{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T10:44:13Z","timestamp":1785408253358,"version":"3.56.0"},"posted":{"date-parts":[[2026,3,13]]},"group-title":"Microbiology","reference-count":70,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,3,13]],"date-time":"2026-03-13T00:00:00Z","timestamp":1773360000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"award":["5T32GM139774-02"],"award-info":[{"award-number":["5T32GM139774-02"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,3,13]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Leptospirosis, caused by pathogenic\n                  <jats:italic>Leptospira<\/jats:italic>\n                  spp. such as\n                  <jats:italic>L. interrogans<\/jats:italic>\n                  , is a bacterial zoonosis of increasing prevalence with no consistently effective treatments in severe cases. We sought to characterize metabolic mechanisms that support\n                  <jats:italic>L. interrogans<\/jats:italic>\n                  infection in the host setting, with the ultimate goal of revealing unexplored therapeutic opportunities. We first established and validated a culture medium, which we refer to as supplemented Human Plasma-Like Medium (sHPLM). sHPLM more closely resembles the physiological environment of the human host than standard culture media, such as the EMJH (Ellinghausen-McCullough-Johnson-Harris) medium typically used for\n                  <jats:italic>Leptospira<\/jats:italic>\n                  culture. To better understand bacterial metabolism, we pioneered metabolomics in sHPLM-cultured\n                  <jats:italic>Leptospira<\/jats:italic>\n                  . Specifically, we developed a liquid chromatography mass spectrometry (LC\/MS) metabolomics-based workflow for both medium analysis and stable isotope tracing with\n                  <jats:italic>L. interrogans<\/jats:italic>\n                  cultures. The application of these innovations revealed that the amino acid glutamine is a major nitrogen source for\n                  <jats:italic>L. interrogans<\/jats:italic>\n                  . A small-molecule inhibitor blocking glutamine utilization, JHU-083, effectively impaired the proliferation of sHPLM cultures. Further, adding glutamine to non-physiological EMJH medium rapidly induced a short-term proliferative boost in\n                  <jats:italic>L. interrogans<\/jats:italic>\n                  and increased biofilm formation. RNA-sequencing after glutamine exposure revealed transcriptional trends for increases in biosynthesis to support these phenotypes. Although ammonium has long been thought to be the sole nitrogen source for\n                  <jats:italic>L. interrogans,<\/jats:italic>\n                  our results demonstrate that glutamine provides a second source of nitrogen for biosynthesis and may act as a metabolite signal to alter\n                  <jats:italic>L. interrogans<\/jats:italic>\n                  physiology in ways that could influence infection. This work highlights that studying\n                  <jats:italic>L. interrogans<\/jats:italic>\n                  under physiological conditions is key to understanding mechanisms supporting infection and points to nitrogen assimilation as a potential target for therapies.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Author Summary<\/jats:title>\n                  <jats:p>\n                    Leptospirosis is a potentially fatal disease transmitted through water and soil contaminated with pathogenic\n                    <jats:italic>Leptospira<\/jats:italic>\n                    bacteria. Much research is currently focused on the idea that an improved understanding of how\n                    <jats:italic>Leptospira<\/jats:italic>\n                    infects hosts and causes disease may inspire the development of improved therapeutics, which are urgently needed. Focusing on\n                    <jats:italic>Leptospira interrogans<\/jats:italic>\n                    , a clinically important pathogenic species, we determined that conventional growth media are inadequate for understanding how the bacterium behaves when inside hosts. Instead, we designed an optimized formulation to mimic human blood, and we applied an underutilized technique for measuring the biochemical reactions that enable pathogen survival. These two innovations revealed that\n                    <jats:italic>L. interrogans<\/jats:italic>\n                    uses glutamine, an abundant nutrient in host blood and tissues, as a source of nitrogen for the production of biomolecules that are required for replication and infection. This discovery is notable as nitrogen demands were previously thought to be met using ammonium. Treating\n                    <jats:italic>L. interrogans<\/jats:italic>\n                    with inhibitors of both glutamine and ammonium metabolism blocked bacterial replication. We also discovered that\n                    <jats:italic>L. interrogans<\/jats:italic>\n                    increases its growth rate, upregulates its expression of biosynthetic pathways when exposed to glutamine, and increases its formation of biofilm. Our results reveal the importance of glutamine in supporting the lifecycle of leptospirosis-causing bacteria.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.03.12.711193","type":"posted-content","created":{"date-parts":[[2026,3,14]],"date-time":"2026-03-14T02:20:12Z","timestamp":1773454812000},"source":"Crossref","is-referenced-by-count":0,"title":["Metabolites from plasma-like medium fuel nitrogen metabolism and influence proliferation in\n                  <i>Leptospira interrogans<\/i>"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7135-5878","authenticated-orcid":false,"given":"Matthew H.","family":"Ward","sequence":"first","affiliation":[{"name":"Department of Chemistry, Washington University in St. Louis, MO, USA, 63130"},{"name":"Department of Medicine, Washington University in St. Louis, MO, USA, 63130"},{"name":"Center for Mass Spectrometry and Metabolic 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