Glyceraldehyde 3-Phosphate Dehydrogenase on the Surface of Candida albicans and Nakaseomyces glabratus Cells-A Moonlighting Protein That Binds Human Vitronectin and Plasminogen and Can Adsorb to Pathogenic Fungal Cells via Major Adhesins Als3 and Epa6.

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.

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.

Release of neutrophil extracellular traps in response to Candida albicans yeast, as a secondary defense mechanism activated by phagocytosis.

Candida albicans is one of the main pathogens responsible for the development of difficult-to-fight fungal infections called candidiasis. Neutrophils are the major effector cells involved in the eradication of fungal pathogens. This group of immune cells uses several mechanisms that enable the rapid neutralization of pathogens. The most frequently identified mechanisms are phagocytosis and the release of neutrophil extracellular traps (NETs). The mechanism for selecting the type of neutrophil immune response is still unknown. In our study, we analyzed the relationship between the activation of phagocytosis and netosis. We detected the presence of two neutrophil populations characterized by different response patterns to contact with C. albicans blastospores. The first neutrophil population showed an increased ability to rapidly release NETs without prior internalization of the pathogen. In the second population, the netosis process was inherently associated with phagocytosis. Differences between populations also referred to the production of reactive oxygen species. Our results suggest that neutrophils use different strategies to fight C. albicans and, contrary to previous reports, these mechanisms are not mutually exclusive.

Candida parapsilosis cell wall proteins-CPAR2_404800 and CPAR2_404780-Are adhesins that bind to human epithelial and endothelial cells and extracellular matrix proteins.

One of the initial steps necessary for the development of Candida infections is the adherence to the host tissues and cells. Recent transcriptomic studies suggest that, in Candida parapsilosis-a fungal infectious agent that causes systemic candidiasis in immunosuppressed individuals-the adhesion is mediated by pathogen cell-exposed proteins belonging to the agglutinin-like sequence (Als) family. However, to date, the actual interactions of individual members of this family with human cells and extracellular matrix (ECM) have not been characterized in detail. In the current study, we focused attention on two of these C. parapsilosis Als proteins-CPAR2_404800 and CPAR2_404780-that were proteomically identified in the fungal cell wall of yeasts grown in the media suitable for culturing human epithelial and endothelial cells. Both proteins were extracted from the cell wall and purified, and using a microplate binding assay and a fluorescence microscopic analysis were shown to adhere to human cells of A431 (epithelial) and HMEC-1 (endothelial) lines. The human extracellular matrix components that are also plasma proteins-fibronectin and vitronectin-enhanced these interactions, and also could directly bind to CPAR2_404800 and CPAR2_404780 proteins, with a high affinity (KD in a range of 10-7 to 10-8 M) as determined by surface plasmon resonance measurements. Our findings highlight the role of proteins CPAR2_404800 and CPAR2_404780 in adhesion to host cells and proteins, contributing to the knowledge of the mechanisms of host-pathogen interactions during C. parapsilosis-caused infections.

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.

The Recruitment and Activation of Plasminogen by Bacteria-The Involvement in Chronic Infection Development.

The development of infections caused by pathogenic bacteria is largely related to the specific properties of the bacterial cell surface and extracellular hydrolytic activity. Furthermore, a significant role of hijacking of host proteolytic cascades by pathogens during invasion should not be disregarded during consideration of the mechanisms of bacterial virulence. This is the key factor for the pathogen evasion of the host immune response, tissue damage, and pathogen invasiveness at secondary infection sites after initial penetration through tissue barriers. In this review, the mechanisms of bacterial impact on host plasminogen-the precursor of the important plasma serine proteinase, plasmin-are characterized, principally focusing on cell surface exposition of various proteins, responsible for binding of this host (pro)enzyme and its activators or inhibitors, as well as the fibrinolytic system activation tactics exploited by different bacterial species, not only pathogenic, but also selected harmless residents of the human microbiome. Additionally, the involvement of bacterial factors that modulate the process of plasminogen activation and fibrinolysis during periodontitis is also described, providing a remarkable example of a dual use of this host system in the development of chronic diseases.

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.

Als3-mediated attachment of enolase on the surface of Candida albicans cells regulates their interactions with host proteins.

