Toxic shock syndrome toxin 1 as an inducer of human tumor necrosis factors and gamma interferon.

We present evidence that toxic shock syndrome toxin 1 (TSST-1) induces the production of high levels of TNF by human blood monocytes. Enriched lymphocyte preparations incubated with the staphylococcal toxin produced significant levels of TNF-like activity that is not neutralized by anti-rHuTNF antibodies and is likely to be lymphotoxin (LT or TNF-beta). We demonstrate also that TSST-1 is a potent inducer of IFN-gamma. When lymphocyte preparations were costimulated with PMA, the TSST-1 effect was strongly potentiated and the levels of cytotoxic factors, IFN-gamma, and IL-2 present in supernatant fluids were comparable to those observed after treatment with PMA and PHA. Thus, TSST-1, which is also known as an inducer of IL-1 and IL-2, stimulates the production of endogenous mediators that could play a role in the physiopathological processes of toxic shock syndrome (TSS). The described results suggest that the discrepancies in the clinical features between TSS and endotoxin shock may be related to qualitative differences in cytokine production.

Biochemical and ultrastructural study of the disruption of blood platelets by streptolysin O.

The membrane-damaging protein toxin, streptolysin O, proved highly lytic on human, guinea-pig and rabbit platelets. About 15 molecules of toxin were sufficient to lyse one cell. Platelet disruption was assessed by electron microscopy, clearing of cell suspensions and assay of lactate dehydrogenase, serotonin, monoamine oxidase and glutathione peroxidase released in the extracellular fluid. This egress reflected the damage of both plasmic and organelle membranes. A quantitative study of lactate dehydrogenase and serotonin liberation taken as respective markers of the cytosol and dense bodies was undertaken as a function of toxin concentration. No platelet aggregation or shape change was elicited by streptolysin O. The ghosts resulting from platelet lysis retained properties of the native membrane such as aggregability and serotonin uptake. Dense bodies were easily separated after gentle disruption of the plasmic membrane by small amounts of toxin. Platelet lysis by streptolysin O proved a useful procedure for the determination of protein content, enzyme activities and serotonin assay on the same lysate in contrast to usual methods.

Interaction of steptolysin O with sterols.

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.

Ganglioside identification on human monocyte membrane with Clostridium perfringens delta-toxin.

Clostridium perfringens delta-toxin was first described as a hemolysin with a restricted lytic spectrum. A selective cytotoxicity of the delta-toxin was then found on rabbit leukocytes: peritoneal and alveolar macrophages were uniformly killed, whereas thymocytes were essentially resistant. The toxin was shown to be specific for GM2 ganglioside or a GM2-like structure. In the present study we report the interaction of delta-toxin with human monocytes. A specific, saturable, and irreversible binding of 125I-delta-toxin was demonstrated. Binding was inhibited by preincubation of the radiolabeled toxin with GM2 and with high amount of GM1 ganglioside. As judged by dye exclusion, no cytotoxicity was observed on freshly isolated monocytes, but when added at the beginning of a culture of human adherent cells, the cytotoxic effect was detected after 48 hours of culture. Taken together, these data indicate the presence of monosialoganglioside(s) at the surface of human monocytes, and suggest a possible reorganisation of such structure into the cell membrane when monocytes mature in vitro toward macrophage-like cells.

Characteristics of guinea-pig immune sera elicited by a synthetic diphtheria toxin oligopeptide.

Several oligopeptides of different lengths contained within the Cys 186-Cys 201 first disulfide loop of the diphtheria toxin molecule have been synthesized by a solid-phase method. 125I-labeled rabbit antibodies raised against diphtheria toxin reacted specifically with oligopeptides linked to m-nitrobenzhydrylamine resin when the amino acid chain length was equal to or greater than 10 residues. The synthetic tetradecapeptide (STDP) corresponding to the sequence Gly 188-Cys 201 was used to immunize guinea-pigs. The immune sera obtained reacted with the whole diphtheria toxin molecule as judged by an antigen-linked immunosorbent assay. Anti-STDP sera exhibited a clear, albeit limited, neutralizing effect against the lethal action of diphtheria toxin on cultivated Vero cells. The anti-STDP sera were also able to partially block the ADP-ribosylation of elongation factor 2 mediated by whole diphtheria toxin. In contrast, anti-STDP sera were almost inactive on the enzymic activities of either toxin fragment A or crm 45, a mutant protein which lacks the 15,000 mol. wt C-terminal sequence of the toxin molecule. On the basis of the results obtained, a possible localization of the Cys 188-Cys 201 loop region on the toxin molecule is proposed.

