Doctoral Degrees (Microbiology)

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Browsing Doctoral Degrees (Microbiology) by Author "Bosch, Caylin"

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    The role of nitrogen limitation in cryptococcal virulence and drug tolerance
    (Stellenbosch : Stellenbosch University, 2021-12) Bosch, Caylin; Botha, Alfred; Volschenk, H.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Cryptococcosis, a disease caused by members of the Cryptococcus neoformans/Cryptococcus gattii species complex, has the highest incidence rate among systemic HIV-associated fungal infections and accounts for more than 15% of all AIDS-related deaths globally. The etiological agents of cryptococcosis are widely distributed within the environment with ecological reservoirs including pigeon guano and the woody debris of numerous tree species. Within its natural habitat, C. neoformans must adapt to severe nutrient stresses. In the present work, we delved into the effects of one such environmental stress, namely nitrogen limitation, as it relates to cryptococcal virulence. Moreover, we explored the clinical relevance of these effects by investigating the role of nitrogen stress in cryptococcal susceptibility to two commonly used antifungal drugs, amphotericin B (AmB) and fluconazole (FLU). By culturing C. neoformans and C. gattii in media with different ecologically relevant nitrogen concentrations, we found that low nitrogen conditions enhanced cryptococcal virulence factor production, as well as the levels of the membrane sterol and antifungal target, ergosterol. Evaluation of drug tolerance using time-kill methodology revealed that nitrogen limited cultures had the highest survival percentages in the presence of both AmB and FLU, suggesting that nitrogen concentration may indeed influence drug tolerance. For a deeper understanding of the effects of nitrogen limitation on pathogenic cryptococci, we investigated the transcriptomic response of C. neoformans to low nitrogen concentrations using RNA-sequencing. It was found that nitrogen limited conditions upregulated the expression of antifungal tolerance- related genes, including those involved in ergosterol biosynthetic processes and cell wall integrity. Low nitrogen conditions were also found to modulate the expression of numerous virulence-associated genes, such as CTR4 and CGP1, which encode a copper transporter and a microtubule-associated protein, respectively. Using gene deletion mutants, we demonstrated for the first time that Ctr4 and Cgp1 are functionally associated with cryptococcal adaptation to nitrogen availability, by contributing to cryptococcal growth in low nitrogen conditions, nitrogen source assimilation, oxidative stress tolerance and antifungal susceptibility. Finally, we evaluated the in vivo effects of cryptococcal pre-adaptation to nitrogen limited environments using a Galleria mellonella infection model and by studying cryptococcal- macrophage interactions. We showed that low nitrogen conditions enhanced the virulence of C. neoformans in an invertebrate host and demonstrated that this nutritional stress influences uptake of the fungus by human macrophages. These findings ultimately highlight the importance of isolate origin in the cryptococcal-host interaction. Altogether, the insights gained from our research greatly enhance our understanding of the role of nitrogen availability in cryptococcal pathogenesis and antifungal tolerance, and partially improve our knowledge on how nitrogen influences the survival of these fungal pathogens in natural and host niches.