Studies on the effect of thymoquinone on oxidative stress and cell wall integrity of Candida glabrata

Thymoquinone is a major active ingredient of black seeds which are produced by the plant, Nigella sativa. Thymoquinone exhibits various chemotherapeutic and chemo-preventative activities including immunostimulation, antimicrobial, anticancer and chemoprotection activities (reviewed by Darakhshan et al., 2015). Despite available data on thymoquinone’s anticancer mechanism of action, there is no description of its antifungal mode of action. This thesis presents data on thymoquinone’s effect on the redox status of C. glabrata and reveals a binding site of TQ on the surface of these cells.<br><br>The data presented in chapter 2 demonstrates for the first time the growth inhibitory effect of TQ on the species C. krusei and C. glabrata beside other Candida species. This chapter also shows that in C. glabrata, thymoquinone exhibited concentration-dependent fungicidal activity on planktonic cells and caused necrotic death. In addition, the antifungal effect of thymoquinone against C. glabrata biofilms was examined. The biofilms were stabilised using a poly propylene biofilm reactor designed to mimic Calgary biofilm device (Almshawit et al., 2014b). The advantages of this novel reactor are described.<br>Thymoquinone may act as anti-oxidant or pro-oxidant depending on the milieu (Mansour et al., 2002). Chapter 3 demonstrated that thymoquinone causes oxidative stress in C. glabrata as a result of increased reactive oxygen species generation. This chapter also reports depletion of reduced glutathione levels and reduced mitochondrial membrane potential. In addition, this chapter provides experimental characterisation of the ALO1 gene and evidence of production of D-erythroascorbic acid by C. glabrata.<br><br>Chapter 4 shows that activation of CWI-MAPK in C. glabrata results following treatment with thymoquinone, hydrogen peroxide, doxycycline or hypo-osmotic treatment. However, CWI-MAPK mutants were neither sensitive nor resistant to thymoquinone. In contrast the downstream kinase mutant (C. glabrata Δslt2) was sensitive to H2O2 (Chapter 5). Activation of CWI-MAPK by hypo-osmolarity was exploited to develop a hypothesis that this mechanism could be used to screen potential CWI-MAPK inhibitors. Survival of cells in water was used as a model to screen such inhibitor. The results give insights into important facts that should be considered when using water suspensions of C. glabrata cells.<br><br>Finally chapter 5 describes for the first time an interaction between thymoquinone and the fungal cell wall. This study demonstrates that thymoquinone induced stress promotes chitin synthesis after induction of CWI-MAPK. Chitin was shown to bind thymoquinone and facilitate its transport through the cell wall inside the cell. This was supported by the observation that inhibition of chitin synthesis led to a decrease in thymoquinone antifungal efficacy. This finding is important in a reconsideration of the use of TQ together with other cell wall active antifungal compounds such as nikkomycin Z.

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