Staphylococcal Exfoliative Toxin B Specifically Cleaves Desmoglein 1
2002; Elsevier BV; Volume: 118; Issue: 5 Linguagem: Inglês
10.1046/j.1523-1747.2002.01751.x
ISSN1523-1747
AutoresMasayuki Amagai, Koji Nishifuji, Takayuki Yamaguchi, Yasushi Hanakawa, Motoyuki Sugai, John R. Stanley,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoStaphylococcal scalded skin syndrome and its localized form, bullous impetigo, show superficial epidermal blister formation caused by exfoliative toxin A or B produced by Staphylococcus aureus. Recently we have demonstrated that exfoliative toxin A specifically cleaves desmoglein 1, a desmosomal adhesion molecule, that when inactivated results in blisters. In this study we determine the target molecule for exfoliative toxin B. Exfoliative toxin B injected in neonatal mice caused superficial epidermal blisters, abolished cell surface staining of desmoglein 1, and degraded desmoglein 1 without affecting desmoglein 3 or E-cadherin. When adenovirus-transduced cultured keratinocytes expressing exogenous mouse desmoglein 1 or desmoglein 3 were incubated with exfoliative toxin B, desmoglein 1, but not desmoglein 3, was cleaved. Furthermore, cell surface staining of desmoglein 1, but not that of desmoglein 3, was abolished when cryosections of normal human skin were incubated with exfoliative toxin B, suggesting that living cells were not necessary for exfoliative toxin B cleavage of desmoglein 1. Finally, in vitro incubation of the recombinant extracellular domains of desmoglein 1 and desmoglein 3 with exfoliative toxin B demonstrated that both mouse and human desmoglein 1, but not desmoglein 3, were directly cleaved by exfoliative toxin B in a dose-dependent fashion. These findings demonstrate that exfoliative toxin A and exfoliative toxin B cause blister formation in staphylococcal scalded skin syndrome and bullous impetigo by identical molecular pathophysiologic mechanisms. Staphylococcal scalded skin syndrome and its localized form, bullous impetigo, show superficial epidermal blister formation caused by exfoliative toxin A or B produced by Staphylococcus aureus. Recently we have demonstrated that exfoliative toxin A specifically cleaves desmoglein 1, a desmosomal adhesion molecule, that when inactivated results in blisters. In this study we determine the target molecule for exfoliative toxin B. Exfoliative toxin B injected in neonatal mice caused superficial epidermal blisters, abolished cell surface staining of desmoglein 1, and degraded desmoglein 1 without affecting desmoglein 3 or E-cadherin. When adenovirus-transduced cultured keratinocytes expressing exogenous mouse desmoglein 1 or desmoglein 3 were incubated with exfoliative toxin B, desmoglein 1, but not desmoglein 3, was cleaved. Furthermore, cell surface staining of desmoglein 1, but not that of desmoglein 3, was abolished when cryosections of normal human skin were incubated with exfoliative toxin B, suggesting that living cells were not necessary for exfoliative toxin B cleavage of desmoglein 1. Finally, in vitro incubation of the recombinant extracellular domains of desmoglein 1 and desmoglein 3 with exfoliative toxin B demonstrated that both mouse and human desmoglein 1, but not desmoglein 3, were directly cleaved by exfoliative toxin B in a dose-dependent fashion. These findings demonstrate that exfoliative toxin A and exfoliative toxin B cause blister formation in staphylococcal scalded skin syndrome and bullous impetigo by identical molecular pathophysiologic mechanisms. desmoglein 1 exfoliative toxin exfoliative toxin A exfoliative toxin B staphylococcal scalded skin syndrome Staphylococcal scalded skin syndrome (SSSS) is a generalized blistering skin disease induced by the exfoliative (epidermolytic) toxin (ET) of Staphylococcus aureus (Melish and Glasgow, 1970Melish M.E. Glasgow L.A. The staphylococcal scalded skin syndrome: development of an experimental model.N Engl J Med. 1970; 282: 1114-1119Crossref PubMed Scopus (300) Google Scholar;Melish et al., 1972Melish M.E. Glasgow L.A. Turner M.D. The staphylococcal scalded-skin syndrome. Isolation and partial characterization of the exfoliative toxin.J Infect Dis. 1972; 125: 129-140Crossref PubMed Scopus (127) Google Scholar). It was also known in the past as Ritter's disease, dermatitis exfoliativa neonatorum, or pemphigus neonatorum. It is a disease primarily affecting infants and young children, but adults can also be affected. Its clinical manifestations begin abruptly with fever, skin tenderness, and erythema, followed by large sheets of epidermal separation involving the entire skin surface within hours to days. The mortality is still 3% in children (Gemell, 1995Gemell C.G. Staphylococcal scalded skin syndrome.J Med Microbiol. 