Albicans Yeast Cells Research Articles

Analysis of fluconazole effect onCandida albicansviability during extended incubations

Fluconazole is an azole agent with primarily fungistatic activity in standard in vitro susceptibility tests. However, recent work has demonstrated that this drug can reduce Candida albicans viability during prolonged incubations under non-growing conditions. The present study was undertaken to examine more closely some of the parameters of this killing activity. Fungicidal effects of 1.0 microg ml-1 of fluconazole were found during 7-14-day exposures in each of two media that prevented proliferation, distilled water and metal-depleted RPMI 1640 tissue-culture medium. Fluconazole appeared to be stable after being incubated at 37 degreesC for either 7 or 14 days. Strains of C. albicans resistant to fluconazole in standard short-term growth-inhibition assays were also found to be resistant to fluconazole's effect on viability in prolonged culture, suggesting similar mechanisms of action for these effects. C. albicans yeast cells pre-incubated for 7 days in distilled water were not more sensitive to the drug in short-term susceptibility assays. Although all proliferation of the organisms in distilled water cultures appeared to cease after 3 days, fluconazole added at 7 days still reduced C. albicans viability. Therefore, the drug appeared to kill the non-proliferating organisms directly rather than preventing growth and thereby the emergence of younger organisms that would live longer. Transmission electron microscopy demonstrated damage to the cell wall-cell membrane complex and interior contents of yeast cells incubated in distilled water alone; fluconazole appeared to increase the percentages of cells so affected. In summary, extended-incubation susceptibility tests demonstrated that fluconazole has direct fungicidal activity of non-proliferating C. albicans yeast cells. These results may be relevant to the manner in which this drug promotes clearance of chronic fungal infections.

Antileukoprotease: an endogenous protein in the innate mucosal defense against fungi.

Previous studies have suggested that endogenous protease inhibitors may participate in the mucosal host defense. Antileukoprotease (ALP) is an important protease inhibitor found on various mucosal surfaces, including those of the respiratory and genital tracts. This study reports on the antimicrobial activity of recombinant (r) ALP toward the human fungal pathogens Aspergillus fumigatus and Candida albicans. rALP expressed pronounced fungicidal activity toward metabolically active A. fumigatus conidia and C. albicans yeast cells; however, metabolically quiescent A. fumigatus conidia were totally resistant. In contrast with the protease inhibitory activity of rALP, the fungicidal activity was localized primarily in the NH2-terminal domain. On a molar base, the fungicidal activity of rALP was comparable with that of human defensins and lysozyme. In addition, rALP caused inhibition of C. albicans yeast cell growth. By exhibiting antifungal activity, ALP may play an important role in the innate mucosal defense against human pathogenic fungi.

Antileukoprotease; An endogenous protein in the innate mucosal defense against fungi

Previous studies have suggested that endogenous protease inhibitors may participate in the mucosal host defense. Antileukoprotease (ALP) is an important protease inhibitor found on various mucosal surfaces, including those of the respiratory and genital tracts. This study reports on the antimicrobial activity of recombinant (r) ALP toward the human fungal pathogens Aspergillus fumigatus and Candida albicans. rALP expressed pronounced fungicidal activity toward metabolically active A. fumigatus conidia and C. albicans yeast cells; however, metabolically quiescent A. fumigatus conidia were totally resistant. In contrast with the protease inhibitory activity of rALP, the fungicidal activity was localized primarily in the NH2-terminal domain. On a molar base, the fungicidal activity of rALP was comparable with that of human defensins and lysozyme. In addition, rALP caused inhibition of C. albicans yeast cell growth. By exhibiting antifungal activity, ALP may play an important role in the innate mucosal defense against human pathogenic fungi.

Ultrastructural alterations of Candida albicans induced by a new imidazole antimycotic omoconazole nitrate.

