Revealing pathogenesis-associated metabolites in Histoplasma capsulatum through comprehensive metabolic profiling

During infection, Histoplasma capsulatum yeasts interact with a variety of phagocytic cells, where macrophages represent an important niche for long-term intracellular fungal survival and replication. In the phagosomes of macrophages, a hostile environment where most microorganisms are killed, Histoplasma not only survives but overcomes several biological challenges and proliferates intracellularly. To better understand the characteristics of intracellular Histoplasma and the phagosomal environment, a metabolomic platform was used to analyze Histoplasma yeasts cultured on different carbon sources and yeasts extracted from macrophages, identifying metabolites associated with pathogenesis. Metabolomic results of in vitro-grown yeasts were further characterized with available transcriptomic data, informing underlying gene expression patterns in response to contrasting milieus. These approaches revealed that Histoplasma yeasts, unlike many other yeasts, do not ferment sugars to ethanol, and, when cultivated on glycolytic versus gluconeogenic carbon sources, produce distinct metabolomes with altered intracellular amino acid, lipid, and sugar contents. Furthermore, analysis of Histoplasma-inoculated media illustrated that Histoplasma secretes mannitol and anthranilates. Lastly, a comparison of the metabolomes derived from in vitro cultivation versus intracellular growth highlighted leucine and cysteine/cystine as amino acids, which may serve as sources of carbon, nitrogen, and sulfur to yeasts within macrophages. These results detail metabolites linked to Histoplasma metabolism during macrophage infection, identifying potential candidates to target for novel histoplasmosis therapeutics.IMPORTANCEIntracellular pathogens reside within host cells, surviving against innate immune responses while exploiting host resources to proliferate. Understanding the mechanisms that underlie their survival and proliferation is critical for developing novel treatments and therapeutics for the diseases these pathogens cause. While Histoplasma is a unique example of a true intra-phagosomal pathogen, insights into its pathogenesis may still inform the study of other intracellular pathogens.