LLO-deficient bacteria remain confined in internalization vacuoles.

<p>JEG3 cells were infected with <i>L</i>. <i>monocytogenes</i> 10403S wild type (WT) or 10403S-Δ<i>hly</i> bacteria (MOI ~ 0.1) and lysed to determine bacterial intracellular loads by CFU counts, or processed for microscopy at the indicated time. <b>A</b>. Bacterial growth curves. Results are mean±SD of triplicate experiments. <b>B</b>. Low magnification micrographs of JEG3 cells infected for 2h or 72h. Images are overlays of <i>Listeria</i> (green) and F-actin (red) signals. Circles highlight a single bacterium within a host cell. Images have been digitally processed to enhance the green fluorescent signal. Bars: 20 μm. <b>C</b>. High magnification micrographs of infected cells at 72h p.i. show a representative LAMP1⁺ compartment encircling a single Δ<i>hly</i> bacterium (arrow), or several LisCVs encircling several WT bacteria (triangles). Overlays show <i>Listeria</i> (green), LAMP1 (red) and Hoechst (blue) signals. Bars: 2 μm. <b>D</b>. Histograms of the number of intracellular bacteria per cell (left) or per LAMP1⁺ compartment (right). At least 1000 cells were examined per experiment. Results represent mean±SD of triplicate experiments.</p

<i>L</i>. <i>monocytogenes</i> switches from actin-based motility to a vacuolar phase in human hepatocytes and trophoblast cells.

<p><b>A-C</b> HepG2 or HeLa cells were infected with <i>L</i>. <i>monocytogenes</i> EGDe (MOI ~ 1–5), counted or lysed to determine bacterial intracellular loads by CFU counts, or processed for microscopy at the indicated time. <b>A</b>. Kinetics of bacterial and cell growth. Results are mean±SD of triplicate experiments. <b>B</b>. Micrographs of HepG2 cells infected for 6h (left panel) or 72h (right panel) with EGDe. Images are overlays of <i>Listeria</i> (green), F-actin or LAMP1 (red) and DAPI (blue) signals. Bars: 10 μm. High magnifications of the squared regions are shown beside with merged signals (on top) or single F-actin or LAMP1 signal (on bottom). Bars: 0.5 μm. <b>C</b>. Histograms of the percentage of intracellular bacteria associated with F-actin (left) or LAMP1 (right). At least 200 bacteria were examined per time-point. Results are mean±SD of triplicate experiments. <b>D</b>. Micrograph of primary human hepatocytes infected with <i>L</i>. <i>monocytogenes</i> 10403S for 72h (MOI ~ 5) and stained with LAMP1 (red in the overlay) and <i>Listeria</i> (green in the overlay) antibodies. Bar: 1 μm. <b>E</b>. Quantification of 10403S bacteria in different phenotypes at 72h p.i. in primary hepatocytes from three human donors. “n” indicates the number of scored bacteria. <b>F</b>. Ultrastructure of representative LisCVs at 72 h p.i. observed by TEM in HepG2 (images 1–3) or JEG3 cells (images 4–9). The nucleus (Nuc.), the nuclear envelope (Nuc. Env.), the membrane of the vacuole (Mb. LisCV), mitochondria (Mito.) and membranous structures (Mbs) are indicated. <u>Images 1–3</u>: a cluster of three <i>Listeria</i> (Lm.) sectioned along their short axis is enclosed within a single-membrane vacuole (LisCV). Three magnifications are shown: scale bars: 1 μm (1), 500nm (2) and 100nm (3). <u>Image 4</u>: three rod-shaped bacteria sectioned along their long axis within a LisCV. Bar: 500nm. <u>Images 5–6</u>: two LisCVs near the nucleus. The septum of a dividing bacterium is pointed with a white arrow (Sept.) and shown at a higher magnification in image 6. Bars: 2 μm and 100nm. <u>Images 7–9</u>: LisCVs in JEG3 cells containing clusters of bacteria, electron-dense heterogeneous materials and membranous structures (shown at a higher magnification in image 9). Altered bacteria are marked with *. Bars: 500 nm. <b>G</b>. Quantification of TEM-observed 10403S bacteria in JEG3 cells at 72h p.i. On the left, % of bacteria in LisCVs, in the cytosol (Cyto) either actin-free “Actin-” or polymerizing actin “Actin+”, and in protusions (PT) or secondary vacuoles (SecV) (also see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006734#ppat.1006734.s002" target="_blank">S2 Fig</a>). On the right, % of intact, degraded or dividing bacteria among vacuolar bacteria. Data are mean±SD of triplicate experiments. “n” indicate the total number of bacteria per category. <b>H</b>. Confocal micrographs of a LisCV. HepG2 cells were infected for 72h with <i>Listeria</i> EGDe-GFP (green) and processed for immunofluorescence with LAMP1 antibodies (red) and DAPI (blue). GFP stains the bacterial cytosol of bacteria in a LAMP1⁺ compartment. Bar: 1 μm. The arrow points the septum of a dividing bacterium, magnified in the black and white image.</p

