Quantitative Organelle Proteomics of Histatin-Treated C. albicans

Salivary histatins are known to exhibit antifungal properties. Several attempts have been made to elucidate the mechanisms operating in fungal cell killing using specifically Candida albicans, a major oral pathogen. While a variety of different antifungal mechanisms have been proposed the precise cascade of events leading to cell death is still not known. Objectives: To study the mechanism of histatin-5-induced cellular alterations in C. albicans at the protein level during cell killing by using quantitative mass spectrometry (MS). Methods: C. albicans cells (strain ATCC 10231) were suspended in 1 mM phosphate buffer, pH 7.0 to a cell density of 5 x 10⁶ cells/mL, and divided into two fractions. Control cells (untreated) and cells treated with histatin 5 (5.9 uM) for 60 min at 37^oC were harvested and then fractionated into mitochondria and cytoplasm containing fractions. Proteins in these subcellular fractions were labeled by isotope-coded-affinity-tags (ICAT), light-ICAT for controls and heavy-ICAT for histatin treated samples. Aliquots containing equal amounts of protein were then mixed, tryptically digested and subjected to nano-flow LC-ESI-MS/MS analysis for protein identification and relative quantitation. Results: The high-throughput MS quantitative proteomic analysis led to the determination of significant alterations at the protein level in both the mitochondrial and the cytoplasmic samples induced by exposure to histatin 5. Histatin treatment led to the down-regulation of mitochondrial proteins predominantly involved in ATP/ADP metabolism, whereas the up-regulated proteins were mostly associated with general respiration. Conclusions: Our work documents large-scale quantitative protein expression differences resulting from the biochemical impact of histatin 5 on C. albicans and has identified critical cellular pathways involved in the histatin killing mechanism. This systems biology approach has the potential to aid in the design and targeting of specific cellular pathways for the generation of novel antifungal agents. Supported by NIH/NIDCR Grants DE05672; DE07652; DE17788 and DE16699.