Necrotic activity of ExhC from Mammaliicoccus sciuri is mediated by specific amino acid residues.

Mammaliicoccus sciuri, a commensal and pathogenic bacterium of significant clinical and veterinary relevance, expresses exfoliative toxin C (ExhC), a specific glutamyl endopeptidase belonging to the chymotrypsin family as the principal virulence factor. However, unlike most members of this family, ETs are inactive against a wide range of substrates and possess exquisite specificity for desmoglein-1 (Dsg1), a cadherin-like adhesion molecule that is crucial to maintain tissue integrity, thereby preventing the separation of skin cells and the entry of pathogens. ExhC is of clinical importance since in addition to causing exfoliation in pigs and mice, it induces necrosis in multiple mammalian cell lines, a property not observed for other ETs. Previous experiments have implicated the ExhC79-128 fragment in causing necrosis. Site-directed mutagenesis of specific residues within this fragment were studied and led to the design of an ExhC variant containing four-point mutations (ExhCmut4) lacking necrotic potential but retaining nearly wild-type (wt) levels of enzymatic activity. Moreover, the determination of the ExhCwt and ExhCmut4 crystal structures identified the conformation in the necrosis-linked region. These results constitute an important step toward the understanding of the mechanisms underlying the necrotic and epidermolytic activity of ExhC.

The Staphylococcus aureus superantigen SElX is a bifunctional toxin that inhibits neutrophil function.

Bacterial superantigens (SAgs) cause Vß-dependent T-cell proliferation leading to immune dysregulation associated with the pathogenesis of life-threatening infections such as toxic shock syndrome, and necrotizing pneumonia. Previously, we demonstrated that staphylococcal enterotoxin-like toxin X (SElX) from Staphylococcus aureus is a classical superantigen that exhibits T-cell activation in a Vß-specific manner, and contributes to the pathogenesis of necrotizing pneumonia. Here, we discovered that SElX can also bind to neutrophils from human and other mammalian species and disrupt IgG-mediated phagocytosis. Site-directed mutagenesis of the conserved sialic acid-binding motif of SElX abolished neutrophil binding and phagocytic killing, and revealed multiple glycosylated neutrophil receptors for SElX binding. Furthermore, the neutrophil binding-deficient mutant of SElX retained its capacity for T-cell activation demonstrating that SElX exhibits mechanistically independent activities on distinct cell populations associated with acquired and innate immunity, respectively. Finally, we demonstrated that the neutrophil-binding activity rather than superantigenicity is responsible for the SElX-dependent virulence observed in a necrotizing pneumonia rabbit model of infection. Taken together, we report the first example of a SAg, that can manipulate both the innate and adaptive arms of the human immune system during S. aureus pathogenesis.

Staphylococcus sciuri exfoliative toxin C is a dimer that modulates macrophage functions.

Staphylococcus sciuri causes multiple infections in humans. Recently, a strain of S. sciuri (HBXX06) carrying exfoliative toxin C (ExhC) was reported to cause fatal exudative epidermal skin pathology in piglets and might be considered as a potential zoonotic agent. However, little is known about the pathogenicity of this bacterium. In this study, we examined the activity of recombinant ExhC-his (rExhC) protein using newborn mice as the model and investigated the effect of rExhC on macrophage functions. Interestingly, we found that both rExhC and S. sciuri ExhC existed as dimers and that rExhC inhibited the phagocytosis of RAW264.7 cell lines but enhanced the production of proinflammatory mediators, such as interleukin-6, interleukin-12, tumor necrosis factor α, and nitric oxide, by murine peritoneal macrophages and RAW264.7 cells. These results suggest that ExhC may play an important role in innate immune response against S. sciuri infection.

Plakoglobin rescues adhesive defects induced by ectodomain truncation of the desmosomal cadherin desmoglein 1: implications for exfoliative toxin-mediated skin blistering.

