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Oxylipin studies expose aspirin as antifungal

Johan L. F. Kock, Olihile M. Sebolai, Carolina H. Pohl, Pieter W. J. van Wyk, Elizabeth J. Lodolo
DOI: http://dx.doi.org/10.1111/j.1567-1364.2007.00273.x 1207-1217 First published online: 1 December 2007


The presence of aspirin-sensitive 3-hydroxy fatty acids (i.e. 3-OH oxylipins) in yeasts was first reported in the early 1990s. Since then, these oxidized fatty acids have been found to be widely distributed in yeasts. 3-OH oxylipins may: (1) have potent biological activity in mammalian cells; (2) act as antifungals; and (3) assist during forced spore release from enclosed sexual cells (asci). A link between 3-OH oxylipin production, mitochondria and aspirin sensitivity exists. Research suggests that: (1) 3-OH oxylipins in some yeasts are probably also produced by mitochondria through incomplete β-oxidation; (2) aspirin inhibits mitochondrial β-oxidation and 3-OH oxylipin production; (3) yeast sexual stages, which are probably more dependent on mitochondrial activity, are also characterized by higher 3-OH oxylipin levels as compared to asexual stages; (4) yeast sexual developmental stages as well as cell adherence/flocculation are more sensitive to aspirin than corresponding asexual growth stages; and (5) mitochondrion-dependent asexual yeast cells with a strict aerobic metabolism are more sensitive to aspirin than those that can also produce energy through an alternative anaerobic glycolytic fermentative pathway in which mitochondria are not involved. This review interprets a wide network of studies that reveal aspirin to be a novel antifungal.

  • antifungal
  • aspirin
  • mitochondria
  • oxylipins
  • yeasts


Oxidized fatty acids (oxylipins) are widely distributed in nature, and have been the subject of various reviews in the past (Needleman et al., 1986; Van Dyk et al., 1994; Kock et al., 2003; Noverr et al., 2003; Erb-Downward & Huffnagle, 2006). This review will, for the first time, interpret a network of oxylipin studies that implicate aspirin as a novel antifungal that also targets yeast mitochondria. This may find application in the control of fungal pathogens.

Extensive bioprospecting to determine whether yeasts can produce acetylsalicylic acid (aspirin)-sensitive oxylipins, such as prostaglandins, was performed in 1988. Prostaglandins are biologically active autacoids, responsible for important functions in mammalian cells (Samuelsson, 1983; Needleman et al., 1986; Kock et al., 1991; Noverr et al., 2003). They are also produced by expensive chemical processes and administered to elicit several responses in humans, such as labor induction and inhibition of blood platelet aggregation. Consequently, because of their high cost, a cheaper biotechnological source for these compounds will have obvious advantages (Dixon, 1991). With radio thin-layer chromatography (TLC) and radioimmunoassay, aspirin-sensitive oxylipins were discovered when the direct precursor, arachidonic acid (AA, a 20 : 4 fatty acid), was fed to the yeast Dipodascopsis uninucleata (Kock et al., 1991, 1992). As a result, the practical applications of this discovery were included in several patents, including one describing the use of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) as antifungals (Kock & Coetzee, 1990). Here, NSAID-sensitive oxylipin production pathways were regarded as the target site.

In these studies, the first prostaglandins in yeasts were discovered (Kock et al., 1991; Noverr et al., 2003). This discovery was later confirmed by Noverr et al. (2001, 2002), when they demonstrated the production of immunomodulatory prostaglandins in the pathogenic yeasts Cryptococcus neoformans and Candida albicans. The presence, biochemistry and possible role as virulence factors of yeast oxylipins, including prostaglandins, were reviewed in 2003 (Noverr et al., 2003). In support of the above observations, Alem & Douglas (2004) demonstrated that biofilms formed by Ca. albicans can be inhibited as much as 95% by aspirin. When prostaglandin E2 (PGE2) was added together with aspirin, the inhibitory effect of aspirin was abolished. In 2005, the same authors suggested that prostaglandin production could be a virulence factor in yeast biofilm-associated infection (Alem & Douglas, 2005). However, all the results reported thus far regarding prostaglandins in yeasts were obtained through immunologic studies, which may be prone to error, as cross-reactions with prostaglandin-like compounds in yeasts may occur. Consequently, more direct evidence obtained using GC-MS and other techniques is needed to finally confirm their presence in yeasts.

