DOI: 10.5061/dryad.8931zcrw0
title: Research data for: Replicative Acinetobacter baumannii strains interfere with phagosomal maturation by modulating the vacuolar pH

description: 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 19606 is eliminated in an autophagy pathway, 398 replicates in ACVs
 and are not degraded. We show that 398 reverts the natural acidification
 of the phagosome by secreting large amounts of ammonia, a by-product of
 amino acid catabolism. We propose that this ability to survive within
 macrophages may be critical for the persistence of clinical A. baumannii
 isolates in the lung during a respiratory infection.
description: 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 <em>A. baumannii</em> 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). <em>A. baumannii </em>suspensions were diluted to
 OD<sub>600</sub> = 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.
 OD<sub>600</sub> values were measured at 30 min intervals
 using a BioTek microplate spectrophotometer. The experiment was performed
 in biological triplicate with three technical replicates per experiment
 for each strain and condition. pH measurements in
 <em>A. baumannii</em> cultures The
 absorbance of phenol red solutions at 560 nm is an indicator of pH, as
 previously described. Phenol red is a pH indicator changing color from
 yellow below pH 6.8 to bright pink above pH 8.2. To analyze the pH changes
 in <em>A. baumannii</em> cultures during bacterial growth,
 indicated strains were maintained overnight in LB broth at 37°C under
 shaking conditions. Bacterial cultures were washed and diluted as
 described for growth assays, with the following modification: non-buffered
 LB pH 5 with phenol red (15 µg/ml) was used. OD<sub>600</sub>
 and OD<sub>560</sub> values were measured every 30 min for 16
 h using a BioTek microplate spectrophotometer. The
 OD<sub>600</sub> absorbance values were subtracted from the
 absorbance at 560 nm data to exclude the effect of bacterial density.
 Finally, the values obtained were used to estimate culture pH by employing
 a standard curve of OD<sub>560</sub> vs pH. The experiment was
 performed in triplicate with three technical replicates per each
 strain. Cell culture conditions
 The J774A.1 mouse macrophage cell line (ATCC TIB-67) was cultured
 in Dulbecco’s Modified Eagle Medium (DMEM) High Glucose (Hyclone,
 SH30022.01) supplemented with 10% heat-inactivated Fetal Bovine Serum
 (FBS, Corning) at 37°C and 5% CO<sub>2</sub>.
 Murine Model of <em>A. baumannii</em>
 pneumonia Colonies from <em>A. baumannii
 </em>strains 398, GFP-398 and GFP-19606 were subcultured from LB
 agar plates and grown in LB broth 16 h at 37°C under shaking conditions.
 Overnight cultures obtained were diluted in sterile LB broth (1:100) and
 incubated for 3 h at 37 °C under shaking conditions. The bacterial inocula
 were prepared by centrifugation at 6500 rpm for 5 min, washed twice
 in PBS, and the pellet resuspended in PBS. Six- to eight-week-old female
 C57BL/6 mice obtained from Charles River Laboratories were intranasally
 inoculated with <em>A. baumannii</em> strains 398, GFP-398, or
 GFP-19606. Briefly, mice were anesthetized by inhalation of 4% isoflurane
 and were infected immediately with 50 μL of bacterial suspension
 (∼1 × 10<sup>8</sup> Colony Forming Units [CFU]) that were
 gradually released with a micropipette into the nares. At 3 and 24 hours
 post-infection (hpi), bronchoalveolar lavage fluid (BALF) was collected.
 For BALF collection, mice were euthanized by CO<sub>2</sub>
 asphyxiation and then, the lungs and trachea were exposed by surgical
 dissection and polyethylene tubing (0.86x1.27mm) was inserted into the
 trachea. Cold PBS containing 1mM EDTA was flushed through the lungs in 1ml
 increments to collect a total volume of 10 ml. All pneumonia studies were
 performed in accordance with the guidelines of the Committee for Animal
 Studies at Washington University School of Medicine, and we have complied
 with all relevant ethical regulations. Antibiotic
 protection assay To analyze the intracellular
 replication of the different strains, J774A.1 cells were seeded 16 h
 before the experiment in 48-well plates (3x10<sup>5</sup>
 cells/well). <em>A. baumannii</em> colonies from LB agar
 plates were inoculated in LB broth and grown overnight at 37°C under
 shaking conditions. Bacterial cultures were centrifugated 5 min at 6500
 rpm and washed twice with PBS. Then, bacterial cultures were normalized to
 OD600 ≈ 1 and appropriate volumes was added to 500 µl of DMEM per well to
 achieve an MOI≈10. Where indicated, bafilomycin A1 (15 nM; Sigma, B1793)
 was added to the growth media and maintained throughout the infection.
