The <i>Salmonella</i> cytoplasm is rapidly acidified upon macrophage infection and requires OmpR.

<p>RAW264.7 macrophages infected with WT <i>Salmonella</i> and the <i>ΔompR</i> null mutant containing I_A488/I_A647 were imaged over time on a Nikon A1R confocal microscope. The D/A ratio in the <i>ΔompR</i> strain showed minimal FRET, indicating <i>ompR</i> was required for cytoplasmic acidification. RAW264.7 macrophages were pre-treated with 25 nM of the V-ATPase inhibitor bafilomycin A1 (BAF) for 30 min prior to infection, and macrophages were infected with I_A488/I_A647-electroporated <i>Salmonella</i> in the presence/absence of BAF for 3 h and imaged as before. BAF neutralized the SCV and prevented <i>Salmonella</i> acidification. <b>(A)</b> Representative images of D/A ratios are shown at 30 and 180 min post-infection and compared to WT <i>Salmonella</i> prior to infection. Infection was carried out as described in Materials and Methods. Scale bar, 3 μm. <b>(B)</b> Prior to infection, the D/A ratios of <i>Salmonella</i> were determined and the intracellular pH was approximately 6.8. The intracellular pH of <i>Salmonella</i> remained fairly constant in macrophages upon BAF treatment (approximately 6.8) or in the <i>ΔompR</i> null strain in macrophages, unlike untreated WT <i>Salmonella</i>. It showed a remarkable intracellular pH drop to 5.65, similar to the <i>ompR</i> null mutant complemented with <i>ompR</i> supplied in <i>trans</i>. Dataset is represented as the mean ± SEM (<i>n</i> = 3).