Periodic Reporting for period 3 - PHAGOSCOPY (PHAGOSCOPY: Dissecting cell-autonomous immunity with ex vivo electron cryo-microscopy)
Période du rapport: 2023-02-01 au 2024-07-31
Many bacterial pathogens acquired the ability to invade and replicate within host cells. Cell-autonomous immunity is a part of the innate immune system that fights off such pathogens. Among the antimicrobial effectors most strongly mobilised by this immune response are the Guanylate-Binding Proteins (GBPs), a family of dynamin-related GTPases that assemble into supramolecular coatomers on the membrane of intracellular pathogen-containing compartments and cytosolic bacteria. These coatomers prime effector functions that lead to pathogen elimination. How nucleotide binding and hydrolysis prime GBPs for membrane targeting and coatomer formation, how they recognise lipopolysaccharides on the bacterial outer membrane and how they mobilise bacterial membrane components for effector activation remains unclear. To address these outstanding questions, we will develop and apply a combination of innovative in situ electron cryo-tomography (cryo-ET), advanced high-resolution fluorescence microscopy and single-molecule biophysics. We will obtain a high-resolution view of dynamic GBP coatomers on pathogen membranes, of non-canonical caspase recruitment to GBP-coated bacteria, and we aim to uncover whether GBP coatomers affect membrane stability and integrity. To achieve this we will combine sophisticated cell-free reconstitution assays with experiments in infected cells. We will systematically examine the structural principles driving the dynamic assembly of the GBP machinery in the complexity of native bacterial membranes, while building up the reaction under conditions that facilitate high-resolution structure determination. While focusing on the biological goals, we will also spearhead technological developments on cryo-ET sample preparation and correlative molecular imaging. This will deliver methodological workflows that will be useful to a broad community of researchers interested in high-resolution analysis of supramolecular biological assemblies.
The project "PHAGOSCOPY" focuses on deciphering the molecular steps and mechanisms that take place when a cell-autonomous immune response involving Guanylate-binding proteins (GBPs) is activated upon cellular invasion by bacterial pathogens. GBPs are dynamin-like GTPases that have recently been recognised as core orchestrators of the cell-autonomous immune response. This function is brought about by the ability of GBPs to coat the membranes of intracellular pathogen replication niches and/or cytosol-invasive gram-negative bacteria, where they are thought to serve as multivalent platforms for the recruitment of downstream effectors that lead to the assembly of non-canonical inflammasomes and inflammatory cell death. GBP1 is the essential unit in the formation of these antimicrobial coatomers and drives the recruitment of other GBP family members and effector molecules. Despite these important roles in maintaining integrity of the host, the molecular mechanisms of these effector functions remain unclear. Our general research goal is to provide a detailed understanding of the molecular processes and mechanisms of GBP activation and assembly on target membranes.
In a major part of the work in this project, we aimed at determining the structural underpinnings of GBP assembly on gram-negative bacterial membranes. We reported the first cryo-EM structure of the full-length GBP1 dimer in its nucleotide-bound state and the molecular ultrastructure of GBP1 coatomers assembled on membranes. Our structure revealed how nucleotide binding induces large-scale conformational changes that activate GBP1 to expose its lipid-modified carboxyl-terminus for association with membranes. Using electron cryotomography along with biochemical and biophysical assays, we demonstrated that the nucleotide-dependent dimer is the critical unit for the assembly of ordered coatomers on liposomes and lipopolysaccharide (LPS)-containing membranes. Our structural data also shed light on the unique evolutionary adaptions that GBPs appear to have acquired to facilitate intercalation into the dense oligosaccharide decoration on the LPS-rich outer membranes of bacterial pathogens. We further demonstrated that GTP hydrolysis drives GBP1-dependent membrane scaffolding and extrusion of tubular protrusions. We showed that this membrane remodelling activity promotes membrane scission or fragmentation, which possibly underlies the ability to recruit and activate caspase4-dependent inflammasomes. Collectively, our results reconciled several conflicting assumptions on the structure and function of GBP assemblies and provide a molecular framework for structure-guided studies into the broad antimicrobial repertoire of GBP-mediated intracellular immunity (Kuhm et al., 2023).
