Environmental former <i>Massilia</i> group bacteria secrete metabolites that promote <i>Leptospira</i> growth

ABSTRACT Understanding pathogen metabolism is critical for identifying key functions for drug targeting, establishing effective in vitro experimental systems, 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 belonging to the former Massilia group produces metabolites to promote the growth of Leptospira interrogans , which has been encountered through an incidental contamination event and analyzed in this study. Gas chromatography–tandem mass spectrometry (GC-MS/MS) analysis demonstrated that cultivation of Massilia sp. strain NBRC 108631 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 strain NBRC 108631, 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. IMPORTANCE Pathogenic Leptospira persist in environmental reservoirs, yet the mechanisms supporting their growth remain poorly defined. Here, we find that metabolites produced by common environmental bacteria belonging to the former Massilia group 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 strategies may facilitate the systematic identification of growth-supporting metabolites and provide a basis for improving selective cultivation for uncultured or difficult-to-culture organisms. The determination of growth-promoting metabolites advances our understanding of pathogen persistence in natural environments and offers a generalized framework to study metabolically dependent microorganisms.