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dc.contributor.advisorKock, J. L. F.
dc.contributor.advisorVan Wyk, P. W. J.
dc.contributor.advisorPohl, C. H.
dc.contributor.authorSebolai, Olihile Moses
dc.date.accessioned2015-11-09T09:57:20Z
dc.date.available2015-11-09T09:57:20Z
dc.date.copyright2007-11
dc.date.issued2007-11
dc.date.submitted2007-11
dc.identifier.urihttp://hdl.handle.net/11660/1529
dc.description.abstractEnglish: 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.en_ZA
dc.description.abstractAfrikaans: Volgens literatuur is Cryptococcus neoformans ‘n belangrike menslike patogeen, verantwoordelik vir ‘n groot aantal sterftes wêreldwyd. Om dit te vererger, was behandeling van cryptococcale infeksies oor die jare moeilik. Dit is grootliks as gevolg van die algemene gebruik van antifungale middels, wat gelei het tot die ontstaan van weerstandbiedende stamme. Die kapsule (met glukuronoxylomannaan as hoof polisakkaried) is die belangrikste virulensiefaktor van dié patogeen, en kan die gasheer se immuunrespons beïnvloed. Verder het onlangse studies nuwe bioaktiewe verbindings, wat ook mag bydra tot die virulensie van patogene soos Cryptococcus neoformans en Candida albicans, geïdentifiseer. Dit sluit verbindings soos oksielipiene (geöksideerde vetsure), wat gerapporteer is om die gasheer se immuunrespons te moduleer gedurende infeksies, in. Dit lê nuwe teikens vir antifungale aksies bloot. In hierdie studie is m.b.v. gaschromatografie-massaspektrometrie die 3- hidroksievetsuur, 3-OH 9:1, in Cryptococcus neoformans var. neoformans UOFS Y-1378 ontdek. Met behulp van immunofluoresensie konfokale laser skandeermikroskopie en immunogoudtransmissie-elektronmikroskopie is aangetoon dat hierdie 3-OH oksielipien in kapsules versamel, vanwaar dit as hidrofobiese druppels deur uitsteeksels (elk ongeveer 30 nm x 400 nm) in die ekstrasellulêre omgewing vrygestel word. Hierdie ontdekking verbreed ons kennis verder oor die bekende spektrum van biologies-aktiewe verbindings wat met die hoof virulensiefaktore van Cryptococcus neoformans geässosieer is. 3-OH 9:1 word in gismitochondria geproduseer, waarskynlik deur β-oksidasie of ‘n tipe II vetsuursinteseweg (FAS II). Getuienis ter stawing van hierdie stelling is verkry nadat die migrasie van 3-OH oksielipienbevattende osmiofilliese material gevolg is gedurende ultrastrukturele studies. Dit is aangetoon dat osmiofilliese materiaal in die mitochondria onstaan en binne die gisselwand neergelê word, van waar dit in die omliggende omgewing vrygestel word d.m.v. kapsulêre uitsteeksels of d.m.v. vrystelling van die kapsule self. As asetielsalisielsuur (ASA, ‘n inhibeerder van mitochondriale funksie – insluitend 3-OH oksielipienproduksie) bygevoeg word, word die migrasie van die osmiofilliese material sowel as vrystelling van die kapsule van die selwand en dus oksielipienvrystelling, opgehef. Hierdie data is in ooreenstemming met literatuur, waar ‘n nuwe vrystellingsmeganisme vir die hoof virulensiefaktor van Cryptococcus neoformans gerapporteer word. Hier word gerapporteer dat virulente polisakkariede, verpak in lipiedvesikels, die selwand en kapsule oorsteek na die omliggende omgewing. Hierdie Ph.D. studie impliseer dat die lipiedvesikels 3-OH oksielipiene bevat.Met behulp van immunofluoresensie konfokale laser skandeermikroskopie (met teenliggaampies teen 3-OH oksielipiene) en gaschromatografie-massaspektrometrie is dit ook gedemonstreer dat 3-OH oksielipiene wyd verspreid is in ander lede van die gisgenus Cryptococcus. In die bestudeerde stamme is hierdie verbindings hoofsaaklik geässosieer met selwandoppervlakke, uitsteeksels, aanhangsels en kragies. Volgens literatuur is giste wat slegs afhanklik is van mitochondriale aerobiese respirasie vir groei meer sensitief vir ASA in vergelyking met giste wat beide aerobiese respirasie en fermentasie kan uitvoer om energie te verkry. In hierdie studie ondersteun in vitro data hierdie hipotese. Hier is gevind dat die groei van alle nie-fermenterende Cryptococcus spesies baie meer sensitief was vir ASA in vergelyking met die fermenterende gis, Saccharomyces cerevisiae (wat soveel as 5 mM ASA kon hanteer). ‘n Afname in die groei van meeste Cryptococcus spesies was reeds duidelik teen ‘n ASA konsentrasie van 2 mM, en teen 3 mM ASA is die groei van alle Cryptococcus spesies beduidend geïnhibeer. Die waargenome effek van ASA mag wees a.g.v. die inhibisie van mitochondriale funksie, insluitend oksidatiewe fosforilering en respiratoriese elektrontransport – belangrike energiegenererende funksies. Hierdie data impliseer dat ASA as antimitochondriale antifungale middel gebruik kan word om die groei van hierdie patogene giste te beheer. Hierdie ontdekking behoort nou verder in vivo ondersoek te word, met inaggenome die toksisiteit van ASA en ander nie-steroïed antiinflammatoriese middels.af
dc.description.sponsorshipNational Research Foundationen_ZA
dc.description.sponsorshipA.W. Mellon Scholarshipen_ZA
dc.language.isoenen_ZA
dc.publisherUniversity of the Free Stateen_ZA
dc.subjectCapsuleen_ZA
dc.subject3-Hydroxy fatty aciden_ZA
dc.subjectLipidsen_ZA
dc.subjectCryptococcus neoformansen_ZA
dc.subjectAspirinen_ZA
dc.subjectThesis (Ph.D. (Microbial, Biochemical and Food Biotechnology))--University of the Free State, 2007en_ZA
dc.subjectAcetylsalicylic aciden_ZA
dc.subjectAntifungal agenten_ZA
dc.subjectElectron microscopyen_ZA
dc.subjectGrowth inhibitionen_ZA
dc.subjectImmunofluorescence confocal laser scanning microscopyen_ZA
dc.subjectMitochondriaen_ZA
dc.subjectYeasten_ZA
dc.titleOxylipins in Cryptococcus neoformans and related yeastsen_ZA
dc.typeThesisen_ZA
dc.rights.holderUniversity of the Free Stateen_ZA


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