Caractérisation d’une phase de persistance intracellulaire du pathogène Listeria monocytogenes

Listeria monocytogenes is a facultative intracellular pathogenic bacterium responsible for a serious disease, listeriosis. Although much work has been done to characterize the virulence mechanisms of this bacterium, there is little data on the mechanisms leading to the asymptomatic carriage of L. monocytogenes in mammalian hosts. One of these mechanisms could be a phase of intracellular persistence. During prolonged infections of human epithelial cells in culture, such as hepatocytes and trophoblast cells, L. monocytogenes changes its intracellular lifestyle. After the active phase of cell-to-cell spread, the bacteria stop polymerizing actin and become trapped in single-membrane vacuoles labeled with the endosomal protein LAMP1.The aim of my thesis was to characterize these "Listeria-Containing Vacuoles" (LisCVs). We have shown that LisCVs are acidic, partially degradative compartments, labeled by the lysosomal protease cathepsin D. Their formation coincides with the disappearance of actin polymerization factor ActA from the bacterial surface and the capture of actin-free cytosolic bacteria by cell membranes. In these compartments, bacterial growth is slowed; a subpopulation is resistant to stress and can survive beyond three days of infection. The use of gentamicin during the infection protocol is not responsible for the formation of LisCVs. However, this antibiotic allows selection of vacuolar bacteria, by specifically inhibiting the growth of cytosolic bacteria. The formation of LisCVs is not specific to laboratory strains. However, the efficacy of the phenomenon could diverge according to the sequence types of L. monocytogenes strains. Vacuolar bacteria have the ability to exit the vacuoles and return to a motile and replicative state during the subculture of infected cells. When expression of the actA gene remains inactive, as in ΔactA mutants, vacuolar Listeria forms persist in host cells in a viable but non-culturable (VBNC) state. These VBNC forms can be transmitted during host cell divisions. All these results reveal a new phase of persistence in the intracellular infectious process of L. monocytogenes during prolonged infections of a subset of epithelial cells. This property could contribute to asymptomatic carriage of this pathogen in epithelial tissues, extend the incubation period of listeriosis, and make bacteria tolerant to antibiotic therapy.Listeria monocytogenes est une bactérie pathogène intracellulaire facultative responsable d’une pathologie grave, la listériose. Si de très nombreux travaux ont permis de caractériser les mécanismes de virulence de cette bactérie, il existe peu de données sur les mécanismes conduisant au portage asymptomatique de L. monocytogenes dans les hôtes mammifères. L’un de ces mécanismes pourrait être une phase de persistance intracellulaire. Lors d’infections prolongées de cellules épithéliales humaines en culture, comme des hépatocytes et des cellules de trophoblastes, L. monocytogenes change de mode de vie intracellulaire. Après la phase active de dissémination de cellule en cellule, les bactéries arrêtent de polymériser l’actine et se retrouvent piégées dans des vacuoles à simple membrane marquées par la protéine endosomale LAMP1. L’objectif de ma thèse était de caractériser ces « Listeria-Containing Vacuoles » (LisCVs). Nous avons montré que les LisCVs sont des compartiments acides, partiellement-dégradatifs, marquées par la protéase lysosomale cathépsine D. Leur formation coïncide avec la disparition du facteur de polymérisation d’actine ActA de la surface bactérienne et la capture des bactéries cytosoliques dépourvues d’actine par des membranes cellulaires. Dans ces compartiments, les bactéries entrent en croissance ralentie ; une sous-population résiste aux stress et peut survivre au-delà de trois jours d’infection. L’utilisation de la gentamicine lors du protocole d’infection n’est pas responsable de la formation des LisCVs. Cependant, cet antibiotique permet la sélection des bactéries vacuolaires, en inhibant spécifiquement la croissance des bactéries cytosoliques. La formation des LisCVs n’est pas spécifique des souches de laboratoire. Toutefois l’efficacité du phénomène pourrait diverger selon les séquençotypes des souches de L. monocytogenes. Les bactéries vacuolaires ont la capacité de sortir des vacuoles et de retourner vers un état motile et réplicatif, après le passage des cellules infectées. Lorsque l’expression du gène actA reste inactive, comme dans les mutants ∆actA, des formes de Listeria vacuolaires persistent dans les cellules hôtes dans un état viable mais non cultivable (VBNC). Ces formes VBNC peuvent être transmises au cours des divisions des cellules hôtes. L’ensemble de ces résultats révèle une nouvelle phase de persistance dans le processus infectieux intracellulaire de L. monocytogenes lors des infections prolongées de certaines cellules épithéliales. Cette propriété pourrait contribuer au portage asymptomatique de ce pathogène dans les tissus épithéliaux, allonger la période d'incubation de la listériose, et rendre les bactéries tolérantes à l’antibiothérapie

Characterization of an intracellular persistence stage on the pathogen Listeria monocytogenes

