Role of the osaA Gene in Aspergillus fumigatus Development, Secondary Metabolism and Virulence

Abstract

Aspergillus fumigatus is the leading cause of aspergillosis, associated with high mortality rates, particularly in immunocompromised individuals. In search of novel genetic targets against aspergillosis, we studied the WOPR transcription factor OsaA. The deletion of the osaA gene resulted in colony growth reduction. Conidiation is also influenced by osaA; both osaA deletion and overexpression resulted in a decrease in spore production. Wild-type expression levels of osaA are necessary for the expression of the conidiation regulatory genes brlA, abaA, and wetA. In addition, osaA is necessary for normal cell wall integrity. Furthermore, the deletion of osaA resulted in a reduction in the ability of A. fumigatus to adhere to surfaces, decreased thermotolerance, as well as increased sensitivity to oxidative stress. Metabolomics analysis indicated that osaA deletion or overexpression led to alterations in the production of multiple secondary metabolites, including gliotoxin. This was accompanied by changes in the expression of genes in the corresponding secondary metabolite gene clusters. These effects could be, at least in part, due to the observed reduction in the expression levels of the veA and laeA global regulators when the osaA locus was altered. Importantly, our study shows that osaA is indispensable for virulence in both neutropenic and corticosteroid-immunosuppressed mouse models.

Keywords: Aspergillus fumigatus; OsaA; WOPR domain; aspergillosis; conidiation; secondary metabolism; virulence.

Figure 1

Alignment of OsaA homologs. Alignment of OsaA of A. fumigatus (AFUB_093810) with homologs of A. flavus (AFLA_004600), A. nidulans (AN6578.2), A. fischeri (XP_001257654.1), A. parasiticus (KAB8200295.1), A. niger (XM_001396613.1), P. rubens (XM_002563660.1), H. capsulatum Ryp1 (ABX74945.1), C. albicans Wor1(Q5AP80), S. pombe Pac2 (BAC54908.1), S. cerevisiae Mit1 (NC_001137), and F. oxysporum Sge1 (XM_018389881). Sequences were obtained from NCBI and aligned using Clustal W Multiple Sequence Alignment tool and ESPript. Two conserved regions of WOPR domain were identified as WOPRa (dash-lined) and WOPRb (dash-dot-lined). Red box with white characters indicates strict identical residues, whereas red characters indicate similarity in a group. Blue frame depicts similarity across groups.

Figure 2

Phylogenetic tree of OsaA homologs from different fungal species. Construction of a phylogenetic tree was carried out using MEGA v11.0. The tree was generated with the maximum likelihood model with a bootstrap value of 1000 (http://megasoftware.net/, 11 June 2023).

Figure 3

osaA influences growth and development in A. fumigatus. (A) The wild-type (WT), ΔosaA, complementation strain (Com), and overexpression strain (OE) were point-inoculated on GMM and grown in the dark at 37 °C. Photographs were taken after 10 days. (B) Colony diameter was measured every 2 days. The error bars represent standard errors. Different letters on the columns indicate values that are statistically different (p < 0.05), as determined using one-way ANOVA with Tukey’s test comparison.

Figure 4

osaA regulates conidial production and spore size. (A) Conidial quantification of top-agar-inoculated plates grown at 37 °C for 24 h, 48 h, and 72 h. Conidia were observed under a bright field microscope and counted with a hemocytometer. (B) Germination rate assessment. Liquid GMM cultures were grown at 37 °C under shaking conditions. Germination was evaluated every 2 h under the microscope using a hemocytometer. (C) Measurements of spore diameter (n = 40 for each strain). Error bars represent the standard error. Different letters on the columns indicate values that are statistically different (p < 0.05). (D) Micrographs of A. fumigatus wild-type (WT), ΔosaA, osaA complementation (Com), and osaA overexpression (OE) conidia. The scale bar represents 10 µm. (E–G) Gene expression analysis of developmental genes brlA (E), abaA (F), and wetA (G). WT, ΔosaA, Com, and OE strains were grown in GMM liquid stationary cultures at 37 °C for 48 h. Expression was calculated using the 2^−ΔΔCT method [68]. Error bars represent the standard error, and different letters on the bar represent significantly different values (p < 0.05). Color key in (B) also applies to (A–C) and (E–G).

