Glutamine from plasma-like medium fuels nitrogen metabolism and influences proliferation in Leptospira interrogans

Leptospirosis, caused by pathogenic Leptospira spp. such as L. interrogans, is a bacterial zoonosis of increasing prevalence with no consistently effective treatments in severe cases. We sought to characterize metabolic mechanisms that support L. interrogans infection, 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 Leptospira culture. We also pioneered metabolomics approaches to study sHPLM-cultured Leptospira. Specifically, we developed a liquid chromatography/mass spectrometry (LC/MS) workflow for both medium metabolomics and stable isotope tracing with L. interrogans cultures. The application of these technologies in L. interrogans revealed that the amino acid glutamine is a major nitrogen source among the many available in sHPLM. Subsequent growth studies in three pathogenic Leptospira strains and the nonpathogenic strain L. biflexa Patoc-1 showed robust proliferation when only glutamine-derived nitrogen was available. Further, a small-molecule inhibitor designed to target glutamine utilization, JHU-083, effectively impaired the proliferation of L. interrogans cultures. Adding glutamine to non-physiological EMJH medium also rapidly induced a short-term proliferative boost in L. interrogans 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 Leptospira spp., our results demonstrate that glutamine provides a second source of nitrogen for biosynthesis in multiple strains and may act as a metabolite signal to alter L. interrogans physiology in ways that could influence infection. This work highlights that studying L. interrogans under physiological conditions is key to understanding mechanisms supporting infection and points to nitrogen assimilation as a potential target for therapies.