Guanylate-binding proteins promote activation of the AIM2 inflammasome during infection with Francisella novicida

Abstract

The AIM2 inflammasome detects double-stranded DNA in the cytosol and induces caspase-1-dependent pyroptosis as well as release of the inflammatory cytokines interleukin 1β (IL-1β) and IL-18. AIM2 is critical for host defense against DNA viruses and bacteria that replicate in the cytosol, such as Francisella tularensis subspecies novicida (F. novicida). The activation of AIM2 by F. novicida requires bacteriolysis, yet whether this process is accidental or is a host-driven immunological mechanism has remained unclear. By screening nearly 500 interferon-stimulated genes (ISGs) through the use of small interfering RNA (siRNA), we identified guanylate-binding proteins GBP2 and GBP5 as key activators of AIM2 during infection with F. novicida. We confirmed their prominent role in vitro and in a mouse model of tularemia. Mechanistically, these two GBPs targeted cytosolic F. novicida and promoted bacteriolysis. Thus, in addition to their role in host defense against vacuolar pathogens, GBPs also facilitate the presentation of ligands by directly attacking cytosolic bacteria.

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Figure 1: Products of ISGs are required for AIM2 activation during infection with F. novicida.
Figure 2: Screening by RNA-mediated interference identifies members of the GBP family as activators of AIM2.
Figure 3: Macrophages from Gbpchr3-deficient mice have deficient activation of AIM2 in response to F. novicida.
Figure 4: GBP2 and GBP5 independently control activation of AIM2 during infection with F. novicida.
Figure 5: The escape of F. novicida from phagosomes is GBP independent.
Figure 6: GBPs promote the activation of AIM2 by inducing bacteriolysis.
Figure 7: GBPs restrict the intracellular replication of F. novicida.
Figure 8: GBPs control host defense against F. novicida in vivo.

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Acknowledgements

We thank N. Gekara (Umea University) for StingGt/Gt mice; M. Roth and S. Hofer for support with mouse experiments; D. Monack (Stanford University) for chicken antibody to F. novicida; N. Kayagaki (Genentech) and V. Dixit (Genentech) for Gbp5−/− and Aim2−/− mice, rat antibody to ASC and rat antibody to caspase-1; L. Gallagher and C. Manoil (University of Washington) for plasmid pFFlp; O. Allatif for statistical analysis; the Imaging and FACS Core Facilities of the Biozentrum, University of Basel, for technical assistance; and the Plateau de Biologie Expérimentale de la Souris and the flow cytometry platform of SFR Biosciences Gerland-Lyon Sud. Supported by Swiss National Science Foundation (PP00P3_139120/1 to P.B.), the University of Basel (ID2153162 to P.B.), the European Research Council (311542 to T.H.) and the Déléguation Générale de l'Armement (M.R.).

Author information

E.M., P.W., T.H. and P.B. conceived of the research; E.M., P.W., R.F.D., S.C., L.A., S.R., S.D., M.S.D., A.K., M.R., T.H. and P.B. performed experiments; D.D., K.P. and M.Y. provided reagents; and T.H. and P.B. wrote the manuscript.

Correspondence to Thomas Henry or Petr Broz.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Induction of Ifnb, Aim2 or Gbp mRNA in dependence of STING and IFNAR signaling.

(a) Induction of Ifnb expression from unprimed wild-type (WT), Tlr2–/–, Myd88–/–, Trif–/– and Stinggt/gt bone-marrow derived macrophages (BMDMs) following infection with wild-type F. novicida U112 for 6 h. (be) Induction of Gbp2, Gbp3, Gbp5 or Aim2 expression from unprimed wild-type, Tlr2–/–, Myd88–/–, Ifnar1–/– and Stinggt/gt BMDMs following infection with wild-type F. novicida U112 for 6 h. Graphs show mean and s.d. of quadruplicate assays and data are representative of two (b-d) or three (a) independent experiments.

Supplementary Figure 2 Gbp knockdown efficiency and real-time cell death after knock-down of Gbps.

(a) Induction of the expression of individual Gbps from LPS/IFNγ-primed wild-type BMDMs treated with Non-Targeting (NT) or the indicated gene-specific siRNA for 22 h. Graph shows mean and s.d. of quadruplicate wells. Gbp1, 4, 6/10, 11 were not tested due to their low expression (see Fig. 2b). (b) Cell death as measured by propidium iodide (PI) influx in real-time in unprimed wild-type BMDMs infected with wild-type F. novicida U112. BMDMs were treated with Non-Targeting (NT) or indicated gene-specific siRNA for 48 h before infection. Graphs show mean and s.d. of triplicate assays and data are representative of three
 independent experiments. *, p<0.05; **, p<0.01; NS, not significant (two-tailed unpaired t-test).

Supplementary Figure 3 Aim2 induction and real-time cell death assay in Gbp-deficient and Ifnar1-deficient cells.

(a) Induction of Aim2 expression from unprimed wild-type, Gbpchr3-deleted and Ifnar1–/– BMDMs infected with wild-type F. novicida U112 for 6 h. *, p<0.01; NS, not significant (two-tailed unpaired t-test). (b) Cell death as measured by propidium iodide influx in real-time in unprimed wild-type, Gbpchr3-deleted and Ifnar1–/– BMDMs left uninfected (UI) or infected with wild-type F. novicida U112. Graphs show mean and s.d. of triplicate assays and data are representative of two (a) and three (b) independent experiments.

