Study of the antifungal mode of action of plant defensins using single cell analysis

Yearly, invasive fungal infections (IFIs), such as candidemia and invasive aspergillosis, cause 1.7 million deaths worldwide and this number is still increasing, which can be mainly explained by the increase of the at-risk population, being immunocompromised patients, and the increased use of modern medical devices, such as implants and catheters. Mortality rates associated with IFIs are high (up to 50%) and the number of currently used antimycotics is limited. Hence, there is a clear need for novel effective antifungal strategies. In this respect, it is of importance to take cellular heterogeneity within fungal populations into account. This heterogeneity can result in a non-responsive subpopulation upon antifungal treatment, which can lead to treatment failure and the recurrence of infections, pointing to the importance of single cell analysis. Therefore, in this PhD thesis, we mainly focused on the usage of single cell technologies to gain a more profound insight in the mode of action of different antifungals.

The first aim in this PhD thesis was to focus on potentiation of currently used antimycotics, as the activity of an antifungal drug can be enhanced by combining it with a non-antifungal, a so-called potentiator. Using an in-house developed digital microfluidic platform, we studied the antifungal mechanism of action of the polyene antifungal amphotericin B (AmB) by analyzing the kinetics of endogenous superoxide radical production and membrane permeabilization in single yeast cells upon treatment. We demonstrated that (i) the nitric oxide donor S-nitrosoglutathione inhibited AmB’s fungicidal action, seemingly functioning as a tolerance mechanism and that (ii) by blocking this pathway using the nitric oxide synthase inhibitor L-NAME, we could increase AmB’s fungicidal activity in Saccharomyces cerevisiae and human pathogens, being Candida albicans and Candida glabrata. Hence, potentiation of AmB resulted in a faster antifungal action and lower required AmB doses.

The second aim of this PhD thesis was to focus on the use of specific plant antimicrobial peptides (AMPs), being plant defensins, which have a different mode of action as compared to currently used antimycotics. Plant defensins have a broad-spectrum antifungal activity, are generally not toxic to human cells and interact with specific fungal targets. At first, we further unravelled the antifungal mode of action of the plant defensin HsAFP1 (Heuchera sanguinea) in bulk. Via transcriptome analysis of HsAFP1-treated C. albicans cells and additional genetic and biochemical tests, we observed that glycosylphosphatidylinositol (GPI)-anchored proteins are involved in HsAFP1's internalization, which is required for its antifungal activity. In addition, we demonstrated that moderate HsAFP1 doses result in autophagy, whereas high doses induce vacuolar dysfunction. We then investigated HsAFP1’s antifungal mode of action at a single cell level using an in-house developed continuous microfluidic platform. We examined the interdependency of reactive oxygen species induction and membrane permeabilization in single yeast cells over time, but within a time frame of 2 minutes, almost all evaluated cells displayed no time between both events, pointing to highly dynamic and correlated processes.

In conclusion, we used two single cell platforms, being a digital microfluidic platform and a continuous microfluidic platform to investigate different antifungal treatment strategies at a higher resolution. Hereby, we gained a more profound insight in fungal killing strategies upon potentiation of currently used antimycotics and using plant defensins, which can be of great interest for the development of novel antifungal drugs.