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Parasexual recombination enables Aspergillus fumigatus to persist in cystic fibrosis

Tobias Engel, Paul E. Verweij, Joost van den Heuvel, Dechen Wangmo, Jianhua Zhang, Alfons J.M. Debets, Eveline Snelders
ERJ Open Research 2020 6: 00020-2020; DOI: 10.1183/23120541.00020-2020
Tobias Engel
1Radboud University Medical Center, Nijmegen, the Netherlands
2Center of Expertise in Mycology Radboudumc/CWZ, Nijmegen, the Netherlands
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  • ORCID record for Tobias Engel
  • For correspondence: eveline.snelders@wur.nl
Paul E. Verweij
1Radboud University Medical Center, Nijmegen, the Netherlands
2Center of Expertise in Mycology Radboudumc/CWZ, Nijmegen, the Netherlands
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Joost van den Heuvel
3Laboratory of Genetics, Wageningen University and Research, Wageningen, the Netherlands
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Dechen Wangmo
3Laboratory of Genetics, Wageningen University and Research, Wageningen, the Netherlands
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Jianhua Zhang
3Laboratory of Genetics, Wageningen University and Research, Wageningen, the Netherlands
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  • ORCID record for Jianhua Zhang
Alfons J.M. Debets
3Laboratory of Genetics, Wageningen University and Research, Wageningen, the Netherlands
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Eveline Snelders
3Laboratory of Genetics, Wageningen University and Research, Wageningen, the Netherlands
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  • ORCID record for Eveline Snelders
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  • FIGURE 1
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    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
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    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
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    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
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    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
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    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

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  • TABLE 1

    Ploidy analysis of Aspergillus fumigatus isolates with a conidial size of ≥2.90 µm

    Isolate numberGroupConidial size µmPloidy
    V132-02CF3.49Diploid
    V124-78CF3.17Diploid
    V104-11CF3.15Diploid
    V208-76CF3.14Diploid
    V108-12CF3.14Diploid
    V130-40CPA3.10Diploid
    V194-19CF3.09Diploid
    V109-60CF3.09Diploid
    V209-70CF3.08Diploid
    V133-16CF3.07Diploid
    V138-11CF3.03Haploid
    V183-71CF3.02Diploid
    V113-61CF3.02Inconclusive ploidy
    V214-14CPA3.00Inconclusive ploidy
    V158-23CF2.98Inconclusive ploidy
    V214-17CPA2.95Haploid
    V172-54CF2.93Haploid
    V224-65CPA2.91Haploid
    V211-47CPA2.91Haploid
    V130-14CPA2.90Haploid

    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.

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      Supplementary material 00020-2020.supplement

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    Parasexual recombination enables Aspergillus fumigatus to persist in cystic fibrosis
    Tobias Engel, Paul E. Verweij, Joost van den Heuvel, Dechen Wangmo, Jianhua Zhang, Alfons J.M. Debets, Eveline Snelders
    ERJ Open Research Oct 2020, 6 (4) 00020-2020; DOI: 10.1183/23120541.00020-2020

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    Parasexual recombination enables Aspergillus fumigatus to persist in cystic fibrosis
    Tobias Engel, Paul E. Verweij, Joost van den Heuvel, Dechen Wangmo, Jianhua Zhang, Alfons J.M. Debets, Eveline Snelders
    ERJ Open Research Oct 2020, 6 (4) 00020-2020; DOI: 10.1183/23120541.00020-2020
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