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Lipopolysaccharide from Gram-negative bacteria is sensed in the host cell cytoplasm by a non-canonical inflammasome pathway that ultimately results in caspase-11 activation and cell death1-3. In mouse macrophages, activation of this pathway requires the production of type-I interferons4,5, indicating that interferon-induced genes have a critical role in initiating this pathway. Here we report that a cluster of small interferon-inducible GTPases, the so-called guanylate-binding proteins, is required for the full activity of the non-canonical caspase-11 inflammasome during infections with vacuolar Gram-negative bacteria. We show that guanylate-binding proteins are recruited to intracellular bacterial pathogens and are necessary to induce the lysis of the pathogen-containing vacuole. Lysis of the vacuole releases bacteria into the cytosol, thus allowing the detection of their lipopolysaccharide by a yet unknown lipopolysaccharide sensor. Moreover, recognition of the lysed vacuole by the danger sensor galectin-8 initiates the uptake of bacteria into autophagosomes, which results in a reduction of caspase-11 activation. These results indicate that host-mediated lysis of pathogen-containing vacuoles is an essential immune function and is necessary for efficient recognition of pathogens by inflammasome complexes in the cytosol.
Previous studies have reported that induction of caspase-11-dependent cell death by Gram-negative bacteria requires Trif-dependent production of type-I interferons (type-I-IFNs)4,5 (Extended Data Fig. 1a). Type-I- IFN production is however not required for pro-caspase-11 induction4,6,7 and is dispensable for caspase-11 activation by transfected lipopolysac- charide (LPS; Extended Data Fig. 1b)2. This indicates that interferon- stimulated genes (ISGs) play a major role in activating caspase-11 in response to intracellular bacteria. To investigate which ISGs were involved in activatingcaspase-11, weusedproteomics-based expression analysis to identify proteins that were highly induced following Salmonella infec- tion. Among the most strongly upregulated proteins were interferon- induced GTPases, such as the large 65-67 kDa guanylate-binding proteins (GBPs) and small 47 kDa immunity-related GTPases (IRGs) (data not shown). These proteinsfunctionin cell-autonomousimmunity, that is, mechanismsthatallowhostcellstokillpathogensorrestricttheirreplication, and have even been associated with the activation of inflammasomes8-10.
Mice have 11 GBPs, which are highly homologous and are clustered in two genomic loci on chromosomes3 and 5,respectively8,11. Recently, GBPs on chromosome 3 have been shown to restrict the replication of Toxoplasma gondiiinperitonealmacrophagesand mice11. We therefore infected bone-marrow-derived macrophages (BMDMs) from Gbpchr3 KO mice, which lack GBP1,2, 3,5 and7 (Extended Data Fig. 2a-e), and wild-typelittermateswithanumberofGram-negativevacuolarpathogens that trigger caspase-11 activation...