Virulent strains of Staphylococcus aureus secrete exfoliative toxins (ETs) that cause the loss of cell‐cell adhesion in the superficial epidermis. S. aureus ETs are serine proteases, which exhibit exquisite substrate specificity, and their mechanisms of action are extremely complex. To date, four different serotypes of ETs have been identified and three of them (ETA, ETB and ETD) are associated with toxin‐mediated staphylococcal syndromes related to human infections leading to diseases of medical and veterinary importance.
- epidermolytic diseases
- Staphylococcus aureus
- exfoliative toxins
- Desmoglein 1
The exfoliative toxins (ETs) also known as epidermolytic toxins, are serine proteases secreted by
In 1878, Baron Gottfried Rotter Von Rittershain described the clinical features of epidermal exfoliation in newborns . The relationship between skin exfoliation and
The principal isoforms of exotoxins implicated in human skin damage are exfoliative toxin A (ETA) and exfoliative toxin B (ETB) . Exfoliative toxin C (ETC) isolated from a horse infection has not been associated with human disease. In 2002, a new exfoliative toxin (ETD) was identified in a clinical sample of
The ETA and ETB serotypes are homologous, have molecular masses of approximately 27 kDa, and contain 242 and 246 amino acids, respectively  and present identical dermatologic symptoms [26, 27].The ETA serotype was described as being heat stable whereas the ETB serotype has been demonstrated to be heat labile. The ETC serotype with a molecular mass of 27 kDa is also heat labile and causes exfoliation in mice and chickens .
2. Exfoliative toxins and associated diseases
ET‐producing strains of
Both ETA and ETB are distinguished by the extent of the damage caused in the epidermis [29, 30]. SSSS clinical manifestations involve fever, skin hypersensitivity, and erythema followed by superficial blister formation and skin separation, leaving long areas of denuded skin [10, 31]. In the localized form, toxin production and formation of flaccid blisters with purulent fluid occur [12, 30]. SSSS occurs mainly in newborns and children with occurrences in adults being rare [11, 32]. The mortality rate in children submitted to immediate treatment is low .
The greater susceptibility of children has been attributed to the immature immune system, weak renal clearance of the toxin, and the fact that children are common carriers of microorganisms . In the most severe cases, exfoliation may affect the entire corporal surface . The quick and sensitive diagnosis of those infections may be performed using radioimmunoassays, enzyme‐linked immunosorbent assays, the reverse passive latex agglutination assay  as well as the polymerase chain reaction (PCR) to amplify the genes that codify ETs.
When the ET serotypes and the clinical forms of the disease were correlated, the ETA toxin was found to be associated with bullous impetigo formation, whereas ETB was found to be associated with SSSS, a generalized manifestation . The ETB plasmid has multiple genes that confer antibiotic resistance, which contributes to the increased resistance of
In addition to
Currently many phylogenetically distant hosts are described as being susceptible to exfoliation caused by the same isoforms of ET, revealing a certain specificity for various host organisms . Among six different ETs (SHETA, SHETB, ExhA, ExhB, ExhC, and ExhD) codified by
In Japan, hospital‐acquired methicillin‐resistant
3. Structural biology and mechanism of exfoliative toxins
The crystal structure of ETA was the first to be determined in atomic detail , followed by ETB  and by ETD  and currently, the atomic coordinates of six ET structures have been deposited with the Protein Data Bank (www.rcsb.org). The crystallographic structures of ETs have revealed much about their mechanisms of action, lack of hydrolytic activity against substrates in the native state, and the susceptibility of certain constituent layers of the epidermis to disruption by ETs.
