Localization of GFP-tagged <i>L. monocytogenes</i> in acidic compartments.

<p><b>A</b>. Cells were infected with the GFP-tagged <i>L. monocytogenes</i> at MOI 50∶1 for 4 hours, and treated with the acidotropic dye Lysotracker Red. Cytospin centrifugation was performed and cells were fixed with 4% paraformaldehyde. Lysotracker Red positive vesicles containing GFP-labeled bacteria are indicated with arrows. Bar represents 5 µm. <b>B</b>. The percentage of Lysotracker Red positive vesicles containing GFP-labeled bacteria was quantified in two independent experiments. A minimum of 200 cells were analyzed. <b>C</b>. Scatter plot analysis showing the correlation between the GFP and Lysotracker Red fluorescence intensities in control cells and TAMs quantified by the Volocity software. A correlation coefficient value of r² equal to 1 indicates 100% bacterial localization with the lysosomal compartments.</p

Escape of <i>L. monocytogenes</i> NF-L327 into the cytosol of infected monocytes.

<p><b>A</b>. Cells were infected with the <i>L. monocytogenes</i> strain NF-L327, that expresses GFP when translocated into the cytosol, at MOI 50∶1 for 30 minutes and medium containing 100 µg/ml gentamicin was added for 1 h to kill extracellular bacteria (0 h). Cells were washed and maintained in RPMI supplemented with 5 µg/ml gentamicin. Cells were lysed after infection (0 h) and 24 h in culture and the colony forming units CFU were quantified, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016960#pone-0016960-g003" target="_blank">Figure 3A</a>. A representative experiment is shown. <b>B</b>. Cells were infected with the <i>L. monocytogenes</i> NF-L327 at MOI 50∶1 as described in A and samples were taken 8 hours post-infection. Cytospin and fluorescence imaging were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016960#s2" target="_blank">Materials and Methods</a>. Bar represents 5 µm. <b>C</b>. Quantification of GFP-labeled <i>Listeria</i> was performed using the Volocity software. The data are presented as number of bacteria in 200 cells (SEM of two independent experiments).</p

Growth curve of untreated control monocytes and TAMs after <i>L. monocytogenes</i> infection.

<p>Cells were infected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016960#s2" target="_blank">Materials and Methods</a>. After the infection, samples were taken at the indicated periods of time, and number of viable cells was assayed by trypan blue exclusion. The data are presented as number of viable cells (panel A) and as percentage of cell recovery, being 100% of recovery at time 0 (panel B). Mean ± SEM of six independent experiments.</p

TAMs control replication of <i>Listeria monocytogenes.</i>

<p><b>A</b>. Intracellular growth curve of <i>L. monocytogenes</i>. Cells were infected for 30 minutes at 37°C with a multiplicity of infection of 25∶1 bacteria per cell, and medium containing 100 µg/ml gentamicin was added for 1 h to kill extracellular bacteria (0 h). Cells were washed and RPMI supplemented with 5 µg/ml gentamicin was added. At the indicated periods of time, 3×10⁵ cells were lysed and colony forming units CFU were quantified, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016960#s2" target="_blank">Materials and Methods</a>. A representative experiment is shown. <b>B</b>. Summary of the growth curves of <i>L. monocytogenes</i>. The data are presented as percentage of the number of CFU recovered in TAMs relative to the number of CFU recovered in control monocytes (mean ± SEM of five independent experiments). <b>C</b>. Cells were infected with <i>L. monocytogenes</i> for 30 minutes at 37°C with a MOI 25∶1 and medium containing 100 µg/ml gentamicin was added for 1 h to kill extracellular bacteria. Intracellular bacteria were detected by immunofluorescence analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016960#s2" target="_blank">Materials and Methods</a> (mean ± SEM of five independent experiments).</p

TAMs express high levels of CD14.

<p>Non-stimulated monocytes and TAMs generated with Trx80 at 100 nM were analyzed for CD14 expression after 24 and 48 hours in culture. MFI: mean fluorescence intensity (mean ± SEM of six independent experiments).</p

Increased recovery of <i>L. monocytogenes</i> in cells treated with the lysosomotrophic agent chloroquine.

<p>Control monocytes and TAMs were infected with <i>L. monocytogenes</i> EGD strain at MOI 25∶1 for 30 minutes in the absence or presence of chloroquine (10 µM) and the treatment was maintained throughout the experiment. <b>A</b>. At the indicated periods of time, cells were lysed and the CFU quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016960#pone-0016960-g003" target="_blank">Figure 3A</a>. Results are shown as ratio between the number of CFU recovered in chloroquine treated cells and the number of CFU recovered in non-treated cells at each time point. <b>B</b>. Number of CFU at time 0 h.</p

TAMs control the replication of <i>Brucella abortus</i>.

<p><b>A</b>. Cells were infected with <i>B. abortus</i> for 45 minutes at 37°C at a MOI 100∶1 and medium containing 100 µg/ml gentamicin was added for 1 hour to kill extracellular bacteria. Intracellular bacteria were detected by immunofluorescence analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016960#s2" target="_blank">Materials and Methods</a>. <b>B</b>. Intracellular growth curve of <i>B. abortus</i>. Cells were infected 45 minutes at 37°C at a MOI 100∶1 and medium containing 100 µg/ml gentamicin was added for 1 h to kill extracellular bacteria (0 h). Cells were washed and RPMI supplemented with 5 µg/ml gentamicin was added. At the indicated periods of time, cells were lysed and the CFU quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016960#pone-0016960-g003" target="_blank">Figure 3A</a>. A representative experiment is shown. <b>C</b>. Summary of growth curves of <i>B. abortus</i>. The data are presented as percentage of the number of CFU in TAMs relative to the number of CFU in control monocytes (SEM of three independent experiments).</p