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      Identification of Host-Targeted Small Molecules That Restrict Intracellular Mycobacterium tuberculosis Growth

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          Abstract

          Mycobacterium tuberculosis remains a significant threat to global health. Macrophages are the host cell for M. tuberculosis infection, and although bacteria are able to replicate intracellularly under certain conditions, it is also clear that macrophages are capable of killing M. tuberculosis if appropriately activated. The outcome of infection is determined at least in part by the host-pathogen interaction within the macrophage; however, we lack a complete understanding of which host pathways are critical for bacterial survival and replication. To add to our understanding of the molecular processes involved in intracellular infection, we performed a chemical screen using a high-content microscopic assay to identify small molecules that restrict mycobacterial growth in macrophages by targeting host functions and pathways. The identified host-targeted inhibitors restrict bacterial growth exclusively in the context of macrophage infection and predominantly fall into five categories: G-protein coupled receptor modulators, ion channel inhibitors, membrane transport proteins, anti-inflammatories, and kinase modulators. We found that fluoxetine, a selective serotonin reuptake inhibitor, enhances secretion of pro-inflammatory cytokine TNF-α and induces autophagy in infected macrophages, and gefitinib, an inhibitor of the Epidermal Growth Factor Receptor (EGFR), also activates autophagy and restricts growth. We demonstrate that during infection signaling through EGFR activates a p38 MAPK signaling pathway that prevents macrophages from effectively responding to infection. Inhibition of this pathway using gefitinib during in vivo infection reduces growth of M. tuberculosis in the lungs of infected mice. Our results support the concept that screening for inhibitors using intracellular models results in the identification of tool compounds for probing pathways during in vivo infection and may also result in the identification of new anti-tuberculosis agents that work by modulating host pathways. Given the existing experience with some of our identified compounds for other therapeutic indications, further clinically-directed study of these compounds is merited.

          Author Summary

          Infection with the bacterial pathogen Mycobacterium tuberculosis causes the disease tuberculosis (TB) that imposes significant worldwide morbidity and mortality. Approximately 2 billion people are infected with M. tuberculosis, and almost 1.5 million people die annually from TB. With increasing drug resistance and few novel drug candidates, our inability to effectively treat all infected individuals necessitates a deeper understanding of the host-pathogen interface to facilitate new approaches to treatment. In addition, the current anti-tuberculosis regimen requires months of strict compliance to clear infection; targeting host immune function could play a strategic role in reducing the duration and complexity of treatment while effectively treating drug-resistant strains. Here we use a microscopy-based screen to identify molecules that target host pathways and inhibit the growth of M. tuberculosis in macrophages. We identified several host pathways not previously implicated in tuberculosis. The identified inhibitors prevent growth either by blocking host pathways exploited by M. tuberculosis for virulence, or by activating immune responses that target intracellular bacteria. Fluoxetine, used clinically for treating depression, induces autophagy and enhances production of TNF-α. Similarly, gefitinib, used clinically for treating cancer, inhibits M. tuberculosis growth in macrophages. Importantly, gefitinib treatment reduces bacterial replication in the lungs of M. tuberculosis-infected mice.

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          Host genotype-specific therapies can optimize the inflammatory response to mycobacterial infections.

          Susceptibility to tuberculosis is historically ascribed to an inadequate immune response that fails to control infecting mycobacteria. In zebrafish, we find that susceptibility to Mycobacterium marinum can result from either inadequate or excessive acute inflammation. Modulation of the leukotriene A(4) hydrolase (LTA4H) locus, which controls the balance of pro- and anti-inflammatory eicosanoids, reveals two distinct molecular routes to mycobacterial susceptibility converging on dysregulated TNF levels: inadequate inflammation caused by excess lipoxins and hyperinflammation driven by excess leukotriene B(4). We identify therapies that specifically target each of these extremes. In humans, we identify a single nucleotide polymorphism in the LTA4H promoter that regulates its transcriptional activity. In tuberculous meningitis, the polymorphism is associated with inflammatory cell recruitment, patient survival and response to adjunctive anti-inflammatory therapy. Together, our findings suggest that host-directed therapies tailored to patient LTA4H genotypes may counter detrimental effects of either extreme of inflammation. Copyright © 2012 Elsevier Inc. All rights reserved.
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            Drug tolerance in replicating mycobacteria mediated by a macrophage-induced efflux mechanism.

