IADR Colgate Student Poster Competition - 4

Mode of action of a novel antifungal

J KOH, K NIIMI, BC MONK
School of Dentistry, University of Otago, Dunedin, New Zealand

New classes of antifungal agent are needed to combat fungal infections and to overcome resistance to the azole drugs. The D-decapeptide BM2 was previously found to be an antifungal inhibitor of the plasma membrane proton pump Pma1p, an essential enzyme in Candida albicans, other pathogenic fungi and the model yeast S.cerevisiae. In order to understand the mode of action of BM2, spontaneous resistant variants of the S. cerevisiae strain T48 were obtained and analysed. Methods. Disk diffusion assays and liquid MIC tests were used to assess the sensitivities of cells to BM2. Plasma membranes were isolated by differential centrifugation of cell lysates from diauxic phase cells in the presence of 2% glucose and by selective removal of contaminating membranes by acid precipitation at pH 5.1. Plasma membrane polypeptide content was assessed by polyacrylamide gel electrophoresis. The ATPase activity of Pma1p, measured in the presence of 5 mM Mg-ATP in a microplate assay in the pH range 5-8.5, was sensitive to 100µM vanadate. Results. Four spontaneous T48/BM2R variants retained their BM2 resistance phenotype in disk diffusion assays and were 4-fold less susceptible compared with T48 in liquid MIC tests. The 100 kDa coomassie-stained Pma1p band of plasma membranes was dramatically reduced in a representative T48/BM2R variant compared with T48. Two additional bands of higher molecular weight were found in the variant strain only. At the optimal pH of 6.5 the ATPase activity of Pma1p in the variant was reduced at least four-fold compared with the T48 and the mutant membranes showed an optimal vanadate sensitive ATPase activity at pH 5.5. Conclusions.The modest (4-fold) resistance and molecular phenotype of the T48/BM2R variant confirm that BM2 targets Pma1p and suggest that the spontaneous mutation(s) affecting the binding of BM2 is functionally related to a component of Pma1p that responds to glucose-dependent phosphoryation.  JK was supported by a summer studentship from the Oral Microbiology and Dental Health Theme.