The multifunctional protein enolase has repeatedly been identified on the surface of numerous cell types, including a variety of pathogenic microorganisms. In Candida albicans-one of the most common fungal pathogens in humans-a surface-exposed enolase form has been previously demonstrated to play an important role in candidal pathogenicity. In our current study, the presence of enolase at the fungal cell surface under different growth conditions was examined, and a higher abundance of enolase at the surface of C. albicans hyphal forms compared to yeast-like cells was found. Affinity chromatography and chemical cross-linking indicated a member of the agglutinin-like sequence protein family-Als3-as an important potential partner required for the surface display of enolase. Analysis of Saccharomyces cerevisiae cells overexpressing Als3 with site-specific deletions showed that the Ig-like N-terminal region of Als3 (aa 166-225; aa 218-285; aa 270-305; aa 277-286) and the central repeat domain (aa 434-830) are essential for the interaction of this adhesin with enolase. In addition, binding between enolase and Als3 influenced subsequent docking of host plasma proteins-high molecular mass kininogen and plasminogen-on the candidal cell surface, thus supporting the hypothesis that C. albicans can modulate plasma proteolytic cascades to affect homeostasis within the host and propagate inflammation during infection.

Fungi-A Component of the Oral Microbiome Involved in Periodontal Diseases.

The human oral cavity is a diverse ecological niche favorable for colonization by hundreds of different species of microorganisms. They include not only bacteria but also numerous species of fungi, many of which are able to cause opportunistic infections when the host's immunity is impaired, predominantly by systemic and chronic diseases like diabetes, pulmonary diseases, renal disorders, or acquired immunodeficiency syndrome. Within the dental biofilm and subgingival sites, fungi of the genus Candida are often found, also in individuals affected with periodontitis. Moreover, fungal species of other genera, including Malassezia, Aspergillus, Penicillium, and Rhodotorula were identified in the oral cavity as well. The wide range of various virulence factors and mechanisms displayed by fungal pathogens allows them effectively invading host tissues during periodontal infections. These pathogenicity-related mechanisms include firstly the fungal ability to adhere successfully to the host tissues closely related to the formation of hyphae, the increase in the surface hydrophobicity, and the surface display of a wide variety of adhesins. Further mechanisms include biofilm formation and secretion of an armory of hydrolytic enzymes and toxins enabling the attack on host cells, modulation of the local inflammatory state, and evading the host immune system. In the pathogenesis of periodontitis, the significant role of fungal co-existence with key bacterial periodontopathogens has been demonstrated, and such interactions were primarily confirmed for Candida albicans and Porphyromonas gingivalis, where the presence of fungi ensured the survival of strictly anaerobic bacteria under unfavorable aerobic conditions. However, several other mechanisms, including those related to the production of quorum sensing molecules, might also be indicated as particularly important for synergistic or antagonistic interactions with a variety of bacterial species within mixed biofilms. These interactions constitute an extraordinary challenge for applying effective methods of combating biofilm-related infections in the periodontium without the risk of the development of drug resistance, the recurrence of disease symptoms, and the progress of life-threating systemic complications.

A Novel Biological Role for Peptidyl-Arginine Deiminases: Citrullination of Cathelicidin LL-37 Controls the Immunostimulatory Potential of Cell-Free DNA.

LL-37, the only human cathelicidin that is released during inflammation, is a potent regulator of immune responses by facilitating delivery of oligonucleotides to intracellular TLR-9, thereby enhancing the response of human plasmacytoid dendritic cells (pDCs) to extracellular DNA. Although important for pathogen recognition, this mechanism may facilitate development of autoimmune diseases. In this article, we show that citrullination of LL-37 by peptidyl-arginine deiminases (PADs) hindered peptide-dependent DNA uptake and sensing by pDCs. In contrast, carbamylation of the peptide (homocitrullination of Lys residues) had no effect. The efficiency of LL-37 binding to oligonucleotides and activation of pDCs was found to be inversely proportional to the number of citrullinated residues in the peptide. Similarly, preincubation of carbamylated LL-37 with PAD2 abrogated the peptide's ability to bind DNA. Conversely, LL-37 with Arg residues substituted by homoarginine, which cannot be deiminated, elicited full activity of native LL-37 regardless of PAD2 treatment. Taken together, the data showed that citrullination abolished LL-37 ability to bind DNA and altered the immunomodulatory function of the peptide. Both activities were dependent on the proper distribution of guanidinium side chains in the native peptide sequence. Moreover, our data suggest that cathelicidin/LL-37 is citrullinated by PADs during NET formation, thus affecting the inflammatory potential of NETs. Together this may represent a novel mechanism for preventing the breakdown of immunotolerance, which is dependent on the response of APCs to self-molecules (including cell-free DNA); overactivation may facilitate development of autoimmunity.

