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Infection and Immunity, October 2002, p. 5835-5845, Vol. 70, No. 10
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.10.5835-5845.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Identification of the Staphylococcus aureus etd Pathogenicity Island Which Encodes a Novel Exfoliative Toxin, ETD, and EDIN-B

Takayuki Yamaguchi,¹ Koji Nishifuji,² Megumi Sasaki,³ Yasuyuki Fudaba,¹ Martin Aepfelbacher,⁴ Takashi Takata,⁵ Masaru Ohara,¹ Hitoshi Komatsuzawa,¹ Masayuki Amagai,² and Motoyuki Sugai^1*

Departments of Bacteriology,¹ Oral Maxillofacial Pathobiology, Hiroshima University Graduate School of Biomedical Sciences, Kasumi 1-2-3, Minami-ku Hiroshima, Hiroshima 734-8553,⁵ Department of Dermatology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582,² Clinical Laboratory, Hiroshima City Hospital, 7-33 Motomachi, Hiroshima, Hiroshima 730-8518, Japan,³ Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, LMU Munich, 80336 Munich, Germany⁴

Received 26 February 2002/ Returned for modification 7 May 2002/ Accepted 25 June 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
We identified a novel pathogenicity island in Staphylococcus aureus which contains open reading frames (ORFs) similar to the exfoliative toxin (ET) gene, glutamyl endopeptidase gene, and edin-B gene in tandem and the phage resistance gene, flanked by hsdM, hsdS (restriction and modification system), and IS256. The protein encoded by the ET-like gene showed 40, 59, and 68% amino acid sequence identities with exfoliative toxin A (ETA), exfoliative toxin B (ETB), and Staphylococcus hyicus ETB (ShETB), respectively. When injected into neonatal mice, the recombinant protein derived from the ET-like gene induced exfoliation of the skin with loss of cell-to-cell adhesion in the upper part of the epidermis as observed in histological examinations, just as was found in neonatal mice injected with ETA or ETB. Western blot analysis indicated that the recombinant protein is serologically distinct from ETA and ETB. Therefore, the product encoded by this new ORF is a new ET member produced by S. aureus and is termed ETD. ETD did not induce blisters in 1-day-old chickens. In the skins of mice injected with ETD, cell surface staining of desmoglein 1 (Dsg1), a cadherin type cell-to-cell adhesion molecule in desmosomes, was abolished without affecting that of desmoglein 3 (Dsg3). Furthermore, in vitro incubation of the recombinant extracellular domains of Dsg1 and Dsg3 with the recombinant protein demonstrated that both mouse and human Dsg1, but not Dsg3, were directly cleaved in a dose-dependent manner. These results demonstrate that ETD and ETA induce blister formation by identical pathophysiological mechanisms. Clinical strains positive for edin-B were suggested to be clonally associated, and all edin-B-positive strains tested were positive for etd. Among 18 etd-positive strains, 12 produced ETD extracellularly. Interestingly, these strains are mainly isolated from other sources of infections and not from patients with bullous impetigo or staphylococcal scalded-skin syndrome. This strongly suggests that ETD might play a pathogenic role in a broader spectrum of bacterial infections than previously considered.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Exfoliative toxin (ET) is an exotoxin produced by staphylococcal species, causing blisters in human and animal skin (29). ET-producing Staphylococcus aureus is involved in staphylococcal scalded-skin syndrome (SSSS), or Ritter disease, and bullous impetigo (33; for a review, see references 21 and 29). SSSS is a generalized blistering skin disease which is primarily a disease of young children and neonates, but adults can also be affected (15). Its clinical manifestations begin abruptly with fever, skin tenderness, and erythema, followed by large sheets of epidermal separation involving the entire skin surface within the next few hours to days. Bullous impetigo is a localized form of SSSS and is frequently seen in older children at the site of infection, especially in the extremities. 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 the circulation and causes exfoliation at remote sites, while in bullous impetigo, the bacteria are present in the lesions. Serologically, ETs involved in human diseases mainly consist of two types, ETA and ETB (6, 27). Both toxins cause intraepidermal cleavage through the granular layer, without epidermal necrolysis or an inflammatory response of the skin (6, 19, 27). Several lines of evidence have suggested that ETs act as serine proteases to induce intraepidermal cleavage: (i) amino acid sequences of ETA and ETB show similarity with the S. aureus V8 serine protease (17), and the catalytic site of V8 protease is conserved in ETA (7); (ii) partially purified ETs preincubated with serine protease inhibitors exhibit delayed skin exfoliation (17); (iii) replacement of the serine residue with glycine in the putative catalytic site of ETA completely abolishes the exfoliative activity of the toxin (35, 38); and (iv) crystal structures of ETA (13, 53) and ETB (52) have recently been determined, and both types were shown to structurally belong to the chymotrypsin family of serine proteases. However, the exact target substrate was a mystery for 30 years, until the pathogenic role of ET was demonstrated in 1970 by using neonatal mice (29). Hints of a breakthrough were 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 blisters similar to those observed with SSSS or bullous impetigo (2, 32, 43). This observation led to the recent identification of Dsg1 as the specific substrate for ETA protease (3).

