Summary Type I interferons (IFNs) are critical mediators of antiviral defense, but their elicitation by bacterial pathogens can be detrimental to hosts. Many intracellular bacterial pathogens, including Mycobacterium tuberculosis, induce type I IFNs following phagosomal membrane perturbations. Cytosolic M. tuberculosis DNA has been implicated as a trigger for IFN production, but the mechanisms remain obscure. We report that the cytosolic DNA sensor, cyclic GMP-AMP synthase (cGAS), is required for activating IFN production via the STING/TBK1/IRF3 pathway during M. tuberculosis and L. pneumophila infection of macrophages, whereas L. monocytogenes short-circuits this pathway by producing the STING agonist, c-di-AMP. Upon sensing cytosolicDNA, cGAS also activates cell-intrinsic antibacterial defenses, promoting autophagic targeting of M. tuberculosis. Importantly, we show that cGAS binds M. tuberculosis DNA during infection, providing direct evidence that this unique host-pathogen interaction occurs in vivo. These data uncover a mechanism by which IFN is likely elicited during active human infections.

Summary Paragraph Mycobacterium tuberculosis (Mtb), a major global health threat, replicates in macrophages (MΦ) in part by inhibiting phagosome-lysosome fusion, until IFN-γ activates the MΦ to traffic Mtb to the lysosome. How IFN-γ elicits this effect is unknown, but many studies suggest a role for macroautophagy (autophagy herein), a cellular process by which cytoplasmic contents are sequestered into an autophagosome and targeted for lysosomal degradation1. The involvement of autophagy has been defined based on studies in cultured MΦ or dendritic cells (DC) where Mtb colocalizes with autophagy (ATG) factors ATG5, ATG12, ATG16L1, p62, NDP52, Beclin1 and LC32–6, stimulation of autophagy increases bacterial killing6–8, and inhibition of autophagy allows for increased bacterial survival1,2,4,6,7. Notably, these studies reveal modest (e.g. 1.5- to 3-fold change) effects on Mtb replication. In contrast, Atg5fl/fl-LysM-Cre mice lacking ATG5 in monocyte-derived cells and neutrophils (polymorphic mononuclear cells, PMN) succumb to Mtb within 30 days4,9, an extremely severe phenotype similar to mice lacking IFN-γ signaling10,11. Importantly, ATG5 is the only ATG factor that has been studied during Mtb infection in vivo and autophagy-independent functions of ATG5 have been described12–18. For this reason, we used a genetic approach to elucidate the role for multiple ATG genes and the requirement for autophagy in resistance to Mtb infection in vivo. We have discovered that, contrary to expectation, autophagic capacity does not correlate with the outcome of Mtb infection. Instead, ATG5 plays a unique role in protection against Mtb by preventing PMN-mediated immunopathology. Furthermore, while ATG5 is dispensable in alveolar MΦ during Mtb infection, loss of Atg5 in PMN can sensitize mice to Mtb. These findings shift our understanding of the role of ATG5 during Mtb infection, reveal a new outcome of ATG5 activity, and shed light on early events in innate immunity that are required to regulate tuberculosis disease pathology and Mtb replication.

Mycobacterium tuberculosis must import iron from its host for survival, and its siderophore-dependent iron acquisition pathways are well established. Here we demonstrate a newly characterized pathway, whereby M. tuberculosis can use free heme and heme from hemoglobin as an iron source. Significantly, we identified the genomic region, Rv0202c – Rv0207c , responsible for the passage of heme iron across the mycobacterial membrane. Key players of this heme uptake system were characterized including a secreted protein and two transmembrane proteins, all three specific to mycobacteria. Furthermore, the crystal structure of the key heme carrier protein Rv0203 was found to have a unique fold. The discovery of a unique mycobacterial heme acquisition pathway opens new avenues of exploration into mycobacterial therapeutics.

The cytokine IL-10 antagonizes pathways that control () infection. Nevertheless, the impact of IL-10 during infection has been difficult to decipher because loss-of-function studies in animal models have yielded only mild phenotypes. We have discovered that the transcription factor basic helix-loop-helix family member e40 (Bhlhe40) is required to repress expression during infection. Loss of Bhlhe40 in mice results in higher expression, higher bacterial burden, and early susceptibility similar to that observed in mice lacking IFN-γ. Deletion of in mice reverses these phenotypes. Bhlhe40 deletion in T cells or CD11c cells is sufficient to cause susceptibility to Bhlhe40 represents the first transcription factor found to be essential during infection to specifically regulate expression, revealing the importance of strict control of IL-10 production by innate and adaptive immune cells during infection. Our findings uncover a previously elusive but significant role for IL-10 in pathogenesis.

