The crystal structure of exfoliative toxin B: a superantigen with enzymatic activity.

The exfoliative toxins (ETs) cause staphylococcal scalded skin syndrome, a disease characterized by specific separation of layers of the skin. Evidence suggests that the toxins act as serine proteases, though the specific substrate and mode of action are not known for certain. The crystal structure of exfoliative toxin A (ETA) was reported earlier and shown to be similar to that of the chymotrypsin-like serine proteases. Here, we report the 2.4 A resolution crystal structure of the other exfoliative toxin, ETB, which is 40% identical to ETA. The overall structures of ETA and ETB are similar including the positions of key residues within the active site. The structure of ETB supports the previous findings that the ETs are serine proteases that cleave substrates after glutamic acid residues. In this study we also discuss a number of structural differences including a large 14 residue loop insertion which may be a key feature involved in the differing biological properties of the ETs, particularly the pyrogenic and lethal activities of ETB not shared by ETA.     

Expression of endothelin receptors in frog, chicken, mouse and human pigment cells

Several reports have shown the participation of vasoactive endothelins (ETs) in the regulation of vertebrate pigment cells. In the present study, we identified ET receptors in pigment cells of vertebrate species by RT-PCR assays, and compared the differential expression of the various subtypes in each species by quantitative PCR. RT-PCR was performed with specific primers for ETC, ETA(X) or ETA in Xenopus laevis melanophores, ETA or ETB(2) in chicken melanocytes, ETA or ETB in murine (B-16, S-91 or Melan-A) or human (SK-Mel 23 or SK-Mel 28) melanoma cells, and the products obtained were confirmed by cloning and sequencing. The results showed the presence of ETA(X), but not ETA mRNA, and confirmed the expression of ETC in X. laevis melanophores. ETA and ETB(2) mRNAs were also demonstrated in chicken melanocytes. ETA and ETB receptor were identified in S-91, B16 and Melan-A murine cells. In human melanoma cells, SK-Mel 23 and SK-Mel 28, we confirmed the presence of ETB mRNA, and also found ETA mRNA. The comparison between the two subtypes present in the pigment cell of each species and among species demonstrated that the expression of ETAs in chicken, mouse, and human melanocytes is negligible, as is the expression of ETA(X) in Xenopus melanophores. The relative expression, as determined by quantitative PCR, was as follows: chicken ETB>SK-Mel 23 ETB>S91 ETB>Xenopus ETC, suggesting that the endothelin system plays a major role in avian and mammalian pigment cell regulation, as compared to lower vertebrates. The phylogenetic analysis revealed that subtype A receptors were probably the most primitive ET receptors, directly deriving from the ancestral type; all the other receptors, B subtypes and C, originated from diverse derivative molecules.     

Phage conversion of exfoliative toxin A production in Staphylococcus aureus

The staphylococcal exfoliative toxins (ETs) are extracellular proteins that cause splitting of human skin at the epidermal layer during infection in infants. Two antigenically distinct toxins possessing identical activity have been isolated from Staphylococcus aureus, ETA and ETB. The gene for ETA (eta) is located on the chromosome, whereas that for ETB is located on a large plasmid. The observation that relatively few clinical isolates produce ETA suggests that the eta gene is acquired by horizontal gene transfer. In this study, we isolated a temperate phage (phiETA) that encodes ETA and determined the complete nucleotide sequence of the phiETA genome. phiETA has a head with a hexagonal outline and a non-contractile and flexible tail. The genome of phiETA is a circularly permuted linear double-stranded DNA, and the genome size is 43 081 bp. Sixty-six open reading frames (ORFs) were identified on the phiETA genome, including eta, which was found to be located very close to a putative attachment site (attP). phiETA converted ETA non-producing strains into ETA producers. Southern blot analysis of chromosomal DNA from clinical isolates suggested that phiETA or related phages are responsible for the acquisition of eta genes in S. aureus.     

The epidermolytic toxins are serine proteases

Certain strains of Staphylococcus aureus usually belonging to phage group II produce epidermolytic toxins (ETA and ETB) which cause intraepidermal splitting in mice, neonates and occasionally adults. Amino acid sequences of ETA and ETB have been reported but the mechanism of epidermolysis remains unknown. A search of the NBRF-PIR computer database showed the toxins to have significant sequence similarity with staphylococcal V8 protease and that the catalytic triad of V8 protease is present in ETA and ETB. Comparison of ETA, ETB and V8 protease with other members of the trypsin-like serine protease family revealed little homology save for the immediate vicinity of the residues constituting the catalytic triad. The toxins, therefore, exhibit a distant relationship to mammalian serine proteases. A potential Ca2(+)-binding loop was identified in ETA (but not ETB) on the basis of sequence similarity with the second calcium-binding loop of rat intestinal calcium-binding protein. Epidermolysis produced by ETA in the mouse bioassay was shown to be inhibited by the presence of EDTA consistent with a Ca2(+)-dependent mechanism.     

