DOI: 10.5061/dryad.95x69p8sr
title: Phenotypic characterization of Histoplasma species

description: 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 between these species: H. capsulatum sensu stricto, H.
 ohiense, H. mississippiense, H. suramericanum, and an African lineage. We
 report diagnostic traits for three species. The other two species can be
 identified by a combination of traits. Our results suggest that 1) there
 are significant phenotypic differences among the cryptic species of
 Histoplasma, and 2) that those differences can be used to positively
 distinguish those species in a clinical setting and for further study of
 the evolution of this fungal pathogen.
description: # 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 Apo PSF
 quality objective on an Olympus BX-61 microscope and collected using a
 QImaging RETIGA 4000R color camera and Volocity 6.3 acquisition software.
   ## Description of the data and file structure We are including four
 files: Three of them are experimental observations, and one of them is the
 code required to analyze the data. halo_radius_miss.csv: ratio sizes of H.
 mississipiense cultures in .5% skim milk media. The variable halo is in
 cms and reflects the ratio of the lysis halo. Isolate refers to the name
 of the Histoplasma strain. od_cells_histo.csv: rates of growth of
 different species of Histoplasma in liquid media. Strain refers to the
 name of the Histoplasma strain. OD refers to the turbidity of the culture
 (reported in Absorbance Units). Cells is the number of cells in the
 culture. Cell_Size_df.csv: cell size for different species of
 Histoplasma. Line refers to the name of the Histoplasma strain. Replicate
 refers to the number of the experimental batch. Area is the ellipse area
 of the cell (in micrometers^2). histoplasma_morphology_code.R: Analytical
 code to generate linear models. ## Sharing/Access information ##
 Code/Software histoplasma_morphology_code.R includes the analytical code
 to generate linear models, and figures in the manuscript.