Background: Mycobacterium tuberculosis has a probable nanocompartment (Mt-Enc). Results: Mt-Enc self-assembles into a 60-subunit cage that encapsulates enzymes via their C-terminal tails, which remain active within Mt-Enc. Conclusion: Cargo proteins are potentially involved in host oxidative stress response, suggesting that enzyme encapulation may be a mechanism to evade host immune assault. Significance: Mt-Enc may be utilized as a novel therapeutic delivery mechanism.

Research in recent years has focused significantly on the role of selective macroautophagy in targeting intracellular pathogens for lysosomal degradation, a process termed xenophagy. In this review we evaluate the proposed roles for xenophagy in controlling bacterial infection, highlighting the concept that successful pathogens have evolved ways to subvert or exploit this defense, minimizing the actual effectiveness of xenophagy in innate immunity. Instead, studies in animal models have revealed that autophagy-associated proteins often function outside of xenophagy to influence bacterial pathogenesis. In light of current efforts to manipulate autophagy and the development of host-directed therapies to fight bacterial infections, we also discuss the implications stemming from the complicated relationship that exists between autophagy and bacterial pathogens.

Streptococcus pneumoniae is a commensal bacterium of the human nasopharynx, but can cause harmful infections if it spreads to other parts of the body, such as pneumonia, sepsis or meningitis. To facilitate pathogenesis studies, we constructed a doxycycline-inducible pooled CRISPR interference (CRISPRi) library targeting all operons in protypical S. pneumoniae strain D39V. Our library design allows fitness within the pool to be assessed by a one-step PCR reaction directly followed by Illumina sequencing (CRISPRi-seq). The doxycycline-inducible CRISPRi system is tightly controllable and suitable for both bottleneck exploration and evaluation of gene fitness in vitro and in vivo. Here, we applied CRISPRi-seq to identify genetic factors important for causing pneumococcal pneumonia. Mice were infected intratracheally with our CRISPRi library and bacteria collected at 24 h (from lung) and 48 h (from both lung and blood) post-infection. CRISPRi-seq showed a critical bottleneck at 48 h after intratracheal infection, with only a few bacteria surviving the brunt of the innate immune response to cause systemic infection. However, earlier at 24 h postinfection, many significant differences in gene fitness cost between in vitro and in vivo conditions were identified, including genes encoding known and putative novel virulence factors, genes essential only in vivo, and genes essential only in vitro. A key advantage of CRISPRi-seq over traditional transposon-based genetic screens is that all genes, including essential genes, can be tested for their role in virulence and pathogenicity. The approaches developed here should be generally applicable to study infection bottlenecks and in vivo fitness for other important human and animal pathogens.

Variation in the presentation of hereditary immunodeficiencies may be explained by genetic or environmental factors. Patients with mutations in HOIL1 (RBCK1) present with amylopectinosis-associated myopathy with or without hyper-inflammation and immunodeficiency. We report that barrier-raised HOIL-1-deficient mice exhibit amylopectin-like deposits in the myocardium but show minimal signs of hyper-inflammation. However, they show immunodeficiency upon acute infection with Listeria monocytogenes, Toxoplasma gondii or Citrobacter rodentium. Increased susceptibility to Listeria was due to HOIL-1 function in hematopoietic cells and macrophages in production of protective cytokines. In contrast, HOIL-1-deficient mice showed enhanced control of chronic Mycobacterium tuberculosis or murine γ-herpesvirus 68 (MHV68), and these infections conferred a hyper-inflammatory phenotype. Surprisingly, chronic infection with MHV68 complemented the immunodeficiency of HOIL-1, IL-6, Caspase-1 and Caspase-1;Caspase-11-deficient mice following Listeria infection. Thus chronic herpesvirus infection generates signs of auto-inflammation and complements genetic immunodeficiency in mutant mice, highlighting the importance of accounting for the virome in genotype-phenotype studies.DOI: http://dx.doi.org/10.7554/eLife.04494.001