Molecular cloning and expression of the epidermolytic toxin A gene of Staphylococcus aureus

Microbial

Pathogenesis

1986, ‘I : 583-594

Molecular cloning and expression epidermolytic toxin A gene of Staphylococcus Paul W. O’Toole Department (Received

aureus and Timothy

of Microbiology, March

of the

Moyne

18, 1986; accepted

J. Foster lnsitute,

Trinity College,

Dublin

2, Ireland

April 30,1986)

O’Toole, P. W. (Dept. of Microbiology, Moyne Institute, Trinity College, Dublin 2, Ireland) and T. J. Foster. Molecular cloning and expression of the epidermolytic toxin A gene of Staphylococcus aureus. Microbial Pathogenesis, 1986; 1: 583-594. The gene coding for serotype A of epidermolytic (exfoliative) toxin has been cloned from Staphylococcus aureus in Escherichia coli phage 1 and plasmid vectors. The coding sequence for eta was localised by subcloning and transposon Tn5 mutagenesis experiments. The eta gene was probably expressed from its natural promoter in E. coli. The protein synthesised in E. coli was located predominantly in the periplasm. It was immunochemically indistinguishable from the toxin purified from S. aureus culture supernatants and had the same molecular weight. Furthermore, subcutaneous injection of this material caused epidermal splitting (the Nikolsky reaction) showing that it was biologically active. An eta shuttle plasmid was transformed into protoplasts of S. aureus. The level of expression of toxin in strain 8325-4 was shown to be dependent on the integrity of the agr gene which is known to be required for the expression of several exoproteins. Key words: Staphylococcus aureus; regulation; scalded skin syndrome.

epidermolytic

toxin

A; molecular

cloning;

exoprotein

Introduction Certain strains of Staphylococcus aureus cause a spectrum of dermatological lesions called the Staphylococcal Scalded Skin Syndrome (SSSS) which are characterised by intraepidermal splitting in the plane of the stratum granulosum. Widespread exfoliation is preceded by erythema and localised bullae (for reviews see references 1 and 2). The identification of the toxins responsible for these symptoms was facilitated by the observation that SSSS-associated strains produced a response very similar to the human syndrome when injected subcutaneously into newborn mice (the Nikolsky sign3). These organisms synthesised an extracellular toxic protein called epidermolytic toxin or exfoliative toxin (ET), the purified forms of which also produced a Nikolsky sign.4,5 Two serologically distinct toxins (ETA and ETs) have been described.6,7f8 They also differ in molecular mass (30 and 29.5 kD, respectively), pl values and heat stability.6*s Recently, highly purified ET was shown to bind specifically to filaggrin proteins contained in the keratohylin granules in the stratum granu/osum.‘” This is thought to prevent binding of filaggrin to keratin (T. Smith and C. Bailey, personal communication). A recent epidemiological study suggested that ETA’ strains involved in the production clinical

of neonatal environment,”

0882-401

O/86/060583+1

pustulosis have the ability to colonise inanimate material but the factor(s) responsible for this are unknown. 2 $03.00/O

6

1986

Academic

Press

Inc.

(London)