Desmoglein 1 (Dsg1) is a desmosomal cadherin that is essential to epidermal integrity. In the blistering diseases bullous impetigo and staphylococcal scalded-skin syndrome, pathogenesis depends on cleavage of Dsg1 by a bacterial protease, exfoliative toxin A, which removes residues 1 to 381 of the Dsg1 ectodomain. However, the cellular responses to Dsg1 cleavage that precipitate keratinocyte separation to induce blister formation are unknown. Here, we show that ectodomain-deleted Dsg1 (Δ381-Dsg1) mimics the toxin-cleaved cadherin, disrupts desmosomes, and reduces the mechanical integrity of keratinocyte sheets. In addition, we demonstrate that truncated Dsg1 remains associated with its catenin partner, plakoglobin, and causes a reduction in the levels of endogenous desmosomal cadherins in a dose-dependent manner, leading us to hypothesize that plakoglobin sequestration by truncated Dsg1 destabilizes other cadherins. Accordingly, a triple-point mutant of the ectodomain-deleted cadherin, which is uncoupled from plakoglobin, does not impair adhesion, indicating that this interaction is essential to the pathogenic potential of truncated Dsg1. Moreover, we demonstrate that increasing plakoglobin levels rescues cadherin expression, desmosome organization, and functional adhesion in cells expressing Δ381-Dsg1 or treated with exfoliative toxin A. Finally, we report that histone deacetylase inhibition up-regulates desmosomal cadherins and prevents the loss of adhesion induced by Dsg1 truncation. These findings further our understanding of the mechanism of exfoliative toxin-induced pathology and suggest novel strategies to suppress blistering in bulbous impetigo and staphylococcal scalded-skin syndrome.

In vitro keratinocyte dissociation assay for evaluation of the pathogenicity of anti-desmoglein 3 IgG autoantibodies in pemphigus vulgaris.

Patients with pemphigus vulgaris (PV) have circulating anti-desmoglein (Dsg) 3 immunoglobulin G (IgG) autoantibodies that induce blister formation. We developed an in vitro quantitative assay to evaluate the pathogenic strength of anti-Dsg3 IgG autoantibodies in blister formation. To obtain intercellular adhesion mediated dominantly by Dsg3, we used primary cultured normal human keratinocytes expressing low level of Dsg2 in the presence of exfoliative toxin A that specifically digests Dsg1. After incubation with various antibodies, monolayers released by dispase were subjected to mechanical stress by pipetting, and the number of cell fragments were counted. When anti-Dsg3 monoclonal antibodies (mAb) obtained from pemphigus model mice were tested, pathogenic AK23 mAb yielded significantly higher number of cell fragments than AK7 or AK20 non-pathogenic mAb. Dissociation scores, defined with AK23 mAb as the positive control, were significantly higher with active stage PV sera (n=10, 77.4+/-21.4) than controls (n=11, 16.0+/-9.6; p=0.003). When pair sera obtained from 6 PV patients in active stage and in remission were compared, the dissociation scores reflected well the disease activity as those in active stage were four to 17 times higher than those in remission. When sera from different patients showing similar ELISA scores but different clinical severity were tested (n=6), the dissociation scores with sera from severe disease activity were significantly higher than those with sera in remission. These findings indicate that this dissociation assay will provide a simple and objective biological method to measure the pathogenic strength of pemphigus autoantibodies.

Desmogleins 1 and 3 in the companion layer anchor mouse anagen hair to the follicle.

