Comparative transcriptomics of infectious spores from the fungal pathogen Histoplasma capsulatum reveals a core set of transcripts that specify infectious and pathogenic states

Abstract

Histoplasma capsulatum is a fungal pathogen that infects both healthy and immunocompromised hosts. In regions where it is endemic, H. capsulatum grows in the soil and causes respiratory and systemic disease when inhaled by humans. An interesting aspect of H. capsulatum biology is that it adopts specialized developmental programs in response to its environment. In the soil, it grows as filamentous chains of cells (mycelia) that produce asexual spores (conidia). When the soil is disrupted, conidia aerosolize and are inhaled by mammalian hosts. Inside a host, conidia germinate into yeast-form cells that colonize immune cells and cause disease. Despite the ability of conidia to initiate infection and disease, they have not been explored on a molecular level. We developed methods to purify H. capsulatum conidia, and we show here that these cells germinate into filaments at room temperature and into yeast-form cells at 37°C. Conidia internalized by macrophages germinate into the yeast form and proliferate within macrophages, ultimately lysing the host cells. Similarly, infection of mice with purified conidia is sufficient to establish infection and yield viable yeast-form cells in vivo. To characterize conidia on a molecular level, we performed whole-genome expression profiling of conidia, yeast, and mycelia from two highly divergent H. capsulatum strains. In parallel, we used homology and protein domain analysis to manually annotate the predicted genes of both strains. Analyses of the resultant data defined sets of transcripts that reflect the unique molecular states of H. capsulatum conidia, yeast, and mycelia.

Fig 1

Purified microconidia and macroconidia were generated from H. capsulatum. Purified macroconidia and microconidia of the G217B (A) and G186AR (B) strains are shown.

Fig 2

Conidia germinate in vitro into filaments at 27°C and into yeast-phase cells at 37°C. A time course of germination of G217B conidia was performed at the indicated temperatures.

Fig 3

Germination of conidia in macrophages and host tissues. PAS-stained images of conidia-infected bone marrow-derived macrophages are shown at 2 hpi (A), 6 hpi (B), 24 hpi (C), and 72 hpi (D). (E) Occasional mycelial forms observed at 24 hpi. (F) A lung section from a conidia-infected mouse was stained with PAS, hematoxylin, and eosin. Arrows indicate conidia, black arrowheads indicate yeast cells, and the white arrowhead indicates a macroconidium.

Fig 4

Relative expression of G217B and G186AR transcripts in yeast, mycelia, and conidia. (A) Heat map of relative conidial (C), yeast (Y), and mycelial (M) expression levels for pairs of orthologous genes with data present in both strains. Intensities are log₂ BAGEL-estimated relative expression levels from 0 (black) to 3 (saturated). Genes are ordered by phase specificity, defined as the average angular coordinate from plots B and C. Phase-specific genes are indicated by the bracketed regions to the left of the heat map. Relative enrichment plots are shown for G217B (B) and G186AR (C). Yeast, mycelial, and conidial axes are drawn in red, green, and blue, respectively; axis ticks indicate log₂ units of enrichment. Genes were plotted by projecting the BAGEL-estimated relative expression values on the corresponding axes (the condition of lowest expression always has a log₂ enrichment of zero). Yeast, mycelial, and conidial enriched genes, based on a 3-fold enrichment criterion, are colored red, green, and blue, respectively. Genes of interest mentioned in the text and/or tables are highlighted with black circles and labeled. (D) Venn diagrams showing conserved expression of the 3-fold differentially expressed conidial-, mycelial-, and yeast-specific transcripts of the G217B and G186AR strains.

Fig 5

Validation of phase-specific enriched expression. (A) Northern blot analysis of CATA expression was performed on total RNAs isolated from G217B conidia (lane 1), yeast (lane 2), and mycelia (shaking culture [lane 3] or still culture [lane 4]). Ethidium bromide visualization of rRNAs from the same samples is shown below the blot as a loading control. Fluorescence microscopy was used to examine strains expressing GFP under the control of either the TYR1 promoter (B) or the CBP1 promoter (C). In panel B, GFP fluorescence was monitored in cells that were undergoing filamentation at room temperature. GFP expression is observed at much higher levels in a filamentous cell than in yeast-form cells, consistent with mycelial enriched expression. In panel C, GFP fluorescence is observed only in the clump of yeast-phase G186AR cells (left), not in G186AR cells undergoing filamentation at room temperature (right), consistent with yeast-specific enriched expression. DIC, differential interference contrast.

Fig 6

The tyrosinase gene family shows phase-specific expression. Relative enrichment plots are shown for G217B (A) and G186AR (B). Yeast, mycelial, and conidial axes are drawn in red, green, and blue, respectively; axis ticks indicate log₂ units of enrichment. Genes were plotted by projecting the BAGEL-estimated relative expression values on the corresponding axes (the condition of lowest expression always has a log₂ enrichment value of zero). Yeast, mycelial, and conidial enriched genes, based on the 3-fold enrichment criterion, are colored red, green, and blue, respectively. Tyrosinases are highlighted with black circles and labeled.

Fig 7

tRNAs are differentially expressed in conidia, yeast, and mycelia. The heat map shows the enriched expression in mycelia and depleted expression in conidia of 40 tRNA transcripts of G217B. Relative transcript levels in conidia (C), mycelia (M), and yeast (Y) are displayed. Intensities are log₂ BAGEL-estimated relative expression levels from 0 (black) to 4 (saturated). tRNAs are labeled with cognate amino acids and anticodons as predicted by tRNAscan-SE.