Supplementary Figure 4 Ectopic expression of GBPs in Ifnar1–/– cells and efficiency of Gbp2 and Gbp5 knockdown.

(ac) Ectopic expression of GBP2 or GBP5 do not complement type-I-IFN receptor deficiency. Ifnar1–/– macrophages were transduced with lentivirus encoding either GFP only (EV = empty vector) and GFP-GBP2 and GFP-GBP5. 48 h post transduction, macrophages were infected with F. novicida at the indicated MOI. IL-1β concentration in the supernatant was determined at 7 h and 10.5 h post infection (a). Specific ectopic expression was verified by quantifying the Gbp3 (control), Gbp2 and Gbp5 transcript levels. Results are expressed as fold induction relative to the transcript level in Ifnar1–/– macrophages transduced with empty vector control (b). Graphs show mean and s.d. of triplicate assays. The percentage of transduced cells was determined by flow cytometry based on GFP expression (c). (d) RT-PCR for Gbp2 and Gbp5 expression from unprimed wild-type, Gbp2–/– and Gbp5–/– BMDMs treated with Non-Targeting or the indicated gene-specific siRNA for 22 h and infected for 8 h with wild-type F. novicida U112. Graphs show mean and s.d. of quadruplicate assays and data are representative of independent two experiments. *, p<0.05; **, p<0.01; NS, not significant (two-tailed unpaired t-test). ND, not detected.

Supplementary Figure 5 Phagosomal rupture assay using the CCF4/β-lactamase system.

Wild-type, Ifnar1–/– and Gbpchr3-deleted BMDMs were primed for 16 h with IFN-β (500 units/ml), infected for 1 h with wild-type F. novicida (FN) U112, a β-lactamase-deficient mutant (Δbla) or a ΔFPI mutant and loaded with CCF4-AM for 1 h before analysis by flow cytometry. Phagosomal rupture is associated with β-lactamase (encoded by FTN_1072) release into the cytosol and cleavage of the CCF4 substrate (maximum emission at 520 nm) into a product which emits with a maximum of fluorescence at 447 nm. FACS plots show pooled data from three independent samples and representative of three independent experiments. Live cells (propidium iodide negative) are shown.

Supplementary Figure 6 GBPs co-localize with irregularly shaped bacteria next to ASC specks.

Wild-type BMDMs infected with wild-type F. novicida for 8 h, stained for DNA (DAPI), GBP2, F. novicida and ASC. Scale bars: 10 μm. Data are representative of three
 independent experiments.

Supplementary Figure 7 Analysis of bacterial replication in infected macrophages.

(a) Quantification of bacterial loads in single cells by high-resolution microscopy in flow over time. Wild-type BMDMs were left uninfected or infected with GFP+-wild-type F. novicida or a ΔFPI mutant at an MOI of 10 for 0-12 h, fixed and analyzed by ImageStreamTM microscopy in flow. Each bar corresponds to the number of cells with the indicated numbers of bacteria per cell grouped by increments of 5... Wild-type UI vs. wild-type MOI 10 3h p>0.9999, wild-type UI vs. wild-type MOI 10 6h p>0.9999, wild-type UI vs. wild-type MOI 10 9h p<0.0001, wild-type UI vs. wild-type MOI 10 12h p<0.0001, wild-type MOI 10 12h vs. ΔFPI MOI 10 12h p<0.0001 Kolmogorov-Smirnov test with Bonferroni correction). (b) Quantification of bacterial loads as determined by microscopy. Wild-type, Aim2–/–, Gbpchr3-deleted and Ifnar1–/– BMDMs were infected with GFP+-wild-type F. novicida at an MOI of 10 for 16 h, fixed and analyzed by confocal microscopy. Graph show pooled data from 2 independent experiments (n>1000 bacteria counted). *, p<0.05; **, p<0.01; ***, p<0.001 (two-tailed unpaired t-test).

Supplementary Figure 8 Bacteriolysis and cell death during F. novicida infection are independent of ROS or NO production.

(a) Quantification of lysed (propidium iodide+ F. novicida) in IFN-γ-primed wild-type and Nos2–/–/Cybb–/–BMDMs infected for 8 h with wild-type F. novicida. Imaging of lysed (propidium iodide+) F. novicida in IFN-γ-primed wild-type and Nos2–/–/Cybb–/–BMDMs infected for 8 h with wild-type F. novicida. Arrowheads indicate region in insets. Scale bars 10 μm. (b) LDH release from naïve or IFN-γ-primed wild-type and Nos2–/–/Cybb–/– BMDMs infected for 8 h with wild-type F. novicida U112. Graphs show mean and s.d. of quadruplicate wells and data are representative of three
 independent experiments. NS, not significant (two-tailed unpaired t-test).

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Supplementary Figures 1–8 and Supplementary Tables 2 and 3 (PDF 1160 kb)

Supplementary Table 1

List of siRNA used for screening (XLS 282 kb)

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Meunier, E., Wallet, P., Dreier, R. et al. Guanylate-binding proteins promote activation of the AIM2 inflammasome during infection with Francisella novicida. Nat Immunol 16, 476–484 (2015). https://doi.org/10.1038/ni.3119

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