Phenotypic characterization of Histoplasma species

Histoplasmosis is an endemic mycosis that often presents as a respiratory infection in immunocompromised patients. Hundreds of thousands of new infections are reported annually around the world. The etiological agent of the disease, Histoplasma, is a dimorphic fungus commonly found in the soil where it grows as mycelia. Humans can become infected by Histoplasma through inhalation of its spores (conidia) or mycelial particles. The fungi transitions into the yeast phase in the lungs at 37°C. Once in the lungs, yeast cells reside and proliferate inside alveolar macrophages. Genomic work has revealed that Histoplasma is composed of at least five cryptic phylogenetic species that differ genetically.  Three of those lineages have received new names. Here we evaluated multiple phenotypic characteristics (colony morphology, secreted proteolytic activity, yeast size and growth rate) of strains from five of the phylogenetic species of Histoplasma to identify phenotypic traits that differentiate b..., , , # Phenotypic characterization of Histoplasma species

**Evaluation of extracellular proteolytic activity:** To evaluate extracellular proteolytic activity in different species of *Histoplasma*, we grew the 27 *Histoplasma* strains (Table S1) in HMM plates supplemented with 1.5% skim milk. Strains with a proteolytic activity show a clear halo around their yeast colonies.

**Optical density and growth curves of *Histoplasma* yeast cultures:** We mixed 600 μl of liquid culture with 300 μl of 3M NaOH in a plastic cuvette, which was covered with Parafilm and vortexed for 10 seconds to separate yeast clumps and measure optical density (OD) in a GENESYS 10vis spectrophotometer (Thermo Spectronic) starting at day 0, and at every 24 hours after that until day 11.

**Yeast Area:** We mixed 10 μl from a yeast culture that had large yeast clumps removed with 10 μl of Lactophenol Cotton Blue on a glass slide. Differential interference contrast (DIC) images were obtained using 100X/1.4 Oil UPlan S Ap...