|De Lucca Ii, Anthony|
Submitted to: Interscience Conference on Antimicrobial Agents & Chemotherapy Proceedings
Publication Type: Proceedings
Publication Acceptance Date: 6/25/2002
Publication Date: 9/27/2002
Citation: De Lucca II, A.J., Palmgren, M.S., Cleveland, T.E., Walsh, T.J. 2002. Biophysical properties of CAY-1, a fungicidal plant-derived saponin. In: Proceedings of 42nd Interscience Conference on Antimicrobial Agents & Chemotherapy. Abstract F-827. p. 205.
Technical Abstract: Background: CAY-1, a plant saponin, is lethal to several medically and agriculturally important fungi. CAY-1 displays synergy with amphotericin B and itraconzaole against fungi. To further elucidate its properties, we studied pH effect on CAY-1 activity and possible fungal wall and membrane binding sites. Methods: (1) pH effect was determined in buffers (pH 3.0 to 9.4). Germinated conidia (25 ul, 10**5/ml in 1% potato dextrose broth [PDB]) of Aspergillus flavus were added to CAY-1 (225 ul, final concentrations: 0-12.4 ug/ml per buffer type). Controls were germinated conidia (25ul) in the proper buffer (225ul). After incubation (30 min, 30 C) sample aliquots were spread on PDA plates, incubated overnight (30 C) and colonies enumerated. Three replicate experiments were performed (n =18). (2) Ergosterol (ERG) and cholesterol (CHOL) were dissolved in ethanol (1mg/2ml). Pantoea agglomerans lipopolysaccharide (LPS) and glucan (GLU) were prepared in PBS (1mg/ml, pH 7.2). Compounds were diluted in PDB before testing. Diluted ERG or CHOL (200 ul) were added to CAY-1(200 ul, 37.2 ug/ml in PDB) with final molar ratios of 1:1, 2:1, and 4:1 (compound: CAY-1). GLU and LPS were added to CAY-1 to give final weight ratios of 16:1, 33:1, and 66:1 (compound: CAY-1). All mixtures were incubated (15 and 45 min, 30 C). Next, germinated conidia (25 ul) of A. flavus were added to mixture aliquots (225 ul) and 225ul PDB (control). Bioassays were performed as above (n= 12). Results: (1) A significant (p less than or equal to 0.05) decrease in viable colonies occurred at all pH values with CAY-1 greater than 1.24 ug/ml. (2) After 15 min incubation, ERG (4:1 ratio) and all mixtures of CHOL, GLU, and LPS had significantly higher (p less than or equal to 0.05) counts than the CAY-1 control. After 45 min incubation, all mixture ratios were higher (p less than or equal to 0.05) than controls. Conclusions: (1) CAY-1 is fungicidal over a wide pH range and (2) Fungal cell membrane and wall constituents block CAY-1 activity indicating CAY-1 binds to them.