Proton translocating ATPase mediated fungicidal activity of a novel complex carbohydrate: CAN-296.

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.