Induction of germ-tube formation by Candida albicans in amino acid liquid synthetic medium at 25°C

Candida albicans (3153A) was found to exhibit extensive germ-tube and mycelial development at 25°C when transferred from amino acid synthetic medium at pH 6 to medium of pH 7. Significant germ-tube formation was detectable after approximately 8 h and in all experimental treatments, the peaks of maximal germination occurred at approximately 40–44 h. Such a transition was not only dependent on the initial pH of the medium but also on the glucose concentration and inoculum size. The optimum initial glucose concentration and inoculum size for maximal germ-tube development was 1.25% and 2×10⁶ cells ml^–1 respectively and above or below these values the extent of germ-tube formation was greatly reduced.     

The first clinical isolates of <Emphasis Type="Italic">Candida dubliniensis</Emphasis> in Slovakia

Candida dubliniensis, yeast closely related to Candida albicans, is a new pathogen associated mainly with infections of immunocompromised hosts. In this study, we report the first isolation of three isolates of C. dubliniensis in Slovakia. The first selection of both C. albicans and C. dubliniensis from the other Candida species was done on the basis of specific green color of primoculture grown on CHROMagar Candida. The presumptive identification was completed by supplemental tests: germ-tube formation, production of chlamydospores, ability or inability to grow at 42 and 45,°C and by commercial set API 20C AUX. Parallely, the discrimination between both species was performed by PCR assay using primers specific for Candida dubliniensis     

Gamma-aminobutyric acid (GABA) increases in vitro germ-tube formation and phospholipase B1 mRNA expression in Candida albicans

Candida albicans is a commensal yeast in humans that disseminates in immunocompromised persons. Its spreading is modulated by melanin, hormones, or some neurotransmitters, among other factors. The neurotransmitter gamma-aminobutyric acid (GABA) is used by bacteria, plants, and fungi as a carbon and nitrogen source. In this article, the in vitro effect of different doses of GABA on germ-tube formation and expression of phospholipase B1 (PLB1) mRNA in two Candida albicans strains was investigated. Results demonstrated that GABA increases both germ-tube formation and PLB1 mRNA expression in the two Candida strains in a dose-dependent manner, which suggests that GABA promotes the growth of C. albicans.     

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.     

Regulation of chitin synthesis during germ-tube formation in Candida albicans

The synthesis of chitin during germ-tube formation in Candida albicans may be regulated by the first and last steps in the chitin pathway: namely l-glutamine-d-fructose-6-phosphate aminotransferase and chitin synthase. Induction of germ-tube formation with either glucose and glutamine or serum was accompanied by a 4-fold increase in the specific activity of the aminotransferase. Chitin synthase in C. albicans is synthesized as a proenzyme. N-acetyl glucosamine increased the enzymic activity of the activated enzyme 3-fold and the enzyme exhibited positive co-operativity with the substrate, UDP-N-acetylglucosamine. Although chitin synthase was inhibited by polyoxin D (K _i =1.2M) this antibiotic did not affect germination. During germ-tube formation the total chitin synthase activity increased 1.4-fold and the expressed activity (in vivo activated proenzyme) increased 5-fold. These results could account for the reported 5-fold increase in chitin content observed during the yeast to mycelial transformation.Non-Standard Abbreviations GlcNac N-acetyl glucosamine - UDP-GlcNac UDP-N-acetyl glucosamine - PMSF phenylmethylsulphonylfluoride     

A simple medium for isolation and identification of Candida albicans directly from clinical specimens

A simple and specific medium consisting of chitosan, trypticase, Tween-80 and agar is devised to isolate the organisms directly from the clinical specimens and to produce germ tubes and chlamydospores for rapid differentiation and identification of Candida albicans from other closely related Candida species. By manipulating the incubating conditions, the specific phase of the organism can be produced in liquid or on solid medium at different time intervals to study the physiology of the organism.Many methods and media have been proposed in the past for identification of Candida albicans and to differentiate this from the closely related species of Candida (5–8, 15). Taschdjian, Burchall&Kozinn (15) showed that C. albicans produces germ tube within an hour or two when it is grown in human or animal serum or serum substitutes. The specificity of this germ tube test was later confirmed by various workers by using different media (3–5). The distinctive feature that differentiates C. albicans from other species is the production of chlamydospores (14). However, in all these studies three types of media were required to isolate the organisms from clinical specimens and to produce germ tubes and chlamydospores for identification. Recently studies have shown that a single medium can be employed to produce both structural components of the organism from the primary isolation medium but the preparation of the medium is more exhaustive (1) and time consuming (13) than the medium to be described here. The present investigation was therefore undertaken to develop a simple and specific medium to isolate the organism directly from the clinical specimens and to produce various morphological phases of Candida albicans to differentiate from other closely related Candida species for clinical diagnosis and to provide a medium to study the physiology and metabolism of the organism under in vitro conditions.Supported in part by Grant CA 20917, National Cancer Institute, National Institutes of Health and ALSAC.     