As part of our efforts toward a mechanistic description of GBP activation and membrane targeting, we developed a cryo-EM sample preparation method based on microelectromechanical system (MEMS). In contrast to conventional methods in which protein samples are embedded in a thin free standing liquid film spanning a cryo-EM support gris, in our method the sample is enclosed in nanofluidic channels formed between ultrathin, electron-transparent membranes. We showed that these devices are suitable for high-resolution 3D structure determination of macromolecular complexes from picoliter sample volumes, which is six orders of magnitude improvement over standard cryo-EM sample preparation. We could demonstrate that our device protects the sample from a destructive air-water interface and provides excellent control of ice thickness across individual imaging supports. Most importantly, the unique flexibility of MEMS engineering allows the integration of arbitrary fluidic components directly on the imaging support and we anticipate that it will provide entirely new opportunities for cryo-EM imaging, ranging from highthroughput screening in structure-based drug design to new frontiers in exploring and resolving structural dynamics (Huber et al., 2022).
The analysis of the GBP coatomer cryo-EM data revealed that non-uniform resolution of the coatomer components is a major impediment to density interpretation. To mitigate this effect, we developed novel algorithms to optimally restore local contrast of such 3D reconstructions (Bharadwaj et al., 2022).
In a major part of the work in this project, we aimed at determining the structural underpinnings of GBP assembly on gram-negative bacterial membranes. We reported the first cryo-EM structure of the full-length GBP1 dimer in its nucleotide-bound state and the molecular ultrastructure of GBP1 coatomers assembled on membranes. Our structure revealed how nucleotide binding induces large-scale conformational changes that activate GBP1 to expose its lipid-modified carboxyl-terminus for association with membranes. Using electron cryotomography along with biochemical and biophysical assays, we demonstrated that the nucleotide-dependent dimer is the critical unit for the assembly of ordered coatomers on liposomes and lipopolysaccharide (LPS)-containing membranes. Our structural data also shed light on the unique evolutionary adaptions that GBPs appear to have acquired to facilitate intercalation into the dense oligosaccharide decoration on the LPS-rich outer membranes of bacterial pathogens. We further demonstrated that GTP hydrolysis drives GBP1-dependent membrane scaffolding and extrusion of tubular protrusions. We showed that this membrane remodelling activity promotes membrane scission or fragmentation, which possibly underlies the ability to recruit and activate caspase4-dependent inflammasomes. Collectively, our results reconciled several conflicting assumptions on the structure and function of GBP assemblies and provide a molecular framework for structure-guided studies into the broad antimicrobial repertoire of GBP-mediated intracellular immunity (Kuhm et al., 2023).
As part of our efforts toward a mechanistic description of GBP activation and membrane targeting, we developed a cryo-EM sample preparation method based on microelectromechanical system (MEMS). In contrast to conventional methods in which protein samples are embedded in a thin free standing liquid film spanning a cryo-EM support gris, in our method the sample is enclosed in nanofluidic channels formed between ultrathin, electron-transparent membranes. We showed that these devices are suitable for high-resolution 3D structure determination of macromolecular complexes from picoliter sample volumes, which is six orders of magnitude improvement over standard cryo-EM sample preparation. We could demonstrate that our device protects the sample from a destructive air-water interface and provides excellent control of ice thickness across individual imaging supports. Most importantly, the unique flexibility of MEMS engineering allows the integration of arbitrary fluidic components directly on the imaging support and we anticipate that it will provide entirely new opportunities for cryo-EM imaging, ranging from highthroughput screening in structure-based drug design to new frontiers in exploring and resolving structural dynamics (Huber et al., 2022).
The analysis of the GBP coatomer cryo-EM data revealed that non-uniform resolution of the coatomer components is a major impediment to density interpretation. To mitigate this effect, we developed novel algorithms to optimally restore local contrast of such 3D reconstructions (Bharadwaj et al., 2022).
Our results so far have already provided novel insight into the mechanism of activation and membrane targeting of GBP1. Moreover, using structural and biophysical methods, we have been able to show that GBP1 possesses GTP-dependent membrane remodeling activity that we could show results in fragmentation of membranes. Our results provide important clues on the mechanism by which GBP polymers may recruit non-canonical cascades to sites of bacterial membrane damage to activate the pyroptotic pathway. In ensuing experiments we will address how other GBP family members are recruited to the GBP1 coatomer and how the composition of the GBP heteropolymers influences effector functions. We will also reconstitute the costumers on complex membranes better resembling vacuolar and bacterial membranes in a native environment to understand how membrane composition defines specificity of GBPs.