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Biomedical subjects

M Thelestam

Publications and source records attributed to M Thelestam.

At least 55 records · Page 3Linked to original sources
PubMed1991

Extravasation of staphylococcal alpha-toxin in normal and injured CNS regions lacking blood-brain barrier function: observations after ventral root replantation.

Staphylococcus aureus plays an important role as a bacterial pathogen after traumatic injury. The majority of isolated strains produces alpha-toxin, a 33-kDa protein, with membrane-damaging and lethal effects. The central nervous system (CNS) has been considered as the possible target for the lethal action of this toxin. A transfer of alpha-toxin across an intact blood-brain barrier (BBB) is however unlikely. The aim of the present study was to determine if alpha-toxin is accumulated in CNS regions which lack the BBB function. The distribution of alpha-toxin after intravascular injections, in normal mice and rats as well as in rats subjected to ventral root replantation, was assessed using immunogold technique. The results show that, although alpha-toxin does not cross the BBB, alpha-toxin-like immunoreactivity could be detected in the area postrema and at the optic nerve-retinal junction. Extravasation of alpha-toxin was also shown to occur in the spinal cord even 22 months after ventral root replantation. This finding suggests that axon regeneration after ventral root replantation takes place in a macromolecular environment which is totally different from the normal CNS. The implications of vascular spread of alpha-toxin to regions devoid of BBB function are discussed in relation to the bacterial infections which might complicate severe spinal injuries.

Animals↗
PubMed1991

Oligomerisation of cell-bound staphylococcal alpha-toxin in relation to membrane permeabilisation.

We have studied the kinetics of staphylococcal alpha-toxin oligomerisation in relation to membrane permeabilisation, using as targets cultured adrenocortical Y1 cells, rabbit red blood cells (RRBC), human platelets, and liposomes prepared of lipids extracted from platelets. After isolation of membranes from toxin-treated cells, oligomeric toxin was detected (i) by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography or Western blotting, and (ii) by electron microscopy of negatively stained specimens. alpha-Toxin was found to oligomerise on all membranes independently of the temperature. On RRBC and Y1 cells most of the membrane associated toxin appeared converted to the oligomeric form. Hexamers were always present along with membrane permeabilisation. However, hexamers were also detected at conditions when membrane permeabilisation did not occur; at low temperature, in the presence of high concentrations of Ca2+, and after pretreatment of cells with concanavalin A (Con A). Addition of a neutralising monoclonal antibody (MAb) to cell-bound toxin collected it into aggregates much larger than the hexamers. By contrast hexameric toxin remained after addition of a non-neutralising MAb. Our data suggest that the active toxin species is not monomeric, and support the hypothesis that alpha-toxin permeabilises membranes by forming hexameric protein-lined transmembrane channels.

Animals↗
PubMed1991

ADP-ribosylation in Clostridium difficile toxin-treated cells is not related to cytopathogenicity of toxin B.

ADP-ribosylation of a protein in human fibroblasts treated with partially purified Clostridium difficile toxin B was previously reported. Here we show that the same protein was ADP-ribosylated also in human fibroblasts exposed to supernatant from a C. difficile strain producing neither toxin A nor toxin B. Furthermore, in Chinese hamster ovary and in Vero cells, showing toxin B-induced cytopathogenic effect, the protein was not significantly ADP-ribosylated. The results indicate that the ADP-ribosylation is unrelated to the cytopathogenic effect of toxin B. It appears to be caused by another unidentified factor from C. difficile, and the substrate may correspond to a protein modified endogenously in cells exposed to stressful situations. Cellular actin was not ADP-ribosylated by toxin B.

Adenosine Diphosphate Ribose↗
PubMed1991

Clostridium difficile toxin A and its effects on cells.

Clostridium difficile toxin A in its native form is a high molecular weight (520-540 K) aggregate with five major biological activities. It is lethal, enterotoxic, cytotoxic and cytotonic, and induces hemagglutination of rabbit red blood cells. Possibly these activities are contained in separate components. A major subunit of c. 230-310 K has been defined but lower molecular weight components cannot be excluded. The major component has been cloned, and sequence analysis indicated a complicated pattern of repeating sequences in the C-terminal third of the molecule. This review deals mainly with the effects of toxin A on cultured cells. Most mammalian cells are sensitive to toxin A whose major effect is to stop cell division irreversibly. The toxin binds via its repeat sequences to a trisaccharide receptor expressed on rabbit red cells and on brush border membranes from hamster intestine. This receptor seems to be functional in the hemagglutination reaction and the enterotoxicity. Its role in the cytotoxic effect of the toxin is not clear, but no other receptor structure has as yet been identified. In order to exert its cytotoxic (antiproliferative) effect toxin A must first be internalized by endocytosis. Thus a latency period of at least 30 min after toxin binding to cells is consistently observed, and all cytotoxic effects can be prevented by blocking the endocytosis pathway. The first microscopically visible signs of cytotoxicity consist in retraction and rounding of intoxicated cells. In addition the nucleus becomes polarized to one side of the cell while other cell organelles are not significantly affected. These morphological changes seem to be the consequence of a cytoskeletal rearrangement, mainly involving some components of the microfilament system. Inhibition of macromolecular syntheses as well as permeabilization of the plasma membrane may follow the early cytoskeletal effects and finally lead to cell death. Attempts to identify metabolic pathways of significance in the cytotoxicity suggest that the cytosolic level of Ca2+ is not important, thus excluding certain mechanisms for cell killing. In this respect the cytotoxic mode of action of toxin A clearly differs from that of toxin B. However, the biochemical basis for the antiproliferative effect of toxin A remains unknown.

