Inhibition of cell cycle-dependent hyphal and biofilm formation by a novel cytochalasin 19,20‑epoxycytochalasin Q in Candida albicans

Abstract

Biofilm-mediated drug resistance is a key virulence factor of pathogenic microbes that cause a serious global health threat especially in immunocompromised individuals. Here, we investigated the antihyphal and antibiofilm activity of 19,20‑epoxycytochalasin Q (ECQ), a cytochalasin actin inhibitor isolated from medicinal mushroom Xylaria sp. BCC1067 against Candida albicans. Remarkably, 256 µg/ml of ECQ inhibited over 95% of C. albicans hyphal formation after 24 h-treatment. Combined ECQ and lipid-based biosurfactant effectively enhanced the antihyphal activity, lowering required ECQ concentrations. Hyphal fragmentation and reduction of biofilm biomass, shown by SEM and AFM visualization of ECQ-treated biofilms, were well corelated to the reduced metabolic activities of young and 24 h-preformed C. albicans biofilms. Induced intracellular accumulation of reactive oxygen species (ROS) also occurred in accompany with the leakage of shrunken cell membrane and defective cell wall at increasing ECQ concentrations. Transcriptomic analyses via RNA-sequencing revealed a massive change (> 1300 genes) in various biological pathways, following ECQ-treatment. Coordinated expression of genes, associated with cellular response to drugs, filamentous growth, cell adhesion, biofilm formation, cytoskeleton organization, cell division cycle, lipid and cell wall metabolisms was confirmed via qRT-PCR. Protein-protein association tool identified coupled expression between key regulators of cell division cyclin-dependent kinases (Cdc19/28) and a gamma-tubulin (Tub4). They coordinated ECQ-dependent hyphal specific gene targets of Ume6 and Tec1 during different phases of cell division. Thus, we first highlight the antihyphal and antibiofilm property of the novel antifungal agent ECQ against one of the most important life-threatening fungal pathogens by providing its key mechanistic detail in biofilm-related fungal infection.

Figure 1

Effect of ECQ and ECQ in combination with sophorolipids against hyphal formation of C. albicans; (a) effect of ECQ in various concentrations against hyphal formation of C. albicans at 37 °C for 0, 1, 2 or 24 h and (b) effect of ECQ in combination with sophorolipids against hyphal formation at 5 h. Cells were observed using a microscope at 400 × magnification. Numbers represent the percentage of hyphal cells normalized with total cells (N > 120 cells).

Figure 2

Antibiofilm activity of ECQ against young and 24 h-preformed biofilm of C. albicans; (a) illustration defines stages of biofilm, (b) XTT staining, (c) CV staining (d) SEM micrographs shown at different magnification (500 × , 2000 × and 10,000 ×) and (e) AFM micrographs showing the variation in height of C. albicans biofilms. Young biofilm and 24 h-preformed biofilm were formed on coated poly-lysine glass cover slips and treated with ECQ for 24 h. The illustration was created with BioRender. Error bars represent the standard error of the mean (SEM) (*p < 0.05, using one-way ANOVA compared to the untreated condition).

Figure 2

Antibiofilm activity of ECQ against young and 24 h-preformed biofilm of C. albicans; (a) illustration defines stages of biofilm, (b) XTT staining, (c) CV staining (d) SEM micrographs shown at different magnification (500 × , 2000 × and 10,000 ×) and (e) AFM micrographs showing the variation in height of C. albicans biofilms. Young biofilm and 24 h-preformed biofilm were formed on coated poly-lysine glass cover slips and treated with ECQ for 24 h. The illustration was created with BioRender. Error bars represent the standard error of the mean (SEM) (*p < 0.05, using one-way ANOVA compared to the untreated condition).

Figure 3

Antifungal ability of ECQ against C. albicans. Fluorescence microscopy images of C. albicans biofilm cells stained with PI or DCFDA. Error bars represent the standard error of the mean (SEM) (*p < 0.05, using one-way ANOVA compared to the untreated condition). Cell survival (%) represents the percentage of surviving cells relative to untreated cells.

Figure 4

Transcriptomic analysis, GO distribution and qRT-PCR analysis of C. albicans genes regulated (> 2-folds) by ECQ treatment; (a) transcriptomic provided numbers of genes regulated more than 2-folds by ECQ treatment, (b,c) the genes were classified GO by biological processes via QuickGO and DAVID bioinformatics resources and (d) relative expression of C. albicans genes analyzed via qRT-PCR. The C. albicans was treated with 128 μg/ml of ECQ for 2 h. The relative mRNA levels of the treated cells were compared to the untreated cells and normalized with a housekeeping gene, ACT1. At least two independent qRT-PCR experiments were performed in triplicates.

Figure 4

Transcriptomic analysis, GO distribution and qRT-PCR analysis of C. albicans genes regulated (> 2-folds) by ECQ treatment; (a) transcriptomic provided numbers of genes regulated more than 2-folds by ECQ treatment, (b,c) the genes were classified GO by biological processes via QuickGO and DAVID bioinformatics resources and (d) relative expression of C. albicans genes analyzed via qRT-PCR. The C. albicans was treated with 128 μg/ml of ECQ for 2 h. The relative mRNA levels of the treated cells were compared to the untreated cells and normalized with a housekeeping gene, ACT1. At least two independent qRT-PCR experiments were performed in triplicates.

Figure 5

Proposed mechanisms of action of ECQ for inhibition of hyphal and biofilm formation; (a) protein–protein interaction network was constructed using the STRING database. In the network, genes serve as nodes and edges serve as protein–protein associations. Line thickness of the network edge indicates the strength of data support. Halo color represents expression of genes from low (red) to high (blue) and (b) a model illustration of mechanisms of action of ECQ. Periodic expression of key note genes for each cell cycle were identified by Côte et al. ECQ dependent keynote genes were labeled as red letter (down-regulated expression), blue letter (up-regulated expression) and black letter (unchanged expression).