Delivery of anti-fungal dsRNA into yeast and filamentous fungi using Laser-Activated Nanoparticles - This multidisciplinary research project will develop a new method to deliver antifungal agents to medically relevant fungi using ultrashort laser pulses and nanoparticles. Our long-term goal is to advance new approaches to treat fungal infections, particularly of the skin and mucous membranes, caused by multi-drug resistant yeasts such as Candida auris or recalcitrant dermatophytes such as Trichophyton rubrum. The objective of this proposal is to develop double-stranded RNA (dsRNA) as an antifungal agent; where the dsRNA will target and inhibit the translation of essential fungal genes leading to cell death. However, dsRNA cannot easily penetrate the fungal cell wall, which is composed of chitin, glucans, mannans, and glycoproteins. The central hypothesis, which was formulated on the basis of our own preliminary data, is that we can generate transient breaks in the fungal cell wall using cavitation and shock waves that result from irradiating gold nanoparticles (AuNPs) with infrared femtosecond (fs) laser pulses. The rationale is that suppression of these essential genes would lead to decreased growth and/or viability of the fungi. At the same time, there is no known mechanism by which fungal cells could develop resistance against dsRNA. The objective of the proposed research will be achieved by three independent specific aims: 1. Delivery of lethal dsRNA through photoporation via Laser-Activated Nanoparticles (LANPs) in C. albicans and T. rubrum; 2. Evaluation of the effect of nanoparticle size and composition on photoporation via LANPs; and 3. Evaluation of LANP toxicity in human keratinocytes, and effectiveness in inhibiting fungal biofilms. This proposal is innovative because it uses an entirely different approach to selectively impede fungal cells by using (a) dsRNA as an antifungal agent and (b) laser activated nanoparticles to facilitate the intracellular delivery of dsRNA. This research is significant because it can lead to the development of new strategies to treat oral, vaginal and skin mycoses. This method can also become a new laboratory tool for the delivery of DNA, dsRNA, or CRISPR/Cas9 gene editing systems to fungal cells, and can be widely used in different biotechnology areas to study and modulate gene function. We will also validate essential genes in C. albicans using RNAi, which has been barely explored in budding yeast. Finally, the multifaceted nature of this research will foster creativity and encourage student collaboration within different disciplines.