Anchorage of the hair to its follicle is of paramount importance for survival of rodents in the wild, and is aberrant in some human alopecias. Little is understood about the mechanisms responsible for hair shaft anchorage. Desmoglein (Dsg)3-/- (knockout) mice lose hair during telogen, but their anagen hairs remain anchored to the follicle. We hypothesized that Dsg1 compensates for the loss of Dsg3 in the anagen hair follicles of these Dsg3-/- mice. Consistent with this hypothesis, we found Dsg1 and Dsg3 expression overlapping in the companion layer. To functionally address this hypothesis, we used exfoliative toxin A (ETA) to inactivate Dsg1 in Dsg3-/- mice. Four hours after injection of ETA, Dsg3-/- mice, but not Dsg3+/+ or Dsg3+/- mice, showed striking loss of anagen hair, which was confirmed and quantitated by gentle tape stripping. Histology of the skin of these mice as well as of the tape-stripped hair showed separation between the outer root sheath and inner root sheath of the hair follicle, at the plane of the companion layer. Immunostaining for trichohyalin and K6, which highlights the companion layer, in skin and stripped hair confirmed the plane of separation. Labeling of proliferating cells with bromodeoxyuridine demonstrated that the matrix keratinocytes responsible for producing the hair shaft were below the split and remained in the follicle after loss of the anagen hair. These findings demonstrate the importance of the companion layer, and particularly the Dsg1 and Dsg3 in this layer, in anchoring the anagen hair to the follicle.

Calcium-dependent conformation of desmoglein 1 is required for its cleavage by exfoliative toxin.

In bullous impetigo, Staphylococcus aureus spreads under the stratum corneum of skin by elaboration of exfoliative toxin, which hydrolyzes only one peptide bond in a highly structured calcium-binding domain of desmoglein 1, resulting in loss of its function. We investigated the basis of this exquisite specificity. Exfoliative toxin cannot cleave desmoglein 1 pretreated at 56 degrees C or higher or at low or high pH, suggesting that the proper conformation of desmoglein 1 is critical for its cleavage. Because cleavage occurs in an area of desmoglein 1 stabilized by calcium, we determined if the conformation necessary for cleavage is calcium-dependent. Depletion of calcium from desmoglein 1 completely inhibited its cleavage by exfoliative toxin, even after calcium was added back. A change in conformation of desmoglein 1 by calcium depletion was shown, with immunofluorescence and enzyme-linked immunoassay, by loss of binding of PF sera, which recognize conformational epitopes. This change in conformation was confirmed by tryptophan fluorometry and circular dichroism, and was irreversible with repletion of calcium. These data suggest that the specificity of exfoliative toxin cleavage of desmoglein 1 resides not only in simple amino acid sequences but also in its calcium-dependent conformation.

Staphylococcal exfoliative toxin B specifically cleaves desmoglein 1.

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.

Insulin-stimulated GLUT4 translocation in adipocytes is dependent upon cortical actin remodeling.

Rhodamine-labeled phalloidin staining of morphologically differentiated 3T3L1 adipocytes demonstrated that F-actin predominantly exists juxtaposed to and lining the inner face of the plasma membrane (cortical actin) with a smaller amount of stress fiber and/or ruffling actin confined to the cell bottom in contact with the substratum. The extent of cortical actin disruption with various doses of either latrunculin B or Clostridium difficile toxin B (a Rho family small GTP-binding protein toxin) directly correlated with the inhibition of insulin-stimulated glucose uptake and GLUT4 translocation. The dissolution of the cortical actin network had no significant effect on proximal insulin receptor signaling events including insulin receptor autophosphorylation, tyrosine phosphorylation of insulin receptor substrate and Cbl, or serine/threonine phosphorylation of Akt. Surprisingly, however, stabilization of F-actin with jasplakinolide also resulted in a dose-dependent inhibition of insulin-stimulated glucose uptake and GLUT4 translocation. In vivo time-lapse confocal fluorescent microscopy of actin-yellow fluorescent protein demonstrated that insulin stimulation initially results in cortical actin remodeling followed by an increase in polymerized actin in the peri-nuclear region. Importantly, the insulin stimulation of cortical actin rearrangements was completely blocked by treatment of the cells with latrunculin B, C. difficile toxin B, and jasplakinolide. Furthermore, expression of the dominant-interfering TC10/T31N mutant completely disrupted cortical actin and prevents any insulin-stimulated actin remodeling. Together, these data demonstrate that cortical actin, but not stress fibers, lamellipodia, or filopodia, plays an important regulatory role in insulin-stimulated GLUT4 translocation. In addition, cortical F-actin does not function in a static manner (e.g. barrier or scaffold), but insulin-stimulated dynamic cortical actin remodeling is necessary for the GLUT4 translocation process.

