Analysis of rS6p phosphorylation in bone marrow-derived macrophages.

<p>Immunoblot analysis of cell lysates from <i>Myd88</i>^-/- <b>(a)</b> or C57BL/6 <b>(b)</b> BMMs that were serum-starved and infected with <i>Legionella</i> (MOI = 20) for 4 hrs as indicated under serum-free conditions. Quantified band intensities are normalized to uninfected conditions (UN) and listed below each blot. Single-cell analysis of rS6p phosphorylation in serum-starved BMMs infected with either <i>ΔflaA</i> <b>(c)</b> of <i>ΔdotA</i> <b>(d)</b>. Graph shows means and 95% confidence intervals of phospho-rS6p fluorescent intensity signal for at least 100 infected cells for each condition. * p<0.05, ** p<0.005 (one-way ANOVA). <b>(a-d)</b> A representative of three biological replicates is shown for each experiment.</p

Loss of MTOR function triggers a host cell death response that requires bacterial replication.

<p><b>(a)</b> Experimental scheme for the results shown in <b>(b-e and g-j)</b>. <b>(b-j)</b> BMMs were serum-starved and infected under serum-free conditions either at MOI = 20 <b>(b-e and g-j)</b> or MOI = 10 <b>(f)</b>. <b>(b)</b> Micrographs showing representative of live and dead macrophages infected with <i>Legionella</i> for 12 hrs and stained with anti-<i>L</i>. <i>pneumophila</i> (L.p), anti-ubiquitinated proteins (FK2) antibodies and Hoechst 33342. Arrowheads indicate LCVs, Bar = 5μm. (*) marks the condensed nucleus of the dead cells. The mean nuclear volumes ± s.d of at least 100 live and 100 dead cells are graphed. <b>(c-d)</b> Quantitation of infected and neighboring uninfected <i>Myd88</i>^-/- <b>(c-d)</b>, C57BL/6 <b>(d)</b> and <i>Mtor</i>^-/- <b>(d)</b> macrophages with condensed nuclei after infections with <i>ΔflaA</i> <b>(c-d)</b> or Δ<i>dotA</i> <b>(d)</b>. Means ± s.d of technical replicates of dead cell as percentage of total cells in each condition are shown. <b>(e)</b> Quantitation of infected <i>Myd88</i>^-/- BMMs with condensed nuclei after infection with <i>L</i>. <i>dumoffii</i> and treatment with inhibitors or vehicle as indicated. <b>(f-g)</b> Kinetics of the cell death response in C57BL/6 BMMs under serum starvation conditions infected as indicated. Quantitation of infected cells with condensed nuclei <b>(f)</b> and LDH released in the culture supernatants <b>(g)</b> are shown. <b>(h-i)</b> Analyses of ubiquitin recruitment <b>(h)</b> and LCV size <b>(i)</b> in live and dead <i>Myd88</i>^-/- BMMs infected with <i>ΔflaA</i> and treated with PP242. <b>(j)</b> Cell death in infected <i>Myd88</i>^-/- BMMs with <i>thyA ΔflaA</i> strain treated with vehicle (DMSO) or PP242 in the presence or absence of thymidine. <b>(c, e, h-j)</b> Rapamycin (250nM), PP242 (2.5μM), LY294002 (10μM), Torin2 (300nM). <b>(c-j)</b> Means ± s.d of technical triplicates for each condition are shown. At least 50 cells <b>(e, h-j)</b> or 200 cells <b>(c-d)</b> were analyzed for each condition. A representative of two <b>(e-j)</b> or three <b>(c-d)</b> biological replicates is shown for each experiment. <b>(b-j)</b> n.s—not significant, ** p<0.005 (unpaired T-test).</p

Serum lipids, SREPB1/2 and MTOR regulate LCV stability.