Virulence factors of staphylococci such as ET are accessory proteins which are not essential for cell growth or division. Genetic determinants for these factors are often associated with mobile genetic elements such as phages, plasmids, and pathogenicity islands (9, 30, 34). For instance, genes for ETA (eta) (58) and enterotoxins A (10) and E (14) are encoded on prophage genomes in the S. aureus chromosome, and genes for enterotoxin D (8), ETB (etb) (59), and EDIN-C (59) have been shown to exist on plasmids. The term "pathogenicity island" was first coined to describe large chromosomal virulence-associated segments in uropathogenic Escherichia coli (11). Since then, the definition has been broadened to indicate regions, ranging in size from an island of less than 2 kb with a single gene (44) up to a 200-kb island of multigene elements, which have been identified in many species (25). In staphylococcal species, at least three types of pathogenicity islands have been reported (28).

In systematic screening of ET genes in clinical S. aureus isolates from diseased patients, we found a strain whose SmaI-digested DNA fragment hybridized with probes for eta, etb, and edin-B (the gene for EDIN-B). Here we report the identification and characterization of a novel ET gene, etd, from this strain. We demonstrate that the etd gene is tandemly encoded in a DNA block with a serine protease gene and the edin-B gene, forming a pathogenicity island on the chromosome. Purified recombinant ETD induced exfoliation of the skin of neonatal mice and specifically cleaved Dsg1 as ETA did.

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Bacterial strains and culture conditions. S. aureus TY114 and TY129 were isolated from pus discharged in a cutaneous wound from a patient in the Clinical Laboratory Department in Hiroshima City Hospital. TY67 was a laboratory stock of a clinical isolate from an impetigo patient. Strains positive for edin-B were from a stock of clinical strains in M. Aepfelbacher’s laboratory in Munich, Germany (16). S. aureus RN4220 and RN450 (edin-B-, eta-, and etb-negative strains) were obtained from Richard Novick and Alexander Tomasz, respectively. Coagulase typing was performed by use of coagulase type-specific antisera (Denka-Seiken, Tokyo, Japan). DNA from S. aureus TY114 was cloned into E. coli XL-II Blue. S. aureus was grown in brain heart infusion broth (Difco Laboratories, Detroit, Mich.) or Trypticase soy broth (TSB) (Becton Dickinson). E. coli was grown in Luria-Bertani broth. When necessary, ampicillin (100 µg/ml) or kanamycin (10 µg/ml) was added for selection or plasmid maintenance.

Materials and chemicals. TSKgel HA1000 and TSKgel G3000 SW_XL were purchased from Tosoh, Tokyo, Japan. Lysostaphin was from SIGMA Genosys Japan, Tokyo, Japan. Recombinant ETA with a His tag on the carboxyl-terminal end was expressed in E. coli DH10B and recovered from the soluble fraction in lysis buffer (20 mM Tris-Cl [pH 8.0], 0.2 M NaCl, 0.2% Triton X-100, and protease inhibitor cocktail [Complete Mini, EDTA free; Roche Diagnostics, Mannheim, Germany]). Extracellular domains of mouse or human Dsg1 and Dsg3 with E tags and His tags on the carboxyl termini were expressed in a baculovirus and collected from culture supernatants as previously described (24). These recombinant ETA and Dsg proteins were purified by using TALON affinity resin (Clontech, Palo Alto, Calif.). Primers used in this study are listed in Table 1. Those for sea through sed, edin-B, and tst-1 were designed according to the nucleotide sequences deposited in GenBank: sea, accession number M18970; seb, M11118; sec, X05815; sed, M94872; edin-B, AJ277173; tst-1, U93688. Long range PCR was performed with a TaKaRa LA PCR kit (TAKARA Bio Inc., Osaka, Japan) according to the following amplification protocol: an initial denaturation step at 96°C for 1 min, 40 s, followed by 30 cycles of 96°C for 20 s of denaturation, 65°C for 16 min, and 72°C for 20 min.