in the

Ltd

584

P. W. O’Toole

and T. J. Foster

Several groups have set out to define the genetic basis of ET production. Expression of ETs was shown to be plasmid associated because curing of a 42 kb plasmid was correlated with loss of toxin production. 12r13However it is not clear if ET8 is actually plasmid-encoded because plasmid transfer has only been achieved into ETB- strains that had previously been cured of the same type of plasmid.‘4,‘5 The production of ET, was not correlated with a plasmid and the gene is thus assumed to be chromosomal although attempts to establish linkage with known chromosomal markers have so far failed.16 In order to facilitate a molecular genetic analysis of ET production we have cloned the gene coding for serotype A of the toxin (eta) from a SSSS-associated strain of S. aureus. The expression of the toxin by phage 1 and plasmid vectors in E. co/i is described. A shuttle plasmid carrying eta was constructed and transferred into S. aureus. The level of ET in culture supernatants of S. aureus was shown to be dependent on the integrity of a locus called agr which has previously been shown to be required for the expression of several different exo-proteins. Results Molecular cloning of the epidermolytic toxin A gene Genomic DNA from S. aureus strain TC16 was cleaved partially with EcoRl and ligated with EcoRI-cut IL47.1 DNA. After packaging in vitro recombinant phages were selected by plating on E. co/i WL95. The chimaeric phages were harvested and about 5000 plaques formed by plating on strain C600 were screened by immunoblotting with specific anti-ETA serum. Positively reacting plaques were picked, replated and tested for their reaction with pre-immune rabbit serum and with anti-ET, serum preadsorbed with purified toxin in order to discriminate between protein A clones and ETA clones. One phage (leta) which failed to react in both tests was kept for further study. Analysis of leta-specified proteins In order to determine if the antigen expressed by Leta corresponded to serotype A epidermolytic toxin, proteins present in a lysate of the phage were concentrated and analysed by Western blotting and by immunodiffusion. Leta produced a polypeptide which co-migrated with the native toxin in SDS-polyacrylamide gels (Fig. 1, tracks D and E). In addition it specified an immunoreactive protein of approximately 2 kD greater molecular mass which was presumed to be the unprocessed precursor of the normally extracellular molecule. Gel immonodiffusion analysis of the Aeta lysate was performed in order to compare the phage-specified antigen(s) with purified ETA. The reaction of identity between the aeta-specified protein and the purified toxin (Fig. 2) shows that the antigens are immunochemically identical. Subcloning eta in plasmid vectors EcoRI-cleaved leta DNA was ligated with pACYC184 DNA cut with the same enzyme and transformed into E. co/i C600. Colonies were screened for ETA production by colony immunoblotting. The 8.3 kb EcoRl fragment of lleta (Fig. 3(a)) was shown to be sufficient to confer the ETA+ phenotype. One such plasmid pETA (Fig. 3(b)) was studied further and a 3.9 kb EcoRI-HindIll fragment was subcloned into pBR322 to give pETA (Fig. 3(c)). The 3.9 kb fragment was also subcloned into EcoRIHindIll-cleaved pUCl8 and pUCl9 vectors to form pETA and pETA4, respectively. These plasmids expressed similar levels of ETA in whole cell lysates suggesting that the

Cloning

30

epidermolytic

toxin

A

585

kl

Fig. 1. Western immunoblotting analysis of ETA expressed by recombinant phage and plasmids in Escherichia co/i. Proteins were separated by polyacrylamide gel electrophoresis (12.5% acrylamide), transferred to nitrocellulose, and incubated with antibodies as described in Materials and Methods. Track A, culture supernatant of S. aureus TC16, 5 pg of protein; track B, periplasmic extract of E. co/i C600 (pETAl), 20 pg protein; tracks C and E purified ET,, 0.5 pg; track D, concentrated lysate of lefa prepared as described in Materials and Methods.

promoter for the eta gene is functional independent of vector plasmid promoters.

in E. co/i and that

Mapping eta by deletion and transposon mutagenesis Several restriction fragments from within the 3.9 kb EcoRI-Hindlll were subcloned into the appropriately cleaved vector plasmids (Fig. 3(c)). None of these expressed ET,.

expression

of ET is

fragment of pETA pUCl8 and pUCl9

Fig. 2. lmmunodiffusion analysis of proteins specified by recombinant phage and plasmids in Escherichia co/i. The central well (X) contains peripheral wells contain antigen preparations; A and E, purified ET, (0.5 pg); B ieta lysate of E. co/i C600 (50 pg protein); C, concentrated aureus TC16 (5 pg protein); D, periplasmic extract of E. co/i C600 (pETA ) (50 pg protein); F, 1L47.1 lysate of E. co/i C600 (50 pg protein).

anti-ET,, antiserum. culture supernatant

The of S.

Cloning

epidermolytic

KK ia)

Aeta

toxin

A

EE

587

s EKE /

t

(XL4711

(b)

pETA

E C

zb

HC

E

H H C

(pAcYC184)

(c)

'/HP

C C,HE

7 Hn Hn H

pETA (PBR322) 2kb'

Cd)

pETAjL$M

2k~ 135 24

6

Fig. 3. Restriction map of recombinant phages and plasmids carrying eta. The open boxes represent vector sequences and the thin horizontal lines the cloned S. aureus sequences. Part A is a map of iela while parts 6, C and D show the recombinant plasmids pETA1, pETA and pETA3, respectively. Note the different scales in each map. The horizontal bars in part C indicate fragments subcloned from pETA into pUC18 and pUCl9 to generate ETA- recombinants. The vertical arrows underneath the map of pETA in part D are the positions of Tn5 insertions which caused an ETA- phenotype. Mutant number 5 was used to generate the Hincll DNA probe used in hybridization experiments. The letters are abbreviations for restriction endonuclease cleavage sites as follows: K, Kpnl; E, EcoRI; S, Sacl; H, Hindill; C, C/al; B, BstEll; A, Accl; Hp, Hpal; Hn, Hindll.

