Phenotypic characterization of Histoplasma species
Data files
May 17, 2024 version files 105.67 KB
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Cell_Size_df.csv
38.55 KB
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halo_radius_miss.csv
673 B
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histoplasma_morphology_code.R
60.64 KB
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od_cells_histo.csv
3.51 KB
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README.md
2.29 KB
Abstract
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.
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.