{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T09:28:57Z","timestamp":1785403737450,"version":"3.56.0"},"posted":{"date-parts":[[2026,7,20]]},"group-title":"Genetics","reference-count":63,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,7,20]],"date-time":"2026-07-20T00:00:00Z","timestamp":1784505600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"name":"Duke Science and Technology Initiative"},{"award":["AI039115-28"],"award-info":[{"award-number":["AI039115-28"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["AI172451-04"],"award-info":[{"award-number":["AI172451-04"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Duke University School of Medicine Summer Research Opportunities Program"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,7,20]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Growth at physiologically relevant temperatures is essential for fungal pathogenesis and is controlled by several cellular factors. The evolution of fungal thermotolerance is concerning as warming environments may promote the emergence of new pathogens. Trehalose, a disaccharide absent in mammals, plays a central role in thermotolerance by stabilizing proteins and membranes during heat stress. Trehalose is synthesized from glucose-6-phosphate (G6P) and uridine-diphosphate-glucose (UDPG) in two steps catalyzed by trehalose-6-phosphate synthase (Tps1) and trehalose-6-phosphate phosphatase (Tps2). Here, we investigated genetic suppression of Tps1 function in\n                  <jats:italic>Cryptococcus deneoformans<\/jats:italic>\n                  , a species in the\n                  <jats:italic>Cryptococcus<\/jats:italic>\n                  pathogenic species complex. Tps1 is essential for growth at 37\u00b0C in\n                  <jats:italic>C. deneoformans<\/jats:italic>\n                  and spontaneous suppressor mutations restored the growth of\n                  <jats:italic>tps1<\/jats:italic>\n                  \u0394 mutants at 37\u00b0C. Whole-genome sequencing followed by variant calling analysis primarily identified loss-of-function mutations in the gene encoding hexokinase 1 (Hxk1). Targeted gene deletion mutants further showed that loss of either\n                  <jats:italic>HXK1<\/jats:italic>\n                  or\n                  <jats:italic>HXK2<\/jats:italic>\n                  can bypass\n                  <jats:italic>tps1<\/jats:italic>\n                  \u0394 in a carbon source-dependent manner. The\n                  <jats:italic>tps1<\/jats:italic>\n                  \u0394 mutant exhibited elevated hexokinase activity, accumulation of G6P and glycogen, and ATP depletion after heat shock. Deletion of\n                  <jats:italic>HXK1<\/jats:italic>\n                  or\n                  <jats:italic>HXK2<\/jats:italic>\n                  restored hexokinase activity and partially restored G6P and ATP levels in the\n                  <jats:italic>tps1<\/jats:italic>\n                  \u0394 mutant, while glycogen remained elevated, indicating that excess glycolytic flux underlies the\n                  <jats:italic>tps1<\/jats:italic>\n                  \u0394 high-temperature growth defect. Overall, our study uncovers a previously unappreciated mechanism of Tps1-mediated heat adaptation in\n                  <jats:italic>C. deneoformans<\/jats:italic>\n                  , by revealing that Tps1 functions as a critical metabolic gatekeeper that safeguards glycolytic flux to sustain growth at elevated temperatures.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Article summary<\/jats:title>\n                  <jats:p>\n                    Trehalose is crucial for fungal thermal adaptation and mutants lacking trehalose are inviable at 37\u00b0C. This study examined how genetic suppressors restore viability at 37\u00b0C in mutants lacking\n                    <jats:italic>TPS1<\/jats:italic>\n                    , which encodes trehalose-6-phosphate synthase. Through whole-genome sequencing of spontaneous suppressor isolates and variant calling analysis, mutations were identified in\n                    <jats:italic>HXK1<\/jats:italic>\n                    . Gene deletion mutants and biochemical assays show these mutations alter glycolytic flux. We show that\n                    <jats:italic>tps1<\/jats:italic>\n                    \u0394 mutants exhibit unbridled glycolysis, and their growth at 37\u00b0C is restored by\n                    <jats:italic>hxk1<\/jats:italic>\n                    \u0394 mutations that reduce glycolytic flux. This study highlights the interdependence between Tps1 and Hxk1, which may have broader relevance across organisms.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.07.15.738695","type":"posted-content","created":{"date-parts":[[2026,7,21]],"date-time":"2026-07-21T03:55:16Z","timestamp":1784606116000},"source":"Crossref","is-referenced-by-count":0,"title":["Trehalose-6-phosphate synthase promotes thermotolerance by governing glycolytic flux in\n                  <i>Cryptococcus deneoformans<\/i>"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2650-9035","authenticated-orcid":false,"given":"Vikas","family":"Yadav","sequence":"first","affiliation":[{"name":"Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-2432-3607","authenticated-orcid":false,"given":"Kahlia A.","family":"Carl","sequence":"additional","affiliation":[{"name":"School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6369-5995","authenticated-orcid":false,"given":"Joseph","family":"Heitman","sequence":"additional","affiliation":[{"name":"Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7503-6492","authenticated-orcid":false,"given":"Erica J.","family":"Washington","sequence":"additional","affiliation":[{"name":"Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA"},{"name":"Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026072219300808000_2026.07.15.738695v1.1","doi-asserted-by":"crossref","first-page":"607","DOI":"10.1038\/s41564-022-01112-0","article-title":"Evolution of the human pathogenic lifestyle in fungi","volume":"7","year":"2022","journal-title":"Nature Microbiology"},{"key":"2026072219300808000_2026.07.15.738695v1.2","doi-asserted-by":"publisher","DOI":"10.1016\/j.smim.2023.101738"},{"key":"2026072219300808000_2026.07.15.738695v1.3","doi-asserted-by":"publisher","DOI":"10.1126\/scitranslmed.3004404"},{"key":"2026072219300808000_2026.07.15.738695v1.4","unstructured":"D. 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