{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,21]],"date-time":"2026-07-21T20:00:26Z","timestamp":1784664026497,"version":"3.55.0"},"posted":{"date-parts":[[2024,11,27]]},"group-title":"Microbiology","reference-count":36,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2024,11,27]],"date-time":"2024-11-27T00:00:00Z","timestamp":1732665600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2024,11,27]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  The obligate intracellular parasite\n                  <jats:italic>Toxoplasma gondii<\/jats:italic>\n                  replicates within a specialized compartment called the parasitophorous vacuole (PV). Recent work showed that despite living within a PV,\n                  <jats:italic>Toxoplasma<\/jats:italic>\n                  endocytoses proteins from the cytosol of infected host cells via a so-called ingestion pathway. The ingestion pathway is initiated by dense granule protein GRA14, which binds host ESCRT machinery to bud vesicles into the lumen of the PV. The protein-containing vesicles are internalized by the parasite and trafficked to the Plant Vacuole-like compartment (PLVAC), where cathepsin protease L (CPL) degrades the cargo and the chloroquine resistance transporter (CRT) exports the resulting peptides and amino acids to the parasite cytosol. However, although the ingestion pathway was proposed to be a conduit for nutrients, there is limited evidence for this hypothesis. We reasoned that if\n                  <jats:italic>Toxoplasma<\/jats:italic>\n                  uses the ingestion pathway to acquire nutrients, then parasites lacking GRA14, CPL, or CRT should rely more on biosynthetic pathways or alternative scavenging pathways. To explore this, we conducted a genome-wide CRISPR screen in wild-type (WT) parasites and \u0394\n                  <jats:italic>gra14<\/jats:italic>\n                  , \u0394\n                  <jats:italic>cpl<\/jats:italic>\n                  , and \u0394\n                  <jats:italic>crt<\/jats:italic>\n                  mutants to identify genes that become more fitness conferring in ingestion-deficient parasites. Our screen revealed a significant overlap of genes that become more fitness conferring in the ingestion mutants compared to WT. Pathway analysis indicated that \u0394\n                  <jats:italic>cpl<\/jats:italic>\n                  and \u0394\n                  <jats:italic>crt<\/jats:italic>\n                  mutants relied more on pyrimidine biosynthesis, fatty acid biosynthesis, TCA cycle, and lysine degradation. Bulk metabolomic analysis showed reduced levels of glycolytic intermediates and amino acids in the ingestion mutants compared to WT, highlighting the pathway\u2019s potential role in host resource scavenging. Interestingly, ingestion mutants showed an exacerbated growth defect when grown in amino acid-depleted media, suggesting a role for the\n                  <jats:italic>Toxoplasma<\/jats:italic>\n                  ingestion pathway during nutrient scarcity.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Importance<\/jats:title>\n                  <jats:p>\n                    <jats:italic>Toxoplasma gondii<\/jats:italic>\n                    is an obligate intracellular pathogen that infects virtually any nucleated cell in most warm-blooded animals. Infections are asymptomatic in most cases but people with weakened immunity can experience severe disease. For the parasite to replicate within the host, it must efficiently acquire essential nutrients, especially as it is unable to make several key metabolites. Understanding the mechanisms by which\n                    <jats:italic>Toxoplasma<\/jats:italic>\n                    scavenges nutrients from the host is crucial for identifying potential therapeutic targets. Our study highlights the function of the ingestion pathway in sustaining parasite metabolites and contributes to parasite replication under amino acid limiting conditions. This work advances our understanding of the metabolic adaptability of\n                    <jats:italic>Toxoplasma<\/jats:italic>\n                    .\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2024.11.27.625683","type":"posted-content","created":{"date-parts":[[2024,11,27]],"date-time":"2024-11-27T21:55:14Z","timestamp":1732744514000},"source":"Crossref","is-referenced-by-count":1,"title":["Metabolic Adaptability and Nutrient Scavenging in Toxoplasma gondii: Insights from Ingestion Pathway-Deficient Mutants"],"prefix":"10.64898","author":[{"given":"Patrick A.","family":"Rimple","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Einar B.","family":"Olafsson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6823-9072","authenticated-orcid":false,"given":"Benedikt M.","family":"Markus","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fengrong","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0430-8216","authenticated-orcid":false,"given":"Leonardo","family":"Augusto","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5237-1095","authenticated-orcid":false,"given":"Sebastian","family":"Lourido","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6859-8895","authenticated-orcid":false,"given":"Vern