Biosynthesis of major phospholipids inCandida albicans

Biosynthesis of major phospholipids was examined by identifying enzymes and in vitro uptake of specific labeled precursors through various pathways inCandida albicans. The presence of PS synthetase, choline kinase, and ethanolamine kinase was demonstrated in this organism. Phosphatidylcholine was found to be synthesized mainly through cytidine diphosphate-choline (CDP)-choline and methylation pathways. The presence of a methylation pathway was further confirmed by blocking methyltransferases with 2-hydroxyethyl hydrazine. Phosphatidylethanolamine was synthesized by all three, CDP-ethanolamine, Phosphatidylserine (PS)-decarboxylase, and base exchange pathways, while PS was formed by PS-synthetase and base exchange mechanisms.     

Evidence of morphology-specific isozymes inCandida albicans

S-Adenosylmethionine (SAM) synthetase of yeast and hyphal-phase cells of the dimorphic fungusCandida albicans was characterized by kinetic analysis and response to inhibitors. The enzyme from yeast-phase cells has a Km of 0.17 mM for methionine, 0.14 mM for ATP, and is inhibited (in vitro) by dimethyl-sulfoxide, methionine sulfone, and methionine sulfoxide. The hyphal-phase SAM synthetase has a Km of 0.06 mM for methionine, 0.02 mM for ATP, and its activity (in vitro) is enhanced by the substances that inhibit the yeast-phase enzyme. These data strongly suggest that isozymes of SAM synthetase are present inC. albicans and that they are possibly morphology specific. In vivo studies revealed that synthesis of the enzyme is repressed by the addition of methionine to the growth medium and that specific activity of the enzyme increases when intracellular SAM levels are lowered. In addition, it was shown that the increase in specific activity seen during yeast hypha morphogenesis and in yeast cells grown in a methionine-free medium involves de novo protein synthesis.     

Glucose influence on germ tube production inCandida albicans

The influence of different glucose concentrations was tested in a minimal synthetic medium onCandida albicans strain. After 18 hours of starvation, germ-tube (GT) production, amount of consumed glucose, oxygen and the pH of the medium were checked every hour from the beginning through the end of the experiment. Optimal GT production was obtained with 1 g/l of glucose. At this concentration the greatest glucose and oxygen consumption were also noted. No pH variations in the medium were observed in all of the glucose concentrations used. At 3 and 5 g/l glucose concentrations a lower GT production were obtained. The Crab-tree effect might interfere with GT production when glucose concentration is higher than 1 g/l. This data may support the hypothesis that GT production is strictly glucose dependent.     

Detection of extracellular phospholipase activity inCandida albicans andRhodotorula rubra

Phospholipases are important pathogenicity determinants inCandida albicans. They play a significant role in damaging cell membranes and invading host cells. High phospholipase production is correlated with an increased ability of adherence and a higher mortality rate in animal models. By means of an egg yolk-containing agar and thePz (= phospholipase activity zone) value according to Price, the present study investigated phospholipase production in 170 strains ofC. albicans. At an incubation temperature of 37 °C,Pz values ranged from 0.395 to 1; no clear relationship was found between clinical origin of the isolates and severity of the disease. In addition toC. albicans, a total of 110 strains of 16 other yeast species were investigated for possible phospholipase production. Only yeasts of the speciesRhodotorula rubra showed phospholipase activity, with mean values exceeding those observed inC. albicans. This result was confirmed by an assay using sterile culture filtrates and phosphatidyl-[3H-methyl]-choline-dipalmitoyl as a substrate. SinceRh. rubra has only rarely been demonstrated as a pathogen in humans, we believe that factors such as reduced growth at 37 °C, absence of dimorphism and low ability of adherence lessen the importance of high phospholipase activity inRh.rubra as a pathogenicity determinant. Therefore, potential virulence factors should always be considered in the context of the whole spectrum of pathogenic determinants.     

Microscopical evidences for the presence of a nucleus inCandida albicans chlamydoconidia

By using the fluorescent, DNA specific stain DAPI (4,6-diamidino-2-phenylindole) some microscopic observations ofCandida albicans pseudomycelium and chlamydoconidia were performed. In this manner blue fluorescent dots were noted both in yeasts, psudomycelium and chlamydoconidia, so evidencing the presence of a nucleus inC. albicans chlamydoconidia.     

Alteration of ergosterol content and chitin synthase activity inCandida albicans

Chitin synthase activity in total membrane fractions from two polyene-resistant, ergosterol-deficient mutants ofCandida albicans was significantly higher in comparison to the parental polyene-sensitive strain. The zymogen component from membrane preparations of stationaryphase cells of ergosterol mutants was more susceptible to trypsin digestion than those from the parental polyene-sensitive strain. Mechanisms that might explain the effect of changes in membrane composition in the mutant strains on chitin synthase activity are discussed.     

