Fig 2. SirRH controls the acidic stress response of S. pneumoniae in both culture media and pneumocytes.

(A) The ΔsirR mutant is susceptible to acidified media. The ΔsirR, wr sirR⁺, atpC^A49T and wt cells were grown in BHI until an OD_620nm 0.3 and then incubated in ABM at pH 4.8 for 1 h. Viable cells were assessed by spreading dilutions in BHI-blood-agar plates and incubating these at 37°C for 16 h. (B) Bafilomycin-A1-induced lysosomal neutralization does not affect the impaired intracellular survival of the ΔsirR mutant in IAV-infected cells. A549 cells were infected with the ΔsirR, atpC^A49T and wt cells and intracellular survival was determined as described in the Fig 1. White bars correspond to non-virus infected cells, black bars to IAV-infected cells and gray bars to Bafilomycin-A1-treated cells. (C) IAV-infected A549 cells showed a diminished percentage of S. pneumoniae colocalizing with lysosomes. Representative confocal micrograph showing colocalization of S. pneumoniae (dyed with DAPI) with acidic vesicles dyed with Lysotracker Deep Red in A549 cells. IAV-infected and mock-infected A549 cells were posteriorly infected with S. pneumoniae for 3 h, and stained with the acidotropic LysoTracker Deep Red dye. The event showed in this panel was localized at z-stack no.21 (out of 35). Scale bar, 5 μm. (D) S. pneumoniae colocalize with acidic vesicles in A549 cells. Multichannel plot profile corresponding to the yellow line in the merge inset in “a” depict the fluorescent intensities (a.u., arbitrary units) of LysoTracker Deep Red and DAPI-stained S. pneumoniae (blue). (E) Quantification of LysoTracker association with DAPI-stained S. pneumoniae in mock-infected and IAV-infected A549 cells at 3 hpi with pneumococci. White bar corresponding to mock-infected cells and gray bar to IAV-infected cells. n ≥ 100 bacteria per coverslip. Values represent the mean ± SEM of at least three replicates. Statistical significance was calculated by Student´s t-test (*; p<0.05).