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 activ ity against a wide spectrum of pathogenic yeasts, including isolates resist ant to azoles and polyenes. The rapid irreversible action of CAN-296 on int act fungal cells and protoplasts suggested a membrane-located target for it s action. The proton translocating ATPase (H+-ATPase) of fungi is an essent ial enzyme required for the regulation of intracellular pH and nutrient tra nsport. Inhibition of H+-ATPase leads to intracellular acidification and ce ll death. We therefore investigated the effect of CAN-296 on H+-ATPase-medi ated proton pumping by intact cells of Candida and Saccharomyces species by measuring the glucose-induced acidification of external medium. CAN-296 in hibited proton pumping of Candida albicans, Candida glabrata, Candida kruse i, Candida guilliermondii and Saccharomyces cerevisiae at low concentration s (0.075-1.25 mg/l). Other commonly used antifungal agents such as amphoter icin B, itraconazole and fluconazole had no effect on H+-ATPase-mediated pr oton primping. A clinical isolate of C. glabrata with reduced in vitro susc eptibility (MIC = 10 mg/l) to CAN-296 also showed resistance to CAN-296 inh ibition of proton pumping. Purified membrane fractions rich in H+-ATPase ac tivity were not inhibited by CAN-296 suggesting that the effect on the H+-A TPase-mediated proton pumping in intact yeast cells is an indirect effect, perhaps mediated by local or global disruption of the plasma membrane. Thes e results suggest that the inhibition of fungal Ht-ATPase is at least partl y responsible for the antifungal activity of CAN-296. (C) 2000 Elsevier Sci ence B.V. and International Society of Chemotherapy. All rights reserved.