Growth ofCandida albicans in a minimal synthetic medium without biotin

Summary Growth ofCandida albicans strain B 311-10 was observed in a minimal synthetic biotin-free medium, using different glucose concentrations, during the first 30 hours of its development at 28 °C. The yeast's growth was observed spectrophotometrically at 675 nm reading its optical density every hour. The minimal medium of Shepherdet al. [12], with glucose (15 g/L) and biotin was modified: this vitamin was eliminated and the concentration of glucose was gradually lowered to 0.5 g/L. At 5 g/L of glucose and without biotin very good growth was obtained. Based on our results during the first 30 hours of growth, biotin has no influence on the yeast's growth. This medium would be useful for the study of the physiology ofC. albicans during the first period of its development.     

Effect of glucose starvation on germ-tube production byCandida albicans

By incubating starved and unstarved yeast cells in synthetic media with a pH of 4.5 or 6.7 at 37°C the effect of a 3 hours' glucose starvation on germ-tube production byCandida albicans was evaluated. In addition the endocellular content of total carbohydrates, glycogen, trehalose and proteins after and before the starvation were dosed. The most interesting result was the overcoming of the pH-regulated dimorphism, thanks to the starvation treatment. Infact the starved cultures produced germtubes indifferently in neutral or acid media, whereas the filamentation of the unstarved cultures was more copious in pH 6.7 medium. The endocellular content of trehalose and protein was unchanged, whereas total carbohydrates and glycogen showed a shortage after the 3 hours' glucose starvation. The possible involvements of these metabolic changes in the regulation of dimorphic transition are discussed.     

Cell-associated collagenolytic activity by Candida albicans

Cell associated collagenolytic activity of Candida albicans was quantified by measuring the degradation of synthetic peptide 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA), which is a specific substrate for collagenase, by the freeze-thaw procedure method. This collagenolytic activity was enhanced by cells cultured in the presence of bovine serum albumin (BSA) in culture medium. However, this activity was inhibited in the presence of ethylenediaminetetraacetic acid disodium salt (EDTA-2Na), but not by the serine proteinase inhibitor p-amidinophenyl methanesulfonyl fluoride (APMSF), nor the aspartyl proteinase inhibitor pepstatin A. These results suggested the presence of a metalloenzyme on pericellular C. albicans. This revised version was published online in June 2006 with corrections to the Cover Date.     

New milk medium for germ tube and chlamydoconidia production byCandida albicans

A new medium consisting of UHT milk, tween 80 and agar is described for the development of both germ tube and chlamydoconidia byCandida albicans. In total 172 isolates from clinical specimens, includingC. albicans (112),C. guilliermondii (4),C. krusei (3),C. parasilopsis (16).C. tropicalis (28),Torulopsis glabrata (6) andTrichosporon beigellii (3), were examined in this medium by using the standard method. A higher percentage (98.2%) of germ tube production byC. albicans was found in this medium than in undiluted serum (90.2%). In addition, onlyC. albicans was found to be able to produce a high percentage of chlamydoconidia (95.5%) after 48 hours' incubation. In comparison with the conventional medium, corn meal tween 80 agar (21.4%), this new medium gives a significantly higher percentage and abundance of chlamydoconidia production. Being simple, cheap and easy to prepare, the new milk medium is proposed as very practical in the clinical mycology laboratory.     

