Host cell responses to Candida albicans biofilm-derived extracellular vesicles

Abstract

Candida albicans is a prevalent fungal pathogen responsible for infections in humans. As described recently, nanometer-sized extracellular vesicles (EVs) produced by C. albicans play a crucial role in the pathogenesis of infection by facilitating host inflammatory responses and intercellular communication. This study investigates the functional properties of EVs released by biofilms formed by two C. albicans strains-3147 (ATCC 10231) and SC5314-in eliciting host responses. We demonstrate the capability of C. albicans EVs to trigger reactions in human epithelial and immune cells. The involvement of EVs in pathogenesis was evidenced from the initial stages of infection, specifically in adherence to epithelial cells. We further established the capacity of these EVs to induce cytokine production in the epithelial A549 cell line, THP-1 macrophage-like cells, and blood-derived monocytes differentiated into macrophages. Internalization of EVs by THP-1 macrophage-like cells was confirmed, identifying macropinocytosis and phagocytosis as the most probable mechanisms, as demonstrated using various inhibitors that target potential vesicle uptake pathways in human cells. Additionally, C. albicans EVs and their cargo were identified as chemoattractants for blood-derived neutrophils. After verification of the in vivo effect of biofilm-derived EVs on the host, using Galleria mellonella larvae as an alternative model, it was demonstrated that vesicles from C. albicans SC5314 increased mortality in the injected larvae. In conclusion, for both types of EVs a predominantly pro-inflammatory effect on host was observed, highlighting their significant role in the inflammatory response during C. albicans infection.

Keywords: Candida albicans; biofilm; candidiasis; extracellular vesicles; host immune response; pathogenic fungi.

Figure 1

Adhesion to A549 epithelial cells of C. albicans strain 3147 (A, C) and SC5314 (B, D), in the absence (A, B) or presence of C. albicans EVs (C, D). The quantitative analysis of the number of fungal cells adhered to A549 cells determined with CFU counting (E) and microscopic counting of C. albicans cells in the field of view (F). A representative result of the three independent experiments is presented. The statistical significance levels were marked with * for p < 0.05 and ** for p < 0.01.

Figure 2

Adhesion to BJ fibroblast cells of C. albicans strain 3147 (A, C) and SC5314 (B, D), in the absence (A, B) or presence of C. albicans EVs (C, D). The quantitative analysis of the number of fungal cells adhered to BJ cell determined with CFU counting (E) and microscopic counting of C. albicans cells in the field of view (F). A representative result of the three independent experiments is presented. The statistical significance levels were marked with * for p < 0.05 and ** for p < 0.01.

Figure 3

The analysis of the A549 epithelial cells response to C. albicans EVs tested on changes in gene expression and cytokine production. The mRNA gene expression of IL-8 after 3h (A) and 24h (D), IL-1β after 3h (B) and 24h (E), and IL-10 after 3h (C) and 24h (F). Untreated cells served as a control. The analysis of cytokine production at the protein level (G). LPS at concentration of 100 ng/ml was used as a positive control. The statistical significance levels were marked with ns for p 0.05, * for p < 0.05, ** for p < 0.01, and *** for p < 0.001.

Figure 4

The chemotaxis of neutrophils in the presence of C. albicans EVs. (A) Microscopic imaging. (B) Counting of neutrophil cells. Untreated neutrophils served as a control. The fMLP at concentration of 10 µM was used as a positive control. The statistical significance levels were marked with * for p < 0.05, and ** for p < 0.01.

Figure 5

Uptake of C. albicans EVs labeled with DiI dye by THP-1 cells differentiated into macrophage-like cells after 24 h of incubation. The control is presented as untreated THP-1 cells incubated with fractions collected after a mock chromatographic separation of DiI dye used for EVs staining. The cell nuclei were stained with Hoechst 33342. As representative results the images showing the plane of the cell nucleus are presented. Visualization was performed using a Leica Stellaris confocal microscope. Scale bar: 10 μm.

Figure 6

The mechanism of internalization of C. albicans EVs by THP-1 cells differentiated into macrophage-like cells. The flow cytometry analysis (A, B) was conducted after 6 h of incubation of THP-1 cells with EVs after treatment of cells with inhibitors. The control of the uptake of EVs by THP-1 cells untreated with any inhibitor was established as 100%. The average values from two independent experiments are presented with the statistical significance levels marked as ns for p 0.05, * for p < 0.05, and ** for p < 0.01 compared to the control. The mechanism of action of the used inhibitors is depicted (C).

Figure 7

The analysis of cytokine production by THP-1 cells differentiated into macrophage-like cells (A–C) and blood-derived monocytes differentiated into macrophages (D–F). The levels of IL-8 (A, D), TNF-α (B, E) and IL-10 (C, F) are shown as representative results from three independent experiments. Untreated cells served as a control. LPS at concentration of 100 ng/ml was used as a positive control. The statistical significance levels were denoted as ns for p 0.05, * for p < 0.05, ** for p < 0.01, and *** for p < 0.001 in comparison to the control.

Figure 8

The survival of Galleria mellonella larvae after administration of 1 × 10⁸ of C. albicans EVs. PBS injection was used as a control. The statistical significance level was denoted as ** for p < 0.01.

Figure 9

The functional properties of C. albicans strain 3147 and SC5314 EVs derived by biofilm forming cells on host cells. The figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license.