4. Similarities and differences among ETs and other serino proteinases
Exfoliative toxins are glutamic‐acid specific trypsin‐like serine proteinases that share 50% sequence identity but display very low sequence identity with other serine proteases. The significant sequence identity of ETs is also reflected in the high structural similarity as evidenced by the low RMSD values of the superposed structures (ETA‐ETB: 0.9, ETA‐ETD: 1.3, and ETB‐ETD: 0.6). Similar to other trypsin‐like serine‐proteinases, the three‐dimensional structures of ETs are characterized by two six‐stranded β‐barrels domains, S1 and S2, whose axes lie roughly perpendicular to each other, a Greek key motif consisting of four antiparallel strands and N‐ and C‐terminal extensions. The amino acids constituting the catalytic triad (His‐Ser‐Asp) and Thr190 and His213 which are characteristic of glutamate‐specific serine proteinases are located at the junction of the S1 and S2 domains [51, 53].
ETs specifically cleave both mouse and human desmoglein 1 following glutamic acid 381, however only the presence of the Glu381–Gly382 bond, highly conserved in desmogleins, does not guarantee hydrolysis. The prerequisites for the exquisite specificity exhibited by ETs involves not only the presence of this cleavage site, but, also (1) the presence of the highly charged N‐terminal alpha‐helix, (2) the calcium dependent conformation of its substrate Dsg‐1, and (3) existence of a specific sequence 110 residues upstream of the cleavage site of the substrate Dsg‐1, characteristics that differentiate them from other typical glutamic‐acid‐specific serine proteinases of the chymotrypsin family.
This N‐terminal extension which is unique to ETs and its deletion results in an inactive protein [53, 54] that interacts with residues in loop 2 thereby coordinating and determining the architecture of the S1 pocket and hence contributing to substrate specificity [51–53] by modifying the pocket entrance. The amino acid sequences (Figure 1D) and the conformations in loop2 (Figure 1E) are different in the ETs. In ETA (Figure 1E), this loop is longer than in ETB and ETD, additionally its Trp14 and Tyr18 present in the N‐terminal helix are buried deeper in the S1 pocket than in ETB which contains Lys and Glu and in ETD with Arg and Lys at these equivalent positions. In the other trypsin‐like serine proteinases, the presence of a disulfide bridge determines the conformation of the pocket (Figure 1A).
5. Tyrosines 157 and 159 are essential for ETB activity
Based on the results of site‐directed mutagenesis, Sakurai et al.  concluded that the substitution of either Tyr 157 or 159 in ETB decreased exfoliative activity and the double mutation resulted in the complete loss of exfoliative activity and antigenicity. Interestingly, ETA does not possess either one of these tyrosines but contains Phe and His at these positions and in ETD these positions are occupied by Tyr and Thr.
6. Why are the exfoliative toxins inactive in the native states?
Gly193 is highly conserved in serine proteinases, however, in structures of ETs the peptide bond between residues 192 and 193 (chymotrypsin numbering) is flipped 180° relative to the other serine proteases. Pro192 in ETA and ETD and Val192 (ETB) form hydrogen bonds with both the amide nitrogen atoms and the hydroxyl oxygen atoms of the catalytic serine residues interrupting the charge‐relay‐network. These enzymes can only be functional if this bond is ruptured and the conformation is restored as in other serine proteinases.
7. Molecular mechanisms of the
exfoliative toxin S. aureus
exfoliative toxins selectively and directly solubilize mouse and human desmoglein 1 S. aureus
In 1970, Melish and Glasgow first investigated mechanisms of action of the exfoliative toxin (ET)‐producing
In 2000, Amagai and colleagues established desmoglein 1 (Dsg1), a desmosomal cadherin‐type adhesion molecule and also known as pemphigus foliaceus autoantigen, as the target of
In humans, there are four subclasses of Dsg with different tissue distributions. Among them, Dsg2 is expressed in all desmosome‐bearing tissues, whereas Dsg1 and Dsg3 are expressed preferentially in stratified squamous epithelia . Dsg1 and Dsg3 are hypothesized to have compensatory effects . For example, if both Dsg1 and Dsg3 express in the same epithelial cells, and adhesive function by Dsg1 is abolished, the loss of adhesive function can be compensated by intact Dsg3. In the epidermis, Dsg1 is expressed in the whole layers, whereas Dsg3 is expressed in basal and immediate suprabasal layers . In contrast, in oral mucous membrane, both Dsg1 and Dsg3 are expressed in the whole layer, but the expression level of Dsg1 is relatively low compared with that of Dsg3 . As Dsg2 and Dsg4 are expressed weakly in basal and upper granular layers, respectively , these molecules may have less ability to compensate the loss of Dsg1 function.