Animals↗
PubMed1991

The structure of Staphylococcus aureus alpha-toxin: effects of trypsin treatment.

Staphylococcus aureus alpha-toxin was treated with trypsin, which inactivates the toxin. Two-dimensional crystals of the modified protein were produced on preformed lipid layers. The projection structure obtained by electron crystallographic analysis of a large number of crystals showed tetragonal p4 symmetry and a resolution of approximately 12 A. The fragments of the toxin, 17 and 18 kDa large, were arranged in a way resembling those observed earlier for the native protein (Olofsson et al., J. Mol. Biol. 214, 299-306, 1990). However, after trypsin treatment the stain-deficient region corresponding to one alpha-toxin monomer shows two separated subdomains of similar size. This separation is probably related to the inability of the modified toxin to undergo the conformational change thought to be essential for the membrane-damaging effect.

Bacterial Toxins↗
PubMed1990

Crystalline layers and three-dimensional structure of Staphylococcus aureus alpha-toxin.

Interaction of the pore-forming protein alpha-toxin from Staphylococcus aureus with lipid components from platelet membranes induces crystal formation of the toxin oligomers. Structure analysis of crystalline areas in either sodium phosphotungstic acid or a sodium phosphotungstic acid/glucose mixture has been performed with electron microscopy and image processing. Ordered domains extending up to a few micrometers were observed, particularly after application of alpha-toxin to pre-formed lipid layers. The crystals, showing tetragonal symmetry, formed either separate two-dimensional sheets or three-dimensional piles of layers. The corresponding unit cell parameter of the single layer was a = b = 109.4 A (standard deviation 2.1 A, n = 21). Incubation of the toxin with intact membranes or extracted lipids as well as application of the lipid layer technique resulted in congruous crystalline properties. The projected averaged alpha-toxin oligomer shows cyclic symmetry with a stain-filled space in the centre. The bulk of the three-dimensional model consists of four asymmetric protein units forming a ring. In addition, a small domain covers the central cavity at the face of the protein opposite to the underlying lipid. The conditions under which the tetragonal arrays are formed on the lipid layers suggest that the alpha-toxin molecule is in a conformation binding to a hydrophobic surface rather than fully inserted into a lipid bilayer.

Bacterial Toxins↗
PubMed1990

Microfilament-disrupting Clostridium difficile toxin B causes multinucleation of transformed cells but does not block capping of membrane Ig.

The effects of Clostridium difficile toxin B on some actin-dependent cellular functions were studied. A three-day incubation of intoxicated B-lymphocytes and transformed 3T3 fibroblasts resulted in dose-dependent multinucleation. Using vimentin-negative Daudi cells we showed that this effect of toxin B does not involve vimentin. As DNA and protein syntheses are not impaired in the cells used, the results suggest that toxin B has an effect on the actin-containing contractile ring during mitosis, in a manner similar to that of the microfilament-disrupting agent cytochalasin B. Toxin B is the first bacterial toxin shown to have this effect. It was also found that the capping of surface IgM on B-lymphocytes was not inhibited by toxin B, whereas cytochalasin B did inhibit capping. These results suggest that capping is dependent on a specific membrane-associated actin structure, which is not affected by toxin B.

Actin Cytoskeleton↗
PubMed1988

Staphylococcal alpha toxin--recent advances.

The elucidation of the amino acid sequence of alpha toxin in 1984 has greatly promoted our understanding of the basic biochemistry and interaction of this toxin with membranes. These aspects are discussed and the concept of alpha toxin as a channel forming protein is critically evaluated. The lethal action of alpha toxin has not yet been clarified, but the previously postulated action as a neurotoxin is not supported by recent observations.

Bacterial Toxins↗
PubMed1988

Oligomerization of 3H-labelled staphylococcal alpha-toxin and fragments on adrenocortical Y1 tumour cells.