In the bioprospecting study, radio TLC in combination with 1H two-dimensional (2D) correlation spectroscopy (COSY) nuclear magnetic resonance (NMR), GC-MS [electron impact (EI) and fast atom bombardment (FAB)] as well as infrared spectroscopy analyses, exposed a novel aspirin-sensitive oxylipin, i.e. 3R-OH 5Z,8Z,11Z,14Z-eicosatetraenoic acid (3-HETE, a 20 : 4 fatty acid with an OH group on carbon 3) in the yeast Dipodascopsis uninucleata (Fig. 1) (Van Dyk et al., 1991, 1993). This compound was only formed in the presence of exogenous AA, a precursor for prostaglandin formation in humans. This was surprising, as only aspirin-sensitive prostaglandins were expected. Later studies showed that this yeast is capable of producing a wide variety of novel 3-OH oxylipins (i.e. 3-OH 14 : 2; 3-OH 14 : 3; 3-OH 20 : 3; 3-OH 20 : 5) when fed with different precursors (Fox et al., 1997; Venter et al., 1997). Furthermore, a maximum 3-HETE yield of 1.9% from AA was obtained. This was achieved by: (1) adding AA at the point of sexual reproduction (ascus formation at its highest); (2) using a medium buffered to pH 7 in the absence of sucrose; and (3) using a fatty acid concentration of 200 mM (Fox et al., 1997). Before this discovery, only the presence of saturated 3-OH oxylipins, with no reference to function, had been reported in yeasts (Kurtzman et al., 1974; Van Dyk et al., 1994). Later, the production of 3-HETE was also reported in Dipodascopsis tothii (Kock et al., 1997), a close relative of Dipodascopsis uninucleata on the basis of rRNA gene comparisons (Kurtzman & Robnett, 1998).


The chemical structures of typical 3-hydroxy oxylipins. (a) R-3-hydroxy-5,8,11,14-eicosatetraenoic acid; (b) S-3-hydroxy-5,8,11,14-eicosatetraenoic acid.

Distribution of 3-OH oxylipins in yeasts

3-OH oxylipins are widely distributed in yeasts (Kock et al., 1998, 2003, 2004, 2006; Noverr et al., 2003; Leeuw et al., 2007). Antibodies directed against chemically synthesized 3-OH oxylipins, and found to be specific for 3-OH oxylipins in general, were of assistance in mapping 3-OH oxylipins with different chain lengths and desaturation levels. This provided a much needed tool with which to visualize the distribution of these oxidized compounds in the life cycles of yeasts and among different yeast species. These studies showed that these oxylipins are mainly associated with yeast sexual stages (asci), where they, among others, coat the outer surfaces of ascospores (Fig. 2) (Bhatt et al., 1998; Kock et al., 1998; Groza et al., 2002, 2004). So far, in addition to antibody mapping, chemical analysis by GC-MS in most cases has confirmed the presence of 3-OH oxylipins.


The life cycle of Dipodascopsis uninucleata and distribution of 3-HETE visualized through immunofluorescence mapping. (a) Liberated ascospores showing high affinity for oxylipin antibody. (b) Hyphae with low oxylipin antibody affinity. (c) Gametangiogamy with tip of adhering gametes showing high affinity for oxylipin antibody. (d) Young ascus with ascospores demonstrating high affinity for oxylipin antibody. (e) Liberated fluorescing ascospores from ascus. (f) Empty ascus protoplast: still with characteristic morphology. (g) Deformed mature ascus protoplast containing fluorescing ascospores mainly at base. (a, b) Asexual vegetative stage. (c, d, e, f, g) Sexual stage. Reprinted by permission of Federation of the European Biochemical Societies from Kock et al., 1998©.

