Browsing by Author "Bhana, Zoe Tyler"

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    Nutritional-associated changes in lipid metabolism affecting the pathogenicity of Cryptococcus neoformans
    (Stellenbosch : Stellenbosch University, 2022-04) Bhana, Zoe Tyler; Volschenk, Heinrich; Botha, Alfred ; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Cryptococcus neoformans is the etiological agent for the disease, cryptococcal meningitis, affecting approximately 250,000 individuals, with an estimated 180,000 attributable deaths each year. The effect of this disease is exacerbated by the ubiquitous distribution of C. neoformans in the natural environment, being widely distributed within ecological reservoirs, including the woody debris of numerous tree species and pigeon guano. Within these habitats, C. neoformans encounters severe nutrient limitation, particularly in bioavailable nitrogen. Since it has been previously demonstrated that markedly low nitrogen concentrations commonly experienced in their arboreal habitats enhance the in vitro virulence factor production of C. neoformans, it became important to further elucidate this yeast’s genetic response to nitrogen stress to better understand its adaptation to the severely nutrient-limited host environment. Lipid metabolism has been identified as a core virulence function of C. neoformans required for its entire infection process and several lipid species have been shown to influence cryptococcal virulence. Although it is known that lipid metabolism and nitrogen limitation (NL) are connected to the virulence of C. neoformans, the tripartite relationship between NL, lipid metabolism, and cryptococcal virulence has not been characterised. In particular, the regulatory role of lipid metabolism in the enhanced virulence phenotype of C. neoformans during growth in nitrogen-limiting conditions relevant to its arboreal habitats has not been defined. Therefore, this study aimed to investigate the nitrogen limiting effect on the transcriptional landscape of C. neoformans, with a focused view on lipid metabolism and the lipid profile and distribution within the cryptococcal membrane. Furthermore, this study aimed to interrogate the influence of lipid metabolism on the in vitro virulence factor production of C. neoformans during NL. Here we show that the lipid metabolism of C. neoformans is affected during nitrogen-limited growth as the differential gene expression data revealed lipid metabolism as one of the major biological processes significantly enriched under NL conditions with several genes involved in lipid metabolic processes being differentially regulated. These included genes were involved in sphingolipid-, carnitine-, inositol phosphate- and fatty acid metabolism. Four lipid-related deletion strains, namely the carnitine carrier, CNAG_00675 (cacΔ), sphingolipid methyltransferase, CNAG_01475 (smtΔ), long-chain fatty acid transporter, CNAG_01191, (lfatΔ) and an inositol polyphosphate phosphatase, CNAG_06065, (sac101Δ) were then selected to experimentally evaluate whether these highly regulated genes related to lipid metabolism confer the enhanced virulent phenotype of C. neoformans associated with NL. Indeed, carnitine, fatty acid, sphingolipid, and inositol phosphate metabolism were implicated in the production of the primary cryptococcal virulence factors, namely the polysaccharide capsule and melanisation. Specifically, capsule sizes and melanisation was significantly reduced in the cacΔ, smtΔ, lfatΔ and the sac101Δ deletion strains at low nitrogen (LN) at 30°C (LN30) condition compared to the H99 wild-type. Our results revealed how these lipid metabolic processes are directly linked to the virulence factor elaboration of C. neoformans during NL. Similarly, capsule sizes were significantly reduced in cacΔ and lfatΔ deletion strains at the LN at 37°C (LN37) condition, however, melanisation was significantly increased in these deletion strains compared to the laccase activity displayed by H99. This suggests that the deletions of CAC and LFAT, together with the compounding effect of LN and high-temperature stress, differentially regulate this virulence factor during growth at 30°C. Ergosterol production was also significantly affected by the deletions of the carnitine carrier and the long-chain fatty acid transporter at LN37, as lower ergosterol levels were measured compared to H99. Similar observations were made for the smtΔ and cacΔ strains at LN30. These results implicate these lipid genes in regulating membrane ergosterol levels in C. neoformans which could be potential drug targets for anticryptococcal therapies. By further analysing the effect of NL on the lipid composition of C. neoformans, it was confirmed that nitrogen stress does influence its lipid profile and distributions. In particular, the phospholipid (PL) species profile of H99 varied significantly during growth at the LN vs HN conditions at both 30°C and 37°C. Specifically, phosphatidylethanolamine (PE) and phosphatidylcholine (PC) were identified as the major PLs, however, their prominence was differently distributed across all the conditions. At LN conditions, PCs were the most abundant at 30°C, whereas PE species were more prominent at 37°C. This suggests that nitrogen and temperature stress strongly influenced the PL profile of C. neoformans. As it is known that the PL composition greatly contributes to the membrane properties and the ultimate function and fitness of a cell, these results indicate that the observed changes in the PL composition could contribute to the virulence factor production of C. neoformans. Although this is yet to be elucidated, this study provides the groundwork for future research on the tripartite relationship between lipids, nitrogen stress, and pathogen fitness/virulence in C. neoformans. Overall, this was the first study to demonstrate that the nitrogen status of the external milieu, commonly found in the arboreal niches of C. neoformans, influences its lipid metabolism and that this relationship directly influences the in vitro virulence factor production of H99. Further research should aim at exploring the regulatory role of lipid metabolism in the virulence of C. neoformans during NL as this would provide potential gene targets for anticryptococcal therapies as well as novel insights into the physiology and pathogenesis of C. neoformans within its primary ecological niches.