Mutational analysis of the superantigen staphylococcal exfoliative toxin A (ETA)

Exfoliative toxin A (ETA) is known to be a causative agent of staphylococcal scalded skin syndrome (SSSS). Although relatively little is known about exactly how the exfoliative toxins (ETs) cause SSSS, much has been discovered recently that may help elucidate the mechanism(s) by which ETA exhibits activities such as lymphocyte mitogenicity and epidermolytic activity. Here, we have shown that highly purified ETA does have T lymphocyte mitogenic activity in that wild-type ETA induced T cell proliferation whereas several single amino acid mutants lacked significant activity. Neither wild-type ETA nor any single amino acid mutants were proteolytic for a casein substrate, yet esterase activity was detected in wild-type ETA and several mutants, but eliminated in other mutants. A mutation in aa 164 (Asp to Ala) showed a 9-fold increase in esterase activity as well. Finally, we correlated esterase activity with epidermolytic activity. All mutants that lost esterase activity also lost epidermolytic activity. Conversely, mutants that retained esterase activity also retained exfoliative activity, implicating serine protease or serine protease-like activity in the causation of SSSS. Moreover, the mutants that displayed markedly reduced T cell superantigenic activity retained their epidermolytic activity (although some of these mutants required higher doses of toxin to cause disease), which suggests an ancillary role for this activity in SSSS causation.     

Endothelin receptors in human parathyroid gland.

We studied whether specific receptors for endothelins (ETs) exist in human parathyroid tissues and whether ETs may have any effect on secretion of PTH from parathyroid cells. Binding studies using [125I]ET-1 to the parathyroid membranes obtained from patients with hyperparathyroidism (2 adenomas, 2 hyperplasias) revealed that ET-1 competitively inhibited the binding of [125I]ET-1 to the membranes (the apparent Kd: 62 +/- 18 pM), whereas ET-3 showed biphasic and less steep inhibition curve than ET-1 in all tissue membranes examined. Northern blot analysis of poly(A)+ RNA from the parathyroid adenoma clearly demonstrated gene expression of both ETA and ETB receptors as well as preproET-1. ET-1 inhibited basal PTH secretion from dispersed adenoma cells more potently than ET-3. The present study clearly demonstrates the presence of both ETA and ETB receptor subtypes in human parathyroid tissues through which ETs may modulate PTH secretion in an autocrine and/or paracrine manner.     

Subtype-specific interactions of type C staphylococcal enterotoxins with the T-cell receptor

The goal of this study was to investigate the molecular interaction between superantigens and the T-cell receptor (TCR). Using a quantitative polymerase chain reaction (PCR) to assess T-cell proliferation profiles, we found that SEB, SEC1, SEC2 and SEC3 expanded human T cells bearing Vβ3, Vβ12, Vβ13.2, Vβ14, Vβ15, Vβ17 and Vβ20. SEC2 and SEC3 have the additional ability to expand T cells bearing Vβ13.1, and their expansion of Vβ3 was markedly reduced compared to SEB and SEC1. Based on the activity of SEC1 mutants containing single amino acid substitutions, we concluded that the differential abilities of these native toxins to stimulate Vβ3 and Vβ13.1 was determined by the residue in position 26, located in the base of the SEC α3 cavity. The SEC1 mutant, in which Val in position 26 was substituted with the analogous SEC2/SEC3 residue (Tyr), generated a Vβ expansion profile that was indistinguishable from those generated by SEC2 and SEC3. Using these findings, the co-ordinates of a recently reported murine TCR β-chain crystal structure, and other documented information, we propose a compatible molecular model for the interaction of SEC3 with the T-cell receptor. In this model complex, the complementarity-determining regions (CDRs) 1 and 2 and the hypervariable loop 4 of the Vβ element contact SEC3 predominantly through residues in the α3 cavity of the toxin. CDR3 of the β chain is not involved in any toxin contacts. The proposed model not only includes contacts identified in previous mutagenesis studies, but is also consistent with the ability of tyrosine and valine in position 26 to differentially affect the expansion of Vβs 3 and 13.1 by the SEC superantigens.     

