Discovery of a sexual cycle in Aspergillus lentulus, a close relative of A. fumigatus

Abstract

Aspergillus lentulus was described in 2005 as a new species within the A. fumigatus sensu lato complex. It is an opportunistic human pathogen causing invasive aspergillosis with high mortality rates, and it has been isolated from clinical and environmental sources. The species is morphologically nearly identical to A. fumigatus sensu stricto, and this similarity has resulted in their frequent misidentification. Comparative studies show that A. lentulus has some distinguishing growth features and decreased in vitro susceptibility to several antifungal agents, including amphotericin B and caspofungin. Similar to the once-presumed-asexual A. fumigatus, it has only been known to reproduce mitotically. However, we now show that A. lentulus has a heterothallic sexual breeding system. A PCR-based mating-type diagnostic detected isolates of either the MAT1-1 or MAT1-2 genotype, and examination of 26 worldwide clinical and environmental isolates revealed similar ratios of the two mating types (38% versus 62%, respectively). MAT1-1 and MAT1-2 idiomorph regions were analyzed, revealing the presence of characteristic alpha and high-mobility-group (HMG) domain genes, together with other more unusual features such as a MAT1-2-4 gene. We then demonstrated that A. lentulus possesses a functional sexual cycle with mature cleistothecia, containing heat-resistant ascospores, being produced after 3 weeks of incubation. Recombination was confirmed using molecular markers. However, isolates of A. lentulus failed to cross with highly fertile strains of A. fumigatus, demonstrating reproductive isolation between these sibling species. The discovery of the A. lentulus sexual stage has significant implications for the management of drug resistance and control of invasive aspergillosis associated with this emerging fungal pathogen.

Fig 1

MAT locus of Aspergillus lentulus. The arrangement of the A. lentulus idiomorph region shows the difference in organization between isolates 78-3 (MAT1-1) and 78-2 (MAT1-2). Colored block arrows indicate MAT1-1-1 (red), MAT1-2-1 (green), and MAT1-2-4 (white) sequences. Colored boxes indicate the α1 domain (salmon pink), HMG domain (light green), and nearly identical flanking regions (gray). Introns are represented by black boxes; lines extending between boxes and arrows represent idiomorph sequence. Smaller red and green segments represent regions between 10 and 29 bp in length with ≥70% MAT1-1-1 and MAT1-2-1 nucleotide conservation, respectively. Black arrowheads (direction indicates 5′ to 3′ sequence) show the positions of primers (see Table S2 in the supplemental material) used for amplification of the idiomorph region.

Fig 2

Sexual reproduction in Aspergillus lentulus. (A) Paired cultures of isolates 78-2 (MAT1-2) × 78-3 (MAT1-1) on oatmeal agar (9-cm-diameter petri dish) with cleistothecia (arrows) along the barrage zones following 3 weeks of incubation at 28°C. (B) Close-up of a barrage zone showing pale yellow cleistothecia. Scale bar, 400 μm. (C) SEM micrograph of a cleistothecium showing the interwoven hyphae that form the peridial wall. Scale bar, 100 μm. (D) SEM micrograph of lenticular ascospores (white arrow) and smaller globose conidia (black arrow). Scale bar, 10 μm.

Fig 3

Evidence for meiotic recombination. The gel shows segregation patterns of a RAPD-PCR amplicon in A. lentulus parental isolates (P1 and P2) and 12 ascospore progeny (lanes 1 to 12) from the cross 78-3 (P1) × 78-2 (P2), using primer OPW08. MM, molecular size marker; C, water control. Arrow indicates the diagnostic RAPD band. Red and green lane headings indicate MAT1-1 and MAT1-2 genotypes, respectively.