            Treatment of tuberculosis, a complex granulomatous disease, requires long-term multidrug therapy to overcome tolerance, an epigenetic drug resistance that is widely attributed to nonreplicating bacterial subpopulations. Here, we deploy Mycobacterium marinum-infected zebrafish larvae for in vivo characterization of antitubercular drug activity and tolerance. We describe the existence of multidrug-tolerant organisms that arise within days of infection, are enriched in the replicating intracellular population, and are amplified and disseminated by the tuberculous granuloma. Bacterial efflux pumps that are required for intracellular growth mediate this macrophage-induced tolerance. This tolerant population also develops when Mycobacterium tuberculosis infects cultured macrophages, suggesting that it contributes to the burden of drug tolerance in human tuberculosis. Efflux pump inhibitors like verapamil reduce this tolerance. Thus, the addition of this currently approved drug or more specific efflux pump inhibitors to standard antitubercular therapy should shorten the duration of curative treatment. Copyright © 2011 Elsevier Inc. All rights reserved.
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              Mycobacterium tuberculosis Eis Regulates Autophagy, Inflammation, and Cell Death through Redox-dependent Signaling

              Introduction Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that can survive and even multiply within host macrophages [1], [2]. Mtb can persist within phagosomes by interfering with intracellular membrane trafficking and by arresting phagosome maturation in infected host cells [3]. Pathogenic mycobacteria have developed several strategies for surviving and growing under nutrient-limited conditions [4]. Autophagy, or the removal of aged organelles, plays a central role in regulating important cellular functions [5], [6] and aids in innate and adaptive immune defense against Mtb and other intracellular pathogens [5], [7]–[9]. Physiological or pharmacological induction of autophagy in macrophages results in increased co-localization of mycobacterial phagosomes and the autophagy effector LC3, and the fusion of the former with lysosomes, which overcomes the blockade of membrane trafficking and increased bactericidal activity [7]. Although autophagy plays key roles in host innate and adaptive immune defenses, it can, under certain circumstances, result in type II programmed cell death [10], [11]. Autophagic processes are activated in response to cellular stresses, such as oxidative stress, and can influence several types of cell death, including autophagy-related cell death [12]. Recently, we showed that the mycobacterial BCG cell wall triggers autophagy-induced cell death in radiosensitized colon cancer cells [13]. Additionally, several viral gene products may be involved in autophagy-induced cell death [14]. However, the genetic basis for mycobacterial induction of autophagy, and its implications for host cell viability, remain to be elucidated. The “enhanced intracellular survival” (eis) gene and its protein product, Eis, a unique protein of 42 kDa, of Mtb H37Rv enhance the survival of the saprophytic M. smegmatis during repeated passage through the human macrophage-like cell line U-937 [15]. Bioinformatic analyses showed that Eis is a member of the GCN5-related family of N-acetyltransferases [16]. Recent studies have revealed that kanamycin resistance is associated with eis promoter mutations that increase Eis transcript and protein levels [17]. Additionally, regulation of eis expression by SigA enhanced intracellular growth of the W-Beijing Mtb strain in monocytic cells [18]. Moreover, Eis inhibited the proliferation of mitogen-activated T cells and, by blocking the phosphorylation of extracellular signal-regulated kinase (ERK), reduced the production of tumor necrosis factor (TNF)-α and interleukin (IL)-4 [19]. Despite being implicated in host-pathogen interactions during Mtb infection, the precise role of Eis in innate immune regulation remains to be determined. In an effort to gain further insight into the role of Eis in host responses, we examined autophagy, inflammatory cytokine production, and reactive oxygen species (ROS) generation in macrophages infected with wild-type (Mtb-WT), eis-deletion (Mtb-Δeis), or complemented (Mtb-c-eis) Mtb strains. Infection with Mtb-Δeis significantly increased autophagy, inflammatory responses, and ROS generation in macrophages. NADPH oxidase (NOX) and mitochondria were found to be the major sources of ROS, which contributed to the induction of autophagy and inflammatory responses in Mtb-Δeis-infected cells. Increased and excessive activation of autophagy in macrophages infected with Mtb-Δeis had no effect on antimicrobial responses, but stimulated caspase-independent cell death (CICD). Mtb-Δeis-induced host cell death was regulated by autophagic pathways and influenced by c-Jun N-terminal kinase (JNK)-dependent ROS generation. Furthermore, we show that the N-acetyltransferase domain of Eis is responsible for its modulation of ROS generation and proinflammatory responses in macrophages. Results Mycobacterium tuberculosis Eis Inhibits Autophagy in Macrophages Previous studies identified a role for the eis gene in enhancing the survival of mycobacteria in human monocytic cells [15]. However, the