The phosphoinositide pathway of lymphoid cells: labeling after permeabilization by alveolysin, a bacterial sulfhydryl-activated cytolysin.

An improved method allowing incorporation of [3H]myo-inositol into the phosphoinositide pool of human lymphoid cells is described. The procedure devised involves cell permeabilization with a thiol-activated membranolytic toxin, alveolysin, and optimization of the phosphoinositide labeling and extraction. In these conditions 4 to 10% of the added [3H]myo-inositol is found intracellularly and half of this amount (2-5%) is incorporated into the phosphoinositide pool in only 1 h as compared to the classical 0.2 to 0.3% incorporation obtained after 10 to 20 h. The integrity of coupling between receptors and phospholipase C was assessed by the inositol phosphate production after cell stimulation by various agonists.

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 47 kDa 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.

[Purification of an extracellular thiol-dependent hemolysin from Bacillus alvei]. / Purification de l'hémolysine thiol-dépendante extracellulaire de Bacillus alvei.

Alveolysin a sulfhydryl-dependent cytolytic extracellular protein released by Bacillus alvei has been purified by salting-out by ammonium sulfate, gel filtration, isoelectric focusing on pH gradient and chromatography on DEAE-cellulose. The purified protein after reduction by thiols (active hemolytic form) proved homogeneous by disc polyacrylamide gel electrophoresis and by gel immunodiffusion. The molecular weight was 60,000 daltons, Two molecular forms of pI 5.1 and 7.0 were detected by gel isoelectrofocusing. The toxin was lethal to the mouse. Lytic activity was inhibited by cholesterol and antistreptolysin O anstisera. Immunological cross-reaction was observed between alveolysin and streptolysin O.

Mitogenicity of streptococcal extracellular products and antagonism with concanavalin A.

Extracellular products have been purified from group A Streptococcus pyogenes culture supernatant fluids and their mitogenicities have been tested on rabbit and mouse lymphocytes. Two fractions were mitogenic: the kappa-fraction (pI = 4.8, mol. wt. = 30,000), a protein which was identified as the erythrogenic toxin, and the epsilon-fraction (pI = 10.3, mol. wt. = 17,000) a glycoprotein, both stimulated rabbit and CBA mouse spleen cells. The stimulation of rabbit thymocytes was weak unless macrophages or 2-mercaptoethanol were added. A third product, the gamma-fraction (a protein, pI = 4.2, mol. wt. = 72,000) was very weakly mitogenic and had the capacity to reduce the stimulation induced by a T-cell mitogen, such as Con A, but not by a B-cell mitogen such as Nocardia.

Induction of interferon by streptococcus pyogenes extracellular products.

Lymphocyte-activating streptococcal exoproteins, which were previously characterized, have been tested for their capacity to induce interferon in vitro. Two out of the 3 different streptococcal fractions, studied on mice splenocytes, were shown to elicit the production of a significant amount of interferon. A large proportion of the interferon detected in the supernatants from mice activated spleen cells was acid-labile interferon. The highest level of interferon titer was obtained with the streptococcal fraction identified as the erythrogenic toxin.

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.

Cytokine production by murine cells activated by erythrogenic toxin type A superantigen of Streptococcus pyogenes.