1995; 43: 318-327Crossref PubMed Scopus (86) Google Scholar), and over 50% in adults with underlying diseases despite antibiotic treatment (Cribier et al., 1984Cribier B. Piemont Y. Grosshans E. Staphylococcal scalded skin syndrome in adults: a clinical review illustrated with a new case.J Am Acad Dermatol. 1984; 30: 319-324Abstract Full Text PDF Scopus (123) Google Scholar). SSSS caused by antibiotic-resistant strains of S. aureus has recently emerged as an even more serious problem (Yokota et al., 1996Yokota S. Imagawa T. Katakura S. Mitsuda T. Arai K. Staphylococcal scalded skin syndrome caused by exfoliative toxin B-producing methicillin-resistant Staphylococcus aureus.Eur J Pediatr. 1996; 155: 722PubMed Google Scholar;Acland et al., 1998Acland K.M. Darvay A. Griffin C. Aali S.A. Russell-Jones R. Staphylococcal scalded skin syndrome in an adult associated with methicillin-resitant Staphylococcus aureus.Br J Dermatol. 1998; 140: 518-520https://doi.org/10.1046/j.1365-2133.1999.02721.xCrossref Scopus (36) Google Scholar). The pathogenic role of ET in SSSS is well established. For example, ET injected into neonatal mice causes extensive blisters similar to the disease manifestations in human neonates (Melish and Glasgow, 1970Melish M.E. Glasgow L.A. The staphylococcal scalded skin syndrome: development of an experimental model.N Engl J Med. 1970; 282: 1114-1119Crossref PubMed Scopus (300) Google Scholar). In SSSS, S. aureus is present at distant foci such as the pharynx, nose, ear, or conjunctiva, and ET produced by S. aureus gets into circulation and causes exfoliation at remote sites, whereas in bullous impetigo, a localized form of SSSS, S. aureus is present in the lesions. ET has two major serotypes A and B (ETA and ETB). In the U.S.A. and Europe more than 80% of toxin-producing S. aureus produce ETA (Cribier et al., 1984Cribier B. Piemont Y. Grosshans E. Staphylococcal scalded skin syndrome in adults: a clinical review illustrated with a new case.J Am Acad Dermatol. 1984; 30: 319-324Abstract Full Text PDF Scopus (123) Google Scholar), whereas in Japan there is a predominance of ETB-producing strains (Murono et al., 1988Murono K. Fujita K. Yoshioka H. Microbiologic characteristics of exfoliative toxin-producing Staphylococcus aureus.Pediatr Infect Dis J. 1988; 7: 313-315Crossref PubMed Scopus (26) Google Scholar). The gene encoding ETA is located on the chromosome whereas the gene encoding ETB is found on a large plasmid. The genes for ETA and ETB have been cloned and their amino acid sequences have been deduced (O'Toole and Foster, 1986O'Toole P.W. Foster T.J. Molecular cloning and expression of the epidermolytic toxin A gene of Staphylococcus aureus.Microb Pathog. 1986; 1: 583-594Crossref PubMed Scopus (26) Google Scholar;Lee et al., 1987Lee C.Y. Schmidt J.J. Johnson-Winegar A.D. Spero L. Iandolo J.J. Sequence determination and comparison of the exfoliative toxin A and toxin B genes from Staphylococcus aureus.J Bacteriol. 1987; 169: 3904-3909PubMed Google Scholar). The mature proteins of ETA and ETB are 242 and 246 residues, respectively, after their signal sequences are cleaved. The ETA and ETB amino acid sequences are about 40% identical to each other. Although previous ultrastructural studies have shown intercellular cleavage with split desmosomes after injection of ET into neonatal mice, it was not clear whether disruption of desmosomes is an initial event or a secondary event that occurs only after edema caused by other intercellular pathology (Lillibridge et al., 1972Lillibridge C.B. Melish M.E. Glasgow L.A. Site of action of exfoliative toxin in the staphylococcal scaled-skin syndrome.Pediatrics. 1972; 50: 728-738PubMed Google Scholar;Melish et al., 1974Melish M.E. Glasgow L.A. Turner M.D. Lillibridge C.B. The staphylococcal epidermolytic toxin: its isolation, characterization, and site of action.Ann NY Acad Sci. 1974; 236: 317-342Crossref PubMed Scopus (31) Google Scholar;Elias et al., 1975Elias P.M. Fritsch P. Dahl M.V. Wolff K. Staphylococcal toxic epidermal necrolysis: pathogenesis and studies on the subcellular site of action of exfoliatin.J Invest Dermatol. 1975; 65: 501-512Crossref PubMed Scopus (45) Google Scholar;Dimond et al., 1977Dimond R.L. Wolff H.H. Braun-Falco O. The staphylococcal scalded skin syndrome: an experimental histochemical and electron microscopic study.Br J Dermatol. 