Candida albicans mutants deficient in respiration are resistant to the small cationic salivary antimicrobial peptide histatin 5

Histatins are a group of small cationic peptides in human saliva which are well known for their antibacterial and antifungal activities. In a previous study we demonstrated that histatin 5 kills both blastoconidia and perm tu bes of Candida albicans in a time- and concentration-dependent manner at 37 degrees C, whereas no killing was detected at 4 degrees C. This indicated that killing activity depends on cellular energy. To test histatin 5 killin g activity at lower cellular ATP levels at 37 degrees C, respiratory mutant s, or so-called petite mutants, of C. albicans were prepared. These mutants are deficient in respiration due to mutations in mitochondrial DNA, Mutant s were initially identified by their small colony size and were further cha racterized with respect to colony morphology, growth characteristics, respi ratory activity, and cytochrome spectra. The killing activity of histatin 5 at the highest concentration was only 28 to 30% against respiratory mutant s, whereas 98% of the wild-type cells were killed. Furthermore, histatin 5 killing activity was also tested on wild-type cells in the presence of the respiratory inhibitor sodium azide or, alternatively, the uncoupler carbony l cyanide m-chlorophenylhydrazone. In both cases histatin 5 killing activit y was significantly reduced. Additionally, supernatants and pellets of cell s incubated with histatin 5 in the presence or absence of inhibitors of mit ochondrial ATP synthesis were analyzed by sodium dodecyl sulfate gel electr ophoresis. II was observed that wild-type cells accumulated large amounts o f histatin 5, while wild-type cells treated with inhibitors or petite mutan ts did not accumulate significant amounts of the peptide. These data showed first that cellular accumulation of histatin 5 is necessary for killing ac tivity and second that accumulation of histatin 5 depends on the availabili ty of cellular energy. Therefore, mitochondrial ATP synthesis is required f or effective killing activity of histatin 5.