Mechanism of the effect of levorin on transport of amino acids inCandida albicans

By use of incubation media of various ionic compositions and pH's, it was possible to reveal that inhibition of the transport of neutral amino acids having long hydrophobic chains (leucine, phenylalanine) inCandida albicans by the polyene antibiotic levorin was mainly due to redistribution—caused by the antibiotic—of monovalent cations in the cells and protoplasts of the yeast. In an acidic medium, levorin was also found to induce a decrease of proton release from the cells, accompanied by some inhibition of leucine transport. Transport of neutral amino acids having short hydrophobic chains (alanine, glycine, proline) was inhibited by this polyene antibiotic, irrespective of the ionic composition or pH of the medium.     

Induction of mycelial type of development inCandida albicans by the antibiotic monorden and N-acetyl-D-glucosamine

Two new effective methods for synchronous germ tube production inCandida albicans have been described. Both are based on the use of stationary grown cultures and their further incubation in an aerated simple mineral medium enriched with vitamins containing either high glucose concentration (100 mmol/l) and the antibiotic monorden being added, or N-acetyl-D-glucosamine (100 mmol/l) as the sole carbon source. On the other hand yeast morphology could be maintained in the medium with high glucose concentration.On the basis of the methods developed it was possible to compare respiration intensity, respiration quotients, and sensitivity against some metabolic inhibitors in both morphological forms. Labeling experiments showed slight differences in the time course of glycine incorporation. The mycelial cell walls contained more chitin than the yeastlike cells. Using light and electron microscopy the interrelationships between concentration of monorden, or N-acetyl-D-glucosamine, physiological state of inoculum and the germ tube frequency were determined.The results are discussed with regard to the induction of germ tubes by low glucose concentration inCandida albicans from the more general aspect of regulation of fungal morphogenesis.     

Influence of carbon and nitrogen sources on glutathione catabolic enzymes inCandida albicans during dimorphism

The effect of carbon sources, glucose and sucrose, and nitrogen sources such as ammonia, glutamate andl-citrulline on the activities of glutathione metabolic enzymes has been studied. Yeast and mycelial cells were used to identify changes in activity levels of glutathione reductase (GSSGR), glutathione transferase (GST), glutathione peroxidase (GPX) and -glutamyl transpeptidase (GGT). Enzyme activities from cells grown in sucrose media were lower than in glucose media regardless of the enzyme tested, morphological form, or the growth interval. In all enzymes except GST, activity was higher in yeast form than in mycelia, regardless of nitrogen source, with lower activity from 24 to 72 h than at 96 h. In citrulline media, yeast form showed the maximum GST, GGT, and GPX activity. In ammonia-amended media, mycelia showed maximum activity in GGT, whereas in glutamate media, mycelia showed the maximum activity in GST. Also, the type of nitrogen source had no effect on GPX activity in the mycelial form. Finally, changing the nitrogen source showed no significant effect on GSSGR activity, either in the yeast or mycelial form.     

Stable transformation and regulated expression of an inducible reporter construct inCandida albicans using restriction enzyme-mediated integration

To allow the regulated expression of cloned genes inCandida albicans, a plasmid was constructed using the inducible promoter of theC. albicans MAL2 gene. To demonstrate that theMAL2 promoter could regulate cloned genes placed under its control, a fusion construct was made with the coding sequence of theC. albicans URA3 gene. This plasmid was introduced into a Ura^? strain ofC. albicans using the process of restriction enzyme-mediated integration (REMI). This procedure involves the transformation of theBamHI-linearized plasmid in the presence ofBamHI enzyme. The majority of transformants generated contained insertions of the plasmid at chromosomalBamHI sites. All transformants examined were inducible forURA3 expression, which was determined by growth analysis and by measuring the level ofURA3 gene product activity. The Ura⁺ phenotype of the transformants was stable during growth under nonselective conditions. This system offers the advantages of stable transformation, easy recovery of integrated DNA, and inducible expression of genes inC. albicans.     

Distribution of secreted aspartyl proteinases using a polymerase chain reaction assay withSAP specific primers inCandida albicans isolates

Secreted aspartyl proteinase (Sap) distribution among different C. albicans isolates was determined using SAP-specific primers in polymerase chain reaction (PCR) assay. SAP1, SAP2, and SAP3 were detected in 13 of 40 (32.5%), SAP4 in 38/40 (95%), SAP5 were detected in 30/40 (75%), SAP6 in 23/40 (57.5%) of C. albicans strains isolated from blood cultures. SAP1-SAP3 were detected in 37 of 40 (92.5%), SAP4 were detected in 3/40 (7.5%), SAP5 in 3/40 (7.5%), SAP6 in 5/40 (12.5%) of C. albicans strains isolated from vaginal swab cultures. Sap1, Sap2 and Sap3 isoenzymes were found to be related to the vaginopathic potential of C. albicans; Sap4, Sap5 and Sap6 isoenzymes were found to be correlated with systemic infections.     

