{"doi":"10.1101/2025.09.25.678451","title":"GBP1 recruitment to actin-rich pedestals induced by extracellular Gram-negative bacteria promotes pyroptosis","abstract":"Abstract The IFNγ-induced GTPase guanylate binding protein 1 (GBP1) binds to lipopolysaccharide (LPS) on cytosolic Gram-negative bacteria and promotes pyroptosis via the recruitment and activation of caspase-4 on the bacterial outer membrane. Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC, respectively) are extracellular pathogens that also induce LPS-and caspase-4-dependent pyroptosis. However, whether GBP1 is involved in this process remains unknown. EPEC and EHEC adhere intimately to intestinal epithelial cells via avid interactions between the bacterial adhesin Intimin and Tir (Translocated intimin receptor), a type 3 secretion system effector protein. Intimin-mediated clustering of Tir triggers actin polymerisation, leading to pedestal-like structures at bacterial attachment sites. Here we show that GBP1 is recruited to actin-rich pedestals in human cells infected with EPEC and EHEC in vitro and mouse colonocytes infected with the EPEC-like murine pathogen Citrobacter rodentium in vivo . GBP1-dependent caspase-4 trafficking to these sites leads to pyroptosis and IL-18 release. GBP1 mutants defective in LPS coatomer formation also localised to EPEC pedestals, pointing to LPS-independent mobilisation of GBP1 by actin polymerisation induced by extracellular A/E pathogens. To further dissect the underlying mechanism, we engineered a chimeric receptor (FcγR-Tir) by combining the intracellular signalling domain of Tir and the extracellular ligand-binding domain of the Fcγ receptor. Clustering of FcγR-Tir with IgG-coated beads produced ‘sterile’ actin-rich pedestals that were sufficient to recruit GBP1 independently of bacteria. Our findings reveal that cytosolic GBP1 is mobilised to sites of pathogen-induced actin remodelling independently of LPS. We establish that GBP1 not only operates as a pattern-recognition receptor but also orchestrates effector-triggered immunity against pathogens that hijack the actin cytoskeleton. 175-word Abstract The IFNγ-induced GTPase guanylate binding protein 1 (GBP1) binds to lipopolysaccharide (LPS) on cytosolic Gram-negative bacteria and promotes pyroptosis via the recruitment and activation of caspase-4 on the bacterial outer membrane. Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC, respectively) are extracellular pathogens that adhere to host cells and stimulate dense actin polymerisation underneath their attachment sites, generating structures called actin-rich ‘pedestals’. Here we show that GBP1 traffics to actin-rich pedestals in human cells infected with EPEC and EHEC in vitro and mouse colonocytes infected with the EPEC-like murine pathogen Citrobacter rodentium in vivo . GBP1 promotes caspase-4 recruitment to actin-rich pedestals, leading to pyroptosis and IL-18 release. GBP1 mutants defective in LPS coatomer formation also localised to EPEC pedestals. Our novel assay that mimics pathogenic effector activity revealed GBP1 recruitment to ‘sterile’ actin polymerisation sites. We conclude that cytosolic GBP1 is mobilised to sites of pathogen-induced actin remodelling independently of LPS. Our study establishes that GBP1 not only operates as a pattern-recognition receptor but also orchestrates effector-triggered immunity against pathogens that hijack the actin cytoskeleton.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":576147,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9573,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1485099,"name":"Ishaan Chaudhary","orcid":null,"position":1,"is_corresponding":false},{"id":1485100,"name":"Dharitri Chaudhuri","orcid":null,"position":2,"is_corresponding":false},{"id":1485101,"name":"Justin Chun Ngai Wong","orcid":null,"position":3,"is_corresponding":false},{"id":1484742,"name":"Priyanka Paul Biswas","orcid":"0000-0003-1932-2352","position":4,"is_corresponding":false},{"id":447363,"name":"Wouter W. Kallemeijn","orcid":"0000-0002-3660-2930","position":6,"is_corresponding":false},{"id":1484743,"name":"Qiyun Zhong","orcid":"0000-0001-7053-9640","position":7,"is_corresponding":false},{"id":1485102,"name":"Marianne Guenot","orcid":null,"position":9,"is_corresponding":false},{"id":469913,"name":"Edward W. Tate","orcid":"0000-0003-2213-5814","position":10,"is_corresponding":false},{"id":1309678,"name":"Sandhya S. Visweswariah","orcid":"0000-0003-2786-7344","position":11,"is_corresponding":false},{"id":459688,"name":"Gad Frankel","orcid":"0000-0002-0046-1363","position":12,"is_corresponding":false},{"id":1484744,"name":"Avinash R. Shenoy","orcid":"0000-0001-6228-9303","position":13,"is_corresponding":false},{"id":1485103,"name":"Tamas Korcsmaros","orcid":null,"position":14,"is_corresponding":false},{"id":1484745,"name":"Arthur M. Talman","orcid":"0000-0003-2584-3672","position":15,"is_corresponding":false},{"id":648046,"name":"Eva‐Maria Frickel","orcid":"0000-0002-9515-3442","position":16,"is_corresponding":false},{"id":1484741,"name":"Daniel J. Bennison","orcid":"0000-0001-9026-9262","position":0,"is_corresponding":true}],"reference_count":84,"raw_metadata":null,"created_at":"2026-07-19T02:57:56.636458Z","pmid":"41040234","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}