Aspergillus fumigatus flbB encodes two basic leucine zipper domain (bZIP) proteins required for proper asexual development and gliotoxin production

Abstract

The opportunistic human pathogen Aspergillus fumigatus reproduces asexually by forming a massive number of mitospores called conidia. In this study, we characterize the upstream developmental regulator A. fumigatus flbB (AfuflbB). Northern blotting and cDNA analyses reveal that AfuflbB produces two transcripts predicted to encode two basic leucine zipper domain (bZIP) polypeptides, AfuFlbBβ (420 amino acids [aa]) and AfuFlbBα (390 aa). The deletion of AfuflbB results in delayed/reduced sporulation, precocious cell death, the lack of conidiophore development in liquid submerged culture, altered expression of AfubrlA and AfuabaA, and blocked production of gliotoxin. While introduction of the wild-type (WT) AfuflbB allele fully complemented these defects, disruption of the ATG start codon for either one of the AfuFlbB polypeptides leads to a partial complementation, indicating the need of both polypeptides for WT levels of asexual development and gliotoxin biogenesis. Consistent with this, Aspergillus nidulans flbB(+) encoding one polypeptide (426 aa) partially complements the AfuflbB null mutation. The presence of 0.6 M KCl in liquid submerged culture suppresses the defects caused by the lack of one, but not both, of the AfuFlbB polypeptides, suggesting a genetic prerequisite for AfuFlbB in A. fumigatus development. Finally, Northern blot analyses reveal that both AfuflbB and AfuflbE are necessary for expression of AfuflbD, suggesting that FlbD functions downstream of FlbB/FlbE in aspergilli.

Fig. 1.

Summary of AfuflbB. (A) Northern blot showing mRNA levels of AfuflbB throughout the life cycle of A. fumigatus WT (AF293) strain. Con, conidia. The time (hours) of incubation in liquid submerged culture (Veg) and of post-asexual developmental induction (Asex) is shown. Equal loading of total RNA was evaluated by ethidium bromide staining of rRNA. A band shown between the two AfuflbB transcripts is due to nonspecific binding of the probe to rRNA. (B) Schematic presentation of a genomic DNA region covering the AfuflbB gene and two transcripts. The AfuflbB ORF (shaded box), transcripts (arrows), and the introns (shown by discontinuity in the arrow) were verified by sequence analysis of AfuflbB cDNA. The start codon (ATG) of AfuFlbBβ is assigned as +1. (C) Alignment of N terminus of FlbB proteins in A. fumigatus (Afu) and A. nidulans (Ani). The predicted N termini (Met) of AfuFlbBβ, AfuFlbBα, and AniFlbB are marked. Note that the 42nd amino acid in A. nidulans FlbB is not methionine but isoleucine. Alignment was done by ClustalW (http://www.ch.embnet.org/software/ClustalW.html) and is presented by BOXSHADE, version 3.21 (http://www.ch.embnet.org/software/BOX_form.html).

Fig. 2.

Requirement of AfuflbB for proper asexual development. (A) Photographs of the colonies of WT (AF293), ΔAfuflbB (TKSS1.01), and complemented (C′; TPX5.10) strains grown on solid MMG with 0.5% YE at 37°C for 3 days (top panels) and the close-up views of the colonies (middle and bottom panels). (B) Progression of synchronized asexual development of the WT and ΔAfuflbB (Δ) strains on solid MMG with 0.1% YE. Numbers indicate the time (hours) postinduction. Note the color differences between the WT and ΔAfuflbB strains. (C) Northern blot analyses for levels of AfubrlA, AfuabaA, AfuwetA, and AfuvosA transcripts in the WT and ΔAfuflbB strains during the life cycle. Numbers indicate the time (hours) in liquid MMG with 0.1% YE (Veg) or of post-asexual developmental induction (Asex). Equal loading of total RNA was demonstrated by ethidium bromide staining of rRNA.

Fig. 3.

Effects of ΔAfuflbB in liquid submerged culture. (A) Photomicrographs of the mycelium of WT (AF293), ΔAfuflbB (Δ; TKSS1.01) and complemented (C′; TPX5.10) strains grown in liquid MMG with 0.1% YE for 18 h at 37°C and 250 rpm. The arrows indicate conidiophore structures. Note that WT and C′ strains started to produce vesicles at 18 h, whereas the ΔAfuflbB mutant fails to develop. (B) Viability of WT, ΔAfuflbB (Δ), and C′ strains grown in liquid MMG with 0.1% YE at 37°C and 250 rpm for the period of 5 days. Data represent the mean values (± standard deviations) of three independent experiments. Note that the ΔAfuflbB mutant exhibits accelerated cell death at days 4 and 5. (C) Apoptotic cell death levels of WT, ΔAfuflbB (Δ), and C′; strains grown in liquid MMG with 0.1% YE at 37°C for 4 days were examined by Evans blue staining. Note the clear differences in the levels of staining among the mycelia.

Fig. 4.