Staphylococcus aureus alpha-toxin has previously been shown to bind to erythrocyte membranes and the isolated membranes contain the toxin in both monomeric and hexameric form. The hexamers are believed to form the ring-shaped structures observed by electron microscopy on toxin-treated erythrocytes. It has not previously been shown that hexamers are formed also on nucleated mammalian cells although it has been assumed that hexamers in both systems create transmembrane channels, responsible for the toxin-induced membrane damage. Here we demonstrate by autoradiography that 3H-alpha-toxin bound to and formed high molecular weight complexes-presumably hexamers-on cultured adrenocortical Y1 tumour cells. The binding kinetics suggested a non-specific association of alpha-toxin with the membrane, rather than specific receptor-binding. The pH during toxin binding did not influence the subsequently induced membrane damage. Non-membrane damaging alpha-toxin fragment preparations also bound firmly to the cell membranes. Upon contact with Y1 cells the fragments formed complexes of the same apparent molecular size as those generated from intact alpha-toxin. Two interpretations are possible: either the fragment oligomers are somehow defective i.e. not able to form transmembrane structures or the functional relevance of toxin oligomerization for alpha-toxin-induced membrane damage must be questioned.

Adrenal Cortex↗
PubMed1988

The projection structure of alpha-toxin from Staphylococcus aureus in human platelet membranes as analyzed by electron microscopy and image processing.

Most strains of Staphylococcus aureus produce alpha-toxin, a 33-kDa membrane active protein which is considered to be an important virulence factor of this bacterium. When alpha-toxin interacts with membranes an oligomeric from of the toxin can be seen by electron microscopy as characteristic ring structures in the membrane. A two-dimensional study of these annular structures, incorporated in membranes of human platelets, was performed, introducing a partly new method for rotational alignment of individual particles. It is shown that the averaged oligomer consists of six subunits. At neutral pH the outer diameter of the ring is about 75 A. The stain-filled pore or cavity in the center has a diameter of about 25 A. The size of the hexamer is increased if the pH is lowered.

Blood Platelets↗
PubMed1987

Characterization of domain borders and of a naturally occurring major fragment of staphylococcal alpha-toxin.

A naturally occurring staphylococcal alpha-toxin fragment with an apparent membrane-binding capacity but without toxic activities is shown to be derived from the C-terminal half of the intact polypeptide chain by cleavage between position 134 and 135 in the parent molecule. The resulting N-terminus is slightly ragged with a fragment start not only at position 135 but also at the adjacent position 136. Another naturally occurring fragment starts at position 9, derived from an original cleavage between position 8 and 9 in the parent molecule. Analysis of non-purified fragment mixtures confirmed these positions and established that only one further region, at positions 71-72, is partly sensitive to proteolysis under natural conditions. Trypsin treatment has limited effects on the native toxin molecule, giving essentially only two initial cleavages with resultant large fragments. One of these cleavages is at the peptide bond between position 131 and 132, thus only three residues away from the position of the major naturally occurring cleavage. The other bond sensitive to trypsin is between position 8 and 9, thus identically positioned to the cleavage occurring naturally. Together, all the cleavages define a region in a central segment of the polypeptide chain that has all the properties of an inter-domain segment. The C-terminal half appears to constitute a membrane-binding domain, and the N-terminal half a structure needed for full biological activity, functionally subdividing the parent polypeptide chain.

Amino Acid Sequence↗
PubMed1987

Calcium and calmodulin in cellular intoxication with Clostridium difficile toxin B.

In cultured human lung fibroblasts treated with Clostridium difficile toxin B, the development of the cytopathogenic effect was inhibited by the proton ionophore monensin but was not affected by some other ionophores. The calcium channel blockers verapamil and LaCl3 protected the cells against intoxication, as did the calmodulin antagonists trifluoperazine, amitriptyline, R 24571, and dansylcadaverine. Since these agents could not prevent intoxication when added after the toxin internalization was completed, we suggest that calmodulin and uptake of extracellular calcium are needed for the internalization but not for the cytosolic action of the toxin.

Bacterial Proteins↗
PubMed1987

Cellular internalisation of Clostridium difficile toxin A.

