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. Author manuscript; available in PMC: 2016 Jun 10.
Published in final edited form as: Cell Host Microbe. 2015 Jun 2;17(6):820–828. doi: 10.1016/j.chom.2015.05.005

Cyclic GMP-AMP Synthase (cGAS) Is an Innate Immune DNA Sensor for Mycobacterium tuberculosis

Angela C Collins 1,2,†, Haocheng Cai 3,†, Tuo Li 3,†, Luis H Franco 4,5, Xiao-Dong Li 3, Vidhya R Nair 1,2, Caitlyn R Scharn 1,2, Chelsea E Stamm 1,2, Beth Levine 1,2,4,5, Zhijian J Chen 3,4,*, Michael U Shiloh 1,2,*
PMCID: PMC4499468  NIHMSID: NIHMS706158  PMID: 26048137

Summary

Activation of the DNA-dependent cytosolic surveillance pathway in response to Mycobacterium tuberculosis infection stimulates ubiquitin-dependent autophagy and inflammatory cytokine production, and plays an important role in host defense against M. tuberculosis. However, the identity of the host sensor for M. tuberculosis DNA is unknown. Here we show that M. tuberculosis activated cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) synthase (cGAS) in macrophages to produce cGAMP, a second messenger that activates the adaptor protein stimulator of interferon genes (STING) to induce type I interferons and other cytokines. cGAS localized with M. tuberculosis in mouse and human cells and in human tuberculosis lesions. Knockdown or knockout of cGAS in human or mouse macrophages blocked cytokine production and induction of autophagy. Mice deficient in cGAS were more susceptible to lethality caused by infection with M. tuberculosis. These results demonstrate that cGAS is a vital innate immune sensor of M. tuberculosis infection.

Introduction

We recently identified the cytosolic DNA sensor cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) synthase (cGAS) as the primary trigger for production of type I interferons and other cytokines in the setting of viral infections including herpes simplex virus type 1, vaccinia virus and HIV (Gao et al., 2013; Li et al., 2013; Sun et al., 2013). When cGAS binds DNA, it is activated to synthesize cGAMP from adenosine triphosphate (ATP) and guanosine triphosphate (GTP) (Wu et al., 2013). cGAMP then binds and activates the endoplasmic reticulum protein STING, which in turn activates the protein kinases IκB kinase (IKK) and TANK-binding kinase 1 (TBK1). IKK and TBK1 then stimulate nuclear translocation of nuclear factor- κB (NF-κB) (Abe and Barber, 2014) and interferon regulatory factor 3 (IRF3) (Liu et al., 2015) to induce transcription of type-I interferons (e.g., IFNβ) and other cytokines (Cai et al., 2014). While the induction of type-I interferons is critical for immune defense against viral infections (Stetson and Medzhitov, 2006), some bacterial pathogens such as M. tuberculosis and Listeria monocytogenes exploit the type-I interferon pathway for their own advantage (Eshleman and Lenz, 2014).

In addition to initiating a signaling cascade leading to IFNβ production, cytosolic DNA is also vital for autophagy induction. Cytosolic DNA can activate a STING-dependent pathway for ubiquitin-dependent autophagy induction (Watson et al., 2012) but also a STING-independent autophagy pathway through dissociation of the negative regulator Rubicon from Beclin 1 (Liang et al., 2014). The DNA-triggered induction of autophagy is important for the host defense against bacterial infections. Thus, in the context of bacterial infections, it is important to determine whether the activation of the cytosolic DNA surveillance pathway (CSP), which induces both autophagy and interferons, is protective or detrimental to the host.

Despite being considered a classic intraphagosomal pathogen, M. tuberculosis can rarely be found residing in the macrophage cytoplasm (van der Wel et al., 2007) where M. tuberculosis pathogen associated molecular patterns can interact with host sensors. M. tuberculosis also activates the cytosolic DNA surveillance pathway via a mechanism requiring phagosome disruption by the mycobacterial protein secretion system ESX-1 (Manzanillo et al., 2012) and the virulence factors ESAT-6 and CFP-10 (De Leon et al., 2012; Simeone et al., 2012). Mycobacterial DNA then escapes into the cytoplasm to activate cytokine production in a STING-, TBK-1- and IRF-3-dependent manner (Manzanillo et al., 2012). Although M. tuberculosis DNA activates the CSP to induce IRF-3 dependent cytokine production including IFNβ (Manzanillo et al., 2012) as well as autophagy induction, the identity of the host sensor for M. tuberculosis DNA is not known. Because the detection of M. tuberculosis infection by macrophages is so vital to the innate immune response (Stamm et al., 2015), we sought to identify the receptor for M. tuberculosis DNA and determine its role in the host response to infection.

