Detection of cytosolic Shigella flexneri via a C-terminal triple-arginine motif of GBP1 inhibits actin-based motility

Abstract

Dynamin-like guanylate binding proteins (GBPs) are gamma interferon (IFN{gamma})-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 amongst 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 co-localize with GBP1 less frequently than wildtype S. flexneri, 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 co-recruitment of four additional GBP paralogs (GBP2, GBP3, GBP4 and GBP6). GBP1-decorated Shigella replicate but fail to form actin tails leading to their intracellular aggregation. Consequentially, wildtype 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 counter-defense to restrict GBP1 recruitment.\n\nImportanceSeveral pathogenic bacterial species evolved to invade, reside and replicate inside the cytosol of their host cells. One adaptation common to most cytosolic bacterial pathogens is the ability to co-opt the hosts actin polymerization machinery, in order to generate force for intracellular movement. This actin-based motility enables Gram-negative bacteria such as Shigella to propel themselves into neighboring cells thereby spreading from host cell to host cell without exiting the intracellular environment. Here, we show that the human protein GBP1 acts as a cytosolic glue trap capturing cytosolic Gram-negative bacteria through a unique protein motif and preventing disseminated infections in cell culture models. To escape from this GBP1-mediated host defense, Shigella employs a virulence factor that prevents or dislodges the association of GBP1 with cytosolic bacteria. Thus, therapeutic strategies to restore GBP1 binding to Shigella may lead to novel treatment options for shigellosis in the future.