Spore morphology of <i>Enterospectra</i> and <i>Pancytospora</i> species by Nomarski optics.

<p>A. Wild <i>Oscheius</i> sp. 3 strain JU408, with <i>Enteropsectra longa</i> infection. Gut lumen was indicated. Arrow indicates spores, here long and thin spores that are aligned along the apical side of intestinal cells. <b>B.</b> Wild <i>O</i>. <i>tipulae</i> strain JU2551, with <i>Enteropsectra breve</i> infection. Arrow indicates spores, here small spores along the apical side of intestinal cells. <b>C.</b> Wild <i>O</i>. <i>tipulae</i> strain JU1505, with <i>Pancytospora philotis</i> infection. Spores are found throughout the intestinal cell. <b>D</b>. Wild <i>Caenorhabditis brenneri</i> strain JU1396, with <i>Pancytospora epiphaga</i> infection. Spores are seen in the epidermal cells in the tail that does not contain any gut tissue (posterior to the rectum). Anterior is to the right. The "fur" on the outside of the cuticle is formed by unidentified bacteria (see [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006093#ppat.1006093.ref037" target="_blank">37</a>] for another example). Scale bar: 10 μm in A-D. <b>E.</b> Two-dimensional diagram of <i>Oscheius</i> sp. 3 intestine infected with <i>Enteropsectra longa</i>. The intestine is formed of polarized epithelial cells. <i>Enteropsectra longa</i> starts to form spores along the apical side of the intestinal cells.</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

Collection of wild nematode-infecting microsporidia strains

<p>Collection of wild nematode-infecting microsporidia strains</p

Responses of <i>C</i>. <i>elegans</i> strains with transcriptional reporters <i>C17H1</i>.<i>6p</i>::<i>GFP</i> and <i>F26F2</i>.<i>1p</i>::<i>GFP</i> to exposure by different microsporidia.

<p>Strains ERT54 carrying <i>C17H1</i>.<i>6p</i>::<i>GFP</i> (<b>A</b>) and ERT72 carrying <i>F26F2</i>.<i>1p</i>::<i>GFP</i> (<b>B</b>) were analyzed for GFP induction at different time points after infection with different microsporidia and the proportion of animals with GFP induction is shown. GFP was reproducibly induced in ERT54 and ERT72 upon infection with <i>N</i>. <i>parisii</i>, <i>N</i>. <i>major</i> and <i>N</i>. <i>homosporus</i>, while GFP signal was rarely observed in ERT54 and ERT72 inoculated with <i>N</i>. <i>ausubeli</i> or <i>E</i>. <i>longa</i> or the negative control. <i>N</i>. <i>ausubeli</i> did infect the <i>C</i>. <i>elegans</i> reporter strains, as monitored by DIC as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006093#ppat.1006093.t005" target="_blank">Table 5</a>. <b>C</b>. Transcript levels for three genes were measured after 4 hours of infection of N2 <i>C</i>. <i>elegans</i> by <i>N</i>. <i>parisii</i> (ERTm1) and <i>N</i>. <i>ausubeli</i> (ERTm2). The fold increase in transcript level was measured relative to uninfected N2 levels. Infection dose was normalized between <i>Nematocida</i> by successful invasion events counted as intracellular sporoplasms at 4 hpi. To independently compare the microsporidian doses in parallel to the transcript quantification, we also measure the levels of <i>Nematocida</i> SSU rRNA after 4 hours of infection of <i>C</i>. <i>elegans</i> in the same experiment: we found that the rRNA level measured after infection with ERTm2 was 1.25-fold higher than that with ERTm1.</p

Ultrastructural observations of <i>Enteropsectra longa</i>.

<p>Transmission electron micrographs of <i>E</i>. <i>longa</i> strain JUm408 after high-pressure freezing/freeze substitution. <b>A.</b> <i>E</i>. <i>longa</i> meront. A nucleus is visible in the cytoplasm full of ribosomes. <b>B.</b> Lower magnification with a multinucleated meront. Two meronts are indicated, one with a single nucleus in the plane of section (left) and one with several nuclei (right). Two host nuclei are visible on the right, with a dark nucleolus. Intestinal cells contain two nuclei. <b>C.</b> Early sporonts with an electron-dense coat indicated by arrowhead. <b>D.</b> Sporont undergoing a cell division (big arrow); small arrow indicates junction of host intestinal cells; the arrowhead indicates a host Golgi apparatus. <b>E.</b> Mitotic spindle (arrows designate microtubules) in a sporont; the spindle plaque is indicated by an arrowhead. <b>F.</b> Nascent polar tube (arrow) in a sporoblast. <b>G.</b> Wrinkled sporoblasts (*). Arrows indicate the host rough endoplastic reticulum folding around the microsporidia. <b>H.</b> Late stage sporoblast in the center, mature spore on the top left; arrows indicate polar tubes. <b>I.</b> Mature spore with the anterior part of the polar tube, including the anchoring disk. <b>J.</b> Cross-section of mature spores. The exospore and endospore layers are shown in the inset. Arrowheads indicate polar tubes. <b>K.</b> Two mature spores in the intestinal lumen that do not show an additional membrane around them. Low magnification inset shows the positions of the two spores in the lumen and arrowhead indicates host microvilli. <b>L.</b> Low magnification view of cross-section of host, with the intestinal lumen in the center. <i>E</i>. <i>longa</i> spores (arrowheads) concentrate around the apical membrane of intestinal cell, while meronts and early sporonts are on the basal side. Scale bar is 500 nm, unless indicated otherwise. A, anchoring disk; Chr: chromatin; Ex, exospore; En, endospore; Lu, lumen; M, meront; Mi: host mitochondrion; Mv, host microvilli; Nu, nucleus; HNu host nucleus; Pt, polar tube; RER, rough endoplastic reticulum; St: sporont.</p

Summary of the interactions between rhabditid nematodes and microsporidia in the wild and in laboratory.

<p>A mosaic green color means that the corresponding natural infection was found. Plain green means that the infection worked in the laboratory and red means that the infection did not work in the laboratory. White: not determined.</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

Nematode-infecting microsporidia specificity.

<p>Nematode-infecting microsporidia specificity.</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