Hemolytic activity of and phagosomal acidification by ΔureC hly⁺ rBCG. (A) RT-PCR analysis of BCG-strains for Hly-secretion. Secretion of Hly was analyzed by RT-PCR using RNA from parental BCG (lane 1), rBCG ΔureC (lane 2), hly⁺ rBCG (lane 3), and ΔureC hly⁺ rBCG (lane 4). Shown is 1 out of 3 representative experiments. (B) Hemolysis by hly⁺ rBCG and ΔureC hly⁺ rBCG but not parental BCG. Sheep red blood cells were incubated with hly⁺ rBCG (open triangles), ΔureC hly⁺ rBCG (closed triangles), parental BCG (closed squares), or L. monocytogenes (open diamonds), or remained untreated (open circles). At indicated time points, aliquots were taken and release of hemoglobin was determined by optical absorption as measurement for lysis of red blood cells. Shown is 1 representative experiment of 4. (C–F) Primary murine macrophages were infected with parental BCG (C and D) or ΔureC hly⁺ rBCG (E and F) for 2.5 hours. Images on the left show BCG stained with DID (signal shown in green). Images on the right show a pseudocolor representation of the fluorescence ratio in the blue and green channels with the Lyso Sensor Yellow/Blue dye. Images are merged with a black-and-white phase contrast image. Note that not all bacteria in each batch were stained due to the poor solubility of DID and its high affinity to the hydrophobic cell wall. Scale bar: 20 μm.