The pUC18eta plasmid pETA was chosen for transposon Tn5 mutagenesis because the production of toxin was more obvious in colony immunoblotting experiments than by the lower copy number pBR322 derivative. Six independent ETAmutants were identified by colony immunoblotting and their ETA- phenotype confirmed by Western immunoblotting of whole cell extracts fractionated by SDSpolyacrylamide gel electrophoresis. The Tn5 insertions covered a 790 bp region spanning the central Hindll site in pETA (Fig. 3(d)) and indicate the location of the coding sequence for the toxin in the cloned DNA. Analysis

of pETA-specified

polypeptides

In order to further characterise the product of the cloned eta gene, a periplasmic extract of E. cob C600 carrying pETA was concentrated and analysed by Western immunoblotting and by gel immunodiffusion. A single polypeptide having the same molecular weight as the native toxin reacted with anti-ET, serum in a Western immunoblotting experiment (Fig. 1, track B). The larger immunoreactive species present in Leta lysates was absent. Also, a reaction of identity occurred between this material and the toxin purified from culture supernatants of S. aureus (Fig. 2). There was no evidence that the toxin was degraded or inactivated during the periplasmic extraction procedure, the only breakdown product detected by Western immunoblotting had the same mass as the freeze-thaw product characteristic of the toxin isolated from S. aureus culture supernatants. About 25 pg toxin was extracted from 2x1O’o cells. The concentrated periplasmic extract which contained about 0.5 pg toxin as estimated by Western immunoblotting and immunodiffusion, when injected subcutaneously into neonatal mice yielded a positive Nikolsky reaction in 3.5 h (Fig. 4)

588

P. W. O’Toole

and T. J. Foster

3

Cloning

epidermolytic

toxin

A

589

showing that the polypeptide specified in E. colihas the biological activity characteristic of epidermolytic toxin. Thus the recombinant phage and plasmids carrying eta in E. colispecify a polypeptide which is immunochemically indistinguishable from the toxin purified from culture supernatants of toxinigenic S. aureus. Construction

of a shuttle

plasmid

and expression

of eta in Staphylococcus

aureus

In order to investigate the factors which regulate the expression of epidermolytic toxin in S. aureus, a shuttle plasmid was constructed from pETA by introducing into its unique Hindlll site (Fig. 3) a 3.1 kb Hindlll fragment from pCW59. This carries an origin of replication which is functional in S. aureus and a chloramphenicol resistance gene that is expressed both in E. co/i and in S. aureus. The shuttle plasmid pETA was selected in E. co/i and then transformed into protoplasts of S. aureus RN4220, ISP546 and 8325-4. Southern hybridisation experiments using an intragenic eta probe showed that strain 8325-4 lacks DNA sequences homologous with the eta gene. Furthermore toxin has not been detected in culture supernatants of this strain (unpublished data). Strain ISP546 has a transposon Tn557 insertion in the agr gene” which is involved in the regulation of expression of several exoprotein genes.18 Strain RN4220 is also thought to have an agr mutation.lg The levels of ETA in 24-h culture supernatants of the pETA5-carrying strains were measured by titration against anti-ETA serum by rocket immunoelectrophoresis. The agr+ strain 8325-4 expressed an extracellular ETA concentration of 3375 ,ug/ml while ISP546 and RN4220 had 35-fold lower and lo-fold lower titres respectively. This shows that the eta gene is regulated by the agr system. In order to demonstrate that differences in plasmid copy number were not responsible for the varying levels of ET* production the pETA plasmid-chromosome ratio was measured densitometrically and shown to be the same in the three strains tested (data not shown).