Structural Insights into the Interactions of Candidal Enolase with Human Vitronectin, Fibronectin and Plasminogen.

Significant amounts of enolase-a cytosolic enzyme involved in the glycolysis pathway-are exposed on the cell surface of Candida yeast. It has been hypothesized that this exposed enolase form contributes to infection-related phenomena such as fungal adhesion to human tissues, and the activation of fibrinolysis and extracellular matrix degradation. The aim of the present study was to characterize, in structural terms, the protein-protein interactions underlying these moonlighting functions of enolase. The tight binding of human vitronectin, fibronectin and plasminogen by purified C. albicans and C. tropicalis enolases was quantitatively analyzed by surface plasmon resonance measurements, and the dissociation constants of the formed complexes were determined to be in the 10-7-10-8 M range. In contrast, the binding of human proteins by the S.cerevisiae enzyme was much weaker. The chemical cross-linking method was used to map the sites on enolase molecules that come into direct contact with human proteins. An internal motif 235DKAGYKGKVGIAMDVASSEFYKDGK259 in C. albicans enolase was suggested to contribute to the binding of all three human proteins tested. Models for these interactions were developed and revealed the sites on the enolase molecule that bind human proteins, extensively overlap for these ligands, and are well-separated from the catalytic activity center.

Plant-Derived Substances in the Fight Against Infections Caused by Candida Species.

Yeast-like fungi from the Candida genus are predominantly harmless commensals that colonize human skin and mucosal surfaces, but under conditions of impaired host immune system change into dangerous pathogens. The pathogenicity of these fungi is typically accompanied by increased adhesion and formation of complex biofilms, making candidal infections challenging to treat. Although a variety of antifungal drugs have been developed that preferably attack the fungal cell wall and plasma membrane, these pathogens have acquired novel defense mechanisms that make them resistant to standard treatment. This causes an increase in the incidence of candidiasis and enforces the urgent need for an intensified search for new specifics that could be helpful, alone or synergistically with traditional drugs, for controlling Candida pathogenicity. Currently, numerous reports have indicated the effectiveness of plant metabolites as potent antifungal agents. These substances have been shown to inhibit growth and to alter the virulence of different Candida species in both the planktonic and hyphal form and during the biofilm formation. This review focuses on the most recent findings that provide evidence of decreasing candidal pathogenicity by different substances of plant origin, with a special emphasis on the mechanisms of their action. This is a particularly important issue in the light of the currently increasing frequency of emerging Candida strains and species resistant to standard antifungal treatment.

Peptidylarginine Deiminase of Porphyromonas gingivalis Modulates the Interactions between Candida albicans Biofilm and Human Plasminogen and High-Molecular-Mass Kininogen.

Microorganisms that create mixed-species biofilms in the human oral cavity include, among others, the opportunistic fungus Candida albicans and the key bacterial pathogen in periodontitis, Porphyromonas gingivalis. Both species use arsenals of virulence factors to invade the host organism and evade its immune system including peptidylarginine deiminase that citrullinates microbial and host proteins, altering their function. We assessed the effects of this modification on the interactions between the C. albicans cell surface and human plasminogen and kininogen, key components of plasma proteolytic cascades related to the maintenance of hemostasis and innate immunity. Mass spectrometry was used to identify protein citrullination, and microplate tests to quantify the binding of modified plasminogen and kininogen to C. albicans cells. Competitive radioreceptor assays tested the affinity of citrullinated kinins to their specific cellular receptors. The citrullination of surface-exposed fungal proteins reduced the level of unmodified plasminogen binding but did not affect unmodified kininogen binding. However, the modification of human proteins did not disrupt their adsorption to the unmodified fungal cells. In contrast, the citrullination of kinins exerted a significant impact on their interactions with cellular receptors reducing their affinity and thus affecting the role of kinin peptides in the development of inflammation.

Candida albicans Shields the Periodontal Killer Porphyromonas gingivalis from Recognition by the Host Immune System and Supports the Bacterial Infection of Gingival Tissue.