<i>L</i>. <i>monocytogenes</i> cycles from vacuolar to cytosolic stages during cell subculturing.

<p><b>A</b>. Experimental design of cell subculturing. (“d”: day). <b>B-C</b>. HepG2 cells containing EGDe-GFP bacteria entrapped in LisCVs were purified by FACS (B), plated and examined by microscopy 1h later (d3+1h, C). (C) GFP-positive bacteria (green) are present in LAMP1⁺ compartments (red) near nuclei (blue). Bar: 10 μm. <b>D-E</b>. The same cells were examined 8h (d3+8h) and 3 days later (d6). Micrographs show representative images of cells stained with <i>Listeria</i> antibodies (red), fluorescent phalloidin to label F-actin or LAMP1 antibodies (green) and DAPI (blue). Bar: 10 μm. The framed regions are shown at a higher magnification in the upper right corner. <b>F</b>. The same cells were examined after another cell passage and 1 day of growth (d7) and labeled with ActA antibodies, fluorescent phalloidin and DAPI. Bar: 10 μm. <b>G</b>. JEG3 cells were infected with <i>Listeria</i> EGDe and grown as in (A) up to d7. The overlay images show confocal micrographs of <i>Listeria</i> or F-actin (green), <i>Listeria</i> or LAMP1 (red) and DAPI (blue). Bacteria heavily replicated in the cytosol, were concentrated at the edge of the host cell and were associated with short actin tails. Bar: 10 μm. A magnified image of the region pointed by an arrow is shown on the right.</p

A high concentration of gentamicin favors the selection of <i>L</i>. <i>monocytogenes</i> persistent forms.

<p><b>A-F</b>. JEG3 cells were infected with <i>L</i>. <i>monocytogenes</i> 10403S (MOI ~ 0.1; without 10-min exposure to gentamicin 100 μg/mL), and incubated 72h in presence of different concentrations of gentamicin (0, 1, 5 or 25 μg/mL). Experiments were performed in triplicates. <b>A</b>. Number of bacteria in the extracellular medium (by CFU counts) and viability of host cells (represented as a percentage of infected versus uninfected live cells scored by a trypan blue assay). <b>B</b>. Representative micrographs of infected cells grown in 1 or 5 μg/mL gentamicin. White arrows show groups of LisCVs; triangles point actin-free cytosolic bacteria. Bar: 10 μm. <b>C-F</b>. Effect of the concentration of gentamicin (5 or 25 μg/mL) on the number of bacteria per cell (<b>C</b>), the number of bacteria per phenotype (<b>D</b>), the number of bacteria per LisCV (<b>E</b>) and the proportion of bacteria in different phenotypes (<b>F</b>) (*** <i>p</i><0.0005, “ns”, non-significant, Student <i>t</i>-test). <b>G</b>. Emergence of VBNC bacteria during the subculture of 10403S-Δ<i>actA</i>-infected cells grown in 5 or 25 μg/mL gentamicin (as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006734#ppat.1006734.g006" target="_blank">Fig 6</a>). Infected cells were propagated for 13 days with passages at d3, d6 and d10. Cell lysates were plated before each passage and at d13. Each dot represents the number of bacteria forming colonies (CFU) in the lysate of a well. nd: not detectable. The results are from triplicate experiments (two wells per experiment).</p

