Candida albicans cell wall comprises a branched beta-D-(1-->6)-glucan with beta-D-(1-->3)-side chains

The structure of immunogenic and immunomodulatory cell wall glucans of Candida albicans is commonly interpreted in terms of a basic polysaccharide consisting of a beta-D-(1-->3)-linked glucopyranosyl backbone possessing beta-D-(1-->6)-linked side chains of varying distribution and length. This proposed molecular architecture has been re-evaluated by the present study on the products of selective enzymolysis of insoluble C. albicans glucan particles (GG). High resolution 1H (400 and 700 MHz) and 13C (100 and 175 MHz) NMR analyses were performed on a soluble beta-glucan preparation (GG-Zym) obtained by GG digestion with endo-beta-D-(1-->3)-glucanase and on its high- (Pool 1) and low-molecular weight (Pool 2) sub-fractions. The resonances typical of uniformly beta-D-(1-->6)- and beta-D-(1-->3)-linked linear glucans, together with additional multiplets assigned to short-chain oligoglucosides, were detected in GG-Zym. Pool 1 (46.3+/-6.4% of GG-Zym content) consisted of beta-D-(1-->6)-linked glucopyranosyl polymers, with short beta-D-(1-->3)-branched side chains of 2.20+/-0.02 units (branching degree (DB)=0.14+/-0.03). Pool 2 was a mixture of glucose and linear short-chain beta-D-(1-->3)-oligoglucosides. Further digestion of Pool 1 by beta-D-(1-->6)-glucanase yielded a mixture of glucose and short beta-D-(1-->6)-linked, either linear or beta-D-(1-->3,6) branched, oligomers. These endoglucanase digestion patterns were consistent with the presence in C. albicans cell wall glucans of beta-D-(1-->6)-linked glucopyranosyl backbones possessing beta-D-(1-->3)-linked side chains, a structure very close to that of beta-D-(1-->6)-glucan from Saccharomyces cerevisiae yeast. This finding may provide the grounds for further elucidation of the cell wall structure and a better understanding of the biological properties of C. albicans beta-glucans.     

Hyphal formation of Candida albicans is controlled by electron transfer system

Most Candida albicans cells cultured in RPMI1640 medium at 37 degrees C grow in hyphal form in aerobic conditions, but they grow in yeast form in anaerobic conditions. The hyphal growth of C. albicans was inhibited in glucose-deficient conditions. Malonic acid, an inhibitor of succinate dehydrogenase, enhanced the yeast proliferation of C. albicans, indicating that the hyphal-formation signal was derived from the glycolysis system and the signal was transmitted to the electron transfer system via the citric acid cycle. Thenoyl trifluoro acetone (TTFA), an inhibitor of the signal transmission between complex II and Co Q, significantly inhibited the hyphal growth of C. albicans. Antimycin, KCN, and oligomycin, inhibitors of complex III, IV, and V, respectively, did not inhibit the hyphal growth of C. albicans. The production of mRNAs for the hyphal formation signal was completely inhibited in anaerobic conditions. These results indicate that the electron transfer system functions upstream of the RAS1 signal pathway and activates the expression of the hyphal formation signal. Since the electron transfer system is inactivated in anaerobic conditions, C. albicans grew in yeast form in this condition.     

The Swi/Snf chromatin remodeling complex is essential for hyphal development in Candida albicans

The ability of dimorphic transition between yeast and hyphal forms in Candida albicans is one of the vital determinants for its pathogenicity and virulence. We isolated C. albicans SWI1 as a suppressor of the invasive growth defect in a Saccharomyces cerevisiae mutant. Expression of C. albicans SWI1 in S. cerevisiae partially complemented the growth defect of a swi1 mutant in the utilization of glycerol. Swi1 is in a complex with Snf2 in C. albicans, and both proteins are localized in the nucleus independent of the growth form. Deleting SWI1 or SNF2 in C. albicans prevented true hyphal formation and resulted in constitutive pseudohypha-like growth in all media examined. Furthermore, swi1/swi1 mutant was defective in hypha-specific gene expression and avirulent in a mouse model of systemic infection. These data strongly suggest the conserved Swi/Snf complex in C. albicans is required for hyphal development and pathogenicity.     

