Fig 1.
Portrait of the cystic fibrosis lung mycobiome.
Relative abundance of fungal species in 89 sputum samples from 28 patients, identified by culture (top) and ITS1 sequencing (bottom). *: Pearson correlation > 0.5.
Fig 2.
Diversity of species and antifungal resistance profiles of 1,603 fungal isolates from cystic fibrosis patients.
Each dot represents an isolate from the corresponding cystic fibrosis patient and colours represent species identity. The vertical dispersion of isolates within each phenotypic class is simply for visual clarity, as with the horizontal dispersion of isolates from an individual patient. (A) Summary of yeast isolates. The isolates are categorized as: “Fluconazole resistant” if their relative growth with fixed concentration of fluconazole at 128 μg/ml was greater than 2 times that of the relative growth of reference C. albicans strain SN95; “Fluconazole susceptible” if their relative growth was less than 2 times the relative growth of SN95; and “Variable” if their resistance profiles were variable over biological and technical duplicates. C. albicans is divided into two groups: isolates that show standard yeast morphology in rich medium at 30°C are in blue; and isolates that show filamentous growth under these conditions are in red. (B) Summary of mold isolates. The isolates are categorized as: “Itraconazole resistant” if their relative growth with fixed concentration of itraconazole at 0.5 μg/ml was greater than 2 times that of the relative growth of reference A. fumigatus strain AF293; and “Itraconazole susceptible” if their relative growth was less than 2 times the relative growth of AF293
Fig 3.
Filamentous growth of C. albicans clinical isolates is caused by non-synonymous mutation in NRG1.
(A) DIC images of C. albicans isolates from patient CF170 cultured in YPD at 30°C. C. albicans isolates Y1 and Y2 grow in the yeast form, while the filamentous isolate F1 grow as a mixture of yeast and filaments. Scale bar represents 20 μm. (B) Schematic representation of NRG1F1, showing the homozygous D271N mutation in the C2H2 zinc finger DNA binding domain (red bar). (C) Functional validation that NRG1F1 confers filamentous growth. Deletion of one allele of NRG1 in Y1 (Y1 NRG1Y1/nrg1Δ) does not alter yeast growth morphology, while replacement of the remaining NRG1 allele in this background with the F1 NRG1 allele (Y1 NRG1F1/nrg1Δ) causes a filamentation phenotype comparable to that observed in F1. This was also true in an independent laboratory strain, SN95 [38]. Replacing one NRG1 allele of F1 with the Y1 allele restores yeast form growth. Finally, a homozygous NRG1 deletion mutant (nrg1Δ/nrg1Δ) in an independent background [39] also has comparable growth morphology to F1.
Fig 4.
Prevalence of NRG1 mutations in filamentous C. albicans isolates from multiple patients.
Schematic representation of NRG1, the C2H2 zinc finger DNA binding domain (red bar), and the mutations found in different filamentous isolates and the corresponding microscopy images of representative isolates. Scale bar represents 20 μm.
Fig 5.
Phenazine methosulfate (PMS) or P. aeruginosa inhibits filamentation of C. albicans with standard growth morphology (Y1), but not of filamentous isolate F1.
The non-inducing condition is synthetic defined (SD) medium at 30°C and the inducing condition is SD + 5 mM N-acetylglucosamine at 37°C for 48 h. Treatments included 5 μM PMS or 100 μl of PA14 P. aeruginosa overnight culture, as indicated. Images of colonies are provided in the first and third rows, and DIC microscopy images of cells from the colony are in the second and fourth rows. Scale bar on spot image represents 2 mm, and scale bar on DIC image represents 10 μm.