The antifungal effects of an imidazole-antimycotic omoconazole nitrate (OMZ) on the morphology and ultrastructure of Candida albicans yeast cells were studied using scanning and transmission electron microscopy. The treatment of growing Candida cultures with fungistatic doses (0.4 to 4 micrograms/ml) of OMZ produced the formation of a chain or cluster of cells. Thickening of the cell wall and accumulation of electrondense vesicles in the wall were clearly observed. Development of Golgi-like complex membranous structures in the cytoplasm was the most prominent finding. The cytological alteration induced by exposure to a higher concentration (40 micrograms/ml) of the drug was characterized by disruption of the intracytoplasmic organelles. Our results confirm the strong antifungal activity of OMZ against fungal cells.

Assessment of a mouse model of neutropenia and the effect of an anti-candidiasis monoclonal antibody in these animals.

As previously reported, monoclonal antibody (MAb) B6.1 increases resistance to hematogenous disseminated candidiasis in normal and SCID mice. In this study, MAb B6.1 was examined in a mouse model of neutropenia. The neutropenia was induced for a short period of time by a single dose of the anti-neutrophil antibody, MAb RB6-8C5, or for a protracted period by doses of MAb RB6-8C5 every other day. At low doses (< or = 25 microg/mouse), neutrophils were primarily affected, but at high doses (> or = 50 microg/mouse), lymphocytes were also depleted. Mice given either single or multiple doses of MAb RB6-8C5 were more resistant to experimental hematogenous disseminated candidiasis if they received MAb B6.1 before and after challenge with Candida albicans yeast cells intravenously. These results show the utility of MAb RB6-8C5 for induction of a protracted neutropenia in mice and demonstrate that MAb B6.1 can enhance resistance against candidiasis under neutropenic conditions.

The highly immunogenic enolase and Hsp70p are adventitious Candida albicans cell wall proteins.

Screening cDNA libraries with polyclonal antibody preparations against Candida albicans yeast or mycelial cell walls resulted in isolation of several positive clones. Some of them encoded enolase; others encoded a protein of the 70 kDa heat-shock protein family (Hsp70p), etc. The presence of these cytosolic proteins in the cell wall of actively growing C. albicans was discovered by analytical (SDS-PAGE and Western blot) and cytological (indirect immunofluorescence) experiments. Supplementation of cell cultures with papulacandin B, an antibiotic that inhibits formation of the beta-glucan skeleton, resulted in the release of enolase to the supernatant fluids; this release was prevented when 0.6 M KCl was present as an osmotic stabilizer. The cell wall of C. albicans incorporated exogenously added proteins (enolase and Escherichia coli and C., albicans cytosolic proteins). The presence in the C. albicans cell wall of enolase, Hsp70p, and probably other intracellular proteins that are highly immunogenic might help the fungal cells to evade the host defences, and consequently could represent a survival mechanism for C. albicans 'in vivo'.

Experimental candidal mastitis in goats: clinical, haematological, biochemical and sequential pathological studies.

The present study, first of its kind, was conducted with the objectives to understand hitherto little known aspects of candidal mastitis, like its sequential pathology, pathogenesis and clinico-biochemical changes. For this purpose, unilateral intramammary inoculation of 10 goats with Candida albicans (1.2 x 10(7) yeast cells) resulted in the development of mastitis, with gross and microscopic lesions being restricted to the infected udder halves only and without dissemination of infection to the opposite uninfected udder halves as well as other organs of the body. The experiment was continued for 40 days and after infection, there was sharp fall in milk yield and Candida albicans was directly demonstrated in the milk and re-isolated from the milk and udder tissues up to 30th day after inoculation. An increase in total immunoglobulins in the milk and plasma along with increase in total plasma proteins were also observed. Haematology revealed leukocytosis and neutrophilia. Microscopically, there was acute purulent mastitis, which later became chronic, nonpurulent and interstitial with formation of granulomas. It was concluded that Candida albicans was highly pathogenic to the lactating goat mammary gland even without immunosuppression or antibiotic treatment, resulting in severe irreversible tissue damage and nearly complete agalactia.

