Native architecture of a human GBP1 defense complex for cell-autonomous immunity to infection

• 成果类型: Article
• 署名作者: Zhu, Shiwei; Bradfield, Clinton J.; Maminska, Agnieszka; Park, Eui-Soon; Kim, Bae-Hoon; Kumar, Pradeep; Huang, Shuai; Kim, Minjeong; Zhang, Yongdeng; Bewersdorf, Joerg; MacMicking, John D.
• 署名单位: Howard Hughes Medical Institute; Yale University; Yale University; Yale University; Yale University; Yale University
• 刊物名称: SCIENCE
• ISSN/ISSBN: 0036-13081
• DOI: 10.1126/science.abm9903
• 发表日期: 2024-03-01
• 关键词: guanylate-binding proteins; higher-order assemblies; innate immune; membrane; protects; gtpases; gtp

摘要

INTRODUCTION The compartmentalized landscape of eukaryotic cells offers a wide variety of intracellular niches for microbial pathogens to hide and replicate. Consequently, eukaryotes have evolved compartment-specific immune surveillance mechanisms that alert the host to infection and recruit antimicrobial proteins that help bring microbial replication under control. Many of these host defense proteins form giant macromolecular complexes when encountering either pathogens or their products to amplify innate immune signaling and spatially localize protein partners at the site of microbial recognition. In some cases, complete signaling cascades are built directly upon the invading pathogen itself, a distinctive situation in which a large foreign object acts as the anchoring platform for assembling the entire host defense machinery. How these massive host-pathogen platforms are initiated and structurally organized at the molecular level remains unknown. RATIONALE Since their discovery in the physical assembly of antimicrobial and innate immune signaling complexes over a decade ago, guanylate-binding proteins (GBPs) have emerged as major organizers of intracellular host defense to a broad array of bacteria, viruses, or parasites in animals and plants. In mammals, these large dynamin-like guanosine triphosphatases (GTPases) relocate to intracellular pathogens, where they can establish macromolecular assemblies on the microbial outer membrane (OM) that serve as interactive hubs for inflammatory proteins or bactericidal effectors as part of the cell-autonomous innate immune response. To better understand the mechanistic details underlying these distinct hybrid structures, we enlisted host and bacterial genetics plus single-particle nanoscopy and cryo-electron tomography (cryo-ET) to visualize GBP defense complexes on the surface of a gram-negative bacterial pathogen, Salmonella enterica serovar Typhimurium (Stm), within the cytosol of human cells. RESULTS We identified a multiprotein defense complex assembled directly on Stm inside human cells. This defense complex comprised four members of the human GBP family (GBP1, GBP2, GBP3, and GBP4) together with human caspase-4 and one of its natural substrates, full-length Gasdermin D (GSDMD). It triggered innate immune signaling through caspase-4 cleavage of GSDMD into its pore-forming subunits, resulting in the extracellular release of an immune cytokine, interleukin-18 (IL-18), and pyroptotic cell death needed for protection against this bacterial pathogen. Notably, human GBP1 was obligate for initiating the entire signaling cascade; its genetic removal prevented the remaining immune proteins being recruited onto the gram-negative bacterial surface. C-terminal anchorage and GTPase-driven self-assembly enabled GBP1 to bind to and polymerize over the surface of cytosolic Stm to establish the recruitment platform. Nearly 30,000 GBP1 molecules were assembled in just a few minutes. Reconstitution of this massive GBP1 defense complex with a bacterial minicell system allowed cryo-ET visualization of the entire coat structure in its native state. Within this native platform, individual GBP1 dimers were found to adopt an open conformation for vertical insertion into the bacterial OM through their extended C-terminal lipidated tail. Anchorage of the upright GBP1 conformer led to OM disruption, which released the gram-negative cell wall component, lipopolysaccharide (LPS), to activate coassembled caspase-4. CONCLUSION An emerging paradigm for innate immune signaling cascades is the higher-order assembly of repetitive protein units that generate large polymers capable of amplifying signal transduction. Our results identify human GBP1 as the principal repetitive unit, numbering thousands of proteins per bacillus, that undergoes dramatic conformational opening to establish a host defense platform directly on the surface of gram-negative bacteria. This platform enabled the recruitment of other immune partners, including GBP family members and components of the inflammasome pathway, that initiate protective responses downstream of activating cytokines such as interferon-gamma. Elucidating this giant molecular structure not only expands our understanding of how human cells recognize and combat infection but may also have implication for antibacterial approaches within the human population. Architecture of a human GBP1 defense complex. (Left) 3D reconstruction of human GBP1 on the outer membrane (OM) of Salmonella bacteria from cryo-ET. IM, inner membrane. Size of scale bar shown in Angstroms. (Right) Pseudomodel showing the extended upright GBP1 conformer inserting into the OM LPS layer. Release of LPS by GBP1 insertion subsequently activated caspase-4 following its coassembly on the same bacterial surface.