Life Cycle Dominates the Volatilome Character of Dimorphic Fungus Coccidioides spp

Abstract

Valley fever (coccidioidomycosis) is an endemic fungal pneumonia of the North and South American deserts. The causative agents of Valley fever are the dimorphic fungi Coccidioides immitis and C. posadasii, which grow as mycelia in the environment and as spherules within the lungs of vulnerable hosts. Current diagnostics for Valley fever are severely lacking due to poor sensitivity and invasiveness, contributing to a 23-day median time to diagnosis, and therefore, new diagnostic tools are needed. We are working toward the development of a breath-based diagnostic for coccidioidomycosis, and in this initial study, we characterized the volatile metabolomes (or volatilomes) of in vitro cultures of Coccidioides Using solid-phase microextraction (SPME) and comprehensive two-dimensional gas chromatography coupled to time of flight mass spectrometry (GC×GC-TOFMS), we characterized the volatile organic compounds (VOCs) produced by six strains of each species during mycelial or spherule growth. We detected a total of 353 VOCs that were at least 2-fold more abundant in a Coccidioides culture than in medium controls and found that the volatile metabolome of Coccidioides is more dependent on the growth phase (spherules versus mycelia) than on the species. The volatile profiles of C. immitis and C. posadasii have strong similarities, indicating that a single suite of Valley fever breath biomarkers can be developed to detect both species.IMPORTANCE Coccidioidomycosis, or Valley fever, causes up to 30% of community-acquired pneumonias in highly populated areas of the U.S. desert southwest where the disease is endemic. The infection is difficult to diagnose by standard serological and histopathological methods, which delays appropriate treatment. Therefore, we are working toward the development of breath-based diagnostics for Valley fever. In this study, we characterized the volatile metabolomes (or volatilomes) of six strains each of Coccidioides immitis and C. posadasii, the dimorphic fungal species that cause Valley fever. By analyzing the volatilomes during the two modes of growth of the fungus-mycelia and spherules-we observed that the life cycle plays a significant role in the volatiles produced by Coccidioides In contrast, we observed no significant differences in the C. immitis versus C. posadasii volatilomes. These data suggest that life cycle, rather than species, should guide the selection of putative biomarkers for a Valley fever breath test.

Keywords: GC×GC; Valley fever; biomarkers; coccidioidomycosis; comprehensive two-dimensional gas chromatography; dimorphic fungus; fungal infections; untargeted metabolomics; volatile metabolites.

FIG 1

Numbers of analytes detected in at least a 2-fold-higher abundance in a Coccidioides culture than in the medium controls that are shared and unique among C. posadasii mycelia (square) versus spherules (circle) (A), C. immitis mycelia (square) versus spherules (circle) (B), mycelia of C. posadasii (red) versus C. immitis (blue) (C), and spherules of C. posadasii (red) versus C. immitis (blue) (D).

FIG 2

Forty-three volatiles significantly different in abundance between life cycles (P < 0.05), expressed as log₂ fold changes in peak abundance. Volatiles marked with asterisks indicate greater statistically significant differences (*, P < 0.001; **, P < 0.0001). Compound identifications: 195, hexanal; 255, 2-hexen-1-ol; 270, cyclohexanone; 386, 1-methyl-3-(1-methylethyl)-benzene; 428, 2,4,6-trimethylpyridine; 554, 2-(2-butoxyethoxy)-ethanol.

FIG 3

Volatiles detected in at least two-thirds of all Coccidioides strains in spherule (A) or mycelial (B) cultures, with VOCs in boldface type detected in both life cycles and those in italic type detected in statistically significantly different abundances between life cycles (P < 0.05). Open shapes indicate VOCs with at least a 2-fold-higher relative abundance than in medium blanks, and solid shapes indicate VOCs with at least a 10-fold-higher relative abundance. Strains are color-coded as blue for C. immitis and red for C. posadasii. The percentages of listed VOCs detected in each strain and the percentages of strains producing each VOC are noted at the bottom and right sides of each chart, respectively. Compound identifications: 97, acetic acid; 239, 1-hexanol; 255, 2-hexen-1-ol; 383, limonene; 428, 2,4,6,-trimethylpyridine; 820, 2-butyl-1-octanol.

FIG 4

Principal-component analysis (PCA) score plot using 353 VOCs as features, produced by C. immitis (blue) and C. posadasii (red) when cultured under conditions that induced mycelial (squares) or spherule (circles) morphologies. Blank media are shown in gray. Fungal cultures and medium blanks were analyzed in triplicate, yielding 72 fungal and 6 blank observations. PCA score plots with the observations colored by strain are shown in Fig. S1 in the supplemental material.

FIG 5

Hierarchical clustering analysis (HCA) of 72 fungal cultures and 6 medium blanks (columns) based on the relative abundances of 353 volatiles (rows). Clustering of fungal and blank samples was performed using Pearson correlations, and clustering of volatiles was performed using Euclidean distance, both with average linkages. Samples are color-coded by strain, population, species, and life cycle, as labeled in the key; medium blanks are in gray.