N-Acetyl-d-Glucosamine Induces Germination in Candida albicans through a Mechanism Sensitive to Inhibitors of cAMP-Dependent Protein Kinase

The present study examines the involvement of cAMP-dependent protein kinase (PKA) in the dimorphic transition of Candida albicans by assessing the in vivo effect of two permeable PKA inhibitors on N-acetyl-d-glucosamine (GlcNAc)- and serum-induced differentiation. The permeable myristoylated derivative of the heat-stable PKA inhibitor (MyrPKI), which inhibited C. albicans PKA in vitro, caused a concentration-dependent inhibition of germ-tube formation in cultures induced to germinate by GlcNAc; germination halted irrespective of the time of addition of the inhibitor. MyrPKI also blocked dibutyryl-cAMP (dbcAMP)- and glucagon-stimulated germination but did not affect serum-induced germination. H-89, another highly specific PKA inhibitor, displayed the same effect on germination. Neither MyrPKI nor H-89 had any effect on budding of yeast cells. In conclusion, our results indicate that cAMP-mediated activation of PKA plays a pivotal role in the biochemical mechanism underlying morphogenesis.     

In vitro modification of Candida albicans invasiveness

Candida albicans produces germ-tubes (GT) when it is incubated in animal or human serum. This dimorphism is responsible for its invasive ability.The purpose of the present paper is (1) to evaluate the ability of rat peritoneal macrophages to inhibit GT production of ingested Candida albicans, obtained from immunized rats and then activated in vitro with Candida-induced lymphokines; (2) to determinate any possible alteration of phagocytic and candidacidal activities.The phagocytes were obtained from rats immunized with viable C. albicans. Some of them were exposed to Candida-induced lymphokines in order to activate the macrophages in vitro. The monolayers of activated, immune and normal macrophages were infected with a C. albicans suspension during 4 hr.Activated macrophages presented not only the highest phagocytic and candidacidal activities but a noticeable inhibition of GT formation and incremented candidacidal activity.     

N-Acetyl-d-Glucosamine Induces Germination in Candida albicans through a Mechanism Sensitive to Inhibitors of cAMP-Dependent Protein Kinase

The present study examines the involvement of cAMP-dependent protein kinase (PKA) in the dimorphic transition of Candida albicans by assessing the in vivo effect of two permeable PKA inhibitors on N-acetyl-d-glucosamine (GlcNAc)- and serum-induced differentiation. The permeable myristoylated derivative of the heat-stable PKA inhibitor (MyrPKI), which inhibited C. albicans PKA in vitro, caused a concentration-dependent inhibition of germ-tube formation in cultures induced to germinate by GlcNAc; germination halted irrespective of the time of addition of the inhibitor. MyrPKI also blocked dibutyryl-cAMP (dbcAMP)- and glucagon-stimulated germination but did not affect serum-induced germination. H-89, another highly specific PKA inhibitor, displayed the same effect on germination. Neither MyrPKI nor H-89 had any effect on budding of yeast cells. In conclusion, our results indicate that cAMP-mediated activation of PKA plays a pivotal role in the biochemical mechanism underlying morphogenesis.     

An effective medium for the selective growth of yeast or mycelial forms of Candida albicans: Biochemical aspects of the two forms

A new synthetic medium, based on a modification of a commercially available tissue culture medium, allows Candida albicans to be grown in the yeast or mycelial form. Salient features of the system are described and comparisons with previous physiological investigations are discussed. A concise biochemical profile of these two forms of C. albicans is also presented. The results indicate vast metabolic differences between the two forms.     

Interactions ofCandida albicans with bacteria and salivary molecules in oral biofilms

The yeastCandida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells.C. albicans andCandida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind toStreptococcus gordonii NCTC 7869 while two otherCandida species (Candida krusei andCandida kefyr) do not. Adherence ofC. albicans was greatest when the yeast had been grown at 30° C to mid-exponential growth phase. For 21 strains ofC. albicans there was a positive correlation between the ability to adhere toS. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence ofC. albicans toS. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins ofC. albicans are co-expressed.     

Optimal concentration of ammonium ion in a minimal synthetic medium for the growth of Candida albicans

Candida albicans strain B 311-10 with and without starvation was cultivated in the minimal synthetic medium of Shepherd et al. [18], modified without biotin, aminoacids, low glucose concentration [20] and with decreasing amounts of (NH₄)₂SO₄, to determine the optimal growth requirement for this strain. All the experiments were carried out under sterile conditions at 25 °C in a thermostat with initial O.D.s (675 nm) of 0.500 and 0.100. Cell growth was generally monitored everyday for six days with a spectrophotometer by determining the absorbance of the cultures at 675 nm. All the experiments were repeated three times and a statistical analysis of the data with a probability of 99% and 1% of error was performed to confirm the validity of the results. Best growth was obtained with starved cells at an initial O.D. of 0.100 and with a 0.1 g/L concentration of (NH₄)₂SO₄. At this concentration, the growth of C. albicans B 311-10 was best between the first and the fourth day with the maximum at the third day. With (NH₄)₂SO₄ concentrations of 0.05 and 0.5 g/L, cell growth was the same.     

