The human salivary peptide histatin 5 exerts its antifungal activity through the formation of reactive oxygen species

Previous studies have shown that the human salivary antifungal peptide hist atin 5 is taken up by Candida albicans cells and associates intracellularly with mitochondria. The purpose of the present study was to investigate the biological consequence of this specific subcellular targeting. Histatin 5 inhibited respiration of isolated C. albicans mitochondria as well as the r espiration of intact blastoconidia in a dose and time-dependent manner. A n early perfect correlation was observed between histatin-induced inhibition of respiration and cell killing with either logarithmic- or stationary-phas e cells, but stationary-phase cells were less sensitive. Because nonrespiri ng yeast cells are insensitive to histatin 5, the potential mechanistic rel ationship between histatin 5 interference with the respiratory apparatus an d cell killing was explored by using an oxygen radical sensitive probe (dih ydroethidium). Fluorimetric measurements showed that histatin 5 induced the formation of reactive oxygen species (ROS) in C. albicans cells as well as in isolated mitochondria and that ROS levels were highly correlated with c ell death. In the presence of an oxygen scavenger (L-cysteine), cell killin g and ROS formation were prevented. to addition, the membrane-permeant supe roxide dismutase mimetic 2,2,6,6-tetramethylpiperidine-N-oxyl, abolished hi statin-induced ROS formation in isolated mitochondria. In contrast to hista tin 5, the conventional inhibitors of the respiratory chain, sodium cyanide or sodium azide, neither induced ROS nor killed yeast cells. These data pr ovide strong evidence for a comprehensive mechanistic model of histatin-5-p rovoked yeast cell death in which oxygen radical formation is the ultimate and essential step.