Involvement of calcium inhibitable binding to the cell wall in the fungicidal activity of CAN-296

CAN-296 is a heat stable, complex carbohydrate (molecular mass 4300 Ca) iso lated from the cell wall of the filamentous fungus Mucor rouxii. It possess es potent in-vitro fungicidal activity against a wide spectrum of pathogeni c yeasts, including azole-resistant isolates of Candida albicans and Candid a glabrata. As a preliminary step in the study of the mode of action of thi s novel antifungal agent, we investigated the effect of various cations on the antifungal activity as well as the binding of CAN-296 to intact cells a nd cell-wall fractions of C.albicans. The antifungal activity of CAN-296 wa s inhibited by low concentrations of calcium, magnesium and lithium and by high concentrations of barium, cobalt and manganese, but not by potassium a nd copper. The calcium-mediated inhibition of the antifungal activity of CA N-296 was readily reversible by the removal of calcium by dialysis, and the fungicidal activity of the inhibited compound was fully restored. The upta ke/binding of CAN-296 to intact cells and to the cell-wall fraction of C. a lbicans was time and concentration dependent. Maximum uptake/binding was ob tained at 5 mg/L within 60 min and was associated with the aggregation of i ntact cells. Washing intact cells and the cell-wall fraction preincubated w ith radiolabelled CAN-296 with 150-fold excess of unlabelled compound faile d to remove CAN-296 associated with the intact cells and the cell-wall frac tion, suggesting that the binding of CAN-296 to C. albicans is tight. The u ptake/binding of CAN-296 and the drug-mediated aggregation of intact cells were inhibited by calcium in a concentration-dependent manner. The fact tha t CAN-296 is a fungicidal agent that binds to intact cells and the cell-wal l fraction of C. albicans very tightly, together with the observation that calcium was able to inhibit the fungicidal activity as well as the uptake/ binding of CAN-296, suggests that the mode of action of this novel antifung al agent may involve interaction with the cell wall of C. albicans.