Discussion Several lines of evidence indicate that the gene which codes for epidermolytic toxin A has been cloned intact. The product which is synthesised in E, co/i by the phage A and plasmid recombinants has the same molecular weight and is immunochemically identical to the toxin purified from S. aureus culture supernatants. Furthermore the plasmid-specified protein has exfoliative activity because it stimulates the Nikolsky reaction in neonatal mice (Fig. 4). In the Aeta lysates two polypeptides reacted with anti-ET, serum in Western immunoblotting experiments. One had the same mass as the secreted staphylococcal protein while the second was about 2 kD larger. The latter protein is probably the unprocessed precursor of the protein which is normally secreted by S. aureus and which is predominantly periplasmic in intact E. co/i cells. Thus the secretory apparatus of E. co/i can recognise the signal sequence of ET, but in leta-infected cells this process cannot occur normally possibly due to the perturbation of the cytoplasmic membrane. The natural promoter for the eta gene seems to have been cloned intact and is functional in E. co/i. The toxin was expressed from plasmid vectors (pBR322, pACYCl84, pUC18 and pUCl9) irrespective of the orientation of the insertion with respect to the vector promoters adjacent to the cloning sites. Other S. aureus exoprotein genes which have been cloned and whose promoters appear to function in E. co/i are

590

P. W. O’Toole

and T. J. Foster

a-haemolysin,20 TSST-1 ,*I staphylokinase,** protein A,23 and DNase.24 In contrast the promoters of the enterotoxin B25 and ET, genes25a do not function in E. co/i. Mapping analysis of the eta clone indicates that at least 790 bp of DNA is required for expression of the ETA” phenotype in E. co/i. Amino acid composition analysis has predicted that there are 230 residues in ETA8whereas molecular weight determinations’ suggest that about 270 residues will be required. This would need 810 bp of DNA. Additional sequences will be required for the secretory signal sequence and the regulatory region. This will be confirmed by DNA sequence analysis. S. aureus strain 8325-4 does not synthesise detectable ETA because it does not carry the eta gene as was shown here by Southern hybridisation experiments. However the toxin was expressed by strains 8325-4, RN4220 and ISP546 carrying the shuttle plasmid pETA5. The levels of ETA in culture supernatants were compatible with a regulatory model for exoprotein synthesis which involves the product of the agr gene acting in trans to stimulate transcription of eta. 18,1s,26This data also suggests that the expression of ETA is regulated by an agr element in its normal host and provides further evidence that the eta locus including the promoter has been cloned intact. The slightly higher levels of expression of ETA by RN4220 compared to ISP546 agrees with our previous studies on the expression of IX-, p- and &haemolysins” (and M. O’Reilly and T.J.F., unpublished data) which suggested that the former strain has a leaky mutation affecting agr while the mutation carried by ISP546 is absolute. It will be interesting to extend these studies to naturally occurring strains which produce both ETA and ETB but express one of the two serotypes at very low levels (J. de Azavedo, personal communication). Materials

and methods

Bacterial strains, plasmids and phages The bacterial strains are listed in Table 1 and the plasmids in Table 2. The replacement vector IL47.1*’ was used to clone the eta gene and to construct Aeta. Nomenclature and abbreviations The structural genes for the epidermolytic toxin serotypes A and B (ETA and ETB) are called eta and etb, respectively. The toxin-producing phenotype is referred to as ETA’.

Table 1 Strain

Bacterial strains Genotype

Staphylococcus TC16 Wild RN4220 agr

aureos type

8325-4 ISP546

agr’ agr:

Escherichia C600

coli lac thr leu thi tonA hspR hspM lac thr leu thi tonA hspR hspM (P2) Alac-pro,,,, ara thi gyrA rpo0 ara thi Alac-pro,,,, rpsL hspR (48Odlacl AM1 5)

WL95 XAcSuTBl

:Tn551

Relevant

characteristics

References

Produces ETA, Phage group II Mutant of 8325-4 capable of accepting shuttle plasmids NCTC8325 cured of prophages Derived from 8325-4

J de Azavedo 19 27 17 28

P2 lysogen

of C600

Non-suppressing mutagenesis with Host for detecting plasmids

29 host used 1467 chimaeric

for Tn5 pUC

30 Bethesda Research Laboratories

Cloning

Table 2

epidermolytic

toxin

591

A

Plasmids

Plasmid

Host

Markers

Other

pBR322 pACYCl84 pUC18

E. coli E. coli E. co/i

Ap’ Tc’ Cm’ Tc’ AP’