Screening of reducing agents for anaerobic growth of Candida albicans SC5314

This study aimed to evaluate the influence of different redox potentials (Eh) on cell growth, whole-cell protein profile and cell surface hydrophobicity (CSH) of Candida albicans SC5314. The yeast was grown in YNB broth enriched with reducing (158 mM sodium sulfite, 4 mM sodium sulfite, 2.5 mM sodium metabisulfite, 1.3 mM 2-mercaptoethanol, 5.5 mM thioglycolic acid, and 3.2 mM l-cysteine hydrochloride) and oxidizing agents (15 mM ammonium persulfate and 80 mM potassium ferricyanide) and incubated in normoxic and anoxic atmospheres at 37 °C, for 48 h. Pre- and post-incubation Eh values were determined and cytoplasm proteins were extracted. Proteins were parted by SDS-PAGE and their profiles were compared. 3.2 mM l-cysteine and 1.3 mM 2-mercaptoethanol promoted and maintained negative Eh values during incubation. No differences were detected among SDS-PAGE profiles. CSH differences only were observed with 4 mM sodium sulfite and 3.2 mM l-cysteine. Results showed that 3.2 mM l-cysteine is a reducing agent that allows maintenance of negative Eh in both anoxic and normoxic conditions and it seems not to interfere in the global expression of plasmatic proteins.     

Antifungal mechanism of an antimicrobial peptide, HP (2--20), derived from N-terminus of Helicobacter pylori ribosomal protein L1 against Candida albicans

The antifungal activity and mechanism of HP (2-20), a peptide derived from the N-terminus sequence of Helicobacter pylori Ribosomal Protein L1 were investigated. HP (2--20) displayed a strong antifungal activity against various fungi, and the antifungal activity was inhibited by Ca(2+) and Mg(2+) ions. In order to investigate the antifungal mechanism(s) of HP (2-20), fluorescence activated flow cytometry was performed. As determined by propidium iodide staining, Candida albicans treated with HP (2-20) showed a higher fluorescence intensity than untreated cells and was similar to melittin-treated cells. The effect on fungal cell membranes was examined by investigating the change in membrane dynamics of C. albicans using 1,6-diphenyl-1,3,5-hexatriene as a membrane probe and by testing the membrane disrupting activity using liposome (PC/PS; 3:1, w/w) and by treating protoplasts of C. albicans with the peptide. The action of peptide against fungal cell membrane was further examined by the potassium-release test, and HP (2-20) was able to increase the amount of K(+) released from the cells. The result suggests that HP (2-20) may exert its antifungal activity by disrupting the structure of cell membrane via pore formation or directly interacts with the lipid bilayers in a salt-dependent manner.     

Structural characterization of a water-soluble beta-(1-->6)-linked D-glucan isolated from the hot water extract of an edible mushroom, Agaricus bitorquis

A water-soluble polysaccharide, isolated from the hot aqueous extract of an edible mushroom, Agaricus bitorquis, was found to consist of d-glucose only. On the basis of total hydrolysis, methylation analysis, and NMR studies (¹H, ¹³C, TOCSY, DQF-COSY, NOESY, ROESY, HMQC, and HMBC), the structure of the repeating unit was established as→6)-β-d-Glcp-(1→     

Immune responses to yeast and mycelial forms of Candida albicans in intraperitoneally infected mice

Candida albicans E-139 produced pure mycelial and yeast cultures in a low sulphate medium at different temperatures. The influence of the morphological phase, dose and viability of the fungi on the kinetic of delayed-type hypersensitivity (DTH) and anti-mycelial and anti-yeast antibodies have been studied in mice injected intraperitoneally. The mycelial form elicited higher DTH levels than the yeast phase. This effect seems to be related to its antigenic properties. The effect of dose on the immune response depends on the viability of the fungus. The mycelial cytoplasmic antigens were more effective than the yeast ones in detecting antibodies induced during the experiments, particularly during the later stages of the observation periods, suggesting that such antigens may be useful in the serodiagnosis of Candida infections.     

CaIPF7817 is involved in the regulation of redox homeostasis in Candida albicans

CaIPF7817, a functionally unknown gene in Candida albicans, was suggested to be involved in the redox system previously, but its exact role is unknown. In this study, ipf7817 null mutant was generated with the URA-blaster method. After the deletion of CaIPF7817, intracellular levels of reactive oxygen species were significantly increased; mitochondrial membrane potential, a direct indicator of mitochondrial function, was elevated; some important redox-related genes, including GLR1, SOD2, and TRR1, were up-regulated; and the GSH/GSSG ratio was raised. These changes indicated that CaIPF7817 played important roles in the regulation of redox homeostasis in C. albicans.     