Further studies reported these oxylipins to be associated mainly with the ascospores of Dipodascopsis uninucleata (Fig. 2) (Kock et al., 1998), Dipodascus (Van Heerden et al., 2005, 2007), many lipomycetaceous species, i.e. Lipomyces doorenjongii, L. kockii, L. kononenkoae, L. starkeyi, L. yamadae, L. yarrowii, Smithiozyma japonica and Zygozyma oligophaga (Smith et al., 2000b), Saturnispora saitoi (Bareetseng et al., 2006), Saccharomycopsis (Sebolai et al., 2001, 2004, 2005), Eremothecium (Bareetseng et al., 2004; Kock et al., 2004; Leeuw et al., 2006, 2007), and Ascoidea (Figs 3 and 4) (Ncango et al., 2006). Although 3-OH oxylipins were reported in lipomycetaceous yeasts, no 3-HETE could be detected when these yeasts were fed with AA (Kock et al., 1992). Furthermore, in contrast to the situation in Dipodascopsis uninucleata, 3-OH oxylipins accumulate mainly on the ascus tip of the closely related Dipodascopsis tothii, as observed by immunofluorescence microscopy (Smith et al., 2000a).


Light micrograph (a), immunofluorescence-only micrograph showing in more detail selectively fluorescing brims surrounding hat-shaped ascospores in circles (b, compare Fig. 4a), light combined with immunofluorescence micrograph (c), and light micrograph of stained ascospores (d) of Ascoidea corymbosa. A, ascus; As, ascospore; AW, ascus wall; FAs, fluorescing ascospores; T, ascus tip. Taken with permission from Ncango et al. (2006).


Scanning electron micrographs of individually released ascospore (a) and aggregated released ascospores (b) in Ascoidea corymbosa. The release of ascospores from the ascus opening (tip) is shown in (c). A, ascus; As, ascospore; B, bowl; Br, brim. Taken with permission from Ncango et al. (2006).

3-OH oxylipins are not limited to ascospores. Electron microscopy, including immunogold labeling and immunofluorescence microscopy, shows that surfaces of flocculating vegetative cells of the baker's yeast Saccharomyces cerevisiae as well as Saccharomycopsis malanga contain 3-OH oxylipins (Kock et al., 2000; Strauss et al., 2005; Speers et al., 2006). These compounds were also found on the surfaces of hyphae of pathogenic yeasts (Deva et al., 2000, 2001, 2003).

Functions of 3-OH oxylipins

The first evidence concerning the biological function of 3-OH oxylipins was presented in the 1990s. It was reported that 3R-HETE affects signal transduction processes in human neutrophils and tumor cells in multiple ways (Nigam et al., 1999) and acts as a strong chemotactic agent, the potency of which is comparable with those of leukotriene B4 or fMet-Leu-Phe. The cell signaling cascade triggered by 3-HETE appears to imply G-protein-dependent processes. A novel 3-OH oxylipin, 3,18-dihydroxy-5,8,11,14-eicosatetraenoic acid, was identified in Ca. albicans, a pathogen involved in vulvovaginal candidiasis (Deva et al., 2000, 2001, 2003). These researchers concluded that the administration of aspirin should be beneficial in the treatment of this disease in two ways: (1) by inhibiting 3-OH oxylipin formation – mainly associated with the hyphal phase; and (2) by inhibiting PGE2 formation in the infected host tissue.

Recently, Ciccoli et al. (2005) uncovered a novel mode of infection of the yeast pathogen Ca. albicans. They found that this yeast converts AA, released from infected or inflamed host cells, to a 3-HETE-like compound. This oxylipin then acts as substrate for the host cyclooxygenase-2 (COX-2), leading to the production of the potent proinflammatory compound 3R-hydroxyprostaglandin E2 (3R-OH-PGE2). They uncovered a cascade of novel bioactive 3R-OH prostaglandins, produced from 3-HETE via mammalian COX-2 (Fig. 5).


A diagram showing the formation of potent inflammatory 3-hydroxy prostaglandins in host cells from 3-HETE produced via incomplete β-oxidation from host-released AA by the yeast Candida albicans. ASA, aspirin; COX-2, cyclooxygenase-2. Taken with permission from Kock et al. (2005).