Listeria monocytogenes est une bactérie pathogène intracellulaire facultative responsable d’une pathologie grave, la listériose. Si de très nombreux travaux ont permis de caractériser les mécanismes de virulence de cette bactérie, il existe peu de données sur les mécanismes conduisant au portage asymptomatique de L. monocytogenes dans les hôtes mammifères. L’un de ces mécanismes pourrait être une phase de persistance intracellulaire. Lors d’infections prolongées de cellules épithéliales humaines en culture, comme des hépatocytes et des cellules de trophoblastes, L. monocytogenes change de mode de vie intracellulaire. Après la phase active de dissémination de cellule en cellule, les bactéries arrêtent de polymériser l’actine et se retrouvent piégées dans des vacuoles à simple membrane marquées par la protéine endosomale LAMP1. L’objectif de ma thèse était de caractériser ces « Listeria-Containing Vacuoles » (LisCVs). Nous avons montré que les LisCVs sont des compartiments acides, partiellement-dégradatifs, marquées par la protéase lysosomale cathépsine D. Leur formation coïncide avec la disparition du facteur de polymérisation d’actine ActA de la surface bactérienne et la capture des bactéries cytosoliques dépourvues d’actine par des membranes cellulaires. Dans ces compartiments, les bactéries entrent en croissance ralentie ; une sous-population résiste aux stress et peut survivre au-delà de trois jours d’infection. L’utilisation de la gentamicine lors du protocole d’infection n’est pas responsable de la formation des LisCVs. Cependant, cet antibiotique permet la sélection des bactéries vacuolaires, en inhibant spécifiquement la croissance des bactéries cytosoliques. La formation des LisCVs n’est pas spécifique des souches de laboratoire. Toutefois l’efficacité du phénomène pourrait diverger selon les séquençotypes des souches de L. monocytogenes. Les bactéries vacuolaires ont la capacité de sortir des vacuoles et de retourner vers un état motile et réplicatif, après le passage des cellules infectées. Lorsque l’expression du gène actA reste inactive, comme dans les mutants ∆actA, des formes de Listeria vacuolaires persistent dans les cellules hôtes dans un état viable mais non cultivable (VBNC). Ces formes VBNC peuvent être transmises au cours des divisions des cellules hôtes. L’ensemble de ces résultats révèle une nouvelle phase de persistance dans le processus infectieux intracellulaire de L. monocytogenes lors des infections prolongées de certaines cellules épithéliales. Cette propriété pourrait contribuer au portage asymptomatique de ce pathogène dans les tissus épithéliaux, allonger la période d'incubation de la listériose, et rendre les bactéries tolérantes à l’antibiothérapie.Listeria monocytogenes is a facultative intracellular pathogenic bacterium responsible for a serious disease, listeriosis. Although much work has been done to characterize the virulence mechanisms of this bacterium, there is little data on the mechanisms leading to the asymptomatic carriage of L. monocytogenes in mammalian hosts. One of these mechanisms could be a phase of intracellular persistence. During prolonged infections of human epithelial cells in culture, such as hepatocytes and trophoblast cells, L. monocytogenes changes its intracellular lifestyle. After the active phase of cell-to-cell spread, the bacteria stop polymerizing actin and become trapped in single-membrane vacuoles labeled with the endosomal protein LAMP1.The aim of my thesis was to characterize these "Listeria-Containing Vacuoles" (LisCVs). We have shown that LisCVs are acidic, partially degradative compartments, labeled by the lysosomal protease cathepsin D. Their formation coincides with the disappearance of actin polymerization factor ActA from the bacterial surface and the capture of actin-free cytosolic bacteria by cell membranes. In these compartments, bacterial growth is slowed; a subpopulation is resistant to stress and can survive beyond three days of infection. The use of gentamicin during the infection protocol is not responsible for the formation of LisCVs. However, this antibiotic allows selection of vacuolar bacteria, by specifically inhibiting the growth of cytosolic bacteria. The formation of LisCVs is not specific to laboratory strains. However, the efficacy of the phenomenon could diverge according to the sequence types of L. monocytogenes strains. Vacuolar bacteria have the ability to exit the vacuoles and return to a motile and replicative state during the subculture of infected cells. When expression of the actA gene remains inactive, as in ΔactA mutants, vacuolar Listeria forms persist in host cells in a viable but non-culturable (VBNC) state. These VBNC forms can be transmitted during host cell divisions. All these results reveal a new phase of persistence in the intracellular infectious process of L. monocytogenes during prolonged infections of a subset of epithelial cells. This property could contribute to asymptomatic carriage of this pathogen in epithelial tissues, extend the incubation period of listeriosis, and make bacteria tolerant to antibiotic therapy

To be cytosolic or vacuolar: the double life of Listeria monocytogenes

Intracellular bacterial pathogens are generally classifie d into two types: those that exploit host membrane trafficking to construct specific nich es in vacuoles (i.e., “vacuolar pathogens”), and those that escape from vacuoles into the cy tosol, where they proliferate and often spread to neighboring cells (i.e., “cytosolic pat hogens”). However, the boundary between these distinct intracellular phenotypes is tenuous and may depend on the timing of infection and on the host cell type. Here, we d iscuss recent progress highlighting this phenotypic duality in Listeria monocytogenes , which has long been a model for cytosolic pathogens, but now emerges as a bacteriu m also capable of residing in vacuoles, in a slow/non-growing state. The ability of L. monocytogenes to enter a persistence stage in vacuoles might play a role during the as ymptomatic incubation period of listeriosis and/or the carriage of this pathogen i n asymptomatic hosts. Moreover, persistent vacuolar Listeria could be less susceptible to antibiotics and more difficult t o detect by routine techniques of clinical biology. These hyp otheses deserve to be explored in order to better manage the risks related to this food-born e pathogen

Phage-mediated dispersal of multicellular bacteria

Streptomyces are renowned for their prolific production of specialized metabolites with applications in medicine and agriculture. These multicellular bacteria present a sophisticated developmental cycle, and play a key role in soil ecology. Little is known about Streptomyces -phage interactions and the impact of phages on Streptomyces physiology. In this study, we investigated the conditions governing the expression and production of ‘Samy’, a prophage found in Streptomyces ambofaciens ATCC 23877. This phage is a siphovirus produced simultaneously with the activation of other mobile genetic elements. We show that Samy production increases bacterial dispersal in stress conditions, facilitating the dissemination of the lineage. Altogether, we unveiled a new property of a bacteriophage infection that it is closely linked to the multicellular community life of Streptomyces bacteria

<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

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

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