Candida spp. yeast-like fungi are opportunistic pathogens in humans and have been recently found to release extracellular vesicles (EVs) that are involved in many vital biological processes in fungal cells. These include communication between microorganisms and host–pathogen interactions during infection. The production of EVs and their content have been significantly characterized in the most common candidal species Candida albicans, including the identification of numerous virulence factors and cytoplasmic proteins in the EV cargo. We have here conducted the isolation and proteomic characterization of EVs produced by the clinically important non-albicans Candida species C. glabrata, C. tropicalis and C. parapsilosis. With the use of ultracentrifugation of the cell-free culture supernatant, the candidal EVs were collected and found to be a heterogeneous population of particles for each species with sizes ranging from 60–280 nm. The proteinaceous contents of these vesicles were analyzed using LC-MS/MS, with particular attention paid to surface-expressed proteins that would come into immediate and direct contact with host cells. We thereby identified 42 extracellular and surface-connected proteins from C. glabrata, 33 from C. parapsilosis, and 34 from C. tropicalis, including membrane-associated transporters, glycoproteins and enzymes involved in the organization of the fungal cell wall, as well as several cytoplasmic proteins, including alcohol dehydrogenase, enolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and pyruvate kinase, for which the vesicular transport is a possible mechanism underlying their non-classical secretion.

Extracellular proteases are key factors contributing to the virulence of pathogenic fungi from the genus Candida. Their proteolytic activities are crucial for extracting nutrients from the external environment, degrading host defenses, and destabilizing the internal balance of the human organism. Currently, the enzymes most frequently described in this context are secreted aspartic proteases (Saps). This review comprehensively explores the multifaceted roles of Saps, highlighting their importance in biofilm formation, tissue invasion through the degradation of extracellular matrix proteins and components of the coagulation cascade, modulation of host immune responses via impairment of neutrophil and monocyte/macrophage functions, and their contribution to antifungal resistance. Additionally, the diagnostic challenges associated with Candida infections and the potential of Saps as biomarkers were discussed. Furthermore, we examined the prospects of developing vaccines based on Saps and the use of protease inhibitors as adjunctive therapies for candidiasis. Given the complex biology of Saps and their central role in Candida pathogenicity, a multidisciplinary approach may pave the way for innovative diagnostic strategies and open new opportunities for innovative clinical interventions against candidiasis.

Numerous probiotic microorganisms have repeatedly been shown to produce nanometer-sized structures named extracellular vesicles (EVs). Recently, it has been suggested that similarly to whole microbial cells, EVs produced by probiotics may also demonstrate health benefits to the host, while their application does not involve the risk of infection caused by live microorganisms. In this work, we isolated EVs from two probiotic species originating from different taxonomic domains — yeast Saccharomyces boulardii CNCM I-745 and bacterium Streptococcus salivarius K12. The diameters of S. boulardii EVs were about 142 nm and for S. salivarius EVs about 123 nm. For S. boulardii EVs, 1641 proteins and for S. salivarius EVs, 466 proteins were identified with a liquid chromatography-coupled tandem mass spectrometry and then functionally classified. In both microbial species, metabolic proteins significantly contributed to the cargo of EVs comprising 25% and 26% of all identified vesicular proteins for fungi and bacteria, respectively. Moreover, enzymes associated with cell wall rearrangement, including enzymatically active glucanases, were also identified in EVs. Furthermore, probiotic EVs were shown to influence host cells and stimulate the production of IL-1β and IL-8 by the human monocytic cell line THP-1, and, at the same time, did not cause any remarkable reduction in the survival rate of Galleria mellonella larvae in this invertebrate model commonly used to evaluate microbial EV toxicity. These observations suggest that the EVs produced by the investigated probiotic microorganisms may be promising structures for future use in pro-health applications.

Although Candida species are widespread commensals of the microflora of healthy individuals, they are also among the most important human fungal pathogens that under certain conditions can cause diseases (candidiases) of varying severity ranging from mild superficial infections of the mucous membranes to life-threatening systemic infections. So far, the vast majority of research aimed at understanding the molecular basis of pathogenesis has been focused on the most common speciesCandida albicans. Meanwhile, other closely related species belonging to the CTG clade, namely, Candida tropicalis and Candida dubliniensis, are becoming more important in clinical practice, as well as a relatively newly identified species, Candida auris. Despite the close relationship of these microorganisms, it seems that in the course of evolution, they have developed distinct biochemical, metabolic, and physiological adaptations, which they use to fit to commensal niches and achieve full virulence. Therefore, in this review, we describe the current knowledge on C. tropicalis, C. dubliniensis, and C. auris virulence factors, the formation of a mixed species biofilm and mutual communication, the environmental stress response and related changes in fungal cell metabolism, and the effect of pathogens on host defense response and susceptibility to antifungal agents used, highlighting differences with respect to C. albicans. Special attention is paid to common diagnostic problems resulting from similarities between these species and the emergence of drug resistance mechanisms. Understanding the different strategies to achieve virulence, used by important opportunistic pathogens of the genus Candida, is essential for proper diagnosis and treatment.

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.

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.