(683e) Investigation of the Cellular Uptake and Cytotoxicity of Cell-Penetrating Peptides in Candida Fungal Pathogens

Cell-penetrating peptides (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 fungi. We have investigated 12 different peptides for their ability to function as CPPs for the important human fungal pathogens Candida albicans and C. glabrata. The peptides were commercially synthesized with an N-terminal 5-carboxyfluorescein label serving as small-molecule cargo and as a fluorescent probe to report cellular uptake. We have used flow cytometry to screen the CPPs for their potential to efficiently enter the Candida cells. Among the 12 CPPs, we have identified several promising CPPs, including pVEC and SynB, that can enter both C. albicans and C. glabrata cells, and the overall uptake process depends on both the salt concentration in the buffer and on the concentration of the peptides. We have also identified CPPs with some specificity; for example, Hst-5 can only translocate into C. albicans, whereas TP-10 has higher uptake in C. glabrata. We found that generally the CPPs with higher uptake also have higher toxicity toward both Candida species, but SynB showed almost no antimicrobial activity at a concentration where nearly 100% of cells have high levels of peptide uptake. Furthermore, we explored the subcellular localization of CPPs at different concentrations with fluorescent microscopy and flow cytometry. Most of the promising CPPs showed a 3-stage behavior, where CPPs evenly distributed in the cytosol at high concentrations, localized inside vacuoles at moderate concentrations, and gathered around the membranes at lower concentration. Cecropin B, in contrast, shows â??all-or-nothingâ? behavior, with the peptide either present in the cytosol or exhibiting no apparent association or uptake. In addition, we have also investigated the uptake at different temperatures to aid understanding of the mechanism of uptake for the peptides. Some peptides (e.g., MPG and (KFF)₃K) are significantly affected by temperature, indicating a potential ATP-dependent uptake mechanism, whereas temperature has minimal impact on other peptides (e.g., pVEC and Cecropin B). Based on these results, we are now exploring the structure-function relationship for translocation of CPPs into fungal cells.