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TABLE 1. Primers used in this study

DNA manipulation. Routine DNA manipulations were performed by standard procedures (39). Preparation of chromosomal DNA from staphylococcal cells and transformation of S. aureus by electroporation were performed as previously described (47). Southern blotting of DNA and hybridization were performed as described previously (46). The nucleotide sequence was determined by using a PRISM Dye Terminator cycle sequencing kit and an automated sequencer (ABI PRISM 310) (both from Perkin-Elmer Japan, Inc., Tokyo, Japan). Pulsed-field gel electrophoresis (PFGE) was performed according to standard protocols by using a CHEF DR-II apparatus (Bio-Rad Japan, Tokyo, Japan) (initial switch time, 5 s; final switch time, 40 s; run time, 22 h; voltage gradient, 200 V in Tris-borate-EDTA at 16°C).

Purification. Recombinant ETD (the orf1 product) expressed in S. aureus was purified from the culture supernatant as follows. S. aureus growing exponentially in TSB was inoculated into 1 liter of the same fresh medium and incubated with continuous agitation by a rotary shaker for 24 h at 37°C. The culture was centrifuged at 10,000 x g for 20 min at 4°C. Concentrated culture filtrate (CCF) was prepared by 80% saturated ammonium sulfate precipitation of the culture supernatant. CCF dialyzed against 10 mM phosphate buffer (pH 6.8) (buffer 1) was applied to a hydroxyapatite column (15 by 95 mm) which was equilibrated with buffer 1. The column was washed with buffer 1 until most of the unbound proteins had passed through. Bound proteins were eluted by stepwise elution with 100, 250, and 500 mM phosphate buffer (pH 6.8). The eluate with 100 mM phosphate buffer (pH 6.8) was dialyzed against buffer 1 and concentrated to 500 µl. The sample was loaded onto a TSKgel SW3000_XL (7.5 by 300 mm; Tosoh) and eluted with buffer 1 at a flow rate of 0.5 ml/min, and the active fraction was collected. The active fraction was further applied to a Bio-Scale CHT2-1 hydroxyapatite column (7 by 52 mm; Bio-Rad) and eluted with a linear gradient from 10 to 500 mM phosphate buffer (pH 6.8). For purification of His₆-tagged ET, primer sets were designed to amplify DNA fragments corresponding to the mature form of ETB or ETD (orf1). Amplified DNA fragments were cloned into pQE70 (Qiagen, Valencia, Calif.) in order to express the fusion protein with a His₆ tag sequence at the C terminus in E. coli. Recombinant proteins were purified by using nickel-nitrilotriacetic acid resin according to the manufacturer's protocol.

Antisera. The purified protein was emulsified with either Freund complete adjuvant or Freund incomplete adjuvant (Difco Laboratories) (50 µg of protein per ml). For each sample, 2-kg rabbits were immunized on day 1 with the sample emulsified with Freund complete adjuvant and every 2 weeks with the sample emulsified with Freund incomplete adjuvant. At 8 weeks, the rabbits were injected intravenously with 20 µg of the protein sample. Antisera were obtained 15 weeks after the first injection.

In vivo assay. To evaluate the exfoliative activity of ETD, neonatal ICR mice (age, <24 h) or 1-day-old chickens were subcutaneously injected with the toxin dissolved in 100 µl of phosphate-buffered saline (PBS), and the skin was analyzed by eye and microscopically at 1 to 18 h after injection.

Immunofluorescence. Cryosections of nonfixed skin from neonatal mice injected with ETD or PBS alone were used for indirect immunofluorescence to localize Dsg1 and Dsg3, as previously described (3). Anti-Dsg1 sera obtained from patients with pemphigus foliaceus and an anti-Dsg3 mouse monoclonal antibody, AK9, which specifically recognizes the extracellular domain of mouse Dsg3 (K. Tsunoda, T. Ota, M. Aoki, T. Yamada, T. Nagai, T. Nakagawa, S. Koyasu, T. Nishikawa, and M. Amagai, unpublished data) were used. Sections were examined under an Eclipse E800 fluorescent microscope (Nikon Corp., Tokyo, Japan).

In vitro assay. Approximately 1 µg of purified mouse or human Dsg1 or Dsg3 was incubated overnight at 37°C with the indicated amount of ETD in 50 µl of PBS with 1 mM CaCl₂. Digested samples were assessed by immunoblot analysis with an anti-E tag mouse monoclonal antibody (Pharmacia Biotech, Uppsala, Sweden) for detection of the recombinant proteins.

