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ABSTRACT: The complete DNA sequence of Candida albicans DIT2, encoding cytochrome P450 family 56 (CYP56), was obtained, and heterologous expression was achieved in Escherichia coli, where CYP56 was targeted to the membrane fraction. In reconstituted assays with the purified enzyme, CYP56 was shown to catalyze the conversion of N-formyl tyrosine into N,N'-bisformyl dityrosine, a reaction that was dependent on cytochrome P450 reductase, NADPH, and oxygen, yielding a turnover of 21.6 min(-1) and a k(s) of 26 microM. The Hill number was calculated as 1.6, indicating that two molecules of the substrate could bind to the protein. Azole antifungals could bind to the heme of CYP56 as a sixth ligand with high affinity. Both chromosomal alleles of CYP56 were disrupted using the SAT1 flipper technique, and CYP56 was found to be nonessential for cell viability under the culture conditions investigated. Susceptibility to azole drugs that bind to cytochromes P450 was tested, and the mutant showed unaltered susceptibility. However, the mutant showed increased susceptibility to the echinocandin drug caspofungin, suggesting an alteration in 1,3-glucan synthase and/or cell wall structure mediated by the presence of dityrosine. Phenotypically, the wild-type and mutant strains were morphologically similar when cultured in rich yeast extract-peptone-dextrose medium. However in minimal medium, the cyp56Delta mutant strain exhibited hyphal growth, in contrast to the wild-type strain, which grew solely in the yeast form. Furthermore, CYP56 was essential for chlamydospore formation.
Antimicrobial Agents and Chemotherapy 08/2008; 52(10):3718-24. · 4.84 Impact Factor
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ABSTRACT: To characterize the interaction of Sclerotinia sclerotiorum and S. minor with strains of the mycoparasite and commercial biocontrol agent Coniothyrium minitans using novel perfusion chamber gasket co-culture.
Sclerotinia were cultured in perfusion chamber gaskets and then flooded with Coniothyrium conidia. After germination, Coniothyrium failed to show any form of directed growth, making contact with Sclerotinia hyphae in a random manner. In turn, some Coniothyrium hyphae coiled round Sclerotinia counterparts and although no intracellular growth was observed, Coniothyrium proliferated, while the hyphae of Sclerotinia became vacuolated and lost the cytoplasm. When co-cultures of Sclerotinia with Coniothyrium were flooded with FITC-lectins, small difference in fluorescence between the fungi was found with FITC-Con A suggesting that cell walls of both the species exposed mannose. In contrast, Coniothyrium fluoresced poorly in comparison with Sclerotinia when FITC-wheat germ agglutinin was used, indicating a marked paucity of N-acetylglucosamine exposure by cell walls of Coniothyrium, hence reduced exposure to chitinolytic enzyme action.
The approach employed supported direct sequential microscopic observation of Coniothyrium and Sclerotinia as well as the utilization of representative fluorescent moieties to characterize relative carbohydrate cell wall exposure.
Letters in Applied Microbiology 07/2008; 47(2):128-33. · 1.62 Impact Factor
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ABSTRACT: To develop a novel procedure for isolating and characterizing cryptococcal cell-surface proteins using biotinylation, fluorescein isothiocyanate (FITC)-streptavidin, flow cytometry and associated ligand-receptor analysis, confocal microscopy and electrophoretic separation.
Cell proteins of both acapsulate and encapsulated Cryptococcus neoformans cells were labelled using sulfo-NHS-biotin which, in turn, was complexed with FITC-streptavidin. Resulting cell population fluorescence supported visualization of cell-surface protein distribution by confocal microscopy, as well as evaluation of protein exposure by flow cytometry and the calculation of the ligand-binding determinants EC(50), F(max) and H(n). Biotinylation of cell-surface proteins also supported their isolation by affinity chromatography and characterization by SDS/PAGE. Ligand-binding determinants, such as EC(50) values, indicated that acapsulate and stationary phase cells have greatest affinity for biotin. F(max) values demonstrated greatest protein exposure among stationary phase cells; in turn, encapsulated cells expose more protein than acapsulate counterparts. H(n) values of below unity potentially confirm the complex multi-receptor nature of biotin binding to cryptococcal cell surfaces under investigation. Fluorescence visualization showed marked but localized fluorescence indicative of protein exposure around sites of cell division. In turn, biotinylation of cell-surface proteins and their release under reducing conditions demonstrated at least two noncovalently linked proteinaceous entities, of 43 and 57 kDa, exposed on acapsulate cryptococcal cell walls.
A novel method for identifying, in situ, cell-surface proteins exposed by C. neoformans was established. This novel technique was successfully implemented using both acapsulate and encapsulated C. neoformans cells, both were found to have dynamic and markedly localized protein distribution around sites of cell division and associated cell wall trauma.
A novel procedure, employing a versatile combination of flow cytometry, ligand-receptor analysis, confocal microscopy and biotinylation, supported the characterization and isolation of cryptococcal cell-surface proteins.
