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

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

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

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

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

<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

Gonococci enter macrophages despite a block in phagocytosis.

(A-E) Gonococci internalization by U937 MFs (A-D, H and J) and hMDMs (E) requires formin-mediated actin polymerization but not canonical phagocytosis. (A-E, H and J) Quantitative analysis of bacteria uptake by macrophages using inside/out microscopy in the presence/absence of different inhibitors added 30 min prior to infection. Gonococcal infections were stopped at 8hpi and the percentage of intracellular colonies in each condition was calculated (green bars). L. pneumophila ΔdotA infections were stopped at 2hpi and the percentage of internalized bacteria was determined (gray bars). The internalization index reflects how each treatment affects uptake compared to vehicle control and was calculated for each condition by dividing the percentage of internalized objects from the treatment condition by the percentage of internalized objects from the respective vehicle control condition, which was then multiplied by 100. Inhibitors targeting: (A) actin polymerization (cytochalasin D, 5μM) and microtubules polymerization (nocodazole, 3μM); (B) the small GTPases Cdc42 (ML-141, 5μM), Rac1/2/3 (EHT 1864, 5μM) and the downstream effectors of Rho GTPase—LIMK1/2 kinases (BMS-5, 1μM) and ROCK1/2 kinases (GSK269962, 1μM); (C) the phagosome regulators Dynamin (dynasore, 30μM) and Phosphoinositide-3 kinase (LY294002, 10μM); (D) actin polymerization regulators formins (SMIFH2, 12.5 or 25μM), Arp2/3 complex (CK869, 10μM), Myosin II ATPases ((-) blebbistatin, from 2.5 to 20μM). (E) Decreased gonococci uptake at 8hpi by hMDMs treated with SMIFH2 as indicated. (F) Immunoblot analysis of the U937 cell line stably expressing Cas9 used for CRISPR genome editing. (G) Immunoblot validation for the loss of Arp2 in the ACTR2 KO and LC3 in the MAP1LC3B KO U937 MFs. (H and J) N.g colony internalization is not affected by the loss of (H) Arp2 or (J) LC3. (I) mRNA expression for the indicated genes in U937 cells from RNAseq presented in normalized transcript per million (nTPMs). Data obtained from the Human Atlas Project [112]: MAP1LC3A (https://www.proteinatlas.org/ENSG00000101460-MAP1LC3A/cell+line); MAP1LC3B (https://www.proteinatlas.org/ENSG00000140941-MAP1LC3B/cell+line); MAP1LC3C (https://www.proteinatlas.org/ENSG00000197769-MAP1LC3C/cell+line). (A-E, H and J) Means from at least three biological replicates ± SD. At least 100 N.g colonies or L.p ΔdotA bacteria for each condition were scored. The statistical significance of the differences between the internalization index from inhibitor-treated cells and the vehicle-treated cells for each condition were calculated using the unpaired T-test (A-C, D for Arp2/3INH, H and J) or one-way ANOVA (D for ForminINH and MyosinIIINH, E), not significant (ns.).</p

FMNL3 co-localizes with polymerized actin at the sites of N.g colony plasma membrane invasion.

Micrographs show U937 MFs expressing FMNL3-V5 (A-B, E-F) or FMN1-V5 (C-D). (A-D) Representative micrographs showing FMNL3-V5 (A-B) but not FMN1-V5 (C-D) localizes at the actin ruffles at the plasma membrane surrounding an invading N.g FA1090 colony. Single plane image is shown in (A and C) from the z-projection image shown in (B and D). Individual channels of the merged image in (A) are pseudo-colored with the Kindlmann color map for range visualization of the fluorescent signal intensity. Fluorescent signal for actin and FMNL3 colocalize at the site of N.g invasion (PCC = 0.922) (B), whereas actin and FMN1 do not (PCC = 0.066). Fluorescent signal intensity profiles are shown in (B and D) for line 1 drawn along the invading colony and line 2 drawn across another area of the cell. (E-G) Plasma membrane region proximal to surface-associated gonococcal colonies (E) or aggregates of heat-killed gonococci (F) did not accumulate FMNL3 and actin microfilaments. (G) Quantitative analysis of FMNL3 accumulation at plasma membrane sites in contact with live gonococcal colonies or with aggregates of heat-killed gonococci. Means from two biological replicates ± SD. The statistical significance was calculated using the unpaired T-test. (A-G) Representative experiments from two (F-G) or at least three (A-E) biological replicates are included.</p

Direct contact with live macrophages is required for gonococcal replication.

(A)N.g FA1090 growth under conditions that permit or restrict direct contact with U937 MFs in PBSG media. Contact separation between bacteria and the macrophages is facilitated by a 0.4μm filter barrier. Total CFUs recovered at 8hpi are shown. (B) N.g FA1090 growth in association with PFA-fixed U937 MFs in PBSG media. Macrophages were fixed for 45min with PFA (4% vol./vol. in PBS) and extensively washed with warm PBS prior to infection. Total CFUs recovered at the indicated time-points are shown. (A-B) Representative experiments from two (B) or three (A) biological replicates are included. (A-B) For each condition means of technical triplicates ± SD are shown, (A) one-way ANOVA analysis, not significant (ns.); (B) two-way ANOVA, ** p = 0.0016.</p