1977; 96: 483-492Crossref PubMed Scopus (25) Google Scholar). ETs have also been reported to be superantigens (Choi et al., 1989Choi Y.W. Kotzin B. Herron L. Callahan J. Marrack P. Kappler J. Interaction of Staphylococcus aureus toxin 'superantigens' with human T cells.Proc Natl Acad Sci USA. 1989; 86: 8941-8945Crossref PubMed Scopus (916) Google Scholar;Kappler et al., 1989Kappler J. Kotzin B. Herron L. et al.V beta-specific stimulation of human T cells by staphylococcal toxins.Science. 1989; 244: 811-813Crossref PubMed Scopus (613) Google Scholar), which bind to major histocompatibility complex class II molecules on antigen-presenting cells and to the variable parts of the T cell receptor, resulting in polyclonal T cell activation. Other investigators have argued, however, that the previous demonstration of superantigenic activity with ETs was probably due to contamination with other staphylococcal enterotoxins (Fleischer and Bailey, 1992Fleischer B. Bailey C.J. Recombinant epidermolytic (exfoliative) toxin A of Staphylococcus aureus is not a superantigen.Med Microbiol Immunol. 1992; 180: 273-278Crossref PubMed Scopus (28) Google Scholar;Fleischer et al., 1995Fleischer B. Gerlach D. Fuhrmann A. Schmidt K.H. Superantigens and pseudosuperantigens of gram-positive cocci.Med Microbiol Immunol. 1995; 184: 1-8Crossref PubMed Scopus (35) Google Scholar). Therefore, until recently, the molecular mechanism of the epidermal separation by ET had been unclear, even 30 y after the pathogenic role of ET was demonstrated using neonatal mice in 1970 (Ladhani et al., 1999Ladhani S. Joannou C.L. Lochrie D.P. Evans R.W. Poston S.M. Clinical, microbial, and biochemical aspects of the exfoliative toxins causing staphylococcal scalded-skin syndrome.Clin Microbiol Rev. 1999; 12: 224-242Crossref PubMed Google Scholar). The clue to how ETA might cause the blister was provided by studies of the pathophysiology of pemphigus foliaceus, an autoimmune blistering disease, in which inactivation of the desmosomal cadherin desmoglein 1 (Dsg1) by autoantibodies was shown to cause clinical and histologic blisters similar to those seen in bullous impetigo and SSSS (Stanley, 1993Stanley J.R. Cell adhesion molecules as targets of autoantibodies in pemphigus and pemphigoid, bullous diseases due to defective epidermal cell adhesion.Adv Immunol. 1993; 53: 291-325Crossref PubMed Scopus (224) Google Scholar;Amagai, 1999Amagai M. Autoimmunity against desmosomal cadherins in pemphigus.J Dermatol Sci. 1999; 20: 92-102Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar;Mahoney et al., 1999Mahoney M.G. Wang Z. Rothenberger K.L. Koch P.J. Amagai M. Stanley J.R. Explanation for the clinical and microscopic localization of lesions in pemphigus foliaceus and vulgaris.J Clin Invest. 1999; 103: 461-468Crossref PubMed Scopus (384) Google Scholar). These observations led us to hypothesize and prove that ETA specifically cleaves Dsg1 (Amagai et al., 2000Amagai M. Matsuyoshi N. Wang Z.H. Andl C. Stanley J.R. Toxin in bullous impetigo and staphylococcal scalded skin syndrome targets desmoglein 1.Nature Med. 2000; 6: 1275-1277Crossref PubMed Scopus (365) Google Scholar). In this study, we determine that the molecular pathophysiology of blistering caused by ETB is identical to that of ETA, namely the direct cleavage of Dsg1. ETB-producing S. aureus TY4 chromosomal DNA was prepared as described previously (Sugai et al., 1998Sugai M. Fujiwara T. Komatsuzawa H. Suginaka H. Identification and molecular characterization of a gene homologous to epr (endopeptidase resistance gene) in Staphylococcus aureus.Gene. 1998; 224: 67-75Crossref PubMed Scopus (28) Google Scholar;Yamaguchi et al., 2001Yamaguchi T. Hayashi T. Takami H. et al.Complete nucleotide sequence of a Staphylococcus aureus exfoliative toxin B plasmid and identification of a novel ADP-ribosyltransferase, EDIN-C.Infect Immun. 2001; 69: 7760-7771Crossref PubMed Scopus (113) Google Scholar) and used as a polymerase chain reaction (PCR) template. A primer set, 5′-AAGCTTCCACCTAATACCCTAATAATC-3′ and 5′-GGATCCACAGAGGTTCAACTCATGGTT-3′, was designed to generate a 1148 bp DNA fragment containing the entire etb gene (M17348) (Jackson and Iandolo, 1986Jackson M.P. Iandolo J.J. Sequence of the exfoliative toxin B gene of Staphylococcus aureus.J Bacteriol. 