title: Proton translocating ATPase mediated fungicidal activity of a novel complex carbohydrate: CAN-296.
abstract: CAN-296 is a complex carbohydrate (approximately 4300 Da) isolated from the cell wall of Mucor rouxii. It exhibits excellent in vitro fungicidal activity against a wide spectrum of pathogenic yeasts, including isolates resistant to azoles and polyenes. The rapid irreversible action of CAN-296 on intact fungal cells and protoplasts suggested a membrane-located target for its action. The proton translocating ATPase (H+-ATPase) of fungi is an essential enzyme required for the regulation of intracellular pH and nutrient transport. Inhibition of H+-ATPase leads to intracellular acidification and cell death. We therefore investigated the effect of CAN-296 on H+-ATPase-mediated proton pumping by intact cells of Candida and Saccharomyces species by measuring the glucose-induced acidification of external medium. CAN-296 inhibited proton pumping of Candida albicans, Candida glabrata, Candida krusei, Candida guilliermondii and Saccharomyces cerevisiae at low concentrations (0.078-1.25 mg/l). Other commonly used antifungal agents such as amphotericin B, itraconazole and fluconazole had no effect on H+-ATPase-mediated proton pumping. A clinical isolate of C. glabrata with reduced in vitro susceptibility (MIC = 10 mg/l) to CAN-296 also showed resistance to CAN-296 inhibition of proton pumping. Purified membrane fractions rich in H+-ATPase activity were not inhibited by CAN-296 suggesting that the effect on the H+-ATPase-mediated proton pumping in intact yeast cells is an indirect effect, perhaps mediated by local or global disruption of the plasma membrane. These results suggest that the inhibition of fungal H+-ATPase is at least partly responsible for the antifungal activity of CAN-296.
authors.affiliation: Department of Medicine, Wayne State University, Detroit, MI 48201, USA.
authors.forename: A M
authors.initials: AM
authors.lastname: Ben-Josef
authors.forename: E K
authors.initials: EK
authors.lastname: Manavathu
authors.forename: D
authors.initials: D
authors.lastname: Platt
authors.forename: J D
authors.initials: JD
authors.lastname: Sobel
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000935:Antifungal Agents / Q000494:pharmacology*; D002175:Candida / Q000187:drug effects* / Q000201:enzymology; D002462:Cell Membrane / Q000201:enzymology; D003470:Culture Media; D005944:Glucosamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008826:Microbial Sensitivity Tests; D017494:Proton Pumps / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors*; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000201:enzymology
chemical_list: D000935:Antifungal Agents; C111294:CAN 296; D003470:Culture Media; D017494:Proton Pumps; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D005947:Glucose; D005944:Glucosamine