.0 lO.O
+
+ ++
++ ++
90
120
+
++ ++ ++ ++ ++
.
++ ++ ++ ++
T h e scoring system indicates: ( - ) , no separation possible; ( + / - ) , separation only achieved at the edge of the disc; (+), complete separation achieved with difficulty; ( + + ) , upper epidermis readily peeled away from the underlying tissue.
TABLE 2. TEMPERATURE-DEPENDENCE OF /~ vitro EPIDERMOLY$1S BY E T A
Assay results* (hr)
Temperature (°C)
1
2
o
-
7 15 20 27 37
-+/++ Each
3
4
-
-
+/-
+/++ ++
+/+ ++ ++ ++
+ ++ ++ ++ ++
5
6
+
++ ++ ++ ++ ++ ++
++ ++ ++ ++ ++
assay contained 10 #g of ETA. Negative controls scored ( - ) at all times up
to 6 hr. *See T a b l e 1
for explanation.
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T.P. SMITH and C. J. BAILEY
time required for complete epidermolysis. The effect of pH was studied at 37°C in the presence of 10/tg ETA. In the pH range 3.8-8.7 (final pH value), epidermolysis occurred exactly as at pH 7.4. Beyond this range, the skin samples began to break up in the absence of toxin, probably because of splitting at the dermal-epidermal junction.
Effects of exogenous substances A large number of substances were tested for their ability to perturb the course of epidermolysis. The respiratory inhibitors fluoride, arsenate, iodoacetate, azide, cyanide and 2,4 dinitrophenol (1 mM of each) were preincubated with the tissue for 30 rain. None of these inhibitors blocked epidermolysis or caused any apparent change in its rate, under the standard conditions. Inhibitors of microtubule assembly (colchicine, l mM), ion transport (ouabain, 1 #M; amiloride, 0.1 mM; bumetanide, 0.1 mM; furosemide, 1 mM) and lysosomal systems (ammonium chloride, 10 mM; chloroquine, 10 mM) were ineffective under standard conditions. To test whether the toxin required a carbohydratedependent uptake system, the lectins conconavalin A (10/~M), pokewood mitogen (1:20 dilution) and phytohaemagglutinin (1:20 dilution) and the sugars D-glucose, N-acetyl-Dglucosamine, D-galactose, N-acetyl-D-galactosamine, D-mannose, D-fructose, D-sorbitol (all at 100mM) of D-glucosamine, D-galactosamine, N-acetylneuraminic acid (all at 50 mM) were each incorporated into the assay system without any effect on the progress of epidermolysis. Inhibitors of the four major classes of proteolytic enzymes were tested. Leupeptin (1 mM); aprotinin (0.5mg/ml); soyabean trypsin inhibitor (0.5mg/ml); phenylmethane sulphonyl fluoride (1 mM); pepstatin (0.1 mM); phosphoramidon (0.1 mg/ml) and ~2macroglobulin (0.5 mg/ml) had no effect on epidermolysis under standard conditions. But in the presence of EDTA (10 mM), a negative response was observed at 30 min incubation under standard conditions. As the two other metalloprotease inhibitors, phosphoramidon and ~2-macroglobulin, had no effect, it seemed possible that EDTA might inhibit epidermolysis by removal of an essential metal ion which is not necessarily associated with a protease.