Isolation of a new superantigen with potent mitogenic activity to murine T cells from Streptococcus pyogenes

Abstract A mitogenic substance on murine lymphocytes was detected in the culture supernate of Streptococcus pyogenes type 12 strain. This substance had a molecular weight of 28 000 and p I 9.2, and was designated as S. pyogenes mitogen (SPM). The proliferative response of C3H/HeN spleen cells began at 1 ng ml⁻¹ and reached a maximal response at 100 ng ml⁻¹ of SPM for 4 days culture. Anti-Thy 1.2 mAb and complement-treated spleen cells abrogated the proliferative response to any dose of SPM. Although the anti-major histocompatibility complex class I mAbs had no blocking effect on proliferation by SPM, this proliferation was substantially inhibited by the addition of either anti-I-A or anti-I-E mAb, and complete inhibition was produced by the addition of both mAbs. Fixed antigen-presenting cells still induced T cell proliferation by SPM. A significant expansion of T cells bearing Vβ13 T-cell receptor was observed up to 73% among the Thy1.2⁺ cells in cultures stimulated with SPM, indicating expansion in a Vβ-specific manner. Immunoblotting of IEF-separated proteins showed that anti-streptococcal pyrogenic exotoxin (SPE) C reacted with a protein of p I 6.9 and anti-SPEB did not show any reactivity. SPEA was reported to expand Vβ8.1 and 8.2 bearing murine T cells, and SPM did not. SPM also exhibited potent mitogenic activity on human T cells and Vβ21⁺ T cells were selectively expanded. These results lead to the conclusion that SPM was neither SPEA, B nor C, but a new protein belonging to a group of streptococcal superantigens with activity on not only human but also murine lymphocytes.     

Superantigens increase the survival of mice bearing T cell lymphomas by inducing apoptosis of neoplastic cells

Superantigens bind to major histocompatibility complex class II molecules and interact with T cells expressing a particular T cell receptor Vβ inducing a strong proliferation/deletion response of the superantigen-reactive T cells. However, there have been no attempts to investigate the ability of Sags to induce apoptosis in neoplastic T cells by signaling through the Vβ region of their TCR. In the present study we show that bacterial and MMTV-encoded superantigens induce the apoptosis of AKR/J cognate lymphoma T cells both in vitro and in vivo. The Fas-Fas-L pathway was shown to be involved in the apoptosis of lymphoma T cells induced by bacterial superantigens. In vivo exposure to bacterial superantigens was able to improve the survival of lymphoma bearing mice. Moreover, the permanent expression of a retroviral encoded superantigen induced the complete remission of an aggressive lymphoma in a high percentage of mice. The possibility of a therapeutic use of superantigens in lymphoma/leukemia T cell malignancies is discussed.     

Fluorescence studies of thermal stability of staphylococcal exfoliative toxins A and B

Abstract The quantum yields of intrinsic fluorescence of staphylococcal exfoliative toxins ETA and ETB were determined after excitation at 295 nm and 275 nm respectively. The variations in the intrinsic fluorescence and degree of fluorescence polarization showed conformational modifications of ETA around 57–59°C and of ETB around 52–54°C. Above these transition temperatures, ETB precipitated out and its biological activity was lost, whereas ETA showed almost no precipitation and no change in its biological activity, despite the structural changes in the molecule.     

Murine AIDS superantigen reactivity of the T cells bearing V beta 5 T cell antigen receptor.

A B cell line, B6-1710, that expresses the defective virus known to induce murine AIDS stimulates a large fraction of nonprimed splenic T cells. Analysis of the T cell population responding to the B6-1710 for TCR V beta-chain usage revealed that, in addition to the previously reported V beta 5-chain-positive T cells, T cells bearing V beta 11 and V beta 12 are also specifically enriched. We have established V beta 5+ T cell lines, clones, and hybridomas expressing identical TCR with different CD4/CD8 phenotypes and demonstrated that T cell reactivity to B6-1710 is, although not absolute, dependent on the presence of CD4 molecules. Further analysis of T cell hybridomas with known J beta-chain usage revealed that D beta- and J beta-chain usage do not play crucial roles in T cell reactivity to B6-1710 B cells. However, T cell hybridomas derived from TCR-V beta gene transgenic mice were found to be heterogeneous for their reactivity to B6-1710, suggesting that the V alpha-chains associating with the transgenic V beta-chain determine T cell responsiveness to B6-1710. These data clearly demonstrate that T cell reactivity to a murine AIDS virus expressing B cell line resembles that previously reported for Mls-like superantigens.     

