QCR7 affects the virulence of Candida albicans and the uptake of multiple carbon sources present in different host niches

Abstract

Background: Candida albicans is a commensal yeast that may cause life-threatening infections. Studies have shown that the cytochrome b-c1 complex subunit 7 gene (QCR7) of C. albicans encodes a protein that forms a component of the mitochondrial electron transport chain complex III, making it an important target for studying the virulence of this yeast. However, to the best of our knowledge, the functions of QCR7 have not yet been characterized.

Methods: A QCR7 knockout strain was constructed using SN152, and BALb/c mice were used as model animals to determine the role of QCR7 in the virulence of C. albicans. Subsequently, the effects of QCR7 on mitochondrial functions and use of carbon sources were investigated. Next, its mutant biofilm formation and hyphal growth maintenance were compared with those of the wild type. Furthermore, the transcriptome of the qcr7Δ/Δ mutant was compared with that of the WT strain to explore pathogenic mechanisms.

Results: Defective QCR7 reduced recruitment of inflammatory cells and attenuated the virulence of C. albicans infection in vivo. Furthermore, the mutant influenced the use of multiple alternative carbon sources that exist in several host niches (GlcNAc, lactic acid, and amino acid, etc.). Moreover, it led to mitochondrial dysfunction. Furthermore, the QCR7 knockout strain showed defects in biofilm formation or the maintenance of filamentous growth. The overexpression of cell-surface-associated genes (HWP1, YWP1, XOG1, and SAP6) can restore defective virulence phenotypes and the carbon-source utilization of qcr7Δ/Δ.

Conclusion: This study provides new insights into the mitochondria-based metabolism of C. albicans, accounting for its virulence and the use of variable carbon sources that promote C. albicans to colonize host niches.

Keywords: Candida albicans; biofilm; hyphae; mitochondrial disorder; virulence.

Figure 1

Complex III plays an important role in the virulence of Candida albicans. (A) Strains were cultured overnight in YPD, followed by washing and serial dilution with PBS. Different rip1Δ/Δ, cor1Δ/Δ, qcr2Δ/Δ, qcr6Δ/Δ, qcr7Δ/Δ, qcr8Δ/Δ, qcr9Δ/Δ, and qcr10Δ/Δ concentrations (5 μl of 10⁷ cells/ml to 10³ cells/ml) were spotted on solid YEP media containing 2% glucose and several alternative carbon sources. Plates were then photographed after incubation at 30°C for 2 days. (B) C albicans suspension of 5 × 10⁶ cells in Spider medium were incubated in 12-well flat-bottom plates at 37°C for 90 min, after which nonattached cells were removed from the wells by washing once with PBS. Fresh corresponding induction medium was added to each well and incubated at 37°C for 48h. Next, the well was washed with PBS and stained with crystal violet; after decolorization, measurements were taken using a microplate reader at 620 nm. “****” represents p < 0.0001 for the WT vs. mutant strains. (C) Cells of each type (1 × 10⁵ cells in 5 µl PBS) were spotted on regular indicated filament-inducing agar plates and incubated at 37°C for 7 days before taking photographs.

Figure 2

QCR7 mutant attenuates the virulence of C albicans infection in vivo. (A) Mice survival following illness initiation with the injection of 5 × 10⁵ C albicans cells. (B) Comparison of mouse kidneys was conducted by injection with WT, qcr7Δ/Δ, and QCR7AB. Mice were sacrificed 48h after the C albicans infection. (C–E) Fungal burden in the kidneys of mice injected with WT, qcr7Δ/Δ, and QCR7AB (p<0.0001 WT vs mutant). The mice were sacrificed 2 days after an intravenous injection with 5 × 10⁵ CFU C albicans. Kidney weights were calculated as each mouse’s weight per gram of body weight (p=0.0122 WT vs mutant). “*” represents p < 0.05 and “**” represents p < 0.01 for the WT vs. mutant strains. (F) Representative hematoxylin–eosin- or periodic acid-Schiff-base-stained sections of kidney-tissue samples from three experiments. (G) Immunofluorescence image showing the biodistribution of F4/80 (red) and Ly6G (green) in the renal of mice infected with Candida albicans. The nuclei were stained blue by DAPI. Scale bar = 50μm. Hyphae (red arrow), pseudo-hyphae (yellow arrow), and spores (black arrow).

