Sebolai, O. M.Pohl, C. H.Madu, Lynda Uju2017-07-132017-07-132017-02http://hdl.handle.net/11660/6464English: In this dissertation; an attempt was made to elucidate the role of 3-hydroxy C9:0 in determining the fate of cryptococcal cells when acted upon by amoebae. First, it was sought to determine the physiological concentrations of 3-hydroxy C9:0 that were secreted by C. neoformans UOFS Y-1378. Through using LCMS, it was established that cells secreted 0.2 mM of these molecules. Thus, in the design of some studies 1 mM was used in order to establish if a dose-dependent response would be established. The direct effect of these molecules on amoebal growth was next investigated. Here, it was clear that at physiological concentrations, these 3-hydroxy fatty acids did not negatively affect the growth of amoebae. This finding was critical as it pointed that these molecules probably effected a signalling mechanism in amoebae that may promote the survival of cryptococcal cells when interacting with amoebae. Could the concerned mechanism involve phagocytosis? To answer this question, co-culture experiments were designed wherein cryptococcal cells were deliberately fed to amoebae and in some experiments 3-hydroxy C9:0 was added to the co-culture media. It became evident that cryptococcal cells, which did not naturally produce 3-hydroxy C9:0, were more readily: 1) internalised, and 2) phagocytosed by amoebae. Interestingly, when 3-hydroxy C9:0 was artificially added (to the co-culture media) the cryptococcal cells became resistant to amoeba. This response was dose-dependent. Additionally, the strain UOFS Y-1378 (naturally produces 3-hydroxy C9:0) was more resistant to amoeba when compared to the other strains that did not produce 3-hydroxy C9:0. These results suggested that these molecules protected cells against amoebal phagocytosis. To investigate the manner/mechanism(s) through which cells were protected, a number of further experiments were designed. In one experiment, fetuin A was analysed. This protein is reported to be pivotal in enhancing the phagocytic efficiency of macrophages. Thus, could such a molecule be present in amoeba and important be manipulated by 3-hydroxy C9:0? The data showed that amoebae produced a fetuin A-like molecule. The levels of this molecule were low, similar to levels obtained in the presence GXM, in the presence of 3-hydroxy C9:0. This suggested that 3-hydroxy C9:0 may lower the number of cryptococcal cells that could potentially be internalised by amoebae. Thus, this molecule may be anti-phagocytic. Additionally, it seems that 3-hydroxy C9:0 may also protect cryptococcal cells that could be internalised and exposed to the harsh internal environment of amoebal food vacuole. This assertion was tested after experiments were designed mimicking the internal food vacuole environment. To be specific, it was demonstrated that an acapsular strain that was devoid of 3-hydroxy C9:0 (which in theory should be susceptible to hydrogen peroxide) was resistant when exposed to hydrogen peroxide in the presence of 3-hydroxy C9:0. Moreover, strains that were devoid of 3-hydroxy C9:0 were more susceptible to amoebapore (amoebal anti-microbial peptide found inside the food vacuole) compared to a strain that naturally produces these 3-hydroxy acids. When considering all these results, it is reasonable to suggest that it seems that 3-hydroxy C9:0 protected cells against amoebal phagocytosis. This, therefore, highlights the production pathway of these molecules as targets for developing drugs that may impair the pathogenesis of cryptococcal cells. It was thus not surprising to note that when the strain UOFS Y-1378 was exposed to aspirin, a known inhibitor of 3-hydroxy fatty acid production, its cells became susceptible to amoebal phagocytosis. The findings recorded in the dissertation also have implications for the fate of cryptococcal cells when acted upon by macrophages. The findings suggest that cells may deploy 3-hydroxy C9:0, possibly in concert with the capsule, in order to evade immuno-processing leading to a diseased-state in a susceptible host. In clinical settings, it is well known that without the prospect of treatment a diseased-host could die within three months when immunocompromised. Thus, the idea would be administering a cheap drug like aspirin could assist in combating cryptococcal infections.Afrikaans: In die verhandeling is 'n poging aangewend om die rol van 3-hidroksie C9:0 in die bepaling van die lot van Cryptococcus-selle, in die teenwoordigheid van amoebae, uit te