Results

M. tuberculosis induces cGAS in human macrophages and during active tuberculosis

To first test if cGAS is expressed during M. tuberculosis infection, we queried the GEO database and found that during mouse infection (Kang et al., 2011), cGAS is induced in the lungs of infected mice (Fig. S1, related to Fig. 1). We next infected the human THP-1 macrophage cell line with M. tuberculosis and measured cGAS mRNA by quantitative PCR. We found that cGAS is upregulated 2 fold by 6 h after infection and 5 fold after 24 h (Fig. 1A). We also determined if cGAS protein is induced in human macrophages by infecting THP-1 cells with M. tuberculosis and measuring cGAS accumulation by western blot (Fig. 1B) and immunofluorescence microscopy (Fig. 1C). We observed that M. tuberculosis infection upregulated cGAS in human macrophages 24 h after infection (Fig. 1B, C). cGAS puncta occurred adjacent to intracellular mycobacteria and in the cytoplasm (Fig. 1C). Quantification of the degree of colocalization revealed that ~30% of intracellular M. tuberculosis colocalized with cGAS (Fig. 1D). We next asked if cGAS is expressed in human tuberculosis lesions by immunohistochemistry (Fig. 1E; Fig. S2, related to Fig. 1) and immunofluorescence microscopy (Fig. 1F) and found that cGAS was expressed diffusely in lung biopsies obtained from patients with human tuberculosis. By immunofluorescence we observed a punctate staining pattern similar to our observations in THP-1 cells (Fig. 1F) with occasional colocalization of M. tuberculosis with cGAS (Fig. 1F). Taken together, M. tuberculosis infection is sufficient to induce cGAS in macrophages and the cGAS protein is expressed in human tuberculous lesions.

Figure 1. M. tuberculosis infection upregulates cGAS expression in macrophages and human lungs.

Figure 1

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A, Quantitative PCR of cGAS in M. tuberculosis infected THP-1 cells. Results are representative of 2 independent experiments. Mean ± S.E.M., *p ≤ 0.05 compared to mock cells by two-way ANOVA with Dunnett’s multiple comparison test. B, Western blot of cGAS in M. tuberculosis infected THP-1 cells 24h after infection. Results are representative of 2 independent experiments. C, D, Confocal microscopy (C) and quantification (D) of cGAS association with mCherry M. tuberculosis in THP-1 cells 24h after infection (Mean ± S.E.M.). Results are representative of >3 independent experiments. Scale bars, 10µm. E, Immunohistochemistry for cGAS in a lung biopsy from a confirmed M. tuberculosis infected individual. Scale bar, 50 µm left, 20 µm right panel. F, Confocal microscopy of cGAS and M. tuberculosis in a lung biopsy as in (D). Scale bars, 5 µm. Arrowheads indicate colocalization.

cGAS is required for IFNβ production by human and mouse macrophages infected with M. tuberculosis

Since cGAS induces IFNβ in response to viral DNA (Gao et al., 2013; Li et al., 2013; Sun et al., 2013), and M. tuberculosis releases its DNA into the macrophage cytoplasm (Manzanillo et al., 2012; Watson et al., 2012), we asked if cGAS is required for M. tuberculosis to induce IFNβ and other cytokines using THP-1 cells stably expressing shRNA against cGAS or STING (Sun et al., 2013) (Fig. S3, related to Fig. 2). M. tuberculosis induced IFNβ transcription in a manner that depended on the multiplicity of infection (MOI; Fig. 2A). Knockdown of cGAS significantly reduced the induction of IFNβ (Fig. 2A) and the chemokine CXCL10 (Fig. 2B) by M. tuberculosis. Likewise, STING knockdown in THP-1 cells blocked IFNβ expression (Fig. 2A), as has been recently reported (Manzanillo et al., 2012). Knockdown of STING or cGAS had no effect on transcription of an NFκB-dependent cytokine, TNFα (Fig. 2C), suggesting that other ligands in M. tuberculosis may activate NF-κB through a cGAS-independent pathway. To obtain definitive genetic evidence for cGAS’s role in sensing M. tuberculosis infection in macrophages, we infected mouse bone marrow derived macrophages (BMDMs) deficient in cGAS or STING and measured IFNβ, CXCL-10, and TNFα by quantitative PCR. As in THP1 cells, IFNβ expression was MOI dependent (Fig. 2D) and both cGAS and STING were required for IFNβ and CXCL-10 but not TNFα transcription (Fig. 2D–F). Thus, we conclude that cGAS is essential for inducing IFNβ in response to M. tuberculosis infection in both human and mouse macrophages.