The mode of pathogenic action of the Steptococcus pyogenes superantigen erythrogenic toxin type A (ETA) in causing toxic shock-like syndrome in humans is thought to be mediated by massive release of cytokines by patients immune cells. The cytokine-inducing capacity of ETA as an extracellular protein was compared with that of lipopolysaccharide (LPS), a component of cell wall of gram-negative bacteria. Peritoneal macrophages and splenocytes of BALB/c and C3H/HeJ mice were stimulated by ETA and LPS. Tumor necrosis factor (TNF), interleukin 3 (IL-3) and interleukin 6 (IL-6) activities in the supernatants of stimulated cells were evaluated. In contrast to LPS, ETA induced only low amounts of IL-6 and no detectable TNF activities in peritoneal macrophage supernatants. ETA-triggered BALB/c and C3H/HeJ splenocytes produced great amounts of IL-6. ETA triggered the production of IL-3 by both mice strains splenocytes in a dose dependent manner. The amounts of IL-3 in supernatants were comparable to those induced by concanavalin A. The simultaneous presence of ETA and LPS in macrophage and splenocyte cultures induced a slight enhancement above an additive value after 72-96 h. Challenge of BALB/c mice with ETA 6 h before the harvest of peritoneal macrophages led to an enhanced production of IL-6 upon stimulation with ETA as well as with LPS. Splenocytes of nude BALB/c mice did not produce IL-6 upon stimulation with ETA, whereas LPS-induced IL-6 production was similar in these mice and in their littermates. The pathogenic effect of ETA on host's immune cells could most likely be explained as a consequence of T cell activation. The results confirm also that LPS- and ETA-induced shock is mediated by different cell types.

Erythrogenic toxins A, B and C: occurrence of the genes and exotoxin formation from clinical Streptococcus pyogenes strains associated with streptococcal toxic shock-like syndrome.

We report the study of 53 clinical isolates of group A streptococci, all from patients with streptococcal toxic shock-like syndrome. The strains were analysed for the occurrence of the genes of erythrogenic toxins (pyrogenic exotoxins) types A, B and C and in vitro production of these toxins. In contrast to reports indicating that 85% of the toxic shock-like syndrome-associated isolates contained the erythrogenic toxin A gene, only 58.5% of our strains harboured this gene. The erythrogenic toxin C gene was detected in 22.6% of the isolates. Erythrogenic toxin A and erythrogenic toxin B were produced by 68.7% and 58.3% of the strains containing either gene. For all group A streptococci, irrespective of clinical association, the erythrogenic toxin B gene was detected in all the isolates tested. Thus, it is difficult to define a specific role for erythrogenic toxin B in toxic shock-like syndrome as there was no clear correlation between this disease and the presence of toxin genes. Our results suggest the existence of other pathogenic factor(s) produced by group A streptococci which may stimulate human peripheral T lymphocytes in a manner similar to that of erythrogenic toxins, thus explaining different observations in previous epidemiological genetic studies.

Purified -SH-activated toxins (streptolysin O, alveolysin): new tools for determination of platelet enzyme activities.

The purpose of this study was to examine the lytic effect on human platelet preparations (washed, gel-filtered and dextran-isolated) of two sulfhydryl-activated bacterial protein toxins, streptolysin 0 and and alveolysin, and to compare their efficacy with that of other disruptive procedures (freezing and thawing, ultrasonic, mechanical, or nystatin-toluene treatment) as a method for the determination of various platelet enzyme activities. The enzymes assayed were alkaline and acid phosphatases, monoamine oxidase, phenolsulfotransferase, N-acetyltransferase, hydroxyindole-O-methyltransferase, glutathione peroxidase and lactate dehydrogenase. In all cases, the lowest activities were found after freezing and thawing and/or ultrasonic disruption. The highest activities were always observed in the platelet lysates obtained after toxin, and in some instances after nystatin-toluene treatment. Intermediate values were obtained for mechanical disruption. The -SH-activated cytolysins thus appear to be appropriate and gentle tools for the assay of platelet enzymes when compared to the physical or chemical procedures generally employed.

Interaction of Clostridium perfringens delta toxin with erythrocyte and liposome membranes and relation with the specific binding to the ganglioside GM2.

