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.     

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.     

Enhancement of cellular protein synthesis sensitivity to diphtheria toxin by interferon.

In attempts to determine whether, by analogy to cholera and tetanus toxins, diphtheria toxin (DT) can also relieve the antiviral effect of interferon (IF), we found that it rather enhanced the inhibitory effect of IF on the replication of murine leukemia virus in chronically infected NIH/3T3 cells. This enhancement was found to be a consequence of an increased sensitivity to DT of cellular protein synthesis in IF-treated cells. IF stimulated the anti-protein synthesis activity of DT in both mouse cells that are known to be highly resistant to this toxin and in human HeLa cells that are highly sensitive to this toxin. This stimulation was dependent on IF dose. The effect of IF on DT action was strictly species specific, indicating that it was not a consequence of the mere binding of IF to the cell membrane, but rather reflected the cellular changes that followed this initial binding. IF was found to be capable of potentiating intact DT, but could not potentiate its fragments in any combination. IF did not have any effect on the in vitro nicotinamide adenine dinucleotide glycohydrolase activity of DT, suggesting that the effect of IF is not due to molecular modification of the toxin.     

Detecting inhibition of coxsackievirus replication by measuring DNA synthesis--effect of proteinase inhibitors

The replication of picornaviruses can be monitored by microscopic examination of the cytopathogenic effect. We found that measuring cellular DNA synthesis gave a more objective and reliable estimate of the viral effect on the cells. Furthermore, by simultaneously measuring DNA synthesis in uninfected cells, we obtained a sensitive indication of whether the agents tested influenced cellular activity. The method was employed to investigate the effect of various proteinase inhibitors on the replication of coxsackie-B3 virus in HEp2 cells. Certain inhibitors of metallo-proteinases had a limited, but consistent, protective effect against viral activity.     

Inhibition of secretion of staphylococcal alpha toxin by cerulenin

Secretion of alpha toxin by Staphylococcus aureus strain Wood 46 was preferentially inhibited by cerulenin, an antibiotic that stops fatty-acid synthesis by inhibiting beta-keto acyl acyl carrier-protein synthetase. At the concentrations used, cerulenin had a negligible effect on cell growth and total protein synthesis, but reduced lipid synthesis by 50%. Extracellular and membrane-associated alpha toxin was absent in cultures treated with cerulenin, but toxin formation was resumed after either removal of the antibiotic or addition of exogenous fatty acids. The apparent absence of toxin precursor in membranes of inhibited cells favours inhibition at an earlier stage in toxin synthesis.     

A rapid microassay for detecting antibodies against poliovirus based on [14C]thymidine uptake of treated cell cultures.

DNA synthesis of mammalian cells propagated in microplates can easily be measured if cell cultures incubated with [14C]thymidine are harvested on the glass fibre filters by a semiautomatic harvesting technique. Soon after infection with poliovirus, [14C]thymidine uptake of U cells (established, human amniotic cell line) is inhibited. This inhibition can be prevented by previous virus neutralization with antibody. Based on this effect a rapid, precise assay method was set up to determine neutralizing antibody titres against poliovirus. There was a good correlation between titres obtained by this assay and those obtained by 50% end point titrations in cytopathogenic effect inhibition assays.     

肾综合征出血热患者早期骨髓细胞结构异常的研究

目的 观察汉坦病毒(HV)对人体骨髓细胞的作用。方法 应用免疫荧光技术、光镜和电镜细胞学检查对骨髓细胞结构尤其是超微结构进行观察研究，并设对照组。结果 检测了27例肾综合征出血热(HFRS)患者骨髓细胞的病毒抗原，20例呈现阳性。发现粒细胞含HV抗原阳性细胞百分数最高(76％)，其他依次为单核细胞(65％)，淋巴细胞(40％)，巨核细胞(20％)和红细胞(3．7％)。利用光镜、透射电镜和扫描电镜进行细胞学观察发现感染病毒的骨髓细胞、细胞膜和细胞器的损伤明显高于对照组。结论 HV可侵入骨髓细胞中，并对人骨髓细胞具有致病变作用。     

Suppressive and Enhancing Effect of T-2 Toxin on Murine Lymphocyte Activation and Interleukin 2 Production

