Contributions of Spore Secondary Metabolites to UV-C Protection and Virulence Vary in Different Aspergillus fumigatus Strains

Abstract

Fungi are versatile organisms which thrive in hostile environments, including the International Space Station (ISS). Several isolates of the human pathogen Aspergillus fumigatus have been found contaminating the ISS, an environment with increased exposure to UV radiation. Secondary metabolites (SMs) in spores, such as melanins, have been shown to protect spores from UV radiation in other fungi. To test the hypothesis that melanin and other known spore SMs provide UV protection to A. fumigatus isolates, we subjected SM spore mutants to UV-C radiation. We found that 1,8-dihydroxynaphthalene (DHN)-melanin mutants of two clinical A. fumigatus strains (Af293 and CEA17) but not an ISS-isolated strain (IF1SW-F4) were more sensitive to UV-C than their respective wild-type (WT) strains. Because DHN-melanin has been shown to shield A. fumigatus from the host immune system, we examined all DHN mutants for virulence in the zebrafish model of invasive aspergillosis. Following recent studies highlighting the pathogenic variability of different A. fumigatus isolates, we found DHN-melanin to be a virulence factor in CEA17 and IF1SW-F4 but not Af293. Three additional spore metabolites were examined in Af293, where fumiquinazoline also showed UV-C-protective properties, but two other spore metabolites, monomethylsulochrin and fumigaclavine, provided no UV-C-protective properties. Virulence tests of these three SM spore mutants indicated a slight increase in virulence of the monomethylsulochrin deletion strain. Taken together, this work suggests differential roles of specific spore metabolites across Aspergillus isolates and by types of environmental stress.IMPORTANCE Fungal spores contain secondary metabolites that can protect them from a multitude of abiotic and biotic stresses. Conidia (asexual spores) of the human pathogen Aspergillus fumigatus synthesize several metabolites, including melanin, which has been reported to be important for virulence in this species and to be protective against UV radiation in other fungi. Here, we investigate the role of melanin in diverse isolates of A. fumigatus and find variability in its ability to protect spores from UV-C radiation or impact virulence in a zebrafish model of invasive aspergillosis in two clinical strains and one ISS strain. Further, we assess the role of other spore metabolites in a clinical strain of A. fumigatus and identify fumiquinazoline as an additional UV-C-protective molecule but not a virulence determinant. The results show differential roles of secondary metabolites in spore protection dependent on the environmental stress and strain of A. fumigatus As protection from elevated levels of radiation is of paramount importance for future human outer space explorations, the discovery of small molecules with radiation-protective potential may result in developing novel safety measures for astronauts.

Keywords: Aspergillus fumigatus; UV-C; melanin; secondary metabolite; space station; spore; virulence determinants.

FIG 1

UV-C sensitivity and secondary metabolite profiles of Af293, ΔakuA mutant, and ΔakuA -mluc mutant strains. (A) Percent viability following exposure to various doses of UV-C radiation of control strains. (B) Secondary metabolite profiles of control strains.

FIG 2

UV-C sensitivity and secondary metabolite profiles of Af293 and Af293 DHN-melanin mutants in the intact and disrupted akuA backgrounds. (A) Percent viability following exposure to various doses of UV-C radiation for Af293 and the ΔpksP, ΔakuA, and ΔpksP ΔakuA mutants. (B) Secondary metabolite profiles of Af293 and the ΔpksP, ΔakuA, and ΔpksP ΔakuA mutants. Asterisks (*) indicate statistical significance using Welch’s corrected t test (details in Table S2).

FIG 3

UV-C sensitivity and secondary metabolite profiles of ISS-isolated IF1SW-F4, CEA17, and their DHN-melanin mutants. (A) Percent viability following exposure to various doses of UV-C radiation for IF1SW-F4 and IF1SW-F4 ΔpksP. (B) Secondary metabolite profiles of IF1SW-F4 and IF1SW-F4 ΔpksP. (C) Percent viability following exposure to various doses of UV-C radiation for CEA17 and CEA17 ΔpksP. (D) Secondary metabolite profiles of CEA17 and CEA17 ΔpksP. Asterisks (*) indicate statistical significance using Welch’s corrected t test (details in Table S2).

FIG 4

UV-C sensitivity and secondary metabolite profiles of conidium-associated SM mutants of Af293 in backgrounds with disrupted and intact akuA. (A to C) Percent viability following exposure to various doses of UV-C radiation for ΔdmaW (A), ΔtpcC (B), and ΔfmqA (C) mutants in Af293 with intact akuA. (D to F) Percent viability following exposure to various doses of UV-C radiation for ΔdmaW (D), ΔtpcC (E), and ΔfmqA (F) in Af293 with disrupted akuA. Asterisks (*) indicate statistical significance using Welch’s corrected t test (details in Table S2).

FIG 5

Virulence of DHN-melanin mutants of three Aspergillus fumigatus strains in a zebrafish model of invasive aspergillosis. (A) Percent survival of zebrafish upon infection with Af293 and the ΔpksP mutant. (B) Percent survival of zebrafish upon infection with CEA17 and the ΔpksP mutant. (C) Percent survival of zebrafish upon infection with IF1SW-F4 and ΔpksP mutant. P values were generated by Cox proportional hazards regression analysis. DPI, days postinfection.

FIG 6

Virulence of three conidial SM mutants of Aspergillus fumigatus in a zebrafish model of invasive aspergillosis. Percent survival of zebrafish upon infection with Af293 WT and the ΔdmaW, ΔtpcC, and ΔfmqA mutants. P values were generated by Cox proportional hazards regression analysis.