1986; 167: 726-728PubMed Google Scholar) with terminal HindIII and BamHI sites. The PCR product was cloned into pGEM-T Easy vector to generate pTY231. The pTY231 was digested with HindIII and BamHI, and the insert was cloned into E. coli-S. aureus shuttle vector pCL8 to generate pTY133. S. aureus RN4220 (NCTC8325-4r-), which is a standard strain with no production of ETA or ETB, was then transformed with pTY133, and one of the transformants was designated TY2134. The physical map and genetic determinants of NCTC8325 indicate that this strain does not possess eta or etb (Iandolo, 2000Iandolo J.J. Genetic and physical map of the chromosome of Staphylococcus aureus 8325.in: Fischetti V.A. Novick R.P. Ferretti J.J. Portnoy D.A. Reed J.I. Gram-positive pathogens. ASM Press, Washington, DC2000: 317-325Google Scholar). The nucleotide sequence of the etb gene was confirmed by DNA sequencing. Staphylococcus aureus TY2134 exponentially growing in Trypticase soy broth (Becton Dickinson Microbiology Systems, Cockeysville, MD) was inoculated in 1 liter of the same fresh medium and incubated with continuous agitation by a rotary shaker for 24 h at 37°C until the cells reached the stationary phase. The culture was centrifuged at 10,000 × g for 20 min at 4°C. Concentrated culture filtrate was prepared by 80% saturated ammonium sulfate precipitation of the culture supernatant. A sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent Coomassie Brilliant Blue staining of concentrated culture filtrate revealed a single major protein band of 27 kDa, which was not present in the concentrated culture filtrate of RN4220 pCL8. We therefore used Coomassie Brilliant Blue staining of SDS-PAGE gel for detection of the ETB-positive fraction with the 27 kDa band as the marker. Concentrated culture filtrate dialyzed against 10 mM phosphate buffer (pH 6.8) (buffer 1) was applied to a hydroxyapatite column (Wako, 15 mm × 90 mm), which was equilibrated with buffer 1. The column was washed with buffer 1 until most of the unbound proteins passed through. Bound proteins were eluted by stepwise elution with 100 mM, 250 mM, and 500 mM phosphate buffer (pH 6.8), respectively. Eluate with 100 mM phosphate buffer (pH 6.8) was dialyzed against 10 mM phosphate buffer (pH 6.8) (buffer 2) and concentrated to 500 µl. The sample was loaded onto TSKgel SW3000XL (Tosoh, 7.5 mm × 300 mm) and eluted with buffer 2 at a flow rate of 0.5 ml per min, and the ETB-positive fractions were collected. Those fractions were collected and further applied to Bioscale CHT2-I hydroxyapatite HPLC column (Bio-Rad, 7.5 mm × 52 mm), and eluted with a linear gradient from 10 mM to 500 mM phosphate buffer (pH 6.8) at a flow rate of 1 ml per min. The ETB-positive fractions were collected and extensively dialyzed against phosphate-buffered saline (PBS). This purified ETB was used for incubation with cryosections of normal human skin (see below) and recombinant Dsg1 and Dsg3 (see below). We also cloned the etb gene by a similar approach using an ETB-producing S. aureus isolated from a patient as a PCR template. A primer set, 5′-ACCCTAATAATCCAAAAACAG-3′ and 5′-CACAGAGGTTCAACTCATGGT-3′, was used to amplify the etb gene and promoter sequence, and the PCR products were ligated into the pCRII vector and then subcloned into the pCE104 vector (a kind gift from Dr. Patrick Schlievert) (Vath et al., 1997Vath G.M. Earhart C.A. Rago J.V. Kim M.H. Bohach G.A. Schlievert P.M. Ohlendorf D.H. The structure of the superantigen exfoliative toxin A suggests a novel regulation as a serine protease.Biochemistry. 1997; 36: 1559-1566Crossref PubMed Scopus (113) Google Scholar). The supernatant from S. aureus RN4220 transformed with this plasmid contained ETB as the major protein, which was greater than 90% pure as determined by SDS-PAGE. This partially purified ETB was used for incubation with transduced HaCaT cells (see below) and injected into neonatal mice prior to immunofluorescence and immunoblotting of epidermis (see below). To evaluate the exfoliative activity of recombinant ETB, neonatal ICR or BALB/C mice (< 24 h of age) were injected subcutaneously with a designated amount of ETB in 20–50 µl of PBS, and the skin was analyzed grossly and microscopically 1–24 h after injection. Mouse back skin was homogenized on dry ice, and then extracted with Laemmli sample buffer. Samples with equal amounts of protein (protein assay kit; Bio-Rad Laboratories, Hercules, CA) were separated by 6% Tris-glycine PAGE (Novex gels; Invitrogen, Carlsbad, CA), and then transferred to nitrocellulose membranes (Trans-Blot; Bio-Rad Laboratories). The membranes were incubated with rabbit antiserum against mouse Dsg1 and Dsg3, and ECCD-2 rat monoclonal antibody against mouse E-cadherin (a kind gift from M. Takeichi) (Shirayoshi et al., 1986Shirayoshi Y. Nose A. Iwasaki K. Takeichi M. N-linked oligosaccharides are not involved in the function of a cell-cell binding glycoprotein E-cadherin.Cell Struct Funct. 