 Infected cells were centrifugated 10 min at 200 x g and incubated at 37°C
 and 5% CO<sub>2</sub>. After 1 h, macrophages were washed
 three times with PBS and treated with DMEM supplemented with 10% FBS and
 colistin (50 µg/mL) to eliminate the extracellular bacteria. At the time
 points indicated, the cells were washed three times with PBS and lysed
 with 500 µl of Triton X-100 (0.05%) per well. Serial dilutions of
 bacterial suspensions obtained were plated on LB agar and incubated
 overnight at 37°C, to determine number of colony forming units
 (CFUs). To determine the number of total and
 intracellular bacteria present in BALF from <em>A.
 baumannii-</em>infected mice, two 500 µl aliquots of lavage fluid
 were centrifuged at 6500 rpm for 10 min. The pellets were incubated for 1
 h at 37°C in PBS (total bacteria) or PBS with colistin (50 mg/mL)
 (intracellular bacteria). Then the cells were washed three times with PBS
 and lysed with 500 µl of Triton X-100 (0.05%) per aliquot. CFUs were
 determined by serial dilutions of the bacterial suspensions.
 Ammonia production Ammonia production by
 <em>A. baumannii</em> strains 19606 and 398 was measured using
 the Ammonia Assay Kit (AA0100, Sigma) according to the manufacturer’s
 instructions. Briefly, the strains were cultured overnight in LB broth at
 37 °C under shaking conditions. Then, bacterial cultures were
 centrifugated and washed with PBS. <em>A. baumannii
 </em>suspensions were diluted to OD<sub>600</sub> = 0.01
 in 150 μl of buffered LB (MES 100 mM, pH 5) in 96 well plates and
 incubated for 6 h at 37°C under shaking conditions. Ammonia levels were
 measured, at the indicated times. The experiment was performed in
 biological triplicate. Intracellular ammonia production
 was measured using the same kit. Briefly, macrophages were infected with
 <em>A. baumannii </em>strains 19606 and 398 in duplicates. 24
 hpi a set of wells was scraped in PBS to determine the number of
 macrophages. The remaining wells were treated with 200 µl of Triton X-100
 (0.05%), centrifuged at 6500 rpm for 10 min and ammonia levels were
 measured in the supernatant. Flow Cytometry
 BALF samples were centrifugated at 300 x g for 5 min and
 incubated in 500 µl Pharm Lyse Buffer (BD Biosciences 555899) for 3 min at
 room temperature to lyse red blood cells. The cells were then washed with
 flow cytometry buffer [PBS + 0.5% Bovine Serum Albumin (BSA) (Fisher
 BioReagents, BP9706100) + 2 mM EDTA] and incubated at 4°C with Fc Block
 (BD Biosciences, 553142) for 10 min. Samples were then stained for 30 min
 with CD45-BV510 (Biolegend, 103138) and Ly6G-BV421 (Biolegend,127628),
 CD11b-Alexa700 (BD Biosciences, 557960), CD11c-APC (BD Biosciences,
 550261), and Siglec-F-BV786 (BD Biosciences, 740956). Finally, cells were
 washed and fixed in 2% paraformaldehyde until acquisition on a Benton
 Dickinson (BD) LSR II Fortessa cytometer. GFP signal was acquired in the
 FITC channel. Total cell counts per mouse were calculated using Precision
 Count Beads (Biolegend, 424902) according to the manufacturer’s
 instructions. Cytospin of BALF cells
 BALF samples were centrifugated at 300 x g for 5 min and the
 pellets were resuspended in 500 µl Pharm Lyse Buffer (BD Biosciences,
 555899) for 3 min at room temperature to lyse red blood cells. The cells
 were resuspended in PBS and total cells and viability was determined using
 Trypan Blue solution (Sigma, T8154) and counted using the TC20 automated
 cell counter (BioRad). Samples were centrifugated at 300 x g for 5 min
 onto CytoPro Poly-L-Lysine Coated Microscope Slides (ELITechGroup,
 SS-118). The slides were air-dried overnight at 4 °C and fixed in 4%
 paraformaldehyde. Samples were incubated with permeabilizing and blocking
 solution [PBS + 0.1% saponin + 0.5% BSA (Fisher BioReagents, BP9706100) +
 10% FBS (Corning)]. Cells were stained with Alexa Fluor 647 Rat Anti-Mouse
 Siglec-F antibody (BD, 562680) and nuclei with
 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) solution
 (Invitrogen, D1306) 1 h at 37°C. After staining, the samples were rinsed
 with washing solution [PBS + 0.1% saponin + 0.5% BSA (Fisher BioReagents,
 BP9706100)], then rinsed with water, and mounted with a coverslip in
 Invitrogen ProLong Gold Antifade Mountant (Invitrogen, P36930). Finally,
 the cells were analyzed by confocal microscopy.
