The mitochondrial protein Bcs1A regulates antifungal drug tolerance by affecting efflux pump expression in the filamentous pathogenic fungus Aspergillus fumigatus

Abstract

Aspergillus fumigatus is the predominant pathogen responsible for aspergillosis infections, with emerging drug-resistant strains complicating treatment strategies. The role of mitochondrial functionality in fungal resistance to antifungal agents is well-documented yet not fully understood. In this study, the mitochondrial protein Bcs1A, a homolog of yeast Bcs1, was found to regulate colony growth, ion homeostasis, and the response to antifungal drugs in A. fumigatus. Microscopic observations revealed substantial colocalization of Bcs1A-GFP fusion protein fluorescence with mitochondria. Bcs1A deletion compromised colony growth and the utilization of non-fermentable carbon sources, alongside causing abnormal mitochondrial membrane potential and reduced reactive oxygen species production. These findings underscore Bcs1A's vital role in maintaining mitochondrial integrity. Phenotypic analysis and determinations of minimum inhibitory concentrations indicated that the Δbcs1A mutant was more resistant to various antifungal agents, such as azoles, terbinafine, and simvastatin, compared to wild-type strain. RNA sequencing and RT-qPCR analysis highlighted an upregulation of multiple efflux pumps in the Δbcs1A mutant. Furthermore, loss of the principal drug efflux pump, mdr1, decreased azole tolerance in the Δbcs1A mutant, suggesting that Bcs1A's modulated of azoles response via efflux pump expression. Collectively, these results establish Bcs1A as essential for growth and antifungal drug responsiveness in A. fumigatus mediated through mitochondrial regulation.IMPORTANCEDrug resistance presents a formidable obstacle in the clinical management of aspergillosis. Mitochondria are integral to various biochemical pathways, including those involved in fungi drug response, making mitochondrial proteins promising therapeutic targets for drug therapy. This study confirms that Bcs1A, a mitochondrial respiratory chain protein, is indispensable for mitochondrial functionality and multidrug tolerance in Aspergillus fumigatus. Mutation of Bcs1A not only leads to a series of drug efflux pumps upregulated but also shows that loss of the primary efflux pump, mdr1, partial reduction in drug tolerance in the Bcs1A mutant, highlighting that Bcs1A's significant influence on mitochondria-mediated drug resistance.

Keywords: Aspergillus fumigatus; Bcs1A; drug resistance; mitochondria; vegetative growth.

Fig 1

Mitochondria-localized Bcs1A is required for hyphal growth in A. fumigatus. (A) Bioinformatic analysis of selected Bcs1 homologs from eukaryotes. The phylogenetic tree was constructed using MEGA 7 software. Domains were analyzed using SMART (http://smart.embl-heidelberg.de/). (B) Subcellular localization of green fluorescent protein (GFP)-tagged Bcs1A. MitoTracker was used to visualize mitochondria. Scale bar, 10 µm. Colony phenotypes (C) and diameters (D) of the wild-type (WT), Δbcs1A, and bcs1A^C strains grown in minimal medium (MM) and yeast extract-glucose (YG) media at 37°C for 48 h. ns, not significant; ***, P < 0.001.

Fig 2

Bcs1A is essential for maintaining optimal mitochondrial function in A. fumigatus. (A) Colony morphologies of the WT, Δbcs1A, and bcs1A^C strains on solid medium containing glucose, glycerol, and ethanol as carbon sources, respectively, at 37°C for 48 h. (B) The indicated strains were cultured in a liquid medium supply with glucose, glycerol, and ethanol as carbon sources, respectively, with shaking at 37°C, 220 rpm for 48 h. (C) The mitochondrial membrane potential was assessed using flow cytometry, with FITC-A on the x-axis indicating the relative fluorescence intensity value. (D) Quantitative and statistical analyses of the mean fluorescence intensity and (E) ROS generation in the WT and Δbcs1A mutant strains. **, P < 0.01; ***, P < 0.001.

Fig 3

Loss of bcs1A reduces susceptibility to antifungal drugs in A. fumigatus. (A) Colony morphology of the strains after cultivation on minimal medium at 37°C for 2 days, as well as after exposure to various concentrations of antifungal drugs (0.15 µg/mL itraconazole, 0.5 µg/mL voriconazole, 1 µg/mL bifonazole, 0.8 µg/mL terbinafine, 10 µg/mL simvastatin, 1 µg/mL amphotericin B, and 0.8 µg/mL caspofungin) at 37°C for 3 days. (B) MIC values of itraconazole and voriconazole were determined using E-test strips on plates containing a mixture of conidia (1 × 10⁵) of the WT or Δbcs1A strains in solid YG medium, followed by incubation at 37°C for 3 days.

Fig 4

RNA-sequencing analysis of the bcs1A mutant and WT strains. (A) Volcano plot showing DEGs in the Δbcs1A mutant compared to the WT strain. (B). Heatmap comparing the RNA-seq data of the Δbcs1A mutant relative to the WT strain. (C) GO analysis of the functional category and (D) KEGG analysis of pathway enrichment of DEGs (P < 0.05 and lg₂FC > 1 or < −1).

Fig 5

Loss of bcs1A leads to upregulating various multidrug resistance-associated transport genes. (A) Heatmap showing RNA-seq data of multidrug transport-related genes. Real-time quantitative PCR analysis of indicated genes in the WT and Δbcs1A strains without ITC treatment (B) and with ITC treatment for 1 h (C). ns, not significant; *, P < 0.05; ***, P < 0.001; **, P < 0.01; ***, P < 0.001. (D). Colony morphologies of the indicated strains cultured in MM containing different ITC concentrations (0, 0.15, 0.2, 0.3 µg/mL).

Fig 6

Addition of metal ions can increase azole tolerance of A. fumigatus. (A) Heatmap showing metal ion transport-related genes in the Δbcs1A mutant and its parental WT strain. (B) Colony morphologies of strains grown on MM in the presence of various metal ions (FeCl₃ 1 mM; CaCl₂ 50 mM; CuSO₄ 100 µM, MgCl₂ 50 mM; ZnCl₂ 1 mM) at 37°C for 2 days or (C) with 0.2 µg/mL ITC at 37°C for 2.5 days.