Effect of chelators EDTA at 10 mM blocked epidermolysis by ETA after pre-incubation of skin plus chelator or pre-incubation of toxin plus chelator. EDTA (10 mM) also inhibited ETB after pre-incubation of skin plus chelator. However the result is complicated by the observation that EDTA alone caused skin-splitting after 2 hr in the assay system. It was only possible to assay for inhibition by EDTA because the action of the toxin is apparent after 30 min. A quantitative distinction between the skin-splitting activities was made by histological examination. Epidermolytic toxin split the skin at the granular layer (Fig. la). Toxin plus EDTA produced a split at the dermal-epidermal boundary (Fig. l b) and it was shown that EDTA alone gave the same cleavage. To confirm that chelation can prevent intoxication, we searched for chelators which did not cause epidermolysis. The results are shown in Table 3 together with representative data for the metal-ion affinities. EGTA (10 mM) prevented epidermolysis by ETA after pre-incubation of chelator with skin or toxin (Fig. lc), and by ETB, after pre-incubation of chelator with skin, and by itself caused no morphological or histological effect on the epidermis. Three other chelators, 1,10-phenanthroline, citrate and oxalate prevented epidermolysis after pre-incubation with skin, EGTA, oxalate and citrate blocked epiderm-
FIG. 1. HISTOLOGYOF NEONATALMOUSESKIN. Tissue sections were processed for microscopy after incubation in phosphate-buffered saline with: (a) ETA (50#g/ml) after 30min; (b) ETA (50pg/ml)+EDTA (10mM), after 2hr; (c) ETA (50 #g/ml) + EGTA (10 mM), after 2 hr. The positions of the stratum corneum (SC); stratum granulosum (SG) and dermis (D) are indicated. In (a) the tissue has split intra-epidermally, just below the SG. In (b) the split is at the junction of the dermis and epidermis, no splitting was observed after 30 min. In (c) no split occurred. The scale bar indicates 10 #m.
680
T. P. S M I T H and C. J. BAILEY TABLE 3. EFFECT OF CHELATORSON it/ vitro EPIDERMOLYS1S
Chelator (mM) E D T A (10) E G T A (I0) 1, 10-Phenanthroline (10) 2,2'-Dipyridyl (I0) Diethyldithiocarbamate (10) Chelex-100 (250 mg/ml) Citrate (50) Oxalate (50) Malonate (50) Succinate (50)
Stability constant (log KApp) Ca 2+ Zn 2÷ (stoichiometry) 7.7 7.5 0.5 0.5 (Mg 2÷) n.a. n.a. 3.6 3.0 1.5 1.2
13.5 (1) 9.4 (I) 17 (3) 13.5 (3) 11.5 (2) n.a. 5.0 (1) 8.2 (3) 4.5 (2) 1.8 (1)
Assay result* 30 min + + + + + + + + + +
The log KApp values are apparent values recalculated at pH 7.4 from the data o f DAWSON et al. (1986). The ratio o f complexed to free metal ion, in the presence of excess chelator, m a y be calculated from the formula: log [Complexed/Free] = log KApp+ (Stoichiometry)X log [Ligand]. The stoichiometry o f the complexes is the molar ratio of chelator to metal ion in the dominant complex at excess chelator. The stoichiometry of all Ca 2+ complexes is 1, but varies as indicated for Zn 2÷ complexes. n.a. not available. *See Table 1 for additional information.
olysis for up to 2hr. 1,10-Phenanthroline caused a rate change so that, although a negative result was obtained at 30 min (Table 3), epidermolysis was apparent ( + / - ) at 60 min and complete (+ +) at 90 min. This chelator on its own did not produce any skinsplitting reaction at times up to 90 min. Three more chelators with high affinities for metal ions (Table 3), 2,2'-dipyridyl, diethyldithiocarbamate and Chelex-100 did not inhibit the toxin. Of these, the resin is a solid which cannot be in intimate contact with the inner layers of the skin and it may be that the skin site is also inaccessible to the other two inactive chelators. Since two of the inhibitors are carboxylic acids, others of that class were tested. Succinate and malonate are without substantial metal-ion binding ability (Table 3) and did not inhibit the toxin. On the basis of the wide variety of different chemical substances which inhibit epidermolysis by ETA, it seems reasonable to conclude that the metal-chelating ability is the cause of inhibition. The test protocol does not indicate whether the required metal ion is sequestered from the skin or the toxin or both. Therefore experiments were carried out in which skin or toxin samples were separated from the chelators after pre-incubation but before assay. Skin punches were incubated in 5 mM EGTA in PBS (200/~1) for 30 min at 37°C, removed, washed in PBS (20 ml) for 1 min and blotted before the test. In the assay system these samples remained entirely unresponsive to 10 #g of ETA for up to 2 hr. To test for the existence of essential metal