Pieter W. J. van Wyk

Effect of marine polyunsaturated fatty acids on biofilm formation of Candida albicans and Candida dubliniensis.

The effect of marine polyunsaturated fatty acids on biofilm formation by the human pathogens Candida albicans and Candida dubliniensis was investigated. It was found that stearidonic acid (18:4 n-3), eicosapentaenoic acid (20:5 n-3), docosapentaenoic acid (22:5 n-3) and docosahexaenoic acid (22:6 n-3) have an inhibitory effect on mitochondrial metabolism of both C. albicans and C. dubliniensis and that the production of biofilm biomass by C. dubliniensis was more susceptible to these fatty acids than C. albicans. Ultrastructural differences, which may be due to increased oxidative stress, were observed between treated and untreated cells of C. albicans and C. dubliniensis with formation of rough cell walls by both species and fibrillar structures in C. dubliniensis. These results indicate that marine polyunsaturated fatty acids may be useful in the treatment and/or prevention of biofilms formed by these pathogenic yeasts.

3-Hydroxy fatty acids found in capsules of Cryptococcus neoformans

Using immunofluorescence confocal laser scanning microscopy, immunogold transmission electron microscopy and gas chromatography--mass spectrometry, we demonstrated the presence of 3-hydroxy fatty acids in Cryptococcus neoformans. Our results suggest that these oxylipins accumulate in capsules where they are released as hydrophobic droplets through tubular protuberances into the surrounding medium.

Polyunsaturated fatty acids cause apoptosis in C. albicans and C. dubliniensis biofilms

BACKGROUND Polyunsaturated fatty acids (PUFAs) have antifungal properties, but the mode by which they induce their action is not always clear. The aim of the study was to investigate apoptosis as a mode of action of antifungal PUFAs (stearidonic acid, eicosapentaenoic acid and docosapentaenoic acid) which are inhibitory towards biofilm formation of C. albicans and C. dubliniensis. METHODS Candida biofilms were grown in the absence or presence of 1mM PUFAs (linoleic acid, stearidonic acid, eicosapentaenoic acid, docosapentaenoic acid) for 48h at 37°C. The effect of these PUFAs on the membrane fatty acid profile and unsaturation index, oxidative stress, mitochondrial transmembrane potential and apoptosis was evaluated. RESULTS When biofilms of C. albicans and C. dubliniensis were exposed to certain PUFAs there was an increase in unsaturation index of the cellular membranes and accumulation of intracellular reactive oxygen species (ROS). This resulted in apoptosis, evidenced by reduced mitochondrial membrane potential and nuclear condensation and fragmentation. The most effective PUFA was stearidonic acid. CONCLUSIONS The resultant cell death of both C. albicans and C. dubliniensis is due to apoptosis. GENERAL SIGNIFICANCE Due to the increase in drug resistance, alternative antifungal drugs are needed. A group of natural antifungal compounds is PUFAs. However, understanding their mechanisms of action is important for further use and development of these compounds as antifungal drugs. This paper provides insight into a possible mode of action of antifungal PUFAs.

Arachidonic acid increases antifungal susceptibility of Candida albicans and Candida dubliniensis

OBJECTIVES During Candida albicans infection, arachidonic acid (AA) is released from phospholipids of infected host cell membranes and used by C. albicans as the sole carbon source and for production of eicosanoids. AA can be incorporated into the phospholipids of yeasts, influencing the saturation level and fluidity of yeast cell membranes. It is suggested that the effectiveness of polyene (e.g. amphotericin B) and imidazole (e.g. clotrimazole) antifungals may depend upon the level of unsaturation and ergosterol in the membrane. Therefore, the aim of this study was to evaluate the effect of AA on the cell membrane and susceptibility of C. albicans and Candida dubliniensis biofilms towards amphotericin B and clotrimazole. METHODS Both yeasts were grown in the presence and absence of AA and the effect of amphotericin B and clotrimazole was examined by confocal laser scanning microscopy, determination of mitochondrial metabolism, unsaturation index of the phospholipid fractions and ergosterol content of the membranes. RESULTS AA had no effect on the viability of the cells in the biofilm; however, there was an increase in ergosterol levels as well as antifungal susceptibility of biofilms grown in the presence of AA. CONCLUSIONS AA influences phospholipid unsaturation and ergosterol content of both yeasts C. albicans and C. dublininensis, increasing susceptibility towards the antifungals. Pretreatment of biofilms with polyunsaturated fatty acids may result in the reduction in antifungal dose needed to inhibit biofilms.