ActA deficiency promotes intracellular persistence of <i>Listeria</i> in a VBNC state.

<p><b>A</b>. JEG3 cells were infected with Δ<i>actA</i> strains (MOI ~ 1) for 3 days (d3) then passed and propagated as indicated. <b>B</b>. Representative micrographs of EGDe-Δ<i>actA</i> or 10403S-Δ<i>actA</i> subcultured for 10 days (d10), with two cell passages (at d3 and d6). The color of each staining is indicated on the panel headlines. Bars: 10 μm. <b>C</b>. Comparison of CFU- and microscopic-count methods for the quantification of intracellular Δ<i>actA</i> bacteria at d10. Data are mean±SD from three wells in two independent experiments. 10403S-Δ<i>actA</i> did not form any colony (nd: not detectable). <b>D</b>. Infected JEG3 cells at d10 were permeabilized with 0.1% Triton X-100 and double-labeled with SYTO9 and PI. Intact <i>Listeria</i> cells are stained in green (arrows), while damaged bacteria (*) and nuclei are stained in red. Bar: 1 μm. Squared boxes show higher magnifications. Images are representative of 3 independent experiments. <b>E</b>. Micrographs of JEG3 cells harboring EGDe-Δ<i>actA</i> at day 13. Two representative fields of independent experiments are shown. The color of each staining is indicated on the panel headlines. Bars: 10 μm. Bacteria pointed by arrows are shown at a higher magnification below. A dividing bacterium is highlighted in white. Bars: 2 μm. The % of each bacterial category is indicated as mean±SD of triplicate experiments. <b>F</b>. JEG3 cells were infected with EGDe-Δ<i>actA</i> or EGDe-Δ<i>actA+actA</i> at MOI ~ 1. Infected cells were propagated for 13 days with cell passages at d3, d6 and d10 and cell lysates were plated before each passage and at d13. Data are mean ± SD of triplicate experiments. EGDe-Δ<i>actA</i> is in a VBNC state from d6 to d13. nd: not detectable. <b>G</b>. Representative micrographs of JEG3 cells infected with EGDe-Δ<i>actA</i> or EGDe-Δ<i>actA+actA</i> at d13, after staining with DAPI (blue) and <i>Listeria</i> antibodies (green). <b>H</b>. Quantification of wild type intracellular <i>L</i>. <i>monocytogenes</i> by CFU or immunofluorescence-labeling of bacteria. HepG2 cells were infected with strain EGDe (same experiment as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006734#ppat.1006734.g001" target="_blank">Fig 1A</a>) and JEG3 cells were infected with strain 10403S (same experiment as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006734#ppat.1006734.s003" target="_blank">S3C Fig</a>). Intracellular bacteria were quantified by CFU counts or microscopy. Data are mean ± SD of triplicate experiments.</p

The formation of LisCVs is associated with ActA deficiency.