Thermotolerance and trehalose accumulation induced by heat shock in yeast cells of Candida albicans

Candida albicans yeast cells growing exponentially on glucose are extremely sensitive to severe heat shock treatments (52.5°C for 5 min). When these cultures were subjected to a mild temperature preincubation (42°C), they became thermotolerant and displayed higher resistance to further heat stress. The intracellular content of trehalose was very low in exponential cells, but underwent a marked increase upon non-lethal heat exposure. The accumulation of trehalose is likely due to heat-induced activation of the trehalose-6-phosphate synthase complex, whereas the external trehalase remained practically unmodified. After a temperature reversion shift (from 42°C to 28°C), the pool of trehalose was rapidly mobilized without any concomitant change in trehalase activity. These results support an important role of trehalose in the mechanism of acquired thermotolerance in C. albicans and seem to exclude the external trehalase as a key enzyme in this process.

Ultrastructural and biochemical studies onCandida albicansafter prolonged incubation in sea water

Candida albicans yeast cells suspended in sterilized sea water and cultivated in Brain Heart Infusion broth were compared. Viability, chemical composition, surface hydrophobicity and ultrastructural characteristics showed variations after incubation in sea water. The yeast cells developed some ultrastructural changes after about a month in sea water. The surface hydrophobicity of the yeast cells was gradually reduced, starting from day 16, and continued to decline throughout the 32 days in sea water. A decrease in total carbohydrate, lipid and protein contents was also observed and corresponded with ultrastructural modifications.

Ultrastructural and biochemical studies on Candida albicans after prolonged incubation in sea water

Candida albicans yeast cells suspended in sterilized sea water and cultivated in Brain Heart Infusion broth were compared. Viability, chemical composition, surface hydrophobicity and ultrastructural characteristics showed variations after incubation in sea water. The yeast cells developed some ultrastructural changes after about a month in sea water. The surface hydrophobicity of the yeast cells was gradually reduced, starting from day 16, and continued to decline throughout the 32 days in sea water. A decrease in total carbohydrate, lipid and protein contents was also observed and corresponded with ultrastructural modifications.

Effect of fluconazole on viability of Candida albicans over extended periods of time.

The treatment of chronic mycoses may expose the infecting organisms to antimicrobial agents for extended periods of time. It is possible that an azole antifungal drug such as fluconazole, with primarily fungistatic activity in standard in vitro susceptibility tests, might be able to damage the fungal cells and reduce their viability over prolonged incubations under nonproliferating conditions. To test this possibility, Candida albicans yeast cells were exposed to various concentrations of fluconazole in RPMI 1640 tissue culture medium for 4 h at 37 degrees C, washed free of the drug, and then incubated at 37 degrees C for a 28-day period; enumeration of the remaining CFU at various times during this period revealed no increased loss of viability for the fluconazole-exposed organisms. However, when fluconazole was added to the organisms maintained in distilled water (with or without pretreatment with the drug), a marked reduction of viability was found. At 14 days of incubation with two strains of C. albicans, negative cultures were found for 7 of 10 and 10 of 11 samples, respectively, containing 1.0 microgram of fluconazole per ml versus 0 of 10 and 1 of 11 control samples (P of < 0.01 and 0.001, respectively). The effect of fluconazole on fungal viability under these conditions became noticeable at approximately 7 days and was greater when the samples were incubated at 37 degrees C rather than 25 degrees C. These findings suggest that fluconazole may have fungicidal effects on fungal cells during prolonged exposures under conditions in which the organisms are prevented from proliferating by lack of nutrients.

Use of a microtiter plate assay to detect the rate of killing of adherent Candida albicans by antifungal agents

Candida albicans may become adherent to prosthetic devices of various kinds and thereby produce infections that are difficult to treat with standard antifungal therapy. The objective of the present work was to study the effectiveness of antifungal agents against adherent C. albicans yeast cells. A microtiter plate assay was developed to assess the time required for killing of the fungal cells by three antifungal agents. The assay initially was validated by demonstrating that the percentage of organisms adhering to the test wells was relatively constant and that exposure to the antifungal agents caused only minimal dislodgement of viable organisms from the plates. In studies that used this assay to determine the time required for killing the adherent yeast cells, chlorhexidine was found to be the most effective; in fact, in comparing the minimal lethal concentrations of the agents for exposures of 2 minutes versus 4 hours, a ratio of 2.9 was obtained for chlorhexidine versus 1050 for amphotericin B and 556 for nystatin. The microtiter plate assay used in these studies may therefore be useful as a screening test to determine which antifungal agents have the most rapid fungicidal effects on adherent fungal organisms.

