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[Preprint]. 2020 Nov 6:2020.11.06.371971.
doi: 10.1101/2020.11.06.371971.

Genomic and phenotypic analysis of COVID-19-associated pulmonary aspergillosis isolates of Aspergillus fumigatus

Jacob L Steenwyk  1 , Matthew E Mead  1 , Patrícia Alves de Castro  2 , Clara Valero  2 , André Damasio  3   4 , Renato A C Dos Santos  2 , Abigail L Labella  1 , Yuanning Li  1 , Sonja L Knowles  5 , Huzefa A Raja  5 , Nicholas H Oberlies  5 , Xiaofan Zhou  6 , Oliver A Cornely  7   8   9   10 , Frieder Fuchs  11 , Philipp Koehler  7   8 , Gustavo H Goldman  2 , Antonis Rokas  1
Affiliations

Genomic and phenotypic analysis of COVID-19-associated pulmonary aspergillosis isolates of Aspergillus fumigatus

Jacob L Steenwyk et al. bioRxiv. .

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Abstract

The ongoing global pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) first described from Wuhan, China. A subset of COVID-19 patients has been reported to have acquired secondary infections by microbial pathogens, such as fungal opportunistic pathogens from the genus Aspergillus . To gain insight into COVID-19 associated pulmonary aspergillosis (CAPA), we analyzed the genomes and characterized the phenotypic profiles of four CAPA isolates of Aspergillus fumigatus obtained from patients treated in the area of North Rhine-Westphalia, Germany. By examining the mutational spectrum of single nucleotide polymorphisms, insertion-deletion polymorphisms, and copy number variants among 206 genes known to modulate A. fumigatus virulence, we found that CAPA isolate genomes do not exhibit major differences from the genome of the Af293 reference strain. By examining virulence in an invertebrate moth model, growth in the presence of osmotic, cell wall, and oxidative stressors, and the minimum inhibitory concentration of antifungal drugs, we found that CAPA isolates were generally, but not always, similar to A. fumigatus reference strains Af293 and CEA17. Notably, CAPA isolate D had more putative loss of function mutations in genes known to increase virulence when deleted (e.g., in the FLEA gene, which encodes a lectin recognized by macrophages). Moreover, CAPA isolate D was significantly more virulent than the other three CAPA isolates and the A. fumigatus reference strains tested. These findings expand our understanding of the genomic and phenotypic characteristics of isolates that cause CAPA.

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Conflict of interest statement

Conflict of Interest

Oliver A. Cornely is supported by the German Federal Ministry of Research and Education, is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy - CECAD, EXC 2030 – 390661388 and has received research grants from, is an advisor to, or received lecture honoraria from Actelion, Allecra Therapeutics, Al-Jazeera Pharmaceuticals, Amplyx, Astellas, Basilea, Biosys, Cidara, Da Volterra, Entasis, F2G, Gilead, Grupo Biotoscana, IQVIA, Janssen, Matinas, Medicines Company, MedPace, Melinta Therapeutics, Menarini, Merck/MSD, Mylan, Nabriva, Noxxon, Octapharma, Paratek, Pfizer, PSI, Roche Diagnostics, Scynexis, and Shionogi. Philipp Koehler has received non-financial scientific grants from Miltenyi Biotec GmbH, Bergisch Gladbach, Germany, and the Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany, and received lecture honoraria from or is advisor to Akademie für Infektionsmedizin e.V., Astellas Pharma, European Confederation of Medical Mycology, Gilead Sciences, GPR Academy Ruesselsheim, MSD Sharp & Dohme GmbH, Noxxon N.V., and University Hospital, LMU Munich outside the submitted work. Antonis Rokas is a Scientific Consultant for LifeMine Therapeutics, Inc.

Figures

Figure 1.
Figure 1.. Inhalation of Aspergillus spores can result in fungal infection.
Inhalation of Aspergillus spores from the environment can travel to the lung and then grow vegetatively and spread to other parts of the body.
Figure 2.
Figure 2.. Phylogenomics confirms CAPA isolates are Aspergillus fumigatus and mutational spectra among genetic determinants of virulence.
(A) Phylogenomic analysis of a concatenated matrix of 4,525 single-copy orthologous groups genes (sites: 7,133,367) confirmed CAPA isolates are A. fumigatus. Furthermore, CAPA isolates are closely related to reference strains A1163 and Af293. Bipartitions with less than 85% ultrafast bootstrap approximation support were collapsed. (B) Genome-wide SNPs, indels, and CN variants were filtered for those present in genetic determinants of virulence. Thereafter, the number of genetic determinants of virulence with high impact polymorphisms were identified. The number known to increase or decrease virulence in null mutants was determined thereafter.
Figure 3.
Figure 3.. CAPA isolates have BGCs encoding the toxic small molecule gliotoxin.
Gliotoxin is known to contribute to virulence of A. fumigatus. The genomes of CAPA isolates of A. fumigatus contain biosynthetic gene clusters known to encode gliotoxin. Note, the BGC of CAPA A was split between two contigs and therefore the BGC is hypothesized to be present.
Figure 4.
Figure 4.. Strain heterogeneity among CAPA isolates.
CAPA isolates and reference strains Af293 and CEA17 virulence significantly varied in the Galleria moth model of disease (p < 0.001; log-rank test). Pairwise examinations revealed CAPA D was significantly more virulent than all other strains (Benjamini-Hochberg adjusted p-value < 0.01 when comparing CAPA isolate D to another isolate; log-rank test). Growth of CAPA isolates and references strains Af293 and CEA17 in the presence of (B) osmotic, (C) cell wall, and (D) oxidative stressors. Growth differences between CAPA isolates and reference strains Af293 and CEA17 were observed across all growth conditions (p < 0.001; multi-factor ANOVA). Pairwise differences were assessed using the post-hoc Tukey Honest Significant Differences test and were only observed for growth in the presence of CFW at 25 μg/mL (p < 0.001; Tukey Honest Significant Differences test) in which the CAPA isolates did not grow as well as the reference isolates. To correct for strain heterogeneity in growth rates, radial growth in centimeters in the presence of stressors was divided by radial growth in centimeters in the absence of the stressor (MM only). Abbreviations of cell wall stressors are as follows: CFW: calcofluor white; CR: congo red; CSP: caspofungin. Growth in the presence of other stressors is summarized in Supplementary Figure 4.
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