Miriam Kutsch

Several human pathogens exploit the kinetic forces generated by polymerizing actin to power their intracellular motility. Human cell-autonomous immune responses activated by the cytokine interferon-gamma (IFNγ) interfere with such microbial actin-based motility, yet the underlying molecular mechanisms are poorly defined. Here, we identify the IFNγ-inducible human giant GTPases GVIN1 as a novel host defense protein that blocks the bacterial pathogen Burkholderia thailandensis from high-jacking the host’s actin polymerization machinery. We found that GVIN1 proteins form a coatomer around cytosolic bacteria and prevent Burkholderia from establishing force-generating actin comet tails. Coatomers formed by a second IFNγ-inducible GTPase, human guanylate binding protein 1 (GBP1), constitute a GVIN1-independent but mechanistically related anti-motility pathway. We show that coating with either GVIN1 or GBP1 displaces the Burkholderia outer membrane protein BimA, an actin nucleator that is essential for actin tail formation. Both GVIN1 and GBP1 coatomers require additional IFNγ-inducible co-factors to disrupt the membrane localization of BimA, demonstrating the existence of two parallel-acting IFNγ-inducible defense modules that evolved to target a virulence trait critical for the pathogenesis of numerous bacterial infectious agents.

Guanylate-binding proteins (GBPs) are interferon-inducible large GTPases that play a central role in cell-autonomous immunity against intracellular bacterial pathogens. A defining feature of GBPs is their ability to translate GTP binding and hydrolysis into large-scale conformational rearrangements that drive self-assembly into higher-order structures, including dimers, polymers, and membrane-associated coatomers. This review integrates insights from biochemical, biophysical, structural, and cell biology studies to summarize current mechanistic models of GBP self-assembly, with a particular focus on human GBP1. We highlight how GTP hydrolysis-driven GBP1 polymers and coatomers act as self-regulating nanomachineries that recognize and remodel the pathogen-associated molecular pattern lipopolysaccharide, thereby fulfilling a dual function as immune sensor and effector in non-canonical inflammasome activation and bacterial membrane disruption. By directly linking nucleotide binding kinetics, enzymatic activity, and assembly dynamics to cellular and infection-related phenotypes, this review places decades of biochemical and biophysical work on GBP1 into a clear physiological context of antibacterial host defense.

The dynamin-related human guanylate-binding protein 1 (GBP1) mediates host defenses against microbial pathogens. Upon GTP binding and hydrolysis, auto-inhibited GBP1 monomers dimerize and assemble into soluble and membrane-bound oligomers, which are crucial for innate immune responses. How higher-order GBP1 oligomers are built from dimers, and how assembly is coordinated with nucleotide-dependent conformational changes, has remained elusive. Here, we present cryo-electron microscopy-based structural data of soluble and membrane-bound GBP1 oligomers, which show that GBP1 assembles in an outstretched dimeric conformation. We identify a surface-exposed helix in the large GTPase domain that contributes to the oligomerization interface, and we probe its nucleotide- and dimerization-dependent movements that facilitate the formation of an antimicrobial protein coat on a gram-negative bacterial pathogen. Our results reveal a sophisticated activation mechanism for GBP1, in which nucleotide-dependent structural changes coordinate dimerization, oligomerization, and membrane binding to allow encapsulation of pathogens within an antimicrobial protein coat.

