Histatins, a group of
histidine-rich proteins in human saliva, exhibit antimicrobial activity and are therefore considered to be important in the prevention of
infections in the oral cavity. Although killing of C. albicans by
histatins has been extensively studied, little is known about the processes responsible for this antifungal activity. Recent studies show the requirement of metabolic activity and
ATP production for
histatin 5 killing activity. Therefore, the goal of this study was to investigate the kinetics of
histatin 5 interaction at different temperatures with C. albicans wild type cells and with respiratory deficient mutants of C. albicans. Synthetic
histatin 5 was labeled with
fluorescein-5-isothiocyanate (
FITC) and its association with C. albicans cells was followed by epi-fluorescence microscopy and fluorescence confocal microscopy. At 37 degrees C,
histatin 5 accumulates intracellularly, and both killing activity and uptake of unlabeled and
FITC-labeled
histatin 5 are time- and concentration-dependent. At 4 degrees C, no killing is observed and
FITC-
histatin 5 is only associated with the cytoplasmic membrane. Internalization and killing activity only occurs after cells are transferred to 37 degrees C. In addition, cellular accumulation of
histatin 5 is concomitant with a moderate alteration of membrane integrity leading to the release of UV-absorbing cell components into the medium. The uptake of
histatin 5, the release of UV-absorbing materials and killing of C. albicans are markedly decreased by the respiratory inhibitor
sodium azide. Concomitantly, respiratory deficient mutants of C. albicans are also less susceptible to
histatin 5. These results indicated that
histatin 5 killing activity could be directly correlated to
histatin 5 internalization. Both of these processes are prevented by modulators of cellular metabolic activity.