Influence of lipid composition on the sensitivity of Candida albicans to antifungal agents

Cerulenin, a specific inhibitor of fatty acid and sterol biosyntheses, inhibited growth and lipid synthesis in C. albicans, which on supplementing the growth medium with optimum concentrations of fatty acids was reversed. Significant changes in the levels of phospholipids and sterols were observed in fatty acid-supplemented cells. Altered phospholipids and their fatty acid profile rendered cells more resistant to miconazole and thereby more permeable to [3H]proline. Thus it appears that fatty acid composition plays an important role in determining the permeability susceptibility of C. albicans to drugs.     

Dimorphism-associated variations in the lipid composition of Candida albicans

Yeast and mycelial forms of Candida albicans ATCC 10231, growing together in 12 h and in 96 h cultures, were separated and their lipids were extracted and characterized. The total lipid content of the yeast forms was always lower than that of the mycelial forms. In 12 h cultures the lipids from the two morphological forms consisted mainly of polar compounds, viz, phospholipids and glycolipids. In 96 h cultures both the yeast and mycelial forms accumulated substantial amounts of apolar compounds, mainly steryl esters and triacylglycerols. The mycelial forms were more active than the yeast forms in this respect. Major differences in the lipid composition between the two morphological forms involved the contents of sterols and complex lipids that contain sterols. As a rule, the yeast lipids contained much larger proportions of free sterols than the mycelial lipids. However, the mycelial lipids contained several times more sterols than the yeast forms but bound as steryl glycosides, esterified steryl glycosides and steryl esters. Steryl glycosides and esterified steryl glycosides occurred in yeast lipids only in traces, if at all. The major steryl glycoside in the mycelial forms was unequivocally identified as cholesteryl mannoside. At both phases of growth the apolar and polar lipid fractions from the mycelial forms contained higher levels of polyunsaturated fatty acids (18:2 and 18:3) but lower levels of oleic acid (18:1) than the corresponding fractions from the yeast forms. The lipid content and composition of 12 h and 96 h yeast and mycelial forms of C. albicans KCCC 14172, a clinical isolate, were almost identical with those of C. albicans ATCC 10231.     

Growth inhibitory effect of antibiotic tetaine on yeast and mycelial forms of Candida albicans

The mycelial (M) form of Candida albicans is more sensitive to the action of the antibiotic tetaine than the yeast (Y) form. Tetaine, at low concentrations about 1 microgram/ml also inhibits Y-M transition. It causes severe deformation of cells, agglutination and inhibits septum formation in the yeast forms. Tetaine action is reversed by dipeptides in both forms and by tripeptides in M form. N-acetyl glucosamine is a powerful antagonist of tetaine action on both morphological forms. Tetaine action on mycelial forms is slightly antagonised by N-acetyl mannosamine and very powerfully by glutamine.     

Isolation of a mycelial mutant of Candida albicans

A mutant of Candida albicans strain MEN, which was unable to produce mycelia in SSV medium and in horse serum at 37 degrees C, was isolated by a physical separation procedure. The mutant was shown to be derived from the parental strain by growth and morphology studies, sugar uptake and fermentation patterns, and the presence of genetic markers.     

Comparison of Candida dubliniensis and C. albicans based on polar lipid composition

AIMS: To test the hypothesis that strains of Candida dubliniensis and C. albicans can be differentiated on the basis of polar lipid profiles. METHODS AND RESULTS: Five isolates of C. dubliniensis and six isolates of C. albicans were tested by growth at 45 degrees C, production of chlamydospores on cornmeal agar, colonial colour on CHROMagar Candida medium and assimilation of DL-lactate, alpha-methyl-D-glucoside and xylose. Polar lipids were then extracted from freeze-dried cultures and analysed using fast atom bombardment mass spectrometry. Isolates were grouped by single linkage clustering based on correlation coefficients for strain pairs calculated with carboxylate and phospholipid molecular species distributions. The most intense carboxylate and phospholipid molecular species anions were of m/z 281 (C(18 : 1)) and m/z 515 (PA 23 : 2). Phosphatidylethanolamine and phosphatidylglycerol were the predominant phospholipid families in C. dubliniensis, compared with phosphatidic acid in C. albicans isolates. All of the C. dubliniensis isolates grouped together in one cluster, whereas all of the C. albicans isolates grouped in a separate cluster. CONCLUSIONS: Fast atom bombardment mass spectrometry can differentiate the two species based on analysis of polar lipid distributions. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings demonstrate that C. dubliniensis and C. albicans have distinct polar lipid profiles.     

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.     

The effect of altered lipid composition on the transport of various amino acids in Candida albicans.

