Cell-penetrating peptides (CPPs) are a class of small peptides with the ability to cross membranes and carry biomolecules with them. We are exploring CPPs as vehicles for delivering biomolecular cargo into the opportunistic fungal pathogen, Candida albicans. We recombinantly produced the CPPs histatin 5 (Hst5) and MPG as fusions to green fluorescent protein (GFP) to explore the intracellular delivery of GFP into C. albicans using CPPs. MPG is a short amphipathic CPP that inserts readily into natural membranes. Hst5, which is found in human saliva, is a histidine-rich antimicrobial peptide that must cross the cell membrane to exert its fungicidal activity. We studied CPP-mediated translocation of GFP using flow cytometry to detect GFP inside cells. Fusion to MPG led to translocation of GFP into 38% of C. albicans cells, which was significantly higher than the 13% cells that exhibit translocation of GFP without a CPP (p ≤ 0.001). In contrast, Hst5 did not translocate GFP into cells, with only 5% of cells showing Hst5-GFP translocation. Our results show that MPG can be used to deliver GFP, while Hst5 is not as promising. To better understand the translocation of MPG and Hst5, we used molecular dynamics simulations to study their interaction with C. albicans cell membranes. Our simulation studies showed that MPG enters the membrane model readily, with the N-terminal residues initiating the entry. In contrast and consistent with experimental results, Hst5 fails to enter the membrane even after 400 ns. Using knowledge gained from simulations, we designed MPG variants with amino acid substitutions to improve translocation into C. albicans cells and are currently testing the variants for enhanced translocation efficiency. Our results highlight the utility of combining experiments and simulations to study the translocation of CPPs and to engineer CPPs for improved delivery of cargo into C. albicans cells.

Studying the transport of CPP-cargo protein fusions into Candida albicans using experiments and simulations

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.