Glucan-associated protein modulations and ultrastructural changes of the cell wall in Candida albicans treated with micafungin, a water-soluble, lipopeptide antimycotic.

The composition of glucan-associated proteins (GAP) in the cell wall of Candida albicans was strongly affected by treatment with a sub-MIC yet beta-glucan synthesis inhibitory concentration (0.01 microg/ml) of FK463 (micafungin). Namely, a decrease in enzymes of glucose metabolism (mostly enolase and a novel 40 kDaltons component, here identified as the enzyme fructose-1,6-biphosphate aldolase) was observed, and this was coupled with an increase in two beta1-3 exo-glucanase isoforms (34 and 44 kDa, respectively). No GAP changes were detected in the same strain of the fungus made resistant to the drug, attesting to the specificity of the observed cell wall protein modulation. In addition, GAP changes were accompanied by marked ultrastructural alterations upon treatment with the sub-MIC dose of the drug, the majority of which was an aberrant cell surface morphology and a derangement of the normal layering of the cell wall. Our data demonstrate that sub-MIC doses of micafungin do critically affect not only the beta-glucan synthetic machinery but also protein composition and the whole cell wall structure of Candida albicans.

Analytical and semipreparative enantiomeric separation of azole antifungal agents by high-performance liquid chromatography on polysaccharide-based chiral stationary phases. Application to in vitro biological studies.

High-performance liquid chromatography (HPLC) was used for the enantiomeric separation of chiral imidazole derivatives endowed with antimycotic activity. Enantioselective columns, containing carbamates of cellulose and amylose, were used. The influence of the nature and content of an alcoholic modifier in the mobile phase was studied. The isolated enantiomers, separated on semipreparative columns, were submitted to in vitro biological investigations.

Identification of a glucan-associated enolase as a main cell wall protein of Candida albicans and an indirect target of lipopeptide antimycotics.

Growth-subinhibitory nonlytic doses of cilofungin (lipopeptide antibiotic affecting (1,3)-beta-D-glucan synthesis) inhibited the incorporation of 46- to 48-kDa glucan-associated (46K) protein into the growing cell wall of Candida albicans. The purified 46K protein constituent strongly reacted with a monoclonal antibody against enolase, a major cytoplasmic enzyme of the fungus. In addition, two internal fragments of 12- and 15-amino acid residues from a tryptic digest of 46K protein showed 100% identity with amino acids in positions 34-45 and 66-80 of enolase. By immunoelectron microscopy with polyclonal and monoclonal anti-enolase antibodies, the 46K protein was clearly detected in the inner layers of the fungal cell wall. Thus, consistent with the proposed immunogenic and diagnostic roles of enolase in candidiasis, biochemical, immunochemical, and ultrastructural evidence strongly suggest that the cilofungin-susceptible 46K protein is a cell wall-associated form of this enzyme.

The activity of cilofungin on the incorporation of glucan associated proteins into hyphal cells of Candida albicans.

The effect of the cilofungin, a beta 1-3 glucan synthase inhibitor, on the incorporation of the glucan associated proteins (GAP) into the mycelial wall of Candida albicans was investigated. For this study sub-inhibitory (< 2 micrograms/ml) doses of cilofungin were employed during the yeast to mycelial transition in a defined chemical medium, at 37 degrees C for 24 hours. Under these conditions, and particularly at the dose of 0.50 micrograms/ml cilofungin exerted a marked effect on GAP incorporation into the mycelial cell wall. The changes were essentially the absence of the two prominent bands of 46 and 31 kDa of the untreated cell wall coupled with an apparent increase in the amount of 55-56 kDa constituent, as well as of a minor constituent of 27-28 kDa. Radiolabel incorporation experiments demonstrated increased synthesis of a 34 kDa GAP, in addition to confirming the absence of the 46 kDa constituent, in mycelial cells under cilofunging treatment. Thus, sub-inhibitory doses of cilofungin may greatly alter the pattern of essential cell wall constituents such as the glucan-associated proteins, suggesting that this drug also has important effects on cell wall structure and fine organization, independent of, or prior to, its principal lytic effect on the fungal organism.

The lipopeptide antimycotic, cilofungin modulates the incorporation of glucan-associated proteins into the cell wall of Candida albicans.