When infected, mammalian cells usually release AA for transformation via aspirin-sensitive COX-1 and COX-2 to proinflammatory eicosanoids such as prostaglandins, thromboxanes, and prostacyclin. These compounds are potent regulators of the host immune responses, and play a role in numerous basic host cell physiologic processes. Ciccoli et al. (2005) have shown that 3-HETE is also an appropriate substrate for COX-2, being almost as effective as AA, and produces novel 3-OH eicosanoids, including 3R-hydroxyprostaglandin B2, 3R-hydroxyprostaglandin D2, 3R-OH-PGE2 and 3R-hydroxyprostaglandin F. These authors showed that 3-OH eicosanoids have strong biological activities similar to and in some cases even more potent than those of the normally produced eicosanoids.

As yeast growth, formation of virulent hyphal stages as well as 3-HETE and COX-2-produced 3R-OH prostaglandins are inhibited by low concentrations of aspirin, this research suggests new targets for the control of yeast infection. Research concerning the applicability of aspirin and other NSAIDs as antifungals in order to control yeast infection should now be addressed – an idea first proposed in 1990 (Kock & Coetzee, 1990).

This is of special importance, as a 3-OH oxylipin has recently been discovered in a strain of the yeast pathogen Cr. neoformans (Sebolai et al., 2007) – a yeast responsible for worldwide morbidity and mortality, especially in immunosuppressed AIDS patients. The results suggest that these oxylipins accumulate on the inside of capsules, from where they are released as hydrophobic droplets (vesicles) through capsular tubular protuberances into the surrounding medium. Strikingly, Rodrigues et al. (2007) also reported recently on a similar release mechanism for the major polysaccharide virulence factor of Cr. neoformans. They found that polysaccharide-packaged lipid vesicles cross the cell wall and the capsule into the surrounding environment. Are 3-OH oxylipins also present in these lipid vesicles, and, if so, do they play a role in pathogenesis? What will the effect of aspirin be on oxylipin production and Cryptococcus pathogenicity? This finding expands the known spectrum of biologically active compounds associated with the capsule of Cr. neoformans and that may serve as targets for new types of antifungals.

Interestingly, studies on flocculating Saccharomyces cerevisiae showed that the strains studied were incapable, under the conditions tested, of producing the 3-HETE (ab initio or from exogenously fed AA) that is necessary for the synthesis of inflammatory COX-2-produced 3-OH prostaglandins in mammalian cells. These results thus affirmed the Generally Regarded as Safe (GRAS) status of biotechnologically important Saccharomyces cerevisiae strains, as no known inflammatory eicosanoids or COX-2 precursors were detected (Strauss et al., 2005).

3-OH oxylipins are not only strong proinflammatory lipid mediators (Nigam et al., 1999; Ciccoli et al., 2005), but also show potent antifungal activity against some molds and yeasts (Sjogren et al., 2003). The literature shows that 3-OH 10 : 0, 3-OH 11 : 0, 3-OH 12 : 0 and 3-OH 14 : 0 have antifungal activity with minimal inhibitory concentrations between 10 and 100 μg mL−1 against some species of Aspergillus, Penicillium, Kluyveromyces, Pichia and Rhodotorula. It will be of interest to determine whether yeasts produce specific 3-OH oxylipins for their own protection against other fungi. Furthermore, 3-OH oxylipins are also found in Gram-negative bacteria as a crucial part of the inflammatory disease-causing lipopolysaccharide endotoxin component (Rietschel et al., 1994; Annane et al., 2005). Here, lipopolysaccharide plays an important role in the development of inflammation, which may eventually lead to septic shock, the most severe complication of sepsis and a deadly disease worldwide.

Oxylipins, mitochondria and aspirin inhibition

3-OH oxylipins are not only found in yeasts. According to the literature, these compounds may also be produced in mitochondria by β-oxidation in mammalian cells (Szponar et al., 2003). So far, indirect evidence suggests that 3-OH oxylipins are probably produced in a similar way, especially in the sexual cells of various yeasts. This is based on the link found between yeast oxylipin production and mitochondria, both of which are inhibited by a known mammalian mitochondrial inhibitor, aspirin (Glasgow et al., 1999). This is contrary to the general belief that β-oxidation occurs only in peroxisomes of yeast (Hiltunen et al., 2003, 2005). In these elegant biochemical studies, mainly Saccharomyces cerevisiae was analyzed without reference to the sexual cell types of the large diversity of nonrelated yeasts.