1986; 11: 245-252Crossref PubMed Scopus (143) Google Scholar). To transduce cells with constructs encoding mouse Dsg1 and mouse Dsg3 with FLAG tag, recombinant adenovirus was constructed. The cosmid cassette pAxCAw, control Ad Ax1w, and the parent virus Ad5-dLX were all kind gifts from Dr. Izumi Saito (Tokyo University, Japan) (Miyake et al., 1996Miyake S. Makimura M. Kanegae Y. et al.Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome.Proc Natl Acad Sci USA. 1996; 93: 1320-1324Crossref PubMed Scopus (781) Google Scholar). cDNA encoding mouse Dsg1-FLAG and mouse Dsg3-FLAG (Amagai et al., 2000Amagai M. Matsuyoshi N. Wang Z.H. Andl C. Stanley J.R. Toxin in bullous impetigo and staphylococcal scalded skin syndrome targets desmoglein 1.Nature Med. 2000; 6: 1275-1277Crossref PubMed Scopus (365) Google Scholar) were subcloned into the Ad cosmid cassette pAxCAw. Adenovirus containing CA promoter and cDNA encoding mouse Dsg1-FLAG and mouse Dsg3-FLAG (AxmDsg1F and AxmDsg3F) were generated by the COS-TPC method (Miyake et al., 1996Miyake S. Makimura M. Kanegae Y. et al.Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome.Proc Natl Acad Sci USA. 1996; 93: 1320-1324Crossref PubMed Scopus (781) Google Scholar) as follows. The cosmid DNA was mixed with the EcoT22I-digested DNA-terminal protein complex of Ad5-dLX and used to cotransfect to 293 cells in which recombinant viruses were generated through homologous recombination. Virus stocks were prepared using standard procedures (Miyake et al., 1996Miyake S. Makimura M. Kanegae Y. et al.Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome.Proc Natl Acad Sci USA. 1996; 93: 1320-1324Crossref PubMed Scopus (781) Google Scholar), and were concentrated by the CsCl gradient method. The virus titer was checked with a plaque formation assay. HaCaT cells were cultured in a 12-well plate and transduced with AxmDsg1F and AxmDsg3F. After 24 h the cells were incubated with 0, 0.05, 0.15, and 0.25 µg per ml of recombinant ETB in culture media and incubated for 10 min or 1 h. Then the cells were washed with PBS, and extracted with 200 µl 2 × SDS Laemmli sample buffer (Bio-Rad Laboratories). The transduced Dsg1 and Dsg3 were visualized by immunoblotting with anti-FLAG tag rabbit antibody (Zymed, San Francisco, CA). Formalin-fixed, paraffin-embedded skin from neonatal mice injected with ETB or saline was used for indirect immunofluorescence to localize Dsg1, Dsg3, and E-cadherin, as previously described (Amagai et al., 2000Amagai M. Matsuyoshi N. Wang Z.H. Andl C. Stanley J.R. Toxin in bullous impetigo and staphylococcal scalded skin syndrome targets desmoglein 1.Nature Med. 2000; 6: 1275-1277Crossref PubMed Scopus (365) Google Scholar). A rabbit antiserum against extracellular domain 5 of mouse Dsg3 was raised and affinity purified on the antigenic peptide. A rabbit antiserum, raised similarly against extracellular domain 5 of mouse Dsg1, and ECCD-2 were also used. Stained sections were photographed using confocal microscopy (Leica TCS 4D, Wetzlar, Germany). Cryosections of nonfixed normal human skin were incubated with 0.1 µg per ml of ETA (Toxin Technology, Sarasota, FL) in PBS with 1 mM CaCl2 (PBS-Ca), 0.1 µg per ml of recombinant ETB in PBS-Ca, or PBS-Ca alone for 1 h at room temperature. The sections were then stained with anti-Dsg1 sera obtained from patients with pemphigus foliaceus, anti-Dsg3 mouse monoclonal antibody, 5H10, which reacts with the extracellular domain (Proby et al., 2000Proby C.M. Ohta T. Suzuki H. et al.Development of chimeric molecules for recognition and targeting of antigen-specific B cells in pemphigus vulgaris.Br J Dermatol. 2000; 142: 321-330Crossref PubMed Scopus (31) Google Scholar), anti-Dsg1 + 2 mouse monoclonal antibody, DG3.10, which reacts with the cytoplasmic domain (Koch et al., 1990Koch P.J. Walsh M.J. Schmelz M. Goldschmidt M.D. Zimbelmann R. Franke W.W. Identification of desmoglein, a constitutive desmosomal glycoprotein, as a member of the cadherin family of cell adhesion molecules.Eur J Cell Biol. 1990; 53: 1-12PubMed Google Scholar), antidesmocollin mouse monoclonal antibody, 52-3D, which reacts with the cytoplasmic domain of Dsc1–3 (a kind gift from Dr. D. R. Garrod) (Collins et al., 1991Collins J.E. Legan P.K. Kenny T.P. MacGarvie J. Holton J.L. Garrod D.R. Cloning and sequence analysis of desmosomal glycoproteins 2 and 3 (desmocollins): cadherin-like desmosomal adhesion molecules with heterogeneous