Figure 2. cGAS is essential for cGAMP production and IFNβ induction by M. tuberculosis.

Figure 2

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A, Quantitative PCR for IFNβ in THP-1 cells stably expressing shRNA against GFP (control), STING or cGAS, 4 hours after infection with M. tuberculosis at various multiplicity of infections (MOI). B, C, THP1 cells as in (A) infected at an MOI of 10 and CXCL10 (B) and TNF (C) expression determined by qPCR. Results are representative of >3 independent experiments. Mean ± S.E.M., **p ≤ 0.005 by one-way ANOVA with Dunnett’s multiple comparison test. D, BMDMs from WT, STINGgt/gt, cGAS−/− mice were infected with M. tuberculosis for 4 hours at various MOIs, the RNA was isolated to measure IFNβ expression by qPCR. E, F, Samples infected at an MOI of 10 were further analyzed for (b) CXCL-10 and (c) TNF induction. Results are representative of 3 independent experiments. Mean ± S.E.M., **p ≤ 0.005 compared to WT infected cells. G, Time course of cGAMP production during M. tuberculosis infection. WT BMDMs were infected with WT M. tuberculosis or M. tuberculosis ΔEccD and cGAMP levels determined by quantitative mass spectrometry. H, BMDMs from WT, STINGgt/gt, and cGAS−/− mice were infected with WT M. tuberculosis, and cGAMP levels determined at 8 h. Red bars represent isotopically-labeled cGAMP internal standard. Green bars represent biological samples.

Another organism that can activate the CSP is Listeria monocytogenes. Infection of THP-1 cells with L. monocytogenes resulted in IFNβ induction in a cGAS and STING dependent manner (Fig. S3 related to Fig. 2), indicating a conserved role for cGAS in detecting cytoplasmic bacterial DNA. However, in contrast to THP-1 cells, IFNβ and CXCL-10 induction by L. monocytogenes in mouse macrophages was cGAS-independent but STING-dependent (Fig. S3 related to Fig. 2), consistent with a role for the bacterial secretion of cyclic di-AMP, a direct STING ligand (Hansen et al., 2014; Woodward et al., 2010). Thus, it appears that cytosolic bacterial DNA plays a dominant role in IFNβ induction in THP1 cells, whereas both the DNA sensing pathway and bacterial c-di-AMP are important in activating STING in the mouse macrophages. Infection with another Gram-positive bacterium reported to induce IFNβ via secretion of DNA, Streptococcus pyogenes (Gratz et al., 2011), demonstrated an intermediate phenotype as IFNβ and CXCL-10 were undetectable in STINGgt/gt BMDM and significantly reduced in BMDM lacking cGAS (Fig. S3 related to Fig. 2). As above, induction of the NFκB-dependent cytokine IL-1β was independent of cGAS and STING (Fig. S3 related to Fig. 2).

cGAMP is produced during M. tuberculosis infection in a cGAS and ESX-1 dependent manner