Deposition of laminin 5 by keratinocytes regulates integrin adhesion and signaling.

Deposition of laminin 5 over exposed dermal collagen in epidermal wounds is an early event in repair of the basement membrane. We report that deposition of laminin 5 onto collagen switches adhesion and signaling from collagen-dependent to laminin 5-dependent. Ligation of laminin 5 by integrin alpha(6)beta(4) activates phosphoinositide 3-OH-kinase (PI3K) signaling. This activation allows for adhesion and spreading via integrin alpha(3)beta(1) on laminin 5 independent of RhoGTPase, a regulator of actin stress fibers. In contrast, adhesion and spreading on collagen via alpha(2)beta(1) is Rho-dependent and is inhibited by toxin B, a Rho inhibitor. Deposition of laminin 5 and ligation of alpha(6)beta(4) increases PI3K-dependent production of phosphoinositide di- and triphosphates, PI3K activity, and phosphorylation of downstream target protein c-Jun NH(2)-terminal kinase. Conversely, blocking laminin 5-deposition with brefeldin A, an inhibitor of vesicle transport, or with anti-laminin 5 monoclonal antibodies abolishes the PI3K-dependent spreading mediated by alpha(3)beta(1) and phosphorylation of c-Jun NH(2)-terminal kinase. Studies with keratinocytes lacking alpha(6)beta(4) or laminin 5 confirm that deposition of laminin 5 and ligation by alpha(6)beta(4) are required for PI3K-dependent spreading via alpha(3)beta(1). We suggest that deposition of laminin 5 onto the collagen substratum, as in wound repair, enables human foreskin keratinocytes to interact via alpha(6)beta(4) and to switch from a RhoGTPase-dependent adhesion on collagen to a PI3K-dependent adhesion and spreading mediated by integrin alpha(3)beta(1) on laminin 5.

Staphylococcal scalded skin syndrome: exfoliative toxin A (ETA) induces serine protease activity when combined with A431 cells.

Staphylococcal scalded skin syndrome is the term used for a spectrum of primarily neonatal blistering skin diseases caused by the exfoliative toxins, ETA and ETB, of Staphylococcus aureus. Despite 25 y of research, the toxins' mechanism of action is still poorly understood, although evidence suggests that they may act as serine proteases. In this study, 0.1 mg purified ETA isolated from a baby with pemphigus neonatorum was incubated with A431 cells (a human squamous cell line) at 37 degrees C for 8, 24 and 48 h and the supernatant tested for protease activity using azocasein as a non-specific substrate. Phosphate-buffered saline was also incubated as negative control. Incubation of ETA with A431 cells for 48 h resulted in a four-fold increase in supernatant azocaseinolytic activity compared with buffer and cells, ETA alone and buffer alone (p < 0.001). Furthermore, this proteolytic activity was inhibited by PMSF (p < 0.001), a specific serine protease inhibitor. These results provide further evidence for the role of the exfoliative toxins as serine proteases. Furthermore, the A431 cell assay provides a simpler, quicker, cheaper and more acceptable alternative to neonatal mouse epidermis to study the mechanism of action of the exfoliative toxins.

Superantigens but not mitogens are capable of inducing upregulation of E-selectin ligands on human T lymphocytes.

Bacterial infections can exacerbate immune mediated dermatoses, possibly via superantigens produced by these bacteria. Therefore, we asked whether superantigens induce the expression of adhesion molecules which may then facilitate invasion of highly activated T cells into different organs. The influence of exfoliative toxin (ET) and toxic shock syndrome toxin-1 (TSST-1) stimulation on the expression of a broad panel of adhesion and costimulatory molecules was investigated by flow cytometry. We found that only the E-selectin ligands cutaneous lymphocyte-associated antigen (CLA) and sialylated Lewis(x) (CD15s) are significantly upregulated by these superantigens but not by mitogen stimulation. In contrast, the mucosal lymphocyte-associated antigen (MLA) recognized by the monoclonal antibody Ber-Act8 was not differentially induced by mitogen or superantigen stimulation. Therefore, T lymphocyte stimulation by bacterial superantigens might directly influence their skin homing capacity. Furthermore, the superantigen-driven induction of CD15s-an adhesion molecule which is absent or only weakly expressed by resting or mitogen stimulated T cells-may indicate a role of this antigen for T cell skin homing. An additional adhesion pathway via E-selectin may thus be available to lymphocytes, comparable to granulocytes which constitutively express CD15s.

