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Title: Analyse van tolerantiemechanismen van biofilm-gistcellen aan antifungale componenten en de in vivo relevantie hiervan.
Other Titles: Analysis of tolerance mechanisms of biofilm yeast cells for antifugal compounds and the in vivo relevance of these mechanisms
Authors: Bink, Anna; S0108119
Issue Date: 3-May-2012
Abstract: Among the pathogenic fungi, Candida albicans is most frequently associated with biofilm formation and is a major cause of device-related infections in most nosocomial diseases. Those infections are particularly serious because biofilm-associated Candida cells are resistant to a wide spectrum of antifungal drugs and the current treatment options for fungal biofilm-related infections are very scarce. The basis of this drug resistance is not clear but could be due to a combined action of different mechanisms including (i) expression of resistance genes, (ii) drug binding to the extracellular matrix, (iii) the change in membrane composition or (iv) the presence of persister cells (cells that can survive high doses of an antimicrobial agent). Therefore, it is necessary to search for new approaches for the treatment of these biofilm-related infections.In this research project, the major aim was to gain more insight into tolerance mechanisms of the human pathogen C. albicans to antifungal compounds, with a focus on biofilm cells, and translation of this information to the in vivo situation. Knowledge of tolerance mechanisms of yeast cells to antifungal compounds is of great importance for antifungal therapy: the activity of an antifungal compound can potentially be increased by the use of the compound in combination with an inhibitor of its tolerance mechanism. The first part of this work was focused on unraveling the mode of action and tolerance mechanisms of various antifungals against planktonic yeast cultures. More specifically, the mode of action and tolerance mechanisms of planktonic Saccharomyces cerevisiae cells to miconazole and a piperazine-1 carboxamidine derivative BAR0329 was unraveled. Miconazole and BAR0329 induce the formation of reactive oxygen species (ROS) in susceptible yeast species and are active against C. albicans planktonic and biofilm cells. Intracellular accumulation of both antifungal compounds in S. cerevisiae is dependent on functional lipid rafts. Moreover, we could demonstrate that BAR0329 induces caspase- and mitochondrial fission-dependent apoptosis in yeast. The second part of this work was dedicated toward gaining more insight in the mode of action and tolerance mechanisms of yeast biofilm cells to conventional ROS-inducing antimycotics like miconazole, amphotericin B and caspofungin. This research lead to the findings that superoxide dismutases (SODs) play an important role in the persistence and tolerance of C. albicans biofilms to miconazole and amphotericin B. This opens up the possibility for novel anti-biofilm therapy, consisting of the combination of a ROS-inducing antifungal with specific SOD inhibitors. Further investigation revealed that the transcription factor Efg1, which is a central regulator of numerous cellular processes in C. albicans, is involved in tolerance mechanisms of C. albicans biofilms to miconazole, amphotericin B and caspofungin. Phenocopying the EFG1 deletion bydiclofenac treatment confirmed the role of Efg1 in tolerance mechanisms of C. albicans biofilms to caspofungin. In summary, the results of this doctoral research contributed to a better fundamental understanding of the tolerance mechanisms of planktonic and biofilm yeast cells against different classes of antifungals. The rational design of specific inhibitors of cellular determinants (like Efg1 and SODs) underlying biofilm tolerance to conventional antimycotics could lead to novel anti-biofilm therapy combining an antifungal compound with such inhibitor. In this respect, the in vivo data of this research project provides clear evidence that modulation of the activity of biofilm tolerance determinants, e.g. via diclofenac, is useful in combination therapy with antifungals like caspofungin to treat C. albicans biofilm-associated infections.
Table of Contents: Table of contents

Een woord vooraf I
Abstract V
Samenvatting VII
List of abbreviations IX
Table of contents XI
Chapter 1 1.1 Outline 1
1.2 References 6
Chapter 2 Anti-biofilm strategies: How to eradicate Candida biofilms? 9
Additional information: overview of (currently) used antifungal agents against systemic 20
mycoses
Chapter 3 Membrane rafts are involved in intracellular miconazole accumulation in yeast cells 29
Additional information: genes that result in miconazole resistance upon deletion in 37
S. cerevisiae
Chapter 4 The mode of fungicidal action of the piperazine-1-carboxamidine BAR0329 45
Additional data: BAR0329 induces ROS and cell death in C. albicans biofilms 53
Supporting information 55
Chapter 5 Superoxide dismutases are involved in Candida albicans biofilm persistence against 59
miconazole
Additional information: functions of the six SOD isoenzymes in C. albicans 65
Chapter 6 Potentiation of anti-biofilm activity of amphotericin B by superoxide dismutase inhibition 69
Chapter 7 Efg1 contributes to tolerance of Candida albicans biofilms against antifungal agents in vitro 75
and in vivo
Chapter 8 The non-steroidal anti-inflammatory drug diclofenac potentiates the in vivo activity of 87
caspofungin against Candida albicans biofilms
Chapter 9 Conclusions and outlook 99
9.1 General conclusions 100
9.1.1 Mode of fungicidal action of miconazole against yeast (Chapter 3) 100
9.1.1.1 Induction of actin stabilization 101
9.1.1.2 Lipid rafts 103

9.1.2 Mode of fungicidal action of the piperazine-1-carboxamidine BAR0329 105
(Chapter 4)
9.1.2.1 BAR0329 induces caspase- and mitochondrial fission-dependent 106
apoptosis in yeast.
9.1.2.2 Anti-biofilm activity of BAR0329 against C. albicans 107
9.1.3 SODs involved in miconazole persistence (Chapter 5) and AmB tolerance of 108
C. albicans biofilms (Chapter 6)
9.1.4 Efg1 involved in tolerance of C. albicans biofilms (Chapter 7) 110
9.1.5 Potentiation by diclofenac of CAS activity against C. albicans biofilms 111
(Chapter 8)
9.1.6 Final conclusions 113

9.2 Novel trends in antifungal/anti-biofilm drug discovery 116
9.3 References 119
List of publications
ISBN: 978-90-8826-239-5
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Centre of Microbial and Plant Genetics

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