B.","family":"Carruthers","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2024120207303529000_2024.11.27.625683v1.1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijpara.2009.04.003"},{"key":"2024120207303529000_2024.11.27.625683v1.2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijpara.2013.09.003"},{"key":"2024120207303529000_2024.11.27.625683v1.3","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M404232200"},{"key":"2024120207303529000_2024.11.27.625683v1.4","doi-asserted-by":"publisher","DOI":"10.1038\/sj.emboj.7601189"},{"key":"2024120207303529000_2024.11.27.625683v1.5","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1008432"},{"key":"2024120207303529000_2024.11.27.625683v1.6","doi-asserted-by":"publisher","DOI":"10.1038\/415926a"},{"key":"2024120207303529000_2024.11.27.625683v1.7","doi-asserted-by":"publisher","DOI":"10.1111\/j.1600-065X.2010.00990.x"},{"key":"2024120207303529000_2024.11.27.625683v1.8","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.91.2.509"},{"key":"2024120207303529000_2024.11.27.625683v1.9","doi-asserted-by":"publisher","DOI":"10.1016\/j.chom.2015.04.003"},{"key":"2024120207303529000_2024.11.27.625683v1.10","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1011543"},{"key":"2024120207303529000_2024.11.27.625683v1.11","first-page":"e03081","article-title":"Two Toxoplasma gondii putative pore-forming proteins, GRA47 and GRA72, influence small molecule permeability of the parasitophorous vacuole","volume":"15","year":"2024","journal-title":"mBio"},{"key":"2024120207303529000_2024.11.27.625683v1.12","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0903831106"},{"key":"2024120207303529000_2024.11.27.625683v1.13","doi-asserted-by":"publisher","DOI":"10.1091\/mbc.E12-11-0827"},{"key":"2024120207303529000_2024.11.27.625683v1.14","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1006362"},{"key":"2024120207303529000_2024.11.27.625683v1.15","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2006.01.056"},{"key":"2024120207303529000_2024.11.27.625683v1.16","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.00782-08"},{"key":"2024120207303529000_2024.11.27.625683v1.17","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1010138"},{"key":"2024120207303529000_2024.11.27.625683v1.18","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-023-36571-4"},{"key":"2024120207303529000_2024.11.27.625683v1.19","doi-asserted-by":"publisher","DOI":"10.1111\/tra.12556"},{"key":"2024120207303529000_2024.11.27.625683v1.20","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.01188-14"},{"key":"2024120207303529000_2024.11.27.625683v1.21","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365-2958.2010.07181.x"},{"key":"2024120207303529000_2024.11.27.625683v1.22","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M112.443697"},{"key":"2024120207303529000_2024.11.27.625683v1.23","doi-asserted-by":"crossref","unstructured":"Warring, S. D. , Dou, Z. , Carruthers, V. B. , McFadden, G. I. & Dooren, G. G. van . Characterisation of the Chloroquine Resistance Transporter homologue in Toxoplasma gondii. Eukaryotic cell (in press) (2014) doi:EC.00027\u201314 [pii].","DOI":"10.1128\/EC.00027-14"},{"key":"2024120207303529000_2024.11.27.625683v1.24","doi-asserted-by":"crossref","unstructured":"Kannan, G. et al. Role of Toxoplasma gondii Chloroquine Resistance Transporter in Bradyzoite Viability and Digestive Vacuole Maintenance. mBio 10, (2019).","DOI":"10.1128\/mBio.01324-19"},{"key":"2024120207303529000_2024.11.27.625683v1.25","doi-asserted-by":"crossref","unstructured":"Markus, B. M. , Bell, G. W. , Lorenzi, H. A. & Lourido, S . Optimizing Systems for Cas9 Expression in Toxoplasma gondii. mSphere 4, (2019).","DOI":"10.1128\/mSphere.00386-19"},{"key":"2024120207303529000_2024.11.27.625683v1.26","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2017.131"},{"key":"2024120207303529000_2024.11.27.625683v1.27","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2016.08.019"},{"key":"2024120207303529000_2024.11.27.625683v1.28","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-020-18991-8"},{"key":"2024120207303529000_2024.11.27.625683v1.29","first-page":"e00060","article-title":"CRISPR Screens Identify Toxoplasma Genes That Determine Parasite Fitness in Interferon Gamma-Stimulated Human Cells","volume":"14","year":"2023","journal-title":"mBio"},{"key":"2024120207303529000_2024.11.27.625683v1.30","doi-asserted-by":"publisher","DOI":"10.1016\/j.chom.2019.09.008"},{"key":"2024120207303529000_2024.11.27.625683v1.31","doi-asserted-by":"publisher","DOI":"10.1038\/nature09022"},{"key":"2024120207303529000_2024.11.27.625683v1.32","doi-asserted-by":"publisher","DOI":"10.3791\/60985"},{"key":"2024120207303529000_2024.11.27.625683v1.33","doi-asserted-by":"publisher","DOI":"10.1101\/2024.07.10.601519"},{"key":"2024120207303529000_2024.11.27.625683v1.34","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-020-01018-x"},{"key":"2024120207303529000_2024.11.27.625683v1.35","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-022-01663-4"},{"key":"2024120207303529000_2024.11.27.625683v1.36","doi-asserted-by":"crossref","unstructured":"White, M. D. et al. Selective host autophagy is induced during the intracellular parasite Toxoplasma gondii infection controlling amino acid levels. mSphere 9, e00369\u201324.","DOI":"10.1128\/msphere.00369-24"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2024.11.27.625683","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T07:32:49Z","timestamp":1768462369000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2024.11.27.625683"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,11,27]]},"references-count":36,"URL":"https:\/\/doi.org\/10.1101\/2024.11.27.625683","relation":{"is-preprint-of":[{"id-type":"doi","id":"10.1128\/msphere.01011-24","asserted-by":"subject"}]},"subject":[],"published":{"date-parts":[[2024,11,27]]},"subtype":"preprint"}}