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

Abstract

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.

Figure 1. cGAS is essential for induction of the cytosolic surveillence response during intracellular bacterial infection

(A) BMDMs were infected with WT or ΔESX-1 M. tuberculosis (Erdman) for 4 h, and IFN-β and IFIT1 transcripts were measured by RT-qPCR. mRNA levels are expressed as percentages relative to infected WT cells. (B) Same as (A) but IFN-β protein was measured by ELISA (left) or with ISRE-luciferase cells (right) 24 h post-infection. (C) Same as (A) but M. tuberculosis strain CDC1551. (D) Same as (A) but TNFα. (E) Same as (A) but with U937 knockdown cell lines. mRNA levels are expressed as percentage relative to infected scramble control cells. (F-H) BMDMs were infected for 4 h and transcript levels were measured during infection with L. pneumophila ΔflaA (L.p.) or ΔflaAΔsdhA (L.p. ΔsdhA) (F), L. monocytogenes (L.m.) (G), or S. Typhimurium (S. T.) (H). n.s., not significant; **p < 0.005 by two-tailed t-test comparing to WT infected with WT bacteria. See also Figure S1.

Figure 2. cGAS is required to target cytosolic DNA to the ubiquitin-mediated selective autophagy pathway

(A) MEFs expressing 3×FLAG-tagged cGAS transfected with Cy3-labeled plasmid DNA for 4 h and immunostained for 3×FLAG or indicated markers. (B) Quantification of cGAS+ Cy3-DNA co-stained with indicated marker from (A). Differences are not statistically significant. (C) RAW 264.7 cells stably expressing 3×FLAG-cGAS transfected with Cy3-DNA for 4 h and immunostained for 3×FLAG and indicated markers. (D) Quantification of ubiquitin and LC3 colocalization with Cy3-DNA 4 h post-transfection in BMDMs. (E) Same as (D) but RAW 264.7 knockdown cell lines. (F) Same as (D) but U937 knockdown cell lines. (G) BMDMs were transfected with interferon-stimulatory DNA (ISD) for 2 h, and LC3-II conversion was analyzed by quantitative Western blot (left) and expressed as a fold-increase in the ratio of LC3-II/Actin. Results are representative of at least three independent experiments. *p < 0.05, **p < 0.005 by two-tailed t-test comparing to SCR unless otherwise indicated. See also Figure S2.

Figure 3. cGAS is required to target Mycobacterium tuberculosis to the ubiquitin-mediated selective autophagy pathway

(A) RAW 264.7 cells stably expressing 3×FLAG-cGAS infected with mCherry M. tuberculosis for 45 min and immunostained for 3×FLAG. (B) Quantification of (A) during the indicated time course. (C) RAW 264.7 cells stably expressing 3×FLAG-cGAS infected with mCherry M. tuberculosis for 45 min and immunostained for 3×FLAG and indicated markers. (D) Quantification of (C). (E) Quantification of (C) during the indicated time course. (F) BMDMs infected with mCherry M. tuberculosis and immunostained for ubiquitin and LC3 4 h post-infection. (G) Quantification of (F). (H) Quantification of ubiquitin- and LC3-positive M. tuberculosis in RAW 264.7 knockdown cell lines. (I) Same as (H) but U937 knockdown cell lines. (J) BMDMs were infected with M. tuberculosis for 2 h, and LC3-II conversion was analyzed by quantitative Western blot (left) and expressed as a fold-change in the ratio of LC3-II/Actin. Results are representative of at least three independent experiments. (K) CFUs from BMDMs infected with M. tuberculosis at 6, 24, 72, and 120 h (left) and normalized to CFUs at 0 h (right). (L-P) WT and cGas^-/- mice were infected with ~100 aerosolized M. tuberculosis CFUs (n = 4 or 6 per group). (L) IFIT1 transcripts in lungs of uninfected and infected mice. (M) Serum IFN-β levels 21 days post-infection as measured by ISRE-luciferase reporter cells. (N-O) CFUs in lungs (N) and spleens (O) 21 and 80 days post-infection. (P) Survival of mice monitored for 102 days (n = 6 WT and 3 cGas^-/-). *p < 0.05, **p < 0.005, n.s. not significant by two-tailed t-test comparing to SCR or WT unless otherwise indicated. See also Figure S3.

Figure 4. cGAS binds to M. tuberculosis-derived DNA during infection

(A) Western blot of 3×FLAG-cGAS after streptavidin pulldown of cell lysates from 3×FLAG-cGAS RAW 264.7 cells transfected with interferon-stimulatory DNA (ISD) or biotinylated-ISD. (B) Western blot of inputs and FLAG IPs from WT or ΔESX-1 M. tuberculosis infected RAW 264.7 cells stably expressing 3×FLAG-GFP or 3×FLAG-cGAS (left panel), or Strep-cGAS as a negative control (right panel). Whole cell lysates (Input) or IPs (Flag-IP) were visualized by Western blot using anti-FLAG (left) or anti-Strep (right) antibodies. (C) qPCR of M. tuberculosis-derived sequences from DNA isolated from IPs in (B). Quantities were normalized to inputs. (D) Same as (C) but mouse Actin. n.s., not significant, **p < 0.005 by two-tailed t-test. See also Figure S4.