ions in the toxin, samples of ET were assayed and analysed before and after EDTA treatment. Samples of ETA and ETB were each incubated with 10 mM EDTA and after 2 hr incubation the toxins were separated from the chelator by Sephadex chromatography. They were found to be as active as untreated toxin: epidermolysis (+ +) occurred after 30 min incubation with 10/ag of either ETA or ETB. Samples of ET were also assayed for the presence of metal ions. Two samples of ETA, isolated by Sephadex chromatography without EDTA pre-treatment, gave 0.3 and 0.7 gram-Atom (gA) per mole protein of copper plus 0.7(2) gA per mole of zinc, together with traces of magnesium; similar samples of ETB contained 0.2 and 0.4 gA per mole of copper and 0,1 and 0.3gA of zinc. An ETA sample was adjusted to 10mM EDTA,
Activity of Epidermolytic Toxin
681
dialysed for three days at 2°C, then isolated by Sephadex chromatography. Zinc and copper was undeteetable in this ETA ( < 0.01 gA of each metal per mole of protein). A large quantity of freeze-dried toxin from the latter preparation was examined for the presence of metal ions using X-ray fluorescence techniques. No X-ray emissions in the region 4-40 keV (covering the range of atomic numbers for all the transition metals) in comparison with pure hen-egg lysozome, a metal-free protein, could be detected. The sensitivity of this technique is very great, although it may vary a little between elements. For zinc, control experiments showed that a level of 0.02 gA per mole was readily detectable. It is concluded that the evidence is consistent with the occurrence of a required metal ion in the skin, that EDTA does not remove essential metal ions from ETA or ETB, and that ETA does not contain any essential ions, although ETA as usually isolated may contain adventitious, non-essential metal ions, at levels approaching 1 gA per mole protein. Attempts were made to restore sensitivity to skin rendered resistant by EGTA pretreatment. After addition of 0.1 mM of any of Ca 2+, Mg 2+, Zn 2+, Co 2+, Fe 2+ (none of which inhibited even at 6 mM), no epidermolysis was evident when the skin was challenged with 10#g ETA at 37°C for 60min. However it was possible to protect the epidermolytic reaction from EGTA inhibition by cation swamping. In the experiments, 5 mM EGTA and 6 mM cation were briefly pre-incubated before the addition of ETA and skin punches. The transition metals, Mn 2÷, Cu 2+, Fe 2+, Zn 2+, and Co 2+ prevented the EGTA-dependent inhibition of epidermolysis, but Ca :+ and Mg 2+ did not. DISCUSSION
The in vitro assay utilized in this report provided a rapid and sensitive end-point assay system. The convenient limit of 0.1 pg at 0.5 #g/ml (2 x 10-SM) could readily be lowered by extending the assay period and by accepting detection at a low level of confidence. In the in vitro assay the lowest confirmed limit is 0.1 #g at 2-4 #g/ml. (WUEPPERet al., 1975; NISHIOKA et al., 1981). The main advantage of the in vitro assay is not an increase in sensitivity but the ability to vary the assay conditions and to test the effects of exogenous additives. By scoring the ease of epidermolysis as a function of dose and incubation time, it is possible to put the assay on a semi-quantitative basis that makes it convenient to search for inhibitory effects. The insensitivity of epidermolysis to a range of variations and supplementations extends the earlier reports (ELIAS et al., 1977; NISHIOKAet al., 1981) and has important implications for the possible mechanism of epidermolytic toxin. For a large proportion of protein toxins, intoxication requires uptake into eukaryotic cells via receptor-mediated endocytosis (MIDDLEBROOKand KOHN, 1981). Such uptake systems involve complex membrane transport events and are expected to be energydependent, temperature-sensitive, pH-dependent and also susceptible to inhibition by lysosomatropic agents and high concentrations of analogues of receptor molecules (typically carbohydrate). The individual properties of the toxins vary so that not all systems are susceptible to all the factors listed. For example, ricin and abrin show a measurable uptake at 5°C, there is a high temperature-dependence, but no critical temperature, of uptake. Lysosomatropic substances do not prevent inhibition at neutral pH; but inhibitors of glycolysis and respiration completely protect cells (reviewed by OLSNESand S^NDVIG, 1983). In the case of diptheria toxin (PAPPENI-mIMER, 1977) intoxication is inhibited at low temperatures, by ammonium chloride and by fluoride but not by other inhibitors of energy metabolism or by uncoupling agents.