To demonstrate that the cGAS product cGAMP is produced during M. tuberculosis infection, we developed and utilized a sensitive mass spectrometry assay to measure cGAMP levels in M. tuberculosis-infected mouse BMDMs. This assay utilizes a heavy isotope 13C1015N5-labeled cGAMP internal standard to quantify the absolute amounts of endogenous unlabeled cGAMP in cell lysates. We first determined a time course of cGAMP production and found that in wild type BMDMs cGAMP peaked at 8 h after infection with wild type M. tuberculosis (Fig. 2G). However, infection with an M. tuberculosis ΔeccD mutant, a critical component of the ESX-1 secretion machine that fails to disrupt the phagosomal membrane and release mycobacterial DNA (Manzanillo et al., 2012; Stanley et al., 2007), did not induce cGAMP production in macrophages (Fig. 2G). Thus, phagosomal rupture via ESX-1 and release of mycobacterial DNA into the cytoplasm is necessary to induce cGAMP production. Next, we quantitated cGAMP in M. tuberculosis-infected BMDM from WT and cGAS−/− (Gao et al., 2013) mice as well as the STING golden ticket mutant mice (STINGgt/gt), which have a loss of expression of STING (Sauer et al., 2011). M. tuberculosis induced cGAMP production in WT and STINGgt/gt BMDM at a level of approximately 6000 molecules per cell, but cGAS deficient macrophages produced no detectable cGAMP (Fig. 2H). Thus, cGAS is required for cGAMP production in M. tuberculosis-infected macrophages.

cGAS is required for LC3 association with M. tuberculosis

Sensing of cytosolic DNA by M. tuberculosis induces recruitment of the autophagy markers LC3, NDP52, p62 and ubiquitination of M. tuberculosis containing organelles (Manzanillo et al., 2013; Manzanillo et al., 2012; Watson et al., 2012); the autophagy induction mediates early inhibition of bacterial growth (Watson et al., 2012). LC3 is a protein component of both autophagy and LC3-associated phagocytosis that marks both classic double membrane enveloped autophagosomes and single membrane enveloped phagosomes (Green and Levine, 2014). Such LC3 marked organelles are targeted to lysosomes for degradation of intracompartmental contents. We first asked if STING or cGAS is required for induction of general autophagy by starvation, and found that neither STING nor cGAS deficiency prevented LC3 starvation induced LC3-puncta formation (Fig. 3A,B). To determine if cGAS is necessary to regulate association of M. tuberculosis with LC3 we transduced mouse BMDMs with a lentivirus to express GFP-LC3 and infected them with mCherry-expressing M. tuberculosis. Absence of STING or cGAS significantly reduced but did not eliminate the association of GFP-LC3 with mCherry-M. tuberculosis (Fig. 3C,D). Thus, we conclude that cGAS is required for early recruitment of LC3, a marker of autophagy and LC3-associated phagocytosis, during M. tuberculosis infection of macrophages.

Figure 3. Selective autophagy of M. tuberculosis requires cGAS and STING.

Figure 3

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A, B, WT, STINGgt/gt, and cGAS−/− BMDM in triplicate on cover slips were placed into starvation medium and 4 hours later fixed and stained for LC3. B, For each cover slip, LC3 puncta were counted for 100 cells. Shown is the mean ± S.E.M. of LC3 puncta per cell for each genotype and treatment. C, D, Mouse BMDMs transduced with GFP-LC3 were infected with mCherry-expressing M. tuberculosis and colocalization quantified (Scale bar, 3 µm). Shown are mean ± S.E.M. *p ≤ 0.05, **p ≤ 0.005 one-way ANOVA with Dunnett’s test.

cGAS is required for early control of M. tuberculosis replication by macrophages

Since activation of the CSP provides early protection from M. tuberculosis infection in macrophages (Watson et al., 2012), we determined the role of cGAS in controlling intracellular replication of M. tuberculosis. We infected BMDM deficient in either STING or cGAS, and quantified intracellular mycobacterial growth. While all macrophages ingested bacteria equally at time zero (Fig. 4A), both STING and cGAS deficient BMDM were more permissive for M. tuberculosis growth (Fig. 4A). However, both cGAS and STING deficient macrophages were able to control intracellular M. tuberculosis growth equally at 3 and 7 days after infection (data not shown), suggesting that additional antimicrobial mechanisms are able to compensate in macrophages later in the course of infection. Because cGAS produces the cyclic dinucleotide cGAMP, and some cyclic dinucleotides such as cyclic di-AMP and cyclic di-GMP can impact bacteria directly (Li and He, 2012; Witte et al., 2013), we tested if cGAMP alone could inhibit M. tuberculosis growth in vitro, and found no effect of cGAMP at a concentration as high as 10 µM (Fig. S4, related to Fig. 4). Thus, cGAS signaling plays a role in early events controlling M. tuberculosis infection in human macrophages.