Figure 3

QCR7 is required for maintaining mitochondrial functions and carbon-source use. (A) Strains were cultured overnight in YPD, followed by washing and serial dilution with PBS. Different WT, qcr7Δ/Δ, and QCR7AB concentrations (5 μl of 10⁷ cells/ml to 10³ cells/ml) were spotted on solid YEP media containing 2% glucose and several alternative carbon sources. Plates were then photographed after incubation at 30°C for 2 days. (B) The intracellular ATP content was measured using a microplate reader. “**” represents p < 0.01 for the WT vs. mutant strains. (C) ROS levels were measured using the dichlorodihydrofluorescein diacetate dye and a multifunctional enzyme-mark instrument. (D) Mitochondrial membrane potential (MMP) was evaluated using the JC-1 assay kit and Cytomics FC500 flow cytometer (Ex/Em of 595/488 nm), and the red/green mean fluorescent intensities were recorded for each sample. The transition of JC-1 dye from red to green fluorescence was used to easily detect the decrease in MMP, after which MMP was determined as the ratio of red to green fluorescence.

Figure 4

Deletion of QCR7 affects biofilm formation and hyphal growth maintenance in Candida albicans under various carbon sources, with the most significant effect under GlcNAc conditions. (A) C albicans suspensions of 5 × 10⁶ cells in Spider medium supplemented with mannitol, glucose, sucrose, maltose, and GlcNAc as sole carbon sources were incubated in 12-well flat-bottom plates at 37°C for 90 min, after which nonattached cells were removed from the wells by washing once with PBS. Fresh corresponding induction medium was added to each well and incubated at 37°C for 48h. Next, each well was washed with PBS and stained with crystal violet; after decolorization, measurements were taken using a microplate reader at 620 nm. “*” represents p < 0.05 and “**” represents p < 0.01 for the WT vs. mutant strains. (B) Each cell type (1 × 10⁵ cells in 5 µl of PBS) was spotted on the Spider medium, containing 2% glucose, 2% mannitol, 2% maltose, 2% sucrose, and 2% GlcNAc at 37°C. Photographs were then taken after 7 days of growth. (C) The wild type, qcr7Δ/Δ, and QCR7AB were cultured overnight in liquid YPD at 30°C, followed by washing and dilution to OD_{600 nm} = 0.1 of PBS, after which resuspension in Spider medium supplemented with GlcNAc as the sole carbon source was conducted, and incubation continued at 37°C. The hyphal morphology was finally visualized through fluorescence microscope (Olympus, Japan). Scale bars are 10 μm. “NS” represents not significant.

Figure 5

Comparative transcriptomic analysis of QCR7 mutants of C albicans. The distribution of significantly downregulated (A) or upregulated (B) genes in the GO category “molecular process” following qcr7Δ/Δ, compared with the wild type. (C) A heatmap displaying the upregulated and downregulated genes that associate partly with the GO category “biofilm formation”. Fold enrichment of these genes is shown in Table S3 . (D) Validation of the genome-wide transcriptional data by qPCR in the qcr7Δ/Δ mutant strain. “*” represents p < 0.05, “**” represents p < 0.01, “***” represents p < 0.001 and “****” represents p < 0.0001 for the WT vs. mutant strains.

Figure 6

Biofilm formation, hyphal growth on solid, and spot assay on cell-wall stressors or multiple alternative carbon sources with WT, qcr7Δ/Δ, HWP1, YWP1, XOG1, SAP6, and HYR1 overexpressing strains in the qcr7Δ/Δ background. (A) Biofilm formation (48h, 37°C) of strains was profiled and statistically compared to reference strain C albicans SN250. (B) Each cell type (1 × 10⁵ cells in 5 µl of PBS) was spotted on the Spider medium at 37°C. Photographs were then taken after 7 days of growth. (C) The sensitivities of wild-type strain, qcr7Δ/Δ strain, and overexpression strain to different carbon-source conditions were observed by spot assay and incubation was conducted at 30°C for 2 days before photographs were taken. “**” represents p < 0.01 and “***” represents p < 0.001 for the WT or overexpressing strains vs. mutant strains.