{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T17:05:49Z","timestamp":1789751149966,"version":"4.0.1"},"posted":{"date-parts":[[2026,9,15]]},"group-title":"Microbiology","reference-count":68,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T00:00:00Z","timestamp":1789430400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,13]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  <jats:italic>Toxoplasma gondii<\/jats:italic>\n                  replicates inside a host-derived parasitophorous vacuole (PV), and interferon-gamma (IFN\u03b3) induces host restriction factors that target this compartment. Previous CRISPR screens identified the dense granule protein GRA66 and the GRA57\/GRA70\/GRA71 complex as required for parasite fitness in IFN\u03b3-stimulated human cells, but the host pathways they oppose were unknown. Here, we show that GRA66, a PV membrane (PVM)-associated protein predicted to be an N-acylphosphatidylethanolamine (NAPE)-hydrolyzing phospholipase D, is required to prevent premature egress driven by the host phospholipase and acyltransferase RARRES3. Loss of GRA66 caused premature parasite egress, host cell death, and impaired replication in human cells. These phenotypes persisted in cells lacking RNF213, the E3 ubiquitin ligase that dominates IFN\u03b3-dependent\n                  <jats:italic>Toxoplasma<\/jats:italic>\n                  restriction in human cells, and\n                  <jats:italic>\u0394gra66<\/jats:italic>\n                  vacuoles recruited less RNF213 and ubiquitin than wild type, indicating an RNF213-independent mechanism rather than an exaggerated RNF213 response. Complementation with a catalytic-site mutant of the GRA66 zinc-binding motif failed to restore any of these phenotypes, indicating a requirement for its predicted enzymatic activity. Deleting RARRES3 rescued the premature egress of \u0394\n                  <jats:italic>gra66<\/jats:italic>\n                  and catalytic-mutant parasites but not of\n                  <jats:italic>\u0394gra70<\/jats:italic>\n                  parasites, whereas the replication defect persisted, revealing a second, RARRES3-independent consequence of GRA66 loss. RARRES3 was recruited to the PVM and intravacuolar network after IFN\u03b3 stimulation, and structural modeling supported assignment of GRA66 to the NAPE-phospholipase D family with an intact di-zinc active site. These findings define a lipid-centered, RNF213-independent arm of human cell-autonomous immunity and identify the parasite effector required to withstand it.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Importance<\/jats:title>\n                  <jats:p>\n                    <jats:italic>Toxoplasma gondii<\/jats:italic>\n                    infects a large fraction of the world\u2019s population and persists for life inside host cells, sheltered within a compartment called a vacuole. The immune signal interferon gamma normally holds the parasite in check, but how human cells attack this vacuole, and how the parasite resists, is only partly understood because humans lack the main anti-vacuolar defenses mice use. Here, we show that human cells recruit a lipid-remodeling enzyme, RARRES3, to this vacuole, and that RARRES3 drives the parasite to exit prematurely, triggering host cell death. The parasite counters with a lipid-modifying enzyme of its own, GRA66, which is required to prevent that premature exit. This host defense works independently of the pathway thought to dominate human control of\n                    <jats:italic>Toxoplasma<\/jats:italic>\n                    . Our findings identify lipid remodeling of the pathogen-containing vacuole as a distinct arm of human cell-intrinsic immunity, and the parasite enzyme that counteracts it as a potential drug target.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.09.12.751112","type":"posted-content","created":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T12:40:13Z","timestamp":1789476013000},"source":"Crossref","is-referenced-by-count":0,"title":["<i>Toxoplasma gondii<\/i>\n                  GRA66 prevents premature egress driven by the host phospholipase RARRES3, independently of RNF213"],"prefix":"10.64898","author":[{"given":"Caroline","family":"de Moraes de Siqueira","sequence":"first","affiliation":[{"name":"Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stephanie","family":"Martinez-Beltran","sequence":"additional","affiliation":[{"name":"Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emma A","family":"Johnston","sequence":"additional","affiliation":[{"name":"Department of Molecular Genetics and Microbiology, School of Medicine, Duke University, Durham, NC, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8707-4608","authenticated-orcid":false,"given":"J\u00f6rn","family":"Coers","sequence":"additional","affiliation":[{"name":"Department of Molecular Genetics and Microbiology, School of Medicine, Duke University, Durham, NC, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0289-7109","authenticated-orcid":false,"given":"Jeroen P. 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