<p><b>A-C</b> Cells were infected with <i>Listeria</i> EGDe for 72h (HepG2, MOI ~ 1; JEG3 MOI ~ 0.1) and labeled with <i>Listeria</i> polyclonal and ActA monoclonal antibodies. <b>A</b>. Histograms of the percentage of ActA–positive bacteria. Data are mean±SD of triplicate experiments. At least 500 bacteria were observed per time-point. <b>B-C</b>. Micrographs show representative cells labeled with antibodies against <i>Listeria</i>, ActA and/or LAMP1 and DAPI to visualize bacterial nucleoid and cell nuclei. For the overlay images, the color of each staining is indicated on the panel headlines. Arrows point groups of ActA-negative bacteria in LisCVs. Stars (*) indicate examples of ActA–positive bacteria. Bars: 10 μm. <b>D-E</b>. JEG3 cells monolayers were infected for 72h with <i>L</i>. <i>monocytogenes</i> 10403S wild type (WT) or 104033S-Δ<i>actA</i> strain (MOI ~ 0.1) in triplicate experiments. <b>D</b>. Intracellular growth of bacteria assessed by CFU counts. <b>E</b>. Representative LisCVs in cells infected with WT or Δ<i>actA</i> bacteria. Arrows point LisCVs showed at a higher magnification on the right. Bars: 2 μm. <b>F</b>. TEM micrographs show EGDe-Δ<i>actA</i> bacteria in a vacuole at three magnifications. Bars: 1 μm; 0.2 μm; 0.1 μm. Black arrows point the single membrane of the vacuole (Mb. LisCV) and the double-membrane of a neighboring mitochondrion (Mb. mito.) The white arrow points the peptidoglycan (PG) of an intact <i>Listeria</i>. Altered bacteria are indicated by *. Nuc., nucleus; Cyt., cytosol. <b>G</b>. LAMP1⁺ bacteria in mitotic cells. Micrographs are overlays of <i>Listeria</i> (green), LAMP1 (red), F-actin (white) and DAPI (blue) signals and are representative of mitotic cells observed in 10 independent experiments. Bars: 2 μm. White arrows point to LAMP1⁺ <i>Listeria</i>.</p

Model for the intracellular life cycle of <i>L</i>. <i>monocytogenes</i> in hepatocytes and trophoblastic cells. 1–4. The active stage.

<p>After bacterial internalization into the host cell and transient residence within a primary vacuole (<b>1</b>), bacteria escape into the cytoplasm, multiply and induce expression of the actin-polymerizing factor ActA (<b>2</b>). Actin recruitment and polymerization promotes bacterial motility <b>(3)</b> and cell-to-cell spread via the generation of membrane protrusions from the primary infected cell to neighbor cells (<b>4)</b>. After resolution of the protrusions (<b>5</b>), bacteria are in double-membrane secondary vacuoles, from which they escape and start a new cycle of infection <b>(6)</b>. <b>7–8. The phenotypic switch</b>. During the dissemination stage, bacteria stop producing ActA by an unknown asynchronous mechanism. ActA-free cytosolic bacteria (beige bacteria) multiply in the cytosol (<b>7</b>). A xenophagy-like process captures actin-free cytosolic bacteria into tertiary <i>Listeria</i>-containing vacuoles (LisCV) (<b>8</b>). <b>9. The LisCV stage</b>. LisCVs are lysosome-like compartments, in which subpopulations of bacteria resist stress and degradation and enter a slow/non-replicative state (dark orange bacteria), others are sensitive to stress and die (white bacteria with a “*”), and others enter a VBNC state (yellow bacteria). <b>10–11. The reactivation stage</b>. Unidentified stimuli induce reactivation of bacteria, which exit from the vacuoles (<b>10</b>). Production of ActA at the bacterial surface re-initiates a novel cycle of actin-polymerization and spreading (<b>11</b>). <b>12–14. Behavior of ActA-deficient bacteria</b>. <i>actA</i> mutants escape from the primary vacuole, replicate in the cytosol (<b>12</b>) and are captured by a xenophagy-like process (<b>13</b>). In LisCVs, bacteria enter a slow/non-replicative state (dark orange), or die (*) or enter a VBNC state (yellow) (<b>14</b>). <b>15. The dormant stage</b>. Δ<i>actA</i> bacteria are propagated during host cell divisions as VBNC contaminants. In absence of a reactivation signal, wild type bacteria may behave similarly to Δ<i>actA</i> bacteria and acquire the VBNC state. <b>16</b>. Gentamicin selects vacuolar and VBNC <i>L</i>. <i>monocytogenes</i> by inhibiting the growth of cytosolic bacteria.</p

LisCVs have lysosomal features.