Desmocollin (Dsc) 1, another desmosomal cadherin is also expressed in superficial epidermis. It is hypothesized that Dsg1 and Dsc1 may have combinational effect on integrity of keratinocyte cell adhesion : Abolishment of either Dsg1 by ETs or genetic ablation of Dsc1 causes dissociation of keratinocytes in the superficial layer of mouse epidermis [10, 13, 59, 67]. If adhesive function of Dsg1 is abolished by ETs, it may cause keratinocyte separation only in spinous‐to‐granular layers of epidermis, in which loss of adhesive function by Dsg1 could not be compensated by other Dsgs. This could be a reasonable explanation why ETs cause only superficial epidermal blisters in SSSS patients, although ETs produced in upper respiratory organs (e.g., tonsils), enter the circulatory system and induce toxemia .
ETs are unique glutamate‐specific serine proteases that hydrolyze a single peptide bond within the extracellular segment of Dsg1 S. aureus
Hanakawa et al. demonstrated that substitution of catalytic serine in ETA, ETB and ETD to alanine causes loss of their functions to solubilize Dsg1 . Kinetic analysis of three ETs revealed kcat/Km values in the range of 2–6 × 104 M-1 s-1, suggesting their efficient enzymatic activity to digest relatively large molecules. These findings indicate that three known
The same group also investigated substrate‐specificity of
7.3. Possible mechanisms of ET‐associated keratinocyte dissociation
Desmosomes composed of two major transmembrane cadherin‐type adhesion molecules (Dsg and Dsc) and cytoplasmic plaque proteins that link between desmosomal cadherins and intracellular cytoskeletons. It has been long debated questions whether disruption of Dsgs alone by pemphigus autoantibodies is sufficient to cause keratinocyte dissociation, or subsequent disorganization of other desmosomal consituents in plasma membrane of keratinocytes is necessary .
To determine whether cleavage of the extracellular segment of Dsg1 by
Meanwhile, Simpson et al. proposed another theory for ET‐induced keratinocyte dissociation through sequestration of plakoglobin (PG), a member of catenin family cytoplasmic protein, by ectodomain‐deleted Dsg1 . When truncated Dsg1, in which amino acids 1–381 were spliced to mimic ET‐cleaved carboxy‐termini of Dsg1, was expressed in primary human keratinocytes, it reduced mechanical strength of keratinocyte sheets in a dose‐dependent manner, implicating a dominant‐negative effect by truncated Dsg1. Truncated Dsg1 localized in close to intercellular borders and reduce endogenous desmosomal cadherin Dsc3 and desmosomal plaque protein desmoplakin in intercellular borders. In the same cells, PG localized in intercellular borders and seem to be associated with truncated Dsg1. Remarkably, triple‐point mutation of the PG‐binding region in the truncated Dsg1 restored mechanical integrity of keratinocyte sheets, implicating that PG binding to truncated Dsg1 is essential in disruption of desmosomes and subsequent keratinocyte dissociation.
Putting all these findings together, the authors advocate a theory that cleavage of Dsg1 by ETs initiate keratinocyte dissociation, while subsequent PG sequestration may contribute to the expansion of intercellular spaces between keratinocytes (Figure 4). Further accumulation of
7.4. How ET‐producing
penetrate the epidermis through firm keratinocyte adhesion in the upper stratum granulosum? S. aureus
The aforementioned theory can satisfactorily explain how ETs cause blistering in SSSS, in which ETs access to the skin from dermal side. However, this theory cannot explain the mechanisms of blistering in bullous impetigo, in which ET‐producing
To address this issue, we recently established a mouse model of bullous impetigo .
Based on these findings, we propose a novel hypothesis for percutaneous entry of ET‐producing