Both polypeptides are required for proper asexual development. (A) Schematics of the three AfuflbB alleles used for complementation. The predicted start codons (ATG) of the AfuflbB⁺ allele were replaced with GCC, leading to the AfuflbBα and AfuflbBβ alleles. (B) Photographs of the colonies of WT (AF293), ΔAfuflbB (Δ; TKSS1.01), C′ (TPX6.01; ΔAfuflbB AfuflbB⁺), α (TPX8.02; ΔAfuflbB AfuflbBα), and β (TPX7.06; ΔAfuflbB AfuflbBβ) strains grown on solid MMG with 0.1% YE for 3 days. (C) Progression of synchronized asexual development of the WT, ΔAfuflbB (Δ), C′, α, and β strains on solid MMG with 0.1% YE. Numbers indicate the time (hours) postinduction. The right panel shows the corresponding Northern blot of the samples taken from developmental induction. Equal loading of total RNA was demonstrated by ethidium bromide staining of rRNA. (D) Photomicrographs of the mycelium of the WT, ΔAfuflbB (Δ), C′, α, and β strains grown in liquid MMG with 0.1% YE at 37°C and 250 rpm for 18 h. Note that the ΔAfuflbB, α, and β strains do not produce any conidiophore structures, whereas the WT and C′ strains start to form vesicles (marked by arrows) at 18 h.

Fig. 5.

Differential requirement of AfuflbB and other developmental regulators for gliotoxin production. (A) Photographs of relevant strains grown in liquid submerged culture for 3 days. Note the differences in pigmentation. (B) TLC of gliotoxin produced after 2 days of liquid submerged culture of the WT (AF293), ΔAfuflbB (Δ; TKSS1.01), C′ (TPX6.01), α (TPX8.02), and β (TPX7.06) strains. Toluene-acetate-formic acid (5:4:1) was used as developing solvent. (C) Effects of ΔAfuflbB on expression of gliZ. Northern blot of gliZ at 2 days is presented. Equal loading of total RNA was demonstrated by ethidium bromide staining of rRNA. (D) TLC of the chloroform extracts of various strains including the ΔAfuflbE (TKSS6.07) and ΔAfubrlA (A1176) mutants grown in liquid MMG with 0.1% YE for 2 days or on solid MMG with 0.5% YE for 5 days. Note the lack of gliotoxin production by ΔAfubrlA. G, gliotoxin standard.

Fig. 6.

Differential suppression of developmental defects by 0.6 M KCl. (A) Photographs of the colonies of WT (AF293), ΔAfuflbB (Δ; TKSS1.01), C′ (TPX6.01), α (TPX8.02), and β (TPX7.06) strains grown on MMG with 0.1% YE and 0.6 M KCl at 37°C for 3 days. (B) Photomicrographs of the mycelium of the WT, ΔAfuflbB (Δ), C′, α, and β strains cultured for 14 h in liquid MMG with 0.1% YE and then transferred into MMG supplemented with 0.6 M KCl. Numbers indicate incubation time posttransfer. Conidiophore structures are marked by arrows. Note that only the ΔAfuflbB mutant failed to develop. (C) TLC of gliotoxin in the presence of 0.6 M KCl in liquid submerged culture. After growth in liquid MMG with 0.1% YE for 14 h, the mycelia of relevant strains were transferred into MMG supplemented with 0.6 M KCl and further cultured at 37°C for 24 h. G, gliotoxin standard.

Fig. 7.

Partial complementation of ΔAfuflbB by AniflbB. (A) Phenotypic analyses of the WT (AF293), ΔAfuflbB (Δ; TKSS1.01), complemented (C′; ΔAfuflbB AfuflbB⁺; TPX5.10) and cross-complemented (Ani; ΔAfuflbB AniflbB⁺; TPX9.03) strains grown on solid MMG with 0.1% YE at 37°C for 3 days or in liquid MMG with 0.1% YE at 37°C and 250 rpm, for 18 h. (B) Northern blot showing mRNA levels of AfuflbB and AniflbB in A. fumigatus WT, ΔAfuflbB, C′, Ani, and A. nidulans WT (FGSC4) strains. C, conidia; V, 12-h vegetative; AS, 12-h asexual induction. Note that AniflbB is highly expressed under the ΔAfuflbB background. (C) TLC for gliotoxin production in liquid submerged culture of relevant strains for 3 days. G, gliotoxin standard. (D) Progression of synchronized asexual development in WT (AF293), ΔAfuflbB (Δ), C′, and Ani strains on solid MMG with 0.1% YE. Numbers indicate the time (hours) postinduction. Right panel shows the corresponding Northern blot for AfubrlA in the samples taken from asexual induction. Equal loading of total RNA was demonstrated by ethidium bromide staining of rRNA.

Fig. 8.

Genetic interactions between upstream developmental regulators in A. fumigatus. (A) Levels of AfuflbB, AfuflbC, AfuflbD, and AfuflbE transcripts in WT (AF293), ΔAfuflbB (TKSS1.01), and ΔAfuflbE (TKSS6.07) strains throughout the life cycle. Numbers indicate the time (hours) in liquid MMG with 0.1% YE (Veg) or of post-asexual developmental induction (Asex). A band shown between the two AfuflbB transcripts is due to nonspecific binding of the probe to rRNA. (B) A genetic model for upstream regulation of asexual development in A. fumigatus (see Discussion).