Candida albicans cells grown on alkanes of different chain lengths (C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, and C₁₈) exhibited a low growth rate and gradual increase in the total lipid content with the increase in the length of alkanes. There was a significant change in the phospholipids and sterols content of various alkane-grown cells compared to glucose-grown cells. In glucose-grown cells, the transport of various amino acids, e.g., proline, glutamic acid, lysine, glycine, phenylalanine, serine, methionine, and leucine was found to be energy dependent and against a concentration gradient. In alkane-grown cells, the transport of lysine, proline, serine, and methionine was reduced, however, there was no effect on the uptake of glycine, glutamic acid, phenylalanine, and leucine. The results were interpreted as different carrier(s) responsible for amino acid uptake responsed differently to the change of lipid environment.     

The synergistic antifungal activity of resveratrol with azoles against Candida albicans

Candida albicans is one of the most common clinical pathogenic microorganisms and it is becoming a serious health threat, particularly to immunocompromised populations. Drug resistance of Candida species has also frequently emerged, and combination therapy for fungal infections has attracted considerable attention. In this study, we established the Qinling Mountains myxobacterial secondary metabolites library and a synergic assay in combination with ketoconazole against C. albicans was introduced for metabolites screening. Two active compounds with synergic anticandidal activities were obtained, which were identified as trans-resveratrol and cis-resveratrol. According to our study, resveratrol can reduce the dosage to 1/64 of ketoconazole as well as itraconazole. Furthermore, synergistic anticandidal activity of resveratrol combined with azoles was verified against a panel of clinical C. albicans isolates, and the combination strategy enhanced the azoles susceptibility of three fluconazole-resistant isolates. These findings suggest that resveratrol enhances the efficacy of azoles and provides a promising application in therapy of C. albicans infection.     

Isolation and morphological characterization of a mycelial mutant of Candida albicans.

In this paper we describe the isolation of a novel strain of Candida albicans which is a mycelium at ambient temperatures. Mutagenesis of C. albicans ATCC 10261 with N-methyl-N-nitro-N-nitrosoguanidine followed by plating on solid media at 28 degrees C yielded colony morphology variants which were characterized by a raised, rough-surfaced colony of irregular outline in marked contrast to the flat, shiny circular colonies of the parental 10261 strain. One mutant colony, hOG301, was studied in detail. Strain hOG301 was stable and exhibited mycelial morphology over a wide temperature range (5 to 40 degrees C) in several media. The hyphae comprising hOG301 mycelium were examined by light microscopy, scanning electron microscopy, and transmission electron microscopy and showed morphological features described in the literature as being typical of both true hyphae and pseudohyphae. In contrast to 10261, hOG301 was not pathogenic after intraperitoneal injection in mice. This is the first report of a mycelial C. albicans that is stable at ambient temperatures.     

Viable quantitative PCR for assessing the response of Candida albicans to antifungal treatment

Propidium monoazide (PMA) or ethidium bromide monoazide (EMA) treatment has been used before nucleic acid detection methods, such as PCR, to distinguish between live and dead cells using membrane integrity as viability criterion. The performance of these DNA intercalating dyes was compared in many studies utilizing different microorganisms. These studies demonstrated that EMA and PMA differ in their abilities to identify nonviable cells from mixed cell populations, depending on the microorganism and the nature of the sample. Due to this heterogeneity, both dyes were used in the present study to specifically distinguish dead from live Candida albicans cells using viable quantitative PCR (qPCR). The viable qPCR was optimized, and the best results were obtained when pre-treating the cells for 10 min in the dark with 25 μM EMA followed by continuous photoactivation for 15 min. The suitability of this technique to distinguish clotrimazole- and fluconazole-treated C. albicans cells from untreated cells was then assessed. Furthermore, the antifungal properties of two commercial essential oils (Thymus vulgaris and Matricaria chamomilla) were evaluated. The viable qPCR method was determined to be a feasible technique for assessing the viability of C. albicans after drug treatment and may help to provide a rapid diagnostic and susceptibility testing method for fungal infections, especially for patients treated with antifungal therapies.     

Immune response in mice infected with Candida albicans in the mycelial form

The effect of the infection with the mycelial form of a Candida albicans strain (Mycology Dept.) upon the immune system in mice was studied.BALB/c mice were infected intraperitoneally in a single dose of a 3×10⁶, 6×10⁶ and 12×10⁶ cell suspension of the strain.Macrophages's activity was studied the days 7,14,21,28, 35, and 42 after inoculation, by the following assays: phagocytosis in vitro, mononucleated phagocytic system by the colloidal carbon clearance technique, the lymphocyte's activity by the direct plaque forming cells technique (PFC) and delayed hypersensitivity (DTH).Infection with the mycelial form did not affect the peritoneal macrophage's phagocytic ability, neither modified the delayed hypersensitivity to sheep red blood cells (SRBC). However, a slight and transient depression of the lymphocyte stimulation was found. Suppression of PFC to SRBC was high when a 12×10⁶ cell suspension was used in contrast to the infection with blastospores.These results suggest that systemic infection by Candida albicans in its mycelial form do not induce a non specific immunosuppression.     