T-2 toxin, a fungal metabolite shown previously to exert potent immunosuppressive effects, was examined for its effects on activation and interleukin 2 (IL 2) production by murine and rat splenocytes. Splenocytes (1 × 10⁶ cells/well) were incubated with 1 μg Concanavalin A (Con A) for 48h at which time cellular protein and DNA synthesis by these cells were ascertained using radiolabeled precursors. IL 2 synthesis was determined from the cell supernatant using the IL 2-requiring cell line CTLL. Spleen cells from mice treated for 4 consecutive days with 2 mg/kg toxin exhibited a 50% reduction in in vitro Con A activation but the supernatant IL 2 levels from these cells was 4-fold higher than cells from control mice. In vitro exposure of Con A-activated normal spleen cells to various toxin doses for 48h resulted in diminished protein and DNA synthesis at 0.4 ng toxin with maximum inhibition at 1 ng (50% inhibition (TC1D₅₀) - 0.5 ng). Enhanced synthesis of both products was observed at lower toxin concentrations. IL 2 production by these cells followed a similar toxin dose response. Rat splenocytes were slightly more resistant and CTLL cells were slightly more sensitive to T-2 toxin than mouse splenocytes. These results indicate the variable effects a cytotoxic agent can have on lymphoid cells and that dosage is an important parameter for these effects.     

Entry of Shigella dysenteriae toxin into HeLa cells

The rate of shigella toxin entry into the cytosol of HeLa S3 cells was estimated from the toxin-induced reduction in protein synthesis. Whereas high toxin concentrations strongly reduced protein synthesis within 30 min, lower concentrations required longer times. The major part of the cell-bound toxin entered only after several hours. Toxin entered cells after incubation at 25 degrees C but not at 20 degrees C, although toxin binding was the same at the two temperatures. Increasing the KCl concentration to 0.2 M protected against toxin. The toxin entry was strongly reduced when the level of ATP in the cells was reduced by incubation with metabolic inhibitors. Lysosomotrophic agents such as NH4Cl and chloroquine had little or no protective effect, but the protonophores carbonyl cyanide p-trifluoromethoxyphenylhydrazone and carbonyl cyanide m-chlorophenylhydrazone and the ionophore monensin protected cells against the toxin. Cells were also protected when the pH was reduced to 6.4. The entry of shigella toxin is discussed in relation to that of other protein toxins with intracellular sites of action.     

Pathomorphological characteristics of experimental toxemia induced by thermostableYersinia pseudotuberculosis toxin

Pathogenic properties of thermostable toxin responsible for pathogenicity of Yersinia pseudotuberculosis were experimentally studied. The toxin exerted a pronounced polyorgan cytopathogenic effect with predominating degenerative destructive changes and membranolytic effect on cell ultrastructure of parenchymatous organs. The toxin is believed to be directly involved in the development of typical pathomorphological picture of pseudotuberculosis, which confirms its pathogenetic role.     

Early events in the action of staphylococcal alpha-toxin on the plasma membrane of adrenocortical Y1 tumor cells.

The early events in staphylococcal alpha-toxin action on mouse adrenocortical (Y1) tumor cells were studied. Cell-bound toxin could be partially neutralized by anti-alpha-toxin and inactivated by trypsin added within 10 min at 37 degrees C after the end of the binding step. Likewise, cell-bound toxin was capable of lysing rabbit erythrocytes (RRBC) added to the cells within 10 min after binding at 37 degrees C. After this time, the Y1 cells could not be rescued from intoxication by antibodies or trypsin, and the toxin was not accessible for lysis of RRBC. However, at 0 to 4 degrees C, the cell-bound toxin remained accessible to antibodies for at least 4 h. CaCl2 (30 mM) did not affect binding of the toxin to Y1 cells but completely prevented the intoxication if added within 10 min at 37 degrees C after the end of the binding step. The intoxication was independent of metabolic energy, active receptor clustering on the cell surface, and endocytosis of the toxin. Therefore, alpha-toxin interacted with the Y1 cell membrane in at least three separable steps: binding, a conformational change at the cell surface, and membrane damage. These early events appear to be similar to those occurring on RRBC treated with alpha-toxin.     

Diphtheria pathogenesis in Guinea pig tracheal organ culture

The effect of diphtheria toxin on guinea pig trachea in organ culture was examined to measure the susceptibility of respiratory epithelial cells to toxin action. Exposure of individual tracheal rings to toxin resulted in cessation of protein synthesis as well as the development of cytopathology within a few hours. Continued incubation led to further inhibition of protein synthesis and extensive disorganization of the epithelial layer. Other inhibitors of protein synthesis were monitored for their effect on the structural integrity of tracheal cells but were found incapable of eliciting similar histopathology. Early after its addition, toxin at minute concentrations possessed cytotoxic properties as well as the ability to inhibit protein synthesis. Interpretation of these data is correlated with current information on the structure and activity of diphtheria toxin.     