682
T.P. SMITH and C. J. BAILEY
The uniformly-negative effects of so m a n y inhibitors of receptor-mediated endocytosis, suggest that epidermolytic toxin does not enter cells by such a mechanism. The strength of this conclusion is limited only by the nature of the skin test assay. It may be that epidermolysis is an effect which occurs at saturation levels of toxin where the effects of inhibition could be less obvious. Positive effects were observed with a group of chelators suggesting that chelation is the unifying property of this group of inhibitors. One of these, E D T A , has been reported to inhibit ETA by removal of a metal ion which could be replaced by Zn 2+, Co 2+, Mg 2÷ and Mn 2+ (KONDO et al., 1976; SAKURAI and KONDO, 1978). In samples of the toxin purified from culture medium, it was reported that copper was present in the native toxin at stoichiometric amounts. Although our experiments confirm that purified ETA routinely contains copper (and zinc) in amounts close to a stoichiometric ratio, they also show that the metal-ions can be removed by E D T A , after which the toxin is active and metal-free. The interpretation of the original experiments of KONDO and collaborators depends critically on the assumption that dialysis has reduced the E D T A concentration from 200 mM to less than 2.5 m M (DIMONO et al., 1976) in order that the chelator does not interfere with the assay. The results in the present paper are consistent with the theory that ETA and ETB have the same binding properties and mechanism, as is expected for two proteins showing a high degree of amino acid sequence homology (O'ToOLE and FOSTER, 1987; LEE et al., 1987). Our observation that E D T A splits the skin at the dermal-epidermal junction is in agreement with previous observations (SCALETTA and MACCALLUM, 1972; DIMOND et al., 1976). Histochemical evidence has been produced to show that the EDTA-separated epidermis may be subsequently split by ET (ELIAS et al., 1976), but there is no gross morphological evidence to support this claim. The major result of the present work is that epidermolysis requires a metal ion which is provided by the tissue, not the toxin. The identity of the metal cannot be established exactly, but the experiments on protective effects imply that the cation must have a higher affinity for E G T A than do Ca 2+ and Mg2÷; that is, a transition metal rather than an alkaline earth metal. Since metal ions are implicated in m a n y biochemical mechanisms it remains uncertain whether the ion is necessary for transport to the putative intracellular target which is profilaggrin (SMITH and BAILEY, 1986; SMITH et aL, 1987) or for some other event. It is, however, known (LOCKHART, unpublished) that E D T A does not interfere with the binding of ET to profilaggrin on western blots. Acknowledgements--We thank DAVIDDOFF, TERRYWILLIAMSand ALBERTMcQUAID for assistance with the metal ion analyses and PAT HARTIGANfor advice on histology. The research was supported by grants from the Irish Medical Research Council.
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B from Staphylococcus aureus. Biochem. J. 203, 775-778. DAWSON,R. M. C., ELLIOT,D. C., ELLIOTT,W. H. and HONES,K. M. (1986) Stability constants for metal ion complexes. In: Data for Biochemical Research (3rd edn), pp 399-415. Oxford: Clarendon Press. DEAZAVEDO, J., BAILEY, C. J. and ARBUTBNOTT,J. P. (1988) Purification of epidermolytic toxins from Staphylococcus aureus. Methods Enzymol. 165, 32-37. DIMOND,R. L., ERICKSON,K. L. and WUEPPER,K. D. (1976) The role of divalent cations in epidermolysis. Brit. J. DermatoL 95, 25-34. ELIAS,P. M., FRITSCn,P., TAPPE1NER,G., MITTERMAYER,H. and WOLFE,K. (1974) Experimental staphylococcal toxic epidermal necrolysis (TEN) in adult humans and mice. J. Lab. Clin. Med. 84, 414~23.