The superantigen exfoliative toxin induces cutaneous lymphocyte-associated antigen expression in peripheral human T lymphocytes.

Several immune-mediated dermatoses including psoriasis and atopic dermatitis can be exacerbated by bacterial infections. Superantigen producing bacteria can be isolated from skin lesions of these dermatoses. Consistent with superantigen effects, skewed T cell receptor variable gene usage has been demonstrated within these lesions. Therefore, the question arises whether superantigen induce a skin-seeking phenotype within peripheral T cells. In this study, we investigated the in vitro influence of the V beta 2-selective superantigen exfoliative toxin from Staphylococcus aureus on the expression of the cutaneous lymphocyte-associated antigen on peripheral T lymphocytes of healthy donors. We demonstrate that exfoliative toxin dramatically upregulates cutaneous lymphocyte-associated antigen expression on T cell receptor V beta 2+ lymphocytes. Up to 69% of V beta 2+ lymphocytes expressed cutaneous lymphocyte-associated antigen after 5 days of in vitro culture. Additionally, exfoliative toxin also increased cutaneous lymphocyte-associated antigen expression in CD3+ T cell receptor V beta 2- lymphocytes indicating a different effect as caused by the superantigen-T cell receptor V beta 2 interaction. Our findings suggest influence of bacterial superantigens on T lymphocyte skin homing in vivo.

Superantigenic staphylococcal exotoxins induce T-cell proliferation in the presence of Langerhans cells or class II-bearing keratinocytes and stimulate keratinocytes to produce T-cell-activating cytokines.

Several staphylococcal toxins are among a growing number of immunostimulatory molecules called "superantigens" because of their ability, when presented by appropriate major histocompatibility complex class II+ accessory cells, to activate essentially all T cells bearing particular T-cell receptor V beta gene segments. We have examined the ability of murine epidermal Langerhans cells and/or keratinocytes to act as accessory cells in the T-cell response to the superantigens staphylococcal enterotoxin B and exfoliative toxin, also known as epidermolysin. Purified murine splenic T cells were stimulated with staphylococcal enterotoxin B or exfoliative toxin in the presence of Langerhans cells--enriched epidermal cells from normal mice or epidermal cells isolated from mice pretreated with recombinant interferon-gamma, a procedure that induces the expression of major histocompatibility complex class II molecules on keratinocytes. The data show that both Langerhans cells and class II-bearing keratinocytes can act as accessory cells in the T-cell response to staphylococcal enterotoxin B and exfoliative toxin. We also observed that both human and murine keratinocytes cultured in the presence of staphylococcal enterotoxin B or exfoliative toxin produce increased amounts of cytokine(s) capable of stimulating thymocytes and D10 cells, and that this toxin activity is independent of the level of expression of class II on keratinocytes. Studies by enzyme-linked immunosorbent assay showed that staphylococcal enterotoxin B stimulates keratinocytes to produce tumor necrosis factor-alpha but not interleukin-1, suggesting tumor necrosis factor-alpha and perhaps other cytokines are responsible for the T-cell proliferative activity. These results demonstrate that two distinct epidermal constituents (i.e. Langerhans cells and keratinocytes) can serve as accessory cells in the responses of T cells to superantigenic bacterial toxins. It is possible that such toxins contribute to the pathogenesis of a variety of skin diseases by either locally activating T cells bearing particular V beta genes and/or enhancing keratinocyte production of immunomodulatory cytokines.

