The frequency of severe systemic fungal diseases has increased in the last few decades. The clinical use of antibacterial drugs, immunosuppressive agents after organ transplantation, cancer chemotherapy, and advances in surgery are associated with increasing risk of fungal infections. Opportunistic pathogens from the genera Candida and Aspergillus as well as pathogenic fungi from the genus Cryptococcus can invade human organism and may lead to mucosal and skin infections or to deep-seated mycoses of almost all inner organs, especially in immunocompromised patients. Nowadays, there are some effective antifungal agents, but, unfortunately, some of the pathogenic species show increasing resistance. The identification of fungal virulence factors and recognition of mechanisms of pathogenesis may lead to development of new efficient antifungal therapies. This review is focused on major virulence factors of the most common fungal pathogens of humans: Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. The adherence to host cells and tissues, secretion of hydrolytic enzymes, phenotypic switching and morphological dimorphism contribute to C. albicans virulence. The ability to grow at 37 degrees C, capsule synthesis and melanin formation are important virulence factors of C. neoformans. The putative virulence factors of A. fumigatus include production of pigments, adhesion molecules present on the cell surface and secretion of hydrolytic enzymes and toxins.

Neutrophils use different mechanisms to cope with pathogens that invade the host organism. The most intriguing of these responses is a release of neutrophil extracellular traps (NETs) composed of decondensed chromatin and granular proteins with antimicrobial activity. An important potential target of NETs is Candida albicans—an opportunistic fungal pathogen that employs morphological and phenotype switches and biofilm formation during contact with neutrophils, accompanied by changes in epitope exposition that mask the pathogen from host recognition. These processes differ depending on infection conditions and are thus influenced by the surrounding environment. In the current study, we compared the NET release by neutrophils upon contact with purified main candidal cell surface components. We show here for the first time that in addition to the main cell wall-building polysaccharides (mannans and β-glucans), secreted aspartic proteases (Saps) trigger NETs with variable intensities. The most efficient NET-releasing response is with Sap4 and Sap6, which are known to be secreted by fungal hyphae. This involves mixed, ROS-dependent and ROS-independent signaling pathways, mainly through interactions with the CD11b receptor. In comparison, upon contact with the cell wall-bound Sap9 and Sap10, neutrophils responded via a ROS-dependent mechanism using CD16 and CD18 receptors for protease recognition. In addition to the Saps tested, the actuation of selected mediating kinases (Src, Syk, PI3K, and ERK) was also investigated. β-Glucans were found to trigger a ROS-dependent process of NET production with engagement of Dectin-1 as well as CD11b and CD18 receptors. Mannans were observed to be recognized by TLRs, CD14, and Dectin-1 receptors and triggered NET release mainly via a ROS-independent pathway. Our results thus strongly suggest that neutrophils activate NET production in response to different candidal components that are presented locally at low concentrations at the initial stages of infection. However, NET release seemed to be blocked by increasing numbers of fungal cells.

The delicate balance between eukaryotic pathogens and their human hosts during the initiation and development of infection is a complex process involving many diverse interactions. Different infectious agents, including pathogenic fungi, parasitic protozoa and multicellular parasites, directly interact through their cell surface with epithelial or endothelial cells of the human host as well as various proteinaceous host ligands such as extracellular matrix or plasma proteins. Eukaryotic pathogens possess a number of virulence factors but a relatively recently recognized and particularly interesting group of factors capable of enhancing virulence is the set of so-called 'moonlighting proteins'. This term was coined for a relatively large collection of housekeeping enzymes lacking special targeting motifs that would determine their extracellular localization, but that are often present at the cell surface of pathogen. Several such enzymes with key metabolic functions in glycolysis, the pentose phosphate cycle or other fundamental intracellular processes perform entirely new, non-catalytic roles often associated with adhesion to host ligands. Our current study summarizes some of the current knowledge of interesting moonlighting proteins which play putative or confirmed roles as virulence factors in pathogenic fungi, parasitic protozoa and multicellular parasites.

