The AIM2 inflammasome detects double-stranded DNA in the cytosol and induces caspase-1-dependent 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.

Significance A major component of the cell envelope of Gram-negative bacteria is LPS, also known as endotoxin. LPS produced during bacterial infections triggers inflammation, which can lead to septic shock and death. Our immune system can recognize LPS both outside and inside of cells. The recognition of extracellular and vacuolar LPS by LPS binding proteins is well described, but little is known about the recognition of cytoplasmic LPS. Here, we show that cytoplasmic LPS derived from the intracellular bacterial pathogen Legionella activated a proinflammatory immune response. We further identified host guanylate binding proteins as critical mediators of immunity triggered by cytoplasmic LPS. These findings are likely to advance our understanding of how cells can sense intracellular LPS.

IFN-␥ orchestrates a potent antimicrobial host response. However, the underlying molecular basis for this immunological defense system is largely unknown. In a systematic approach to identify IFN-␥-regulated host effector molecules, a notable number of transcripts with consensus GTP-binding motives were obtained. Further extensive transcriptome and genome analyses identified five novel family members of murine guanylate-binding proteins (mGBPs) now designated mGBP6, 7, 8, 9, and 10. Moreover, in this study, all 10 mGBP members (mGBP1-10) were extensively characterized. mGBPs are selectively up-regulated in vitro by a set of proinflammatory cytokines and TLR agonists as well as in vivo after Listeria monocytogenes and Toxoplasma gondii infection. After IFN-␥ stimulation, mGBP1, 2, 3, 6, 7, and 9 are associated with intracellular Toxoplasma parasites and, interestingly, virulent Toxoplasma interfere with mGBP recruitment. Taken together, mGBPs comprise an important set of host defense molecules.

IFN-γ orchestrates the host response against intracellular pathogens. Members of the guanylate binding proteins (GBP) comprise the most abundant IFN-γ-induced transcriptional response. mGBPs are GTPases that are specifically up-regulated by IFN-γ, other proinflammatory cytokines, toll-like receptor agonists, as well as in response to Listeria monocytogenes and Toxoplasma gondii infection. mGBP2 localizes at the parasitophorous vacuole (PV) of T. gondii; however, the molecular function of mGBP2 and its domains in T. gondii infection is not known. Here, we show that mGBP2 is highly expressed in several cell types, including T and B cells after stimulation. We provide evidence that the Cterminal domain is sufficient and essential for recruitment to the T. gondii PV. Functionally, mGBP2 reduces T. gondii proliferation because mGBP2-deficient cells display defects in the replication control of T. gondii. Ultimately, mGBP2-deficient mice reveal a marked immune susceptibility to T. gondii. Taken together, mGBP2 is an essential immune effector molecule mediating antiparasitic resistance.pathogen defense | cell-autonomous immunity | host-pathogen interaction

Pathogenic and commensal Gram-negative bacteria produce and release outer membrane vesicles (OMVs), which present several surface antigens and play an important role for bacterial pathogenesis. OMVs also modulate the host immune system, which makes them attractive as vaccine candidates. At the cellular level, OMVs are internalized by macrophages and deliver lipopolysaccharide (LPS) into the host cytosol, thus activating the caspase-11 non-canonical inflammasome. Here, we show that OMV-induced inflammasome activation requires TLR4-TRIF signaling, the production of type I interferons, and the action of guanylate-binding proteins (GBPs), both in macrophages and Mechanistically, we find that isoprenylated GBPs associate with the surface of OMVs or with transfected LPS, indicating that the key factor that determines GBP recruitment to the Gram-negative bacterial outer membranes is LPS itself. Our findings provide new insights into the mechanism by which GBPs target foreign surfaces and reveal a novel function for GBPs in controlling the intracellular detection of LPS derived from extracellular bacteria in the form of OMVs, thus extending their function as a hub between cell-autonomous immunity and innate immunity.