Fungicidal and Cytotoxic Activity of a Capsicum chinense Defensin Expressed by Endothelial Cells

Plant defensins are antimicrobial peptides that exhibit mainly antifungal activity against a broad range of plant fungal pathogens. However, their actions against Candida albicans have not been extensively studied. The mRNA for γ-thionin, a defensin from Capsicum chinense, has been expressed in bovine endothelial cells. The conditioned medium of these cells showed antifungal activity on germ tube formation (60–70% of inhibition) and on the viability of C. albicans (70–80% of inhibition). Additionally, C. albicans was not able to penetrate transfected cells. Conditioned medium from these cells also inhibited the viability (80%) of the human tumor cell line, HeLa.     

Effects of Antifungal Agents in Sap Activity of <Emphasis Type="Italic">Candida albicans</Emphasis> Isolates

Some antifungal agents have shown to exert effects on expression of virulent factors of Candida as the production of secretory aspartyl proteinase (Sap). In this study, we sought to determine and to compare the influence of fluconazole and voriconazole in proteinase activity of this microorganism. Thirty-one isolates obtained from oral mucosa of human immunodeficiency virus positive (HIV⁺) patients were used in this study. The minimal inhibitory concentrations (MIC) of fluconazole and voriconazole were determined using the broth microdilution method with RPMI 1640 medium and with yeast carbon base–bovine serum albumin (YCB–BSA) medium. The Sap activity following by digestion of BSA as substrate was determined for four Candida albicans strains arbitrarily chosen according to susceptibility (susceptible or resistant) to fluconazole or voriconazole. Besides, the SAP1 to SAP7 genes were screened by PCR for the same isolates that were determined by the Sap activity. In vitro susceptibility testing using the two media presented similar MIC values. Increased Sap activity was observed in resistant isolates on presence of drugs, but the Sap activity by susceptible isolates to azoles showed different behavior on the presence of drug. We detected the presence of SAP1 to SAP7 genes from all susceptible or resistant C. albicans isolates. The present study provides important data about the proteinase activity and the presence of genes of SAP family in fluconazole and voriconazole susceptible or resistant C. albicans isolates.     

Comparative activities of glycolytic enzymes in yeast and mycelial forms of Candida albicans

Through use of a synthetic defined medium which allows for the exclusive growth of yeast or mycelial forms of Candida albicans the activity of several major glycolytic enzymes in these forms were examined and compared. The results indicate vast metabolic differences between the forms. These data are discussed in relationship to the phenomenon of morphogenesis in C. albicans which in turn relates to problems in immunology and pathogenics of this important opportunistic organism.     

A viability assay for Candida albicans based on the electron transfer mediator 2,6-dichlorophenolindophenol

Candida albicans is an opportunistic fungal pathogen with comparably high respiratory activity. Thus, we established a viability test based on 2,6-dichlorophenolindophenol (DCIP), a membrane-permeable electron transfer agent. NADH dehydrogenases catalyze the reduction of DCIP by NADH, and the enzymatic activity can be determined either electrochemically via oxidation reactions of DCIP or photometrically. Among the specific respiratory chain inhibitors, only the complex I inhibitor rotenone decreased the DCIP signal from C. albicans, leaving residual activity of approximately 30%. Thus, the DCIP-reducing activity of C. albicans was largely dependent on complex I activity. C. albicans is closely related to the complex I-negative yeast Saccharomyces cerevisiae, which had previously been used in DCIP viability assays. Via comparative studies, in which we included the pathogenic complex I-negative yeast Candida glabrata, we could define assay conditions that allow a distinction of complex I-negative and -positive organisms. Basal levels of DCIP turnover by S. cerevisiae and C. glabrata were only 30% of those obtained from C. albicans but could be increased to the C. albicans level by adding glucose. No significant increases were observed with galactose. DCIP reduction rates from C. albicans were not further increased by any carbon source.