Oxylipins in Cryptococcus neoformans and related yeasts

[摘要] English: Literature shows that Cryptococcus neoformans is an important human pathogenresponsible for many deaths worldwide. To compound this, treatment of cryptococcalinfections has over the years been difficult. This is largely due to the widespread use ofantifungals, leading to the emergence of drug resistant strains. The capsule (withglucuronoxylomannan as major polysaccharide) is the principal virulence factor of thispathogen, and can influence the hosts' immune response. Moreover, recent studieshave identified novel bioactive compounds, which can also contribute to the virulence ofpathogens such as Cryptococcus neoformans and Candida albicans. These includecompounds such as oxylipins (oxidized fatty acids), which have been reported tomodulate the hosts' immune response during infections. This exposes new targets forantifungal action.In this study, the 3-hydroxy fatty acid, 3-OH 9:1, has been discovered inCryptococcus neoformans var. neoformans UOFS Y-1378 using gas chromatographymassspectrometry. Immunofluorescence confocal laser scanning microscopy andimmunogold transmission electron microscopy revealed that this 3-OH oxylipinaccumulates in capsules, where it is released as hydrophobic droplets throughprotuberances (each about 30 nm x 400 nm) into the extracellular environment. Thisdiscovery further expands our knowledge of the known spectrum of biologically activecompounds associated with this main virulence factor of Cryptococcus neoformans.3-OH 9:1 is produced in yeast mitochondria probably through β-oxidation or fattyacid synthesis pathway type II (FAS II). Evidence supporting this statement, wasprovided after mapping the migration of 3-OH oxylipin-containing osmiophilic material during ultrastructural studies. Here, osmiophilic material was shown to originate inmitochondria and is deposited inside the yeast cell wall, from where it is released intothe surrounding medium, along capsule protuberances or through capsule detachment.When acetylsalicylic acid (ASA, an inhibitor of mitochondrial function �?including 3-OHoxylipin production) was added, the migration of the osmiophilic material as well ascapsule detachment from cell walls and hence oxylipin release was abrogated. This datais in accordance with literature, where a novel release mechanism for the majorvirulence factor of Cryptococcus neoformans is reported. Here, virulent polysaccharidepackaged lipid vesicles are reported to cross the cell wall and the capsule into thesurrounding environment. This Ph.D. study implicates the lipid vesicles to contain 3-OHoxylipins.It was also demonstrated that 3-OH oxylipins are widely distributed in othermembers of the pathogenic yeast genus Cryptococcus, following immunofluorescenceconfocal laser scanning microscopy (using antibodies directed towards 3-OH oxylipins)and gas chromatography-mass spectrometry. In the examined strains these compoundswere mainly associated with cell wall surfaces, protuberances, appendages andcollarettes. According to literature, yeasts that are dependent only on mitochondrialaerobicrespiration for growth, are more sensitive to ASA compared to yeasts thatpossess both energy production pathways i.e. aerobic respiration and fermentation. Inthis study, in vitro data corroborate this hypothesis. Here, the growth of all nonfermentingCryptococcus species was much more sensitive to ASA compared to thefermentative yeast, Saccharomyces cerevisiae (which could tolerate as much as 5 mMASA). 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 Cryptococcusspecies was significantly inhibited. The observed ASA effect may be due to inhibition ofmitochondrial function, which includes inhibition of oxidative phosphorylation andrespiratory electron transport chain �?functions important for energy generation. Thesedata suggest that ASA can be used as an antimitochondrial antifungal agent to combatgrowth of these pathogenic yeasts. This discovery should now be further researched invivo taking into account the toxicity of ASA and other non steroidal anti-inflammatorydrugs.

[发布机构] University of the Free State