Caged-hypocrellin mediated photodynamic therapy induces chromatin remodeling and disrupts mitochondrial energy metabolism in multidrug-resistant Candida auris

Abstract

Candida auris is a fungal pathogen with frequent development of multidrug-resistance or pan-drug resistance. Currently, the treatment options for Candida auris are limited. Therefore, there is an urgent need for alternative therapeutic strategies. Antimicrobial photodynamic therapy (aPDT), which generates reactive oxygen species (ROS) through light-activated photosensitizers, has shown promise against C. auris; however, its molecular mechanism remains unclear. To investigate COP1T-HA-mediated PDT-induced genomic alterations, we constructed a 3D genome map of Candida species, which uncovered the reorganization of chromatin architecture in response to PDT treatment. Our data showed that low-dose PDT causes subtle local adjustments in chromatin topology, whereas high-dose PDT leads to more pronounced changes in A/B compartmentalization, topologically associating domain (TAD) organization, and chromatin looping associated with key genes related to mitochondrial energy metabolism. Confocal imaging confirmed that high-dose COP1T-HA-mediated PDT induces localized ROS accumulation near the nucleus and a temporally ordered cellular stress response. Furthermore, functional validation through QCR10, NDUFA5, and MP knockouts confirmed the essential roles of these genes in mitochondrial integrity, ATP synthesis, ROS homeostasis, and biofilm formation. Mutants showed altered mitochondrial membrane potential, intracellular pH imbalance, and enhanced glycolytic compensation, highlighting the impact of electron transport disruption on energy metabolism. This study provides the first comprehensive insight into COP1T-HA-mediated PDT-induced chromatin reorganization in C. auris and establishes a direct connection between 3D genome remodeling and fungal energy metabolism, offering a foundation for chromatin-targeted antifungal strategies.

Keywords: 3D genome architecture; Chromatin remodeling; Mitochondrial energy metabolism; Multidrug-resistant Candida auris; Photodynamic therapy (PDT).

Graphical abstract

Fig. 1

COP1T-HA intracellular localization and the spatiotemporal sequence of PDT-induced oxidative and nuclear responses in Candida auris. (A) Confocal microscopy showing subcellular localization of COP1T-HA (red autofluorescence) in C. auris following low-dose (LD-PDT) or high-dose photodynamic treatment (HD-PDT). MitoTracker Green was used to label mitochondria and DAPI to stain nuclei. Scale bars represent 50 μm (B) Confocal co-staining of SOSG and Hoechst under different PDT conditions. Scale bars represent 50 μm (C) Fluorescence microscopy showing sequential changes in intracellular ROS production (SOSG), nuclear morphology (DAPI), and DNA fragmentation (TUNEL) after HD-PDT treatment. Scale bars represent 50 μm(SOSG), 25 μm(DAPI), 50 μm(TUNEL).

Fig. 2

PDT-induced chromatin remodeling in C. auris. (A) 3D genome structures of C. auris under control, LD-PDT, and HD-PDT conditions. Chromosomes are color-coded, showing spatial organization and compaction differences in these conditions. (B) Hi-C contact maps at 10 kb resolution for control, LD-PDT, and HD-PDT conditions, illustrating chromatin interaction changes following PDT treatment. (C) Circos plot displaying A/B compartment shifts, TAD organization, and chromatin loop alterations across different conditions. (D) Differential chromatin interaction maps comparing control vs. LD-PDT (left) and control vs. HD-PDT (right), highlighting regions with significant changes in chromatin interactions. Warmer colors indicate increased interactions, while cooler colors indicate decreased interactions.

Fig. 3

PDT-induced A/B compartment reorganization and functional implications in C. auris. (A) A/B compartment eigenvector heatmaps in chromosome 1 for control, LD-PDT, and HD-PDT. (B–D) Boxplots illustrating differences in eigenvector values (B), compartment A/B distribution (C), and compartment length distribution (D) across conditions, highlighting shifts in chromatin activity. (E) Pie charts depicting the number of genomic regions undergoing compartment transitions between control and PDT-treated samples, categorized as A2A (stable active), A2B (active-to-inactive), B2A (inactive-to-active), and B2B (stable inactive). (F) KEGG pathway enrichment analysis of genes located in regions undergoing compartment transitions.

