AbstractKiller yeasts secrete toxins which are lethal or inhibitory to sensitive yeasts but to which they themselves are immune. Williopsis mrakii is a killer yeast but very little is known about the physiology of the organism and the characteristics of its toxin. This research was concerned with an investigation into killer toxin production by W.mrakii, purification of the killer factor and its action against medically important Candida species.
A total of 30 presumptive killer yeast strains were tested in vitro for killing activity against a range of sensitive yeast strains, including clinical isolates of Candida, using a standard agar diffusion bioassay. Several yeasts, of the genus Pichia, showed widespread activity against the sensitive strains assayed but W.mrakii was demonstrated as showing the greatest anti-Candida activity.
Crude W.mrakii killer toxin showed a differential killing action against strains of Candida isolated from clinical specimens. No direct correlation between the site of isolation and susceptibility to the killer toxin was found. The status of the host and species-type would also contribute to the toxin-receptor interaction. It was found that at critical concentrations the killer factor of W.mrakii exerted a greater effect on stationary phase cells than cells from an exponential phase of growth. At low concentrations, the killer toxin produced a fungistatic effect on sensitive yeasts but at higher concentrations there was evidence to suggest that membrane damage accounted for the fungicidal effects of the killer factor. The cidal nature of the toxin was reflected in a rapid decrease in sensitive cell viability.
W.mrakii is a ‘microaerophilic’ yeast with simple nutritional requirements. In a gently agitated system the killer factor was produced in a minimal medium as a consequence of cell growth and its activity reached constant levels as the cells entered stationary phase. Toxin activity was lost at several stages during processing of the culture supernatants because of membrane-binding and losses to the ultrafiltrate. PhastSystem analysis and gel filtration chromatography suggested that the active toxin molecule was an acidic polypeptide with a molecular weight of 1800-5000 Da. Rapid purification of the toxin using FPLC techniques was hampered by problems in accurately assessing toxin activity in the presence of high salt concentrations. This was overcome by the development of a novel colourimetric assay of toxin activity.
|Date of Award||Aug 1993|
|Sponsors||Glaxo Group Research|
|Supervisor||Graeme Walker (Supervisor)|