Figure 4.

Systemic effects of wild-type and His351 EF mutants. (a) Survival rate of groups of three C57BL/6 mice challenged with wild-type or mutant EF (50 μg) and PA (100 μg). Mice challenged with EF (wild-type) + PA died within 12 h, while none died in the groups challenged with EF(H351R) + PA or EF(H351A) + PA, or in the control groups (EF or PA only). Mice were injected intravenously with PBS, EF (100 μg) and PA (200 μg), or EF(H351A) (100 μg) and PA (200 μg); (b) Histology of liver and lung tissues of mice treated with ET or ET(H351A). Mice treated with EF in combination with PA died within 3 h, and were immediately necropsied. Mice treated EF(H351A) in combination with PA were euthanized 6 h later by CO₂ inhalation and immediately necropsied. In the liver, sinusoid stenosis (left arrow), scattered necrotic lesions, and inflammatory cell infiltration (right arrow) were found in mice treated with EF(H351A) in combination with PA, while more marked necrotic lesions and inflammatory cell infiltration (right arrow) accompanied by hemorrhaging (left arrow) were identified in mice treated with EF in combination with and PA. In the lungs, hemorrhaging, edema, and inflammatory cell infiltration (arrows) were found around the trachea and bronchus in mice treated with EF in combination with PA but not in those treated with EF(H351A) in combination with PA; (c) and (d) cAMP levels in liver and lung tissues of mice treated with ET or ET (H351A). Mice were euthanized by CO₂ inhalation 2 h after injection and immediately necropsied. Significant differences between the groups are indicated by p-values. The p-values of the indicated groups versus the solvent control group are shown. Data represent the mean ± standard error of the mean based on n = 3 mice per treatment. At least two biological replicates were performed for each experiment. (** p < 0.01, * p < 0.05).