role of eis in autophagy activation in macrophages, which plays a key role in defense and cellular homeostasis [5], is not fully understood. We first infected bone marrow-derived macrophages (BMDMs) with the Mtb-WT, Mtb-Δeis, and Mtb-c-eis strains of Mtb H37Rv and examined the kinetics of autophagosome formation by immunostaining for LC3. As shown in Figure 1A, in BMDMs infected with Mtb-Δeis we observed the recruitment of endogenous LC3 in punctate structures the formation of which peaked 24 h after infection, before decreasing substantially by 48 h post-infection (Fig. 1A, right). In contrast, autophagosome formation was not increased in BMDMs infected with Mtb-WT or Mtb-c-eis (Fig. 1A). Additionally, RAW 264.7 macrophages transfected with green fluorescent protein (GFP) fused to the autophagosome protein LC3 (GFP-LC3) [20] showed a significant increase in GFP-LC3 puncta formation when infected with Mtb-Δeis at a multiplicity of infection (MOI) of 10 (over levels in cells infected with Mtb-WT or Mtb-c-eis at the same bacterial load; Fig. S1A). Moreover, Mtb-Δeis-induced formation of LC3 punctae in BMDMs (Fig. 1B) and RAW 264.7 cells (Fig. S1B) was abrogated by treatment for 4 h with 3-methyladenine (3-MA), a classical inhibitor of autophagy [21]. 10.1371/journal.ppat.1001230.g001 Figure 1 Mtb Eis modulates autophagy in macrophages. (A) BMDMs were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis (MOI = 10) for 4 h (as described in the Materials and Methods), and then incubated for 24 h (left) or the indicated periods of time (right). Cells were fixed, stained with DAPI to visualize nuclei (blue), and immunolabeled with an anti-LC3 antibody. Primary antibody was detected using an Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). Left: representative immunofluorescence images of LC3 punctae; right: quantification of data (LC3-punctated cells were counted manually). ***p 99%) were removed, as determined through staining with auramine-rhodamine (Merck, Darmstadt, Germany). After washing, the cells were incubated in fresh medium for a further 3 days. They were then lysed in autoclaved distilled water to allow intracellular bacteria to be collected [8]. The lysates were then re-suspended and sonicated for 5 min in a preheated 37°C water bath sonicator (Elma, Singen, Germany). Aliquots of the resulting sonicates were serially diluted in 7H9 broth, plated separately on 7H10 agar plates, and incubated at 37°C in 5% CO2 for 12 d. Colony counting was then performed in triplicate. Mice were challenged by aerosol exposure with Mtb-WT, Mtb-Δeis, or Mtb-c-eis using an inhalation device (Glas-Col, Terre Haute, IN, USA) calibrated to deliver approximately 50 bacteria into the lungs. Five mice per group were sacrificed at 4 weeks post-challenge, and bacteria in lung and spleen homogenates were counted. Numbers of viable bacteria in lung/spleen were determined by plating serial dilutions of whole organ homogenates on Middlebrook 7H11 agar (Difco, Detroit, MI, USA). Colonies were counted after 3–4 weeks of incubation at 37°C. Reagents, DNA, Abs, and Transfection DPI (a NOX inhibitor), NAC (an antioxidant), catalase, and z-VAD-fmk (a pan-caspase inhibitor) were purchased from Calbiochem (San Diego, CA, USA). 3-MA, tiron (a commercial deflocculant), and DAPI were purchased from Sigma. DMSO (Sigma) was added to cultures at a concentration of 0.1% (v/v) as a solvent control (SC). The plasmid that encoded EGFP-LC3 [20] was a gift from Tamotsu Yoshimori (Osaka University, Japan). pCMV-Eis-WT and pCMV-Eis-ΔAT constructs were created by subcloning the whole eis gene (WT), or an acetyltransferase domain-deletion mutant (lacking the sequence encoding residues 61–137 of the 402-amino-acid Eis protein) from pET21a. This was achieved by cutting at the BamHI and SacI restriction sites and then ligating the resulting inserts into the pCMV-Tag1 mammalian expression vector (Stratagene Co., USA). Anti-LC3 antibodies (Abs) used for Western blotting and immunofluorescence analysis were purchased from Novus Biologicals and MBL International (Woburn, MA, USA), respectively. Anti-rabbit IgG-Alexa488 and IgG-TRITC, and anti-mouse IgG-Cy2, were purchased from Jackson Immunoresearch (West Grove, PA, USA). siRNAs specific for mBeclin 1 (sc-29798), mAtg5 (sc-41446), and mJNK1 (sc-29381), each a pool of five target-specific 19–25 nt siRNAs, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cells were transfected with plasmids and/or siRNAs using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Measurement of ROS Production Intracellular ROS levels were measured by DCFH-DA and DHE assays as described previously [61]. Briefly, BMDMs were differentiated in culture dishes and infected with bacterial strains (MOI = 10) for 30 min. Cells were then incubated with either DCFH-DA (5 µM) or DHE (10 µM; Molecular Probes) for 30 min at 37°C in 5% CO2 and then washed with Krebs-Hepes buffer (for DHE staining) or HBSS (for DCFH-DA staining). Total intracellular levels of ROS were determined by FACS analyses of the oxidative conversion of cell-permeable DCFH-DA (Molecular Probes) to fluorescent DHE (Molecular Probes), using the FACSCanto II system (Becton Dickinson, San Jose, CA, USA). A mitochondrion-specific hydroethidine-derivative fluorescent dye (MitoSOX; M36008; Calbiochem) was used to determine relative mitochondrial O2 − levels in BMDMs. Cells were incubated for 30 min in PBS containing 5 µM MitoSOX. They were then washed twice and analyzed using the FACSCanto II system. All FACS data were collected using 50,000 to 100,000 cells and analyzed using FlowJo software (Tree Star, Ashland, OR, USA). Cell Viability and Apoptosis Assays Cell viability was assessed by PI staining and then examined by fluorescence microscopy or flow cytometric analysis. Trypan blue-stained cells were counted using a ViCell counter (Beckman Coulter, Fullerton, CA, USA). Apoptosis was examined by TdT-mediated dUTP Nick-End Labeling (TUNEL; Promega), according to the manufacturer's instructions. Labeled cells were examined under a laser-scanning confocal microscope (model LSM 510; Zeiss). Each condition was assayed in triplicate, and at least 200 cells per well were counted. To analyze in vivo cell death, single-cell suspensions were prepared in RPMI 1640 medium by passing cell populations through a nylon mesh with 50 µm pores and were subjected to further analysis. Western Blotting, RT-PCR and ELISA Treated BMDMs were processed for analysis by sandwich ELISA, Western blotting, and RT-PCR as described previously [2]. For Western blot analysis, primary Abs were diluted 1∶1000. Membranes were developed using a chemiluminescent reagent (ECL; Pharmacia-Amersham, Freiburg, Germany) and subsequently exposed to film (Pharmacia-Amersham). Supernatant TNF-α and IL-6 levels were measured by sandwich ELISA using Duoset Ab pairs (Pharmingen, San Diego, CA, USA) [2]. To provide RNA for RT-PCR analysis, paraffin-embedded tissue sections were first deparaffinized in octane [62]. After vigorous vortexing, 150 µL of methanol were added. Samples were vortexed again and the tissue was pelleted by centrifugation (10,000×g, 2 min). Supernatants were removed, and the remaining tissue was vacuum-dried for 20 min. Next, pellets were resuspended in digestion buffer (20 mM Tris-HCl, pH 7.6, 0.5% N-laurylsarcosine, 1 M guanidine thiocyanate, 25 mM 2-mercaptoethanol) containing proteinase K (5 mg/mL; Sigma). After overnight digestion at 55°C, RNA was extracted using TRIzol (Invitrogen) according to the manufacturer's instructions. For quantitative RT-PCR analysis was performed by using SYBR Green (Molecular Probes) PCR core reagents (Applied Biosystems), and transcript levels were quantified by using an ABI 7900 Sequence Detection System (Applied Biosystems). The mean value of triplicate reactions was normalized against the mean value of β-actin. Primers were used at 400 nM. Autophagy Analysis Autophagosome formation was measured by LC3 punctate staining, as described previously [8]. To quantitate autophagy, we used fluorescence microscopy to count the percentages of GFP-LC3-positive autophagic vacuoles in transfected cells or the numbers of endogenous LC3 punctate dots in primary cells. Each condition was assayed in triplicate, and at least 200 cells per well were counted. LC3 conjugation was evaluated by Western blot analysis using an antibody raised to LC3-I/II. Transmission Electron Microscopy Infected and stimulated RAW 264.7 macrophages were washed with PBS and then fixed with 3% formaldehyde, 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 1 h. They were then post-fixed in 1% osmium tetroxide, 0.5% potassium ferricyanide in cacodylate buffer for 1 h; embedded in straight resin; and cured at 80°C for 24 h. Ultrathin sections (70–80 nm), cut using an ultramicrotome (RMC MT6000-XL), were stained with uranyl acetate and lead citrate and examined using a Tecnai G2 Spirit Twin transmission electron microscope (FEI Company, USA) and a JEM ARM 1300S High Voltage electron microscope (JEOL, Japan). Immunofluorescence Immunofluorescence analysis was performed as described previously [8]. Briefly, cells were fixed with 4% paraformaldehyde in PBS at 4°C for 10 min and permeabilized with 0.01% Triton X-100 in PBS for 10 min. Cultures were then stained for 2 h at room temperature with primary antibodies, including rabbit anti-mouse LC3 (1∶400; MBL International). After washing, to remove excess primary antibody, cultures were then incubated for 1 h at room temperature with an anti-rabbit IgG-Alexa488 secondary antibody (Jackson Immunoresearch). Nuclei were stained by incubation with DAPI for 5 min. Slides were examined using a laser-scanning confocal microscope (model LSM 510; Zeiss). Statistical Analyses Data obtained from independent experiments (presented as mean±SD) were analyzed by the paired Student's t-test with Bonferroni correction or analysis of variance (for multiple comparisons). A p value<0.05 was deemed to indicate statistical significance. Accession Numbers The GenBank accession number for the eis gene is AF144099. Supporting Information Figure S1 Autophagic vesicles are increased in macrophages infected with Mtb-Δeis, but not in cells infected with Mtb-WT or Mtb-c-eis. (A and B) Formation of GFP-LC3 vacuoles (dots) was determined in RAW 264.7 cells transfected with GFP-LC3 cDNA. Transfected cells were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis (MOI = 10) for 24 h (A) or Mtb-Δeis (MOI = 10) for 24 h in the presence or absence of 3-MA (B). Top, representative immunofluorescence images; bottom, percentage of GFP-LC3 cells with punctae. (C) Co-localization of autophagosomes (endogenous LC3, red) and lysosomes (lamp-1, green) was increased