Figure 4. cGAS is required for controlling intracellular bacterial replication and protects mice from M. tuberculosis mortality.

Figure 4

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A, BMDMs from WT, STINGgt/gt, cGAS−/− mice were infected with M. tuberculosis in triplicate at an MOI of 1. CFU values obtained at day 0 and 24 h after infection. Mean ± S.E.M., *p ≤ 0.05 by one way ANOVA B, C, WT, STINGgt/gt, and cGAS−/− mice were infected with a low dose (200) M. tuberculosis. B, CFU was enumerated in the lungs of infected mice (Mean ± S.E.M., n=5 mice per genotype per time point). C, Infected mice were monitored for survival. Shown is the combined survival from two independent experiments (N=14–18 mice per group). Results from each independent experiment are provided in the supplementary material (Fig. S7). The Gehan-Breslow-Wilcoxon test was performed to determine statistical significance.

cGAS is protective in mice chronically infected with M. tuberculosis

To test the functional role of DNA sensing mediated by cGAS and STING during M. tuberculosis infection, we performed a low-dose aerosol infection of cGAS−/−, STINGgt/gt and congenic C57Bl/6 mice. Of note, to date no differences in survival have been observed in aged cGAS−/− uninfected mice compared to control wild type littermates (data not shown). All mice were infected with approximately 200 colony-forming units (CFU) via aerosol. CFUs in lung (Fig. 4B), liver, and spleen (Fig. S5, related to Fig. 4) were similar among the different genotypes at days 21 and 42 after infection as well as at the time of death (Fig. S4, related to Fig. 4). We observed an ~0.7 log decrease in all mouse groups from day 21 to day 42, which is consistent with multiple prior studies in similarly performed aerosol challenge models (Converse et al., 2009; Darwin and Nathan, 2005; Manzanillo et al., 2012). Gross inflammation as assessed by pathology at days 21 and 42 and inflammatory cytokines such as IFN-γ, IL-1β, IL-12, and TNF-α were also similar (Fig. S4, related to Fig. 4). IFNβ was reduced about 2-fold in the serum of cGAS deficient mice compared to wild type, but this result was not statistically significant (Fig. S4, related to Fig. 4). However, M. tuberculosis-infected cGAS−/− mice succumbed to infection significantly earlier than their wild type or STING deficient counterparts (Fig. 4C, Fig. S4 related to Fig. 4), suggesting a vital role for cGAS in chronic tuberculosis infection.

Discussion

Here we show that cGAS plays an important role in inducing IFNβ, autophagy and macrophage antimicrobial activity during M. tuberculosis infection in vitro, as well as in resistance to chronic tuberculosis in vivo. The cGAS product cGAMP is detected in M. tuberculosis infected macrophages only when the phagosome is ruptured by the activity of the M. tuberculosis ESX-1 system and when cGAS is expressed. cGAS is expressed in mouse and human macrophages, and in primary human tuberculous lesions, consistent with its important role in regulating the innate immune response during tuberculosis.

M. tuberculosis infection induced upregulation of cGAS as visualized by punctate cytoplasmic staining within infected cells. Likewise, Wassermann et al. (Wassermann et al., 2015) also found punctate cGAS staining during M. tuberculosis infection. Transfection of DNA is known to induce similar punctate staining, and transfected DNA can be colocalized with cGAS in many of these areas of cGAS aggregation (Sun et al., 2013). cGAS has recently been found to be a key interferon stimulated gene (ISG) (Schoggins et al., 2014), and increased cGAS staining during M. tuberculosis infection is also visualized in adjacent non-infected cells likely due to paracrine type I interferon signaling (Stetson and Medzhitov, 2006). Additionally, cGAMP produced in one cell can be transferred via gap junctions to neighboring cells where it activates STING and the production of type I interferon (Ablasser et al., 2013). This suggests that another mechanism for the upregulation of ISGs such as IFNβ and cGAS in uninfected cells may be transfer of cGAMP from M. tuberculosis infected cells to adjacent cells, and indeed Wassermann et al. (Wassermann et al., 2015) demonstrated that during M. tuberculosis infection cGAMP can translocate between cells via connexin channels to induce IFNβ. Furthermore, in vivo, IFNβ may also be induced in macrophages and other cells in a TLR4-dependent manner (Liu et al., 2015).

