Figures
- FIGURE 1
Aspergillus fumigatus isolates selection diagram. Isolates meeting the inclusion criteria are shown for all four groups. If isolates were excluded from the final selection, the reason for exclusion is indicated in the figure. The total number of isolates per group used for conidial size measurement is indicated per group as well as the origin of sample type. a) Cystic fibrosis (CF) patients; b) chronic lung disease; c) acute invasive aspergillosis; and d) environmental. BAL: bronchoalveolar lavage; EORTC/MSG: European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group.
- FIGURE 2
Boxplot analysis of 799 Aspergillus fumigatus isolates screened for conidial size. On the left boxplots are shown for each isolate group. On the right summary statistics from the boxplot analysis are depicted for each group (group size n, median, mean, standard deviation and interquartile range (IQR)). Due to the presence of diploids in the chronically colonised cystic fibrosis patients group and chronically colonised chronic lung disease patients group, the mean conidial size and the standard deviation is largest for these two groups.
- FIGURE 3
Conidial size confirmation by a and b) scanning electron microscopy (SEM), c and d) nuclear staining and e and f) benomyl susceptibility testing. The conidial size was assessed by using SEM and fluorescence microscopy, and both confirm a conidial size difference between the haploid and diploid isolate, while excluding the presence of bi-nucleated cells in the diploid isolate (d). The diploid isolate shows a reduced growth on benomyl and the so-called sectoring of the colony (f) (which are the haploidising conidia from an original diploid) that are both absent in the haploid isolate (e).
- FIGURE 4
Genotyping results of sequential Aspergillus fumigatus isolates of a cystic fibrosis patient. a) Microsatellite genotypes are each represented with a unique colour. The genotype marked in red with black stripes differs in one of the nine genetic markers from the red only. One isolate of the red with black stripes genotype developed voriconazole resistance due to an unknown mechanism. The four selected isolates of the red genotype that were analysed by using whole genome sequencing are shown. b) The arrows indicate the average number of single-nucleotide polymorphisms (SNPs) per year difference between the sequential isolates. c) The unique SNPs are indicated for each isolate with heterozygous accumulating SNPs in the second diploid isolate V209-70 segregating in the voriconazole-resistant isolate V230-14.
- FIGURE 5
The parasexual cycle in Aspergillus fumigatus. Parasexual recombination can be achieved for most fungi in laboratory experiments and is characterised by heterokaryon formation, heterozygous diploid formation, recombination and haploidisation. Heterokaryon incompatibility normally prevents parasexual recombination between non-isogenic strains in A. fumigatus (dashed black line), but in this study we show that during long-lasting fungal colonisation of the human lung, sufficient isogenic genetic variation is generated to make parasexual recombination of A. fumigatus effective. Somatic mutation may yield a heterokaryon or fusion of isogenic nuclei may yield a homozygous diploid which can both subsequently evolve into a heterozygous diploid by de novo mutations. A heterozygous diploid can continue to accumulate mutations or finally after haploidisation segregate into new recombinant genotypes.
Tables
- TABLE 1
Ploidy analysis of Aspergillus fumigatus isolates with a conidial size of ≥2.90 µm
Isolate number Group Conidial size µm Ploidy V132-02 CF 3.49 Diploid V124-78 CF 3.17 Diploid V104-11 CF 3.15 Diploid V208-76 CF 3.14 Diploid V108-12 CF 3.14 Diploid V130-40 CPA 3.10 Diploid V194-19 CF 3.09 Diploid V109-60 CF 3.09 Diploid V209-70 CF 3.08 Diploid V133-16 CF 3.07 Diploid V138-11 CF 3.03 Haploid V183-71 CF 3.02 Diploid V113-61 CF 3.02 Inconclusive ploidy V214-14 CPA 3.00 Inconclusive ploidy V158-23 CF 2.98 Inconclusive ploidy V214-17 CPA 2.95 Haploid V172-54 CF 2.93 Haploid V224-65 CPA 2.91 Haploid V211-47 CPA 2.91 Haploid V130-14 CPA 2.90 Haploid After conidial size measurements, ploidy level was confirmed by benomyl susceptibility and fluorescence-activated cell sorting (FACS) analysis. Ploidy levels were based on benomyl susceptibility and FACS ratio results. If the two markers were not in agreement, inconclusive ploidy was concluded. Diploid isolates were only detected in the cystic fibrosis (CF) and chronic pulmonary aspergillosis (CPA) groups. No isolates with a conidial size ≥2.90 µm were detected in the acute invasive aspergillosis or environmental group.
Supplementary Material
Please note: supplementary material is not edited by the Editorial Office, and is uploaded as it has been supplied by the author.
Supplementary material 00020-2020.supplement