Inhibition of protein synthesis in intact HeLa cells by Shigella dysenteriae 1 toxin.

Shiga toxin purified to near homogeneity from cell lysates of Shigella dysenteriae 1 inhibited protein and deoxyribonucle acid syntheses in intact HeLa cells. Inhibition was dependent on toxin concentration and time of incubation. A minimal latent period of 30 min was observed with saturating doses of toxin. Ribonucleic acid synthesis, uptake of alpha-aminoisobutyric acid, and maintenance of intracellular K+ concentrations were not affected until well after maximal inhibition of protein and deoxyribonucleic acid syntheses. The inhibitory effect of toxin was sensitive to heat inactivation and was prevented by antibody neutralization. Several cytotoxic components were separated by polyacrylamide gel electrophoresis of the purified toxin preparations; all inhibited protein and deoxyribonucleic acid syntheses equally.     

The C terminus of component C2II of Clostridium botulinum C2 toxin is essential for receptor binding

The binary Clostridium botulinum C2 toxin consists of two separate proteins, the binding component C2II (80.5 kDa) and the actin-ADP-ribosylating enzyme component C2I (49.4 kDa). For its cytotoxic action, C2II binds to a cell membrane receptor and induces cell entry of C2I via receptor-mediated endocytosis. Here we studied the structure-function relationship of C2II by constructing truncated C2II proteins and producing polyclonal antisera against selective regions of C2II. An antibody raised against the C terminus (amino acids 592 to 721) of C2II inhibited binding of C2II to cells. The antibody prevented pore formation by C2II oligomers in artificial membranes but did not influence the properties of existing channels. To further define the region responsible for receptor binding, we constructed proteins with deletions in C2II; specifically, they lacked amino acid residues 592 to 721 and the 7 C-terminal amino acid residues. The truncated proteins still formed sodium dodecyl sulfate-stable oligomers but were unable to bind to cells. Our data indicate that the C terminus of C2II mediates binding of the protein to cells and that the 7 C-terminal amino acids are structurally important for receptor binding.     

Effect of bacterial toxins on human B cell activation. I. Mitogenic activity of pertussis toxin

Pertussis toxin (PT) was found to elicit an increased thymidine uptake in resting B lymphocytes purified from human peripheral blood. A significant mitogenic effect was detected for toxin concentrations greater than 100 ng/ml (1nM) and a plateau of stimulation was reached at 1000 ng/ml (10 nM). B cell blasts, activated by a first signal such as Staphylococcus aureus Cowan I or insolubilized anti-mu chain antibody, were also stimulated to DNA synthesis by PT in the same range of concentrations. At lower sub-mitogenic concentrations, the toxin potentiated the response to the low-molecular weight B cell growth factor (LMW-BCGF or 12-kDa BCGF), a progression factor for activated B cells. The "A" or catalytic subunit was devoid of any activity on B cells, suggesting the stimulatory effect of the toxin might be associated with the binding or "B" subunit, as it has been shown for T cells. This hypothesis was strengthened by the observation that, as in T cell, the whole toxin but not the "A" promoter, was able to induce calcium influx in these cells. In addition, the purified "B" oligomer alone was found to promote DNA synthesis in B cells. Finally, a fragment of the soluble cleaved form of the CD23 molecule (Fc epsilon RII) could be involved in the process of PT mitogenicity for B cells.     

Inhibition of in vitro human lymphocyte response by the pneumococcal toxin pneumolysin

The effects of pneumolysin, a sulfhydryl-activated cytolytic toxin produced by Streptococcus pneumoniae, on the in vitro human lymphocyte response was examined. The toxin, at concentrations of one to five hemolytic units per ml, caused marked inhibition of the response of lymphocytes to concanavalin A, phytohemagglutinin, pokeweed mitogen, and protein A. The response was assessed by measuring both [3H]thymidine incorporation and the ability of lymphocytes to produce immunoglobulins and lymphokine activity. The effects of pneumolysin were irreversible, could be prevented by pretreatment of the toxin with cholesterol, and were not related to a direct cytotoxic effect on the lymphocytes. Pneumolysin appeared to act at the initiation phase of the immune response and had no effect on lymphocytes committed to DNA synthesis or to the synthesis and secretion of immunoglobulins. Furthermore, pneumolysin-mediated inhibition of the lymphocyte response was not due to the inhibition of binding of mitogens to leukocytes and is likely to be related to effects on membrane-mediated signals essential for lymphocyte triggering. This may be one means by which pneumolysin plays a role in the pathogenesis of pneumococcal infections.     