Some intracellular pathogens such as L. monocytogenes and C. trachomatis secrete their own cyclic nucleotides such as c-di-AMP into the host cytoplasm, thus bypassing cGAS activation to directly trigger STING-dependent IFNβ production (Woodward et al., 2010). We found that L. monocytogenes could induce IFNβ in the absence of cGAS in mouse BMDM but not in human THP-1 cells, suggesting a role for L. monocytogenes secreted c-di-AMP in mouse but not human cells. Hansen et al. recently reported similar results using RNAi of cGAS (Hansen et al., 2014). It is not clear whether listeria produces significant levels of c-di-AMP to stimulate IFNβ in THP1 cells.

Whether M. tuberculosis produces any cyclic di-nucleotide is a subject of debate. In one study, deletion of the M. tuberculosis di-adenylate cyclase gene disA (Rv3586) had no effect on IFNβ production during M. tuberculosis infection of BMDMs (Manzanillo et al., 2012). However in another recent study, deletion of disA reduced IFNβ production four-fold whereas deletion of the diadenylate cyclic di-AMP phosphodiesterase gene (cnpB) increased IFNβ production 10-fold during BMDM infection (Yang et al., 2014). In our hands, both shRNA mediated depletion and genetic ablation of cGAS prevented IFNβ expression by macrophages, except at very high MOI. Likewise, both Watson et al. (Watson et al., 2015) and Wassermann et al. (Wassermann et al, 2015) also failed to detect a cGAS-independent induction of interferon-dependent genes, suggesting that if c-di-AMP is indeed secreted by M. tuberculosis, it plays only a minor role compared to mycobacterial dsDNA in triggering IFNβ production. Since STING can respond to a variety of cyclic di-nucleotides, an alternative explanation for the induction of a residual amount of IFNβ in cGAS deficient BMDM at high MOI would be the action of an accessory host enzyme producing cyclic dinucleotides in response to bacterial infection.

Identification of cGAS in human tuberculosis lesions and cGAMP in infected macrophages provides a possible explanation for the interferon driven transcriptional signature in human tuberculosis (Berry et al., 2010). Emerging data suggests that successful outcomes in human tuberculosis occur when there is an appropriate balance between bacterial death and inflammation (BoseDasgupta and Pieters, 2014; Tobin et al., 2012). To that end, modulating cGAS activity could be a productive approach to host-directed therapy.

We observed two competing roles for cGAS in regulating host defense against M. tuberculosis in macrophages, namely, inducing type I interferon production and facilitating early control of intracellular replication through the induction of autophagy. Thus, the increased sensitivity of cGAS−/− mice to M. tuberculosis–induced lethality may reflect an imbalance between decreases in detrimental IFNβ signaling (Teles et al., 2013) and activation of protective autophagy/LC3-associated phagocytosis. Studies of M. tuberculosis infection of IRF-3- (Manzanillo et al., 2012) or Parkin-deficient mice (Manzanillo et al., 2013) support this hypothesis. IRF-3 deficiency prevents IFNβ production during M. tuberculosis infection (Manzanillo et al., 2012), though autophagic targeting is not affected (Watson et al., 2012). Notably, IRF-3 mutant mice do not have a defect in bacterial growth in M. tuberculosis infected macrophages in vitro or a profound difference in bacterial growth compared to control mice during low dose aerosol infection, yet they are dramatically protected from mortality during M. tuberculosis infection (Manzanillo et al., 2012), which may reflect IRF-3’s central role as signaling hub (Liu et al., 2015). In contrast, Parkin deficient mice are defective in M. tuberculosis induced autophagy but have no type I interferon defect, and M. tuberculosis infected Parkin deficient mice succumb earlier to infection associated with a modest increase in CFU burden (Manzanillo et al., 2013). In that regard, cGAS-deficient mice had defective LC3 recruitment to M. tuberculosis containing organelles and reduced intracellular killing ability in BMDMs, which might explain why they were more susceptible to chronic infection. Consistent with this notion, Atg5-deficient mice are also more susceptible to lethality induced by M. tuberculosis infection (Castillo et al., 2012; Watson et al., 2012), though the early mortality may in part be explained by a hyperinflammatory state in these mice (Castillo et al., 2012; Watson et al., 2012).