Other procedures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (immunoblotting) were carried out as described previously (49). For detection of ETD and EDIN-B in culture supernatants of clinical strains, S. aureus was grown in TSB (20 ml) with continuous agitation by a rotary shaker for 48 h at 37°C. CCF was prepared as described above and dissolved in 200 µl of PBS. An aliquot (5 µl) of CCF dialyzed against PBS was subjected to SDS-PAGE followed by immunoblotting. Protein was immunodetected by using Renaissance 4CN Plus (Dupont, NEN Research Products, Boston, Mass.). Protein concentrations were determined with the bicinchoninic acid protein assay reagent (Pierce, Rockford, Ill.) with bovine albumin as the standard. Production of enterotoxin and TSST-1 was assayed by using a SET-RPLA enterotoxin detection kit and a TST-RPLA TSST-1 detection kit (both from Denka-Seiken).

Nucleotide sequence accession number. The nucleotide sequence data presented in this report will appear in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession number AB057421.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Identification of a new cluster of virulence-related genes. In a systematic screening study for the ET gene and the edin gene in S. aureus clinical isolates from Hiroshima City Hospital, we found two strains showing PFGE bands faintly hybridizing with eta and etb probes. Interestingly, the same PFGE band could hybridize with the edin-B probe. Therefore, we prepared chromosomal DNAs from these strains and tested them by PCR amplification using mixed primers designed on the basis of the homology observed in the ETA, ETB, and ShETB (Staphylococcus hyicus ETB) genes. A single product of approximately 430 bp was amplified. Since this was close to the expected size of 434 bp, the PCR product was subjected to direct nucleotide sequencing. The nucleotide sequence obtained indicated that the relevant portion of the ORF similar to the ET gene was amplified. Since etb and edin-C are located close together on the ETB plasmid (59), we hypothesized that the ORF and edin-B were closely associated. Therefore, we designed specific primer sets for the ORF and edin-B, and we tested our hypothesis by PCR amplification using different combinations of the primers. Among these, a combination of ET-14 and ednB-2 generated a PCR product of ca. 2 kb. Sequencing of the DNA fragment revealed three ORFs (orf1, orf2, and orf3). The orf1 product exhibited homology to the C-terminal regions of various ETs, and the orf3 product exactly matched the N-terminal 224 amino acids of EDIN-B, suggesting that a potential ET gene and edin-B coexist at the same locus. An inverse PCR strategy was used to identify the flanking region of the 2-kb DNA fragment. Use of primers ET-15 and ednB-1 on EcoRI digests of chromosomal DNA allowed amplification of 1.7 kb of DNA. Sequencing and assembly of the DNA fragments resulted in a contiguous sequence of 2,943 bp which covers the full length of orf1, orf2, and orf3 (edin-B). Comparison of the initial sequence data to genome sequences of S. aureus Mu50 and N315 indicated that the DNA flanking orf1 and edin-B was not present in Mu50 and N315. The identification of possible virulence-related ORFs in such a small DNA fragment led us to search for flanking regions of the 3.3-kb DNA for relevant ORFs. Use of primers ET28 and ET29 on XbaI digests and of primers ET15 and ET40 on HindIII digests amplified 6.1- and 6.2-kb DNA fragments, respectively. Sequencing analysis of these DNA fragments and the concatenation of sequences obtained resulted in a contiguous sequence of 14,849 kb containing 9,054 kb of DNA unique to strain TY114 (Fig. 1). The G+C content of the 9,054 kb of unique DNA was 30.67%.

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FIG. 1. Schematic representation of the etd pathogenicity island. The 9,054 bp of DNA unique to the strain TY114 chromosome, including orf1, edin-B, and identified ORFs (shaded arrows), is presented. The genetic organization of the putative insertion region of the island on strain N315 chromosomal DNA is shown at the top. Solid arrows, ORFs found in N315.