Immunofluorescence localization of the epidermolytic toxin target in mouse epidermal cells and tissue.

An epidermolytic toxin target was observed in keratohyalin granules of sectioned epidermis by a 'direct' fluorescence procedure using FTC-toxin, but not by an 'indirect' procedure using sequential reaction with toxin, anti-toxin and FTC-secondary antibody. The investigation of the two procedures was extended to keratinocytes. A dispase digestion procedure yielded three fractions which corresponded to basal, spinous and granular cells according to biochemical and morphological criteria. It was shown that the 'direct' and 'indirect' procedures both detected the toxin target in the keratohyalin granules of granular cells, but that the 'indirect' procedure was very insensitive. In control experiments, the profilaggrin of keratohyalin granules was detected readily in cells by a 'direct' procedure using FTC-antiprofilaggrin but only weakly by an 'indirect' double antibody procedure. Insensitivity to 'indirect' procedures thus appears to be a particular property of the keratohyalin granule site. It was shown that the toxin target was readily accessible in permeable (trypsin-isolated) granular cells but inaccessible in impermeable (dispase-isolated) cells.

Susceptibility of various animals and cultured cells to exfoliative toxin produced by Staphylococcus hyicus subsp. hyicus.

In piglets inoculated with partially purified exfoliative toxin (pp-shET) produced by Staphylococcus hyicus subsp. hyicus, exfoliation was observed at 12 h after injection. Chickens inoculated with the same dose of pp-shET also showed exfoliation within 30 min of injection. However, exfoliation was not demonstrated in mouse, rat, guinea pig, hamster, dog or cat inoculated with pp-shET until 24 h after injection. In cultured cell lines, especially L-929 and Hep-2, the rounding effect occurred after incubation with pp-shET for 1 h. The rounding effect was also seen in five other cultured cells (NCTC 2544, HeLa/S3, HmLu-1, CHO and BHK-21) 6-24 h after exposure to pp-shET. These round cells survived for 72 h after inoculation and formed a monolayer 24 h after changeover to a toxin-free medium. The rounding effect was observed in cells after the formation of the monolayer, but not before. It was suggested that the rounding effect was not caused by the increase in cyclic AMP in cells inoculated with pp-shET but by the cleavage of intracellular contacts.

Fluid replacement protection of rabbits challenged subcutaneous with toxic shock syndrome toxins.

Toxic shock syndrome toxin 1 (TSST-1) and streptococcal pyrogenic exotoxin A (SPE A) belong to a family of pyrogenic toxins produced by Staphylococcus aureus and Streptococcus pyogenes, respectively. Both toxins are responsible for causing toxic shock syndrome (TSS) and related illnesses, clinically characterized by multiorgan involvement. The most severe TSS symptom is acute hypotension and shock after the initial febrile response. In this study, we examined possible mechanisms of shock development in TSS, particularly the role of T-cell proliferation, endotoxin enhancement by toxins, and capillary leakage. American Dutch belted rabbits, with subcutaneously implanted miniosmotic pumps filled with either TSST-1 or SPE A, served as the animal model. For both TSST-1 and SPE A-treated rabbits, administration of cyclosporin A prevented toxin-induced T-cell proliferation but failed to protect the rabbits. Polymyxin B treatment of rabbits, to neutralize endogenous endotoxin, partially protected rabbits from challenge with either exotoxin; two of six rabbits survived on day 2 when treated with only TSST-1, whereas six of six animals survived after challenge with TSST-1 and polymyxin B. Similarly, with SPE A-treated rabbits, only 1 of 10 animals without polymyxin B treatment survived on day 8, but 4 of 6 rabbits survived on day 8 when given polymyxin B. Fluid replacement was successful in preventing lethality. Twelve of 14 rabbits survived when given TSST-1 with fluid, and all rabbits treated with SPE A and fluid survived. Finally, by using miniosmotic pumps, staphylococcal exfoliative toxin A and concanavalin A were administered to rabbits in an attempt to induce lethality. These two T-cell mitogens caused T-cell proliferation but failed to induce lethality in rabbits. The data suggest that toxin interactions causing vascular leakage and to some extent endotoxin enhancement are of major importance in development of hypotension and shock in TSS. It appears that T-cell proliferation may not contribute significantly to the induction of shock and death.

