Prolonged Subculturing of Aspergillus fumigatus on Galleria Extract Agar Results in Altered Virulence and Sensitivity to Antifungal Agents

Author:

Aaron Curtis (IE)

Abstract:

Background:
Prolonged colonisation by Aspergillus fumigatus may lead to an altered phenotype in vivo. The aim of the work presented here was to examine the effect of serially subculturing A. fumigatus on agar generated using Galleria mellonella larvae extract in order to characterize the alterations in the phenotypes that may occur.
Methods:
Gallerial extract agar (termed GEA20) was produced by grinding 20 G. mellonella larvae in 20 mL of sterile phosphate-buffered saline (PBS) using a sterile mortar and pestle. The extract was centrifuged at 538× g for 5 min to remove particulate matter, and 20 mL of the supernatant was added to 80 mL of autoclaved agar (2 g w/v) supplemented with 0.1 g (w/v) of glucose, allowed to cool prior to addition, and 100 μL of penicillin–streptomycin (pen–strep) A. fumigatus conidia were point inoculated onto GEA20 plates and incubated at 37 °C until growth reached the edge of the agar plate, which took an average of 9.24 days before subculturing onto fresh GEA20 plates. The isolated strains were assessed for gliotoxin production via HPLC analysis, antimicrobial susceptibility in 96 well plate assays and proteomic alterations through label free mass spectrometry.
Results: After 25 passages over 231 days A. fumigatus showed alterations in virulence, antifungal susceptibility, and in protein abundances. Passaged strains demonstrated reduced virulence in G. mellonella larvae and increased tolerance to hemocyte-mediated killing, hydrogen peroxide, itraconazole, and amphotericin B. A label-free proteomic analysis of control and passaged A. fumigatus strains revealed a total of 3329 proteins, of which 1902 remained following filtration, and 32 proteins were statistically significant and differentially abundant. Proteins involved in the response to oxidative stress (e.g. (S)-S-oxide reductase (+2.63-fold), developmental regulator FlbA (+2.27-fold), and histone H2A.Z (−1.82-fold)) were altered in abundance in the passaged strain. These results indicate that the prolonged subculturing of A. fumigatus on Galleria extract agar results in alterations in the susceptibility to antifungal agents and in the abundance of proteins associated with the oxidative stress response.
Conclusions:
Prolonged subculturing of A. fumigatus on GEA20 plates resulted in an alteration in the phenotype and proteome. The passaged strains demonstrated reduced virulence in vivo and increased tolerance to hemocyte-mediated killing, hydrogen peroxide, itraconazole, and amphotericin B. This tolerance may be due to the proteomic alterations evident in the passaged strains conferring tolerance to oxidative stress. Prolonged A. fumigatus colonization in vivo may also lead to strains better adapted to the pulmonary environment as well as those that display enhanced tolerance to antifungal agents. Such a process may have implications for human health, as inadvertent selection for drug-tolerant and persistent strains could complicate therapy.

Abstract Number: 76

Conference Year: 2024

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