Anti-inflammatory drugs selectively target sporangium development in Mucor

It is known that acetylsalicylic acid, an anti-inflammatory and anti-mitochondrial drug, targets structure development and functions of yeasts depending on elevated levels of mitochondrial activity. Using antibody probes, we previously reported that sporangia of Mucor circinelloides also contain increased mitochondrial activity, yielding high levels of 3-hydroxyoxylipins. This was, however, not found in Mortierella alpina (subgenus Mortierella). In this study we report that acetylsalicylic acid (aspirin) also targets sporangium development of Mucor circinelloides selectively, while hyphae with lower levels of mitochondrial activity are more resistant. Similar results were obtained when the anti-inflammatory compounds benzoic acid, ibuprofen, indomethacin, and salicylic acid were tested. The anti-inflammatory drugs exerted similar effects on this dimorphic fungus as found under oxygen-limited conditions. Interestingly, sporangium development of Mortierella alpina was found not to be selectively targeted by these drugs. Mortierella alpina, which could not exhibit dimorphic growth under oxygen-limited conditions, was also more sensitive to the anti-inflammatory drugs when compared with Mucor circinelloides. These results prompt further research to assess the applicability of these antimitochondrial antifungals to protect plants and animals against Mucor infections.

Gas bubble formation in the cytoplasm of a fermenting yeast.

Abstract Current paradigms assume that gas bubbles cannot be formed within yeasts although these workhorses of the baking and brewing industries vigorously produce and release CO2 gas. We show that yeasts produce gas bubbles that fill a significant part of the cell. The missing link between intracellular CO2 production by glycolysis and eventual CO2 release from cells has therefore been resolved. Yeasts may serve as model to study CO2 behavior under pressurized conditions that may impact on fermentation biotechnology.

Oxylipin and mitochondrion probes to track yeast sexual cells.

When oxylipin and mitochondrion probes, i.e., fluorescing antibodies specific for 3-hydroxy fatty acids (3-OH oxylipins) and rhodamine 123 (Rh123), were added to yeast cells, these probes accumulated mainly in the sexual cells (i.e., both associated with ascospores) and not in the vegetative cells. This suggests increased mitochondrial activity in asci, since 3-OH oxylipins are mitochondrially produced and it is known that Rh123 accumulates selectively in functional mitochondria that maintain a high transmembrane potential (Delta Psi m). This increased activity may be necessary for the production and effective release of the many spores found in single-celled asci. These results may be useful in the rapid identification of asci and in yeast sexual spore mechanics, which may find application in yeast systematics as well as hydro-, aero-, and nano-technologies.

Variation in yeast mitochondrial activity associated with asci

An increase in mitochondrial membrane potential (DeltaPsim) and mitochondrially produced 3-hydroxy (3-OH) oxylipins was experienced in asci of the nonfermentative yeasts Galactomyces reessii and Lipomyces starkeyi and the fermentative yeasts Pichia farinosa and Schizosaccharomyces octosporus. Strikingly, asci of Zygosaccharomyces bailii showed no increase in mitochondrial activity (DeltaPsim and oxylipin production). As expected, oxygen deprivation only inhibited ascus formation in those yeasts with increased ascus mitochondrial activity. We conclude that ascus formation in yeasts is not always dependent on mitochondrial activity. In this case, fermentation may provide enough energy for ascus formation in Z. bailii.

Yeast sensors for novel drugs: chloroquine and others revealed.

In this study the mitochondrion is regarded as a target to reveal compounds that may be used to combat various diseases. Consequently, the sexual structures of yeasts (with high mitochondrial activity) were identified as sensors to screen for various anti-mitochondrial drugs that may be toxic to humans and that are directed, amongst others, against fungal diseases and cancer. Strikingly, these sensors indicated that chloroquine is a potent pro-mitochondrial drug which stimulated yeast sexual reproduction. In addition, these sensors also showed that some Non-Steroidal Anti-Inflammatory drugs (NSAIDs), anti-malarial drugs, antifungal and anticancer drugs are anti-mitochondrial. These yeast sensor bio-assays may fast track studies aimed at discovering new drugs as well as their mechanisms and should now be further evaluated for selectivity towards anti-/ pro-mitochondrials, fertility drugs and contraceptives, using in vitro, in vivo, in silico and omics research.

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit growth of yeast pathogens

Studies on yeasts exposed non-steroidal anti-inflammatory drugs (NSAIDs) such as acetylsalicylic acid (aspirin) as potential anti-mitochondrial antifungals. In this study, various NSAIDs were tested for antifungal activity on the human yeast pathogens Candida albicans and Cryptococcus neoformans. Our results suggest a dual action for these drugs, that is, antifungal as well as anti-inflammatory. These results could be useful in the treatment of fungal infections.