Research data for: Replicative Acinetobacter baumannii strains interfere with phagosomal maturation by modulating the vacuolar pH

Bacterial pneumonia is a common infection of the lower respiratory tract that can afflict patients of all ages. Multidrug-resistant strains of Acinetobacter baumannii are increasingly responsible for causing nosocomial pneumonias, thus posing an urgent threat. Alveolar macrophages play a critical role in overcoming respiratory infections caused by this pathogen. Recently, we and others have shown that new clinical isolates of A. baumannii, but not the common lab strain ATCC 19606 (19606), can persist and replicate in macrophages within spacious vacuoles that we called Acinetobacter Containing Vacuoles (ACV). In this work, we demonstrate that the modern A. baumannii clinical isolate 398, but not the lab strain 19606, can infect alveolar macrophages and produce ACVs in vivo in a murine pneumonia model. Both strains initially interact with the alveolar macrophage endocytic pathway, as indicated by EEA1 and LAMP1 markers; however, the fate of these strains diverges at a later stage. While 1..., Bacterial strains and culture conditions
The bacterial strains employed in this work are described in Table S1. Bacteria were seeded on lysogeny broth (LB) agar plates and incubated for 16 h at 37 °C. Thereafter, colonies were grown in LB broth under shaking conditions (200 rpm) for 16 h at 37°C. When appropriate, antibiotics were added to the cultures at the following concentrations: 50 μg/ml zeocin, 30 μg/ml kanamycin, and 15 μg/ml chloramphenicol.
Growth assays
A. baumannii strains 19606 and 398 were cultured overnight in LB broth at 37 °C under shaking conditions. Bacterial cultures were centrifugated at 6500 rpm for 5 min and the pellets were washed with Phosphate-buffered saline (PBS, Sigma, D8662). A. baumannii suspensions were diluted to OD600 = 0.01 in 150 μl of LB buffered at pH 5 (MES 100 mM), 6 (MES 100 mM), or 7 (HEPES 100 mM) in 96-well plates and incubated 16 h at 37°C under shaking conditions. OD600 values were measured at 30 min intervals using a BioTek microplate spect...,