Candida albicans is a pathogenic fungus capable of switching its morphology between yeast-like cells and filamentous hyphae and can associate with bacteria to form mixed biofilms resistant to antibiotics. In these structures, the fungal milieu can play a protective function for bacteria as has recently been reported for C. albicans and a periodontal pathogen-Porphyromonas gingivalis. Our current study aimed to determine how this type of mutual microbe protection within the mixed biofilm affects the contacting host cells. To analyze C. albicans and P. gingivalis persistence and host infection, several models for host-biofilm interactions were developed, including microbial exposure to a representative monocyte cell line (THP1) and gingival fibroblasts isolated from periodontitis patients. For in vivo experiments, a mouse subcutaneous chamber model was utilized. The persistence of P. gingivalis cells was observed within mixed biofilm with C. albicans. This microbial co-existence influenced host immunity by attenuating macrophage and fibroblast responses. Cytokine and chemokine production decreased compared to pure bacterial infection. The fibroblasts isolated from patients with severe periodontitis were less susceptible to fungal colonization, indicating a modulation of the host environment by the dominating bacterial infection. The results obtained for the mouse model in which a sequential infection was initiated by the fungus showed that this host colonization induced a milder inflammation, leading to a significant reduction in mouse mortality. Moreover, high bacterial counts in animal organisms were noted on a longer time scale in the presence of C. albicans, suggesting the chronic nature of the dual-species infection.

Moonlighting proteins are variably exposed at the cell surfaces of Candida glabrata, Candida parapsilosis and Candida tropicalis under certain growth conditions.

BACKGROUND: Adaptability to different environmental conditions is an essential characteristic of pathogenic microorganisms as it facilitates their invasion of host organisms. The most external component of pathogenic yeast-like fungi from the Candida genus is the multilayered cell wall. This structure is composed mainly of complex polysaccharides and proteins that can undergo dynamic changes to adapt to the environmental conditions of colonized niches. RESULTS: We utilized cell surface shaving with trypsin and a shotgun proteomic approach to reveal the surface-exposed proteins of three important non-albicans Candida species-C. glabrata, C. parapsilosis and C. tropicalis. These proteinaceous components were identified after the growth of the fungal cells in various culture media, including artificial saliva, artificial urine and vagina-simulative medium under aerobic conditions and anaerobically in rich YPD medium. Several known proteins involved in cell wall maintenance and fungal pathogenesis were identified at the cell surface as were a number of atypical cell wall components-pyruvate decarboxylase (Pdc11), enolase (Eno1) and glyceraldehyde-3-phosphate dehydrogenase (Tdh3) which are so-called 'moonlighting' proteins. Notably, many of these proteins showed significant upregulation at the cell surface in growth media mimicking the conditions of infection compared to defined synthetic medium. CONCLUSIONS: Moonlighting proteins are expressed under diverse conditions at the cell walls of the C. glabrata, C. parapsilosis and C. tropicalis fungal pathogens. This indicates a possible universal surface-associated role of these factors in the physiology of these fungi and in the pathology of the infections they cause.

Inactivation of the antifungal and immunomodulatory properties of human cathelicidin LL-37 by aspartic proteases produced by the pathogenic yeast Candida albicans.

Constant cross talk between Candida albicans yeast cells and their human host determines the outcome of fungal colonization and, eventually, the progress of infectious disease (candidiasis). An effective weapon used by C. albicans to cope with the host defense system is the release of 10 distinct secreted aspartic proteases (SAPs). Here, we validate a hypothesis that neutrophils and epithelial cells use the antimicrobial peptide LL-37 to inactivate C. albicans at sites of candidal infection and that C. albicans uses SAPs to effectively degrade LL-37. LL-37 is cleaved into multiple products by SAP1 to -4, SAP8, and SAP9, and this proteolytic processing is correlated with the gradual decrease in the antifungal activity of LL-37. Moreover, a major intermediate of LL-37 cleavage-the LL-25 peptide-is antifungal but devoid of the immunomodulatory properties of LL-37. In contrast to LL-37, LL-25 did not affect the generation of reactive oxygen species by neutrophils upon treatment with phorbol esters. Stimulating neutrophils with LL-25 (rather than LL-37) significantly decreased calcium flux and interleukin-8 production, resulting in lower chemotactic activity of the peptide against neutrophils, which may decrease the recruitment of neutrophils to infection foci. LL-25 also lost the function of LL-37 as an inhibitor of neutrophil apoptosis, thereby reducing the life span of these defense cells. This study indicates that C. albicans can effectively use aspartic proteases to destroy the antimicrobial and immunomodulatory properties of LL-37, thus enabling the pathogen to survive and propagate.