<p>Serum-starved <i>Myd88</i>^-/- <b>(a-f)</b> or C57BL/6 <b>(g)</b> BMMs or were infected by the indicated strains (MOI = 20) in synchronized infections for 12 hrs <b>(a-f)</b> or for the indicated time periods <b>(g)</b> in the absence <b>(a-g)</b> or presence <b>(f-g)</b> of FBS. <b>(a-b)</b> Galectin 3 accumulation onto LCVs harboring <i>ΔsdhA ΔflaA</i>. <b>(a)</b> projection micrograph of a representative infected cell with the inset showing the LCV <b>(b)</b> Kinetic analysis of Galectin 3+ LCVs harboring <i>ΔsdhA ΔflaA</i>. <b>(c,e)</b> Representative projection micrographs of Galectin 3 positive <b>(c,e)</b> or negative <b>(c)</b> LCVs harboring Δ<i>flaA</i> after treatments with inhibitors <b>(e)</b> or vehicle alone <b>(c)</b>. The insets show all individual planes of the projection image. Quantitation of Galectin 3+ LCVs after the indicated treatments in the absence <b>(d</b> and <b>f)</b> or presence <b>(f)</b> of FBS. <b>(g)</b> Kinetics of emergence of Galectin 3 positive LCV under serum starvation or replete conditions. <b>(c-g)</b> PP242 (2.5μM), fatostatin (4μM), FBS (10%). <b>(b,d,f</b> and <b>g)</b> Means ± s.d of technical triplicates for each condition are shown. At least 100 LCVs were analyzed for each condition. A representative of two <b>(b, f-g)</b> or three <b>(a, c-e)</b> biological replicates is shown for each experiment. <b>(b, d, f</b> and <b>g)</b> n.s—not significant, ** p<0.005 (unpaired T-test) <b>(a, c</b> and <b>e)</b> Cells were stained with anti-galectin3, anti-<i>Legionella</i> antibodies and Hoechst 33342. Arrowheads indicate the LCVs. Bar = 5μm.</p

MTOR inhibition destabilizes LCVs.

<p><b>(a)</b> Schematic for detection of leaky LCVs by selective plasma membrane permeabilization of infected cells. Single-positive bacteria reside in stable LCVs; double-positive bacteria reside in disrupted LCVs <b>(b-f)</b> Serum-starved <i>Myd88</i>^-/- macrophages infected with <i>ΔflaA L</i>. <i>pneumophila</i> (MOI = 20) for 7hrs. PP242 was added at the time of synchronization– 60min p.i. <b>(b-c)</b> Micrographs of representative stable <b>(b)</b> or leaky <b>(c)</b> LCVs are shown. Cells were stained as indicated in <b>(a)</b>. Arrowheads indicate LCVs. Bar = 5μm <b>(d)</b> Quantitation of destabilized LCVs in cells treated as indicated. <b>(e)</b> Micrographs of representative destabilized LCVs (double-positive) in BMM with aberrant (A) nuclear morphology and stable LCVs (single-positive) in a neighboring cell with normal (N) nuclear morphology Bar = 5μm <b>(f)</b> Quantitation of destabilized LCVs in BMMs with normal or aberrant nuclear morphology. BMMs treated as indicated. <b>(b-f)</b> PP242 (2.5μM). <b>(d-f)</b> Means ± s.d of technical triplicates for each condition are shown. At least 100 LCVs were analyzed for each condition. <b>(b-f)</b> A representative of three biological replicates is shown. <b>(d</b> and <b>f)</b> ** p<0.005 (unpaired T-test).</p

Host lipids dictate the LCV housing capacity.

<p><b>(a-c, f)</b> Size analysis of LCVs harbored by C57BL/6 BMMs infected with <i>ΔflaA</i> bacteria (MOI = 20) for 15 hrs after 60 min synchronization. Cells were serum-starved prior to infection for 10hrs. LCV sizes were measured through 3D microscopy analysis of infected cells as detailed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006088#ppat.1006088.s008" target="_blank">S8 Fig</a>. <b>(a)</b> Relative distributions of sizes of LCV harbored by live and dead cells produced by infections in the presence/absence of FBS. Live/dead distinction was determined morphologically by nuclear condensation. <b>(b-c)</b> Size analysis of the 50 largest LCVs produced by Δ<i>flaA</i> infections shown in <b>(a)</b>. <b>(d)</b> <i>Legionella</i> growth in axenic cultures supplemented with FBS or delipidated FBS (dFBS) (10% v/v). <b>(e)</b> Kinetic analysis of LCVs that support bacterial replication in C57BL/6 BMMs infected with <i>ΔflaA</i> bacteria. FBS was added or omitted after the infection synchronization at 30min post infection. At least 200 LCVs were scored for each condition. <b>(f)</b> Size analysis of LCVs harbored by cells with condensed nucleus from <b>(a)</b>. <b>(g)</b> Percentage of <i>Myd88</i>^-/- BMMs harboring large LCVs (bacteria>20) produced by 12 hrs synchronized infections with Δ<i>flaA</i> bacteria. Cell treatments were initiated at 4 hrs post infection as indicated. <b>(h)</b> <i>L</i>. <i>pneumophila</i> intracellular growth in <i>Acanthamoeba castellanii</i> over 48hrs in the presence of DMSO or Torin2 (300 nM), MOI = 5. <b>(i)</b> Percentage of <i>Myd88</i>^-/- BMMs with condensed nuclei uninfected or infected with <i>ΔflaA</i> bacteria for 9hrs. Infections were synchronized at 60min and various treatments were added at 6hrs as indicated. <b>(j)</b> Model for MTOR-dependent regulation of LCV homeostasis through the lipogenesis and serum-derived lipids. Abbreviations: ubiquitin ligase (UBL), pathogen-associated molecular patterns (PAMPs) <b>(b, f-g, h-i)</b> Means ± s.d of technical triplicates for each condition are shown. <b>(a</b> and <b>g)</b> At least 100 LCVs were analyzed for each condition. A representative of two <b>(d, h-i)</b> or three <b>(a-c, e-g)</b> biological replicates is shown for each experiment. <b>(a, g</b> and <b>h)</b> PP242 (2.5 μM), Brefeldin A (17.8 μM), Nocodazole (20 μM), FBS (10% v/v), dFBS (10% v/v) <b>(b, f-i)</b> * p<0.05, ** p<0.005 (unpaired T-test).</p

