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.

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.

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.

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.

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.

The effect on rhino mouse skin of agents which influence keratinization and exfoliation.

The skin of the rhino mouse, an allelic variant of the hariless mouse, contains deep dermal cysts and huge numbers of hornfilled utriculi which resemble comedones. Chemicals which influence either differentiation or desquamation of horny cells were applied topically twice daily for up to 6 weeks. Except for the dermal cysts, the gross epithelial abnormalities were almost completely corrected by retinoic acid in a dose-dependent fashion. Salicylic acid caused partial emptying of the horny masses, but the utriculi did not regress. Lactic acid, propylene glycol and benzoyl peroxide had minor effects on keratinization and exfoliation. The rhino mouse is a suitable model for assessing chemicals which affect epithelial differentiation (retinoids)or which promote loss of cohesion between horny cells (descaling agents).

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.

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.

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.

Action of staphylococcal epidermolysin: further observations on its species specificity.

Sensitivity to epidermolysin was tested in a number of species of the families of mice (muridae) and hamsters (cricetidae) and in hedgehogs. None of the animals proved sensitive except mus musculus (house mouse) and mesocricetus auratus (golden hamster).

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.

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.

Staphylococcal scalded skin syndrome I. Purification of exfoliatin and maternal transmission of neutralizing ability against exfoliatin.

Exfoliatin, an exotoxin produced by phage group 2 staphylococci, was separated and was analyzed. The toxin was separated in a single peak by Sephadex G-100 gel filtration and subsequent DEAE Sephadex chromatography. By acrylamide gel disc electrophoresis this single peak was found to contain 2 components both of which posessed exfoliative activity and the same antigenic properties. Their molecular weight, however, differed; the major component had a molecular weight of 30,000 and the minor component had 27,000. The toxin was stable when heated at 60 degrees C for 40 minutes, and labile at 100 degrees C for 20 minutes. The toxin was immunogenic to mice and produced antibody which could be transmitted to neonatal mice to neutralize exfoliative activity of the toxin. Innoculation of the bacteria caused the production of antibodies against both exfoliatin and alpha-hemolysin in mice. The antibodies were also transmitted to neonatal mice to neutralize the exfoliative activity of exfoliatin.

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.

Further observations of Corynebacterium renale as an indicator organism in the detection of the exfoliation-positive strains of Staphylococcus aureus.

Exfoliatin-positive S. aureus strains of known phenotypes were examined for bacteriocin production using S. aureus 209P, C. pseudodiphtheriticum, or C. renale as indicator strains. Strains of the phenotype Bac+ Tox+ had a positive result when the simultaneous method was used, and produced a wide inhibition of the indicator strains, especially of C. renale, if the deferred method was used. The strains of the phenotype Bac- Tox+ were negative upon examination by the simultaneous method, but they caused narrow inhibition zones of the indicator strains to appear with the deferred method, especially with C. renale. S. aureus strains of diverse origin used as controls were in both methods negative with regard to inhibition of indicator strains. C. renale was found to be very suitable for the detection of bacteriocins of the exfoliatin-positive staphylococci, particularly when using the deferred method.

[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.

Relationship between susceptibility and immune response to staphylococcal exfoliative toxin A in mammalian species.

Staphylococcal exfoliative toxin A (ETA) had a splitting effect at the granular layer of skin in humans and neonatal mice, but not in rabbits, guinea pigs, golden hamsters, or rats. Besides its splitting effect, ETA could stimulate productions of neutralizing antibody to ETA in rabbits, rats and B10D2 mice, but not in golden hamsters, guinea pigs, or ICR, HRS/J, and C57BL/10 mice. In our epidemiological investigation of human sera, the percentage of antibody to ETA in sera obtained from patients with impetigo (8%) was lower than those in sera of healthy males (23%) and females (29%). The relationship between susceptibility and immune response to ETA in these mammalians could be divided into three groups: the possession of resistant skin and high production of antibody to ETA; the possession of resistant skin and low production of antibody to ETA; the possession of sensitive skin and various titers of antibody to ETA.