Dataset Information

IRG1 and Inducible Nitric Oxide Synthase Act Redundantly with Other Interferon-Gamma-Induced Factors To Restrict Intracellular Replication of Legionella pneumophila.

ABSTRACT: Interferon gamma (IFN-?) restricts the intracellular replication of many pathogens, but the mechanism by which IFN-? confers cell-intrinsic pathogen resistance remains unclear. For example, intracellular replication of the bacterial pathogen Legionella pneumophila in macrophages is potently curtailed by IFN-?. However, consistent with prior studies, no individual genetic deficiency that we tested completely abolished IFN-?-mediated control. Intriguingly, we observed that the glycolysis inhibitor 2-deoxyglucose (2DG) partially rescued L. pneumophila replication in IFN-?-treated macrophages. 2DG inhibits glycolysis and triggers the unfolded protein response, but unexpectedly, it appears these effects are not responsible for perturbing the antimicrobial activity of IFN-?. Instead, we found that 2DG rescues bacterial replication by inhibiting the expression of two key antimicrobial factors, inducible nitric oxide synthase (iNOS) and immune-responsive gene 1 (IRG1). Using immortalized and primary macrophages deficient in iNOS and IRG1, we confirmed that loss of both iNOS and IRG1, but not individual deficiency in either gene, partially reduced IFN-?-mediated restriction of L. pneumophila Further, using a combinatorial CRISPR/Cas9 mutagenesis approach, we found that mutation of iNOS and IRG1 in combination with four other genes (CASP11, IRGM1, IRGM3, and NOX2) resulted in a total loss of L. pneumophila restriction by IFN-? in primary bone marrow macrophages. Our study defines a complete set of cell-intrinsic factors required for IFN-?-mediated restriction of an intracellular bacterial pathogen and highlights the combinatorial strategy used by hosts to block bacterial replication in macrophages.IMPORTANCE Legionella pneumophila is one example among many species of pathogenic bacteria that replicate within mammalian macrophages during infection. The immune signaling factor interferon gamma (IFN-?) blocks L. pneumophila replication in macrophages and is an essential component of the immune response to L. pneumophila and other intracellular pathogens. However, to date, no study has identified the exact molecular factors induced by IFN-? that are required for its activity. We generated macrophages lacking different combinations of IFN-?-induced genes in an attempt to find a genetic background in which there is a complete loss of IFN-?-mediated restriction of L. pneumophila We identified six genes that comprise the totality of the IFN-?-dependent restriction of L. pneumophila replication in macrophages. Our results clarify the molecular basis underlying the potent effects of IFN-? and highlight how redundancy downstream of IFN-? is key to prevent exploitation of macrophages by pathogens.

SUBMITTER: Price JV

PROVIDER: S-EPMC6851286 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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IRG1 and Inducible Nitric Oxide Synthase Act Redundantly with Other Interferon-Gamma-Induced Factors To Restrict Intracellular Replication of Legionella pneumophila.

Price Jordan V JV Russo Daniel D Ji Daisy X DX Chavez Roberto A RA DiPeso Lucian L Lee Angus Yiu-Fai AY Coers Jörn J Vance Russell E RE

mBio 20191112 6

Interferon gamma (IFN-γ) restricts the intracellular replication of many pathogens, but the mechanism by which IFN-γ confers cell-intrinsic pathogen resistance remains unclear. For example, intracellular replication of the bacterial pathogen <i>Legionella pneumophila</i> in macrophages is potently curtailed by IFN-γ. However, consistent with prior studies, no individual genetic deficiency that we tested completely abolished IFN-γ-mediated control. Intriguingly, we observed that the glycolysis in ...[more]

PMID: 31719183

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Project description: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.

Dataset Information

IRG1 and Inducible Nitric Oxide Synthase Act Redundantly with Other Interferon-Gamma-Induced Factors To Restrict Intracellular Replication of Legionella pneumophila.

Publications

IRG1 and Inducible Nitric Oxide Synthase Act Redundantly with Other Interferon-Gamma-Induced Factors To Restrict Intracellular Replication of Legionella pneumophila.

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