In a groundbreaking study, Botha et al. (1992) analyzed the life cycles of the nonfermenting yeasts Dipodascopsis tothii and Dipodascopsis uninucleata, as well as the inhibitory effect of the NSAIDs aspirin and indomethacin. When the yeasts were grown in synchronous culture, the life cycles of both were characterized by similar consecutive asexual and sexual reproductive stages (Fig. 2). In the presence of different concentrations of aspirin (i.e. 0.1, 0.2, 0.5 and 1.0 mM), dose-dependent inhibition of the asexual vegetative stage was observed in both yeasts, although 0.1 and 0.2 mM aspirin did not inhibit this stage in Dipodascopsis uninucleata. The sexual stages were found to be more sensitive to these NSAIDs, and spore liberation was completely inhibited by a concentration of aspirin as low as 0.1 mM in Dipodascopsis tothii. Similar results were obtained with indomethacin, although at much lower concentrations. Later studies reported some liberation of spores by Dipodascopsis uninucleata after 40 h of growth in the presence of 0.1 mM aspirin, which also indicates dose-dependent inhibition of ascospore release, although at much lower concentrations than those needed to inhibit asexual vegetative cells (Kock et al., 1999). Consequently, these results suggest that both aspirin and indomethacin inhibit both asexual and sexual stages in yeast, although the sexual stage proved to be much more sensitive.

It was also shown that Dipodascopsis uninucleata produces 3-OH oxylipins that are inhibited by aspirin in a dose-dependent manner. This hinted at the possibility that these oxylipins are mainly produced during the sexual cycle (Van Dyk et al., 1991, 1993). This was proven with immunofluorescence microscopy, which showed that 3-OH oxylipins accumulate in sexual cells (asci, including gametangia), whereas only limited amounts are associated with the filamentous vegetative stage (Fig. 2) (Kock et al., 1998).

According to the literature, 3-OH oxylipins in Dipodascopsis uninucleata may be produced by β-oxidation (Ciccoli et al., 2005). It was found that Dipodascopsis uninucleata, during its sexual stage, is capable of synthesizing the oxylipins 3R-OH-5Z,8Z-tetradecadienoic acid from exogenously fed linoleic acid (9Z,12Z-octadecadienoic acid) and 3R-OH-5Z,8Z,11Z-tetradecatrienoic acid from exogenously fed 11Z,14Z,17Z-eicosatrienoic acid after, probably, several cycles of β-oxidation (Venter et al., 1997).

Evidence supporting a link between oxylipins and yeast mitochondria was presented by Strauss et al. (2007). Mitochondrial function is generally accepted as being important for expression of flocculation in yeasts. This has been demonstrated by the use of drugs such as antimycin A and ethionine (Nishihara et al., 1976; Egilsson et al., 1979; Iung et al., 1999), which inhibit mitochondrial function, cells that carry deletions in mitochondrial genes (Hinrichs et al., 1988), and petite (respiratory-deficient) mutants (Holmberg & Kielland-Brandt, 1978; Ernandes et al., 1993). In a recent study by Strauss et al. (2007), a link between mitochondrial activity, oxylipin production and flocculation was demonstrated in a flocculating strain of Saccharomyces cerevisiae. Here, strongly flocculating cells showed both increased mitochondrial activity and oxylipin production as compared to weakly flocculating cells. Also, in the presence of aspirin, flocculation, mitochondrial activity and oxylipin production declined sharply. This suggests that aspirin, also a mitochondrial inhibitor in mammalian cells (Somasundaram et al., 1997; Glasgow et al., 1999), inhibits mitochondrial function in yeasts, resulting in the decrease of flocculation and probably oxylipin levels as well. Whether flocculation decrease is due to general mitochondrial or only oxylipin inhibition is not clear.

