Mycobacteria cultured under Pi deprivation conditions are phagocytosed with higher efficiency by macrophages.
The phagocytosis assays were carried out incubating J774A.1 macrophage-like cells with mycobacteria labeled with the lipophilic red fluorescent dye PKH-26 at 1:2, 1:5 and 1:20 MOI. Immunofluorescence microscopy revealed that the number of MOs with engulfed or bound bacilli was significantly higher with Pi-deprived mycobacteria (Fig. 1). Phagocytosis was quantitated by FACS with PKH-26 stained bacilli using a Calibur cytometer (Beckton Dickinson, San Diego, CA, USA). Phagocytosis expressed in mean fluorescence intensity (MFI) and percent was dose-dependent and considerably higher with Pi-deprived bacilli (Fig. 1). At a 1:20 MOI, the percent of phagocytosis of bacilli grown without Pi was 80.2% and 38.7% of bacilli grown with usual amounts of Pi.
Figure. 1. Phosphate deprivation increases M. bovis/BCG phagocytosis. J774A.1 MOs (5 x105) were incubated with M. bovis/BCG bacilli labeled red with the PKH-26; nuclei were stained blue with DAPI. Immunofluorescence microscopy shows a marked difference in the degree of phagocytosis between bacilli grown with and without Pi (a, b). FACS analysis considering the percent and the mean fluorescence index (MFI), showed dose-dependent phagocytosis that was significantly higher with Pi-deprived mycobacteria (c, d, e, f). At a 1:20 MOI, 80.2% of MOs phagocytosed bacilli cultured without Pi while the percent phagocytosis of bacilli cultured with Pi was 38.7. p < 0.05; unpaired t Student’s test. Results of 5 independent experiments are shown.
Assays to verify the viability of phagocytosed mycobacteria grown with and without phosphate.
After 4 h of MO phagocytosis at 1:10 and 1:20 MOI the viability of ingested bacilli was assessed using the LIVE/DEAD Baclight bacterial viability Kit (Molecular Probes, Invitrogen, Carlsbad, CA EUA) following manufacturer’s instructions. Bacilli were fixed in 4 % paraformaldehyde for 20 min and rinsed in TBS. Viability was analyzed by flow cytometry separating bacilli in two regions of the log-integrated red fluorescence propidium iodide versus Syto 9; the number of bacilli found within these regions were used to estimate the percentage of viability (Fig. 2). Viability is defined by the equation: measured % live bacilli = (# of bacilli in live region/# of bacilli in dead region) x 100. The results showed that at 1:10 MOI the viability of the Pi-deprived bacilli was significantly increased (Fig. 2).
Figure 2. Phosphate deprivation increases the viability of mycobacteria within macrophages. After 4 h phagocytosis, mycobacteria viability was analyzed by FACS using the LIVE/DEAD viability assay. A dot plot was set to differentiate living from dead cells (a). The survival rate was calculated using the formula: measured % live bacteria = (# of bacteria in live region / # of bacteria in dead region) x 100) (b). p< 0.05 Mann Whitney test. Results of 3 experiments are presented.
High phagocytosis of microbeads coated with plasma membrane proteins of phosphate-deprived mycobacteria
To study the role of mycobacteria proteins in the phagocytosis of bacilli grown under Pi starvation conditions, we carried out assays with fluorescent microbeads coated with proteins obtained from plasma membranes isolated from whole mycobacteria grown with and without Pi. For phagocytosis assays, MOs (5 x 106) cultured in RPMI 1640 with 10% heat-inactivated fetal bovine serum (FBS) were placed on acid-washed glass coverslips. Microbeads were added to cells at 1:5, 1:20 and 1:100 rate for 4 h at 37°C in 5% CO2. To quantitate phagocytosis of microbeads, the slides were analyzed with a Nikon epifluorescence microscope examining at least 400 cells in random fields of 3 slides at each experimental time (Fig. 2). MOs were scored positive regardless of the number of bound or engulfed microbeads. Binding/phagocytosis of microbeads was dose-dependent and significantly higher with microbeads coated with membrane proteins obtained from bacilli grown without Pi (Fig. 2); at 1:100 rate, 84.44% of MOs bound or engulfed one or more microbeads. With microbeads coated with proteins from bacilli grown with Pi, binding/phagocytosis of microbeads was 45.4%.