Endothelin 1 and 3 enhance neuronal nitric oxide synthase activity through ETB receptors involving multiple signaling pathways in the rat anterior hypothalamus

We have previously reported that endothelin 1 and 3 (ET-1, ET-3) through the ETB receptor decrease norepinephrine release in the anterior hypothalamus and activate the nitric oxide (NO) pathway. In the present work we sought to establish the receptors and intracellular mechanisms underlying the increase in nitric oxide synthase (NOS) activity stimulated by ET-1 and ET-3 in the rat anterior hypothalamus. Results showed that ETs-stimulated NOS activity was inhibited by a selective ETB antagonist (BQ-788), but not by a selective ETA antagonist (BQ-610). In addition, NOS activity was not altered in the presence of an ETA agonist (sarafotoxin 6b), but it was enhanced in the presence of a ETB agonist (IRL-1620). Both Nomega-nitro-L-arginine methyl ester (NOS inhibitor), and 7-nitroindazole (neuronal NOS inhibitor) diminished ETs-stimulated NOS activity. The stimulatory effect of ETs on NOS activity was inhibited in the presence of PLC, PKC, PKA and CaMK-II inhibitors (U-73122, GF-109203X, H-89 and KN-62, respectively), and the IP3 receptor selective antagonist, 2-APB. Our results showed that both ET-1 and ET-3 modulate neuronal NOS activity through the ETB receptor in the rat anterior hypothalamus involving the participation of the PLC-PKC/IP3 pathway as well as PKA and CaMK-II.     

Role of endothelins in septic,cardiogenic, and hemorrhagic shock

Shock is a condition where blood flow is inadequate for tissue needs. In all forms of shock, the concentrations of endothelins (ETs) are elevated, and they are especially high in septic shock. The rise in ETs plasma levels may initially have some positive homeostatic effects, for ETs can help restore normal vascular tone. However, high levels of ETs compromise the appropriate matching of flow to tissue needs and contribute to the pathophysiology of shock. Attempts at regulating the effects of ETs by the use of pharmacological blockers is made complicated by important interactions between the ETA and ETB receptors and potentially different effects on different tissues. We conclude that antagonism of ET receptors is unlikely to be helpful for cardiogenic or hemorrhagic shock. Furthermore, selective blockade is unlikely to be helpful. However, moderate doses of a mixed ET receptor antagonist may be of use for the management of septic patients.     

An evolutionary conserved mechanism of T cell activation by microbial toxins. Evidence for different affinities of T cell receptor-toxin interaction.

The enterotoxins produced by Staphylococcus aureus are the most potent mitogens known. They belong to a group of distantly related mitogenic toxins that differ in other biologic activities. In this study we have compared the molecular mechanisms by which these mitogens activate human T lymphocytes. We used the staphylococcal enterotoxins A to E, the staphylococcal toxic shock syndrome toxin, the streptococcal erythrogenic toxins A and C (scarlet fever toxins, erythrogenic toxins (ET)A, ETC), and the soluble mitogen produced by Mycoplasma arthritidis. We found that all these toxins can activate both CD4+ and CD8+ T cells and require MHC class II expression on accessory and target cells. However, T cells could be activated in the absence of class II molecules if the toxins ETA or SEB were co-cross-linked on beads together with anti-CD8 or anti-CD2 antibodies. Enterotoxins, toxic shock syndrome toxin and scarlet toxins stimulate a major fraction of human T cells, and show preferential, but not exclusive, stimulation of T cells carrying certain TCR V beta. In contrast, the mitogen of M. arthritidis, a pathogen for rodents stimulates only a minority of human T cells but activates a major fraction of murine T cells. Analysis of human T cell clones expressing V beta 5 or V beta 8 TCR showed that these clones responded also to those toxins that did not stimulate V beta 5+ and V beta 8+ T cells in bulk cultures. These results indicate that different TCR bind to these toxins with different affinities and that the specificity of the TCR-V beta-toxin interaction is quantitative rather than qualitative in nature. Taken together our findings suggest that these toxins use a common mechanism of T cell activation. They are functionally bivalent proteins crosslinking MHC class II molecules with variable parts of the TCR. Besides V beta, other parts of the TCR must be involved in this binding. The finding that murine T cells responded more weakly to the toxins produced by the human-pathogenic bacteria than to the Mycoplasma mitogen could indicate that the toxins have been adapted to the host's immune system in evolution.     