Cloning Cloning Cloning

puc19

E. coli

AP’

pETA

E. coli

Tc’ ETA+

pETA

E. co/i

Ap’ ETA+

pETA

E. coli

Ap’ ETA+

pETA

E. coli

Ap’ ETA+

pcw59 pETA

S. aureus Shuttle plasmid

Cm’ Tc’ Ap’ Cm’ ETA+

Cloning vector, polylinker cloning site in opposite orientation to pUC18 8.3 kb EcoRl fragment from lefa cloned in pACYCl84 3.9 kb EcoRI-Hindlll fragment from pETA cloned in pBR322 3.9 kb EcoRI-Hindlll fragment from pETA cloned in pUC18 3.9 kb EcoRI-HindIll fragment from pETA cloned in pUC19 Cloning vector in S. aureus pETA with 3 kb HindIll fragment (Cm’and replication origin) cloned into its single HindIll site

Abbreviations: toxin A.

Ap, ampicillin;

Cm, chloramphenicol;

relevant

properties

vector vector vector

Tc, tetracycline;

r, resistant;

Source

and references

31 32 Bethesda Research Laboratories Bethesda Research Laboratories This

study

This

study

This

study

This

study

33 This

study

ETA, epidermolytic

Bacteriological media, chemicals, enzymes and antibiotics Bacteria were routinely grown in L broth and L agar.34 ;1 phages were propagated in i base and top agar. 34 ET production by S. aureus was monitored by concentrating supernatants of cultures grown in Bernheimer-Schwartz medium.35 Chemicals were obtained from the Sigma Chemical Co (St Louis, Missouri, USA) or were the Analar grade from British Drug House (Poole, Dorset, UK). Ampicillin (Ap) was a gift from Beechams, chloramphenicol (Cm) and tetracycline (Tc) were purchased from Sigma. Restriction enzymes and T4-DNA ligase were obtained from the Boehringer Corporation and were used according to the manufacturer’s instructions. Antisera Anti-ET, and anti-ET, sera were a generous gift from Dr J. de Azavedo (Microbiology Department, Trinity College, Dublin). The antibodies were raised in rabbits to purified toxins. Before use in tests with recombinant plasmids or phages in E. co/i the sera were adsorbed with a concentrated lysate of E. co/i C600. Peroxidase-conjugated swine anti-rabbit globulin was obtained from Dakopatts (Glostrup, Denmark) and peroxidase-conjugated protein A was purchased from Sigma. Molecular cloning and transposon mutagenesis High molecular weight genomic DNA was purified from S. aureus strain TC16 as described.36 Phage AL47.1 was prepared by the high titre method, purified by two block CsCl gradients, and phage DNA released from capsids by formamide extraction.34 Partial EcoRl digests of TCI 6 genomic DNA were prepared by cleaving 5 pg aliquots with varying amounts of EcoRI. A sample having fragments in the IO-I 5 kb range

592

P. W. O’Toole

and T. J. Foster

was ligated with 5 pg IL47.1 DNA cleaved with the same enzyme. About 1 pg ligated DNA was mixed with packaging extracts prepared as described.37 Spi- recombinant phages were selected by plating on E. co/i WL95. These were harvested and stored at 4°C. Subcloning and restriction mapping experiments were carried out by standard procedures. 35 The use of plasmid pCW59 in constructing shuttle plasmids has been described before.‘g*3g Transposon mutagenesis was performed by infecting strain XAcSu- carrying pETA with 1467 carrying Tn5 as described previously.2g*30 DNA hybridisation was performed by the method of Southern38,40 using as a probe the 680 bp Hincll fragment of transposon insertion 5 which extends from the Hincll site in eta to the Hincll site in the inverted repeat of Tn5. (Controls showed that Tn5 itself has no homology with S. aureus DNA.) Plasmid copy number measurement Total cell DNA of S. aureus strains carrying the shuttle plasmid pETA was isolated as described.lg About 2 pg was fractionated by electrophoresis in a 0.8% agarose gel.38 A photographic print was scanned by densitometry and the ratio of plasmid : chromosome DNA was estimated. Transformation Plasmids were introduced into cells of E. co/i C600 treatment38 and into protoplasts of S. aureus.3g