Activity of Epidermolytic Toxin
683
ELIAS, P. M., FRITSCH,P. and MITTERMAYER,H. (1976) Staphylococcal toxic epidermal necrolysis: species and tissue susceptibility and resistance. J. Invest. Dermatol. 66, 80-89. ELIAS, P. M., FRITSCH, P. and EPSTE,N, E. H. (1977) Staphylococcal scalded skin syndrome: clinical features, pathogenesis, and recent microbiological and biochemical developments. Arch. Dermatol. 113, 207-219. FREER, J. H. and ARnUTHNOTI',J. P. (1983) Toxins of Staphylococcus aureus Pharmacol. Therapeut. 19, 55-106. JOHNSON,A. D., SPERO,L., CADES,J. S. and DE CIcco, B. T. (1979) Purification and characterization of different types of exfoliative toxin from Staphylococcus aureus. Infect. lmmun. 24, 679-684. KONDO, I., SAKURAI, S. and SAg1, Y. (1976) Staphylococcal exfoliatin A and B. In: Staphylococci and Staphylococcal Diseases, pp. 489-498 (JEI.ZASZEWlCZ,J., Ed.) Stuttgart: Gustav Fischer. LAEMMLI,U. K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage 1"4. Nature 227, 680-685. LEE, C. Y., SCHMIDT, J. J., JOHtqSON-WIh~GAR, A. D., SPERO, L. and IANOOt.O, J. J. (1987) Sequence determination and comparison of the exfoliative toxin A and B from genes from Staphylococcus aureus. J. Bacteriol. 169, 3904-3909. LILLmRIOGE, C. B., MELISH, M. E. and GLASC,OW, L. A. (1972) Site of action of exfoliative toxin in the staphylococcal scalded skin syndrome. Pediatrics 50, 728-738. MCCALLUM,H. M. (1972) Action of staphylococcal epidermolytic toxin on mouse skin in organ culture. Br. J. Dermatol. 86 (Suppl 8), 40-41. MELlSH, M. E., GLASGOW, L. A. and TuR~a~R, M. D. (1970) Staphylococcal scalded skin syndrome (SSSS). Experimental model and isolation of a new exfoliative toxin (ET). Pediatr. Res. 4, 378-379. MIDDLEnROOK, J. L. and KOHN, L. D. (1981) Receptor-mediated binding and internalisation of toxins and hormones. New York, London: Academic Press. NISH1OKA,K., KATAYAMA,I. and SANO,S. (I 981) Possible binding of epidermolytic toxin to a subcellular fraction of the epidermis. J. Dermatol. 8, 7-12. OLSh'ES, S. and SANDWG, K. (1983) Entry of toxic proteins into cells In: Receptor-mediated endocytosis, pp. 187-236 (CuATRECASAS,P. and Ro'm, T., Eds). London: Chapman and Hall. O'TOOLE, P. W. and FOSTER,T. J. (1987) Nucleotide sequence of the epidermolytic toxin serotype A gene of Staphylococcus aureus. J. Bacteriol. 169, 3910-3915. PAPPE~a-IEIMER,A. W. (1977) Diphtheria Toxin. Ann. Rev. Biochem. 46, 69-94. SAgURAI,S. and KONDO, I. (1978) Characterisation of staphylococcal exfoliatin A as a metallotoxin with special reference to determination of the contained metal by radioactivation analysis. Jpn. J. Med. Sci. Biol. 31, 208-211. SCALETTA,L. S. and MACCULLUM,D. K. (1972) A fine structural study of divalent cation-mediated epithelial union with connective tissue in human oral mucosa. Amer. J. Anat. 133, 431-454. SMm~, T. P. and BAILEY,C. J. (1986) Epidermolytic toxin from Staphylococcus aureus binds to filaggrins. FEBS Left. 194, 309-312. SMITH, T. P., JOHN, D. A. and BAILEY,C. J. (1987) The binding of epidermolytic toxin from Staphylococcus aureus to mouse epidermal tissue. Histochem. J. 19, 137-149. SNEDECOR, G. W. and COCHRAtq,W. G. (1967) Statistical Methods, 6th edn, pp. 125-128. Ames: Iowa State University Press. WUEPPER, K. D.. DIMOND,R. L. and KNUTSON,D. D. (1975) Studies on the mechanism of epidermal injury by a staphylococcal epidermolytic toxin. J. Invest. Dermatol. 65, 191-200.