Journal of Applied Microbiology 09/2007; 103(2):390-9. · 2.34 Impact Factor
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ABSTRACT: Aims: Characterization of the representative protozoan Acanthamoeba polyphaga surface carbohydrate exposure by a novel combination of flow cytometry and ligand–receptor analysis.Methods and Results: Trophozoite and cyst morphological forms were exposed to a panel of FITC–lectins. Population fluorescence associated with FITC–lectin binding to acanthamoebal surface moieties was ascertained by flow cytometry. Increasing concentrations of representative FITC–lectins, saturation binding and determination of Kd and relative Bmax values were employed to characterize carbohydrate residue exposure. FITC–lectins specific for N-acetylglucosamine, N-acetylgalactosamine and mannose/glucose were readily bound by trophozoite and cyst surfaces. Minor incremental increases in FITC–lectin concentration resulted in significant differences in surface fluorescence intensity and supported the calculation of ligand-binding determinants, Kd and relative Bmax, which gave a trophozoite and cyst rank order of lectin affinity and surface receptor presence.Conclusions: Trophozoites and cysts expose similar surface carbohydrate residues, foremost amongst which is N-acetylglucosamine, in varying orientation and availability.Significance and Impact of the Study: The outlined versatile combination of flow cytometry and ligand–receptor analysis allowed the characterization of surface carbohydrate exposure by protozoan morphological forms and in turn will support a valid comparison of carbohydrate exposure by other single-cell protozoa and eucaryotic microbes analysed in the same manner.
Journal of Applied Microbiology 11/2004; 97(6):1319 - 1325. · 2.34 Impact Factor
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ABSTRACT: Characterization of the representative protozoan Acanthamoeba polyphaga surface carbohydrate exposure by a novel combination of flow cytometry and ligand-receptor analysis.
Trophozoite and cyst morphological forms were exposed to a panel of FITC-lectins. Population fluorescence associated with FITC-lectin binding to acanthamoebal surface moieties was ascertained by flow cytometry. Increasing concentrations of representative FITC-lectins, saturation binding and determination of K(d) and relative B(max) values were employed to characterize carbohydrate residue exposure. FITC-lectins specific for N-acetylglucosamine, N-acetylgalactosamine and mannose/glucose were readily bound by trophozoite and cyst surfaces. Minor incremental increases in FITC-lectin concentration resulted in significant differences in surface fluorescence intensity and supported the calculation of ligand-binding determinants, K(d) and relative B(max), which gave a trophozoite and cyst rank order of lectin affinity and surface receptor presence.
Trophozoites and cysts expose similar surface carbohydrate residues, foremost amongst which is N-acetylglucosamine, in varying orientation and availability.
The outlined versatile combination of flow cytometry and ligand-receptor analysis allowed the characterization of surface carbohydrate exposure by protozoan morphological forms and in turn will support a valid comparison of carbohydrate exposure by other single-cell protozoa and eucaryotic microbes analysed in the same manner.
Journal of Applied Microbiology 02/2004; 97(6):1319-25. · 2.34 Impact Factor
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ABSTRACT: Three metabolizing systems (rat, heterologously expressed CYP3A4 and human liver) were used to evaluate 12 analogues of dapsone (4,4'diaminodiphenylsulphone) in-vitro. Methaemoglobin formation in a two-compartment and cytotoxicity in a single-compartment model were studied using human erythrocytes and neutrophils, respectively, as target cells. In the two-compartment system using rat microsomes as a generating system and methaemoglobin as an endpoint, the least potent methaemoglobin formers tested were the 2-methyl-4-propylamino (AXDD14), 2-hydroxy-4-4'amino (ABDD5) derivatives and a sulphone/trimethoprim derivative (K-130). Dapsone itself, a 2-methoxy-4-ethylamino (W10) and a 2-hydroxyl-4-ethylamino compound (ABDD39) were the most toxic. In the single-compartment cytotoxicity test using rat microsomes, AXDD14 was again among the least toxic, as was a 2-methyl 4-cyclopentyl derivative (AXDD17) and surprisingly ABDD39. The most cytotoxic compounds again included dapsone itself as well as two 2-trifluoromethyl derivatives. The only significant methaemoglobin formation and cytotoxicity shown with the heterologously expressed human CYP 3A4 was with AXDD14, which was extensively activated. Interestingly, metabolism of dapsone was low using the expressed CYP 3A4. In the two-compartment system using human liver microsomes, AXDD14, K-130 and ABDD5 were oxidized to a significantly lesser extent compared with dapsone and these preliminary findings indicate that future development of these compounds may be worthwhile.