Antifungal action of human cathelicidin fragment (LL13-37) on Candida albicans

Human cathelicidin LL37 and its fragments LL13–37 and LL17–32 exhibited similar potencies in inhibiting growth of the yeast Candida albicans. After treatment with 0.5 μM and 5 μM LL13–37, the hyphae changed from a uniformly thick to an increasingly slender appearance, with budding becoming less normal in appearance and cell death could be detected. Only the yeast form and no hyphal form could be observed following exposure to 50 μM LL13–37. LL13–37 at a concentration of 5 μM was able to permeabilize the membrane of yeast form as well as hyphal form of C. albicans since the nuclear stain SYTOX Green was localized in both forms. Mycelia treated with LL13–37 stained with SYTOX Green, but did not stain with MitoTracker deep red, indicating that the mitochondria were adversely affected by LL13–37. Bimane-labeled LL13–37 was able to enter some of the hyphae, but not all hyphae were affected, suggesting that LL37impaired membrane permeability characteristics in some of the hyphae. Reactive oxygen species was detectable in the yeast form of C. albicans cells after treatment with LL13–37 but not in the untreated cells. The results suggest that the increased membrane permeability caused by LL13–37 might not be the sole cause of cell death. It might lead to the uptake of the peptide, which might have some intracellular targets.     

Functional expression and characterization of cytochrome P450 52A21 from Candida albicans

Candida albicans contains 10 putative cytochrome P450 (CYP) genes coding for enzymes that appear to play important roles in fungal survival and virulence. Here, we report the characterization of CYP52A21, a putative alkane/fatty acid hydroxylase. The recombinant CYP52A21 protein containing a 6x(His)-tag was expressed in Escherichia coli and was purified. The purified protein, reconstituted with rat NADPH-cytochrome P450 reductase, omega-hydroxylated dodecanoic acid to give 12-hydroxydodecanoic acid, but to a lesser extent also catalyzed (omega-1)-hydroxylation to give 11-hydroxydodecanoic acid. When 12,12,12-d(3)-dodecanoic acid was used as the substrate, there was a major shift in the oxidation from the omega- to the (omega-1)-hydroxylated product. The regioselectivity of fatty acid hydroxylation was examined with the 12-iodo-, 12-bromo-, and 12-chlorododecanoic acids. Although all three 12-halododecanoic acids bound to CYP52A21 with similar affinities, the production of 12-oxododecanoic acid decreased as the size of the terminal halide increased. The regioselectivity of CYP52A21 fatty acid oxidation is thus consistent with presentation of the terminal end of the fatty acid chain for oxidation via a narrow channel that limits access to other atoms of the fatty acid chain. This constricted access, in contrast to that proposed for the CYP4A family of enzymes, does not involve covalent binding of the heme to the protein.     

Expression of the CDR1 efflux pump in clinical Candida albicans isolates is controlled by a negative regulatory element

Resistance to azole antifungal drugs in clinical isolates of the human fungal pathogen Candida albicans is often caused by constitutive overexpression of the CDR1 gene, which encodes a multidrug efflux pump of the ABC transporter superfamily. To understand the relevance of a recently identified negative regulatory element (NRE) in the CDR1 promoter for the control of CDR1 expression in the clinical scenario, we investigated the effect of mutation or deletion of the NRE on CDR1 expression in two matched pairs of azole-sensitive and resistant clinical isolates of C. albicans. Expression of GFP or lacZ reporter genes from the wild type CDR1 promoter was much higher in the azole-resistant C. albicans isolates than in the azole-susceptible isolates, reflecting the known differences in CDR1 expression in these strains. Deletion or mutation of the NRE resulted in enhanced reporter gene expression in azole-sensitive strains, but did not further increase the already high CDR1 promoter activity in the azole-resistant strains. In agreement with these findings, electrophoretic mobility shift assays showed a reduced binding to the NRE of nuclear extracts from the resistant C. albicans isolates as compared with extracts from the sensitive isolates. These results demonstrate that the NRE is involved in maintaining CDR1 expression at basal levels and that this repression is overcome in azole-resistant clinical C. albicans isolates, resulting in constitutive CDR1 overexpression and concomitant drug resistance.     