Figure 5

Thermotolerance is affected by osaA in A. fumigatus. A. fumigatus wild-type (WT), ΔosaA, complementation (Com), and overexpression (OE) strains were point-inoculated on GMM and incubated in the dark at 25 °C, 30 °C, 37 °C, or 42 °C. Photographs were taken 5 days after inoculation. The experiment was carried out with three replicates.

Figure 6

osaA is necessary for resistance to oxidative stress. Plates of solid GMM and GMM supplemented with increasing concentrations of menadione (from 0 to 30 µM) were point-inoculated with A. fumigatus wild-type (WT), ΔosaA, complementation (Com) and overexpression (OE) strains and incubated at 37 °C for 5 days. The experiment was conducted with three replicates.

Figure 7

Effect of osaA in response to cell wall stress. A. fumigatus wild-type (WT), ΔosaA, complementation (Com), and overexpression (OE) strains were point-inoculated on GMM and GMM supplemented with different concentrations of Congo red. Plates were incubated at 37 °C for 72 h. The experiment was carried out in triplicates.

Figure 8

osaA regulates the production of (A) fumiquinazoline C, (B) helvolic acid, (C) pyripyropene A and (D) fumagillin in A. fumigatus. The A. fumigatus wild-type (WT), ΔosaA, complementation (Com), and overexpression (OE) strains were top-agar-inoculated on GMM and incubated at 37 °C for 72 h. Extracts were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS). On the right in each panel, the expression of key genes in the corresponding secondary metabolite genes clusters, fmqD, pdsA, pypC, and fumR, respectively, were selected as indicators of cluster activation. Samples were obtained from GMM liquid stationary cultures incubated for 48 h. Expression was calculated following the 2^−ΔΔCT method [68]. Error bars represent the standard error. Different letters on the columns indicate values that are statistically different (p < 0.05), as determined using one-way ANOVA with Tukey’s test comparison. All the experiments were performed in triplicates.

Figure 9

osaA affects the gene expression of the global regulators veA and laeA in A. fumigatus. A. fumigatus wild-type (WT), ΔosaA, complementation (Com) and overexpression (OE) strains were grown on GMM stationary cultures for 48 h. The relative gene expression of (A) veA and (B) laeA was calculated using the 2^−ΔΔCT method [68]. Error bars represent the standard error. Different letters on the columns indicate values that are statistically different (p < 0.05).

Figure 10

osaA is indispensable for A. fumigatus virulence in both neutropenic and corticosteroid-induced immune-repressed murine models. The A. fumigatus wild-type (WT), ΔosaA, complementation (Com), and overexpression (OE) strains (2 × 10⁶ spores/40 μL PBS) were used to infect female outbred CD-1 mice. (A) For the neutropenic model, mice received an intraperitoneal injection of cyclophosphamide and Kenalog, as detailed in the Section 2. Uninfected controls were treated with cyclophosphamide and Kenalog but not fungal spores. Post-infection, mice were observed three times daily for the first 7 days and once daily from days 8 to 14. Each group included 10 mice. Statistical analysis was performed utilizing GraphPad PRISM and the Mantel–Cox test. Different letters indicate that values are statistically different. Survival was significantly different between ΔosaA and WT (p < 0.0001) and OE and WT (p < 0.0001). (B) For the non-neutropenic model, mice received only Kenalog one day prior to the infection. Uninfected controls were treated with Kenalog but not fungal spores. Mice were monitored for survival for 14 days. Statistical analysis was performed using GraphPad PRISM statistics. This was significantly different between ΔosaA and WT (p < 0.0001) and between OE and WT (p ≤ 0.001).