Journal of Pharmacy and Pharmacology 10/1996; 48(9):945-50. · 2.17 Impact Factor
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ABSTRACT: Cell walls were isolated from stationary-phase cultures of Candida albicans grown at 25 degrees C or 37 degrees C, in iron-depleted and iron-sufficient conditions. Proteins solubilised from cell-wall fractions were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Approximately 40 protein bands were detected by Coomassie blue staining in all wall extracts, regardless of temperature or other growth condition. Sera from patients with oral or systemic candidosis, from whom the isolates were obtained, and pooled normal human serum were examined for the presence of IgG and IgM antibodies to cell-wall proteins by Western blotting. Patient sera recognised more antigens than pooled normal human serum. In particular, an antigen of 44 kda was detected by IgG antibodies in the sera of patients and two antigens of 41 and 14 kda were detected by their IgM antibodies when the sera were used as probes against walls from iron-depleted cells, but not from iron-sufficient cells, grown at 25 degrees C. Two antigens of 45 and 40 kda were detected by IgM antibodies in the sera of patients tested against walls from iron-depleted but not from iron-sufficient cells grown at 37 degrees C. IgG antibodies did not distinguish between these wall preparations from cells grown at 37 degrees C. These results suggest that the specific cell-wall proteins induced during growth in iron-depleted conditions, as well as other proteins, were immunogenic and were recognised by the patients' antibodies.
Journal of Medical Microbiology 03/1989; 28(2):93-100. · 2.50 Impact Factor
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ABSTRACT: Cell surface hydrophobicity (CSH) of Candida albicans was significantly enhanced following growth under iron depletion and was also influenced by growth phase and yeast strain. Polyene antifungals reduced CSH of yeasts grown in iron-sufficient media, however, the triazole fluconazole significantly increased CSH regardless of growth conditions.
Mycological Research.
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ABSTRACT: The numbers of zoospores produced by a pathogenic strain of Saprolegnia diclina and their behaviour are markedly influenced by a variety of environmental variables including temperature, pH, oxygen tension and the presence of biocides. The use of the latter is not recommended, as fish readily succumb to equivalent concentrations of biocides. Analysis of the pattern of the distribution of resulting zoospore cysts demonstrates that zoospores become dispersed by random movement even while in the proximity of the parent colony's nutrient source. However, the presence of amino acids, in particular aspartic and glutamic acid, at concentrations which occur in fish tissue promotes the directed movement of zoospores towards the nutrient source thereby encouraging the colonization of fresh sites.
Transactions of the British Mycological Society.
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ABSTRACT: The effect of increasing culture age on cell surface hydrophobicity (CSH) and cell surface electrostatic charge (measured as zeta potential) of conidia from five isolates of Coniothyrium minitans representing three different morphological types was examined. Conidial CSH of three isolates (A2 960/1, CH1 and CH2) decreased with culture age, whereas CSH of two others (B 1300/2 and IMI 134523) remained high for the whole 42 day experimental period. In contrast, cell surface electrostatic charge decreased uniformly in conidia of all five isolates for the first 34 d and then rose slightly at 42 d. The variation in cell surface electrostatic charge (spectrum width) of the sampled conidia decreased with age for all five isolates. In all five isolates cell surface electrostatic charge of conidia became increasingly negative as the pH of the buffer used to suspend conidia was increased from pH 3.0 to 9.0. No relationship between colony morphology of C. minitans and conidial CSH and cell surface electrostatic charge was found.
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ABSTRACT: The avidity of conidia and 48-h-old germlings of Coniothyrium minitans for FITC-conjugated lectins was characterised by flow cytometry and digital microscopy. Six isolates of C. minitans representing three morphological types were compared. Binding of Con A, SBA and WGA by conidial populations varied markedly in extent and pattern between isolates, however, with increasing culture age, conidia from all isolates demonstrated a significant reduction in lectin avidity. Germling isolates bound significantly different amounts of lectins and lectin binding differed significantly with locality. Spore walls of all germlings from all isolates bound more Con A compared with hyphal apices and mature hyphal walls. In contrast, hyphal apices of the majority of germling isolates, readily bound SBA and mature hyphal walls of germling isolates bound more WGA than other regions of the germlings. Such differential lectin binding by conidia and germlings may influence their specific surface interactions and adherence characteristics.
Mycological Research.
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ABSTRACT: Saprolegnia spp. effectively utilized the protein casein as a sole source of carbon, nitrogen and sulphur, indicating considerable proteolytic activity. In the presence of a more simple carbon source such as glucose, which was readily assimilated, catabolite repression was not observed and casein exploitation was enhanced. Free proteinase activity was not detected by a number of methods, irrespective of culture conditions. However, clearing by mycelia of skimmed milk agar or agar amended with bacteria demonstrated a close association between proteinases and hyphae, suggestive of natural immobilization of proteinase. Casein breakdown was accompanied by release of individual amino acids and ammonia. The latter, indicative of amino acid assimilation and metabolism, was also associated with an increase in pH of culture medium. Single amino acids did not support growth of Saprolegnia but in combination with other amino acids, methionine encouraged greatest biomass production. Certain groupings of amino acids affected growth in a manner which departed from that expected, as assessed by multifactoral analysis of variance, and either enhanced or reduced growth.
Mycological Research.
