Human GBP1 differentially targetsSalmonellaandToxoplasmato license recognition of microbial ligands and caspase-mediated death

Abstract

Human guanylate binding proteins (GBPs), a family of IFNγ-inducible GTPases, promote cell-intrinsic defence against pathogens and host cell death. We previously identified GBP1 as a mediator of cell death of human macrophages infected with Toxoplasma gondii (Tg) or Salmonella Typhimurium (STm). How GBP1 targets microbes for AIM2 activation during Tg infection and caspase-4 during STm infection remains unclear. Here, using correlative light and electron microscopy and EdU labelling of Tg-DNA, we reveal that GBP1-decorated parasitophorous vacuoles (PVs) lose membrane integrity and release Tg-DNA for detection by AIM2-ASC-CASP8. In contrast, differential staining of cytosolic and vacuolar STm revealed that GBP1 does not contribute to STm escape into the cytosol but decorates almost all cytosolic STm leading to the recruitment of caspase-4. Caspase-5, which can bind LPS and whose expression is upregulated by IFNγ, does not target STm pointing to a key role for caspase-4 in pyroptosis. We also uncover a regulatory mechanism involving the inactivation of GBP1 by its cleavage at Asp192 by caspase-1. Cells expressing non-cleavable GBP1 D192E therefore undergo higher caspase-4-driven pyroptosis during STm infection. Taken together, our comparative studies elucidate microbe-specific spatiotemporal roles of GBP1 in inducing cell death by leading to assembly and regulation of divergent caspase signalling platforms.

The zebrafish as a novel model for the in vivo study of Toxoplasma gondii replication and interaction with macrophages

“…6 ; ). Consistent with this, and reports of vacuole breakage in human macrophages ( Fisch et al, 2020 preprint), FIB SEM also captured incomplete vacuole membranes around type I tachyzoites within a macrophage ( Fig. 7 ; ).…”

“…Using ssTEM and FIB SEM, we observed incomplete PV membrane in zebrafish brain cells and macrophages. PV breakage has been observed in vitro after IFN-γ stimulation of murine cells and human macrophages, and is thought to be part of the cell-intrinsic host defense mechanism against Toxoplasma tachyzoites ( Martens et al, 2005 ; Zhao et al, 2008 ; Yamamoto et al, 2012 ; Selleck et al, 2015 ; Fisch et al, 2020 preprint). Zebrafish possess an ortholog of IFN-γ together with a related gene (IFN-γ-rel), and studies have suggested that zebrafish IFN-γ (also known as Ifng1) expression is associated with host protection against bacterial infection, while IFN-γ-rel expression is associated with host protection against viral challenge ( Igawa et al, 2006 ; López-Muñoz et al, 2009 , 2011 ; Sieger et al, 2009 ; Yoon et al, 2016 ).…”

Human guanylate binding proteins: nanomachines orchestrating host defense

“…Similarly, antibodies specific for the inner core/ lipid A portion of LPS are able to bind to GBP1-coated but not uncoated bacteria in vitro, albeit not very efficiently[31]. These in vitro findings thus provide a molecular explanation for observations made by numerous research groups reporting the GBP1-dependent recruitment of the cytosolic lipid A sensor caspase-4 to the surface of the gram-negative bacteria S. flexneri and Salmonella enterica Thyphimurium in the host cell cytosol[31,32,63,83,84].Caspase-4 binds directly to lipid A via its caspase activation and recruitment domain (CARD)[85][86][87]. LPS or lipid A aggregates promote caspase-4 oligomerization via homotypic CARD interaction and lead to the induction of pyroptotic cell death through the proteolytic cleavage and activation of the pore-forming protein gasdermin D (GSDMD)…”

“…GBP-dependent microbial lysis results in the release of pathogen-associated molecular patterns (PAMPs) including microbial DNA, which is sensed by the pattern recognition receptor absent in melanoma 2 (AIM2) in response to F. novicida or Toxoplasma gondii infections ( Figure 5A). Accordingly, AIM2-dependent induction of host cell death by F. novicida or the protozoan Toxoplasma is alleviated inside GBP-deficient cells [12,14,83,84,97].…”

“…Toxoplasma PCVs bound by GBPs undergo vacuolar rupture in a GBP-dependent manner in both mouse and human cells [7,83,84] ( Figure 5A). The importance of GBPs in the disruption of bacterial PCVs is less clear.…”