Inactivation of human kininogen-derived antimicrobial peptides by secreted aspartic proteases produced by the pathogenic yeast Candida albicans.

Ten secreted aspartic proteases (Saps) of Candida albicans cleave numerous peptides and proteins in the host organism and deregulate its homeostasis. Human kininogens contain two internal antimicrobial peptide sequences, designated NAT26 and HKH20. In our current study, we characterized a Sap-catalyzed cleavage of kininogen-derived antimicrobial peptides that results in the loss of the anticandidal activity of these peptides. The NAT26 peptide was effectively inactivated by all Saps, except Sap10, whereas HKH20 was completely degraded only by Sap9. Proteolytic deactivation of the antifungal potential of human kininogens can help the pathogens to modulate or evade the innate immunity of the host.

Fibronectin-, vitronectin- and laminin-binding proteins at the cell walls of Candida parapsilosis and Candida tropicalis pathogenic yeasts.

BACKGROUND: Candida 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. METHODS: The fungal proteins involved in interactions with extracellular matrix proteins were isolated from mixtures of ß-1,3-glucanase- or ß-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. RESULTS: In 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. DISCUSSION: The 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. CONCLUSIONS: Our results reveal new insight into host-pathogen interactions during infections by two important, recently emerging, fungal pathogens.

Kinin release from human kininogen by 10 aspartic proteases produced by pathogenic yeast Candida albicans.

BACKGROUND: Candida albicans yeast produces 10 distinct secreted aspartic proteases (Saps), which are some of the most important virulence factors of this pathogenic fungus. One of the suggested roles of Saps is their deregulating effect on various proteolytic cascades that constitute the major homeostatic systems in human hosts, including blood coagulation, fibrinolysis, and kallikrein-kinin systems. This study compared the characteristics of the action of all 10 Saps on human kininogens, which results in generating proinflammatory bradykinin-related peptides (kinins). RESULTS: Recombinant forms of Saps, heterologously overexpressed in Pichia pastoris were applied. Except for Sap7 and Sap10, all Saps effectively cleaved the kininogens, with the highest hydrolytic activity toward the low-molecular-mass form (LK). Sap1-6 and 8 produced a biologically active kinin-Met-Lys-bradykinin-and Sap3 was exceptional in terms of the kinin-releasing yield (>60% LK at pH 5.0 after 24 hours). Des-Arg(1)-bradykinin was released from LK by Sap9 at a comparably high yield, but this peptide was assumed to be biologically inactive because it was unable to interact with cellular B2-type kinin receptors. However, the collaborative actions of Sap9 and Sap1, -2, -4-6, and -8 on LK rerouted kininogen cleavage toward the high-yield release of the biologically active Met-Lys-bradykinin. CONCLUSIONS: Our present results, together with the available data on the expression of individual SAP genes in candidal infection models, suggest a biological potential of Saps to produce kinins at the infection foci. The kinin release during candidiasis can involve predominant and complementary contributions of two different Sap3- and Sap9-dependent mechanisms.

Thiamine increases the resistance of baker's yeast Saccharomyces cerevisiae against oxidative, osmotic and thermal stress, through mechanisms partly independent of thiamine diphosphate-bound enzymes.

Numerous recent studies have established a hypothesis that thiamine (vitamin B1 ) is involved in the responses of different organisms against stress, also suggesting that underlying mechanisms are not limited to the universal role of thiamine diphosphate (TDP) in the central cellular metabolism. The current work aimed at characterising the effect of exogenously added thiamine on the response of baker's yeast Saccharomyces cerevisiae to the oxidative (1 mM H2 O2 ), osmotic (1 M sorbitol) and thermal (42 °C) stress. As compared to the yeast culture in thiamine-free medium, in the presence of 1.4 µM external thiamine, (1) the relative mRNA levels of major TDP-dependent enzymes under stress conditions vs. unstressed control (the 'stress/control ratio') were moderately lower, (2) the stress/control ratio was strongly decreased for the transcript levels of several stress markers localised to the cytoplasm, peroxisomes, the cell wall and (with the strongest effect observed) the mitochondria (e.g. Mn-superoxide dismutase), (3) the production of reactive oxygen and nitrogen species under stress conditions was markedly decreased, with the significant alleviation of concomitant protein oxidation. The results obtained suggest the involvement of thiamine in the maintenance of redox balance in yeast cells under oxidative stress conditions, partly independent of the functions of TDP-dependent enzymes.