The specific interaction of the cytolytic Clostridium perfringens delta toxin with membrane GM2 was indicated by: (i) characterization of this glycolipid in the membrane of sheep and goat erythrocytes, which are lysed by the toxin, whereas GM2 was undetectable in insensitive rabbit erythrocytes, (ii) demonstration of 125I-toxin binding to GM2, by autoradiography, following incubation with thin-layer chromatograms containing separated neuroblastoma gangliosides, and (iii) toxin fixation by phospholipid-cholesterol unilamellar vesicles containing either sheep gangliosides or GM2. In order to investigate the intramembrane events leading to membrane disruption following toxin binding, the photoreactive probe 12(4-azido-2-nitrophenoxy)stearoyl 1-14C glucosamine, which inserts into the outer layer and labels integral membrane proteins, was used to establish whether delta toxin penetrates into target cell membrane. No toxin labeling was found, suggesting that toxin action takes place at the membrane surface. This contention is supported by the observation that despite toxin binding, GM2 liposomes did not release entrapped 14C-glucose. Treatment of toxin with carboxypeptidases, but not aminopeptidases, abolished both toxin binding capacity onto erythrocytes and its combination with antitoxin neutralizing antibodies, suggesting that the carboxy terminal end of the toxin is critical for binding to cell membrane.

Two different modes of membrane damaging action by bacterial thiol-activated haemolysins.

The mode of action to cause membrane damage to human diploid fibroblasts by streptolysin 0 is not typical for that of all thiol-activated cytolysins. Theta-toxin and alveolysin, on the other hand, share all properties investigated and may thus, with regard to mechanisms of membrane damaging action, constitute a subgroup among thiol-activated haemolysins.

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.

A survey of bacterial toxins involved in food poisoning: a suggestion for bacterial food poisoning toxin nomenclature.

There is at present no accepted nomenclature for bacterial protein toxins, although there have been several attempts at dividing them into groups by their mode of action. In this paper we will not try to describe all known bacterial protein toxins, but concentrate on the toxins involved in food poisoning. Although most of these toxins are enterotoxins (protein exotoxins with the site of action on the mucosal cells of the intestinal tract) there are also other toxins involved in food poisoning, like the neurotoxins. In Table 1 the most important food pathogens in Europe are listed. For most, but not all, of these food pathogens, toxins are virulence factors. Generally, we divide food poisoning into infections and intoxications, where Salmonella spp. and Shigella spp. are typical examples of infections and Clostridium botulinum and Staphylococcus aureus for intoxications. We consider it better to make four different groups of food pathogenic bacteria, according to Table 2. Today the first three groups are all defined as infections, although for both group 2 and 3 the bacterium itself does not harm the host directly. The bacterium in such locations is like an 'enterotoxin factory'. The bacteria belonging to group 3 do not even interact with the epithelial cells in the intestine, while the bacteria of group 2 must colonise the epithelial cells prior to enterotoxin production.

Molecular features of the cytolytic pore-forming bacterial protein toxins.

The repertoire of the cytolytic pore-forming protein toxins (PFT) comprises 81 identified members. The essential feature of these cytolysins is their capacity to provoke the formation of hydrophilic pores in the cytoplasmic membranes of target eukaryotic cells. This process results from the binding of the proteins on the cell surface, followed by their oligomerization which leads to the insertion of the oligomers into the membrane and formation of protein-lined channels. It impairs the osmotic balance of the cell and causes cytolysis. In this review the molecular aspects of a number of important PFT and their respective encoding structural genes will be briefly described.

[Rectovaginal colonization by group B streptococcus]. / Colonisation recto-vaginale par le streptocoque du groupe B.

The influence of various factors, including age, pregnancy, contraceptive methods, menstrual cycle and use of vaginal tampons, on rectal and vaginal colonization with group B streptococci was investigated in 81 women, 25 of whom were pregnant. The organism was found more frequently in the anus (22.2%) than in the vagina (16%) or the pharynx (2.4%). The colonization rate was higher in women younger than 20, and higher in non pregnant (30%) than in pregnant (12%) women. It was also higher during the first phase of the menstrual cycle (45.4%) than during the second phase (21.8%). No significant difference was observed between users and non-users of intrauterine devices, oral contraceptives and vaginal tampons.