The fungal pathogen Aspergillus fumigatus regulates growth, metabolism, and stress resistance in response to light

Abstract

Light is a pervasive environmental factor that regulates development, stress resistance, and even virulence in numerous fungal species. Though much research has focused on signaling pathways in Aspergillus fumigatus, an understanding of how this pathogen responds to light is lacking. In this report, we demonstrate that the fungus does indeed respond to both blue and red portions of the visible spectrum. Included in the A. fumigatus light response is a reduction in conidial germination rates, increased hyphal pigmentation, enhanced resistance to acute ultraviolet and oxidative stresses, and an increased susceptibility to cell wall perturbation. By performing gene deletion analyses, we have found that the predicted blue light receptor LreA and red light receptor FphA play unique and overlapping roles in regulating the described photoresponsive behaviors of A. fumigatus. However, our data also indicate that the photobiology of this fungus is complex and likely involves input from additional photosensory pathways beyond those analyzed here. Finally, whole-genome microarray analysis has revealed that A. fumigatus broadly regulates a variety of metabolic genes in response to light, including those involved in respiration, amino acid metabolism, and metal homeostasis. Together, these data demonstrate the importance of the photic environment on the physiology of A. fumigatus and provide a basis for future studies into this unexplored area of its biology.

FIG 1

Characterization of the LreA and FphA photoreceptors in A. fumigatus. (A) Cartoon depiction of the domain architectures for the WC-1/LreA and phytochrome proteins in N. crassa, A. nidulans, and A. fumigatus. The A. fumigatus models are based upon the predicted protein sequences found in GenBank. Relative differences in protein length (aa, amino acids) and domain separation are depicted but are not shown to scale. Domain functions are described in the text. p-Q, polyglutamine stretch; Zn, zinc finger domain; HK, histidine kinase; ATP, ATPase; RRD, response regulator domain. (B) qRT-PCR analysis of lreA and fphA from a representative time course experiment. Bars reflect the 2^−∆∆CT values relative to the 0-min time point (plus standard deviations [SD] [error bars] of 3 technical replicates). (C) (Top) Split-marker deletion strategy of lreA and fphA. The lreA and fphA genes were replaced with hph (hygromycin resistance) for the single deletion mutants. The fphA gene was replaced with bleR (phleomycin/bleomycin resistance) in the ∆lreA background to generate the double mutant. (Bottom) RT-PCR demonstrating the expected loss of transcript(s) in the respective deletion strains.

FIG 2

A. fumigatus photopigmentation response. (A) (Left) A. fumigatus Af293 grown in an alternating 12-h dark/12-h white light environment for 6 days. (Right) Af293 after 72 h of constant darkness or constant illumination conditions, as indicated. (B) WT or deletion mutants (∆lreA, ∆fphA, and ∆lreA ∆fphA [∆∆] mutants) after 72 h of constant darkness [D] or constant white light illumination [L]. All experiments were performed on GMM plates and incubated at 37°C. All pictures are scans of the plate bottoms shown as grayscale images.

FIG 3

Light regulation of conidial germination in A. fumigatus. (A) Germination rates of Af293 conidia under constant illumination conditions. Each light condition was performed at different times, and each light condition was run concurrently with a dark time course experiment. The averages of the three dark time course experiments are shown (±SD). (B) (Left) Germination rates of WT or mutant strains in constant darkness. (Right) Percentage of germination after 8 h in constant darkness [D] or constant red plus blue light illumination [L]. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) Germination rates of the WT versus ∆lreA mutant in constant darkness versus constant blue light illumination. Abbreviations: D, constant darkness; L, constant blue light illumination. (D) Proposed model for light-regulated germination. Red light inhibits FphA directly, whereas blue light inhibits FphA via an unknown blue light sensor. All germination experiments were performed in liquid GMM and incubated at 37°C.

FIG 4

Light induction of stress resistance in A. fumigatus. (A) qRT-PCR analysis of DNA repair genes in a representative time course experiment. Bars reflect the 2^−∆∆CT values, relative to the 0-min time point for that strain (plus SD of 3 technical replicates). (B) Schematic of the UV or H₂O₂ stress resistance assays performed. All cultures were incubated at 37°C on GMM plates. (C) (Left) Comparison of white, blue, and red light treatment regimens in the UV stress assay using strain Af293.(Right) The UV stress assay, using white light, with the ∆lreA ∆fphA mutant. (D) Comparison of WT versus the mutant strains in the H₂O₂ assay. Each of the mutants strains was tested separately, and each strain was tested with its own WT control. The average of the three WT experiments is shown (plus SD). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 5

Influence of light on cell wall homeostasis. (A) Susceptibility profiles of WT A. fumigatus to Congo red under the indicated light conditions. (B) Comparison of WT, ∆fphA, and ∆fphA R’ strains grown in the dark versus white light. All pictures were taken after 48-h incubation at 37°C.