Methionine-defective mutants inCandida utilis

Ethionine-resistant mutants ofCandida utilis CCY-158 overproducing methionine have been isolated. In these mutants the intracellular methionine concentration decreased significantly during the stationary phase. The wild-type strain CCY-158 and the ethionine-resistant mutants isolated were able to use methionine as the nitrogen source but not as the carbon source. From these ethionine-resistant mutants we isolated mutants unable to use methionine as nitrogen source (Mec^- mutants), the principal alteration being at the level of methionine uptake. Some of the Mec mutants lost also the ability to use other amino acids as nitrogen source.     

Methionine degradation inCandida utilis

Incubation of cell-free extracts ofC. utilis and its ethionine-resistant mutants with methionine, aspartate and alanine, and with various acceptor oxo-acids showed the presence of several transamination activities (Glu-Asp, Ala-Glu, Met-Glu, Met-Ala, Ala-Asp, but not Met-Asp). The lack of utilization of methionine by mec mutants appears to be due to a transport lesion.     

Inhibition of germ tube formation, filamentation and ergosterol biosynthesis inCandida albicans treated with 6-amino-2-n-pentylthiobenzothiazole

Three representativeCandida albicans strains were selected out of 26 clinical isolates and strains from culture collections on the basis of their high level of conversion to germ tubes and mycelial form at mycelium-promoting culture conditions, and on their different sensitivity to 6-amino-2-n-pentylthiobenzothiazole (APB). When these strains were treated with APB at mycelium-promoting culture conditions, a concentration-dependent decrease in the proportion of germ tubes and hyphae was observed, while the proportion of the yeast form increased. When non-saponifiable lipids were extracted from these cultures and analyzed, a concentration-dependent decrease in ergosterol and an increase in 4-methylated sterols was observed. However, the sensitivity of sterol biosynthesis did not directly relate to the sensitivity of the morphological conversion, and was exhibited at higher concentrations of APB. On the basis of these results it is suggested that the inhibition of germ tube formation and filamentation is not a consequence of inhibition of ergosterol biosynthesis in APB-treatedC. albicans.     

Location of monitorial fluorophores inCandida utilis

Summary The fluorescent spectra of whole broth, cell-free broth and resuspended cells were compared during the fermentation ofCandida utilis growing in ethanol for the purposes of identifying the location of monitorial fluorophores in cellular systems. Four cellular fluorophores, tryptophan, pyridoxine, NADH and riboflavin were examined. The results indicated that the fluorescence signal of tryptophan and NADH came mainly from intracellular fluorophores, and the fluorescence signal of pyridoxine and riboflavin mainly came from extracellular fluorophores. The contributions of intracellular and extracellular fluorophores to culture fluorescence signals were found to change during the fermentation.     

Uphill transport of monosaccharides inCandida beverwijkii

The yeastCandida beverwijkii was found to transport several monosaccharides against a concentration gradient. The process is mediated by a mobile carrier and shows a pronounced pH dependence. It is tightly coupled with metabolism and only potassium sorbate uncoupled the equilibrating from the active transport component. Kinetic analysis of uptake and efflux at high monosaccharide concentrations indicates an active transport operating either into or out of cells. T. Deák carried out the work in Prague while supported by the Hungarian Academy of Sciences.     

Transport kinetics of 6-deoxy-D-glucose inCandida parapsilosis

The strictly aerobic yeastCandida parapsilosis transports the nonmetabolizable monosaccharide 6-deoxy-D-glucose by an active process (inhibition by 2.4-dinitrophenol and other uncouplers but not by iodoacetamide), the accumulation ratio decreasing with increasing substrate concentration. Measured accumulation ratios are in agreement with those predicted from kinetic constants for influx and efflux. Energy for transport is probably required in the translocation step. The maximum rate is temperature-dependent with a transition point at 21 °C. the accumulation ratio is not, The uptake is most active at pH 4.5–8.5. It appears not to involve stoichiometric proton symport. The transport system is shared by D-glucose, D-mannose, D-galactose and possibly maltose but not by fructose, sucrose or pentoses. The apparent half-life of the transport system was 3.5–4 h.     

CUG codons inCandida spp.

Codon CUG is used for serine instead of for leucine, its usual assignment, in several yeasts of the genusCandida. We propose a series of steps for the reassignment, including disappearance of leucine CUG and its anticodon CAG, formation of a new serine tRNA, with anticodon CAG, from a duplication of the gene for serine tRNA (IGA), and then production of CUG codons by mutation at sites that are mostly nonessential.