Figure. 3. Phagocytosis of membrane protein-coated microbeads is higher with phosphate deprived bacilli proteins. Assays were carried out with J774A.1 cells and green fluorescent microbeads coated with plasma membrane proteins obtained from mycobacteria grown with and without Pi. MOs (5 x 105) were placed on acid-washed glass coverslips. Microbeads were added to cells at a 1:5, 1:20 and 1:100 rate for 4 h at 37°C in 5% CO2. The percentage of cells with ingested or bound microbeads was estimated counting at least 400 MOs (a, b). At 1:100 rate the phagocytosis of microbeads coated with proteins of Pi-deprived bacilli was 84.44% and of bacilli grown with Pi it was 45.4% (a, c). p<0.05; unpaired t Student’s test. Results of 5 experiments are presented.
Phosphate-deprived mycobacteria alter phagosome acidification
An outstanding ability of virulent mycobacteria is to inhibit phagosome acidification and phagolysosome fusion [2]. We studied th phagosomal acidification process by immunofluorescence microscopy using M. bovis/BCG grown with or without Pi labelled green with FITC and MOs labelled with LysoTracker, a marker that turns red at low pH (Fig.4). Bacilli located in Lysotracker positive vacuoles were yellow fluorescent due to overlapping of FITC and the red fluorescence of Lysotracker. The engulfed bacilli emitted a FITC green fluorescence when were located in Lysotracker negative vacuoles (Fig.4). When infected with bacilli grown with usual amounts of Pi, 80.75 % of cells were Lysotracker positive and with Pi-deprived bacilli the percent of Lysotracker labeled MOs was reduced to 73.06% FACS analysis of the phagocytosis assays confirmed the immunofluorescence findings showing that Pi-deprived bacilli phagocytosis is associated with decreased Lysotracker labeling (Fig. 4).
Figure. 4. Decreased acidification in MOs that engulfed mycobacteria grown without phosphate. Phagocytosis assays were performed with MOs labelled with LysoTracker red and FITC-labelled bacilli cultured with or without Pi. Immunofluorescence microscopy was performed counting randomly at least 400 MO in 3 different slides. MO emitting red fluorescence were scored as Lysotracker positive (a). It was found that 80.75% of MO incubated with mycobacteria grown with usual amount of Pi were LysoTracker labelled while incubation with Pi-deprived bacilli reduced the number of Lysotracker marked MO (76.06%, b). The degree of Lysotracker labelling was also analyzed by FACS which showed that the MIF was less in MO that phagocytosed Pi-deprived mycobacteria (c). The immunofluorescence patterns are shown; bacilli were located in non-acidified compartments (LysoTracker negative) are green fluorescent (arrows). In acidified vacuoles (LysoTracker positive) bacilli emitted yellow fluorescence due to overlapping of FITC -labelled bacilli and Lysotracker red fluorescence (asterisks). Four independent experiments were performed.
Phosphate-starved mycobacteria increment the expression of adhesins in isolated plasma membranes.
We have previously shown by immunoelectron microscopy that PstS-1 is a cell surface located adhesin that triggers the phagocytosis of mycobacteria [10]. To study its location in Pi- starved mycobacteria, we isolated cell walls and plasma membranes to carry out 1-D electrophoresis and immunoblot (Fig. 5). By Coomasie blue staining and immunoblot with a polyclonal anti-M.bovis/BCg antiserum the number of protein bands was higher in cell-walls of Pi-deprived mycobacteria. In the isolated plasma membranes immunobloting with mAb available in our laboratory that recognizes adhesins that participate in the phagocytosis of mycobacteria 5 reactive bands of 38, 19, 23, and 47 kDa that correspond to PstS-1, LpqH, LprG, and the APA antigen, respectively (Fig. 5). In the membranes of bacilli grown with Pi band mild APA reactivity was observed.
Figure 5. Cell membrane located PstS-1, LpqH, LprG, and APA are virulence factors expressed by Pi-deprived mycobacteria. M.bovis/BCG bacilli were cultured with and without Pi for 24 h. The mycobacteria were sonicated and by centrifugation the cell walls and the plasma membranes were obtained. The cell walls were resolved in 12% SDS-PAGE and transferred to PVDF for immunoblot. Coomasie blue stain and immunoblotting with an anti-M.bovis/BCG antiserum detected many protein bands more numerous in cell walls of Pi-deprived bacilli (a,b). Using mAbs available in our laboratory the expression of PstS-1, LpqH, LprG, and APA were demonstrated in isolated plasma membranes of Pi-deprived mycobacteria (c-f). These proteins are recognized virulence factors that participate as adhesins in the infection of host cells.