When long-chain fatty acids such as AA were exogenously fed to asexual vegetative cells of Saccharomyces cerevisiae, no hydroxylation to 3-HETE or shorter-chain oxylipins could be detected, as was evident in sexual cells of Dipodascopsis uninucleata. Only a short-chain 3-OH 8 : 0, produced ab initio in the presence or absence of AA, could be identified (Strauss et al., 2005). Is it possible that this oxylipin is produced via the fatty acid synthesis type II (FAS II) route in mitochondria of vegetative cells (Hiltunen et al., 2005)? Will such a route also be followed in sexual cells of this yeast?

Strikingly, a recent study further strengthens the link between oxylipins and mitochondria. Here, concomitant increases in mitochondrial activity as well as 3-OH oxylipins in sexual cells of nonrelated fermentative and nonfermentative yeasts were reported (Ncango, unpublished data). These were found in the yeasts Ascoidea africana, Asc. corymbosa, Asc. rubescens, Dipodascopsis uninucleata and Pichia anomala.

Also, when aspirin was added to Ascoidea, the sexual stage proved to be most susceptible to inhibition (Ncango et al., 2007), similar to what was found for Dipodascopsis (Botha et al., 1992; Kock et al., 1998), Dipodascus (Van Heerden et al., 2007) and Eremothecium (Leeuw et al., 2007). This is to be expected, as aspirin is known to inhibit β-oxidation in mammalian mitochondria and therefore also 3-OH oxylipin synthesis (Glasgow et al., 1999). This is ascribed to aspirin metabolites having structural similarities to the acyl portions of the substrate and product of the 3-hydroxyacyl-CoA dehydrogenase activity of the β-oxidation pathway. In addition to the above, aspirin may also inhibit mitochondrial activity by uncoupling mitochondrial oxidative phosphorylation and/or inhibiting electron transport (Somasundaram et al. 1997; Norman et al. 2004). It is therefore not surprising that the yeast sexual cycle, which has previously been found to be dependent on mitochondrial activity (Marmiroli et al., 1983; Codon et al., 1995), is more susceptible to aspirin than are asexual vegetative cells (Kock et al., 2003). This is particularly true for Ascoidea, Dipodascopsis, Dipodascus and Eremothecium, in which high mitochondrial activity is presumably necessary to produce sufficient energy through aerobic respiration to sustain high production and assembly throughput during the formation of numerous ascospores within a single enlarged sexual cell.

As expected, aspirin addition and oxygen deprivation yielded similar results in inhibiting the sexual cycle of Dipodascopsis. When this yeast was grown under anoxic conditions, the sexual cycle was completely inhibited, whereas limited asexual growth was still observed. This further emphasizes the importance of aerobic respiration in sexual cell development of this yeast (Botha et al., 1993). Any disruption in mitochondrial activity by low concentrations of aspirin or oxygen will surely negatively affect the proper development of the many sexual spores per sexual cell rather than the relatively less productive vegetative cells during sexual reproduction.

The clear link between oxylipin production, mitochondria and aspirin sensitivity reported in various nonrelated yeasts and different cell types calls for further biochemical studies to determine whether β-oxidation in yeast may occur in cell inclusions other than peroxisomes. The effect of aspirin on peroxisomal β-oxidation and possible oxylipin production via FAS II should also be further researched.


As mitochondrial dependence seems to be linked to aspirin sensitivity in yeasts, it can be concluded that yeast with a mitochondrion-dependent strict aerobic metabolism will be more sensitive to this NSAID than those that can also produce energy through an alternative anaerobic glycolytic fermentative pathway in which mitochondria are not involved. This has been suggested recently (Leeuw et al., 2007) by growth experiments with several yeasts with both energy generation options, such as the aspirin-sensitive, nonfermentative Asc. africana, Asc. corymbosa, Asc. rubescens, Eremothecium ashbyi, E. coryli (weakly fermentative), E. cymbalariae, E. gossypii, E. sinecaudum, Cr. neoformans, Dipodascus albidus, Dipodascopsis uninucleata, Rhodotorula glutinis, and L. starkeyi, and the more resistant fermenting yeasts Ca. magnoliae, Ca. tropicalis, Kluyveromyces marxianus, Pic. anomala, Saccharomyces cerevisiae, Schizosaccharomyces octosporus, Sc. pombe, Zygosaccharomyces baillii.