The signal peptide NPFSD fused to ricin A chain enhances cell uptake and cytotoxicity in Candida albicans

Microorganisms possess stringent cell membranes which limit the cellular uptake of antimicrobials. One strategy to overcome these barriers is to attach drugs or research reagents to carrier peptides that enter cells by passive permeation or active uptake. Here the short endocytosis signal peptide NPFSD was found to efficiently deliver both FITC and GFP into Saccharomyces cerevisiae and Candida albicans with uptake into the majority of cells in a population. The NPFSD signal is itself non-toxic, but when fused to the ricin A chain toxin (RTA) the peptide enhanced both cell uptake and toxicity against C. albicans, which like other yeasts is resistant to naked RTA. Cell entry required at least 1 h incubation, temperatures above 4 degrees C, and an energy source, and uptake was out-competed with free peptide. Therefore, the NPFSD peptide can carry a range of compounds into yeasts and this delivery route holds promise to enhance the activity of antifungals.     

Comparison between allicin and fluconazole in Candida albicans biofilm inhibition and in suppression of HWP1 gene expression

Candida albicans is an opportunistic human pathogen with the ability to differentiate and grow in filamentous forms and exist as biofilms. The biofilms are a barrier to treatment as they are often resistant to the antifungal drugs. In this study, we investigated the antifungal activity of allicin, an active compound of garlic on various isolates of C. albicans. The effect of allicin on biofilm production in C. albicans as compared to fluconazole, an antifungal drug, was investigated using the tetrazolium (XTT) reduction-dependent growth and crystal violet assays as well as scanning electron microscopy (SEM). Allicin-treated cells exhibited significant reduction in biofilm growth (p<0.05) compared to fluconazole-treated and also growth control cells. Moreover, observation by SEM of allicin and fluconazole-treated cells confirmed a dose-dependent membrane disruption and decreased production of organisms. Finally, the expression of selected genes involved in biofilm formation such as HWP1 was evaluated by semi-quantitative RT-PCR and relative real time RT-PCR. Allicin was shown to down-regulate the expression of HWP1.     

Essential Role for Vacuolar Acidification in Candida albicans Virulence

Fungal infections are on the rise, with mortality above 30% in patients with septic Candida infections. Mutants lacking V-ATPase activity are avirulent and fail to acidify endomembrane compartments, exhibiting pleiotropic defects in secretory, endosomal, and vacuolar pathways. However, the individual contribution of organellar acidification to virulence and its associated traits is not known. To dissect their separate roles in Candida albicans pathogenicity we generated knock-out strains for the V₀ subunit a genes VPH1 and STV1, which target the vacuole and secretory pathway, respectively. While the two subunits were redundant in many vma phenotypes, such as alkaline pH sensitivity, calcium homeostasis, respiratory defects, and cell wall integrity, we observed a unique contribution of VPH1. Specifically, vph1Δ was defective in acidification of the vacuole and its dependent functions, such as metal ion sequestration as evidenced by hypersensitivity to Zn²⁺ toxicity, whereas stv1Δ resembled wild type. In growth conditions that elicit morphogenic switching, vph1Δ was defective in forming hyphae whereas stv1Δ was normal or only modestly impaired. Host cell interactions were evaluated in vitro using the Caco-2 model of intestinal epithelial cells, and murine macrophages. Like wild type, stv1Δ was able to inflict cellular damage in Caco-2 and macrophage cells, as assayed by LDH release, and escape by filamentation. In contrast, vph1Δ resembled a vma7Δ mutant, with significant attenuation in host cell damage. Finally, we show that VPH1 is required for fungal virulence in a murine model of systemic infection. Our results suggest that vacuolar acidification has an essential function in the ability of C. albicans to form hyphae and establish infection.     