Given the shared roles of cGAS and STING in inducing IFNβ and recruiting LC3 to M. tuberculosis containing organelles, it was unexpected that cGAS−/− mice would be more susceptible than STINGgt/gt mice to M. tuberculosis infection. In a parallel study, Watson et al. (Watson et al., 2015) did not find a mortality defect when comparing wild type and cGAS−/− mice also infected with ~200 CFU of the M. tuberculosis Erdman strain via aerosol. Of note, whereas the Watson et al. survival study lasted only 100 days, in our hands in two independent experiments cGAS−/− mice did not significantly succumb to disease until after 100 days. Consistent with the fact that wild type C57BL/6 mice are relatively resistant to aerosol M. tuberculosis infection (Manzanillo et al., 2012; Medina and North, 1998), wild type mice did not start to succumb until about 175 days after infection.

Several recent studies have emerged highlighting unique STING independent functions for cGAS, particularly in the induction of autophagy. cGAS, independent of its catalytic activity, interacts directly with the autophagy protein Beclin-1 to block its association with the negative regulator Rubicon (Liang et al., 2014). Thus, we speculate that the increased susceptibility of cGAS−/− mice to tuberculosis may reflect in vivo effects on cGAS mediated autophagy. Indeed, autophagy is not only important for control of intracellular replication of mycobacteria (Castillo et al., 2012), but also for antigen presentation (Deretic et al., 2013), suggesting that during a long-term infection, cGAS deficiency may impact the ability of mice to develop an appropriate adaptive immune response. Taken together, we propose that cGAS is a central signaling hub that is vital for immunity against viruses and bacteria.

Experimental Procedures

Strains and media

The Erdman wild type strain of M. tuberculosis was grown in Middlebrook 7H9 medium or on Middlebrook 7H11 agar plates supplemented with 10% oleic acid-albumin-dextrose-catalase. Tween-80 was added to liquid medium to a final concentration of 0.05%

Mice and macrophages

cGAS−/− mice (Li et al., 2013) were generated and maintained at UTSW. A detailed description of their ontogeny is available in the Supplementary methods and online (Li et al., 2013). STINGgt/g (Sauer et al., 2011) mice purchased from Jackson lab were bred at UTSW. Wild type C57BL/6 mice were bred in the same facility and all mice were housed in specific pathogen-free conditions. All experiments were carried out according to experimental protocols approved by Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center at Dallas.

Immunohistochemistry and immunofluorescence of human specimens

Paraffin embedded specimens were deparaffinized in xylene, subjected to heat mediated antigen-retrieval in 10 mM sodium citrate (pH 6.0), permeabilized in 0.2% Triton-100 (Sigma) and blocked in 5% donkey sera. For immunohistochemistry, human sections were stained with rabbit anti-C6orf150 antibody (cGAS; 1:100, Abcam) or rabbit polyclonal IgG (1:100, Abcam). HRP conjugated secondary antibodies were obtained from Jackson Immunochemicals and used at 1:250. Staining was amplified with AB reagent (Vecta-stain) and detected using DAB reagent (Thermo Scientific). Images were acquired using a Zeiss Axioplan 2 microscope. For immunofluorescence in THP-1 cells and human tissue, cGAS was identified using rabbit anti-MB21D1 antibody (Sigma 1:100) and an HRP conjugated donkey anti-rabbit secondary (1:100, Jackson Immunochemicals) followed by amplification with tyramide (1:50, Perkin Elmer). M. tuberculosis was identified using guinea pig anti-M. tuberculosis (1:25, BEI Resources, NR-13818) and an Alexa 488 conjugated donkey anti-guinea pig secondary (1:100, Jackson Immunochemicals). Hoechst stain was used to visualize nuclei. Sections were mounted using Prolong Gold, allowed to cure for 24 hours in the dark, and then visualized. Images were acquired using a Leica TCS SP5 confocal microscope. This study was approved by the Institutional Review Board of the University of Texas Southwestern Medical Center.