<i>Legionella</i>-induced phosphorylation of rS6p requires MTOR and PI3K activity.

<p><b>(a)</b> Simplified schematics of the PI3K/MTOR signaling axis indicating the inhibitors used in this study. PI3K is inhibited by LY294002. Rapamycin, PP242 and Torin2 act on MTOR and Hanks' Balanced Salt Solution (HBSS) blocks MTOR by starving cells for amino acids. <b>(b-d)</b> Analyses of serum-starved <i>Myd88</i>^-/- BMMs unstimulated or infected with <i>ΔflaA</i> (MOI = 20) for 5hrs under serum-free conditions. Inhibitors—Rapamycin (200nM), PP242 (5μM), LY294002 (10μM)—or HBSS were added at the time of infection synchronization at 60 min post infection. <b>(b)</b> Immunoblot analysis of S6K1 and rS6p phosphorylation from cell lysates showing quantified band intensities normalized to uninfected conditions (UN). <b>(c)</b> Single cell immunofluorescence analysis of phospho-rS6 positive (MFI>300) <i>Myd88</i>^-/-macrophages exposed to <i>ΔflaA</i> (MOI = 20). Graphed are the means and standard deviations (s.d) of technical triplicates for the two distinct groups within the cell population—infected (LCV present) and uninfected (LCV absent) for each condition. At least 100 cells were analyzed for each condition. ** p<0.005 (one-way ANOVA) <b>(d)</b> Immunofluorescense micrographs of representative infected cells from each condition stained with anti-<i>L</i>. <i>pneumophila</i> (L.p), anti-p-rS6p (S235/236), anti-ubiquitinated proteins (FK2) antibodies and Hoechst 33342. Arrowheads indicate <i>Legionella</i>-containing vacuoles, Bar = 5μm. <b>(b-d)</b> A representative of three biological replicates is shown for each experiment.</p

<i>Legionella</i>-induced MTOR activation is a Dot/Icm effector-driven process independent of intracellular niche biogenesis.

<p><b>(a-h)</b> Show results from synchronized infections (MOI = 20) under serum-free conditions of serum-starved <i>Myd88</i>^-/- BMMs treated as indicated. <b>(a)</b> Immonofluorescent micrograph of <i>ΔflaA</i>-infected (**) or neighboring uninfected (*) cells at 6 hrs post-infection stained with anti-<i>L</i>. <i>pneumophila</i> (L.p), anti-p-rS6p (S235/236) antibodies and Hoechst 33342. Arrowheads indicate <i>Legionella</i>-containing vacuoles, Bar = 10μm. <b>(b-c</b> and <b>g)</b> Single cell immunofluorescence analysis of phospho-rS6 positive (MFI>300) BMMs exposed to various <i>Legionella</i> strains for the indicated times <b>(b)</b> or for 8hrs <b>(c</b> and <b>g)</b>. Means ± s.d of technical triplicates for the two distinct groups within the cell population—infected (LCV present) and uninfected (LCV absent) for each condition are shown. <b>(d)</b> Micrographs of cells infected with the indicated strains for 8hrs and stained with anti-<i>L</i>. <i>pneumophila</i> (L.p), anti-p-rS6p (S235/236), anti-ubiquitinated proteins (FK2) antibodies and Hoechst 33342. Arrowheads indicate intracellular bacteria, Bar = 5μm. <b>(e)</b> Quantitation of p-rS6p positive cells (MFI>300) in synchronized infections with a <i>thyA ΔflaA</i> strain in the presence (+thy) or absence (-thy) of thymidine for the indicated time periods. Means ± s.d of technical triplicates are shown. <b>(f-g)</b> Synchronized macrophages infections with different <i>Legionella</i> species are shown. <b>(f)</b> Representative micrographs of cells infected with <i>L</i>. <i>pneumophila</i> or <i>L</i>. <i>dumoffii</i> for 8hrs (MOI = 20) and stained as in <b>(d)</b>, which are quantitated in <b>(g)</b>. <b>(h-i)</b> Cells were treated with cytochalasin D (5μM) or vehicle (DMSO) for 15 min prior to and for the duration of infections (3hrs) with the indicated bacterial strains. <b>(i)</b> Quantitation of p-rS6p positive cells (MFI>300) in contact with bacteria (<i>ΔflaA</i> and <i>ΔdotA</i>) or uninfected cells (UNi). Means ± s.d of technical triplicates are shown. <b>(h)</b> Representative micrograph of macrophages treated with cytochalasin D (5μM) and infected with <i>ΔflaA</i> and stained as in <b>(d)</b>. Arrowhead indicates a bacterium in contact with the macrophage, Bar = 1μm. At least 50 cells <b>(i)</b> or 100 cells <b>(b, c, e</b> and <b>g)</b> were analyzed for each condition. A representative of two <b>(f-h)</b> or three <b>(a-g)</b> biological replicates is shown for each experiment. <b>(b, c, e, g</b> and <b>i)</b> n.s—not significant, ** p<0.005 (unpaired T-test).</p

