Insight Into the Properties and Immunoregulatory Effect of Extracellular Vesicles Produced by Candida glabrata, Candida parapsilosis, and Candida tropicalis Biofilms

Abstract

Currently, non-albicans Candida species, including C. tropicalis, C. glabrata, and C. parapsilosis, are becoming an increasing epidemiological threat, predominantly due to the distinct collection of virulence mechanisms, as well as emerging resistance to antifungal drugs typically used in the treatment of candidiasis. They can produce biofilms that release extracellular vesicles (EVs), which are nanometric spherical structures surrounded by a lipid bilayer, transporting diversified biologically active cargo, that may be involved in intercellular communication, biofilm matrix production, and interaction with the host. In this work, we characterize the size and protein composition of these structures for three species of non-albicans Candida fungi forming biofilm, indicating considerable heterogeneity of the investigated population of fungal EVs. Examination of the influence of EVs on cytokine production by the human monocytic cell line THP-1 differentiated into macrophage-like cells revealed that the tested vesicles have a stimulating effect on the secretion of tumor necrosis factor α and interleukin 8, while they reduce the production of interleukin 10. This may indicate the proinflammatory nature of the effect of EVs produced by these species on the host immune cells. Moreover, it has been indicated that vesicles may be involved in C. tropicalis biofilm resistance to fluconazole and caspofungin. This reveals the important role of EVs not only in the physiology of C. tropicalis, C. glabrata, and C. parapsilosis fungi but also in the pathogenesis of infections associated with the production of fungal biofilm.

Keywords: biofilm; candidiasis; cytokines; extracellular vesicles; pathogenic fungi.

Figure 1

Size characteristics of Candida glabrata, Candida parapsilosis, and Candida tropicalis biofilm-derived extracellular vesicles (EVs). TEM images of EVs (A, C, E) and NTA particle size distribution analysis (B, D, F); representative histograms of the average size distribution from three measurements of a single sample (black line). Numbers show the maxima of particular peaks and red areas indicate the standard deviation (SD) between measurements. Tables include EVs’ size parameters measured by NTA; factors D10, D50, and D90 mean that 10%, 50%, and 90% of the EV population had a diameter of less than or equal to the given value. Data are presented as means ± SEM.

Figure 2

The Venn diagram representing the numbers of individual and common proteins among EVs from C. glabrata, C. parapsilosis, and C. tropicalis biofilms.

Figure 3

Functional classification of proteins identified for C. glabrata, C. parapsilosis, and C. tropicalis biofilm-originated EVs.

Figure 4

The analysis of cytokine production by THP-1 cells stimulated with Candida EVs. The amount of a particular cytokine was shown for stimulation with C. glabrata EVs (CG), C. parapsilosis EVs (CP), and C. tropicalis EVs (CT). A representative result of three independent experiments is presented. To analyze the statistical significance versus control, an unpaired t-test was performed with GraphPad Prism software version 7.0 (GraphPad Software, La Jolla, CA, USA). The statistical significance levels were marked with * for p < 0.05, ** for p < 0.01, *** for p < 0.001, and **** for p < 0.0001.

Figure 5

Candida tropicalis biofilm formation in the presence of C. tropicalis biofilm-derived EVs and the antifungal drugs fluconazole (FLU) and caspofungin (CASP). The thickness of the biofilm was investigated by measuring optical density at 600 nm in the area scan mode (A), the metabolic activity of cells in the biofilm by the XTT test (B), and the viability of the cell by counting CFUs (C). A representative result of three independent experiments is presented. To analyze the statistical significance between samples, an unpaired t-test was performed with GraphPad Prism software version 7.0. The statistical significance levels were marked with * for p < 0.05 and ns when not significant.