In the unique sequence obtained, we identified seven protein-coding regions, including orf1 and edin-B (tentatively named orf1 to orf7) (Table 2). The orf1 product was found to display sequence similarity to ETA and ETB; it showed 40% sequence identity to ETA and 59% sequence identity to ETB (Table 3; Fig. 2). Recently, Sato et al. (40) identified a novel ET from S. aureus Horse-1, isolated from a horse with phlegmon, and cloned the gene. They designated it ETC. The orf1 product showed only 13% sequence identity to ETC. On the other hand, S. hyicus has been shown to produce several ETs (5, 41). Of these, genes for two serologically distinct toxins, ShETA and ShETB, have been cloned (54). The orf1 product showed 16% sequence identity to ShETA and 63% sequence identity to ShETB. Phylogenetic comparison of the orf1 product with known ETs revealed that the orf1 product is most similar to ShETB in its primary structure (Fig. 3). The orf2 product showed a predicted amino acid similarity with glutamyl endopeptidase of Bacillus intermedius (37). The orf7 product exhibited high sequence similarities to phage resistance proteins of Lactococcus lactis, such as AbiK (20), and it probably functions as an abortive infection protein against certain phages of S. aureus. In the 5' extremity, a single copy of IS256, which is 1,173 bp long and has terminal inverted repeats of 26 bp (positions 988 to 1013 and 2286 to 2311), is present. The presence of direct repeats of 8 bp in both boundaries of IS256 (positions 980 to 987 and 2312 to 2319) suggests that it was inserted as a single insertion element into the chromosomal DNA. ORFs just downstream of IS256 showed predicted amino acid similarities to the hsdS and hsdM products, the sequence specificity and modification functions of the type-IC restriction-modification system (26).

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TABLE 2. Features of etd pathogenicity island ORFs

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TABLE 3. Percent amino acid sequence identity among ETs and ORF1

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FIG. 2. Alignment of ETA, ETB, ETC, ORF1, ShETA, and ShETB. Conserved and identical amino acids are shaded. The multiple alignment was constructed by MacVector software (Genetics Computer Group, Oxford, England) using Clustal W. Amino acids corresponding to the catalytic triad of the S2 family of serine proteases (36) are boxed. Accession numbers are P09331 (ETA), AAA26628 (ETB), BAA99412 (ETC), BAB08178 (ShETA), and BAA99411 (ShETB).

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FIG. 3. Phylogenetic tree showing relationships among ETA, ETB, ETC, ORF1, ShETA, and ShETB. The multiple alignment was constructed by MacVector software (Genetics Computer Group) using Clustal W.

Junctions of unique DNA sequence. The junctions of the unique DNA sequences were defined by BLAST searches and PCR. BLAST searches determined that the unique DNA fragment of 9,054 bp (between positions 851 and 9904) was inserted into the chromosome between the ORFs SA2004 and SA2005 on the left and right sides, respectively, and a stretch of 98 bp between positions 2276975 and 2277072 on the N315 chromosome is missing in the unique DNA region (Fig. 4). At both ends of the unique DNA sequence, direct repeats of the pentamer AATTC were present, suggesting that integration of the DNA occurred through a recombination event. We next performed PCR using primers TY85 (on the left of the left junction) and TY86 (on the right of the right junction) based on the known sequences of N315 and Mu50. With a sample of chromosomal DNA from strain TY114 as a template, a PCR product with the expected fragment size of 9.4 kbp was generated by using the LA PCR kit (TAKARA Bio Inc.) (data not shown). In a control PCR using chromosomal DNA from RN450 as a template, a nucleotide product with the expected size of 475 bp appeared (data not shown). These results further support the finding that the unique DNA fragment is present in the chromosome at the specific site between ORFs SA2004 and SA2005 on the left and right sides, respectively, in TY114.

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FIG. 4. Left and right junction sequences. The etd pathogenicity island starts at the first arrow (position 2276975 in the N315 chromosome and position 851 in the deduced sequence) and ends at the second arrow (position 2277072 in the N315 chromosome and position 9904 in the deduced sequence). Direct repeats of the pentamer AATTC are boxed.