<p>JEG3 cells monolayers were infected for 72h with the indicated <i>L</i>. <i>monocytogenes</i> strain (MOI ~ 0.1). <b>A</b>. Cells infected with 10403S were fixed and labeled with Hoechst or <i>Listeria</i> antibody (to detect bacteria), LysoTracker or cathepsin D antibody, and a LAMP1 antibody. Histograms represent the % of LisCVs positive or negative for the indicated marker. Results are mean±SD of triplicate experiments. Representative micrographs are shown beside. The color of each staining is indicated on the panel headlines (bars: 5 μm). A framed LisCV is shown at a higher magnification in the upper right corner. The arrow points a cathepsin D-negative bacterium. <b>B</b>. Cells were infected with the indicated WT or Δ<i>actA</i> strains, permeabilized with 0.1% Triton X-100, double-labeled with SYTO9 and Propidium Iodide (PI) and examined under the microscope. Phase contrast shows groups of cells. Bacteria with intact membranes are stained in green, while the host cell nuclear DNA and damaged bacteria are stained in red. Bar: 5 μm. High-magnifications of regions pointed by arrows are shown on the right. Images are representative of 3 independent experiments. Bar: 1 μm.</p

Intracellular HIV-1 Gag localization is impaired by mutations in the nucleocapsid zinc fingers-2

<p><b>Copyright information:</b></p><p>Taken from "Intracellular HIV-1 Gag localization is impaired by mutations in the nucleocapsid zinc fingers"</p><p>http://www.retrovirology.com/content/4/1/54</p><p>Retrovirology 2007;4():54-54.</p><p>Published online 3 Aug 2007</p><p>PMCID:PMC1976323.</p><p></p>ained for the detection of Gag with an anti-MAp17; with an anti-CD63/Lamp3 for late endosomes, and with an anti-Lamp1 for lysosomes, as indicated. Zoomed-1 picture shows wild-type Gag colocalization with CD63/Lamp3 late endosomal marker (26 ± 6%) and zoomed-2 picture with the Lamp1 marker (6 ± 2%). In contrast, zoomed-3 picture shows less colocalization of this mutant with Lamp3 in comparison to wt (6 ± 4%). Zoomed-4 picture shows an accumulation of NC(ΔZF1ZF2)-Gag mutant at the PM, and less or equal with Lamp1(+) intracytoplasmic vesicles (3 ± 1%)

Intracellular HIV-1 Gag localization is impaired by mutations in the nucleocapsid zinc fingers-0

<p><b>Copyright information:</b></p><p>Taken from "Intracellular HIV-1 Gag localization is impaired by mutations in the nucleocapsid zinc fingers"</p><p>http://www.retrovirology.com/content/4/1/54</p><p>Retrovirology 2007;4():54-54.</p><p>Published online 3 Aug 2007</p><p>PMCID:PMC1976323.</p><p></p>ΔZF1 and ΔZF2 correspond to a complete deletion of the zinc fingers (ZF). ΔNC was described elsewhere [24]. . HelaP4 cells were transfected with the pNL4.3 DNA (wild-type or either one of the NC mutants) and subsequently harvested and lysed. Viral proteins were analyzed by SDS-PAGE and revealed by immunoblotting with anti-CAp24. Immunodetection of the Gag maturation products. Wild-type HIV-1 and NC mutants are indicated. Lanes "V" and "Cell" representing pelletable virions from culture medium and cell lysates, respectively. Pr55Gag, p41(MA-CA), p49(MA-CA-p2-NC) and CAp24/p25 are indicated by arrows