Guanylate-binding proteins promote AIM2 inflammasome activation during Francisella novicida infection by inducing cytosolic bacteriolysis and DNA release

The AIM2 inflammasome detects double-stranded DNA in the cytosol and induces caspase-1dependent pyroptosis as well as release of the inflammatory cytokines IL-1β and IL-18. AIM2 is critical for host defense against DNA viruses and bacteria that replicate in the cytosol, such as Francisella novicida. AIM2 activation by F. novicida requires bacteriolysis, yet whether this process is accidental or a host-driven immune mechanism remained unclear. Using siRNA screening for nearly 500 interferon-stimulated genes, we identified guanylate-binding proteins GBP2 and GBP5 as key AIM2 activators during F. novicida infection. Their prominent role was validated in vitro and in a mouse model of tularemia. Mechanistically, these two GBPs target cytosolic F. novicida and promote bacteriolysis. Thus, besides their role in host defense against vacuolar pathogens, GBPs also facilitate the presentation of ligands by directly attacking cytosolic bacteria. The innate immune system detects invading pathogens through membrane-bound and cytosolic pattern recognition receptors (PRRs), which recognize microbialand damageassociated molecular patterns (MAMPs and DAMPs) and induce conserved signaling Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms Co-corresponding authors: Petr Broz, Ph.D. Assistant Professor Focal Area Infection Biology Biozentrum, University of Basel Klingelbergstrasse 50/70 CH-4056 Basel / Switzerland [email protected] phone: +41 61 267 23 42 fax: +41 61 267 21 18 or [email protected] (TH) . AUTHOR CONTRIBUTIONS E.M, P.W., T.H. and P.B. conceived of the research. T.H. and P.B. wrote the manuscript; E.M., P.W., R.F.D., L.A., S.C., S.R., M.S.D., S.D., A.K., M.R., T.H. and P.B. performed experiments; D.D., K.P. and M.Y. provided reagents. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Europe PMC Funders Group Author Manuscript Nat Immunol. Author manuscript; available in PMC 2015 November 01. Published in final edited form as: Nat Immunol. 2015 May ; 16(5): 476–484. doi:10.1038/ni.3119. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts pathways. Nucleic acids and their derivatives are detected by RIG-I-like receptors, cGAS, DAI and RNA polymerases, resulting in type-I-interferon (type-I-IFN) induction via STING and TBK11-3. Cytosolic microbial and host DNA also induces inflammasome formation through the PYHIN family member AIM2 (absent in melanoma 2)4-7. AIM2 binds doublestranded DNA through its HIN-200 domain8 and recruits the inflammasome adaptor protein ASC. ASC rapidly oligomerizes to form a macromolecular inflammasome complex known as an ASC speck that activates caspase-1. Active caspase-1 promotes the maturation and release of pro-inflammatory cytokines interleukin (IL)-1β and IL-18. In addition it induces pyroptosis, a lytic form of cell death that restricts pathogen replication. The AIM2 inflammasome mediates recognition of DNA viruses as well as a number of Gram-negative and Gram-positive cytosolic bacteria like Listeria monocytogenes, Legionella pneumophila, Mycobacterium spp. and Francisella tularensis subspecies novicida (hereafter referred to as F. novicida)8-14. Importantly, several studies have shown that AIM2 activation by these bacteria requires bacteriolysis and the subsequent release of bacterial chromosomal DNA into the cytosol10, 12, 15. Yet, whether bacteriolysis is accidental or an active, host-directed mechanism is unclear. AIM2 activation by transfection of synthetic DNA or during DNA virus infection is independent of Toll-like receptor (TLR)or interferon-signaling9, 13, 16. In stark contrast, AIM2 activation during F. novicida infection requires the production of type-I-IFNs, which are induced as a result of the recognition of a yet undefined F. novicida-derived nucleic ligand in the cytosol 9, 10, 17, 18, 19, 20. Consistent with this AIM2 inflammasome activation in F. novicida-infected cells requires signaling through STING and the transcription factor IRF39, 10, 17. It was speculated that IFN-signaling is necessary to increase cellular AIM2 in order to detect F. novicida DNA9, yet IFN-mediated AIM2 induction is contested and even low amounts of transfected DNA efficiently trigger AIM2 activation in an IFN-independent manner9. Therefore it is likely that one or several IFN-inducible factors are required for efficient activation of AIM2 during bacterial infections. Type-Iand Type-II-IFNs are potent cytokines that exert anti-microbial effects through the induction of a broad transcriptional program involving ~2000 genes, so-called IFNstimulated genes (ISGs), many of which remain uncharacterized. Prominent among these ISGs are several families of interferon-inducible GTPases, such as the 47-kD immunityrelated GTPases (IRGs) and the 65to 73-kD guanylate-binding proteins (GBPs)21, 22. GBPs are conserved among vertebrates, with 11 GBPs in mice and 7 in humans, and exhibit anti-microbial effects against intracellular bacteria and protozoa23. GBP1 and GBP7 restrict Mycobacterium bovis BCG and Listeria monocytogenes by recruiting antimicrobial effectors to the pathogen-containing vacuole (PCV)24. Several GBPs are recruited onto the Toxoplasma parasitophorous vacuole25 and most are also required for restricting T. gondii replication23, 26-28. In addition, GBPs on murine chromosome 3 promote innate immune recognition of the vacuolar, Gram-negative bacterium Salmonella typhimurium by destabilizing its PCV, leading to the egress of bacteria into the cytosol and subsequent detection of lipopolysaccharide (LPS) by the caspase-11 inflammasome29. In this study we found that GBPs on murine chromosome 3 were a key factor for AIM2 activation during F. novicida infection. In particular GBP2 and GBP5 controlled AIM2 activation by targeting Meunier et al. Page 2 Nat Immunol. Author manuscript; available in PMC 2015 November 01. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts cytosolic F. novicida and inducing their lysis by a yet uncharacterized mechanism. We demonstrate that GBP-deficient mice are unable to control F. novicida infection in vivo. Altogether our data reveal a function for GBPs during microbial infections, in that GBPs promote bacteriolysis in the cytosol and the exposure of bacterial DNA to cytosolic innate immune sensors.