The gamma-interferon (IFNγ)-inducible guanylate-binding proteins (GBPs) promote host defense against gram-negative cytosolic bacteria in part through the induction of an inflammatory cell death pathway called pyroptosis. To activate pyroptosis, GBPs facilitate sensing of the gram-negative bacterial outer membrane component lipopolysaccharide (LPS) by the noncanonical caspase-4 inflammasome. There are seven human GBP paralogs, and it is unclear how each GBP contributes to LPS sensing and pyroptosis induction. GBP1 forms a multimeric microcapsule on the surface of cytosolic bacteria through direct interactions with LPS. The GBP1 microcapsule recruits caspase-4 to bacteria, a process deemed essential for caspase-4 activation. In contrast to GBP1, closely related paralog GBP2 is unable to bind bacteria on its own but requires GBP1 for direct bacterial binding. Unexpectedly, we find that GBP2 overexpression can restore gram-negative-induced pyroptosis in GBP1 ^KO cells, without GBP2 binding to the bacterial surface. A mutant of GBP1 that lacks the triple arginine motif required for microcapsule formation also rescues pyroptosis in GBP1 ^KO cells, showing that binding to bacteria is dispensable for GBPs to promote pyroptosis. Instead, we find that GBP2, like GBP1, directly binds and aggregates “free” LPS through protein polymerization. We demonstrate that supplementation of either recombinant polymerized GBP1 or GBP2 to an in vitro reaction is sufficient to enhance LPS-induced caspase-4 activation. This provides a revised mechanistic framework for noncanonical inflammasome activation where GBP1 or GBP2 assembles cytosol-contaminating LPS into a protein-LPS interface for caspase-4 activation as part of a coordinated host response to gram-negative bacterial infections.

Sensing and killing of intracellular bacterial pathogens are important features of cell-autonomous immunity. The cytokine gamma-interferon (IFNγ) enhances cell-autonomous immunity through upregulation of interferon stimulated genes such as guanylate binding proteins (GBPs). GBPs promote defense against Gram-negative cytosolic bacteria in part through the induction of an inflammatory cell death pathway called pyroptosis. To activate pyroptosis, GBPs facilitate caspase-4 sensing of the Gram-negative bacterial outer membrane component lipopolysaccharide (LPS). There are seven human GBP paralogs and it is unclear how each GBP contributes to LPS sensing and pyroptosis induction. GBP1 forms a multimeric microcapsule on the surface of cytosolic bacteria through direct interactions with LPS and recruits caspase-4 to bacteria, a process deemed essential for caspase-4 activation. In contrast to GBP1, closely related paralog GBP2 is unable to bind bacteria on its own but requires GBP1 for direct bacterial binding. Unexpectedly, we find that GBP2 overexpression can restore Gram-negative-induced pyroptosis in GBP1 ^KO cells, without GBP2 binding to the bacterial surface. A mutant of GBP1 that lacks the triple arginine motif required for microcapsule formation also rescues pyroptosis in GBP1 ^KO cells, showing that binding to bacteria is dispensable for GBPs to promote pyroptosis. Instead, we find that GBP2, like GBP1, directly binds and aggregates ‘free’ LPS through protein polymerization. This provides a novel mechanistic framework for non-canonical inflammasome activation where GBP1 or GBP2 assemble cytosol-contaminating LPS into a protein-LPS interface for caspase-4 activation as part of a coordinated host response to Gram-negative bacterial infections. Significance Statement: Sensing Gram-negative bacterial lipopolysaccharide by human caspase-4 is critical for host defense to intracellular Gram-negative bacterial pathogens. Human guanylate binding proteins (GBPs) facilitate caspase-4 activation in response to Gram-negative infections by a poorly understood mechanism. The prevailing model suggests GBP1 binding to bacteria and consequential recruitment of caspase-4 to the bacterial surface are essential for triggering this host response. Here, we show GBP1 binding to bacteria is dispensable for caspase-4 activation and identify GBP2 as an additional lipopolysaccharide-binding protein that can functionally replace GBP1. We demonstrate that GBP1 and GBP2 share the ability to form lipopolysaccharide-protein complexes, which, we propose, allow caspase-4 activation. Our study provides a new mechanistic framework for cytosolic LPS sensing.

Many cytosolic bacterial pathogens hijack the host actin polymerization machinery to form actin tails that promote direct cell-to-cell spread, enabling these pathogens to avoid extracellular immune defenses. However, these pathogens are still susceptible to intracellular cell-autonomous immune responses that restrict bacterial actin-based motility. Two classes of cytosolic antimotility factors, septins and guanylate-binding proteins (GBPs), have recently been established to block actin tail formation by the human-adapted bacterial pathogen Shigella flexneri. Both septin cages and GBP1 microcapsules restrict S. flexneri cell-to-cell spread by blocking S. flexneri actin-based motility. While septins assemble into cage-like structures around immobile S. flexneri, GBP1 forms microcapsules around both motile and immobile bacteria. The interplay between these two defense programs remains elusive. Here, we demonstrate that GBP1 microcapsules block septin cage assembly, likely by interfering with the function of S. flexneri IcsA, the outer membrane protein that promotes actin-based motility, as this protein is required for septin cage formation. However, S. flexneri that escape from GBP1 microcapsules via the activity of IpaH9.8, a type III secreted effector that promotes the degradation of GBPs, are often captured within septin cages. Thus, our studies reveal how septin cages and GBP1 microcapsules represent complementary host cell antimotility strategies.

