Parasexual recombination enables Aspergillus fumigatus to persist in cystic fibrosis

Contains fulltext : 229467.pdf (publisher's version ) (Open Access)Aspergillus fumigatus is a saprobic fungus that causes a range of pulmonary diseases, some of which are characterised by fungal persistence such as is observed in cystic fibrosis (CF) patients. Creation of genetic variation is critical for A. fumigatus to adapt to the lung environment, but biofilm formation, especially in CF patients, may preclude mutational supply in A. fumigatus due to its confinement to the hyphal morphotype. We tested our hypothesis that genetic variation is created through parasexual recombination in chronic biofilms by phenotypic and genetic analysis of A. fumigatus isolates cultured from different origins. As diploids are the hallmark of parasex, we screened 799 A. fumigatus isolates obtained from patients with CF, chronic pulmonary lung disease and acute invasive aspergillosis, and from the environment for spore size. Benomyl sensitivity, nuclear content measurements through fluorescence-activated cell sorting and scanning electron microscopy were used to confirm the diploid state of large size spores. Whole genome sequencing was used to characterise diploid-associated genetic variation. We identified 11 diploids in isolates recovered from six of 11 (55%) CF patients and from one of 24 (4%) chronic aspergillosis patients, but not in 368 isolates from patients with acute Aspergillus infection and the environment. Diploid formation was associated with accumulation of mutations and variable haploid offspring including a voriconazole-resistant isolate. Parasexual recombination allows A. fumigatus to adapt and persist in CF patients, and plays a role in azole resistance development. Our findings are highly significant for understanding the genetics and biology of A. fumigatus in the human lung

Asexual sporulation facilitates adaptation: The emergence of azole resistance in Aspergillus fumigatus.

<p>Understanding the occurrence and spread of azole resistance in Aspergillus fumigatus is crucial for public health. It has been hypothesized that asexual sporulation, which is abundant in nature, is essential for phenotypic expression of azole resistance mutations in A. fumigatus facilitating subsequent spread through natural selection. Furthermore, the disease aspergilloma is associated with asexual sporulation within the lungs of patients and the emergence of azole resistance. This study assessed the evolutionary advantage of asexual sporulation by growing the fungus under pressure of one of five different azole fungicides over seven weeks and by comparing the rate of adaptation between scenarios of culturing with and without asexual sporulation. Results unequivocally show that asexual sporulation facilitates adaptation. This can be explained by the combination of more effective selection because of the transition from a multicellular to a unicellular stage, and by increased mutation supply due to the production of spores, which involves numerous mitotic divisions. Insights from this study are essential to unravel the resistance mechanisms of sporulating pathogens to chemical compounds and disease agents in general, and for designing strategies that prevent or overcome the emerging threat of azole resistance in particular.</p

Bioconjugation with strained alkenes and alkynes

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The Medical Triazole Voriconazole Can Select for Tandem Repeat Variations in Azole-Resistant Aspergillus Fumigatus Harboring TR(34)/L98H Via Asexual Reproduction

Contains fulltext : 229515.pdf (publisher's version ) (Open Access)Azole-resistant Aspergillus fumigatus isolates recovered at high frequency from patients, harbor mutations that are associated with variation of promoter length in the cyp51A gene. Following the discovery of the TR(34)/L98H genotype, new variations in tandem repeat (TR) length and number of repeats were identified, as well as additional single nucleotide polymorphisms (SNPs) in the cyp51A gene, indicating that the diversity of resistance mutations in A. fumigatus is likely to continue to increase. Investigating the development routes of TR variants is critical to be able to design preventive interventions. In this study, we tested the potential effects of azole exposure on the selection of TR variations, while allowing haploid A. fumigatus to undergo asexual reproduction. Through experimental evolution involving voriconazole (VOR) exposure, an isolate harboring TR(34)(3)/L98H evolved from a clinical TR(34)/L98H ancestor isolate, confirmed by whole genome sequencing. TR(34)(3)/L98H was associated with increased cyp51A expression and high VOR and posaconazole MICs, although additional acquired SNPs could also have contributed to the highly azole-resistant phenotype. Exposure to medical azoles was found to select for TR(34)(3), thus supporting the possibility of in-host selection of TR(34) variants

Evolution of cross-resistance to medical triazoles in Aspergillus fumigatus through selection pressure of environmental fungicides

Resistance to medical triazoles in Aspergillus fumigatus is an emerging problem for patients at risk of aspergillus diseases. There are currently two presumed routes for medical triazole-resistance selection: (i) through selection pressure of medical triazoles when treating patients and (ii) through selection pressure from non-medical sterol-biosynthesis-inhibiting (SI) triazole fungicides which are used in the environment. Previous studies have suggested that SI fungicides can induce cross-resistance to medical triazoles. Therefore, to assess the potential of selection of resistance to medical triazoles in the environment, we assessed cross-resistance to three medical triazoles in lineages of A. fumigatus from previous work where we applied an experimental evolution approach with one of five different SI fungicides to select for resistance. In our evolved lines we found widespread cross-resistance indicating that resistance to medical triazoles rapidly arises through selection pressure of SI fungicides. All evolved lineages showed similar evolutionary dynamics to SI fungicides and medical triazoles, which suggests that the mutations inducing resistance to both SI fungicides and medical triazoles are likely to be the same. Whole-genome sequencing revealed that a variety of mutations were putatively involved in the resistance mechanism, some of which are in known target genes

Metal-free triazole formation as a tool for bioconjugation

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In-host adaptation and acquired triazole resistance in Aspergillus fumigatus: a dilemma for clinical management.

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