Cellular cholesterol licenses Legionella pneumophila intracellular replication in macrophages

Host membranes are inherently critical for niche homeostasis of vacuolar pathogens. Thus, intracellular bacteria frequently encode the capacity to regulate host lipogenesis as well as to modulate the lipid composition of host membranes. One membrane component that is often subverted by vacuolar bacteria is cholesterol – an abundant lipid that mammalian cells produce de novo at the endoplasmic reticulum (ER) or acquire exogenously from serum-derived lipoprotein carriers. Legionella pneumophila is an accidental human bacterial pathogen that infects and replicates within alveolar macrophages causing a severe atypical pneumonia known as Legionnaires’ disease. From within a unique ER-derived vacuole L. pneumophila promotes host lipogenesis and experimental evidence indicates that cholesterol production might be one facet of this response. Here we investigated the link between cellular cholesterol and L. pneumophila intracellular replication and discovered that disruption of cholesterol biosynthesis or cholesterol trafficking lowered bacterial replication in infected cells. These growth defects were rescued by addition of exogenous cholesterol. Conversely, bacterial growth within cholesterol-leaden macrophages was enhanced. Importantly, the growth benefit of cholesterol was observed strictly in cellular infections and L. pneumophila growth kinetics in axenic cultures did not change in the presence of cholesterol. Microscopy analyses indicate that cholesterol regulates a step in L. pneumophila intracellular lifecycle that occurs after bacteria begin to replicate within an established intracellular niche. Collectively, we provide experimental evidence that cellular cholesterol promotes L. pneumophila replication within a membrane bound organelle in infected macrophages

Quantifying Absolute Neutralization Titers against SARS-CoV-2 by a Standardized Virus Neutralization Assay Allows for CrossCohort Comparisons of COVID-19 Sera

The global coronavirus disease 2019 (COVID-19) pandemic has mobilized efforts to develop vaccines and antibody-based therapeutics, including convalescent-phase plasma therapy, that inhibit viral entry by inducing or transferring neutralizing antibodies (nAbs) against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (CoV2-S). However, rigorous efficacy testing requires extensive screening with live virus under onerous biosafety level 3 (BSL3) conditions, which limits high-throughput screening of patient and vaccine sera. Myriad BSL2-compatible surrogate virus neutralization assays (VNAs) have been developed to overcome this barrier. Yet, there is marked variability between VNAs and how their results are presented, making intergroup comparisons difficult. To address these limitations, we developed a standardized VNA using CoV2-S pseudotyped particles (CoV2pp) based on vesicular stomatitis virus bearing the Renilla luciferase gene in place of its G glyco-protein (VSVDG); this assay can be robustly produced at scale and generate accurate neutralizing titers within 18 h postinfection. Our standardized CoV2pp VNA showed a strong positive correlation with CoV2-S enzyme-linked immunosorbent assay (ELISA) results and live-virus neutralizations in confirmed convalescent-patient sera. Three independent groups subsequently validated our standardized CoV2pp VNA (n . 120). Our data (i) show that absolute 50% inhibitory concentration (absIC50), absIC80, and absIC90 values can be legitimately compared across diverse cohorts, (ii) highlight the substantial but consistent variability in neutralization potency across these cohorts, and (iii) support the use of the absIC80 as a more meaningful metric for assessing the neutralization potency of a vaccine or convalescent-phase sera. Lastly, we used our CoV2pp in a screen to identify ultrapermissive 293T clones that stably express ACE2 or ACE2 plus TMPRSS2. When these are used in combination with our CoV2pp, we can produce CoV2pp sufficient for 150,000 standardized VNAs/week. IMPORTANCE Vaccines and antibody-based therapeutics like convalescent-phase plasma therapy are premised upon inducing or transferring neutralizing antibodies that inhibit SARS-CoV-2 entry into cells. Virus neutralization assays (VNAs) for measuring neutralizing antibody titers (NATs) are an essential part of determining vaccine or therapeutic efficacy. However, such efficacy testing is limited by the inherent dangers of working with the live virus, which requires specialized high-level biocontainment facilities. We there-fore developed a standardized replication-defective pseudotyped particle system that mimics the entry of live SARS-CoV-2. This tool allows for the safe and efficient measurement of NATs, determination of other forms of entry inhibition, and thorough investigation of virus entry mechanisms. Four independent labs across the globe validated our standardized VNA using diverse cohorts. We argue that a standardized and scalable assay is necessary for meaningful comparisons of the myriad of vaccines and antibody-based therapeutics becoming available. Our data provide generalizable metrics for assessing their efficacy.Fil: Oguntuyo, Kasopefoluwa. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Stevens, Christian S.. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Hung, Chuan Tien. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Ikegame, Satoshi. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Acklin, Joshua A.. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Kowdle, Shreyas S.. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Carmichael, Jillian C.. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Chiu, Hsin Ping. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Azarm, Kristopher D.. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Haas, Griffin D.. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Amanat, Fatima. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Klingler, Jéromine. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Baine, Ian. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Arinsburg, Suzanne. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Bandres, Juan C.. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Siddiquey, Mohammed N. A.. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Schilke, Robert M.. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Woolard, Matthew D.. State University of Louisiana; Estados UnidosFil: Zhang, Hongbo. State University of Louisiana; Estados UnidosFil: Duty, Andrew J.. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Kraus, Thomas A.. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Moran, Thomas M.. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Tortorella, Domenico. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Lim, Jean K.. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Gamarnik, Andrea Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Hioe, Catarina E.. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Zolla Pazner, Susan. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Ivanov, Stanimir S.. State University of Louisiana; Estados UnidosFil: Kamil, Jeremy. State University of Louisiana; Estados UnidosFil: Krammer, Florian. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Lee, Benhur. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Ojeda, Diego Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: González López Ledesma, María Mora. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Costa Navarro, Guadalupe Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Pallarés, H. M.. No especifíca;Fil: Sanchez, Lautaro Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Perez, P.. No especifíca;Fil: Ostrowsk, M.. No especifíca;Fil: Villordo, S. M.. No especifíca;Fil: Alvarez, D. E.. No especifíca;Fil: Caramelo, J. J.. No especifíca;Fil: Carradori, J.. No especifíca;Fil: Yanovsky, M. J.. No especifíca

