Soil bacterium Massilia secretes metabolites that promote Leptospira growth

Understanding pathogen metabolism is critical for identifying key functions for drug targeting, establishing effective in vitro experimental systems, etc., particularly for metabolically unique organisms such as Leptospira. Pathogenic Leptospira are thought to infect humans from environmental sources; however, direct isolation from environmental samples remains technically challenging and is not yet well established. Here, we report that a ubiquitous environmental bacterium, Massilia sp., produces metabolites that promote the growth of Leptospira interrogans, encountered through an incidental contamination event, and identified in this study. Gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis showed demonstrated that cultivating of Massilia sp. in R2A medium resulted in the accumulation of metabolites, including branched-chain amino acid (BCAA) intermediates, compared to fresh medium. By combining genome-scale metabolic modeling with experimental validation using cell-free culture supernatant supplementation assays, we demonstrate that BCAA intermediates, particularly 2-ketoisocaproic acid (4-methyl-2-oxopentanoate; 4MOP), a leucine biosynthetic intermediate produced by Massilia sp., enhance Leptospira growth. To investigate the metabolic role of 4MOP, we incorporated transcriptomic data into a genome-scale metabolic network model to generate condition-specific models. Resulted flux distributions indicated that Leptospira catabolized imported 4MOP to produce acetyl-CoA. Our results reveal a previously unrecognized metabolic interaction where metabolites produced by environmental bacteria support the growth of pathogenic Leptospira, offering mechanistic insight into its metabolic requirement. These findings have implications to understand the environmental persistence of Leptospira through its metabolic dependencies on coexisting microbes, and they also help develop better strategies for this pathogen. ImportancePathogenic Leptospira persist in environmental reservoirs, yet the mechanisms supporting their growth remain poorly defined. Here, we find that metabolites produced by common environmental bacteria, Massilia sp., can promote Leptospira growth, suggesting a previously unrecognized metabolic dependency on coexisting microbes. Importantly, this study indicates that combining genome-scale metabolic modeling with experimental validation provides a useful framework for identifying metabolic interactions that are otherwise difficult to resolve using conventional culture-based approaches. Current strategy may facilitate the systematic identification of growth-supporting metabolites and provide a basis for improving selective cultivation for uncultured or difficult to culture organisms. Determination of growth promoting metabolites advances our understanding of pathogen persistence in natural environments and offers a generalized framework to study metabolically dependent microorganisms.