Carolina H. Pohl

The distribution of 3-hydroxy oxylipins in fungi

One of the best-kept secrets by fungi especially yeast is the function of the different shapes and surface structures of their vegetative and sexual cells. They definitely do not produce these shapes (e.g. round, elongated, kidney, needle, hat, saturnoid, etc.) and surfaces (e.g. smooth, rough, hairy, warty, etc.) for our curiosity or to be classified, but surely produce these for their own benefit. This mini-review will show that a large variety of 3-hydroxy oxylipins are widely distributed in the fungal domain and closely associated with these surface ornamentations. In concert with nano-scale surface structures, they probably play a role in cell aggregation as well as spore release from sexual structures such as asci.

Candida albicans or Candida dubliniensis

Candida albicans is increasing as an opportunistic pathogen causing candidemia and candidiasis worldwide. In addition, other non‐albicans Candida species are now also associated with pertinent infections. These include the closely related C. dubliniensis, which shares many phenotypic similarities with C. albicans. These similarities pose problems in the identification of isolates and have previously led to misidentification of these species. As a result, several identification techniques based on phenotypic and genotypic characteristics have been developed to differentiate between these Candida species. This review will focus on the similarities and differences between these two Candida species highlighting different identification methods and their advantages and disadvantages.

Oxidized Fatty Acids as Inter-Kingdom Signaling Molecules

Oxylipins or oxidized fatty acids are a group of molecules found to play a role in signaling in many different cell types. These fatty acid derivatives have ancient evolutionary origins as signaling molecules and are ideal candidates for inter-kingdom communication. This review discusses examples of the ability of organisms from different kingdoms to “listen” and respond to oxylipin signals during interactions. The interactions that will be looked at are signaling between animals and plants; between animals and fungi; between animals and bacteria and between plants and fungi. This will aid in understanding these interactions, which often have implications in ecology, agriculture as well as human and animal health.

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.

Effect of inhibitors of arachidonic acid metabolism on prostaglandin E2 production by Candida albicans and Candida dubliniensis biofilms

Arachidonic acid (AA) is released from infected host cells during Candida albicans infection and may serve as carbon source for yeast growth and as precursor for the production of biologically active eicosanoids, such as prostaglandin E2 (PGE2) by C. albicans. However, the mechanism involved in this production is still unclear. Therefore, it was of interest to investigate the effect of different arachidonic acid metabolism inhibitors on PGE2 production by biofilms of C. albicans and the closely related C. dubliniensis. This was done by growing Candida biofilms in the presence of AA as well as cytochrome P450 (CYP), multicopper oxidase, cyclooxygenase or lipoxygenase inhibitors. The concentration of PGE2 was determined by a monoclonal PGE2 enzyme-linked immunosorbent assay and verified with LCMS/MS. The results obtained indicate the ability of C. albicans and C. dubliniensis biofilms to produce PGE2 from exogenous AA. The use of different inhibitors suggested that CYPs and multicopper oxidases are involved in PGE2 production by these Candida biofilms.

Acetylsalicylic acid as antifungal in Eremothecium and other yeasts

Interesting distribution patterns of acetylsalicylic acid (ASA, aspirin) sensitive 3-hydroxy (OH) oxylipins were previously reported in some representatives of the yeast genus Eremothecium—an important group of plant pathogens. Using immunofluorescence microscopy and 3-OH oxylipin specific antibodies in this study, we were able to map the presence of these compounds also in other Eremothecium species. In Eremothecium cymbalariae, these oxylipins were found to cover mostly the spiky tips of narrowly triangular ascospores while in Eremothecium gossypii, oxylipins covered the whole spindle-shaped ascospore with terminal appendages. The presence of these oxylipins was confirmed by chemical analysis. When ASA, a 3-OH oxylipin inhibitor, was added to these yeasts in increasing concentrations, the sexual stage was found to be the most sensitive. Our results suggest that 3-OH oxylipins, produced by mitochondria through incomplete β-oxidation, are associated with the development of the sexual stages in both yeasts. Strikingly, preliminary studies on yeast growth suggest that yeasts, characterized by mainly an aerobic respiration rather than a fermentative pathway, are more sensitive to ASA than yeasts characterized by both pathways. These data further support the role of mitochondria in sexual as well as asexual reproduction of yeasts and its role to serve as a target for ASA antifungal action.

Mapping 3-hydroxy oxylipins on ascospores of Eremothecium sinecaudum

Abstract3-Hydroxy oxylipins were uncovered on ascospores of Eremothecium sinecaudum using immunofluorescence microscopy. This was confirmed by gas chromatography mass spectrometry. These oxylipins were observed only on ascospore parts characterised by nano-scale surface ornamentations simulating a corkscrew as demonstrated by scanning electron microscopy. Conventional ascospore staining further confirms its hydrophobic nature. Using confocal laser scanning microscopy we found that the corkscrew part with spiky tip of needle-shaped ascospores may play a role in rupturing the ascus in order to affect its release. Through oxylipin inhibition studies we hypothesise a possible role for 3-hydroxy oxylipins in facilitating the rupturing process.