[Experimental studies on the mechanism of the action of staphylococcal epidermolytic toxin A utilizing recombinant toxin].

The pathogenic role of proteinases and Ca++ in the action of staphylococcal epidermolytic toxin A (ETA) was investigated using recombinant ETA (rETA). rETA was released from the periplasmic space of E. coli transformed with the plasmid carrying ETA gene and purified by high performance liquid chromatography. The epidermolytic activity of the purified rETA was 5,000 epidermolytic unit per mg of protein. Pieces of newborn mouse skin were cultured in minimum essential medium containing rETA. Various concentrations of alpha 2-macroglobulin, N-ethylmaleimide, leupeptin, L-transepoxysuccynyl-leucylamide (4-guanidino) butane, phenylmethylsulfonyl fluroide, pepstatin A, ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis (2-aminoethylether) tetraacetic acid (EGTA) and 8-(N,N-diethylamino) octyl 3,4,5-trimethoxybenzoate (TMB-8) were added to the medium. Splitting in the upper epidermis occurred after 4 hr of incubation in the presence of 10 micrograms/ml rETA and was not inhibited by the proteinase inhibitors except EDTA and EGTA. EDTA, EGTA and TMB-8 inhibited the splitting completely at concentrations of 0.1-1 mM. The inhibitions caused by these agents were restored by the addition of Ca++ to the medium. These results strongly suggest that the action of ETA is mediated by the increase in cytoplasmic Ca++ concentration resulting from Ca++ influx and/or intracellular Ca++ mobilization.

Action of staphylococcal exfoliative toxins on epidermal cell cultures and organotypic skin.

In the staphylococcal scalded skin syndrome, spontaneous intraepithelial cleavages are due to the exfoliative toxins A or B (ETA or ETB). Until now, these toxins have been studied either on epidermis or on organotypic skin cultures. In the present study, we compare the effects of these toxins on human keratinocyte cell cultures to those on human and mouse organotypic skin cultures. With concentrations of ETA or ETB of 1 mg/ml for 3 hours, spontaneous intraepithelial cleavages were noted in both cell and organotypic cultures. Keratinocyte cell cultures were as sensitive as organotypic skin cultures to these toxins. Since keratohyaline granules may represent a possible binding site for ETA or ETB, we tried to correlate the expression of keratohyaline granules with the appearance of intraepithelial clefts due to the toxins. However, when cultured in liquid medium, epithelia were not differentiated enough to allow the detection of the binding site of ETA-ETB.

Partial purification and some characteristics of proteinase(s) induced by staphylococcal exfoliative toxin.

After 8 hrs incubation with epidermis of newborn mice and exfoliative toxin, a marked increase in caseinolytic activity was detected, which reached a maximum at 12 hrs. Casein-hydrolyzing enzyme(s) induced by ET were partially purified by chromatography. A substantial increase in caseinolytic activity was detected in the fractions obtained from Sephadex G-50, while practically no caseinolytic activity was observed when the extract obtained from the epidermis incubated without ET was applied. The caseinolytic activity appeared as a single peak eluted from DEAE-Sepharose CL-6B and Sephadex G-75. However, on SDS-PAGE, the partially purified fractions exhibited several protein bands. The molecular weights of these band were estimated as 78 KD, 68 KD, 45 KD, 14.5 KD and 8.8 KD. When the partially purified enzyme(s) was preincubated with EGTA or EDTA, substantial inhibition of the activity was observed; however, no recovery of the activity was detected after the addition of CaCl2. Treatment of the enzyme(s) with PMSF and NEM caused little inactivation of the activity. Enzyme activity retained about 57% of the initial activity following 3 min incubation at 60 degrees C, but was completely inactivated after 4 min.