cytoplasmic domains.J Cell Biol. 1991; 113: 381-391Crossref PubMed Scopus (148) Google Scholar), and antidesmoplakin mouse monoclonal antibody, 11-5F (a kind gift from Dr. D. R. Garrod). Staining with DG3.10 in this study represents the expression of Dsg1 because there is no detectable expression of Dsg2 in normal human skin. The entire extracellular domain of mouse and human recombinant Dsg1 and Dsg3, with an E-tag on the carboxyl terminus, were produced as a secreted protein by baculovirus expression as previously described (Ishii et al., 1997Ishii K. Amagai M. Hall R.P. et al.Characterization of autoantibodies in pemphigus using antigen-specific ELISAs with baculovirus expressed recombinant desmogleins.J Immunol. 1997; 159: 2010-2017PubMed Google Scholar;Amagai et al., 2000Amagai M. Matsuyoshi N. Wang Z.H. Andl C. Stanley J.R. Toxin in bullous impetigo and staphylococcal scalded skin syndrome targets desmoglein 1.Nature Med. 2000; 6: 1275-1277Crossref PubMed Scopus (365) Google Scholar). These recombinant human Dsg1 and Dsg3 proteins have been shown to retain their native conformations enough to adsorb out all immunoreactivity of pemphigus foliaceus and vulgaris sera, respectively (Ishii et al., 1997Ishii K. Amagai M. Hall R.P. et al.Characterization of autoantibodies in pemphigus using antigen-specific ELISAs with baculovirus expressed recombinant desmogleins.J Immunol. 1997; 159: 2010-2017PubMed Google Scholar). High Five cells (Invitrogen, San Diego, CA) cultured in serum-free EX Cell 405 medium (JRH Bioscience, Lenexa, KS) were infected with the recombinant viruses and incubated for 3 d. Culture supernatant containing each recombinant Dsg was incubated with the indicated amount of ETB for 1 h at 37°C, and subsequently subjected to immunoblot analysis with anti-E-tag mouse monoclonal antibody (Pharmacia Biotech, Uppsala, Sweden) for detection of the intact as well as digested recombinant proteins. Culture supernatant of RN4220 transfected with etb, but not that of RN4220 itself, digested recombinant Dsg1, indicating that RN4220 did not produce any ET (data not shown). cDNA for ETB was PCR-amplified from ETB-producing S. aureus TY4, subcloned into an E. coli-S. aureus shuttle vector (pTY133), and used to transform S. aureus RN4220 (TY2134). TY2134 produced ETB in culture supernatant, which was recognized as a single predominant band of 27 kDa by Coomassie Blue. N-terminal sequencing of the protein band identified that it was the correctly processed form of recombinant ETB (data not shown). Finally 360 µg recombinant ETB was purified to homogeneity from 1 liter of culture supernatant as described in Materials and Methods (Figure 1a). To confirm the exfoliative activity of the recombinant ETB, 20 µg of ETB in 100 µl of PBS was subcutaneously injected into neonatal mice. The mice started to show gross blisters as soon as 2–3 h after injection around the injected site (Figure 1b; compare to control Figure 1c). To investigate the molecular mechanism of the blister formation by ETB and to determine whether ETB specifically affects Dsg1 as does ETA (Amagai et al., 2000Amagai M. Matsuyoshi N. Wang Z.H. Andl C. Stanley J.R. Toxin in bullous impetigo and staphylococcal scalded skin syndrome targets desmoglein 1.Nature Med. 2000; 6: 1275-1277Crossref PubMed Scopus (365) Google Scholar), we examined the skin from neonatal mice injected with ETB by immunofluorescence with antibodies to Dsg1, Dsg3, and E-cadherin. When the skin was examined 1 h after ETB injection, the cell surface staining of Dsg1 was markedly diminished (Figure 2b; compare to control Figure 2a). In the same area, the cell surface staining of Dsg3 or E-cadherin was unaffected (Figure 2c–f). To determine whether this change in Dsg1 staining was caused by degradation of Dsg1 in the neonatal mouse skin, extracts of the skin from the mice injected with ETB or PBS were subjected to immunoblot analysis for Dsg1, Dsg3, and E-cadherin. A 160 kDa band for Dsg1 was degraded into a band of approximately 113 kDa in all three mice injected with ETB, whereas Dsg3 and E-cadherin were not degraded (Figure 3). These results indicate that Dsg1, but not Dsg3 or E-cadherin, was cleaved in vivo in neonatal mouse skin after injection with ETB. To further demonstrate specific cleavage of Dsg1 by ETB, we transduced HaCaT cells, a human keratinocyte cell line, with recombinant adenovirus containing cDNA encoding mouse Dsg1 or Dsg3 with a FLAG epitope tag on their C-termini. When 50 µg per ml of ETB was added to the culture medium of these cells and incubated for 1 