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Title: Siderophore and pigment production by Candida albicans
Author: Altabet, Altaher Ibrahim
ISNI:       0000 0001 3418 5515
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1997
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The aim of this project was to study siderophore and pigment production by yeast and hyphal forms of Candida albicans. The ability to form true hyphal cells is virtually unique to C. albicans and the first objective was to identify the best liquid medium capable of yielding pure yeasts or pure hyphae for use in this study. Seven liquid media were tested and overall the results demonstrated that the optimal growth conditions for hyphae are high temperature (37°C) and neutral pH. On the other hand, pure-yeast form cultures were obtained by growing at a lower temperature (25°C). Three media (glucose glycine broth, NYP medium and Lee's medium) gave a high percentage of hyphae or yeasts by simply a change in incubation temperature. One mechanism of iron acquisition is the secretion of ferric-specific chelators termed siderophores, whose function is to solubilize and transport iron into the cell. Generally, two types of siderophores exist: the phenolate and hydroxamate types. In this study, four strains of C albicans were examined for the production of siderophores after growth in glucose glycine broth at 37 °C (for hyphae) or 25°C (for yeasts). Siderophore production in both liquid and solid media was determined by the universal chemical assay for siderophores. This assay utilises a dye complex of Chrome azurol S (CAS) and hexadecyltrimethyl ammonium bromide (HDTMA) that has a high affinity for iron. The iron-dye complex is blue with an absorption maximum at 630 nm. When a strong chelator removes the iron from the dye, its colour turns from blue to red. The dye was incorporated into yeast nitrogen base agar to detect siderophore production via the formation of pink zones around Candida colonies. Specific assays for phenolate- or hydroxamate-type siderophores were also used. Phenolate compounds were determined by the Amow method with catechol as a positive control. Hydroxamates were detected by the method of Holzberg and Artis (1983) with desferal as a positive control. The results showed that siderophores are produced by both yeast and hyphal forms of C. albicans. When siderophore production was considered in relation to cell dry weight, secretion by hyphal forms was greater than that of yeast forms. Only hydroxamate-type chelators were found; phenolate-type siderophores were not detected. Green pigment production by C. albicans on plates of blood agar was first observed by Jones and Peck in 1940. A later report (McCourtie and Douglas, 1985) indicated that the pigment was produced by C. albicans during prolonged incubation in medium containing 500 mM galactose. Subsequently, Sweet and Douglas (1991) showed that green pigment synthesis was regulated by the availability of iron. In this study, green pigment production by strains of C. albicans was affected by various environmental factors. All strains tested produced green pigment; however, the amount produced varied from strain to strain. Green pigment synthesis was affected by factors such as carbon source, and increased when galactose was used instead of glucose. However, glucose induced pigment synthesis under conditions of iron limitation whereas galactose did not. Pigment production was also increased at a growth temperature of 37°C as compared with 25 or 30°C; by yeast morphology rather than hyphal morphology; by prolonged incubation periods; and finally, by shaking rather than static growth conditions. All strains inoculated on to Sabouraud dextrose blood agar showed a very dark grey zone around the colonies after incubation at 37°C for 24h. Green pigment production was not induced by growing C. albicans in medium with low concentrations of phosphate , magnesium, manganese, zinc and copper, in contrast to the induction seen with a low concentration of iron. The highest pigment production was noted when the organism was grown at normal concentrations of these components with either carbon source (glucose or galactose). Generally, very low pigment production was observed in media which had no added phosphorus, magnesium, manganese, zinc and copper and which therefore contained only traces of these elements associated with the other chemicals which comprise yeast nitrogen base. When cultured in a chemically defined medium having tryptophan as a major nitrogen source, C. albicans 'Outbreak' strain produced a pink pigment; this was only noted with media containing iron. No pigment was observed with media containing only proline as a nitrogen source even in the presence of exogenous iron. These results indicate that tryptophan is the constituent responsible for the production of a pink pigment and that iron is necessary for pigment formation. The two pigments (green and pink) are quite different from each other. The pink pigment consists of an indole derivative whereas the green pigment comprises two unusual fluorescent materials which give it the yellow-greenish colour. The green pigment shows absorption at 360 nm and 440 nm, but no absorption at 520 nm. Exposure to ordinary light had a marked effect on the green pigment, which is unstable and becomes colourless after few days. Exposure of the pink pigment to light for four months caused a change in colour eventually to light orange. The addition of either acid or alkali had no effect on the green pigment in culture supernates which had an initial pH value of 2.29; the yellow-greenish colour was unchanged. On the other hand, a substantial effect was observed with the pink pigment in culture supernates with an initial pH 3.5 ; the pink pigment was sensitive to alkali and on addition of sodium hydroxide it converted to a light orange, returning to pink with the addition of acid Fluorescence spectra of green and pink pigments confirmed that the green pigment consists of two components, one yellow and the other blue; the yellow component fluoresced at 520 nm (in the middle of the green, not yellow, colour zone) and the blue component fluoresced at 440 nm. The fluorescence spectra of the green and pink pigments showed no similarities. There were also no similarities between the spectra of those of chemicals which comprise YNB medium.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Candidiosis; Siderphores; Yeast