This review prompts the following holistic hypothesis (Fig. 6): (1) the asexual vegetative reproductive phase of strictly aerobic yeasts are more sensitive to aspirin than are yeasts with an additional fermentative pathway; (2) the sexual reproductive phase of yeasts is more sensitive to aspirin than is the asexual vegetative growth phase; (3) flocculation in fermentative yeasts is partially inhibited by aspirin; (4) these phenomena are probably attributable to mitochondrial inhibition by aspirin, which in turn may be linked to the inhibition of products such as 3-OH oxylipins – not necessarily indicating oxylipin function; and (5) mitochondrial respiration and β-oxidation are more pronounced during the sexual phase of yeasts than in their asexual vegetative phase. The general validity of this hypothesis in the fungal domain should now be assessed.


A visual representation of a hypothesis suggesting a possible link between 3-OH oxylipin production, mitochondrial activity, and aspirin sensitivity. x-axis, top: increase in aspirin concentration from left to right. x-axis, bottom: decrease in mitochondrial activity and 3-OH oxylipin levels from left to right. y-axis, left: decrease in mitochondrial activity and 3-OH oxylipin levels from sexual reproductive to asexual growth phases in both strictly aerobic yeasts (RESP.) and yeasts with both aerobic and fermentative pathways (RESP.+FERM.). y-axis, right: different phases of yeast life cycles, i.e. sexual, asexual as well as asexual/sexual flocculation (FLOC.). Middle block: response surface showing the relative sensitivities of different yeast phases towards increasing levels of aspirin.

When interpreting the literature, it is important to realize that aspirin may have additional effects. As well as inhibiting mitochondrial β-oxidation (Glasgow et al., 1999) and uncoupling mitochondrial oxidative phosphorylation and/or inhibiting electron transport (Somasundaram et al., 1997; Norman et al., 2004), aspirin may also cause side effects in mitochondria as well as whole cells. For instance, aspirin may induce apoptosis in many cell types by caspase activation through mitochondrial cytochrome c release (Pique et al., 2000). In the cell, aspirin may cause acetylation of COX-1, resulting in the cessation of the production of physiologically important prostaglandins (Cena et al., 2003). Research on aspirin also suggests that this NSAID has beneficial antioxidant properties by reducing O2 production through lowering of NADPH oxidase activity (Wu et al., 2002).

Concluding remarks

Studies since the late 1980s have revealed mitochondria, linked to the production of 3-OH oxylipins, as new target sites for the development of novel antifungals (Leeuw et al., 2007). To further evaluate the validity of this hypothesis, the existing database should be expanded to include more members of presently acknowledged fermentative and nonfermentative yeast genera (Kurtzman & Fell, 1998) as well as pathogenic molds. These observations should be expanded to in vivo studies to eventually evaluate the applicability of the hypothesis to humans with fungal infections. In addition, other NSAIDs, such as indomethacin, should be tested on their own and in combination with known antifungals for antifungal activity. In vitro studies have shown that the NSAID ibuprofen alone, and in combination with azoles, is a potent, medically useful antifungal in the treatment of candidosis, particularly when applied topically (Pina-Vaz et al., 2000). In this study, ibuprofen at 5 mg mL−1 inhibited growth, and at 10 mg mL−1 showed rapid fungicidal activity against Ca. albicans. These actions are attributed to metabolic alterations and cytoplasmic membrane damage at 5 and 10 mg mL−1, respectively. Unfortunately, no details are available in this study regarding metabolic changes induced by this NSAID.

We conclude that aspirin selectively inhibits mitochondrial activity at low concentrations in yeasts, whereas fermentation is less affected, thereby producing enough energy for growth. This can be demonstrated with comparative growth inhibition studies in which aspirin is added at different concentrations to respiring and respiratory-deficient yeasts. The use of petite mutants by inducing, for example, deletions in yeast mtDNA would be a place to start such research (Moller et al., 2001). The effect of aspirin on the growth of petite phenotype-positive vs. petite phenotype-negative yeasts should also clarify this matter. These studies may further assess the application of low-cost and commonly used NSAIDs such as aspirin as antifungals in human, animal and crop disease.

