IRG1 and iNOS act redundantly with other interferon gamma-induced factors to restrict intracellular replication of Legionella pneumophila

Abstract

Interferon gamma (IFNγ) restricts the intracellular replication of many pathogens, but how IFNγ confers cell-intrinsic pathogen resistance remains unclear. For example, intracellular replication of the bacterial pathogen Legionella pneumophila in macrophages is potently curtailed by IFNγ, but consistent with prior results, no individual genetic deficiency we tested compromised IFNγ-mediated control. Intriguingly, however, we observed that the glycolysis inhibitor 2-deoxyglucose (2DG) partially rescued L. pneumophila replication in IFNγ-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 IFNγ. Instead, we found that 2DG rescues bacterial replication predominantly by inhibiting the induction 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 confirm that loss of both iNOS and IRG1, but not individual deficiency in each gene, partially reduces IFNγ-mediated restriction of L. pneumophila. Further, using a combinatorial CRISPR/Cas9 mutagenesis approach, we find that mutation of iNOS and IRG1 in combination with four other genes (CASP11, IRGM1, IRGM3 and NOX2) results in a total loss of L. pneumophila restriction by IFNγ in primary bone marrow macrophages. There are few, if any, other examples in which the complete set of cell-intrinsic factors required for IFNγ-mediated restriction of an intracellular bacterial pathogen have been genetically identified. Our results highlight the combinatorial strategy used by hosts to block the exploitation of macrophages by pathogens. 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 (IFNγ) 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 determined the exact molecular factors induced by IFNγ that are required for its activity. We generated macrophages lacking different combinations of IFNγ-induced genes in an attempt to find a genetic background in which there is a complete loss of IFNγ-mediated restriction of L. pneumophila. We successfully identified six genes that comprise the totality of the IFNγ-dependent restriction of L. pneumophila replication in macrophages. Our results clarify the molecular basis underlying the potent effects of IFNγ and highlight how redundancy downstream of IFNγ is key to prevent exploitation of the macrophage niche by pathogens.