Disease‐causing microorganisms not only breach anatomical barriers and invade tissues but also frequently enter host cells, nutrient‐enriched environments amenable to support parasitic microbial growth. Protection from many infectious diseases is therefore reliant on the ability of individual host cells to combat intracellular infections through the execution of cell‐autonomous defense programs. Central players in human cell‐autonomous immunity are members of the family of dynamin‐related guanylate binding proteins (GBPs). The importance of these interferon‐inducible GTPases in host defense to viral, bacterial, and protozoan pathogens has been established for some time; only recently, cell biological and biochemical studies that largely focused on the prenylated paralogs GBP1, GBP2, and GBP5 have provided us with robust molecular frameworks for GBP‐mediated immunity. Specifically, the recent characterization of GBP1 as a bona fide pattern recognition receptor for bacterial lipopolysaccharide (LPS) disrupting the integrity of bacterial outer membranes through LPS aggregation, the discovery of a link between hydrolysis‐induced GMP production by GBP1 and inflammasome activation, and the classification of GBP2 and GBP5 as inhibitors of viral envelope glycoprotein processing via suppression of the host endoprotease furin have paved the way for a vastly improved conceptual understanding of the molecular mechanisms by which GBP nanomachines execute cell‐autonomous immunity. The herein discussed models incorporate our current knowledge of the antimicrobial, proinflammatory, and biochemical properties of human GBPs and thereby provide testable hypotheses that will guide future studies into the intricacies of GBP‐controlled host defense and their role in human disease.

Human guanylate‐binding protein 1 (hGBP1) is a key player in innate immunity and fights diverse intracellular microbial pathogens. Its antimicrobial functions depend on hGBP1’s GTP binding‐ and hydrolysis‐induced abilities to form large, structured polymers and to attach to lipid membranes. Crucial for both of these biochemical features is the nucleotide‐controlled release of the C terminally located farnesyl moiety. Here, we address molecular details of the hGBP1 membrane binding mechanism by employing recombinant, fluorescently labeled hGBP1, and artificial membranes. We demonstrate the importance of the GTPase activity and the resulting structural rearrangement of the hGBP1 molecule, which we term the open state. This open state is supported and stabilized by homodimer contacts involving the middle domain of the protein and is further stabilized by binding to the lipid bilayer surface. We show that on the surface of the lipid bilayer a hGBP1 monolayer is built in a pins in a pincushion‐like arrangement with the farnesyl tail integrated in the membrane and the N‐terminal GTPase domain facing outwards. We suggest that similar intramolecular contacts between neighboring hGBP1 molecules are responsible for both polymer formation and monolayer formation on lipid membranes. Finally, we show that tethering of large unilamellar vesicles occurs after the vesicle surface is fully covered by the monolayer. Both hGBP1 polymer formation and hGBP1‐induced vesicle tethering have implications for understanding the molecular mechanism of combating bacterial pathogens.Databases: Structural data are available in RCSB Protein Data Bank under the accession numbers: 6K1Z, 2D4H.

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Many infectious diseases are caused by microbes that enter and survive within host cells. Guanylate binding proteins (GBPs) are a group of immune proteins which recognize and inhibit a variety of intracellular pathogenic microbes. We discovered that a short sequence within GBPs required for the detection of bacteria, the polybasic motif (PBM), has been rapidly evolving between primate species. By swapping PBMs between primate GBP1 genes, we were able to show that specific sequences can both reduce and improve the ability of GBP1 to target intracellular bacteria. We also show that the ability to envelop bacteria has independently evolved in GBP2 of South American monkeys. Taking the results together, this report illustrates how primate GBPs have adapted to defend against infectious pathogens.