The cytopathogenic effect of toxin A from Clostridium difficile was studied in cultured human lung fibroblasts. The final effect was dependent on toxin concentration and exposure time. Binding of the toxin to cells occurred at 0 degrees C as well as at 37 degrees C. The latency before appearance of the cytopathogenic effect was dose-dependent with a minimum of 45 min. The appearance of a cytopathogenic effect in toxin-treated cells was prevented by the addition of trypsin, antitoxin, lysosomotropic agents, inhibitors of the energy metabolism, 200 mM KCl, 20 mM benzyl alcohol and by incubation at 18 degrees C. Several inhibitors of lysosomal proteases did not prevent the appearance of the cytopathogenic effect. When the extracellular pH was lowered to 4.5 for 5 min immediately after toxin binding the period of latency was significantly shortened. Likewise, the protective effects of lysosomotropic agents were abolished by lowering the extracellular pH. Chinese hamster ovary cell mutants, defective in acidification of their endosomes, were less sensitive to toxin A than wildtype cells. The results indicate that cellular internalisation of toxin A is necessary for intoxication. Moreover, we postulate that the toxin needs some sort of enzymatic activation which can take place only after exposure of the toxin to a low pH.

Animals↗
PubMed1987

Distribution of 3H-labeled staphylococcal alpha-toxin and a toxin fragment in mice.

Staphylococcal alpha-toxin and a toxin fragment were labeled with N-succinimidyl[2,3-3H]propionate. The labeled compounds retained greater than 95% biological activity. The distribution of labeled staphylococcal alpha-toxin and alpha-toxin fragment after intravenous administration to BALB/c mice was studied with whole-body and microautoradiography. The animals were divided into three groups that received (i) labeled alpha-toxin only, labeled alpha-toxin after prior injection of unlabeled fragment, or labeled fragment only. After 5 min, the distribution patterns were similar in groups 1 and 2, with the highest amounts of radioactivity found in the blood vessels, liver, spleen, lungs, and kidneys, whereas the labeled fragment alone showed no initial accumulation in the lungs. The kidneys continued to show a high concentration of radioactivity, whereas the levels at 60 min had decreased in the other organs. The toxin showed continued stable binding to the proximal tubuli, whereas the toxin fragment seemed to dissociate and was found only in small amounts in the glomeruli. No radioactivity was found in the central nervous system.

Animals↗
PubMed1987

Modulation of granulocyte functions by bacterial exotoxin and endotoxins.

The modulation of granulocyte functions by bacterial exotoxins (Streptolysin O, alveolysin, theta toxin) and endotoxins from salmonella and lipid A is described here. Incubation of polymorphonuclear granulocytes with thiol-activated toxins resulted in an increased leukotriene generation. Toxin-pretreated PMNs revealed an increased omega oxidation of LTB4, which may explain why toxin-stimulated cells release more LTC4 than LTB4. Furthermore, toxin-pretreated PMNs showed a decreased leukotriene generation on subsequent stimulation with the Ca-ionophore A 23187 or opsonized zymosan.

Bacterial Proteins↗
PubMed1986

ADP-ribosylation in cultured cells treated with Clostridium difficile toxin B.

In cultured fibroblasts intoxicated with Clostridium difficile toxin B, a radioactive moiety was transferred from [14C-adenosine]NAD, but not from [14C-nicotinamide] NAD, into a cellular protein (MW 90,000). No labeling was detected in toxin-treated cultures not yet showing any toxin-induced cytopathogenic effect, whereas maximal labeling was obtained in cultures with about half of the cells showing a cytopathogenic effect. The radioactivity was removed from the substrate by treatment with snake venom phosphodiesterase. The results suggest that ADP-ribosylation of a cellular protein occurs in toxin B-treated cells and that this reaction may be responsible for development of the cytopathogenic effect.

Adenosine Diphosphate Ribose↗
PubMed1986

Lysosomal involvement in cellular intoxication with Clostridium difficile toxin B.

The process of internalisation of Clostridium difficile toxin B into human lung fibroblasts was further studied, with the aim of elucidating the fate of endocytosed toxin. Development of the toxin-induced cytopathogenic effect was reversibly inhibited at 18 degrees C and in the presence of 200 mM KCl or 1-20 mM benzyl alcohol, i.e. at conditions when the fusion between endosomes and lysosomes is prevented. Fibroblasts treated with toxin at 37 degrees C but transferred to 18 degrees C within 10 min were also completely protected, whereas transfer to 18 degrees C later during the latency resulted in only partial protection. KCl was also protective upon addition after the toxin binding step. Inhibitors of lysosomal proteases, such as chymostatin, leupeptin and antipain, prevented the appearance of the cytopathogenic effect, when present during toxin exposure or added after the toxin binding step. Chinese hamster ovary cell mutants, defective in acidification of their endosomes, were resistant to toxin B, whereas wildtype cells were sensitive. The resistance was not overcome by applying a low extracellular pH. The results suggest that exposure to a low pH compartment is necessary but not sufficient for entry of active toxin B to the cytosol. In addition to a low pH, a fusion of toxin-containing endosomes with lysosomes and a further processing of the toxin by lysosomal proteases is required for cellular intoxication.

Animals↗