The orf1 product is a new member of the ETs. To confirm that the orf1 product actually possesses toxin activity, orf1 was cloned into the S. aureus-E. coli shuttle vector pCL8 to generate pTY179, and the gene product was purified from the culture supernatant of TY2182, a derivative of S. aureus RN4220 which was transformed with pTY179. The molecular mass of the purified protein is 27.2 kDa, and the N-terminal sequence, NTYEE, exactly matches the sequence starting from the amino-terminal residue at position 33 of the orf1 product. The sequence starting from the ATG codon with an extension of 32 amino acids is assumed to be a signal sequence, since the protein is extracellularly secreted. We injected 20 µg of the purified protein (mORF1) subcutaneously into neonatal mice. The mice started to show gross blisters around the injection site within 1 h after injection (Fig. 5). Consequently, we constructed a DNA fragment corresponding to the processed form of orf1 by PCR and placed it in frame into a plasmid to express a His₆-tagged fusion protein; then this His₆-tagged protein was purified. Similar results were obtained with injection of the purified His₆-tagged protein into the mice (data not shown). Previous studies demonstrated that ShETs exhibited exfoliative activity in a 1-day-old chicken but not in a neonatal mouse (42). Since the orf1 product was most similar to ShETB in primary structure, we inoculated mORF1 into the back skins of 1-day-old chickens to see whether it showed exfoliative activity toward chickens. However, no exfoliation was observed upon injection of as much as 50 µg of the protein, even after 24 h postinoculation. We prepared a mORF1-specific antibody by using the mORF1 purified from the culture supernatant of TY2182 as described in Materials and Methods, and we compared the serological properties of mORF1 with those of ETA and ETB. As shown in Fig. 6, anti-mORF1 reacted specifically with mORF1 and reacted very weakly with ETB but not at all with ETA. Taken together, these results clearly demonstrated that mORF1 possesses exfoliative activity toward neonatal mice and that it is serologically distinct from ETA and ETB. We therefore designated this protein ETD, as a new member of the ETs produced by S. aureus.

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FIG. 5. Exfoliative activity of mORF1 in neonatal mice. Neonatal mice injected with mORF1 show extensive blisters within 1 h after injection (b), while neonatal mice injected with saline alone do not show blisters (a). Histological examination of the mouse injected with mORF1 shows the characteristic splitting at the granular layer (d), while that of the mouse injected with saline shows intact skin (c).

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FIG. 6. Immunodetection of ET by the anti-mORF1 antibody. Purified recombinant ETA, ETB, and mORF1 were resolved on an SDS-12% PAGE gel and either stained with Coomassie brilliant blue (a) or transferred to a nitrocellulose membrane (b through d). The membrane was subjected to immunodetection by an anti-ETA antibody (b), anti-ETB antibody (c), or anti-mORF1 antibody (d). Lanes 1, ETA (0.7 µg); lanes 2, ETB (0.7 µg); lanes 3, mORF1 (0.6 µg).

ETD selectively digests Dsg1 but not Dsg3. ETA has been shown to specifically target the mouse and human desmosomal cadherin Dsg1, but not Dsg3, and to cleave Dsg1 in vitro and vivo (3). To determine whether ETD affects Dsg1 as does ETA, we examined skin from neonatal mice injected with purified ETD by immunofluorescence with antibodies to Dsg1 and Dsg3. When skin was examined 1 h after injection of 2 µg of the protein, the immunofluorescence of Dsg1 was markedly diminished, whereas the immunofluorescence of Dsg3 was not affected (Fig. 7). These results clearly indicated that Dsg1 was selectively affected by ETD in vivo. To demonstrate the direct proteolysis of the extracellular domain of Dsg1 by ETD, we incubated the recombinant protein representing the entire extracellular domain of Dsg1 and Dsg3 with purified ETD in vitro. It cleaved the 81-kDa recombinant mouse Dsg1 down to a 29-kDa peptide in a dose-dependent fashion, but it did not cleave mouse Dsg3 at all (Fig. 8A). It also cleaved recombinant human Dsg1 in the same manner, but not human Dsg3 (Fig. 8B). These findings clearly indicate that ETD selectively recognizes and cleaves the extracellular domain of mouse and human Dsg1 as does ETA.

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FIG. 7. Immunofluorescence for desmogleins in the epidermis of mice injected with ETD. Neonatal mice were injected with PBS (A and C) or ETD (B and D) and stained for Dsg1 (A and B) or Dsg3 (C and D). Cell surface staining of Dsg1 in mice injected with ETD is much weaker than that in mice injected with saline alone, whereas the staining of Dsg3 is not affected by injection with ETD. Arrowheads indicate epidermal basement membrane.

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FIG. 8. In vitro treatment of baculovirus-expressed recombinant desmogleins by ETD. Approximately 1 µg of the purified extracellular domain of mouse (left) or human (right) Dsg1 or Dsg3 produced by a baculovirus was incubated with various concentrations of ETD. The extracellular domain of Dsg1, but not that of Dsg3, was cleaved by ETD in a dose-dependent fashion. Arrows point to the 29-kDa degraded band of Dsg1. Bars on the left side indicate molecular size standards of 150, 100, 50, and 25 kDa (from top to bottom).

