Mapping carbon utilization pathways in Histoplasma capsulatum through ¹³C-metabolic flux analysis

Histoplasma capsulatum is a human fungal pathogen that survives and proliferates within phagocytic immune cells. To sustain growth in the nutrient-limited phagosome environment, the pathogenic yeast scavenges available carbon sources, which must be metabolized through central carbon metabolism for respiration and biomass synthesis. However, Histoplasma carbon metabolic pathways operating in the pathogenic yeast phase have not been extensively mapped. To address this gap, we employed a fluxomic platform using stable isotope tracers to quantify the cellular reaction rates of central carbon metabolism. This approach revealed that, in Histoplasma yeasts, carbon resides within five main reservoirs: fatty acids, proteins, mannitol, nucleic acids, and cell wall components. Carbon conversion efficiency, or biomass yield, was approximately 50%, indicating substantial CO₂ loss from supplemented carbon substrates, glucose, and glutamate. ¹³C-labeling analysis demonstrated simultaneous glycolysis and gluconeogenesis, and enriched serine labeling confirmed threonine aldolase activity in serine biosynthesis. Compartmentalization of pyruvate metabolism was evident from the labeling of amino acids derived from pyruvate, with the methylcitrate cycle identified as the primary source of labeled pyruvate. Notably, malic enzyme and pyruvate carboxylase exhibited negligible fluxes, while mitochondrial reactions, particularly CO₂-producing ones, were the most active. These results offer insight into key metabolic reactions, alternative pathways, and metabolite/enzyme compartmentalization in Histoplasma yeast metabolism. This foundational framework supports future studies aimed at identifying metabolic targets for novel histoplasmosis therapeutics.IMPORTANCETo our knowledge, this study represents the first application of ¹³C-metabolic flux analysis to a human fungal pathogen, where we identified carbon reservoirs and quantified the metabolic fluxes of pathogenic Histoplasma yeasts. Our findings demonstrated that Histoplasma metabolizes carbon toward cellular respiration to robustly produce CO₂ and energy but also uses alternative pathways within central metabolism for biosynthesis. Given the potential for other pathogenic fungi to share similar metabolic features, especially biomass, our study offers a comprehensive framework for deciphering fungal metabolism, providing insights into their infection-enabling metabolism and offering a foundation for identifying new therapeutic targets.