made competent

by CaCI,

Plaque and colony immunoblotting 1 plaques producing ET were detected by layering 82-mm nitrocellulose discs (Schleicher and Schuell) onto phage overlay plates. The filters were incubated successively with bovine albumin (Sigma), rabbit anti-ET* serum and peroxidase conjugated swine anti-rabbit immunoglobulin serum.41 Expression of ETA by E. coli colonies was detected by the procedure of Helfman et al.42 Release of periplasmic proteins Periplasmic proteins were released by a chloroform shock procedure.43 The cells from a 100 ml mid-exponential LB culture were resuspended in residual broth after centrifugation and decanting the supernatant. Chloroform (50 ~1) was added, the mixture was vortexed and incubated at room temperature for 15 min. 5 ml of 10 IYtM Tris-HCI pH 8.0 containing 2 mM phenylmethylsulphonyl fluoride and 2 mrvt benzamidine was added and the cells were removed by centrifugation at 45000 g for 60 min. The supernatant was decanted and concentrated ten-fold using a CX-10 immersible filter (Millipore). Immunochemical analysis of ETA produced in E. coli Proteins produced in deta lysates were concentrated by precipitation with trichloracetic acid (5% w/v, final concentration) in the presence of 10 pg bovine albumin. This was subjected to SDS-polyacrylamide gel electrophoresis” and Western immunoblotting.45 Filters were incubated with peroxidase-conjugated protein A (Sigma) instead of the second antibody. Material for immunodiffusion analysis46 was dialysed against distilled water, lyophilised and dissolved in a small volume of phosphate buffered saline pH 7.5. Rocket immunoelectrophoresis was performed as described by Owen47 using purified toxin as the standard.

Cloning epidermolytic

toxin A

593

Neonatal mouse assay for epidermolytic activity Three day old Sha-Sha mice weighing between 1.8-2.2 g were injected subcutaneously in triplicate in the dorsal region with a volume not exceeding 100 ~1 of the test material. The a-haemolysin present in S. aureus culture supernatants was inactivated by heating at 65°C for 15 min. The animals were examined at hourly intervals for the development of the Nikolsky sign, i.e. wrinkling of the skin when lightly pinched.5 Note added

in proof

The physical map of the sequence analysis (P.O’T. 242 residues. In addition, also: Jackson MP, landolo This project the following; Mary O’Reilly communicating

eta gene in Fig. 3 has recently been confirmed by DNA and T.J.F., unpublished data). The mature polypeptide has etb has recently been shown to be plasmid encoded25a (see JJ. 1986, J. Bacterial. 166: 574-580).

is funded by the Medical Research Council of Ireland. We would like to thank Joyce de Azavedo for a gift of antisera and for help with animal experiments, and John Arbuthnott for their interest and encouragement and Chris Bailey for results prior to publication.

References 1. 2. 3. 4. 5. 6.

7. 8. 9. 10. 11.

12. 13. 14. 15.

16. 17. 18. 19.

