Fungal infections caused by Candida species, particularly C. albicans and C. glabrata, have become a serious threat to public health. The rising drug resistance has prevented effective treatment and increased the mortal rate. Novel approaches to improve the therapeutic effects of antifungal agents and allow delivery of agents that are not normally cell-permeable are in demand.

In order to improve the intracellular delivery of antifungal agents, we have investigated using cell-penetrating peptides as drug carriers for treating fungal infections. CPPs have been widely studied as tools for delivering a variety of molecular cargo into cells, including DNA, RNA, proteins, and nanoparticles. Previous work with CPPs has mainly focused on their uptake in mammalian cells, but CPPs also have potential as drug delivery and research tools in other organisms, including Candida pathogens.

We have explored various well-studied CPPs to identify peptides that retain their translocation capability with Candida cells, including pVEC, penetratin, MAP, MPG, SynB, TP-10 and cecropin B. The CPPs pVEC, penetratin, MAP and cecropin B show a higher level in the cytosol adopt direct translocation mechanisms and exhibit toxicity towards C. albicans. Our peptide localization and mechanistic studies allow better understanding of the mode of translocation for different CPPs, which is related to the potential toxicity towards Candida pathogens.

To further understand the molecular mechanisms of translocation of CPP, we investigated the biophysical properties of the peptides. CPPs that previously were shown to use direct translocation mechanisms (pVEC, MAP, and cecropin B) exhibit helical conformations upon interaction with cells due to the hydrophobic interaction with the core of bilayers. Membrane associations of peptides that entered cells via endocytosis were controlled by electrostatic forces. Our novel structure characterization methods using circular dichroism with live fungal cells, along with Monte Carlo simulations, allow us to understand how CPPs interact with cell membranes and how the membrane association affects the translocation mechanisms.

After beginning to understand the structure-function relationships of CPPs, we engineered two CPPs, pVEC and SynB, to enable better translocation efficacy and manipulation of translocation mechanisms. We tuned the properties of the peptides, including the net charge and the hydrophobicity, to alter intracellular fates and the level of antifungal activity. These results are promising and motivate better peptide engineering for specific purposes.

Our work with CPPs and fungal pathogens contributes to the understanding of structure-function relationship of CPPs in Candida species. We have provided the foundation for further peptide engineering and explorations into applications of CPPs in treating fungal infections.