FIG. 2.

LLO mutants permeabilize the plasma membrane. C57BL/6 bone marrow-derived macrophages were infected for 4 h without gentamicin and then stained with the membrane-impermeant dye propidium iodide (PI), which increases fluorescence when it passes through the membrane and interacts with host DNA. A total of 2.5 × 10⁴ cells were examined by flow cytometry; the results for half of the cells are shown. The gray histogram represents uninfected cells. The fluorescence range for cells considered permeabilized, as indicated by the marker M1, was defined by adding 10⁶ hemolytic units of purified LLO L461T to the macrophages, as shown in Fig. 1A. The infecting strain and the percentage of cells falling within the marker M1 range are indicated.