in Mtb-Δeis-infected BMDMs. Data are representative of three separate experiments. Scale bars: 10 µm. (D) BMDMs were infected with Mtb-Δeis (MOI = 10) for 24 h in the presence or absence of 3-MA (10 mM) or Baf-A1 (100 nM). Quantitation of the percentages of cells with LC3 punctae. Each condition was assayed in triplicate, and at least 250 cells per well were counted. ***p<0.001, vs. Mtb-WT-infected condition (A); SC (B and D). UI, uninfected; SC, solvent control (0.1% distilled water (B), 0.1% DMSO (D)). (0.66 MB TIF) Click here for additional data file. Figure S2 Activation of autophagy negatively impacts the secretion of proinflammatory cytokines by Mtb-Δeis-infected macrophages. RAW 264.7 cells transfected with siRNAs specific for Beclin-1 (siBec-1) or Atg5 (siAtg5) were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis (MOI = 10) for 24 h. Supernatants were assessed by ELISA for levels of TNF-α and IL-6. Data are presented as the mean±SD of five experiments. ***p<0.001, vs. Mtb-WT-infected condition. UI, uninfected. (0.12 MB TIF) Click here for additional data file. Figure S3 Reactive nitrogen species are not involved in the elevation of ROS generation in Mtb-Δeis-infected macrophages. BMDMs were infected with Mtb-Δeis (MOI = 10) in the presence or absence of L-NAME (0.1, 1, 5 mM) or L-NMMA (0.1, 1, 5 mM). Cells were stained with DHE (for superoxide) or DCFH-DA (for H2O2) and subjected to flow cytometry analysis. Data represent densitometric analyses (mean±SD) of three separate experiments. UI, uninfected; SC, solvent control. (0.07 MB TIF) Click here for additional data file. Figure S4 Intracellular ROS and NOX2 are required for autophagy and proinflammatory responses in Mtb-Δeis-infected macrophages. (A) RAW 264.7 cells transfected with GFP-LC3 cDNA were infected with Mtb-Δeis (MOI = 10) in the presence or absence of DPI (10 µM), NAC (20 mM), catalase (Cat, 1 mU/mL), or tiron (5 mM). Formation of GFP-LC3 vacuoles (dots) was determined in transfected cells, and at least 250 cells per well were counted. Left: representative immunofluorescence images; right: percentage of LC3-punctated cells. (B) BMDMs from WT and NOX2-KO mice were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis (MOI = 10). After 30 min, ROS production (DHE staining) was determined by flow cytometry (left). Quantitative analysis of ROS generation in WT- and NOX2-deficient BMDMs (right). Data represent the mean±SD of three independent experiments. (C) BMDMs from WT and NOX2 KO mice were treated with rapamycin (Rapa; 20 µg/mL) or staurosporine (STS; 500 nM), or nutrient-starved (Starv; maintained in HBSS) for 8 h. Numbers of LC3-punctated cells (counted manually) are shown. Data are presented as the mean±SD of at least three separate experiments, each performed in triplicate. (D) BMDMs from WT and NOX2 KO mice were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis for 6 h and then subjected to RT-PCR analysis. A gel representative of three independent replicates is shown. *** p<0.001, vs. SC (A); WT mice (B). UI, uninfected; SC, solvent control (0.1% DMSO). (0.38 MB TIF) Click here for additional data file. Figure S5 Enhanced cell death in Mtb-Δeis-infected macrophages is regulated by autophagic pathways. (A) BMDMs were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis at the indicated MOIs for 4 h, washed to remove unbound mycobacteria, and then incubated in complete DMEM at 37°C in 5% CO2 for the indicated periods of time. Cells were stained with PI and then examined by fluorescence microscopy. (B) Cell death was determined in RAW 264.7 cells transfected with specific siRNA for beclin-1, atg5, or non-specific scrambled siRNA (siNS) before infection with Mtb-WT, Mtb-Δeis, or Mtb-c-eis, as described in the Materials and Methods. After 36 h, cells were stained with PI and examined by fluorescence microscopy, as described in the Materials and Methods. Transfection efficiency was assessed by RT-PCR (inset). (C) Experimental conditions were identical to those outlined in panel A. Cell viability was assessed by trypan blue staining. Data are presented as the mean±SD of three separate experiments, each performed in duplicate. *p<0.05, ***p<0.001, vs. Mtb-WT-infected condition (A). (0.16 MB TIF) Click here for additional data file.
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                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                February 2014
                20 February 2014
                : 10
                : 2
                : e1003946
                Affiliations
                [1 ]The Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America
                [2 ]Division of Infectious Disease and Vaccinology, School of Public Health, University of California, Berkeley, Berkeley, California, United States of America
                [3 ]Division of Infectious Disease, Massachusetts General Hospital, Boston, Massachusetts, United States of America
                [4 ]Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts, United States of America
                [5 ]Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, United States of America
                [6 ]Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, United States of America
                Weill Medical College of Cornell University, United States of America
                Author notes

