An LaeA- and BrlA-Dependent Cellular Network Governs Tissue-Specific Secondary Metabolism in the Human Pathogen Aspergillus fumigatus

Abstract

Biosynthesis of many ecologically important secondary metabolites (SMs) in filamentous fungi is controlled by several global transcriptional regulators, like the chromatin modifier LaeA, and tied to both development and vegetative growth. In Aspergillus molds, asexual development is regulated by the BrlA > AbaA > WetA transcriptional cascade. To elucidate BrlA pathway involvement in SM regulation, we examined the transcriptional and metabolic profiles of ΔbrlA, ΔabaA, and ΔwetA mutant and wild-type strains of the human pathogen Aspergillus fumigatus. We find that BrlA, in addition to regulating production of developmental SMs, regulates vegetative SMs and the SrbA-regulated hypoxia stress response in a concordant fashion to LaeA. We further show that the transcriptional and metabolic equivalence of the ΔbrlA and ΔlaeA mutations is mediated by an LaeA requirement preventing heterochromatic marks in the brlA promoter. These results provide a framework for the cellular network regulating not only fungal SMs but diverse cellular processes linked to virulence of this pathogen. IMPORTANCE Filamentous fungi produce a spectacular variety of small molecules, commonly known as secondary or specialized metabolites (SMs), which are critical to their ecologies and lifestyles (e.g., penicillin, cyclosporine, and aflatoxin). Elucidation of the regulatory network that governs SM production is a major question of both fundamental and applied research relevance. To shed light on the relationship between regulation of development and regulation of secondary metabolism in filamentous fungi, we performed global transcriptomic and metabolomic analyses on mutant and wild-type strains of the human pathogen Aspergillus fumigatus under conditions previously shown to induce the production of both vegetative growth-specific and asexual development-specific SMs. We find that the gene brlA, previously known as a master regulator of asexual development, is also a master regulator of secondary metabolism and other cellular processes. We further show that brlA regulation of SM is mediated by laeA, one of the master regulators of SM, providing a framework for the cellular network regulating not only fungal SMs but diverse cellular processes linked to virulence of this pathogen.

Keywords: biosynthetic gene cluster; conidia; hyphal growth; hypoxia; mycelial growth; specialized metabolism; srbA; velvet protein complex.

FIG 1

Genes underexpressed in the ΔbrlA mutant are involved in a diverse set of cellular processes. Shown are results from Gene Ontology (GO) enrichment analysis for genes underexpressed in (A) ΔbrlA (selected), (B) ΔabaA, and (C) ΔwetA mutant strains compared to the wild type. The percentage of underexpressed genes in each GO category was calculated by dividing the number of genes in the category that are underexpressed by the total number of genes in the category. Only a representative subset of categories is shown for the ΔbrlA mutant; the full list of statistically enriched categories is provided in Table S2.

FIG 2

BrlA, AbaA, and WetA transcriptionally regulate many biosynthetic gene clusters (BGCs) involved in secondary metabolism. Shown is expression of all secondary metabolic BGCs in Aspergillus fumigatus in all strains tested. (A) Expression in the ΔbrlA, ΔabaA, and ΔwetA mutants. BGCs in which half or more of the genes are overexpressed (overexpressed clusters) are shown in blue, BGCs in which half or more of the genes BGC are underexpressed (underexpressed clusters) are shown in red, and BGCs in which half or more of the genes were differentially expressed but did not have half or more genes either overexpressed or underexpressed (mixed expression) are shown in purple. (B) Overlap between BGCs underexpressed in the ΔbrlA, ΔabaA, and ΔwetA mutants.

FIG 3

Secondary metabolites are produced at lower levels in the ΔbrlA strain relative to the wild type. Summary of metabolite production in ΔbrlA, ΔabaA, and ΔwetA mutants relative to the wild-type strain. Heat map colors represent log₂ fold change in peak area intensity, and gray indicates no metabolite was detected.

FIG 4

Levels of secondary metabolites produced by wild-type and ΔbrlA, ΔabaA, and ΔwetA mutant cultures. Shown is peak area intensity representing total production of representative metabolites from differentially expressed BGCs in A. fumigatus. Metabolite analysis was performed using the same two replicate samples from the RNA-seq experiment. Error bars depict standard deviation. Asterisks depict statistical significance compared with the wild type: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

FIG 5

Chromosomal location of all biosynthetic gene clusters involved in secondary metabolism regulated by BrlA and by LaeA. White chromosomes depict BGCs regulated by LaeA, and gray chromosomes depict BGCs regulated by BrlA.

FIG 6

LaeA activity impacts brlA transcript levels via chromatin modification. (A) Northern analysis depicting expression of brlA and laeA in the ΔbrlA mutant compared to the WT. (B) Northern analysis depicting expression of brlA and laeA in the ΔlaeA mutant compared to the WT. (C) Chromatin immunoprecipitation examining histone modifications of the brlA promoter. H3 depicts total histone 3 occupancy, H3K4me3 (histone H3 trimethyl K4) depicts euchromatic marks, and H3K9me3 (histone H3 acetyl K9) depicts heterochromatic marks. Error bars depict standard deviation. Three biological replicates were performed. (D) Predicted transcription factors in A. fumigatus with >0.5 log₂ fold change in the ΔlaeA mutant (gray bars). Log₂ fold changes of the same transcription factors in the ΔbrlA mutant are shown by blue bars.

FIG 7

Model framework for the cellular network regulating fungal secondary metabolism and diverse cellular processes. Under our proposed model, the chromatin modifier LaeA, by epigenetically regulating the transcription factor BrlA, controls secondary metabolism in the context of fungal vegetative growth and asexual development, as well as additional cellular processes, such as the hypoxia response.