Effect of sub-inhibitory concentration of chlorhexidine on lipid and sterol composition of shape Candida albicans

The effect of a sub-inhibitory concentration of chlorhexidine on lipid and sterol composition of Candida albicans was investigated. The total lipid content of this yeast grown in the presence of chlorhexidine was reduced whilst the total sterol content was increased compared with control-grown cells. Lipids and sterol analyses of this yeast grown in the presence and absence of chlorhexidine are presented. Chlorhexidine-grown yeast had a higher level of phosphatidylethanolamine, phosphatidylcholine and monogalactosyldiacylglycerol. Lower proportions of phosphatidylinositol plus phosphatidylserine, phosphatidic acid and cardiolipin were found in C. albicans grown in the presence of the drug when compared with control-grown yeast. The major fatty acids in control-grown cells were C16 and C18. Drug grown-cells had higher proportions of palmitic acid (16 : 0) and stearic acid (18 : 0), but lower proportions of palmitoleic acid (16 : 1) and oleic acid (18 : 1). Chlorhexidine also decreased the unsaturated-to-saturated fatty acid ratio, while the C16/C18 ratios increased compared to control-grown cells. Differences in the fatty acid composition of major phospholipids and neutral lipids between drug and control-grown yeast were also detected. Sterol analysis of control-grown cells showed that the major sterol present was ergosterol (55.4% wt). A significant increase in ergosterol and obtusifoliol was observed in chlorhexidine-treated cells and a significant decrease in squalene and lanosterol. Our results suggested that chlorhexidine affected the lipid and sterol composition of C. albicans. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of antifungal agents on protein composition of Candida albicans studied by capillary electrophoresis and chip technology

In the present study protein profile of a Candida albicans strain had been examined by chip technology and conventional capillary electrophoresis (CE). Profiles could be characterised by the presence of ten dominating protein peaks. These proteins could be distinguished by both techniques, but their quantity showed significant differences in the electropherograms obtained by CE and chip method. Changes in the protein profile were induced by administration of different antifungal agents. Fluconazole and amphotericin B treatment was able to induce similar changes in the pattern, appearance of a 40-kDa protein and up-regulation of a 60-kDa protein was observed by chip technology. Increase in the quantity of these proteins under stress effect (antifungal treatment) might refer to their stress function in the fungal cell. Treatment of C. albicans cells with MK 94 (fused cyclic Mannich ketone) antifungal compound induced not only the previously mentioned changes, but further specific alterations, appearance of a 19-kDa protein and up-regulation of the low molecular weight proteins. This might refer to the different mode of action of this agent on the fungal cells. Conventional capillary electrophoresis was suitable to detect the appearance of the 19-kDa peak, and up-regulation of the 60 kDa protein, but the other changes could not be detected by this technique. Shorter running time, more effective and baseline separation of proteins refer to the advantages of microchip-based method in the analysis of complex biological samples.     

The composition of Camembert cheese-ripening cultures modulates both mycelial growth and appearance

The fungal microbiota of bloomy-rind cheeses, such as Camembert, forms a complex ecosystem that has not been well studied, and its monitoring during the ripening period remains a challenge. One limitation of enumerating yeasts and molds on traditional agar media is that hyphae are multicellular structures, and colonies on a petri dish rarely develop from single cells. In addition, fungi tend to rapidly invade agar surfaces, covering small yeast colonies and resulting in an underestimation of their number. In this study, we developed a real-time quantitative PCR (qPCR) method using TaqMan probes to quantify a mixed fungal community containing the most common dairy yeasts and molds: Penicillium camemberti, Geotrichum candidum, Debaryomyces hansenii, and Kluyveromyces lactis on soft-cheese model curds (SCMC). The qPCR method was optimized and validated on pure cultures and used to evaluate the growth dynamics of a ripening culture containing P. camemberti, G. candidum, and K. lactis on the surface of the SCMC during a 31-day ripening period. The results showed that P. camemberti and G. candidum quickly dominated the ecosystem, while K. lactis remained less abundant. When added to this ecosystem, D. hansenii completely inhibited the growth of K. lactis in addition to reducing the growth of the other fungi. This result was confirmed by the decrease in the mycelium biomass on SCMC. This study compares culture-dependent and qPCR methods to successfully quantify complex fungal microbiota on a model curd simulating Camembert-type cheese.