Fig. 4

PDT-induced TAD reorganization and its genomic features in C. auris. (A) Hi-C contact maps of chromosome 1 (Ch1) under control, LD-PDT, and HD-PDT conditions, with corresponding insulation scores indicating TAD boundary locations. (B–C) Boxplots comparing gene density (B) and GC content (C) between TAD boundaries and TAD interiors across different conditions, showing their distribution in relation to structural features. (D) Aggregate Hi-C maps showing TAD boundary enrichment across control, LD-PDT, and HD-PDT samples. (E) Pileup analysis of TAD domains under different conditions. (F) Boxplot comparing TAD boundary strength across conditions.

Fig. 5

PDT-induced chromatin looping changes and functional enrichment analysis in C. auris. (A) Top 100 chromatin interactions shown as Circos plots for control, LD-PDT, and HD-PDT conditions. Red lines represent long-range chromatin loops, while blue lines indicate interactions within the same chromosomal region. (B) Loop pileup heatmaps for control, LD-PDT, and HD-PDT conditions, displaying the distribution and intensity of chromatin loops across the genome. (C) Venn diagrams showing the overlap of differentially expressed chromatin loops between control vs. LD-PDT and control vs. HD-PDT conditions, with the number of significant loops in each category. (D) Gene Ontology (GO) enrichment analysis for genes associated with differentially regulated chromatin loops between control and LD-PDT (left) and control and HD-PDT (right).

Fig. 6

Transcriptomic changes following PDT treatment in C. auris. (A) Principal Component Analysis (PCA) comparing control and LD-PDT conditions. (B) PCA for control and HD-PDT conditions. (C) Volcano plot comparing control vs. LD-PDT conditions, highlighting significantly upregulated (red) and downregulated (blue) genes, with the number of differentially expressed genes indicated. (D) Volcano plot for control vs. HD-PDT conditions, illustrating differential gene expression between control and HD-PDT, with genes categorized by fold-change and statistical significance. (E) Venn diagrams showing the overlap of differentially expressed genes between control vs. LD-PDT and control vs. HD-PDT, with the number of unique and shared genes across conditions. (F) GO enrichment analysis for genes differentially expressed in control vs. LD-PDT. (G) GO enrichment analysis for genes differentially expressed in control vs. HD-PDT.

Fig. 7

PDT-induced chromatin reorganization affects energy metabolism genes in C. auris. (A–B) TAD structures, insulation scores, and chromatin loops in control vs. LD-PDT (A) and control vs. HD-PDT (B). Red arcs represent chromatin loops, with specific enrichment near energy metabolism genes. (C–E) Hi-C contact maps, RNA-seq expression, and chromatin loop interactions for QCR10 (C), NDUFA5 (D), and MP (E), key genes involved in mitochondrial function and energy metabolism.

Fig. 8

Phenotypic and ultrastructural analysis of QCR10 and NDUFA5 mutants in C. auris. (A) Growth of wild-type (WT) and mutant strains (qcr10Δ and ndufa5Δ) on solid medium after 36, 48 and 60 h, showing colony morphology differences. (B) Liquid culture growth of WT and mutants after 36 h. (C) Growth curves of WT and mutant strains in liquid culture over 72 h. (D) Scanning electron microscopy (SEM) images of WT and mutant cells at 5000 × and 20000 × magnification. Scale bars represent 20 μm and 5 μm. (E) Transmission electron microscopy (TEM) images of WT and mutant cells at 12000 × and 40000 × magnification. bars represent 1 μm and 500 nm.

Fig. 9

Impact of QCR10 and NDUFA5 deletions on biofilm formation, oxidative stress, ATP levels, and gene expression in C. auris. (A) Biofilm formation of wild-type (WT) and mutant (qcr10Δ and ndufa5Δ) strains observed under scanning electron microscopy (SEM) at 5000 × and 20000 × magnification. Scale bars represent 20 μm and 5 μm. (B) Quantification of biofilm formation using OD₄₉₂ measurements in qcr10Δ and ndufa5Δ mutants compared to WT. (C) Intracellular ROS levels detected by DCFH-DA and SOSG in qcr10Δ and ndufa5Δ mutants compared to WT. (D) Mitochondrial function assessed by ATP production, mitochondrial membrane potential (ΔΨm, JC-1 staining), and intracellular pH (ΔpH, BCECF-AM staining). FCCP (15 μM) and oligomycin (15 μM) were used to define lower and upper ΔΨm thresholds, respectively. (E) Relative mRNA expression of genes associated with biofilm formation (ALS4, EAP1, FKS1), stress response (CRZ1, HSP90), and iron metabolism (FET3) in WT and mutant strains by qRT-PCR. Error bars represent mean ± SD. Statistical significance: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.