Photodynamic inactivation for controlling Candida albicans infections

Antimicrobial photodynamic therapy (APDT) combines a non-toxic dye, termed photosensitizer, which is activated by visible light of appropriate wavelength which will produce reactive oxygen species (ROS). These ROS will react with cellular components inducing oxidative processes, leading to cell death. A wide range of microorganisms, have already showed susceptibility to APDT. Therefore, this treatment might consist in an alternative for the management of fungal infections that is mainly caused by biofilms, since they respond poorly to conventional antibiotics and may play a role in persistent infections. Biofilms are the leading cause of microbial infections in humans, thus representing a serious problem in health care. Candida albicans is the main type of fungi able to form biofilms, which cause superficial skin and mucous membrane infections as well as deep-seated mycoses, particularly in immunocompromised patients. In these patients, invasive infections are often associated with high morbidity and mortality. Furthermore, the increase in antifungal resistance has decreased the efficacy of conventional therapies. Treatments are time-consuming and thus demanding on health care budgets. Additionally, current antifungal drugs only have a limited spectrum of action and toxicity. The use of APDT as an antimicrobial topical agent against superficial and cutaneous diseases represents an effective method for eliminating microorganisms.     

Essential Oils,Silver Nanoparticles and Propolis as Alternative Agents Against Fluconazole Resistant Candida albicans,Candida glabrata and Candida krusei Clinical Isolates

Development of effective and safe therapeutic treatment of fungal infections remains one of the major challenge for modern medicine. The aim of presented investigation was to analyze the in vitro antifungal activity of selected essential oils, ethanolic extracts of propolis and silver nanoparticles dropped on TiO₂ against azole-resistant C. albicans (n = 20), C. glabrata (n = 14) and C. krusei (n = 10) clinical isolates. Among tested essential oils, the highest activity has definitely been found in the case of the oil isolated from the bark of Cinnamomum cassia, with MIC and MFC values for all tested strains in the range of 0.0006–0.0097 % (v/v) and 0.0012–0.019 % (v/v), respectively. High activity was also observed for the Lemon, Basil, Thyme, Geranium and Clove (from buds) essential oils. Significant differences in fungicidal activity have been observed in the case of four tested propolis samples. Only one of them revealed high activity, with MFC values in the range from 0.156 to 1.25 % (v/v). Satisfactory fungicidal activity, against C. albicans and C. glabrata isolates, was also observed in the case of silver nanoparticles, however C. krusei isolates were mostly resistant. We also revealed that constituents of most of essential oils and propolis as well as silver nanoparticles are not substrates for drug transporters, which belong to the most important factors affecting resistance of Candida spp. clinical isolates to many of conventional antimycotics. To conclude, the results of our investigation revealed that essential oils, propolis and silver nanoparticles represent high potential for controlling and prevention candidiasis.     

Candida albicans strain-dependent modulation of pro-inflammatory cytokine release by in vitro oral and vaginal mucosal models

Candida albicans is a commensal organism at several sites and is a versatile, opportunistic pathogen. The underlying factors of pathogen and host associated with commensalism and pathogenicity in C. albicans are complex and their importance is largely unknown. We aimed to study the responses of oral epithelial (OEM) and vaginal epithelial models (VEM) to infection by oral and vaginal C. albicans strains to obtain evidence of inter-strain differences in pathogenicity and of site-specificity. Following inoculation of models, proinflammatory cytokines IL-1α, IL-1β, IL-6, IL-8 and prostaglandin E2 (PGE2) release were monitored and histological staining undertaken. Striking differences in strain behaviour and epithelial responses were observed. IL-1α, IL-1β and IL-8 release were significantly increased from the OEM in response to denture stomatitis strain NCYC 1467. Increased IL-8 release also followed infection of the OEM with both vaginal strains. Overall the VEM was relatively unresponsive to infection with either oral or vaginal strains under these conditions. Adherence and hyphal development were observed for all strains on both models although extensive, uniform tissue penetration was seen only with stomatitis strain NCYC 1467 on the OEM. Candidal strains were assayed for phospholipase (PL) and secreted aspartyl proteinase (SAP) activities where phospholipase (PL) activity was highest for strain NCYC 1467 although highest SAP activity was observed for vaginal strain NCPF 8112 in this assay. This is the first study to concurrently investigate cytokine production from oral and epithelial models using candidal strains originating from these respective mucosal sites from healthy and disease states. These data demonstrate significant differences in inflammatory responses of host epithelia to individual C. albicans strains.     

Detection of Candida albicans in disseminated candidosis by immunofluorescence staining

An indirect immunofluorescence (IF) method using rabbit anti-Candida albicans was used to detect C. albicans in blood samples of 12 patients with systemic candidosis defined clinically, histologically and by blood cultures. Positive staining of C. albicans could be detected in all of the patients. The findings suggest that IF-method offers a more rapid method in the diagnosis of disseminated candidosis.