Tolerance of yeast biofilm cells towards systemic antifungals

Rasmus Kenneth Bojsen

    Research output: Book/ReportPh.D. thesis

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    Abstract

    Fungal infections have become a major problem in the hospital sector in the past decades due to the increased number of immune compromised patients susceptible to mycosis. Most human infections are believed to be associated with biofilm forming cells that are up to 1000-fold more tolerant to antimicrobial agents compared to their planktonic counterparts. Antifungal treatment of biofilms will therefore often result in treatment failure. Consequently, there is a basic requirement to understand the underlying tolerance mechanisms and to development of novel anti-biofilm treatment strategies.
    The focus of this thesis has been to explore the tolerance mechanisms of yeast biofilms to systemic antifungal agents and to identify the molecular target of a novel peptidomimetic with anti-biofilm activity. The genetic tractable S. cerevisiae was used as biofilm model system for the pathogenic Candida species in an attempt to take advantage of the molecular tools available for S. cerevisiae.
    Mature biofilms containing mainly growth arrested cells were shown to be tolerant to three out of four tested antifungals, while all drugs had inhibitory activity against proliferating biofilm cells, demonstrating that drug treatment efficacy of biofilm cells is highly dependent on cellular growth phase. Similar results were obtained for planktonic cells, showing that ceased proliferation is a shared tolerance mechanism between biofilm and planktonic cells.
    It was found that the membrane pore-forming agent amphotericin B was the only tested drug with activity against both growth arrested biofilm and planktonic cells but was found to only kill ~95 % of the cells. By using a collection of barcode tagged deletion mutants, we were identified that defects in protein synthesis, intracellular transport, cell cycle and lipid metabolism resulted in increased amphotericin B tolerance in both biofilm and planktonic cells. We furthermore observed that the tolerance level could be enhanced by nutrient starvation and inhibition of the TOR pathway.
    In conclusion, antifungal tolerance is the combined effect of the physiological state of the cell and the mechanism of action of the drug, and this is independent of mode of growth. Based on these results, it can be suggested that future drug treatment strategies should focus on targeting growth arrested cells, rather than distinguishing between modes of growth.
    At last, we analyzed the antifungal activity of the novel peptidomimetic LTX-109. We showed that this molecule rapidly killed yeast cells and that cell death was associated with release of protons, potassium and amino acids to the extracellular environment. Screening a yeast deletion collection for LTX-109 resistance indicated that complex sphingolipids were involved in fungicidal activity of LTX-109. The sphingolipids may therefore represent a unique antifungal target with therapeutic potential for future drug development.
    Original languageEnglish
    PublisherTechnical University of Denmark
    Number of pages111
    Publication statusPublished - 2014

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