Previous studies show that acetylsalicylic acid (aspirin) at low concentrations affects yeast sexual structure development in a similar fashion than oxygen depletion. This is ascribed to its anti-mitochondrial action. In this study, we report the same for other anti-inflammatory (i.e. ibuprofen, indomethacin, salicylic acid, benzoic acid) as well as anticancer (Lonidamine) drugs, also known for inhibiting mitochondrial activity in mammalian cells. This is shown by a unique yeast bio-assay, with the mitochondrion-dependent sexual structure, riboflavin production, and hyphal morphology of the yeast Eremothecium ashbyi serving as indicators. These drugs affect this yeast in a similar way as found under oxygen limitation conditions by inhibiting sexual structure development (most sensitive), riboflavin production, and yielding characteristically wrinkled and granular hyphae, presenting a unique "anoxic" morphological pattern for this yeast. Only drugs associated with anti-mitochondrial activity presented such a pattern. This bio-assay may find application in the screening for novel drugs from various sources with anti-mitochondrial actions.

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 beta-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.

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.

We previously implicated 3-hydroxy oxylipins and ascospore structure in ascospore release from enclosed asci. Using confocal laser scanning microscopy on cells stained with fluorescein-coupled, 3-hydroxy oxylipin-specific antibodies, we found that oxylipins are specifically associated with ascospores and not the vegetative cells or ascus wall of Ascoidea corymbosa. Using gas chromatography--mass spectrometry the oxylipin 3-hydroxy 17:0 could be identified. Here, we visualize for the first time the forced release of oxylipin-coated, hat-shaped ascospores from terminally torn asci, probably through turgor pressure. We suggest that oxylipin-coated, razor-sharp, hat-shaped ascospore brims may play a role in rupturing the ascus to affect release.

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.