h, Dsg1 was degraded whereas Dsg3 was not (Figure 4a). When these transduced HaCaT cells were incubated with various amounts of ETB (0, 0.05, 0.15, and 0.25 µg per ml) for 10 min or 1 h, Dsg1 was cleaved in a dose- and time-dependent fashion to a 113 kDa product (Figure 4b). The in vivo findings described above do not prove that ETB directly degrades Dsg1, as opposed to, for example, degradation through activation of other proteases in living cells. Therefore, to first demonstrate that living cells are not necessary for this inactivation of Dsg1, we incubated cryosections of normal human skin with ETB, ETA, or PBS, as a control, and stained them with antibodies against various desmosomal components (Figure 5). The cell surface staining of Dsg1 by pemphigus foliaceus sera, which react with the extracellular domain of Dsg1 (Amagai et al., 1995Amagai M. Hashimoto T. Green K.J. Shimizu N. Nishikawa T. Antigen-specific immunoadsorption of pathogenic autoantibodies in pemphigus foliaceus.J Invest Dermatol. 1995; 104: 895-901Crossref PubMed Scopus (225) Google Scholar), was removed by ETB, whereas that of Dsg3 by 5H10, which reacts with the amino terminal extracellular domain of Dsg3 (Proby et al., 2000Proby C.M. Ohta T. Suzuki H. et al.Development of chimeric molecules for recognition and targeting of antigen-specific B cells in pemphigus vulgaris.Br J Dermatol. 2000; 142: 321-330Crossref PubMed Scopus (31) Google Scholar), was not altered at all (Figure 5a, l, d, f). These effects by ETB on the Dsg1 and Dsg3 staining were identical to those by ETA (Figure 5b, e). ETB treatment did not affect the staining pattern by monoclonal antibody DG3.10, which recognizes the cytoplasmic domain of Dsg1 (Figure 5g). ETB treatment did not alter the staining of desmocollin by 52-3D nor that of desmoplakin by 11-5F (Figure 5h, i). ETA treatment did not change the staining by DG3.10, 52-3D, or 11-5F, either (data not shown). These findings indicate that ETB as well as ETA specifically affect the extracellular domain of Dsg1 in the absence of living cells, presumably by cleavage. To demonstrate direct proteolysis of the extracellular domain of Dsg1 by ETB, we incubated a soluble recombinant form of the extracellular domain of Dsg1 and Dsg3 with ETB in vitro (Figure 6). ETB cleaved the 81 kDa recombinant mouse Dsg1 down to a 34 kDa peptide in a dose-dependent fashion, whereas ETB did not cleave mouse Dsg3 at all. In the same way, ETB cleaved the recombinant human Dsg1, but not human Dsg3. These findings indicate that ETB specifically recognizes and cleaves the extracellular domain of both mouse and human Dsg1. The major physiologic function of skin is to form a protective barrier that hampers the penetration of microorganisms and inhibits the loss of water. This barrier has been shown to reside in the stratum corneum. In bullous impetigo, S. aureus is found in a blister cavity just beneath the stratum corneum, thus circumventing the barrier. This blister cavity is known to be caused by ET, released by the pathologic organisms. A similar blister occurs in patients with the autoimmune disease pemphigus foliaceus. In that disease IgG autoantibodies against Dsg1 block the cell adhesion function of Dsg1 with resultant superficial blisters in the epidermis where Dsg1 is expressed without coexpressed Dsg3 (Amagai, 1999Amagai M. Autoimmunity against desmosomal cadherins in pemphigus.J Dermatol Sci. 1999; 20: 92-102Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar;Mahoney et al., 1999Mahoney M.G. Wang Z. Rothenberger K.L. Koch P.J. Amagai M. Stanley J.R. Explanation for the clinical and microscopic localization of lesions in pemphigus foliaceus and vulgaris.J Clin Invest. 1999; 103: 461-468Crossref PubMed Scopus (384) Google Scholar). Although Dsg1 is also expressed in the deep epidermis and mucous membranes, blisters do not occur in these areas because Dsg3 is coexpressed and can compensate for the antibody-induced loss of function of Dsg1. Because pemphigus foliaceus shows identical tissue specificity and histology to SSSS and bullous impetigo, which are caused by ET, we previously suspected that the target molecule for a major type of ET, ETA, might be Dsg1 and proved that ETA specifically cleaves Dsg1 (Amagai et al., 2000Amagai M. Matsuyoshi N. Wang Z.H. Andl C. Stanley J.R. Toxin in bullous impetigo and staphylococcal scalded skin syndrome targets desmoglein 1.Nature Med. 2000; 6: 1275-1277Crossref PubMed Scopus (365) Google Scholar). In this study, we hypothesized that another type of ET that causes SSSS and bullous