The effect of the beta 1-3 glucan synthase inhibitor, cilofungin, on the incorporation of 35S-methionine-labelled glucan associated proteins (GAP) in the cell wall of Candida albicans was investigated in a susceptible strain C. albicans 3153 and resistant strain C. albicans CA-2. Cilofungin exerted a marked effect on the GAP composition of the cell wall at 0.25 mg/L, a concentration which reduced beta 1-3 glucan synthesis by approximately 50% and also inhibited the growth of the susceptible strain C. albicans 3153. A 46 kDa protein was present in large amounts in C. albicans 3153 but not in strain CA-2. This protein was probably not mannosylated and its incorporation was greatly reduced by cilofungin. In addition, a well defined 34 kDa protein was identified together with a distinct band of high molecular mass polydisperse material of between 65 and 96 kDa and another of > 200 kDa. These proteins were strongly reactive to concanavalin A indicating that they were mannosylated, and treatment with cilofungin caused an increase in their production which was also confirmed by immunoblotting with rabbit anti-Candida serum. In contrast, exposure of the drug-resistant strain CA-2 to cilofungin did not result in changes in the composition of the GAP constituents. Only the mannosylated proteins of 34 kDa and the high molecular mass polydisperse material 65-96 kDa were present in the cell wall. The pulse-chase labelling experiments showed that the 46 kDa protein was the first of the GAPs to be incorporated into the cell wall, and that this was suppressed in the presence of cilofungin whereas there was a concomitant increase in the incorporation of the 34 kDa and the high-molecular weight polydisperse material. Thus, cilofungin causes a profound imbalance in GAP incorporation into the growing cell wall which is possibly related to changes in the amount and type of glucan being synthesized at sub-inhibitory concentrations of the antimycotic.

Glucan synthesis and its inhibition by cilofungin in susceptible and resistant strains of Candida albicans.

The lipopeptide antimycotic agent, cilofungin, at a dose of 20 micrograms ml-1, inhibited beta 1-3 glucan synthesis in a drug-susceptible strain (3153; minimum inhibitory concentration (MIC) < 1 microgram ml-1) as well as in a drug-resistant strain of Candida albicans (CA-2, derived from 3153 by nitrosoguanidine mutagenesis; MIC > 50 micrograms ml-1). This was demonstrated for both whole cells under growing and non-growing conditions, and during protoplast regeneration. However, time-effect experiments, during growth of a CA-2 culture initially exposed to an inhibitory dose of cilofungin, showed that this strain was able to progressively regain both glucan synthesis and a growth rate comparable to that of cultures that had not been treated with the drug. This recovery was not attributable to cilofungin instability or degradation within the CA-2 culture. Our study suggests the existence of an as yet unknown drug-related and/or cell-related factor(s) modulating the inhibition of glucan synthesis, and then contributing to the actual inhibitory effects of cilofungin in C. albicans.

The effect of antimycotics on secretory acid proteinase of Candida albicans.

The effect of antimycotics on secretory aspartate (acid) proteinase, a virulence enzyme of Candida albicans, was investigated. The conditions of the study were such as to induce proteinase production in the stationary phase of growth (25-40 hours), when no antifungal tested, except the polyene derivative methyl partricin, significantly reduced the viability of the culture. Among azole derivatives, fenticonazole (FZ) but not miconazole, fluconazole or ketoconazole, exerted strong inhibition on proteinase, in typical dose-diphasic pattern, (0.01 microgram/ml; 1-10 micrograms/ml). 5-fluorocytosine (5-FC) was also inhibitory at a dose interval 1-10 micrograms/ml. In all cases, the inhibition concerned the synthesis of the enzyme rather that its activity as suggested by the results of comparative ELISA, SDS-PAGE and spectrophotometric methods of proteinase detection. Finally, the inhibition of proteinase production by FZ and 5-FC mainly reflected the effect of these antimycotics on general protein synthesis.

Protein synthesis and amino acid pool during yeast-mycelial transition induced by N-acetyl-D-glucosamine in Candida albicans.