Preliminary characterization of the material released to the culture medium by Candida albicans yeast and mycelial cells.

Culture filtrate concentrates were obtained from Candida albicans yeast and mycelial cells grown in the presence of 14C-protein hydrolysate for radioactive labeling of cellular polypeptides. Both growth forms released to the medium minor but significant amounts of proteinaceous materials. The analysis of culture filtrate concentrates by means of SDS-polyacrylamide gel electrophoresis and fluorography revealed a similar and complex electrophoretic pattern, though some qualitative and quantitative differences between samples obtained from yeast and mycelial cells were observed. Materials released, mostly composed of mannoproteins as shown by their affinity towards concanavalin A, presented (i) cross-reactivity (by Western immunoblotting and ELISA) against polyclonal antisera to genuine cell wall components (among them the 58-kilodalton fibrinogen-binding mannoprotein) and (ii) high affinity for polystyrene-latex microbeads. Results presented suggest a possible common identity for the molecules shed to the medium and the cell wall protein and mannoprotein constituents.

Adhesion of Candida albicans to brain tissue of Macaca mulata in an ex vivo assay

An ex vivo adhesion assay was used to examine adhesion of Candida albicans yeast cells to brain tissue of the primate Macaca mulata. Tissues from frontal lobes and striatum (caudate, putamen, and portions of the globus pallidus) were used in the assay. Yeast cells adhered to gray matter at about six times the level of adhesion to white matter. The fungus was able to bind to different cell types within the cortex, basal ganglia, and white matter. Binding to neurons, small neurons or glia, endothelial cells, and neuropil was observed.

Candida albicans ALS1: domains related to a Saccharomyces cerevisiae sexual agglutinin separated by a repeating motif

Transfer of budding Candida albicans yeast cells from the rich, complex medium YEPD to the defined tissue culture medium RPMI 1640 (RPMI) at 37 degrees C and 5% CO2 causes rapid onset of hyphal induction. Among the genes induced under these conditions are hyphal-specific genes as well as genes expressed in response to changes in temperature, CO2 and specific media components. A cDNA library constructed from cells incubated for 20 min in RPMI was differentially screened with yeast (YEPD)- and hyphal (RPMI)-specific probes resulting in identification of a gene expressed in response to culture conditions but not regulated by the yeast-hyphal transition. The deduced gene product displays significant identity to Saccharomyces cerevisiae alpha-agglutinin, encoded by AG alpha 1, an adhesion glycoprotein that mediates mating of haploid cells. The presence of this gene in C. albicans is curious since the organism has not been observed to undergo meiosis. We designate the C. albicans gene ALS1 (for agglutinin-like sequence). While the N- and C-termini of the predicted 1260-amino-acid ALS1 protein resemble those of the 650-amino-acid AG alpha 1, ALS1 contains a central domain of tandem repeats consisting of a highly conserved 36-amino-acid sequence not present in AG alpha 1. These repeats are also present on the nucleotide level as a highly conserved 108 bp motif. Southern and Northern blot analyses indicate a family of C. albicans genes that contain the tandem repeat motif; at least one gene in addition to ALS1 is expressed under conditions similar to those for ALS1 expression. Genomic Southern blots from several C. albicans isolates indicate that the number of copies of the tandem repeat element in ALS1 differs across strains and, in some cases, between ALS1 alleles in the same strain, suggesting a strain-dependent variability in ALS1 protein size. Potential roles for the ALS1 protein are discussed.

