Detection of CytosolicShigella flexnerivia a C-Terminal Triple-Arginine Motif of GBP1 Inhibits Actin-Based Motility

Abstract

Dynamin-like guanylate binding proteins (GBPs) are gamma interferon (IFN-γ)-inducible host defense proteins that can associate with cytosol-invading bacterial pathogens. Mouse GBPs promote the lytic destruction of targeted bacteria in the host cell cytosol, but the antimicrobial function of human GBPs and the mechanism by which these proteins associate with cytosolic bacteria are poorly understood. Here, we demonstrate that human GBP1 is unique among the seven human GBP paralogs in its ability to associate with at least two cytosolic Gram-negative bacteria, Burkholderia thailandensis and Shigella flexneri. Rough lipopolysaccharide (LPS) mutants of S. flexneri colocalize with GBP1 less frequently than wild-type S. flexneri does, suggesting that host recognition of O antigen promotes GBP1 targeting to Gram-negative bacteria. The targeting of GBP1 to cytosolic bacteria, via a unique triple-arginine motif present in its C terminus, promotes the corecruitment of four additional GBP paralogs (GBP2, GBP3, GBP4, and GBP6). GBP1-decorated Shigella organisms replicate but fail to form actin tails, leading to their intracellular aggregation. Consequentially, the wild type but not the triple-arginine GBP1 mutant restricts S. flexneri cell-to-cell spread. Furthermore, human-adapted S. flexneri, through the action of one its secreted effectors, IpaH9.8, is more resistant to GBP1 targeting than the non-human-adapted bacillus B. thailandensis. These studies reveal that human GBP1 uniquely functions as an intracellular “glue trap,” inhibiting the cytosolic movement of normally actin-propelled Gram-negative bacteria. In response to this powerful human defense program, S. flexneri has evolved an effective counterdefense to restrict GBP1 recruitment.

Components of the endocytic and recycling trafficking pathways interfere with the integrity of the Legionella ‐containing vacuole

“…In the absence of SdhA, the LCV becomes disrupted and L. pneumophila is detected in the cytoplasm of the infected macrophage (Creasey & Isberg, 2012;Laguna et al, 2006). Bacteria exposed in this fashion to the cytosol are susceptible to recruitment of the interferon-regulated guanylate-binding proteins (GBP) family of anti-microbial proteins, resulting in leakage of lipopolysaccharide (LPS) and activation of caspase-11 (Creasey & Isberg, 2012;Liu et al, 2018;Piro et al, 2017). As a consequence, gasdermin D is activated and pyroptotic cell death ensues (Aachoui et al, 2013;Pilla et al, 2014;Shi et al, 2015).…”

The species-spanning family of LPX-motif harbouring effector proteins

“…Furthermore, it was found that IpaH9.8 synthesises a ubiquitin-coat on S. flexneri, which in contrast to the host-generated ubiquitin coat does not have antimicrobial effects on the cytosolic bacteria but rather supports the pathogen, possibly through antagonising GBP recruitment (Wandel et al, 2017). All in all, these studies point out that targeting of GBPs by IpaH9.8 is a prerequisite for S. flexneri to counteract restriction of replication and suppress GBP-mediated immunity (Li et al, 2017;Piro et al, 2017;Wandel et al, 2017).…”

“…Poly-ubiquitinylation of NEMO leads to its degradation in a proteasome-dependent manner, thereby dampening NF-κB activation (Ashida et al, 2010). Recently, several independent studies found that IpaH9.8 is implicated in IFN-induced antimicrobial defences and counteracts cell-autonomous immunity mediated by the family of GBPs (guanylate-binding proteins; Li et al, 2017;Piro et al, 2017;Wandel et al, 2017). Upon invasion into human IFNγ-activated cells, S. flexneri is initially targeted by human GBP1 (hGBP1), which recognises cytosol-invading Gram-negative bacteria via the LPS O-antigen and then promotes the hierarchical recruitment of additional GBP paralogs.…”

Homo and hetero dimerisation of the human guanylate‐binding proteins hGBP‐1 and hGBP‐5 characterised by affinities and kinetics

“…Also a coiled-coil interface between hGBP-1 and hGBP-5 might be interesting when recalling the recently published study in which a triple arginine motif in the polybasic region (located at the end of the GED) is responsible for the specific targeting of hGBP-1 to S. flexneri [12]. The isoform hGBP-5 was not under the hGBPs guided to S. flexneri by hGBP-1 and some fraction of hGBP-1 seems to reside in the cytoplasm [11][12][13]. One could speculate that hGBP-5 might inhibit hGBP-1 from targeting the bacterium by forming a coiled-coil interface during hetero interaction and thus blocking the triple arginine motif in…”

“…The hGBPs exhibit diverse biological functions which mark them as key players in the innate immune response [6][7][8][9][10]. Most recently, it was demonstrated that hGBP-1 prevents cell-to-cell spread of Shigella flexneri by inhibiting the actin-dependent motility of the gram-negative bacterium [11,12]. Interestingly, the bacterium evolved a mechanism to counteract this host defence [11][12][13].…”