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-gamma) restricts the intracellular replication of many pathogens, but the mechanism by which IFN-gamma confers cell-intrinsic pathogen resistance remains unclear. For example, intracellular replication of the bacterial pathogen Legionella pneumophila in macrophages is potently curtailed by IFN-gamma. However, consistent with prior studies, no individual genetic deficiency that we tested completely abolished IFN-gamma-mediated control. Intriguingly, we observed that the glycolysis inhibitor 2-deoxyglucose (2DG) partially rescued L. pneumophila replication in IFN-gamma-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-gamma. 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-gamma-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-gamma in primary bone marrow macrophages. Our study defines a complete set of cell-intrinsic factors required for IFN-gamma-mediated restriction of an intracellular bacterial pathogen and highlights the combinatorial strategy used by hosts to block bacterial replication in macrophages.

Publisher

American Society for Microbiology

Publication Date

11-1-2019

Publication Title

MBIO

Department

Biology

Document Type

Article

DOI

https://dx.doi.org/10.1128/mBio.02629-19

Keywords

Legionella pneumophila, Host-pathogen interactions, Innate immunity, Interferons, Macrophages

Language

English

Format

text

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