Protein synthesis at different stages of yeast-mycelial transition induced by N-acetyl-D-glucosamine in Candida albicans was evaluated by following incorporation of radioactive amino acids into the acid-insoluble cellular material. In passing from the early germ-tube formation (60-90 min) to the mature hyphal cell (240-270 min) there was a marked decrease in the capacity for protein synthesis. Apparently, this decrease was not due to a decreased amino acid uptake into the soluble cellular pool or to exhaustion of carbon/energy source in the inducing medium with consequent arrest of growth. Protein synthesis, however, did not decay when amino acids at high concentration were added to the medium fostering the yeast-mycelial transition and this effect was potentiated by glucose. Analysis of the intracellular amino acid pool showed that both germ-tubes and hyphal cells were relatively depleted of several amino acids as compared to the yeast-form cells, whereas in the hyphae there was a higher concentration of glutamic acid/glutamine, the latter being the predominant component. These modulations in amino acid pool composition were not seen when yeasts were converted to hyphae in an amino acid-rich induction medium. This study emphasizes that yeast-form cells of C. albicans may efficiently convert to the mycelial form even under a progressively lowered rate of protein synthesis, and suggests that initiation of hyphal morphogenesis in the presence of N-acetyl-D-glucosamine is somehow separated from cellular growth.

31P nuclear magnetic resonance study of growth and dimorphic transition in Candida albicans.

A 31P NMR study of the fungal pathogen Candida albicans was carried out. Yeast-form cells at different phases of growth, as well as germ tubes and hyphae were examined. In all cases, the NMR spectra showed well separated resonance peaks arising from phosphorus-containing metabolites, the most prominent being attributable to inorganic phosphate (Pi) polyphosphates, sugar phosphates and mononucleotides, NAD, ADP and ATP. Relevant signals were also detected in the phosphodiester region. The intensity of most signals, as measured relative to that of Pi, was clearly modulated both at the different phases of growth and during yeast-to-mycelium conversion, suggesting significant changes in the intracellular concentration of the corresponding metabolites. In particular, the intensity of the polyphosphate signal was high in exponentially growing, yeast-form cells, then progressively declined in the stationary phase, was very low in germ tubes and, finally, undetectable in hyphae. NMR spectral analysis of the Pi region showed that from early-stationary phase, Pi was present in two different cellular compartments, probably corresponding to the cytoplasm and the vacuole. From the chemical shift of Pi, the pH values of these two compartments could be evaluated. The cytoplasmic pH was generally slightly lower than neutrality (6.7-6.8), whereas the vacuolar pH was always markedly more acidic.

Induction of germ tube formation by N-acetyl-D-glucosamine in Candida albicans: uptake of inducer and germinative response.

A number of strains of Candida albicans were tested for germ tube formation after induction by N-acetyl-D-glucosamine (GlcNAc) and other simple (proline, glucose plus glutamine) or complex (serum) compounds. A proportion of strains (high responders) were induced to form germ tubes evolving to true hyphae by GlcNAc alone or by proline or glucose plus glutamine mixture. The majority of strains were low responders because they could be induced only by serum or GlcNAc-serum medium. Two strains were found to be nonresponders: they grew as pseudohyphae in serum. Despite minor quantitative differences, all strains efficiently utilized GlcNAc for growth under the yeast form at 28 degrees C. They also had comparable active, inducible, and constitutive uptake systems for GlcNAc. During germ tube formation in GlcNAc, the inducible uptake system was modulated, as expected from induction and decay of GlcNAc kinase. Uranyl acetate, at a concentration of 0.01 mM, inhibited both GlcNAc uptake and germ tube formation and was reversed by phosphates. Germinating and nongerminating cells differed in the rapidity and extent of GlcNAc incorporation into acid-insoluble and alkali-acid-insoluble cell fractions. During germ tube formation induced by proline, GlcNAc was almost totally incorporated into the acid-insoluble fraction after 60 min. Moreover, hyphal development on induction by either GlcNAc or proline was characterized by an apparent "uncoupling" between protein and polysaccharide metabolism, the ratio between the two main cellular constituents falling from more than 1 to less than 0.5 after 270 min of development. The data suggest that utilization of the inducer for wall synthesis is a determinant of germ tube formation C. albicans but that the nature and extent of inducer uptake is not a key event for this phenomenon to occur.