In vitro M. tuberculosis infections

For macrophage infections, liquid M. tuberculosis cultures were washed twice with PBS, resuspended in PBS and centrifuged to remove clumps. The supernatant was sonicated twice to generate a single cell suspension. Bacteria were resuspended in RPMI media plus 10% horse serum. Bacterial dilutions or plain media for uninfected controls were added to the macrophages and plates centrifuged for 10 minutes at 1500 RPM to initiate infection. Macrophages were then washed with PBS and fresh macrophage media applied.

cGAMP extraction and quantification

cGAMP was measured in TB- or mock-infected BMDMs as we recently described (Rongvaux et al., In Press). Briefly, cells were lysed in cold 80% methanol/2% acetic acid solution, into which 13C1015N5-labeled cGAMP internal standard was immediately supplemented. After a short incubation on ice, insoluble debris was cleared by centrifugation, and extracted twice in 2% acetic acid. cGAMP was enriched by solid phase extraction using HyperSep Aminopropyl SPE Columns (Thermo Scientific), and eluted in 4% ammonium hydroxide/80% methanol. The eluents were vacuum-dried, resuspended in LC/MS-grade water, cleared by centrifugation, and transferred to autosampler vials. Metabolite extracts were separated by hydrophilic chromatography (HILIC) with Xbridge Amide phase (3.5 µm, 3.5 mm ID × 100 mm L, Waters) on a Dionex Ultimate 3000 Rapid Separation Liquid Chromatography system (Thermo Scientific). Mobile phase A was 20 mM ammonium bicarbonate with 20 mM ammonium hydroxide in water, and mobile phase B acetonitrile. The separation ran at a flow of 400 µL/min for the first 14.5 min and 800 µL/min for the remaining 8.5 min, through the following gradient: 0 min 85% B, 3 min 85% B, 10 min 2% B, 14 min 2% B, 14.5 min 85% B and 23 min 85% B.

The HILIC eluent was sprayed online into a TSQ Quantiva triple quadruple mass spectrometer (Thermo Scientific), which performed multiple reaction monitoring experiments for detecting cGAMP and the internal standard. The technical details of the data acquisition and analysis were recently described (Rongvaux et al. Cell. 2014), with the exception that the spray was at default setting for 400 µL/min, and that the scan dwell time was 50 ms.

Statistical analysis

Statistical analysis was performed using GraphPad Prism. For in vitro studies, one-tailed Analysis of Variance (ANOVA) tests were used for experiments with multiple comparisons using Dunnett’s test. For experiments with single comparisons, two-tailed unpaired student’s t-test was used. For experiments containing samples with non-normal distributions such as cytokine and in vivo CFU measurements, the Kruskal-Wallis non-parametric test was used. The Gehan-Breslow-Wilcoxon test was used to analyze survival studies.

Supplementary Material

Supplemental Information

Acknowledgements

We thank D. Marciano for help with figure preparation and critical discussion and members of the Chen and Shiloh labs for critical discussions. This work was supported by NIH grants R01 AI099439 (M.U.S.), U19 AI109725 (B.L and M.U.S.), AI093967 (Z.J.C.), T32 AI007520 (C.E.S.) and T32AI005284 (C.R.S.) and the Cancer Research Prevention Institute of Texas RP120718 (B.L. and Z.J.C.). B.L. and Z.J.C. are investigators of the Howard Hughes Medical Institute. M.U.S. is a Disease Oriented Clinical Scholar at UT Southwestern.

Footnotes

Supplementary Information including detailed supplemental methods is linked to the online version of the paper at Cell Host and Microbe.

Author Contributions

A.C.C., L.H.F. and C.R.S. performed macrophage infections with M. tuberculosis for RNA, CFU, and microscopic analysis. A.C.C., V.R.N. and C.R.S. performed immunohistochemistry and immunofluorescence microscopy. A.C.C., L.H.F. and V.R.N. performed mouse aerosol infections with M. tuberculosis. H.C. performed qPCR of the bacteria-infected samples as well as the infection experiments with Listeria and Streptococcus. T. L. developed the cGAMP mass spectrometry assay and analyzed the data. H. C. and X. L. provided mice and bone marrow macrophages. C.E.S. performed in vitro M. tuberculosis experiments. B.L. provided reagents and was involved in study design. A.C.C., H.C., Z.J.C. and M.U.S. designed the study, analyzed data and wrote the paper. All authors discussed the results and commented on the manuscript.

The authors declare no competing financial interests.

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