Jacqueline M. Kimmey

The Cytosolic Sensor cGAS Detects Mycobacterium tuberculosis DNA to Induce Type I Interferons and Activate Autophagy

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 cytosolic DNA, 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.

Discovery and characterization of a unique mycobacterial heme acquisition system

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.

Unique role for ATG5 in neutrophil-mediated immunopathology during M. tuberculosis infection.

Mycobacterium tuberculosis, a major global health threat, replicates in macrophages in part by inhibiting phagosome–lysosome fusion, until interferon-γ (IFNγ) activates the macrophage to traffic M. tuberculosis to the lysosome. How IFNγ elicits this effect is unknown, but many studies suggest a role for macroautophagy (herein termed autophagy), a process by which cytoplasmic contents are targeted for lysosomal degradation. The involvement of autophagy has been defined based on studies in cultured cells where M. tuberculosis co-localizes with autophagy factors ATG5, ATG12, ATG16L1, p62, NDP52, BECN1 and LC3 (refs 2, 3, 4, 5, 6), stimulation of autophagy increases bacterial killing, and inhibition of autophagy increases bacterial survival. Notably, these studies reveal modest (~1.5–3-fold change) effects on M. tuberculosis replication. By contrast, mice lacking ATG5 in monocyte-derived cells and neutrophils (polymorponuclear cells, PMNs) succumb to M. tuberculosis within 30 days, an extremely severe phenotype similar to mice lacking IFNγ signalling. Importantly, ATG5 is the only autophagy factor that has been studied during M. tuberculosis infection in vivo and autophagy-independent functions of ATG5 have been described. For this reason, we used a genetic approach to elucidate the role for multiple autophagy-related genes and the requirement for autophagy in resistance to M. tuberculosis infection in vivo. Here we show that, contrary to expectation, autophagic capacity does not correlate with the outcome of M. tuberculosis infection. Instead, ATG5 plays a unique role in protection against M. tuberculosis by preventing PMN-mediated immunopathology. Furthermore, while Atg5 is dispensable in alveolar macrophages during M. tuberculosis infection, loss of Atg5 in PMNs can sensitize mice to M. tuberculosis. These findings shift our understanding of the role of ATG5 during M. tuberculosis infection, reveal new outcomes of ATG5 activity, and shed light on early events in innate immunity that are required to regulate disease pathology and bacterial replication.

Phenotypic complementation of genetic immunodeficiency by chronic herpesvirus infection

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

Characterization of a Mycobacterium tuberculosis nanocompartment and its potential cargo proteins

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. Mycobacterium tuberculosis has evolved various mechanisms by which the bacterium can maintain homeostasis under numerous environmental assaults generated by the host immune response. M. tuberculosis harbors enzymes involved in the oxidative stress response that aid in survival during the production of reactive oxygen species in activated macrophages. Previous studies have shown that a dye-decolorizing peroxidase (DyP) is encapsulated by a bacterial nanocompartment, encapsulin (Enc), whereby packaged DyP interacts with Enc via a unique C-terminal extension. M. tuberculosis also harbors an encapsulin homolog (CFP-29, Mt-Enc), within an operon with M. tuberculosis DyP (Mt-DyP), which contains a C-terminal extension. Together these observations suggest that Mt-DyP interacts with Mt-Enc. Furthermore, it has been suggested that DyPs may function as either a heme-dependent peroxidase or a deferrochelatase. Like Mt-DyP, M. tuberculosis iron storage ferritin protein, Mt-BfrB, and an M. tuberculosis protein involved in folate biosynthesis, 7,8-dihydroneopterin aldolase (Mt-FolB), have C-terminal tails that could also interact with Mt-Enc. For the first time, we show by co-purification and electron microscopy that mycobacteria via Mt-Enc can encapsulate Mt-DyP, Mt-BfrB, and Mt-FolB. Functional studies of free or encapsulated proteins demonstrate that they retain their enzymatic activity within the Mt-Enc nanocompartment. Mt-DyP, Mt-FolB, and Mt-BfrB all have antioxidant properties, suggesting that if these proteins are encapsulated by Mt-Enc, then this nanocage may play a role in the M. tuberculosis oxidative stress response. This report provides initial structural and biochemical clues regarding the molecular mechanisms that utilize compartmentalization by which the mycobacterial cell may aid in detoxification of the local environment to ensure long term survival.

Bacterial Pathogens versus Autophagy: Implications for Therapeutic Interventions

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.