edin-B-positive strains are positive for etd. We analyzed the genomic fingerprints and genetic and phenotypic characteristics of edin-B-positive strains from Germany and Japan, and we screened edin-B-positive strains for the presence of etd. As shown in Fig. 9, we confirmed that their DNA patterns by PFGE analysis were very similar, as reported previously (16). A DNA fragment of ca. 140 kb hybridized with both the edin-B and etd probes, indicating that all edin-B-positive strains were positive for etd. By performing immunoblotting using antibodies against ETD, we found that the 12 edin-B- and etd-positive strains produced ETD (Fig. 9). Of 18 strains, 16 strains belonged to coagulase type 2 and 2 strains were nontypeable. Southern blot analysis demonstrated that six strains were sec positive and two strains were seb positive (Fig. 9). This is in contrast with the previous observation that strains positive for eta or etb were negative for enterotoxin genes (sea through sed) (60).

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FIG. 9. PFGE and genotypic and phenotypic characterizations of edin-B-positive S. aureus strains. SmaI-digested DNA of edin-B-positive strains was subjected to PFGE and was run on agarose gels. Gels were subjected to Southern hybridization with probes for detecting genes of interest. DNA fragments that hybridized with the probe for detecting the gene of interest are boxed in green (etd and edin-B), yellow (seb), or red (sec). The hybridization signals for edin-B and etd corresponded to the same ca. 140-kb DNA fragment, and that for an enterotoxin gene (seb or sec) corresponded to a 320- to 350-kb DNA fragment. At the bottom, results of coagulase serotyping and data on production of ETD, EDIN-B, enterotoxin, and TSST-1 are summarized. Production of ETD and EDIN-B was assessed by immunoblotting as described in Materials and Methods. Production and serotyping of enterotoxins and TSST-1 were assayed by using the SET-RPLA and TST-RPLA detection kits, respectively. NT, nontypeable. Lanes: 1, TY114; 2, TY129; 3, TY67; 4, TY130; 5, TY134; 6, TY136; 7, TY137; 8, TY140; 9, TY132; 10, TY133; 11, TY135; 12, TY138; 13, TY139; 14, TY141; 15, TY142; 16, TY143; 17, TY144; 18, TY131. Lanes 1 to 3 show results for Japanese strains; lanes 4 to 18 show results for German strains.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we have identified a novel ET gene, etd, in a clinical isolate of S. aureus and demonstrated that the etd gene is located together with a putative glutamyl endopeptidase gene and edin-B gene within a unique region of DNA not found in N315, Mu50, or NCTC 8325. According to the concept previously presented (22), we designated this region the etd pathogenicity island for the following reasons: (i) the region contains at least three loci possibly associated with virulence, the etd gene, the edin-B gene, and orf2, which shows a predicted amino acid similarity with the glutamyl endopeptidase gene of B. intermedius (37); (ii) the etd and edin-B genes are present in pathogenic but not laboratory strains of S. aureus; (iii) IS256 is present near the 5' border of the DNA region; and (iv) two ORFs, hsdS and hsdM, encoding the sequence specificity and modification functions (26), are present. hsdS and hsdM are also present in the reported pathogenicity islands in the chromosomes of S. aureus N315 (SaPIn2 and SaPIn3) and Mu50 (SaPIm2 and SaPIm3) (28). It has been suggested that such restriction-modification systems essentially stabilize the maintenance of the linked region (26). As in those pathogenicity islands, the hsdR homologue was not present in the etd pathogenicity island. The etd pathogenicity island was inserted at a chromosomal site between ORF SA2004 and ORF SA2005 of strain N315 (Fig. 1). The G+C content of the island (30.67%) was lower than that of the N315 chromosome, 32.8%, supporting the notion that the DNA region is acquired through a horizontal transfer event.

