Joseph E. Alouf

Staphylococcal and streptococcal superantigens: molecular, biological and clinical aspects.

Superantigens (SAgs) include a class of certain bacterial and viral proteins exhibiting highly potent lymphocyte-transforming (mitogenic) activity towards human and or other mammalian T lymphocytes. Unlike conventional antigens, SAgs bind to certain regions of major histocompatibility complex (MHC) class II molecules of antigen-presenting cells (APCs) outside the classical antigen-binding groove and concomitantly bind in their native form to T cells at specific motifs of the variable region of the beta chain (Vbeta) of the T cell receptor (TcR). This interaction triggers the activation (proliferation) of the targeted T lymphocytes and leads to the in vivo or in vitro release of high amounts of various cytokines and other effectors by immune cells. Each SAg interacts specifically with a characteristic set of Vbeta motifs. The review summarizes our current knowledge on S. aureus and S. pyogenes superantigen proteins. The repertoire of the staphylococcal and streptococcal SAgs comprises 24 and 8 proteins, respectively. The staphylococcal SAgs include (i) the classical enterotoxins A, B, C (and antigenic variants), D, E, and the recently discovered enterotoxins G to Q, (ii) toxic shock syndrome toxin-1, (iii) exfoliatins A and B. The streptococcal SAgs include the classical pyrogenic exotoxins A and C and the newly identified pyrogenic toxins, G, H, I, J, SMEZ, and SSA. The structural and genomic aspects of these toxins and their molecular relatedness are described as well as the available 3-D crystal structure of some of them and that of certain of their complexes with MHC class II molecules and the TcR, respectively. The pathophysiological properties and clinical disorders related to these SAgs are reviewed.

Production of Thiol-dependent Haemolysins by Listeria monocytogenes and Related Species

Twenty-six strains belonging to the five main species of the genus Listeria were examined for production of thiol-dependent exotoxins. All strains of L. monocytogenes cultured in charcoal-treated broth secreted a haemolytic factor at a level ranging from 200 to 800 haemolytic units (HU) ml-1, except for the strain EGD (1500 HU ml-1) and the type strain CIP 82110T (10 HU ml-1). The haemolytic activity reached a maximum level by 8-10 h and then rapidly declined as soon as bacterial exponential growth ceased. The titres of haemolytic activity were markedly reduced when bacteria were grown in charcoal-untreated broth. The haemolytic factor produced by L. monocytogenes strains was characterized as listeriolysin O (Mr about 60,000), a member of the group of thiol-dependent exotoxins. Strains of Listeria ivanovii also produced high levels of thiol-dependent exotoxin (about 2500 HU ml-1), in both charcoal-treated and untreated broth. Small amounts of haemolytic factor (about 9-30 HU ml-1) were also produced by Listeria seeligeri in charcoal-treated broth. The haemolysin produced by L. seeligeri was identified for the first time as a thiol-dependent exotoxin of Mr about 60,000, antigenically related to listeriolysin O. As expected, we failed to detect thiol-dependent exotoxin in the two nonhaemolytic species, Listeria innocua and Listeria welshimeri.

Interaction of streptolysin O with sterols

Summary A quantitative study of the specific inhibitory power of cholesterol and other sterols on the hemolytic properties of streptolysin O is reported. This streptococcal exocellular protein is a cytolytic toxin which disrupts cytoplasmic membranes of eukaryote cells. The structural characteristics, particularly the stereochemical ones required for a steroid molecule to inhibit the cytolytic activity of streptolysin O, have been investigated in detail. By immunodiffusion techniques, in agar gel plates or tubes containing sterols, the formation of hydrophobic complexes between streptolysin O and inhibitory steroids, but not non-inhibitory steroids except lanosterol, is shown. Upon interaction with inhibitory steroids streptolysin O loses its immunoreactive properties towards neutralizing and precipitating homologous antibodies. An interpretation of the mechanism of biomembrane disorganization by streptolysin O is discussed in the light of its steroid binding properties.

Superantigen bacterial toxins: state of the art.

Superantigens (SAgs) are viral and bacterial proteins exhibiting a highly potent polyclonal lymphocyte-proliferating activity for CD4(+), CD8(+) and sometimes gammadelta(+) T cells of human and (or) various animal species. Unlike conventional antigens, SAgs bind as unprocessed proteins to invariant regions of major histocompatibility complex (MHC) class II molecules on the surface of antigen-presenting cells (APCs) and to particular motifs of the variable region of the beta chain (Vbeta) of T-cell receptor (TcR) outside the antigen-binding groove. As a consequence, SAgs stimulate at nano-to picogram concentrations up to 10 to 30% of host T-cell repertoire while only one in 10(5)-10(6) T cells (0.01-0.0001%) are activated upon conventional antigenic peptide binding to TcR. SAg activation of an unusually high percentage of T lymphocytes initiates massive release of pro-inflammatory and other cytokines which play a pivotal role in the pathogenesis of the diseases provoked by SAg-producing microorganisms. We briefly describe in this review the molecular and biological properties of the bacterial superantigen toxins and mitogens identified in the past decade.

Purification of RNA-core Induced Streptolysin S, and Isolation and Haemolytic Characteristics of the Carrier-free Toxin

RNA-core (RNAase-resistant fraction of yeast RNA) induced streptolysin S (SLS) was purified (40% recovery) to apparent electrophoretic homogeneity by hydroxylapatite chromatography followed by gel filtration on Sephadex G-100 in the presence of 6 M-guanidine. HCl. The specific activity of the purified toxin was 3 X 10(6) haemolytic units (mg protein)-1. The Mr of the toxin was below 4000 on the basis of SDS-PAGE and 20 000 by gel filtration in guanidine. HCl. High-voltage isoelectric focusing of the purified toxin allowed the isolation of the carrier-free SLS peptide for the first time. This peptide was basic (pI 9.2) as compared to native SLS (pI 3.6). The native toxin and the peptide had similar haemolytic properties except for the high lability of the peptide, which was stabilized by RNA-core. The Mr of the denatured peptide was about 1800, as estimated by gel filtration.

