Browsing by Author "Samie, Shakier"

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    The impact of nitrogen limitation on virulence and the quorum sensing response in Cryptococcus neoformans
    (Stellenbosch : Stellenbosch University, 2020-04) Samie, Shakier; Volschenk, Heinrich; Trollope, Kim; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Cryptococcus neoformans may cause life-threatening meningitis in immune-compromised populations, with high mortality rates despite measures currently put in place to treat infections. It is, therefore, critical that new and effective anticryptococcal drugs and treatment strategies are developed. Using an antipathogenic approach, which does not directly kill the yeast but rather targets key virulence factors or quorum sensing (QS), is a promising, novel strategy. However, the regulation of cryptococcal QS, and how this process influences virulence factor expression, has not been characterised. In particular, the dynamics of these processes under nitrogen-limiting conditions, which the pathogen experiences in its natural environment and during host infection, has not been defined. This study aimed to characterise the expression of cryptococcal virulence factors during nitrogen limitation (NL) and whether this was being influenced by QS. Furthermore, this study aimed to engineer a C. neoformans QS biosensor to monitor QS responses during NL and to screen for potential compounds that could interfere with cryptococcal QS. After growth in nitrogen-limiting conditions relevant to the Cryptococcus natural niche, C. neoformans H99 displayed a thicker polysaccharide capsule, as well as higher urease and laccase activities. Melanisation and capsule thickness were specifically increased during both NL and temperature stress. Ergosterol biosynthesis also appeared to be induced during NL and temperature stress. A C. neoformans CK0289 QS mutant also expressed thicker capsules and increased urease activity during NL. However, capsule thickness, together with melanisation and ergosterol biosynthesis, were expressed notably weaker than C. neoformans H99. This suggests that NL does influence the virulence phenotype, but that QS may be required to signal for increased virulence factor and cell membrane ergosterol production during NL. Garlic-derived organosulfides and synthetic derivatives thereof were selected as compounds to screen for potential anti-QS activity against C. neoformans. The natural organosulfides showed potent antifungal activity and affected the expression of C. neoformans virulence factors. Specifically, capsule thickness was reduced and melanisation completely inhibited. Despite having no inhibitory effect on urease activity or ergosterol biosynthesis, these compounds still serve as possible candidates for the development of antipathogenic drugs against C. neoformans. To determine whether these compounds have quorum quenching activity, a biosensor responsive to cryptococcal QS and able to monitor changes in this process was designed. Although the biosensor was successfully constructed in Saccharomyces cerevisiae, no signal could be detected. This study has demonstrated that NL is an important signal that influences the virulence of C. neoformans, and that this relationship is potentially affected by QS. Further research is needed to comprehensively characterise this phenomenon as this would provide valuable information in understanding the physiology and pathogenicity of C. neoformans.