Purified ETD induced intraepidermal cleavage through the granular layer of the epidermis of neonatal mice which is pathologically indistinguishable from that induced by ETA or ETB. Furthermore, ETD was shown to target Dsg1 but not Dsg3 and to specifically cleave the extracellular domain of Dsg1. ETB has recently been demonstrated to target and cleave Dsg1 but not Dsg3 in vitro and in vivo (4). These results, taken together, indicate that the three ETs share a common target, Dsg1, as the substrate, although their primary amino acid sequences diverged considerably. Therefore, formation of superficial blisters by these toxins can be explained by an identical molecular pathophysiological mechanism through specific digestion of Dsg1 by these toxins. In the epidermis, two isotypes of desmosomal cadherin, Dsg1 and Dsg3, are present, and these proteins possess overlapping functions in cell-to-cell attachment (31, 57). Dsg1 is expressed throughout the epidermis, while Dsg3 is in the lower portion of epidermis (31, 57). Consequently, blistering formation through Dsg1 digestion by ET occurs in a superficial region, namely, in the granular layer, and the lower epidermis is functionally protected by the coexpressed Dsg3. Like ETA and ETB, ETD shows amino acid identity (23%) to the staphylococcal V8 protease. This identity includes the catalytic triad of the S2 family of serine proteases (36), Ser67-His115-Asp189, which forms the active site of alpha-lytic endopeptidases (12, 18, 36) (Fig. 2). This suggests that ETD is also a glutamyl endopeptidase, but final identification awaits the determination of the cleavage site of Dsg1. It is intriguing that this catalytic triad was not conserved in ETC and ShETA (Fig. 2). Understanding the common mechanism of recognition and digestion of Dsg1 by ETs requires further investigation.

Southern blot analysis of edin-B-positive clinical isolates from Germany and Japan demonstrated that all of the edin-B-positive strains were positive for etd. Furthermore, most of them secreted both ETD and EDIN-B (16) into the culture supernatant. EDIN is another exotoxin produced by S. aureus. There are at least three types of EDIN isoforms, EDIN-A (23), EDIN-B (C3stau) (56), and EDIN-C (59), produced by S. aureus. The prototype of this toxin family, EDIN-A, was initially discovered as an inhibitor of morphological differentiation of epidermal keratinocytes in vitro and was designated epidermal cell differentiation inhibitor (EDIN) (45). It was later shown to be a mono-ADP-ribosyltransferase that specifically inhibits eukaryotic small GTP-binding proteins belonging to the Rho family (48) and is now regarded as a member of the large family of bacterial Rho-specific mono-ADP-ribosyltransferases, the C3 family (55). Rho GTPases are central regulators of the eukaryotic actin cytoskeleton (32, 50, 51), and their inactivation by toxins and modulins of virulent bacteria has been shown to block important cellular functions (1). It has previously been demonstrated that etb and edin-C are colocated on an ETB plasmid (59). In this study, edin-B and etd are shown to be localized in the same locus on the chromosome. These results suggest the close association of these genes during evolution. Despite its presumed importance in virulence, the exact role of EDIN in the pathogenesis of S. aureus remains to be elucidated.

DNA fingerprinting and coagulase typing further suggested that etd-positive and edin-B-positive strains are clonally associated. ET has been considered a relatively simple virulence factor that causes blistering, and it has been well accepted that ET-producing strains are associated with bullous impetigo and SSSS. However, to our surprise, all of the German strains in this study were isolated not from impetigo lesions but from other sources of infection (16). Two of the three isolates from Japan, including the original strain, TY114, were from pus discharged from skin wounds, and the other isolate was from an impetigo patient. We screened 88 S. aureus strains isolated from lesions of impetigo patients for etd and found only one strain positive for etd and edin-B (data not shown). These results suggest that the strains belonging to this clonal group are not strongly associated with the onset of bullous impetigo, although most of them produce an active ET, ETD. Our study raises the possibility that ET might play a broader pathogenic role in a variety of infections than previously considered, e.g., it might function in such a way that it destroys epithelial barriers, thus helping the bacteria to spread or to invade the tissues for the exacerbation of infection. Further epidemiological study of ETD- and EDIN-B-producing S. aureus and a comparative genomic study of this clonal group with those causing blistering diseases in humans may identify the pathological function of ETD in S. aureus infection.

 
We are grateful to Yukiko Hada for technical assistance and to Chia Y. Lee for the S. aureus-E. coli shuttle plasmid. We are also grateful to Neil Ledger for editorial assistance. We thank the Research Center for Molecular Medicine, the Research Facility for Laboratory Animal Science, and the Research Facility of the Hiroshima University Faculty of Dentistry for allowing us to use their facilities.

This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture, a research grant from the Tsuchiya Foundation, and Health Science Research grants for research on specific diseases from the Ministry of Health and Welfare of Japan.

 
* Corresponding author. Mailing address: Department of Bacteriology, Hiroshima University Graduate School of Biomedical Sciences, Kasumi 1-2-3, Minami-ku, Hiroshima City, Hiroshima 734-8553, Japan. Phone: (81) 82 257 5635. Fax: (81) 82 257 5639. E-mail: sugai{at}hiroshima-u.ac.jp.

Editor: V. J. DiRita

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Infection and Immunity, October 2002, p. 5835-5845, Vol. 70, No. 10
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