Enzymatic and molecular characteristics of the efficiency and specificity of exfoliative toxin cleavage of desmoglein 1

Exfoliative toxins (ETs) from Staphylococcus aureus blister the superficial epidermis by hydrolyzing a single peptide bond, Glu381-Gly382, located between extracellular domains 3 and 4 of desmoglein 1 (Dsg1). Enzyme activity is dependent on the calcium-stabilized structure of Dsg1. Here we further define the characteristics of this cleavage. Kinetic studies monitoring the cleavage of Dsg1 by ETA, ETB, and ETD demonstrated kcat/Km values of 2-6 x 10(4) m(-1) s(-1), suggesting very efficient proteolysis. Proteolysis by ETA was not efficiently inhibited by broad spectrum serine protease inhibitors, suggesting that the enzyme cleavage site may be inactive or inaccessible before specific binding to its substrate. Using truncated mutants of human Dsg1 and chimeric molecules between human Dsg1 and either human Dsg3 or canine Dsg1, we show that for cleavage, human-specific amino acids from Dsg1 are necessary in extracellular domain 3 upstream of the scissile bond. If these residues are canine rather than human, ETA binds, but does not cleave, canine Dsg1. These data suggest that the exquisite specificity and efficiency of ETA may depend on the enzyme's binding upstream of the cleavage site with a very specific fit, like a key in a lock.     

Dipeptide sulfonamides as endothelin ETA/ETB receptor antagonists

Endothelin-1 (ET-1) is a potent mitogen and modulator of vascular tone. It is synthesized and released from endothelial cells and acts upon two receptor subtypes designated as ETA and ETB. In this study, a series of potent dipeptide sulfonamide dual-endothelin ETA/ETB receptor antagonists were prepared to investigate their potential benefit in vascular diseases. CGS 31398 inhibited [125I]ET-1 binding to human ETA and ETB receptors expressed in Chinese hamster ovary (CHO) cells (ETA/CHO, ETB/CHO) with respective IC50 values of 0.26 and 0.12 nM. However, in anesthetized rats, this compound markedly potentiated ET-1-induced renal vascular resistance, a response normally observed with selective ETB receptor antagonists. To determine whether species differences account for these results, a direct comparison was made between binding to rat and rabbit aortic membranes versus functional antagonism in isolated rat aortic rings. It was found that CGS 31398 had potent affinity for the ETA receptor in rat and rabbit aorta with IC50 values of 0.87 and 0.79 nM, respectively. Inhibition of ET-1-induced contractions of rat aorta by the compound was considerably weaker than expected (pKB = 6.4), while that of sarafotoxin S6c induced contraction of dog saphenous vein (100% inhibition at 100 nM) was consistent with corresponding binding data. These results suggest that although CGS 31398 is a potent dual inhibitor of ETA/ETB receptor binding, it surprisingly displays potent ETB and weak ETA receptor antagonism in functional assays.     

Selective stimulation of human T cells with streptococcal erythrogenic toxins A and B

Streptococcal exotoxins have been implicated in the pathogenesis of a toxic shock-like syndrome and scarlet fever. Previous studies have demonstrated that these toxins are potent stimulators of human T cells and have structural homology to staphylococcal enterotoxins. In the current study, we investigated the mechanism by which streptococcal erythrogenic toxins type A (SPEA) and B (SPEB) activate T cells and compared it with anti-CD3 and the known "superantigen" staphylococcal enterotoxin B. SPEA was found to selectively activate T cells bearing V beta 8, V beta 12, and V beta 14, whereas SPEB selectively activated T cells bearing V beta 2 and V beta 8. Furthermore, fibroblasts transfected with MHC class II molecules were capable of presenting SPEA and SPEB to purified T cells. The T cell response to these toxins, however, was not MHC-restricted. Although the streptococcal exotoxins stimulated both CD4+ and CD8+ T cells, SPEA but not SPEB stimulated the CD4+ T cell subset proportionately more than the CD8+ T cell subset. Our results indicate that SPEA and SPEB, like the staphylococcal enterotoxins, are superantigens and suggest a mechanism by which they may mediate particular systemic syndromes associated with streptococcal infections.     

Venous smooth muscle contains vasoconstrictor ETB-like receptors.

Two endothelin (ET) receptor subtypes have been identified to date: the ETA receptor which preferentially binds ET-1 over ET-3, and the ETB receptor which is non-selective. This study characterized the ET receptor subtypes present in several vascular smooth muscle preparations using standard in vitro techniques. In all but one of the arteries tested, ET-3 was significantly less potent than ET-1. In contrast, the potency of ET-3 was very similar to that of ET-1 in all of the veins. The selective ETA receptor antagonist BQ-123 blunted the ET-1 contractions in rabbit carotid artery, but not in saphenous vein. The selective ETB receptor ligand sarafotoxin S6c contracted the rabbit saphenous vein, but not the carotid artery. These data suggest that vascular smooth muscle cells express ETA and ETB receptors. Stimulation of either receptor subtype can result in force development.