Secreted Aspartic Proteinases: Key Factors in Candida Infections and Host-Pathogen Interactions.

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.

Stress Conditions Affect the Immunomodulatory Potential of Candida albicans Extracellular Vesicles and Their Impact on Cytokine Release by THP-1 Human Macrophages.

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.

Candida albicans Biofilm-Derived Extracellular Vesicles Are Involved in the Tolerance to Caspofungin, Biofilm Detachment, and Fungal Proteolytic Activity.

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.

Living together: The role of Candida albicans in the formation of polymicrobial biofilms in the oral cavity.

The oral cavity of humans is colonized by diversity of microbial community, although dominated by bacteria, it is also constituted by a low number of fungi, often represented by Candida albicans. Although in the vast minority, this usually commensal fungus under certain conditions of the host (e.g., immunosuppression or antibiotic therapy), can transform into an invasive pathogen that adheres to mucous membranes and also to medical or dental devices, causing mucosal infections. This transformation is correlated with changes in cell morphology from yeast-like cells to hyphae and is supported by numerous virulence factors exposed by C. albicans cells at the site of infection, such as multifunctional adhesins, degradative enzymes, or toxin. All of them affect the surrounding host cells or proteins, leading to their destruction. However, at the site of infection, C. albicans can interact with different bacterial species and in its filamentous form may produce biofilms-the elaborated consortia of microorganisms, that present increased ability to host colonization and resistance to antimicrobial agents. In this review, we highlight the modification of the infectious potential of C. albicans in contact with different bacterial species, and also consider the mutual bacterial-fungal relationships, involving cooperation, competition, or antagonism, that lead to an increase in the propagation of oral infection. The mycofilm of C. albicans is an excellent hiding place for bacteria, especially those that prefer low oxygen availability, where microbial cells during mutual co-existence can avoid host recognition or elimination by antimicrobial action. However, these microbial relationships, identified mainly in in vitro studies, are modified depending on the complexity of host conditions and microbial dominance in vivo.

Isolation and Characteristics of Extracellular Vesicles Produced by Probiotics: Yeast Saccharomyces boulardii CNCM I-745 and Bacterium Streptococcus salivarius K12.

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-1ß 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.

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

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.

The Role of Candida albicans Virulence Factors in the Formation of Multispecies Biofilms With Bacterial Periodontal Pathogens.

Periodontal disease depends on the presence of different microorganisms in the oral cavity that during the colonization of periodontal tissues form a multispecies biofilm community, thus allowing them to survive under adverse conditions or facilitate further colonization of host tissues. Not only numerous bacterial species participate in the development of biofilm complex structure but also fungi, especially Candida albicans, that often commensally inhabits the oral cavity. C. albicans employs an extensive armory of various virulence factors supporting its coexistence with bacteria resulting in successful host colonization and propagation of infection. In this article, we highlight various aspects of individual fungal virulence factors that may facilitate the collaboration with the associated bacterial representatives of the early colonizers of the oral cavity, the bridging species, and the late colonizers directly involved in the development of periodontitis, including the "red complex" species. In particular, we discuss the involvement of candidal cell surface proteins-typical fungal adhesins as well as originally cytosolic "moonlighting" proteins that perform a new function on the cell surface and are also present within the biofilm structures. Another group of virulence factors considered includes secreted aspartic proteases (Sap) and other secreted hydrolytic enzymes. The specific structure of the candidal cell wall, dynamically changing during morphological transitions of the fungus that favor the biofilm formation, is equally important and discussed. The non-protein biofilm-composing factors also show dynamic variability upon the contact with bacteria, and their biosynthesis processes could be involved in the stability of mixed biofilms. Biofilm-associated changes in the microbe communication system using different quorum sensing molecules of both fungal and bacterial cells are also emphasized in this review. All discussed virulence factors involved in the formation of mixed biofilm pose new challenges and influence the successful design of new diagnostic methods and the application of appropriate therapies in periodontal diseases.

Characteristics of Extracellular Vesicles Released by the Pathogenic Yeast-Like Fungi Candida glabrata, Candida parapsilosis and Candida tropicalis.

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.