LaeA, a regulator of secondary metabolism in Aspergillus spp

Abstract

Secondary metabolites, or biochemical indicators of fungal development, are of intense interest to humankind due to their pharmaceutical and/or toxic properties. We present here a novel Aspergillus nuclear protein, LaeA, as a global regulator of secondary metabolism in this genus. Deletion of laeA (DeltalaeA) blocks the expression of metabolic gene clusters, including the sterigmatocystin (carcinogen), penicillin (antibiotic), and lovastatin (antihypercholesterolemic agent) gene clusters. Conversely, overexpression of laeA triggers increased penicillin and lovastatin gene transcription and subsequent product formation. laeA expression is negatively regulated by AflR, a sterigmatocystin Zn2Cys6 transcription factor, in a unique feedback loop, as well as by two signal transduction elements, protein kinase A and RasA. Although these last two proteins also negatively regulate sporulation, DeltalaeA strains show little difference in spore production compared to the wild type, indicating that the primary role of LaeA is to regulate metabolic gene clusters.

FIG. 1.

Overview of LaeA. (A) Schematic of laeA gene. Although LaeA contains the exact SAM motif found in histone methyltransferases and arginine methyltransferases, it lacks other conserved domains (e.g., a SET domain and a double E loop) typically found in these proteins. In addition, likely histone methyltransferase and arginine methyltransferase candidates are found in the Aspergillus database (1e⁻⁴² and 1e⁻⁹⁴). Therefore, LaeA appears to be a unique protein methyltransferase. (B) Amino acid comparison of A. nidulans, A. fumigatus, N. crassa, Magnaporthe grisea, C. immitis, and F. sporotrichioides LaeA proteins showing conserved protein methyltransferase SAM binding sites in red. (C) A. nidulans LaeA protein localizes to the nucleus. GFP was fused to the N-terminal end of LaeA. Nuclei were stained with the DNA-specific dye 4,6-diamidino-2-phenylindole (DAPI).

FIG. 2.

Phenotypes of laeA. (A) Asexual sporulation (top) and mycelial pigmentation (bottom) patterns of A. nidulans wild-type (RDIT2.3) (WT) and ΔlaeA (RJW46.4) strains after 5 days of cultivation on GMM. A. fumigatus ΔlaeA presented a similar loss of mycelial pigmentation (data not shown). (B) TLC analysis of chloroform extracts of RDIT2.3 and RJW46.4 after 5 days of cultivation on solid GMM. (C) TLC analysis of chloroform extracts of A. fumigatus AF293 (WT) and TJW54.2 (ΔlaeA) grown in liquid shaking GMM for 3 days. The experiment was performed in triplicate. Std, gliotoxin standard.

FIG. 3.

laeA regulation of secondary metabolism. (A) aflR, stcU, and ipnA gene expression in A. nidulans wild-type (WT) (RDIT2.3) and ΔlaeA (RJW46.4) strains grown in liquid shaking GMM for 12, 24, 48, and 72 h at 37°C. Ethidium bromide-stained rRNA is indicated as a loading control. (B) laeA, lovE, and lovC gene expression in A. nidulans WT/lov+ (RJW51) and ΔlaeA/lov+ (RJW53) strains grown in liquid shaking GMM for 12, 24, 48, and 72 h at 37°C. Ethidium bromide-stained rRNA is indicated as a loading control. MONJ was extracted from WT/lov+ (RJW51) and ΔlaeA/lov+ (RJW53) A. nidulans strains grown in liquid shaking GMM for 3 days. The experiment was performed in triplicate. (C) laeA, ipnA, stcU, lovE, and lovC gene expression in A. nidulans wild-type (WT) (RDIT2.3), OE::laeA (RJW47.3), WT/lov+ (RJW51), and OE::laeA/lov+ (RJW52) strains grown in liquid shaking GMM for 14 h at 37°C and then transferred to liquid shaking TMM for the induction of laeA expression. Time points were 0, 6, 12, and 24 h after transfer. ST and MONJ were extracted from A. nidulans WT (RDIT2.3), OE::laeA (RJW47.3), WT/lov+ (RJW51), and OE::laeA/lov+ (RJW52) strains grown in liquid shaking GMM for 14 h at 37°C and then transferred to liquid shaking TMM for 24 h. ST, ST standard. The MONJ standard was extracted from A. nidulans strain WMH1739. The experiment was performed in triplicate. (D) PN bioassay. Wild-type (FGSC26), ΔlaeA (RJW40.4), and OE::laeA (RJW44.2) strains were grown in liquid shaking GMM for 14 h at 37°C and then transferred to LMM amended with 30 mM cyclopentanone for the induction of laeA for 24 h at 37°C. (E) TLC examination of LOV production in A. terreus laeA overexpression strains. The wild type (ATCC 20542; lane 1), TJW58.9 (hygB resistance gene-containing transformant used as a control; lane 2), and OE::laeA strains containing hygB (TJW58.2, TJW58.4, TJW58.7, TJW58.8, and TJW58.14, in lanes 3 to 7, respectively) were grown in liquid shaking GMM for 18 h at 32°C and then transferred to LMM with 30 mM cyclopentanone for the induction of laeA for 36 h at 32°C. Std, LOV standard. The experiment was performed in duplicate.

FIG. 4.

Regulation of laeA. Effects of overexpression of aflR, pkaA, and ras^G17V on laeA expression. Wild-type (RKIS1), OE::aflR (TJH34.10), OE::pkaA (TKIS20.1), and OE::ras^G17V (RKIS28.5) strains were grown in liquid shaking GMM for 14 h at 37°C and then transferred to TMM. Time points were 0, 6, 12, and 24 h after transfer.

FIG. 5.

laeA expression is not affected in ΔflbA, ΔsfaD, and ΔaflR strains. laeA, aflR, and stcU gene expression was examined in A. nidulans wild-type (TPK1.1 and RKIS1), ΔflbA (TBN39.5), ΔsfaD (TSRB1.38), and ΔaflR (RMFV2) strains grown in liquid shaking GMM for 12, 24, 48, and 72 h at 37°C. Ethidium bromide-stained rRNA is indicated as a loading control.

FIG. 6.

Proposed model of secondary metabolite regulation by LaeA. Gliotoxin is formed from serine and alanine and is proposed to be produced by a nonribosomal peptide synthase (NRPS). Fungal pigments belong to several chemical classes, including polyketides, terpenes, and DOPA melanins.