title: Germ tube adhesins of Candida albicans
Creators: name: Unlu, Gulhan Vardar
Publisher: name: University of Glasgow
Year: 1996
Subjects: subject: Microbiology
subject: FOS: Biological sciences

description: The object of the research described in this thesis was to characterize the germ tube adhesins of Candida albicans. Except for a few experiments with a germ tube- deficient mutant and its corresponding wild-type strain, all the experiments were done with C. albicans strain GDH 2346, already extensively used for adhesion studies in this laboratory. From the numerous culture media for germ tube production, described in the literature, three were chosen for comparative study. Medium 199 and Glucose-Glycine Medium at 37°C were highly satisfactory and gave C. albicans 90% conversion of yeast cells to hyphal-form cells. Sucrose-Gelatine medium gave only C. albicans 15% conversion. Temperature and pH also were influential variables: medium 199 at pH 6.7 and 37°C gave germ tubes, while the same medium at 22°C yielded only budding yeast cells. The same medium at pH 4.0 and 37°C likewise gave only yeast cells. The first experiments on adhesins focused on the production and detection of mannoprotein adhesin (MPA) of the hyphal-form cells on plastic petri dishes. This allowed the isolation of MPA and the demonstration that it did not inhibit the adhesion of yeast-form C. albicans to buccal epithelial cells. This observation had not previously been reported in the literature. Using Concanavalin A-coated latex microspheres, MPA was detected on the plastic surface on which the C. albicans produced germ tubes. The adhesins were extracted with dithiothreitol and iodoacetamide treatment. This suggested that MPA of the hyphal-form cells on the plastic was not identical to that on yeast cells which in turn was involved in attachment to epithelial cells. The main part of the research was directed at investigation of the lectin-like adhesins of hyphal-form C. albicans with fluorescent probes. These were neoglycoproteins consisting of sugars (fucose, mannose, glucose, galactose, lactose) convalently linked to BSA, which itself was labelled with fluorescein. Qualitative observations were made by conventional fluorescence microscopy and quantitative observations by fluorescence microscopy with image analysis, and also by spectrofluorimetry and flow cytometry. This is believed to be first study in which all of these methods have been applied to C. albicans. In conventional fluorescence microscopy, the hyphal-form cells bound both the fucose and mannose probes, but the latter only weakly. The fucose probe bound to both the yeast and germ tube portions of hyphal-form cells. Probes with glucose, galactose or lactose did not label the hyphal-form cells. Semi-quantitation of the lectin-like adhesins by fluorescence microscopy with image analysis confirmed the efficient binding of the fucose probe. Germ tube portions of the hyphal-form cells, especially the central region, had higher fluorescence than yeast-cell portions. As before, the mannose probe bound less effectively than the fucose probe, while the glucose, galactose and lactose probes showed little or no binding. Application of spectrofluorimetry to the study of fucose-probe binding allowed the quantitation of lectin-like adhesins during germ tube production to be monitored. Future work in this area should focus on the molecular biology of adhesin molecules on the yeast and hyphal-form cells. The isolation and characterization of lectins may provide information on the possibility of preventing infection by blocking adhesion in humans. Neoglycoproteins could possibly be used as inhibitors to prevent colonization before microorganisms have had the chance to overwhelm the host. (Abstract shortened by ProQuest.).