Investigation of the antifungal activity and mode of action of proline-rich antimicrobial peptides against Cryptococcus neoformans and Cryptococcus gattii

• Applicant: Dr. Alexandra Brakel
• Subject Area: Biological and Biomimetic Chemistry
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 563387543

The incidence of invasive fungal infections has increased significantly over the past decade, resulting in approximately 1.9 million deaths annually. Of particular concern is the emergence of resistance to existing antimycotics, underscoring the urgent need for the development of new antifungal agents. A potent class for this are the proline-rich antimicrobial peptides (PrAMPs), which were originally isolated mainly from insects, antifungal activity shall be investigated in more detail in this project. Their antibacterial activity has already been extensively studied by many methods, including in vivo efficacy and pharmacokinetics, providing a solid foundation for this application. A particular feature is their very low toxicity to mammalian cells, which has been confirmed in several animal models, an aspect that has often been identified as a problem with conventional antifungals. My initial investigations showed, that the PrAMPs exhibited remarkable efficacy against the human-pathogenic fungus Cryptococcus (C.) neoformans, with minimum inhibitory concentrations down to 1.6 µM. Furthermore, confocal laser scanning microscopy and scanning electron microscopy indicated an intracellular mechanism. Based on these results, this application will investigate the mode of action of PrAMPs in C. neoformans and C. gattii. The antifungal activity will be correlated with the peptide-specific properties in order to enable subsequent optimization and to obtain information on the necessity of certain residues. Based on an initial activity screening, suitable lead compounds will be selected for further studies, taking into account time- and concentration-dependent effects. These studies are designed to validate the hypothesis that PrAMPs exhibit an intracellular mode of action without compromising the integrity of the fungal membrane and cell wall, thereby conferring the advantage of non-toxicity to human cells, utilizing established microscopic and flow cytometric techniques. Preliminary data and the analogy to the known and published mode of action in bacteria indicate that the 80S ribosome acts as the main target of PrAMPs. This will be validated in further binding and inhibition studies on isolated ribosomes. Cross-linking studies will identify potential secondary targets. Fungal resistance induced by stepwise transfer of fungi to medium containing higher concentrations of PrAMP may result in resistance promoting mutations that affect membrane structure, reducing PrAMP uptake, or PrAMP-target binding, reducing target inhibition. Gene mutation analysis will be employed to identify the mutation sites, which will allow to better understand with the mode of action and to optimize the peptide structure to reduce the risk of resistance-inducing mutations. This project lays a foundation for understanding the intracellular mode of action of innovative peptide-based antimycotics, enabling future optimizations and detailed in vivo studies.

• DFG Programme: Research Grants