Elias PM, Fritsch P, Epstein EH. Staphylococcal scalded skin syndrome: clinical features, pathogenesis and recent microbiological and biochemical developments. Arch Dermatol 1977; 113: 207-I 9. Curran JP, Al-Salihi FL. Neonatal staphylococcal scalded skin syndrome: massive outbreak due to an unusual phage type. Pediatrics 1980; 66: 28590. Melish ME, Glasgow LA. The staphylococcal scalded skin syndrome; development of an experimental model. N Engl J Med 1970; 282: 1114-g. Arbuthnott JP, Kent J, Lyell A, Gemmell CG. Toxic epidermal necrolysis produced by an extracellular product of .Staphy/ococcus aureus. Br J Dermatol 1971; 85: 145-9. Arbuthnott JP, Kent J, Noble WC. The response of hairless mice to staphylococcal epidermolytic toxin. Br J Dermatol 1973; 88: 481-5. Kondo I, Sakurai S, Serai Y. New type of exfoliatin obtained from staphylococcal strains, belonging to phage groups other than group II, isolated from patients with impetigo and Ritter’s disease. Infect lmmun 1974; 10: 85161. Arbuthnott JP, Billcliffe 8. Qualitative and quantitative methods for detecting staphylococcal epidermolytic toxin. J Med Microbial 1976; 9: 191-201. Johnson AD, Spero L, Cades JS, de Cicco BT. Purification and characterization of different types of exfoliative toxin from Sfaphylococcus aureus. Infect lmmun 1979; 24: 679-84. Bailey CJ, de Azavedo J, Arburthnott JP. A comparative study of two serotypes of epidermolytic toxin from Staphylococcus aureus. Biochim Biophys Acta 1980; 624: 11 I-20. Smith TP, Bailey CJ. Epidermolytic toxin from Staphy/ococcus aureus binds to filaggrins. Febs Lett 1986; 194: 309-l 2. Kaplan MH, Chmel H, Hsieh HC, Stephens A, Brinsko V. Importance of exfoliatin toxin A produced by Staphylococcusaureusstrains isolated from clustered epidemics of neonatal pustulosis. J Clin Microbial 1986; 23: 83-91. O’Reilly M, Dougan G, Foster TJ, Arbuthnott JP. Plasmids in epidermolytic strains of Staphylococcus aureus. J Gen Microbial 1981; 124: 99-l 97. Rogolsky M, Wiley BB, Glasgow LA. Phage group II staphylococcal strains with chromosomal and extrachromosomal genes for exfoliative toxin production. Infect lmmun 1976; 13: 44-52. O’Reilly M. Plasmids of epidermolytic strains of Staphylococcus aureus. Ph.D. Thesis, University of Dublin, 1984. Masterson R, von David W, Wiley BB, Rogolsky M. Mutagenesis of extrachromosomal determinants for exfoliative toxin B and bacteriocin R, synthesis in Staphylococcus aureus after plasmid transfer by protoplast fusion. Infect lmmun 1983; 42: 973-9. Martin SM. Shoham SC, Alsup M, Rogolosky M. Genetic mapping in phage group II Staphylococcus aureus. Infect lmmun 1980; 27: 532-41. Brown DR, Pattee PA. Identification of a chromosomal determinant of alpha-toxin production in Staphylococcus aureus. Infect lmmun 1980; 30: 3642. Recsei P, Kreiswirth B, O’Reilly M, Schievert P, Gruss A, Novick RP. Regulation of exoprotein gene expression in Staphylococcus aureus by agr. Mol Gen Genet 1986; 202: 58-61. O’Reilly M, de Azavedo JCS, Kennedy S, Foster TJ. Inactivation of the alpha-haemolysin of

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and T. J. Foster

Sfaphylococcu.s aureus 8325-4 by site-directed mutagenesis and studies on expression of its haemolysins. Microbial Pathogenesis 1986; 1: 125-38. 20. Kehoe M, Duncan J, Foster T, Fairweather N, Dougan G. Cloning, expression, and mapping of the Staphylococcus aureus a-haemolysin determinant in Escherichia co/i K12. Infect lmmun 1983; 4: 1105 11. 21. Kreiswirth BN, Lofdahl S, Betley MJ, O’Reilly M, Shlievert PM, Bergdoll MS, Novick RP. The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage. Nature 1983; 305: 709-l 2. 22. Sako T, Sawaki S, Sakurai T, Ito S, Yoshizawa Y, Kondo I. Cloning and expression of the staphylokinase gene of Staphylococcus aureus in Escherichia co/i. Mol Gen Genet 1983; 190: 271-7. 23. Lofdahl S, Guss B, Uhlen M, Philipson L, Lindberg M. Gene for staphylococcal protein A. Proc Natl Acad Sci USA 1983; 80: 697-701. 24. Shortle D. A Genetic system for analysis of staphylococcal nuclease. Gene 1983; 22: 181-9. 25. Ranelli DM, Jones CL, Johns MB, Mussey GJ, Kahn SA. Molecular cloning of staphylococcal enterotoxin I3 gene in Escherichia co/i and Staphylococcus aureus. Proc Natl Acad Sci USA 1985; 82: 58504. 25a. O’Toole PW, Foster TJ. Epidermolytic toxin serotype B of Staphylococcus aureus is plasmid-encoded. FEMS Microbial Lett 1986; 36: 311-314. 26. Bjorklind A, Arvidson S. Mutants of Staphylococcus aureus affected in the regulation of exoprotein synthesis. FEMS Microbial Lett 1980; 7: 203-6. 27. Novick RP. Properties of a cryptic high-frequency transducing phage in Staphylococcus aureus. Virology 1963; 33: 155-66. 28. Appleyard RK. Segregation of new lysogenic types during growth of a double lysogenic strain derived from Escherichia co/i K12. Genetics 1954; 39: 440-52. 29. Loenen WAM, Brammar WJ. A bacteriophage vector for cloning large restriction fragments made with several restriction enzymes. Gene 1980; 10: 249-59. 30. Coleman DC, Foster TJ. 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