                Sarah Stanley received a Helen Hay Whitney postdoctoral fellowship from 2008–2011 that was sponsored by Novartis. This does not alter our adherence to PLOS Pathogens policies on sharing data and materials.

                Conceived and designed the experiments: SAS AKB NS EJR DTH. Performed the experiments: SAS AKB MRS SSL KS NS. Analyzed the data: SAS AKB MRS SSL KS MV MAB AEC CBM DTH. Contributed reagents/materials/analysis tools: CBM. Wrote the paper: SAS AKB DTH.

                Article
                PPATHOGENS-D-13-02179
                10.1371/journal.ppat.1003946
                3930586
                24586159
                647bd92f-7548-44c4-b2e5-6ba063b5cda7
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 19 August 2013
                : 1 January 2014
                Page count
                Pages: 16
                Funding
                SAS gratefully acknowledges Novartis and the Helen Hay Whitney Foundation for funding. AKB gratefully acknowledges funding through NIH K08 AI080944. AEC gratefully acknowledges funding through NIH R01 GM089652. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Microbiology
                Bacterial pathogens
                Emerging infectious diseases
                Host-pathogen interaction
                Pathogenesis
                Molecular cell biology
                Signal transduction
                Chemistry
                Chemical biology
                Medicine
                Infectious diseases
                Bacterial diseases
                Tuberculosis

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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