The asexual pathogen aspergillus fumigatus expresses functional determinants of Aspergillus nidulans sexual development

Abstract

The major fungal pathogen of humans, Aspergillus fumigatus, lacks a defined sexual cycle, although the presence of genes encoding putative mating type idiomorphs and regulators of Aspergillus sexual development heightens the potential for cryptic sexuality in this deuteromycete. To test the functionality of these genetic determinants, we transferred the alpha box-encoding mat1-1 idiomorph from an A. fumigatus isolate to the homothallic fertile species Aspergillus nidulans. Abundant formation of fruiting bodies (cleistothecia) containing viable ascospores establishes functionality of this mating type gene product in the transgenic strain. Using a similar approach, we also established that the conserved transcriptional regulator from A. fumigatus, the nsdD gene product, can act as a functional, positively acting factor for A. nidulans cleistothecium development; moreover, high-level expression of NsdD in the endogenous host A. fumigatus profoundly alters hyphal development by triggering the formation of coiled hyphae. Our findings demonstrate that the presumably asexual pathogen A. fumigatus encodes functional regulators of mating and sexual development, thereby potentiating the case for cryptic sexuality in this fungal pathogen.

FIG. 1.

The A. fumigatus mat1-1 ORF supports sexual development in A. nidulans. (A) Schematic presentation of matB/mat1-1 ORF replacement procedure showing the native matB locus from A. nidulans and the genome architecture after replacement with the A. fumigatus α-box protein-encoding mat1-1 ORF from isolate D141. Homologous regions are depicted by rhombic gray areas; BglII (B) and HindIII (H) recognition sites are indicated. The black bar represents the hybridizing probe in Southern analyses (B) of recipient strain TNO2A3 and one representative carrying the A. fumigatus mat1-1 gene after digestion of chromosomal DNA with BglII or HindIII; M, DNA molecular weight marker. (C) Colony appearance and cleistothecium (Cl) formation of strains AnS30 (mat1-1) and AnS31 (mat1-1^Δα-box), expressing a functioning and malfunctioning allele of the A. fumigatus mat1-1 ORF, respectively: mature fruiting bodies (arrowheads) were formed only when functional MatB or Mat1-1 proteins were expressed from the endogenous matB locus in A. nidulans, and as estimated from cleistothecial densities, the A. fumigatus mat1-1 ORF supported fruiting body formation to an extent similar to that for its A. nidulans orthologue (scale bar, 500 μm; n. a., not applicable; Cl densities were determined from five different 0.5-cm² areas of two independently inoculated culture plates). (D) Crushed cleistothecia from 8-day-old fruiting bodies of strain TNO2A3 (left) or AnS30 (right): ascosporogenesis of the mat1-1 strain appeared delayed, as indicated by the presence of unripe asci and ascogenous hyphae (cw, cleistothecial wall; as, ascospores). Yet after prolonged incubation, equal amounts of fertile ascospores were formed by the two strains.

FIG. 2.

The NsdD orthologue of A. fumigatus is functional in A. nidulans. (A) Pairwise alignment of NsdD proteins from A. fumigatus and A. nidulans. The alignment was created based on accession number sequences Afu3g13870 (A. fumigatus) and AAB16914 (A. nidulans); identical residues are shown in black and nonconserved ones in gray, and the GATA zinc finger domain is boxed with the Ala455 residue of A. fumigatus NsdD highlighted. (B) Colony appearance of A. nidulans FGSC A4 derivatives expressing A. fumigatus (Af) or A. nidulans (An) nsdD from the nitrate-inducible niiA promoter under repressing and inducing conditions: overexpression of either nsdD gene results in repression of asexual sporulation and formation of numerous nests and fruiting bodies. (C) Complementation of deletant KHH52: the recipient strain is unable to form cleistothecia but develops mature fruiting bodies (arrows) that harbor fertile ascospores when either nsdD gene is expressed. Scale bar, 300 μm (upper panels) or 50 μm (lower panel).

FIG. 3.

Forced expression of nsdD in the deuteromycete A. fumigatus results in the formation of coiled hyphal structures. (A) Validation of forced nsdD overexpression in the A. fumigatus host strain AfS45 by Northern analysis. Autoradiographs derived from Northern analysis of RNA samples obtained under repressing (NH₄⁺) or inducing (NO₃⁻) conditions to demonstrate nsdD steady-state transcript levels expressed from the endogenous locus or the niiA promoter, respectively, are shown. Ethidium bromide-stained rRNA serves as a loading control. (B) Colony appearance of strains expressing the nsdD gene from A. fumigatus (Af) or A. nidulans (An). Expression constructs were introduced in the recipient strain AfS45, and confirmed transformants were inoculated on ammonium- or nitrate-containing medium. Reduced sporulation and altered hyphal extension at the colony periphery are characteristic for the transgenic strains expressing either nsdD gene at high levels. (C) Hyphal morphology resulting from forced expression of nsdD in A. fumigatus. The formation of coiled hyphal structures (arrows) resulting from curling tip extension is induced specifically by high nsdD expression, whereas expression of an nsdD loss-of-function allele does not support coil formation. (D) Coil formation in A. nidulans upon overexpression of nsdD. Strain FGSC A4 transformed with either construct for forced expression of the nsdD gene from A. fumigatus (AnS24) or A. nidulans (AnS25) displays fortified hyphal coil formation (arrowheads) on inducing medium. Scale bar, 50 μm.

FIG. 4.

Ultrastructure of hyphal coils resulting from nsdD overexpression in A. fumigatus. Images are representative of A. fumigatus background (mat1-1 or mat1-2) and source of the nsdD gene (A. fumigatus or A. nidulans). (A) Differential interference contrast (DIC) and fluorescence microscopy images of strain AfS54 reveal nuclear distribution and cell wall composition of the induced coils by 4′,6′-diamidino-2-phenylindole (DAPI) staining and calcoflour white (CW) incorporation, respectively; no obvious differences from vegetative, straight-line hyphae are evident. (B) Representative images of AfS54 from scanning electron microscopy to illustrate size and structure of the nsdD-induced morphological alterations. Scale bar, 10 μm.