Characterisation of Candida albicans adhesion to epithelial cells

Cameron, Brian J. (1995) Characterisation of Candida albicans adhesion to epithelial cells. PhD thesis, University of Glasgow.

Abstract

The aims of this study were (i) to analyse an adhesin from C. albicans and (ii) to identify the epithelial cell molecule(s) to which the adhesin binds. Extracellular polymeric material (EP), composed mainly of mannoprotein, is thought to originate from the cell surface when yeasts are grown in medium containing a high concentration of galactose. Previous work has shown that this material contains a proteinaceous adhesin. More recently, a complete purification scheme for the yeast adhesin was devised (Tosh and Douglas, 1992) which made use of the findings from earlier studies on adhesion mechanisms. The purification protocol involves the stepwise treatment of EP with N-glycanase, papain, and dilute alkali followed by affinity adsorption with the trisaccharide determinant of the H (type 2) blood group antigen on inert silica beads. Adhesin purified in this way is devoid of carbohydrate, can inhibit adhesion of yeasts to buccal epithelial cells (BEC) by up to 80%, and consists of one major component as determined by reverse-phase fast protein liquid chromatography (FPLC). Purified adhesin was prepared in this study and it was found to inhibit adhesion of C. albicans GDH 2346 cells to BEC by up to 74.81%. EP was subjected to some of the treatments previously found to have an effect on its adhesion-inhibition activity, prior to analysis by reverse-phase high performance liquid chromatography (RP-HPLC). Each of the treatments produced some alteration in the HPLC profile of EP and the combined treatment of EP with all the above reagents (N-glycanase, papain, and dilute alkali) resulted in the production of a number of protein fragments. The fragments appeared to be more hydrophobic than the parent molecule. The purified adhesin was analysed further. Amino acid analysis showed that it was mainly composed of the amino acids Thr, Pro, Ala, Ser, Val, Gly, Asx and Glx, which made up around 80% of the total amino acids. No Cys was detected and there was very little Met, Phe and Arg. This composition is remarkably similar to a large number of microbial adhesins which would appear to indicate a relationship between the amino acid composition of lectin-like proteins and their function as carbohydrate binding molecules. In previous studies, the visualisation of EP and purified adhesin on SDS-PAGE proved difficult. However, in this study, an alternative approach to protein visualisation was employed. Purified adhesin was biotinylated prior to SDS-PAGE and Western blot analysis. The adhesin was then probed for using 125I-streptavidin and, after autoradiography, was seen as a strong band with a molecular mass of approximately 15.7 kDa. Its successful visualisation meant that partial amino acid sequencing could be attempted. The following sequence was obtained; Phe-Asp-Tyr-Glu-His-Val-Asp-X- Ala-Val. There was no complete match found in the protein database but the purified adhesin sequence contains an interesting motif, FDYE, that is found in a wide range of proteins from a variety of sources. The partial amino acid sequence obtained for the adhesin is most probably an internal sequence, produced as a result of enzymic cleavage by the action of papain. The second aim of this study was to identify receptors for C. albicans on buccal epithelial cells. BEC glycolipids were prepared and a glycolipid chromatogram overlay system was employed. This system has been used to characterise a number of microbial adhesin-receptor relationships. Six strains in total were examined for their ability to bind to separated glycolipids. (Abstract shortened by ProQuest.).

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Cellular biology.
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Supervisor's Name: Douglas, Dr. L.J.
• Date of Award: 1995
• Depositing User: Enlighten Team
• Unique ID: glathesis:1995-71532
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 17 May 2019 09:31
• Last Modified: 20 Aug 2021 12:43
• Thesis DOI: 10.5525/gla.thesis.71532
• URI: https://theses.gla.ac.uk/id/eprint/71532