<i>Legionella</i>-dependent cell death triggered by MTOR suppression is blocked by lipids supplementation.

<p><b>(a)</b> Experimental scheme for the results shown in <b>(b-d, f)</b>. <b>(b-c)</b> <i>Myd88</i>^-/- BMMs with condensed nuclei <b>(b)</b> or positive for phospho-rS6p (MFI>300) <b>(c)</b> produced by infections with <i>ΔflaA</i> (MOI = 20) and the indicated treatments. Infected and neighboring uninfected cells were quantified for each category. <b>(d)</b> Infected and neighboring uninfected BMMs with condensed nuclei after infections with <i>ΔflaA</i> or <i>ΔdotA</i> (MOI = 20) in the presence/absence of FBS. <b>(e)</b> Kinetics of LDH release by C57BL/6 BMMs infected as indicated (MOI = 10) in the presence of FBS or dFBS. <b>(f)</b> Infected <i>Myd88</i>^-/- BMMs with condensed nuclei produced by <i>ΔflaA</i> infection (MOI = 20) and the indicated treatments. <b>(b-f)</b> PP242 (2.5μM), LY294002 (10μM), FBS (10%), dFBS (10%), human LDL (10mg/ml). <b>(b-f)</b> Means ± s.d of technical triplicates for each condition are shown. At least 50 cells <b>(c,f)</b> or 100 cells <b>(b,d)</b> were analyzed for each condition. A representative of two <b>(e-f)</b> or three <b>(b-d)</b> biological replicates is shown for each experiment. <b>(b-f)</b> n.s—not significant, ** p<0.005 (unpaired T-test).</p

PCR amplification of polymorphic <i>Alu</i> insertions in Lemuriformes.

<p>Gel photographs displaying the methodology for establishing evolutionary relationships using <i>Alu</i> elements. The presence and absence of elements, supplemented by sequencing to eliminate the possibility of confounding events, is used to determine which species are more closely related. A total of 5 gel electrophoresis results on a 24-species primate panel are shown with <i>H. sapiens</i> and <i>G. senegalensis</i> as outgroups. <b>A:</b> Amplification of locus Str71B, an <i>Alu</i> insertion shared by the infraorder Lemuriformes. <b>B:</b> Amplification of locus MmA39, an <i>Alu</i> insertion shared by the family Cheirogaleidae. <b>C:</b> Amplification of locus MmA27, an <i>Alu</i> insertion shared by the sister genera <i>Microcebus</i> and <i>Mirza</i>. <b>D:</b> Amplification of locus Str59, an <i>Alu</i> insertion specific to the genus <i>Microcebus</i>. <b>E:</b> Amplification of locus Em6, an <i>Alu</i> insertion affirming the monophyly of the family Lemuridae to the exclusion of other lemur species and outgroups.</p

DNA samples of all species examined in this study.

a<p>Coriell Institute for Medical Research, 403 Haddon Avenue, Camden, NJ 08103, USA.</p>b<p>Duke Lemur Center (DLC), Duke University, Durham, NC 27708, USA.</p>c<p>Integrated Primate Biomaterials and Information Resource (IPBIR), <a href="http://ccr.coriell.org/Sections/Collections/" target="_blank">http://ccr.coriell.org/Sections/Collections/</a>.</p>d<p>Frozen Zoo, San Diego Zoo (SDFZ), <a href="http://conservationandscience.org" target="_blank">http://conservationandscience.org</a>.</p>e<p>Gene Bank of Primates (GBP), German Primate Center, Göttingen, Germany.</p>f<p>Batzer: Adenovirus 12 SV40-transformed fibroblasts maintained in the lab of Dr. Mark Batzer.</p>g<p>From cell lines provided by American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, VA 20108, USA.</p><p>DNA samples of all species examined in this study.</p