Cell death in the human pathogen Candida albicans: effects of garlic (Allium sativum), and garlic constituents allyl alcohol and diallyl disulphide

Garlic extract is very complex, yielding a number of organic sulphur constituents that are thought to be responsible for its anticandidal properties. Many of these are now being investigated in an attempt to determine the mechanisms by which they act. The effects of fresh and freeze dried extracts of Allium sativum (garlic) on the physiology and morphology of Candida albicans were compared. Inhibition of growth and loss of structural integrity was observed for both fresh garlic extract (FGE) has a greater efficacy than garlic powder extract (GPE) as indicated both by its effects on morphology and inhibition of growth. Gas chromatography-mass spectrometry of extracts was employed to separate and quantify putative inhibitory sulphur-containing components fresh and freeze-dried extracts yielded the same components but fresh garlic yielded ten times more sulphur constituents. Cell death mechanisms were investigated by flow cytometry. Low concentrations of allyl alcohol (AA) triggered a necrotic response, whereas an apoptotic type of cell death was observed at higher concentrations (>6mM). Conversely, low concentrations of diallyl disulphide (DADS) induced apoptosis, whereas higher concentrations (>6mM) resulted in a necrotic response. Further investigations with using 2-photon microscopy determined that a short 30 min exposure to 0.5mM DADS and then removal, induced 70% cell death (50% necrotic, 20% apoptotic) within 2h this figure increased to 75% after 4h. Intracellular levels of reactive oxygen species (ROS), were increased with >10mM menadione, 2mg ml"1 GPE, ImM AA or DADS as measured using dihydrofluorescein and detected by flow cytometry. Two-photon laser scanning microscopy was employed to monitor the intracellular responses of individual C. albicans cells after treatment. Changes typical of oxidative stress NADH oxidation, glutathione depletion and increased reactive oxygen species (ROS), were observed. Additionally, DADS induced a marked enhancement of mitochondrial membrane potential and low respiration rates as could be verified in cell suspensions. The plasma membrane was monitored by use of the Bis-oxonol dye, DiBaC4(3). Calculation of the electrochemical potential was achieved by application of the Nernst equation. Complete depolarisation was observed with low concentrations of AA, suggesting that for this constituent, the plasma membrane may be a primary target. Effects of garlic extract and diallyl disulphide on plasma membrane were less obvious. Putative targets for DADS are glutathione-S-transferase as determined by in vitro kinetics using cell-free extracts additional targets are likely to be a component prior to Site II in the respiratory electron transport chain as well as ATPsynthase as determined by decreased oxygen consumption and proton production respectively. Known targets for allyl alcohol are alcohol dehydrogenases Adhl and 2 (in the cytosol) and Adh3 (mitochondrial), although the significant decrease in NAD(P)H after addition of AA is indicative of another mechanism of action.