MTOR-Driven Metabolic Reprogramming Regulates Legionella pneumophila Intracellular Niche Homeostasis.

Vacuolar bacterial pathogens are sheltered within unique membrane-bound organelles that expand over time to support bacterial replication. These compartments sequester bacterial molecules away from host cytosolic immunosurveillance pathways that induce antimicrobial responses. The mechanisms by which the human pulmonary pathogen Legionella pneumophila maintains niche homeostasis are poorly understood. We uncovered that the Legionella-containing vacuole (LCV) required a sustained supply of host lipids during expansion. Lipids shortage resulted in LCV rupture and initiation of a host cell death response, whereas excess of host lipids increased LCVs size and housing capacity. We found that lipids uptake from serum and de novo lipogenesis are distinct redundant supply mechanisms for membrane biogenesis in Legionella-infected macrophages. During infection, the metabolic checkpoint kinase Mechanistic Target of Rapamycin (MTOR) controlled lipogenesis through the Serum Response Element Binding Protein 1 and 2 (SREBP1/2) transcription factors. In Legionella-infected macrophages a host-driven response that required the Toll-like receptors (TLRs) adaptor protein Myeloid differentiation primary response gene 88 (Myd88) dampened MTOR signaling which in turn destabilized LCVs under serum starvation. Inactivation of the host MTOR-suppression pathway revealed that L. pneumophila sustained MTOR signaling throughout its intracellular infection cycle by a process that required the upstream regulator Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and one or more Dot/Icm effector proteins. Legionella-sustained MTOR signaling facilitated LCV expansion and inhibition of the PI3K-MTOR-SREPB1/2 axis through pharmacological or genetic interference or by activation of the host MTOR-suppression response destabilized expanding LCVs, which in turn triggered cell death of infected macrophages. Our work identified a host metabolic requirement for LCV homeostasis and demonstrated that L. pneumophila has evolved to manipulate MTOR-dependent lipogenesis for optimal intracellular replication

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

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

<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