{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T21:01:43Z","timestamp":1790802103059,"version":"4.1.0"},"posted":{"date-parts":[[2023,11,15]]},"group-title":"Microbiology","reference-count":27,"publisher":"openRxiv","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2023,11,15]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  <jats:italic>Toxoplasma gondii<\/jats:italic>\n                  , a medically important intracellular parasite, uses GRA proteins, secreted from dense granule organelles, to mediate nutrient flux across the parasitophorous vacuole membrane (PVM). GRA17 and GRA23 are known pore-forming proteins on the PVM involved in this process, but the roles of additional proteins have remained largely uncharacterized. We recently identified\n                  <jats:italic>GRA72<\/jats:italic>\n                  as synthetically lethal with\n                  <jats:italic>GRA17<\/jats:italic>\n                  . Deleting\n                  <jats:italic>GRA72<\/jats:italic>\n                  produced similar phenotypes to\n                  <jats:italic>\u0394gra17<\/jats:italic>\n                  parasites, and computational predictions suggested it forms a pore. To understand how GRA72 functions we performed immunoprecipitation experiments and identified GRA47 as an interactor of GRA72. Deletion of\n                  <jats:italic>GRA47<\/jats:italic>\n                  resulted in an aberrant \u2018bubble vacuole\u2019 morphology with reduced small molecule permeability, mirroring the phenotype observed in\n                  <jats:italic>GRA17<\/jats:italic>\n                  and\n                  <jats:italic>GRA72<\/jats:italic>\n                  knockouts. Structural predictions indicated that GRA47 and GRA72 form heptameric and hexameric pores, respectively, with conserved histidine residues lining the pore. Mutational analysis highlighted the critical role of these histidines for protein functionality. Validation through electrophysiology confirmed alterations in membrane conductance, corroborating their pore-forming capabilities. Furthermore, \u0394\n                  <jats:italic>gra47<\/jats:italic>\n                  parasites and parasites expressing GRA47 with a histidine mutation had reduced\n                  <jats:italic>in vitro<\/jats:italic>\n                  proliferation and attenuated virulence in mice. Our findings show the important roles of GRA47 and GRA72 in regulating PVM permeability, thereby expanding the repertoire of potential therapeutic targets against\n                  <jats:italic>Toxoplasma<\/jats:italic>\n                  infections.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>IMPORTANCE<\/jats:title>\n                  <jats:p>\n                    <jats:italic>Toxoplasma gondii<\/jats:italic>\n                    is a parasite that poses significant health risks to those with impaired immunity. It replicates inside host cells shielded by the parasitophorous vacuole membrane (PVM), which controls nutrient and waste exchange with the host. GRA72, previously identified as essential in the absence of the GRA17 nutrient channel, is implicated in forming an alternative nutrient channel. Here we found that GRA47 associates with GRA72 and is also important for the PVM\u2019s permeability to small molecules. Removal of GRA47 leads to distorted vacuoles and impairs small molecule transport across the PVM, resembling the effects of GRA17 and GRA72 deletions. Structural models suggest GRA47 and GRA72 form distinct pore structures, with a pore-lining histidine critical to their function.\n                    <jats:italic>Toxoplasma<\/jats:italic>\n                    strains lacking GRA47, or those with a histidine mutation, have impaired growth and reduced virulence in mice, highlighting these proteins as potential targets for new treatments against Toxoplasmosis.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2023.11.15.567216","type":"posted-content","created":{"date-parts":[[2023,11,15]],"date-time":"2023-11-15T22:25:12Z","timestamp":1700087112000},"source":"Crossref","is-referenced-by-count":2,"title":["GRA47 and GRA72 are\n                  <i>Toxoplasma gondii<\/i>\n                  pore-forming proteins that influence small molecule permeability of the parasitophorous vacuole"],"prefix":"10.64898","author":[{"given":"Mebratu A.","family":"Bitew","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pablo S.","family":"Gaete","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christopher","family":"Swale","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Parag","family":"Maru","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9203-1602","authenticated-orcid":false,"given":"Jorge E.","family":"Contreras","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0289-7109","authenticated-orcid":false,"given":"Jeroen P. 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