Binding of pertussis toxin to eucaryotic cells and glycoproteins.

The binding of pertussis toxin and its subunits to cell surface receptors and purified glycoproteins was examined. The interaction of pertussis toxin with components of two variant Chinese hamster ovary (CHO) cell lines was studied. These cell lines are deficient in either sialic acid residues (LEC 2) or sialic acid and galactose residues (LEC 8) on cell surface macromolecules. The binding of pertussis toxin to components of these cells differed from the binding of the toxin to wild-type components. Although the toxin bound to a 165,000-dalton glycoprotein found in N-octylglucoside extracts of wild-type cells, it did not bind to components found in extracts of LEC 2 cells. In contrast, the toxin bound to components found in extracts of LEC 8 cells, which are variant cells that contain increased amounts of terminal N-acetylglucosamine residues on cell surface macromolecules. These results suggest that the receptor for pertussis toxin on CHO cells contains terminal acetamido-containing sugars. The cytopathic effect of the toxin on both types of variant cells was much reduced compared with its effects on wild-type cells. Thus, optimal functional binding of pertussis toxin appears to require a complete sialyllactosamine (NeuAc----Gal beta 4GlcNAc) sequence on surface macromolecules. In addition to studying the nature of the eucaryotic receptor for pertussis toxin, we examined corresponding binding sites for glycoproteins on the toxin molecule. Binding of both S2-S4 and S3-S4 dimers of the toxin to cellular components and purified glycoproteins was observed. The two dimers bound to a number of glycoproteins containing N-linked oligosaccharides but not O-linked oligosaccharides, and differences in the binding of the two dimers to some glycoproteins was noted. These data indicate that the holotoxin molecule contains at least two glycoprotein-binding sites which may have slightly different specificities for glycoproteins.     

Clostridium perfringens iota toxin: binding studies and characterization of cell surface receptor by fluorescence-activated cytometry

The binding characteristics of iota toxin, a binary enterotoxin produced by Clostridium perfringens type E, were studied by fluorescence-activated cytometry. The proteolytically activated binding component of iota toxin, iota b (Ib), bound to various cell types when incubated at 4, 25, or 37 degrees C for 10 min. The binding of Ib was inhibited by antisera against C. perfringens type E or Clostridium spiroforme culture supernatants, but not C. perfringens types C or D. Pretreatment of Vero cells with glycosidases or lectins did not affect Ib interactions, while pronase effectively prevented Ib binding to the cell surface. The Ib protomer (Ibp) bound to the cell surface, but trypsinization of Ibp was necessary for docking of the ADP-ribosylating component, iota a (Ia). Ia attached to cell-bound Ib within 10 min at 37 degrees C, but surface levels of Ia decreased 90% after 30 min and were undetectable by 60 min. Detectable surface levels of Ib also diminished over time, and Western blot analysis suggested internalization or embedment of Ib into the membrane.     

Direct cytotoxic action of Shiga toxin on human vascular endothelial cells.

To help explain a role of the Shiga toxin family in hemorrhagic colitis and hemolytic-uremic syndrome in humans, it has been hypothesized that these toxins cause direct damage to the vascular endothelium. We now report that Shiga toxin purified from Shigella dysenteriae 1 does indeed have a direct cytotoxic effect on vascular endothelial cells in cultures. Human umbilical vein endothelial cells (HUVEC) in confluent monolayers were reduced 50% by 10(-8) M Shiga toxin after a lag period of 48 to 96 h. In comparison, nonconfluent HUVEC were reduced 50% by 10(-10) M Shiga toxin within a 24-h period. These data suggest that dividing endothelial cells are more sensitive to Shiga toxin than are quiescent cells in confluent monolayers. Both confluent and nonconfluent HUVEC specifically bound 125I-Shiga toxin. However, in response to the toxin, rates of incorporation of [3H]leucine into protein were more severely reduced in nonconfluent cells than in confluent cells. Toxin inhibition of protein synthesis preceded detachment of cells from the substratum. The specific binding of 125I-Shiga toxin to human endothelial cells and the cytotoxic response were both toxin dose dependent and neutralized by anti-Shiga toxin antibody. Heat-denatured Shiga toxin was without the cytotoxic effect. In addition, the complete culture system contained less than 0.1 ng of bacterial endotoxin per ml, as measured by the Limulus amoebocyte lysate test.