Oxylipins in Cryptococcus neoformans and related yeasts

English: Literature shows that Cryptococcus neoformans is an important human pathogen responsible for many deaths worldwide. To compound this, treatment of cryptococcal infections has over the years been difficult. This is largely due to the widespread use of antifungals, leading to the emergence of drug resistant strains. The capsule (with glucuronoxylomannan as major polysaccharide) is the principal virulence factor of this pathogen, and can influence the hosts’ immune response. Moreover, recent studies have identified novel bioactive compounds, which can also contribute to the virulence of pathogens such as Cryptococcus neoformans and Candida albicans. These include compounds such as oxylipins (oxidized fatty acids), which have been reported to modulate the hosts’ immune response during infections. This exposes new targets for antifungal action. In this study, the 3-hydroxy fatty acid, 3-OH 9:1, has been discovered in Cryptococcus neoformans var. neoformans UOFS Y-1378 using gas chromatographymass spectrometry. Immunofluorescence confocal laser scanning microscopy and immunogold transmission electron microscopy revealed that this 3-OH oxylipin accumulates in capsules, where it is released as hydrophobic droplets through protuberances (each about 30 nm x 400 nm) into the extracellular environment. This discovery further expands our knowledge of the known spectrum of biologically active compounds associated with this main virulence factor of Cryptococcus neoformans. 3-OH 9:1 is produced in yeast mitochondria probably through β-oxidation or fatty acid synthesis pathway type II (FAS II). Evidence supporting this statement, was provided after mapping the migration of 3-OH oxylipin-containing osmiophilic material during ultrastructural studies. Here, osmiophilic material was shown to originate in mitochondria and is deposited inside the yeast cell wall, from where it is released into the surrounding medium, along capsule protuberances or through capsule detachment. When acetylsalicylic acid (ASA, an inhibitor of mitochondrial function – including 3-OH oxylipin production) was added, the migration of the osmiophilic material as well as capsule detachment from cell walls and hence oxylipin release was abrogated. This data is in accordance with literature, where a novel release mechanism for the major virulence factor of Cryptococcus neoformans is reported. Here, virulent polysaccharide packaged lipid vesicles are reported to cross the cell wall and the capsule into the surrounding environment. This Ph.D. study implicates the lipid vesicles to contain 3-OH oxylipins. It was also demonstrated that 3-OH oxylipins are widely distributed in other members of the pathogenic yeast genus Cryptococcus, following immunofluorescence confocal laser scanning microscopy (using antibodies directed towards 3-OH oxylipins) and gas chromatography-mass spectrometry. In the examined strains these compounds were mainly associated with cell wall surfaces, protuberances, appendages and collarettes. According to literature, yeasts that are dependent only on mitochondrialaerobic respiration for growth, are more sensitive to ASA compared to yeasts that possess both energy production pathways i.e. aerobic respiration and fermentation. In this study, in vitro data corroborate this hypothesis. Here, the growth of all nonfermenting Cryptococcus species was much more sensitive to ASA compared to the fermentative yeast, Saccharomyces cerevisiae (which could tolerate as much as 5 mM ASA). Already at an ASA concentration of 2 mM, a decrease in growth of most Cryptococcus species was evident, and at 3 mM ASA, the growth of all Cryptococcus species was significantly inhibited. The observed ASA effect may be due to inhibition of mitochondrial function, which includes inhibition of oxidative phosphorylation and respiratory electron transport chain – functions important for energy generation. These data suggest that ASA can be used as an antimitochondrial antifungal agent to combat growth of these pathogenic yeasts. This discovery should now be further researched in vivo taking into account the toxicity of ASA and other non steroidal anti-inflammatory drugs.