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.

The mode of action of Shiga toxin on peptide elongation of eukaryotic protein synthesis.

The effect of Shiga toxin, from Shigella dysenteriae 1, on the component reactions of peptide elongation were investigated. Enzymic binding of [3H]phenylalanine-tRNA to reticulocyte ribosomes was inhibited by 50% at 7 nM toxin. Elongation factor 1 (eEF-1)-dependent GTPase activity was also inhibited. Both reactions were not restored by addition of excess eEF-1 protein. In contrast, toxin concentrations of 200 nM were required to inhibit by 50% the elongation factor 2 (eEF-2)-dependent translocation of aminoacyl-tRNA on ribosomes. Addition of excess eEF-2 restored translocation activity. The eEF-2-dependent GTPase activity was unaffected at toxin concentrations below 100 nM, and Shiga-toxin concentrations of up to 1,000 nM did not affect either GTP.eEF-2.ribosome complex-formation or peptidyltransferase activity. Thus Shiga toxin closely resembles alpha-sarcin in action, both being primary inhibitors of eEF-1-dependent reactions. In contrast, the 60 S ribosome inactivators ricin and phytolaccin are primary inhibitors of eEF-2-dependent reactions of peptide elongation.

Shiga toxin from Shigella dysenteriae 1 inhibits protein synthesis in reticulocyte lysates by inactivation of aminoacyl-tRNA binding.

Inhibition of the peptide elongation cycle of eukaryotic protein synthesis by Shiga toxin from Shigella dysenteriae 1 was examined in toxin-treated reticulocyte lysate mixtures. Peptidyl transferase activity of toxin-treated ribosomes was measured by following the decrease in peptidyl-tRNA concentrations when puromycin was added after incubation with toxin. Concentrations of [3H]leucine-labeled peptidyl-tRNA were measured by extraction with cetyltrimethylammonium bromide. The data suggest that Shiga toxin inhibited aminoacyl-tRNA binding. Toxin-treated ribosomes retained peptidyl transferase activity, and toxin did not block translocation. Furthermore, no inhibition of initiation of protein synthesis could be observed. Finally, Shiga toxin had no detectable nucleolytic effect on polysomal 28S rRNA, nor was hydrolysis of 5.8S or 5S rRNA observed.

Ribonuclease activity associated with the 60S ribosome-inactivating proteins ricin A, phytolaccin and Shiga toxin.

All purified preparations of the ribosome-inactivating proteins ricin A, phytolaccin and Shiga toxin were shown to exhibit ribonuclease activity with 5S or 5.8S rRNA substrates. These toxin species generated reproducible patterns of RNA fragments distinct for each toxin species while multiple preparations of a single toxin species yielded similar RNA fragment patterns. The heat inactivation profile of Shiga toxin was identical for its RNase and protein synthesis inhibitory activities. These data are the first to indicate that the ribosome-inactivating catalytic toxins, in addition to alpha-sarcin, exhibit RNase activity. These results suggest RNase activity may be responsible for ribosome-inactivation catalyzed by ricin, phytolaccin and Shiga toxin proteins.

Effect of Pseudomonas aeruginosa cytotoxin on thymidine incorporation by murine splenocytes.

The interaction of highly purified Pseudomonas aeruginosa cytotoxin (PAC) with murine splenocytes was examined. Added at culture initiation, PAC (0.1 to 0.5 microgram/ml) inhibited subsequent [3H]deoxythymidine incorporation measured between 42 to 48 h. Incorporation of [3H]deoxythymidine was inhibited 50% in lipopolysaccharide-, phytohemagglutinin-, and concanavalin A-stimulated cultures by 0.20, 0.32, and 0.39 microgram of PAC per ml, respectively. It is concluded that PAC exhibits a narrow inhibitory concentration response range of 0.1 to 0.5 microgram/ml which, secondarily, is affected by the presence of mitogens. Antitoxin added at splenocyte culture initiation, directly after PAC, yielded greater than or equal to 86% protection against PAC inhibition of [3H]deoxythymidine incorporation. Addition of antitoxin to cultures at different times after PAC demonstrated a time-dependent loss of antitoxin protective effect over a 12-h period, indicating that PAC became cell associated and refractory to antitoxin within this time period. PAC preincubated with splenocytes at 4 degrees C for less than or equal to 1 h could not be removed by washing of cells and was fully inhibitory to [3H]deoxythymidine incorporation when these cells were cultured at 37 degrees C. This finding was confirmed by demonstrating that 125I-labeled PAC bound immediately to cells. It is concluded that PAC action on splenocytes is dose- and time-dependent and consists of a two-phase process: (i) a very rapid binding of PAC to the cell surface available to antitoxin, and (ii) a slower toxicity development phase of ca. 12 h, during which PAC becomes refractory to antitoxin.