Transcriptomics Uncovers Key Genes for Photodynamic Killing on Trichosporon asahii Biofilms

Abstract

Background: The escalating threat of antifungal resistance stemming from Trichosporon asahii (T. asahii) biofilms necessitates the pursuit of innovative therapeutic strategies. Among these approaches, 5-aminolevulinic acid (ALA) photodynamic therapy (PDT), an emerging therapeutic modality, has exhibited promising potential in eradicating T. asahii biofilms.

Methods: The inhibitory activity was evaluated by confocal laser scanning microscopy. To delve deeper into the efficacy of ALA-PDT in eliminating T. asahii biofilms, we conducted a comprehensive transcriptional analysis utilizing transcriptome sequencing.

Results: ALA-PDT demonstrated a profound inhibitory effect on the viability of T. asahii biofilms. Our investigation unveiled 2720 differentially expressed genes following exposure to ALA-PDT. Subsequent meticulous scrutiny allowed for the annotation of genes with a ≥ twofold change in transcription, focusing on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways. Particularly noteworthy were the upregulated genes associated with oxidation-reduction processes, oxidoreductase activity, and catalytic activity. Conversely, the downregulated genes were linked to ATP binding, protein phosphorylation, and protein kinase activity. Additionally, we observed a surge in the transcription of genes that may be involved in oxidative stress (e.g., A1Q1_05494) as well as genes that may be involved in morphogenesis and biofilm formation (e.g., A1Q1_04029, A1Q1_01345, A1Q1_08069, and A1Q1_01456) following ALA-PDT treatment.

Conclusions: Our findings underscore the substantial impact of ALA-PDT on the transcriptional regulation of genes related to oxidative stress, morphogenesis, and biofilm formation, paving the way for novel therapeutic avenues in combating T. asahii biofilms.

Keywords: Trichosporon asahii (T. asahii); 5-aminolevulinic acid (ALA); Biofilm; Photodynamic therapy (PDT).

Fig. 1

Confocal laser scanning microscopy (CLSM) was employed to visualize T. asahii biofilm specimens under various ALA-PDT conditions. Live cells were green and dead cells were red. a group P⁻L⁻ (untreated control group); b group P⁻L¹⁰⁰; c group P⁻L²⁰⁰; d group P⁺L⁻; e group P⁺L¹⁰⁰; f group P⁺L²⁰⁰

Fig. 2

Dead/live Ratio of T. asahii Biofilms Following ALA-PDT Exposure. Significant Differences Observed Between ALA-PDT Groups (group P⁺L¹⁰⁰ and group P⁺L²⁰⁰) and untreated control group (*p < 0.05, **p < 0.01). No statistically significant differences between the control group, the ALA group (group P⁺L⁻), and the light group (group P⁻L¹⁰⁰,_group P⁻L²⁰⁰)

Fig. 3

DEGs Analysis in Various Group Comparisons

Fig. 4

a A Volcano Plot: Differential Expression Analysis Revealed Distinctive Gene Expression Patterns between group P⁻L⁻ and group P⁺L¹⁰⁰ in T. asahii biofilms. b Top 10 GO terms enriched among the up-regulated DEGs. c Top 10 GO terms enriched among the down-regulated DEGs. d Top 25 KEGG pathways

Fig. 5

Genes potentially involved. a beta-glucosidase genes; b chitinase genes; c glucan genes; d lipase genes; e phospholipase genes; f serine genes; g sterol genes; h ubiquitin genes

Fig. 6

Quantitative real-time PCR (qRT-PCR) was performed to validate the gene expression levels identified in the RNA-Seq data, specifically for A1Q1_05494, A1Q1_01456, A1Q1_01345, A1Q1_05711, A1Q1_02782, A1Q1_08069, and A1Q1_04029 genes in both the control group (group P⁻L⁻) and the ALA-PDT group (group P⁺L¹⁰⁰). The relative expression levels of differentially expressed genes (DEGs) were represented using a log2 fold change index. The values are presented as the mean ± standard deviation, and statistical significance was indicated as **, or *** for p < 0.01 and p < 0.001, respectively