Werkingsmechanismen van antifungale componenten: Antibiofilm en ionkanaal inhiberende eigenschappen van plantdefensinen, en 'single cell' analyse van amfotericine B geïnduceerde oxidatieve stress

摘要

Invasive fungal infections (IFIs), such as candidemia and invasive aspergillosis, are a major threat to immunocompromised patients and patients with respiratory disorders. IFIs are associated with high morbidity and mortality rates, and are mainly caused by Candida spp. The latter are increasingly associated with medical device-related infections, as they can form biofilms on the surface of for instance catheters, orthopaedic implants and implantable electronic devices (such as cardiovascular pacemakers). C. albicans biofilm cells are resistant towards a broad range of antifungal drugs and current treatment options for fungal biofilm-related infections are limited. To date, only miconazole, caspofungin, anidulafungin and liposomal formulations of amphotericin B are effective against these biofilms. Moreover, treatment with these antifungal drugs can result in severe side effects, including hepatotoxicity and nephrotoxicity. Hence, there is a need for novel antifungal treatment options.Antimicrobial peptides (AMPs) are of great interest in the search for novel therapeutics, as their multiple modes of action reduce the ability of microorganisms to develop resistance. Moreover, antifungal AMPs are characterized by fungicidal activity and induce rapid killing of a range of microorganisms. One particular family of such AMPs are plant defensins. Plant defensins are in general nontoxic to human cells, as they specifically target fungal membrane compounds, and were shown to induce production of reactive oxygen species and apoptosis in C. albicans, Saccharomyces cerevisiae and/or other fungi.A first aim in this research project was to further elucidate the mechanisms of action of three antifungal plant defensins, i.e. HsAFP1, RsAFP2 and AtPDF2.3, using C. albicans and S. cerevisiae as models. Insight in the mechanisms of action of these defensins and elucidation of the tolerance mechanisms used by fungal pathogens to resist their action will improve our understanding on how to effectively kill these pathogens. In this work, the antibiofilm potential of HsAFP1 and RsAFP2 was investigated towards C. albicans biofilms. Both HsAFP1 and RsAFP2 can prevent formation of C. albicans biofilms, however, they cannot eradicate them. Further structure-activity relationship analysis of HsAFP1 revealed the importance of the γ-core and adjacent regions in antibiofilm activity. This is the first report on plant defensins possessing antibiofilm activity. Next, the ion channel inhibitory activity of AtPDF2.3 was investigated, as in silico analysis revealed that the AtPDF2.3 amino acid sequence carries a partial toxin signature. The latter was previously assigned to scorpion toxins active on ion channels. Electrophysiological recordings indicated that this plant defensin blocks potassium channels in a similar way as scorpion toxins. In S. cerevisiae, AtPDF2.3 antifungal action triggers activation of a tolerance mechanism involving potassium transport and/or homeostasis. As such, a link was found between potassium channel inhibitory activity and antifungal activity involving potassium transport and/or homeostasis.The second aim of this project was to further unravel the mechanism of action of the conventional antifungal drug amphotericin B, and as such improve our understanding of amphotericin B-induced killing. Amphotericin B interacts with ergosterol in the fungal membrane, which ultimately leads to fungal cell death. However, information on amphotericin B-induced events leading to fungal cell death is very limited. Several reports already indicated the importance of analysing cellular heterogeneity with respect to dynamic cell responses towards various stimuli, which hinted us to also investigate amphotericin B-induced events with spatiotemporal resolution. To this end, a novel digital microfluidic (DMF) platform for single cell analysis of S. cerevisiae cells was developed and implemented. This device allows for monitoring cells with spatiotemporal resolution, which is not possible using bulk methods. In a first instance, a proof of concept for using the DMF platform to monitor membrane permeabilization in yeast cells during antifungal treatment with amphotericin B was designed. The device was validated by comparing results obtained on the DMF platform with those obtained in bulk by flow cytometry. Similar results were found for both experimental designs, and therefore, the DMF device was found suitable for single cell analysis of yeast cells during antifungal treatment. Further research focused on investigating the role of superoxide and nitric oxide radicals in amphotericin B’s fungicidal action. Superoxide radicals were found to be important in amphotericin B’s fungicidal action, whereas nitric oxide radicals seem to mediate a tolerance mechanism toward this agent. In addition, a detailed kinetic study revealed that inhibition of nitric oxide radical production increases and accelerates superoxide radical production, membrane permeabilization and loss of reproduction capacity in yeast.In summary, the results of this doctoral research contributed to a better understanding of the mechanisms of action of and the tolerance mechanisms to HsAFP1, RsAFP2, AtPDF2.3 and amphotericin B. For the first time, plant defensins were reported to have antibiofilm activity, thereby expanding the knowledge on plant defensin biological activities. Furthermore, this thesis describes plant defensins as potential novel antifungal lead molecule for further development into novel antifungal and/or antibiofilm drugs to combat fungal infections. In addition, it demonstrates the use of a novel DMF platform in the identification of compounds’ mechanisms of action and characterization of potential synergistic interactions.