The Mtb vacuole in BEAS-2B cells acquires Lamp1, but does not acidify.

A–C) BEAS-2B cells were infected with dsRED-expressing Mtb or RFP-expressing Msm at MOI:5 for 4 or 18 hours. Infected cells were fixed and stained for the lysosomal associated protein, Lamp1. Over 200 intracellular bacteria were counted from 4 independent experiments. Mtb-containing vacuoles were categorized as positive or negative, the mean percent positive and standard error were determined, and a Student's t-test was used to determine statistical significance between groups. Error bars represent the mean and SEM for all events (**p<0.01) D) Primary LAEC were infected with dsRED-expressing Mtb at MOI:5 for 18 hours and analyzed as described above. Scale bar = 10 uM. Images are representative of Lamp1-positive (A,D) or Lamp1-negative (B) Mtb containing compartments. E-F) DC or BEAS-2B cells were infected with GFP-expressing Mtb or GFP-expressing Msm linked to the pH sensitive dye pHrodo Red. Cells were fixed 18 hours after infection and images were acquired on a DeltaVision Core DV wide-field microscope. E) pHrodo signal from perpendicular transection on a single 0.2 µm z-stack was used to generate the relative fluorescence unit (RFU) data point for each individual bacterium. The image on the left is an example of a GFP+ bacterium (green) associated with low pHrodo Red (red) signal, while the image on the right is an example of a bacterium with low GFP signal, but high pHrodo Red signal. Lines on the image indicate the perpendicular transection of the bacterium used to generate the RFU for GFP and pHrodo Red indicated in the panels below the images. F) The pHrodo Red RFU for at least 200 individual bacteria from 4 independent experiments for each condition was plotted on a log scale. Error bars represent the mean RFU and SEM for all events. (*p<0.001)