Functional, transcriptomic, and lipidomic studies of the choC gene encoding a phospholipid methyltransferase in Aspergillus fumigatus

Abstract

This study explored the phospholipid metabolic pathway in A. fumigatus and its relationship with fungal growth, metabolism, and pathogenicity. ChoC, based on its critical roles in many aspects of the fungus and relatively conserved characteristics in filamentous fungi with low similarity with mammalian ones, can be a novel target of new antifungal drugs.

Keywords: Aspergillus fumigatus; ChoC; lipidomics; phosphatidylcholines; transcriptomics.

Fig 1

Summary and function of choC. (A) Identification of the gene choC. (B) Levels of choC mRNA during vegetative growth and asexual development. (C) ChoC is conserved among most fungi. (D) Phenotypes of the colony of wild-type (WT), ΔchoC, and complemented strains on solid minimal medium (MM), MM + serine, MM + ethanolamine (EA), MM + monoethanolamine (MAE), MM + diethanolamine (DAE), and MM + choline upon incubation at 37°C for 3 days. Scale bar: 10 µm. (E) Hyphae of WT, ΔchoC, and complemented strains grown in solid MM. Scale bar = 10 µm.

Fig 2

Roles of choC in cell viability, cell death, and cell wall integrity. (A) Mycelial dry weights of WT and ΔchoC strains grown in liquid MM, MM + DAE, and MM + choline at 37°C for 9 days. Data are the mean values of three independent experiments. (B) Phenotypes of mycelial aggregates formed in liquid MM at days 2 and 6. Scale bar: 20 µm (middle panels) and 10 µm (bottom panels). (C) Alamar blue reduction rates of WT and ΔchoC strains grown in liquid MM, MM + DAE, and MM + choline at 37°C for 9 days. Data points are the mean of three independent experiments. (D) Apoptosis analyses of WT and ΔchoC strains grown in liquid MM by Evans blue staining. Scale bar is 5 µm. The photographs were taken on days 2 and 6. (E) Sensitivity to osmotic pressure of WT and ΔchoC strains grown in liquid MM with 1.2 M sorbitol. Scale bar is 10 µm. (F) Effects of Congo Red on the growth of the ΔchoC mutant. Scale bar: 10 and 20 µm.

Fig 3

DEGs and gene ontology (GO) enrichment analyses. (A) Heatmap analysis shows that statistical significance in some important genes in phospholipid metabolism, cell cycle, autophagy, and other metabolic pathways in WT-2,ΔchoC-2 and WT-4,ΔchoC-4 strains. WT-2/ΔchoC-2 means WT/ΔchoC strains were cultured in MM for 2 days. WT-4/ΔchoC-4 means WT/ΔchoC strains were cultured in MM for 4 days. Triplicates were analyzed per strain per time point. (B) GO enrichment analysis of DEGs between ΔchoC-2 and WT-2. (C) GO enrichment analysis of DEGs between ΔchoC-4 and WT-4. (D) GO enrichment analysis of DEGs between ΔchoC-4 and ΔchoC-2. (E) GO enrichment analysis of DEGs between WT-4 and WT-2. The ordinate is an enriched GO term, and the abscissa is the number of DEGs in the term. Different colors are used to distinguish three major categories: biological processes, cell components, and molecular functions. "*" is a significant enrichment of GO term.

Fig 4

Statistical analysis of KEGG pathway enrichment. (A) The metabolic pathways of significantly upregulated DEGs in the ΔchoC mutant at 2 days. (B) The metabolic pathways of significantly downregulated DEGs in the ΔchoC mutant at 2 days. (C) The metabolic pathways of significantly upregulated DEGs in the ΔchoC mutant at 4 days. (D) The metabolic pathways of significantly downregulated DEGs in the ΔchoC mutant at 4 days. Rich factor refers to the ratio of the number of differentially expressed genes enriched in the pathway to the number of annotated genes. The greater the “Rich factor,” the greater is the degree of enrichment; q-value is the P-value after multiple hypothesis test corrections. The q-value ranges within (0, 1), and the closer to 0, the more significant the enrichment is. The first 20 entries that show the most significant enrichment are shown.

Fig 5

Influence of choC on lipid metabolism in A. fumigatus. (A) The abundance of PC in WT and ΔchoC strains at days 2 and 4. (B) The abundance of PE in WT and ΔchoC strains at day 2. N = 6, *P < 0.05; **P < 0.01; and ***P < 0.001. (C) The abundance of PE in WT and ΔchoC strains at day 4. N = 6, *P < 0.05; **P < 0.01; and ***P < 0.001. (D) The abundance of TAG in WT and ΔchoC strains at day 4. (E) The abundance of DAG in WT and ΔchoC strains at days 2 and 4.

Fig 6

Requirement of ChoC for the pathogenesis of A. fumigatus. (A) Survival curve of mice infected with A. fumigatus in 3 weeks. (B) Fungal burden of the liver in mice infected with A. fumigatus at days 3, 10, and 17. (C) Fungal burden of the spleen in mice infected with A. fumigatus at days 3, 10, and 17. (D) Fungal burden of the kidney in mice infected with A. fumigatus at days 3, 10, and 17. (E) Representative images of periodic acid-Schiff (PAS) and hematoxylin-eosin (HE)-stained kidney sections (200×) on day 23. Bar = 100 µm for 200.