BackgroundCandida parapsilosis and C. tropicalis increasingly compete with C. albicans—the most common fungal pathogen in humans—as causative agents of severe candidiasis in immunocompromised patients. In contrast to C. albicans, the pathogenic mechanisms of these two non-albicans Candida species are poorly understood. Adhesion of Candida yeast to host cells and the extracellular matrix is critical for fungal invasion of hosts.MethodsThe fungal proteins involved in interactions with extracellular matrix proteins were isolated from mixtures of β-1,3-glucanase– or β-1,6-glucanase–extractable cell wall-associated proteins by use of affinity chromatography and chemical cross-linking methods, and were further identified by liquid chromatography-coupled tandem mass spectrometry.ResultsIn the present study, we characterized the binding of three major extracellular matrix proteins—fibronectin, vitronectin and laminin—to C. parapsilosis and C. tropicalis pseudohyphae. The major individual compounds of the fungal cell wall that bound fibronectin, vitronectin and laminin were found to comprise two groups: (1) true cell wall components similar to C. albicans adhesins from the Als, Hwp and Iff/Hyr families; and (2) atypical (cytoplasm-derived) surface-exposed proteins, including malate synthase, glucose-6-phosphate isomerase, 6-phosphogluconate dehydrogenase, enolase, fructose-1,6-bisphosphatase, transketolase, transaldolase and elongation factor 2.DiscussionThe adhesive abilities of two investigated non-albicans Candida species toward extracellular matrix proteins were comparable to those of C. albicans suggesting an important role of this particular virulence attribute in the pathogenesis of infections caused by C. tropicalis and C. parapsilosis.ConclusionsOur results reveal new insight into host–pathogen interactions during infections by two important, recently emerging, fungal pathogens.

The oral cavity contains different types of microbial species that colonize human host via extensive cell-to-cell interactions and biofilm formation. Candida albicans—a yeast-like fungus that inhabits mucosal surfaces—is also a significant colonizer of subgingival sites in patients with chronic periodontitis. It is notable however that one of the main infectious agents that causes periodontal disease is an anaerobic bacterium—Porphyromonas gingivalis. In our study, we evaluated the different strategies of both pathogens in the mutual colonization of an artificial surface and confirmed that a protective environment existed for P. gingivalis within developed fungal biofilm formed under oxic conditions where fungal cells grow mainly in their filamentous form i.e. hyphae. A direct physical contact between fungi and P. gingivalis was initiated via a modulation of gene expression for the major fungal cell surface adhesin Als3 and the aspartic proteases Sap6 and Sap9. Proteomic identification of the fungal surfaceome suggested also an involvement of the Mp65 adhesin and a “moonlighting” protein, enolase, as partners for the interaction with P. gingivalis. Using mutant strains of these bacteria that are defective in the production of the gingipains—the proteolytic enzymes that also harbor hemagglutinin domains—significant roles of these proteins in the formation of bacteria-protecting biofilm were clearly demonstrated.

Cell wall proteins of Candida albicans, besides their best known role in the adhesion of this fungal pathogen to host's tissues, also bind some soluble proteins, present in body fluids and involved in maintaining the biochemical homeostasis of the human organism. In particular, three plasma factors - high-molecular-mass kininogen (HK), factor XII (FXII) and prekallikrein (PPK) - have been shown to adhere to candidal cells. These proteins are involved in the surface-contact-catalyzed production of bradykinin-related peptides (kinins) that contribute to inflammatory states associated with microbial infections. We recently identified several proteins, associated with the candidal cell walls, and probably involved in the binding of HK. In our present study, a list of potential FXII- and PPK-binding proteins was proposed, using an affinity selection (on agarose-coupled FXII or PPK) from a whole mixture of β-1,3-glucanase-extrated cell wall-associated proteins and the mass-spectrometry protein identification. Five of these fungal proteins, including agglutinin-like sequence protein 3 (Als3), triosephosphate isomerase 1 (Tpi1), enolase 1 (Eno1), phosphoglycerate mutase 1 (Gpm1) and glucose-6-phosphate isomerase 1 (Gpi1), were purified and characterized in terms of affinities to the human contact factors, using the surface plasmon resonance measurements. Except Gpm1 that bound only PPK, and Als3 that exhibited an affinity to HK and FXII, the other isolated proteins interacted with all three contact factors. The determined dissociation constants for the identified protein complexes were of 10(-7) M order, and the association rate constants were in a range of 10(4)-10(5) M(-1)s(-1). The identified fungal pathogen-host protein interactions are potential targets for novel anticandidal therapeutic approaches.

No abstract

The increased incidence of severe disseminated infections caused by the opportunistic yeast-like fungi Candida spp. highlights the urgent need for research into the major virulence factors of these pathogens-extracellular aspartic proteinases of the candidapepsin and yapsin families. Classically, these enzymes were considered to be generally destructive factors that damage host tissues and provide nutrients for pathogen propagation. However, in recent decades, novel and more specific functions have been suggested for extracellular candidal proteinases. These include contributions to cell wall maintenance and remodeling, the formation of polymicrobial biofilms, adhesion to external protective barriers of the host, the deregulation of host proteolytic cascades (such as the complement system, blood coagulation and the kallikrein-kinin system), a dysregulated host proteinase-inhibitor balance, the inactivation of host antimicrobial peptides, evasion of immune responses and the induction of inflammatory mediator release from host cells. Only a few of these activities recognized in Candida albicans candidapepsins have been also confirmed in other Candida species, and characterization of Candida glabrata yapsins remains limited.