The impact of ISGylation during Mycobacterium tuberculosis infection in mice

Mycobacterium tuberculosis infection results in 1.5 million deaths annually. Type I interferon (IFN) signaling through its receptor IFNAR correlates with increased severity of disease, although how this increases susceptibility to M. tuberculosis remains uncertain. ISG15 is one of the most highly induced interferon stimulated genes (ISGs) during M. tuberculosis infection. ISG15 functions by conjugation to target proteins (ISGylation), by noncovalent association with intracellular proteins, and by release from the cell. Recent studies indicated that ISG15 can function via conjugation-independent mechanisms to suppress the type I IFN response. These data raised the question of whether ISG15 may have diverse and sometimes opposing functions during M. tuberculosis infection. To address this, we analyzed ISGylation during M. tuberculosis infection and show that ISGylated proteins accumulate following infection in an IFNAR-dependent manner. Type I IFN and ISG15 both play transient roles in promoting bacterial replication. However, as the disease progresses, ISGylation deviates from the overall effect of type I IFN and, ultimately, mice deficient in ISGylation are significantly more susceptible than IFNAR mice. Our data demonstrate that ISGs can both protect against and promote disease and are the first to report a role for ISGylation during M. tuberculosis infection.

Analysis of the contribution of MTP and the predicted Flp pilus genes to Mycobacterium tuberculosis pathogenesis

Mycobacterium tuberculosis (Mtb) is one of the worlds most successful pathogens. Millions of new cases of tuberculosis occur each year, emphasizing the need for better methods of treatment. The design of novel therapeutics is dependent on our understanding of factors that are essential for pathogenesis. Many bacterial pathogens use pili and other adhesins to mediate pathogenesis. The recently identified Mycobacterium tuberculosis pilus (MTP) and the hypothetical, widely conserved Flp pilus have been speculated to be important for Mtb virulence based on in vitro studies and homology to other pili, respectively. However, the roles for these pili during infection have yet to be tested. We addressed this gap in knowledge and found that neither MTP nor the hypothetical Flp pilus is required for Mtb survival in mouse models of infection, although MTP can contribute to biofilm formation and subsequent isoniazid tolerance. However, differences in mtp expression did affect lesion architecture in infected lungs. Deletion of mtp did not correlate with loss of cell-associated extracellular structures as visualized by transmission electron microscopy in Mtb Erdman and HN878 strains, suggesting that the phenotypes of the mtp mutants were not due to defects in production of extracellular structures. These findings highlight the importance of testing the virulence of adhesion mutants in animal models to assess the contribution of the adhesin to infection. This study also underscores the need for further investigation into additional strategies that Mtb may use to adhere to its host so that we may understand how this pathogen invades, colonizes and disseminates.

Bhlhe40 is an essential repressor of IL-10 duringMycobacterium tuberculosisinfection

The cytokine IL-10 antagonizes pathways that control Mycobacterium tuberculosis (Mtb) infection. Nevertheless, the impact of IL-10 during Mtb 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 Il10 expression during Mtb infection. Loss of Bhlhe40 in mice results in higher Il10 expression, higher bacterial burden, and early susceptibility similar to that observed in mice lacking IFN-&ggr;. Deletion of Il10 in Bhlhe40−/− mice reverses these phenotypes. Bhlhe40 deletion in T cells or CD11c+ cells is sufficient to cause susceptibility to Mtb. Bhlhe40 represents the first transcription factor found to be essential during Mtb infection to specifically regulate Il10 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 Mtb pathogenesis.

LysMD3 is a type II membrane protein without an in vivo role in the response to a range of pathogens

Germline-encoded receptors recognizing common pathogen-associated molecular patterns are a central element of the innate immune system and play an important role in shaping the host response to infection. Many of the innate immune molecules central to these signaling pathways are evolutionarily conserved. LysMD3 is a novel molecule containing a putative peptidoglycan-binding domain that has orthologs in humans, mice, zebrafish, flies, and worms. We found that the lysin motif (LysM) of LysMD3 is likely related to a previously described peptidoglycan-binding LysM found in bacteria. Mouse LysMD3 is a type II integral membrane protein that co-localizes with GM130+ structures, consistent with localization to the Golgi apparatus. We describe here two lines of mLysMD3-deficient mice for in vivo characterization of mLysMD3 function. We found that mLysMD3-deficient mice were born at Mendelian ratios and had no obvious pathological abnormalities. They also exhibited no obvious immune response deficiencies in a number of models of infection and inflammation. mLysMD3-deficient mice exhibited no signs of intestinal dysbiosis by 16S analysis or alterations in intestinal gene expression by RNA sequencing. We conclude that mLysMD3 contains a LysM with cytoplasmic orientation, but we were unable to define a physiological role for the molecule in vivo.