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Defective autophagy is associated with chronic inflammation. Loss-of-function of the core autophagy gene Atg16l1 increases risk for Crohn’s disease by enhancing innate immunity in macrophages. However, autophagy also mediates clearance of intracellular pathogens. These divergent observations prompted a re-evaluation of ATG16L1 in antimicrobial immunity. In this study, we found that loss of Atg16l1 in macrophages enhanced the killing of virulent Shigella flexneri (S.flexneri), an enteric bacterium that resides within the cytosol by escaping all membrane-bound compartments. Quantitative multiplexed proteomics revealed that ATG16L1 deficiency significantly upregulated proteins involved in the glutathione-mediated antioxidant response to compensate for elevated oxidative stress, which also promoted S.flexneri killing. Consistently, myeloid cell-specific deletion of Atg16l1 accelerated bacterial clearance in vivo. Finally, pharmacological modulation of oxidative stress by suppression of cysteine import conferred enhanced microbicidal properties to wild type macrophages. These findings demonstrate that control of oxidative stress by ATG16L1 regulates antimicrobial immunity against intracellular pathogens.Impact statementMaculins et al utilize multiplexed mass spectrometry to show that loss of the autophagy gene Atg16l1 in macrophages enhances antimicrobial immunity against intracellular pathogens via the oxidative stress response.

Cdk8 of the RNA Polymerase II mediator complex regulates genes by phosphorylating sequence specific transcription factors. Despite conserved importance for eukaryotic transcriptional regulation, the signals regulating Cdk8 are unknown. Full induction of the yeastGALgenes requires phosphorylation of Gal4 by Cdk8, and we exploited this requirement for growth ofgal3yeast on galactose to identify mutants affecting Cdk8 activity. Several mutants from the screen produced defects in TOR signaling. A mutant designatedgalfour throttle (gft) 1,gft1, was identified as an allele ofhom3, encoding aspartokinase. Defects ingft1/hom3caused hypersensitivity to rapamycin, and constitutive nuclear localization of Gat1. Furthermore, mutations oftor1ortco89, encoding TORC1 components, also preventedGALexpression ingal3yeast, andtco89was determined to be allelic togft7. Disruption ofcdc55, encoding a subunit of PP2A regulated by TOR signaling, suppressed the effect ofgft1/hom3, gft7/tco89, andtor1mutations onGALexpression ingal3yeast, but not ofcdk8/srb10disruptions or Gal4 S699A mutation. Mutations ofgft1/hom3andtor1did not affect kinase activity of Cdk8in vitro, but caused loss of Gal4 phosphorylationin vivo. These observations demonstrate that TOR signaling regulatesGALinduction through the activity of PP2A/ Cdc55, and are consistent with the contention that Cdk8-dependent phosphorylation of Gal4 S699 is opposed by PP2A/ Cdc55 dephosphorylation. These results provide insight into how induction of transcription by a specific inducer can be modulated by global nutritional signals through regulation of Cdk8-dependent phosphorylation.