klaar. Eerstens is gepoog om die fisiologiese konsentrasies van 3-hidroksie C9:0, wat afgeskei word deur C. neoformans UOVS Y-1378, te bepaal. Deur gebruik te maak van LCMS, is daar vasgestel dat 0.2 mM van hierdie molekule uitskei word deur die selle. In die studie is 1 mM gebruik ten einde vas te stel of daar 'n dosis-afhanklike reaksie is. Die direkte effek van hierdie molekules op die groei van amoebas is vervolgens ondersoek. Hieruit het dit geblyk dat 3-hidroksie vetsure, teen fisiologiese konsentrasies, nie ʼn negatiewe invloed op die groei van amoebas het nie. Hierdie bevinding is van kritieke belang aangesien dit daarop dui dat hierdie molekules waarskynlik 'n sein-meganisme bewerkstellig in amoebas, wat die oorlewing van Cryptococcus-selle kan bevorder, wanneer hulle interaksie met amoebas ondergaan. Kan die betrokke meganisme fagositose behels? Om hierdie vraag te beantwoord, is ko-kultuur eksperimente ontwerp waarin Cryptococcus-selle doelbewus vir amoebas gevoer is in die teenwoordigheid en afwesigheid van 3-hidroksie C9:0 in die media. Dit het geblyk dat Cryptococcus-selle, wat nie van nature 3-hidroksie C9:0 produseer nie, meer geredelik: 1) geïnternaliseer, en 2) gefagositeer word deur amoebas. Dit is interessant dat wanneer 3-hidroksie C9:0 kunsmatig by die ko-kultuur media gevoeg word, die Cryptococcus-selle weerstand bied teen amoeba en die respons was dosis-afhanklike. Daarbenewens was stam UOVS Y-1378 (wat natuurlik 3-hidroksie C9:0 produseer) meer bestand teen amoeba in vergelyking met die ander stamme wat nie 3-hidroksie C9:0 produseer nie. Hierdie resultate het aangedui dat hierdie molekules selle teen fagositose deur amoebas beskerm. Om die wyse/meganisme(s) waardeur dit selle beskerm word te ondersoek, is 'n aantal verdure eksperimente ontwerp. In een eksperiment, is Fetuïen A ontleed. Hierdie proteïen is deurslaggewend in die verbetering van die fagositiese doeltreffendheid van makrofae. Kan so 'n molekule ook in amoebas teenwoordig wees en gemanipuleer word deur 3-hidroksie C9:0? Die data het getoon dat amoebae ʼn Fetuïen A-agtige molekule produseer. Die vlakke van hierdie molekule was laag in die teenwoordigheid van 3-hidroksie C9:0, soortgelyk aan vlakke wat in die teenwoordigheid GXM opgemerk is. Dit dui daarop dat 3-hidroksie C9:0 die aantal Cryptococcus-selle wat potensieel geïnternaliseer kan word deur amoebae mag verlaag. Dus, kan hierdie molekuul antifagosities wees. Verder blyk dit dat 3-hidroksie C9:0 ook Cryptococcus-selle kan beskerm wat geïnternaliseer en blootgestel is aan die harde interne omgewing van amoeba se voedselvakuool. Hierdie stelling is getoets nadat eksperimente ontwerp is wat die interne voedselvakuool omgewing naboots. Om spesifiek te wees, dit is bewys dat 'n kapsellose stam wat sonder 3-hidroksie C9:0 was (wat in teorie vatbaar vir waterstofperoksied moet wees) bestand was wanneer dit blootgestel is aan waterstofperoksied in die teenwoordigheid van 3-hidroksie C9:0. Verder was stamme sonder 3-hidroksie C9:0 meer vatbaar vir amoeba-porieë (anti-mikrobiese peptied gevind in die voedselvakuool) in vergelyking met 'n stam wat natuurlik hierdie 3-hidroksie vetsure produseer. By die oorweging van al hierdie resultate is dit redelik om voor te stel dat dit blyk dat 3-hidroksie C9: 0 beskerm selle teen fagositose deur amoebas. Dit beklemtoon dus die produksie weg van hierdie molekules as teikens vir die ontwikkeling van middels wat die patogenese van Cryptococcus-selle kan verminder. Dit was dus nie 'n verrassing om te sien dat wanneer die stam UOVS Y-1378 blootgestel is aan aspirien, 'n bekende inhibeerder van 3-hidroksie vetsuur produksie, dit vatbaar geraak het vir fagositose deur amoeba nie. Die bevindinge in die verhandeling het ook implikasies vir die lot van Cryptococcus-selle se interaksie met makrofae. Die bevindinge dui daarop dat selle 3-hidroksie C9:0, moontlik in samewerking met die kapsel, mag gebruik ten einde immuno-verwerking te ontduik, wat kan lei tot 'n siektetoestand in 'n sensitiewe gasheer. Uit ʼn kliniese oogpunt is dit algemeen bekend dat, sonder die vooruitsig van die behandeling, 'n immuunonderdrukte siek gasheer binne drie maande kan sterf. Dus, is die idee dat die toediening van 'n goedkoop middel soos aspirien, kan help in die stryd teen Cryptococcus-infeksies.en3-Hydroxy C9:0AmoebaAmoebaporeAspirinCapsule (GXM)CryptococcusFetuin AHydrogen peroxideInternalisationPhagocytosisDissertation (M.Sc. (Microbial, Biochemical and Food Biotechnology))--University of the Free State, 2017DissertationUniversity of the Free State