Fig 2.

SecDF contributes to phagosomal escape. (A, left) Confocal fluorescence microscope images of digitonin-treated BMD macrophages infected with mCherry expressing WT L. monocytogenes or ΔsecDF bacteria at 3 h p.i. Cytosolic bacteria were labeled with fluorescein-conjugated anti-Listeria antibody (green), and macrophage nuclei were labeled with DAPI (blue). (Right) percentage of bacteria that escaped macrophage phagosomes at 3 h p.i. as determined by a microscopic fluorescence assay based on selective digitonin membrane permeabilization. The results are representative of 10 microscopic images from 2 independent biological repeats for each strain (in each repeat between 300 and 400 bacteria were counted). The P value was calculated using a chi-square test. (B, left) Confocal fluorescence microscope images of BMD macrophages infected with WT L. monocytogenes or ΔsecDF mutant bacteria at 3 h p.i. Bacteria were labeled with fluorescein-conjugated anti-Listeria antibody (green), macrophage nuclei were labeled with DAPI (blue), and macrophage actin filaments were labeled with rhodamine phalloidin (red). (Right) Percentage of bacteria that escaped macrophage phagosomes at 3 h p.i. as determined by a microscopic fluorescence assay based on actin labeling. The results are representative of 10 microscopic images from 3 independent biological repeats for each strain (in each image, between 50 and 100 bacteria were counted). The P value was calculated using a chi-square test. (C) RT-qPCR transcription analysis of different sec-related genes in intracellularly grown (cytosolic) bacteria (WT L. monocytogenes) in macrophages at 6 h p.i. Transcription levels were measured as relative quantity (RQ), i.e., relative to their levels in bacteria grown exponentially in BHI laboratory medium. Data are presented as log₂ values of the RQ. The data represent 3 biological repeats (n = 3). Error bars represent a 95% confidence interval. (D) RT-qPCR transcription analysis of different sec-related genes in phagosomally trapped bacteria (Δhly mutant) in macrophages at 2.5 h p.i. Transcription levels were measured as the RQ relative to their levels in bacteria grown exponentially in BHI laboratory medium. Data are presented as log₂ values of RQ. The data represent 3 biological repeats (n = 3). Error bars represent a 95% confidence interval, i.e., P ≪ 0.01. (E) Intracellular growth curves of WT L. monocytogenes and the ΔyidC1 and ΔsecA2 mutants in BMD macrophages. Error bars represent the standard deviations from triplicate trials. The data represent 3 independent biological repeats (n = 3).