Microbial, Biochemical and Food Biotechnology
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Browsing Microbial, Biochemical and Food Biotechnology by Subject "3-Hydroxy C9:0"
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Item Open Access The role of cryptococcal 3-hydroxy fatty acids in mediating Cryptococcus-amoebae interactions(University of the Free State, 2017-02) Madu, Lynda Uju; Sebolai, O. M.; Pohl, C. H.English: 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.