Representatives of the Mucorales should also be further studied, especially for their sensitivity towards NSAIDs. The literature suggests the presence of 3-OH oxylipins in Mucor genevensis and Pilobolus, where they are mainly associated with the highly productive asexual sporulation structures such as sporangia and sporangiospores (Strauss et al., 2000; Kock et al., 2001). What is the effect of aspirin on molds?

Recent genetic studies have shown that oxylipins called the precocious sexual inducers or ‘psi factors’ [i.e. secreted mixtures of mainly C8-hydroxylated oleic (18 : 1) and linoleic acid (18 : 2)] play a role in fungal life cycle control. These products of fatty acid oxygenases (PpoA, PpoB, and PpoC) change the ratio of asexual to sexual sporulation in the filamentous mold Aspergillus nidulans (Tsitsigiannis et al., 2005). Studies to determine whether 3-OH oxylipins exert similar effects should now be performed. However, the inhibition of 3-OH oxylipins by aspirin may only be a secondary linked event during mitochondrial inhibition, in which the latter is the main factor contributing to the bias towards asexual reproduction, i.e. through a lack of sufficient energy production to fuel the high production and assembly rate necessary for enlarged sexual cells to form spores. It has also been reported that aspirin induces changes in mitochondrial energy production through the uncoupling of oxidative phosphorylation and/or inhibition of electron transport in the rat intestine (Somasundaram et al., 1997). Is the same true in yeasts?

In addition, oxylipins produced by the Ssp1 protein (a ssp1 gene product) in Ustilago maydis, which is similar to the fatty acid dioxygenase (linoleate diol synthase), were found to be localized on lipid bodies in germinating teliospores. This is probably needed for the mobilization of storage lipids during spore germination (Huber et al., 2002). These studies suggest that oxylipins with different structures (hydroxyl groups on different carbons), chain lengths and origins are associated with sexual/asexual reproduction modes in fungi. It is interesting to note that lipid globules present in the asci of the yeast Dipodascopsis uninucleata also contain 3-OH oxylipins, as demonstrated by immunogold labeling (Smith et al., 2000b). This has probably been produced in mitochondria and then deposited in the lipid globules.

Mitochondria presumably evolved from Gram-negative bacteria such as the rickettsias through endosymbiosis many millions of years ago (Gray et al., 2001). Interestingly, these bacteria also produce 3-OH oxylipins as part of their extracellular lipopolysaccharide layer (Rietschel et al., 1994; Amano et al., 1998). This intriguing correlation raises several important questions. Is it possible that this characteristic was maintained over these years? Can the types of 3-OH oxylipin produced by these bacteria today, as well as those of yeasts, assist in elucidating the origin of yeast mitochondria? Can 3-OH oxylipins, which are necessary for bacterial endotoxicity, also be inhibited by aspirin (Kock et al., 2005)?

Various types of 3-OH oxylipin, probably including novel ones, are produced by yeasts. Although a major fragment with a molecular mass of 175 obtained by EI MS is indicative of a methylated-silylated 3-OH oxylipin (hydroxyl group on carbon 3), this method is in many cases insufficient to identify the total structure of such a compound (Van Dyk et al., 1991). Consequently, more sophisticated GC-MS protocols (Ciccoli et al., 2005) and NMR analysis (Van Dyk et al., 1993) should now be used to further characterize these oxylipins.

In the light of this review, the recent prediction that oxylipin research in fungi will find practical application over the next 10 years seems realistic (Erb-Downward & Huffnagle, 2006), especially as oxylipin studies enabled the identification of an antifungal target site for aspirin, a cheap and commonly used anti-inflammatory drug. However, aspirin administered to humans over extended periods of time and at high dosages can be toxic, sometimes leading to significant morbidity and mortality (Wolfe et al., 1999; Cena et al., 2003; Wikipedia, 2006). For instance, long-term therapeutic use of aspirin may be harmful and result in gastrotoxicity (Wolfe et al., 1999). Will it be possible to combat systemic fungal infections in humans, animals and plants with NSAIDs at nontoxic levels and with prolonged usage? It will be interesting to assess the strategy followed by nonfermentative and fermentative fungi in developing resistance against these new types of antifungal (Ghannoum & Rice, 1999).


The authors wish to thank the National Research Foundation in South Africa for financial support.


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