The sat1 Gene Is Required for the Growth and Virulence of the Human Pathogenic Fungus Aspergillus fumigatus

Abstract

Aspergillus fumigatus is an important opportunistic pathogenic fungus that causes invasive aspergillosis in immunocompromised humans. Regulated fungal growth is essential for disease development and progression. Thus, screening for genes that regulate fungal growth may lead to the identification of potential therapeutic targets for invasive aspergillosis (IA). Screening of the transfer DNA (T-DNA) random-insertion A. fumigatus mutants identified a severe growth deficiency mutant AFM2954 and featured sat1 as the mutated gene described as a putative intracellular protein transporter of unknown function. The deletion of sat1 exhibited severe growth defects and significantly increased the nematode and mouse survival rates and decreased the fungal loads and histopathological damages in mouse lungs. Transcriptomic analyses revealed expression changes associated with the cell wall synthesis, the tricarboxylic acid cycle (TCA cycle), and oxidative phosphorylation genes in the sat1 mutant. Deletion of the gene resulted in resistance to cell wall-perturbing agents and thickened cell wall as well as reduced ATP contents and mitochondrial membrane potential, suggested that sat1 affected the cell wall synthesis and mitochondrial function of A. fumigatus. All together, our study uncovered novel functions of sat1 in growth and virulence of A. fumigatus and provided a theoretical basis for the development of new therapeutic target for treating IA patients. IMPORTANCE Aspergillus fumigatus is the main causative agent of invasive aspergillosis in immunocompromised hosts, with up to 90% lethality. Nevertheless, the fungal factors that regulate the pathogenesis of A. fumigatus remain largely unknown. Better understanding of the mechanisms controlling growth of A. fumigatus may provide novel therapeutic targets. In the present study, we characterized sat1 in the opportunistic pathogen A. fumigatus. The function of sat1 remains unknown. We proved its important role in growth and virulence, likely because of its effects on cell wall synthesis and mitochondrial functions.

Keywords: Aspergillus fumigatus; cell wall; growth; mitochondrion; sat1; virulence.

FIG 1

Disruption of sat1 affected colony morphology of A. fumigatus. Strains were inoculated on PDA medium at 37°C for 3 days.

FIG 2

sat1 is conserved among common pathogenic filamentous fungi. We performed a multiple-sequence alignment of sat1-like proteins using MUSCLE (https://www.ebi.ac.uk/Tools/msa/muscle/), and we showed a partial of the alignment result. Candida albicans, GenPept accession no. XP_721015.2; Cryptococcus neoformans, GenPept accession no. XP_777267.1; Alternaria alternate, GenPept accession no. XP_018390575.1; Bipolaris sorokiniana, GenPept accession no. XP_007694569.1; Talaromyces marneffei, GenPept accession no. XP_002148790.1; Paecilomyces varioti, GenPept accession no. XP_028487758.1; Aspergillus nidulans, GenPept accession no. XP_663893.1; Aspergillus fumigatus, GenPept accession no. XP_755570.1; Aspergillus terreus, GenPept accession no. XP_001208535.1; Aspergillus niger, GenPept accession no. XP_025460961.1; Aspergillus flavus, GenPept accession no. KAF7620608.1; Histoplasma capsulatum, GenPept accession no. XP_001541714.1; Paracoccidioides brasiliensis, GenPept accession no. XP_010762527.1; Microsporum gypseum, GenPept accession no. XP_003174688.1; Trichophyton rubrum, GenPept accession no. XP_003236425.1.

FIG 3

Disruption of sat1 affected the micromorphology of A. fumigatus. (A) Phenotypes of conidial head. Strains were inoculated on PDA for 24 h. Bar, 50 μm. (B) Hyphal morphology. Strains were inoculated in PDB for 12 h. Bar, 50 μm. They were observed by Fluorescent Brightener 28 staining (35 μg/mL).

FIG 4

Deletion of sat1 significantly reduced the virulence of A. fumigatus. (A) Loss of the sat1 gene significantly increased the survival rates of the infected Caenorhabditis elegans. The nematodes were infected by 100 μL of 2.0 × 10⁸ CFU spore suspension. Survival rates over a 72-h observation period are shown. (B) Loss of the sat1 gene significantly increased the survival rates of the infected mice. We challenged BALB/c mice with 30 μL 6 × 10⁶ CFU spore suspension via intranasal instillation. The survival rates over a 14-day postinfection period. On day 3 after infection, a pulmonary lobe of each mice for fungal burden (C), and the other was used for histopathologic analysis (D). Lung tissue sections were stained with hematoxylin and eosin (H&E) and periodic acid-Schiff stain (PAS), respectively. Bar, 200 μm. *, P < 0.05; **, P < 0.01; ns, not significant.

FIG 5

Genome-wide transcriptome analysis of WT and AFM2954 strains. (A) Volcano plot of differentially expressed genes (DEGs). (B) Pathway enrichment of DEGs. Only the top 10 enriched pathway terms are presented. (C) Relative expression levels of development genes, which were obtained by qRT-PCR and normalized against the 18S rRNA gene. *, P < 0.05; **, P < 0.01; ns, not significant.

FIG 6

Disruption of sat1 affected mitochondrial function of A. fumigatus. (A) Relative expression levels of TCA cycle and OXPHOS genes, which were obtained by qRT-PCR and normalized against the 18S rRNA gene. (B) Loss of the sat1 gene reduced intracellular ATP production. (C) Loss of the sat1 gene decreased mitochondrial membrane potential, determined by the ratio of green and red fluorescence using JC-1. **, P < 0.01; ***, P < 0.001; ns, not significant.

FIG 7

Deletion of sat1 enhances resistance to cell wall-perturbing agent and increased wall thickening of A. fumigatus. (A) Relative expression levels of cell wall synthesis genes, which were obtained by qRT-PCR and normalized against the 18S rRNA gene. (B) Deletion strains were resistant to cell wall-damaging agents. Two microliters of 1 × 10⁶ CFU spore suspension were grown on PDA medium supplemented with 200 μg/mL Congo red (CR) or 0.02% SDS at 37°C for 3 days. (C) Loss of the sat1 gene significantly increased cell wall thickness. Transmission electron microscopy (TEM) of hyphal sections of the A. fumigatus WT and AFM2954 strains were grown for 24 h in PDA at 37°C. Bar, 1 μm. The cell wall thickness was measured using ImageJ software. Length of three random points on the cell wall was measured, and the average of these values was the result. **, P < 0.01; ***, P < 0.001; ns, not significant.