Pyrogenicity and Cytokine-Inducing Properties of Streptococcus pyogenes Superantigens: Comparative Study of Streptococcal Mitogenic Exotoxin Z and Pyrogenic Exotoxin A

ABSTRACT Streptococcal mitogenic exotoxin Z (SMEZ), a superantigen derived from Streptococcus pyogenes, provoked expansion of human lymphocytes expressing the Vβ 2, 4, 7 and 8 motifs of T-cell receptor. SMEZ was pyrogenic in rabbits and stimulated the expression of the T-cell activation markers CD69 and cutaneous lymphocyte-associated antigen. A variety of cytokines was released by human mononuclear leukocytes stimulated with SMEZ, which was 10-fold more active than streptococcal pyrogenic exotoxin A. Th2-derived cytokines were elicited only by superantigens and not by streptococcal cells.

Induction of heat-shock proteins by bacterial toxins, lipid mediators and cytokines in human leukocytes.

We studied the influence of a lipid mediator (12-hydroxyeicosatetraenoic acid, 12-HETE), cytokines (IL-6 and TNF-alpha) and different bacterial toxins (alveolysin; exfoliative toxin; toxic shock syndrome toxin 1, TSST-1 and erythrogenic toxin A, ETA) on the expression of heat shock proteins (hsps) in isolated human leucocytes. 12-HETE induces the expression of individual heat shock proteins (65- and 83 kDa) protein in human leukocytes (lymphocytes, monocytes, basophilic granulocytes; LMBs). As was shown by Western blotting (anti-hsp72), IL-6 or TNF-alpha induced hsps preferentially in human LMBs and PMNs, respectively. Among the toxins, ETA and TSST-1 were potent inducers of hsps at low toxin concentrations (10 ng/ml). Alveolysin led to the expression of hsps at hemolytic concentrations (1 HU; 700 ng/ml) whereas at subhemolytic concentrations (7 ng/ml), no heat shock response was observed. The induction of heat shock proteins was also accompanied by increased mRNA levels for hsp70 as determined by PCR analysis. In contrast, exfoliative toxin led to a reduction of the hsp signal in PMNs as determined by Western blotting. Finally, it was demonstrated that PMNs which had been pretreated with TNF-alpha and therefore expressed intracellular hsps were more resistant to cytolytic attack by leukocidin than untreated cells.

Purification and some properties of streptolysin O.

Summary Streptolysin O, a potent membrane-disrupting protein endowed with lethal, cardiotoxic, cytolytic and hemolytic properties, produced by Streptococcus pyogenes, as an exocellular toxin has been purified from culture supernates. Two forms of purified Streptolysin O are obtained with molecular weights of about 110,000 and 55,000 daltons respectively, the heavy form being the major one. This form proved homogeneous by ultracentrifugation, gel immunodiffusion and dise gel electrophoresis. It exhibited immunological identity with the light form. Several electrophoretic forms of Streptolysin O are found upon electrofocusing but some of them appear to be constituted by molecules in a partial toxoidation state. The results obtained and preliminary ultracentrifugation experiment of the 110,000 dalton form in guanidinium hydrochloride lead to consider that Streptolysin O may occur as an oligomeric protein constituted by two protomers of 55,000 daltons each. Some biological properties of the purified toxin are determined.

The sulphydryl-activated cytolysin and a sphingomyelinase C are the major membrane-damaging factors involved in cooperative (CAMP-like) haemolysis of Listeria spp.

The negative mutant approach was used in this study to identify listerial cytolytic factors involved in cooperative haemolysis (CAMP-like phenomenon) with Staphylococcus aureus and Rhodococcus equi. A Listeria monocytogenes non-haemolytic mutant specifically impaired in listeriolysin O (LLO) production gave no CAMP reaction with S. aureus, and was virtually CAMP-negative with R. equi, indicating that the listerial sulphydryl-activated toxin played a major role in cooperative haemolysis. This was confirmed by direct evidence using purified LLO and alveolysin (from Bacillus alvei) in diffusion CAMP assays. To our knowledge, this is the first evidence of involvement of a sulphydryl-activated toxin in cooperative lytic processes. Phosphatidylcholine- and phosphatidylinositol-specific phospholipases C from L. monocytogenes did not seem to significantly contribute to cooperative haemolysis, as the corresponding mutants displayed wild-type CAMP reactions. In contrast, the sphingomyelinase C from Listeria iva-novii was the cytolytic factor responsible for the characteristic shovel-shaped CAMP reaction shown by this listerial species with R. equi. Possible mechanisms of lytic cooperation are discussed.

Pertussis toxin facilitates the progesterone-induced maturation of Xenopus oocyte: Possible role of protein phosphorylation

Progesterone triggers the first meiotic cell division of Xenopus oocyte and inhibits cAMP synthesis. The effect of pertussis toxin purified from Bordetella pertussis was tested on the maturation of Xenopus oocyte. The toxin did not inhibit progesterone‐induced resumption of meiosis or the hormone‐induced drop in cAMP level. This indicates that progesterone action is not mediated by the Ni subunit of the oocyte adenylate cyclase. Furthermore, pertussis toxin caused a reduction in the time course of maturation correlated with the precocious appearance of an alkali stable 47kDa phosphoprotein, a marker of the maturation promoting factor (MPF) activity. Pertussis toxin effects mimicked those of 2‐glycerophosphate suggesting that both agents act on the steady‐state level of phosphorylation implicated in MPF activity.