Killing susceptibility of Candida albicans towards histatin 5 and poly-L-lysine polymers

PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please click Download and log in with a valid BU account to access. If you are the author of this work and would like to make it publicly available, please contact open-help@bu.edu.Thesis (M.S.D.)--Boston University, Henry M. Goldman School of Dental Medicine, 2003.Includes bibliographical references (leaves 104-124).Histatins are a group of small, cationic, histidine-rich proteins secreted by human parotid and submandibular glands. Salivary histatin 5 is a potent antifungal peptide with great promise as a therapeutic agent against Candida albicans. Although the precise mechanism by which histatins kill yeast cells is not known, recent studies showed that histatins bind to the cell surface and are subsequently taken up by yeast cells. Therefore the aim of this study was to determine whether the interaction between histatin 5 and Candida albicans results in damage to the cell envelope and consequent release of intracellular components into the medium. For this purpose the effect of histatin 5 on Candida albicans membrane integrity was compared with the well known effect of poly-L-lysine, which is known to cause yeast cell lysis. Furthermore, the effect of histatin 5 on a respiratory deficient mutant of Candida albicans cells was also tested. Three different experimental approaches were followed to investigate the relationship between killing activity and release of intracellular components. First, a killing assay was performed with histatin 5 (MW 3 kDa) and three different poly-L-lysine peptides (MW 3, 10, 30 kDa) varying either the peptide concentration (0 to 131 nmol/ml) or the incubation time (0 to 90 min). Second, the release of UV-absorbing materials that occurs upon incubation of cells with histatin 5 (33 nmol/ml) was compared with that released upon incubation with three different poly-L-lysine (33 nmol/ml) as monitored by spectrophotometry at 260 nm. The resulting supernatants were also analyzed by SDS-PAGE on 15% gels for protein content. Third, anion-exchange chromatography was employed to determine the nucleotides released from cells into the medium. We found that histatin 5 and poly-L-lysine kill Candida albicans Wild-type cells in a time and concentration dependant manner. During the killing process histatin 5 disappears from the supernatant and intracellular components are released. In contrast, the effect of histatin 5 on the petite mutant #2 of Candida albicans showed little release of intracellular components concomitant with a negligible killing activity. Poly-L-lysine on the other hand exhibited a significant effect on petite mutant viability and release of intracellular components under the same conditions. These results indicate that killing activity correlates with the release of intracellular components and that the mechanism of histatin 5 killing is different from that of poly-L-lysine