Maria Rapala-Kozik

Candida albicans and other closely related pathogenic yeast-like fungi carry on their surface numerous loosely adsorbed “moonlighting proteins”—proteins that play evolutionarily conserved intracellular functions but also appear on the cell surface and exhibit additional functions, e.g., contributing to attachment to host tissues. In the current work, we characterized this “moonlighting” role for glyceraldehyde 3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) of C. albicans and Nakaseomyces glabratus. GAPDH was directly visualized on the cell surface of both species and shown to play a significant part in the total capacity of fungal cells to bind two selected human host proteins—vitronectin and plasminogen. Using purified proteins, both host proteins were found to tightly interact with GAPDH, with dissociation constants in an order of 10−8 M, as determined by bio-layer interferometry and surface plasmon resonance measurements. It was also shown that exogenous GAPDH tightly adheres to the surface of candidal cells, suggesting that the cell surface location of this moonlighting protein may partly result from the readsorption of its soluble form, which may be present at an infection site (e.g., due to release from dying fungal cells). The major dedicated adhesins, covalently bound to the cell wall—agglutinin-like sequence protein 3 (Als3) and epithelial adhesin 6 (Epa6)—were suggested to serve as the docking platforms for GAPDH in C. albicans and N. glabratus, respectively.

Human immune cells possess the ability to react complexly and effectively after contact with microbial virulence factors, including those transported in cell-derived structures of nanometer sizes termed extracellular vesicles (EVs). EVs are produced by organisms of all kingdoms, including fungi pathogenic to humans. In this work, the immunomodulatory properties of EVs produced under oxidative stress conditions or at host concentrations of CO2 by the fungal pathogen Candida albicans were investigated. The interaction of EVs with human pro-monocytes of the U-937 cell line was established, and the most notable effect was attributed to oxidative stress-related EVs. The immunomodulatory potential of tested EVs against human THP-1 macrophages was verified using cytotoxicity assay, ROS-production assay, and the measurement of cytokine production. All fungal EVs tested did not show a significant cytotoxic effect on THP-1 cells, although a slight pro-oxidative impact was indicated for EVs released by C. albicans cells grown under oxidative stress. Furthermore, for all tested types of EVs, the pro-inflammatory properties related to increased IL-8 and TNF-α production and decreased IL-10 secretion were demonstrated, with the most significant effect observed for EVs released under oxidative stress conditions.

It has been repeatedly reported that the cells of organisms in all kingdoms of life produce nanometer-sized lipid membrane-enveloped extracellular vesicles (EVs), transporting and protecting various substances of cellular origin. While the composition of EVs produced by human pathogenic fungi has been studied in recent decades, another important challenge is the analysis of their functionality. Thus far, fungal EVs have been shown to play significant roles in intercellular communication, biofilm production, and modulation of host immune cell responses. In this study, we verified the involvement of biofilm-derived EVs produced by two different strains of Candida albicans—C. albicans SC5314 and 3147 (ATCC 10231)—in various aspects of biofilm function by examining its thickness, stability, metabolic activity, and cell viability in the presence of EVs and the antifungal drug caspofungin. Furthermore, the proteolytic activity against the kininogen-derived antimicrobial peptide NAT26 was confirmed by HPLC analysis for C. albicans EVs that are known to carry, among others, particular members of the secreted aspartic proteinases (Saps) family. In conclusion, EVs derived from C. albicans biofilms were shown to be involved in biofilm tolerance to caspofungin, biofilm detachment, and fungal proteolytic activity.