impetigo, ETB, also cleaves Dsg1. We have shown that injection of recombinant ETB into neonatal mice caused the specific removal of cell surface staining of Dsg1 and the degradation of Dsg1 in vivo with resultant blister formation, whereas Dsg3 and E-cadherin were not affected. Similarly, when ETB was added to cultured keratinocytes expressing tagged Dsg1 or Dsg3, ETB digested Dsg1 without affecting Dsg3. Specific loss of immunofluorescence of Dsg1, but not other cell surface molecules, after incubation of normal human skin with ETB, suggested that inactivation of Dsg1 by ETB did not require living cells and was probably a direct effect. Finally, we have demonstrated that ETB specifically cleaved the recombinant extracellular domain of Dsg1, but not that of Dsg3, in vitro. These findings indicate that ETB recognizes the extracellular domain of Dsg1 and cleaves it directly. Histologic studies suggested that ET binds to a receptor in the granular layer of the epidermis, although to keratohyalin granules, not to the cell surface (Smith et al., 1989Smith T.P. John D.A. Bailey C.J. Epidermolytic toxin binds to components in the epidermis of a resistant species.Eur J Cell Biol. 1989; 49: 341-349PubMed Google Scholar). Other studies have suggested binding to an epidermal GM4-like glycolipid (Sakurai and Kondo, 1979Sakurai S. Kondo I. A possible receptor substance for staphylococcal exfoliatin isolated from mice.Jpn J Med Sci Biol. 1979; 32: 85-88PubMed Google Scholar;Tanabe et al., 1995Tanabe T. Sato H. Ueda K. et al.Possible receptor for exfoliative toxins produced by Staphylococcus hyicus and Staphylococcus aureus.Infect Immun. 1995; 63: 1591-1594PubMed Google Scholar). Our studies demonstrate direct cleavage of recombinant Dsg1 in solution, however, suggesting that any epidermal receptor other than Dsg1 is unnecessary for activation of ET's proteolytic activity. It is hypothesized that ETA and ETB are serine proteases. ETA and ETB are 25% identical to the staphylococcal serine protease V8. Structural studies show particularly striking homology in the region of the serine-aspartic acid-histidine catalytic triad that forms the active site of trypsin-like serine proteases (Dancer et al., 1990Dancer S.J. Garratt R. Saldanha J. Jhoti H. Evans R. The epidermolytic toxins are serine proteases.FEBS Lett. 1990; 268: 129-132Abstract Full Text PDF PubMed Scopus (73) Google Scholar). In addition, the X-ray crystal structure of ETA and ETB showed significant structure similarity with known glutamate-specific trypsin-like serine proteases (Vath et al., 1997Vath G.M. Earhart C.A. Rago J.V. Kim M.H. Bohach G.A. Schlievert P.M. Ohlendorf D.H. The structure of the superantigen exfoliative toxin A suggests a novel regulation as a serine protease.Biochemistry. 1997; 36: 1559-1566Crossref PubMed Scopus (113) Google Scholar;Vath et al., 1999Vath G.M. Earhart C.A. Monie D.D. Iandolo J.J. Schlievert P.M. Ohlendorf D.H. The crystal structure of exfoliative toxin B: a superantigen with enzymatic activity.Biochemistry. 1999; 38: 10239-10246Crossref PubMed Scopus (58) Google Scholar). It is interesting that ETA and ETB share identical binding specificity and cleavage specificity although overall identity is only 40%. We suspect that amino acid residues that are responsible for this specificity should be conserved. The final identification of ETA and ETB as glutamate-specific serine proteases has to await the determination of the cleavage site of Dsg1 by ETA and ETB. Dsg1 is targeted by ETA as well as ETB in SSSS and bullous impetigo, resulting in a blister just below the stratum corneum, the major barrier in skin. These toxins, then, allow the bacteria to circumvent this barrier and spread just beneath it. These findings provide an important framework to understand the molecular mechanism for blister formation in these diseases as well as cell-cell adhesion of keratinocytes in the epidermis. We especially thank Dr. Takeji Nishikawa for insightful discussion on this project. We are grateful to Dr. Patrick Schlievert for providing the pCE104 vector. Our kind thanks also go to Dr. Izumu Saito for the cosmid cassette pAxCAw, control adenovirus Ax1w, and parent virus Ad5- dLX, and to Dr. Masatoshi Takeichi and Dr. David R. Garrod for monoclonal antibodies. This work was supported by Health Science Research Grants for Research on Specific Disease from the Ministry of Health and Welfare, Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan, and grants from the National Institutes of Health.
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