 Immunofluorescence staining
 1.3x10<sup>5</sup> J774A.1 cells were plated onto
 glass coverslips in 24 well plates and incubated for 16 to 18 h at 37 °C
 and 5% CO<sub>2</sub>. Inocula of the indicated <em>A.
 baumannii</em> strains were prepared by centrifugation of overnight
 cultures at 6500 rpm for 5 min, washing twice in PBS, and resuspension of
 the pellet in PBS. Bacterial suspensions were normalized to OD≈1 and an
 appropriate volume was used to infect the cells (MOI≈10). Afterward, the
 plates were centrifugated for 10 min at 200 x g to enhance bacterial
 contact with the host cells and incubated for 1 h at 37 °C and 5%
 CO<sub>2</sub>. Cells were washed three times with PBS, and
 extracellular bacteria were killed by treatment of the cells with colistin
 (50 µg/mL) for 1 h. When necessary, cells were incubated with DQ Green BSA
 (10 µg/ml; Invitrogen, D12050) 1 h prior to the end of the infection. At
 the indicated time points, samples were fixed with 4% paraformaldehyde for
 15 min at 37°C and then stored in permeabilizing and blocking solution
 [PBS + 0.1% saponin + 0.5% BSA (Fisher BioReagents, BP9706100) + 10% FBS
 (Corning)]. The glass coverslips were incubated with the indicated primary
 antibodies produced in rabbit: anti-LC3 (Sigma, L7543), anti-EEA1
 (Invitrogen, PA1-063A), anti-LAMP1 (Abcam, ab24170) or anti-<em>A.
 baumannii</em> at a 1:100 dilution for 1 h at 37 °C. The cells were
 then washed 3 times with washing solution and incubated with the indicated
 secondary antibody goat anti-rabbit: Alexa Fluor 647 (Invitrogen, A-21244)
 at a 1:250 dilution, Alexa Fluor 555 phalloidin (0.33 µM; CST, #8953) and
 DAPI for 1 h at 37°C. Afterwards, samples were washed with PBS, rinsed
 with water, and mounted with a coverslip in Invitrogen ProLong Gold
 Antifade Mountant (Invitrogen, P36930). Stained samples were analyzed by
 confocal microscopy.  Confocal microscopy
 Infected cells were analyzed with a Zeiss LSM880 laser scanning
 confocal microscope (Carl Zeiss Inc.) equipped with 405nm diode, 488nm
 Argon, 543nm HeNe, and 633nm HeNe lasers. A Plan-Apochromat 63X (NA 1.4)
 DIC objective and ZEN black 2.1 SP3 software were used for image
 acquisition. Live images were acquired with a Zeiss spinning disk confocal
 microscope (Carl Zeiss Inc.) equipped with 488nm and 560nm lasers. A
 Plan-Apochromat 63X/1.3 Oil Ph 3 (UV) VIS-IR M27 objective and ZEN black
 2.1 SP3 software were employed for image acquisition. Images were analyzed
 using ImageJ software (NIH, USA). Live
 imaging 5x10<sup>5</sup> J774A.1
 macrophages were plated in a 10 mm Glass Bottom Culture 35 mm petri dish
 (MATEK corporation, P35G-0-14-C) and incubated 12-16 h at 37°C and 5%
 CO<sub>2</sub>. The next day, cells were infected with
 mCherry-398 at an MOI≈10 as described above. 15 minutes prior to the
 indicated infection time points, cells were washed twice with PBS and
 incubated in DMEM with LysoSensor Green DND-189 (1µM; Molecular Probes,
 Invitrogen, L7535). The cells were then rinsed twice in PBS and were
 immediately analyzed by confocal microscopy. During image acquisition, the
 cells were maintained at 37°C and 5% CO<sub>2</sub> in a
 temperature-controlled CO<sub>2</sub> chamber on the
 microscope. Transmission electron
 microscopy Cells from BALF were centrifuged at 300 x g
 for 5 minutes and incubated in 500µl Pharm Lyse Buffer (BD Biosciences
 555899) for 3 minutes at room temperature to lyse red blood cells. The
 cells were centrifugated and resuspended in PBS. Total cell number and
 viability were measured using Trypan Blue solution (Sigma T8154) and
 counted with TC20 automated cell counter (BioRad). At least 1x106 cells
 were fixed in 2% paraformaldehyde/2.5% glutaraldehyde (Polysciences Inc.,
 Warrington, PA) in 100 mM sodium cacodylate buffer pH 7.2 for 1 h at room
 temperature and subsequently incubated to 4˚C overnight. Samples were
 washed in sodium cacodylate buffer at room temperature and postfixed in 1%
 osmium tetroxide (Polysciences Inc.) for 1 h. The cells were then rinsed
 in distilled water, and bloc-stained for 1 h with 1% aqueous uranyl
 acetate (Ted Pella Inc., Redding, CA). Subsequently, the samples were
 rinsed in distilled water several times, dehydrated in a graded series of
 ethanol, and finally embedded in Eponate 12 resin (Ted Pella Inc.).
 Sections of 95 nm were cut with a Leica Ultracut UCT ultramicrotome (Leica
 Microsystems Inc., Bannockburn, IL), stained with uranyl acetate and lead
 citrate, and viewed on a JEOL 1200 EX transmission electron microscope
 (JEOL USA Inc., Peabody, MA) equipped with an AMT 8-megapixel digital
 camera and AMT Image Capture Engine V602 software (Advanced Microscopy
 Techniques, Woburn, MA). Images were processed using ImageJ
 software. Statistical analysis
 Statistical analyses were performed using GraphPad Prism 8.0
 (GraphPad Software Inc., La Jolla, CA). Datasets were analyzed by a
 Mann–Whitney test, Welch's t-test, one-way ANOVA-test or two-way
 ANOVA-test, as indicated.