Effect of Abscess Fluid Supernatants on the Kinetics of Candida albicans Growth

Abscess fluid supernatants have been found to inhibit microbial growth, an effect that appears to be due to a protein originating in the cytoplasm of neutrophils. The antimicrobial activity of calprotectin, the responsible protein, is reversible by the addition of zinc to the culture medium. To more carefully analyze this type of antimicrobial activity supernatants of fluids from experimental subcutaneous Candida albicans abscesses in mice were studied to determine how they affect the in vitro growth kinetics of C. albicans. The abscess fluid supernatants inhibited growth in a dose-dependent fashion and more at early times than at later ones. Instability of the abscess fluid antimicrobial activity did not appear to explain the timing of the growth inhibition. A marked lengthening of the lag phase of growth was observed in cultures containing the supernatants (from approximately 6 hr in the control cultures to 15-20 hr in those with the supernatants). On the other hand, the abscess fluid supernatants had only minimal effects on the generation times of actively proliferating C. albicans yeast cells. In addition, these supernatants did not appear to significantly affect the percentage of inoculated organisms undergoing cell division, as determined by a limiting dilution assay. Therefore, these results indicate that abscess fluid supernatants extend the lag phase of C. albicans growth, an effect similar to that seen with zinc-deprived organisms.

Binding of plasma fibronectin to Candida albicans occurs through the cell binding domain

Candida albicans yeast cells bind soluble human plasma fibronectin (Fn) through a glycoprotein receptor (adhesin) located on the cell surface. This work demonstrates that a 120 kDa proteolytic fragment of Fn encompassing the cell binding domain binds more avidly to the yeast cell adhesin than does the parent Fn molecule. The presence of binding of Fn fragments containing heparin- and gelatin-binding domains of Fn could not be detected. The binding of the 120 kDa fragment is inhibited by a monoclonal antibody to the cell binding domain containing the amino acid sequence, Arginine-Glycine-Aspartic acid (RGD) as well as by an RGD-containing ∼23-mer Fn peptide, but not with heparin or GRGDSPL. The fact that the cell binding domain of soluble Fn binds more avidly than does the parent molecule may explain the difference in the interaction of soluble Fn and immobilized Fn with Candida. It is possible that, upon immobilization, Fn may expose domains of the molecule previously unexposed when the molecule is in the soluble state.

Murine tissues exposed to cytotoxic drugs display altered patterns of Candida albicans adhesion

An ex vivo adhesion assay was used to examine the binding of Candida albicans yeast cells to tissues from mice treated with cytotoxic drugs such as lipopolysaccharide and the clinically used anticancer drugs doxorubicin, cisplatin, and vincristine. No major differences were observed in binding of the fungal cells to liver and kidney tissues from treated or untreated animals. All drug-treated spleens displayed altered patterns of C. albicans adhesion compared with the control group, with yeast cells bound not only to the marginal zone but also to the white and red pulp. Immunostaining for macrophages, which are proposed as the site of normal adhesion, showed no apparent differences between the control and the experimental spleens that could account for the change in adhesion patterns. Scanning electron microscopy images suggested that yeast binding to the white pulp of treated tissue is mediated through fibers, perhaps extracellular matrix components exposed as result of the cytotoxic treatment. Exposure of new attachment sites for C. albicans in treated tissues may facilitate initiation of infection.

The contribution of electrostatic forces to the process of adherence of Candida albicans yeast cells to substrates

The contribution of electrostatic forces to the process of adherence of Candida albicans yeast cells to substrates

The contribution of electrostatic forces to the process of adherence of Candida albicans yeast cells to substrates.

The contribution of electrostatic interactions to the adherence process of Candida albicans and other Candida species was investigated by mixing cationic or anionic exchange resins possessing free -COO- or -NH+4 groups, respectively, on their surface. The adherence process of yeast cells to the anionic exchange resin is a saturable event that is essentially complete by 60 min. There is no measurable interaction of yeast cells with the cationic exchange resin. All clinical isolates of C. albicans, C. pseudotropicalis, one isolate each of C. tropicalis and Torulopsis glabrata possessed electrostatic charge as defined by this method, whereas two clinical isolates of C. parapsilosis, C. krusei and one isolate of C. tropicalis did not have measurable electrostatic surface charge. The adherence process to the exchange resin with the free -NH+4 group was dependent upon the pH of the suspending medium and varied from one isolate to another. Fixing yeast cells, or alternatively, endothelial cells, in such a manner as to change the surface charge of both and then measuring adherence of yeast cells to the target cells was an event that was not controlled by electrostatic interactions as they are defined herein. It appears that whatever contribution electrostatic charges make to the adherence process, that at best, it is a minor contribution.