Doctoral Degrees (Microbiology)

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Browsing Doctoral Degrees (Microbiology) by Author "Botes, Angela"

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    Insights into the lignocellulosic physiology of the yeast pathogen Cryptococcus neoformans var. grubii
    (Stellenbosch : Stellenbosch University, 2014-04) Botes, Angela; Botha, Alfred; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Cryptococcus neoformans (Sanfelice) Vuillemin is an opportunistic pathogen that causes cryptococcal meningitis, predominantly in immuno-compromised individuals, particularly in those suffering from human immuno virus (HIV) or acquired immuno-deficiency syndrome (AIDS). This basidiomycetous yeast species is sub-divided into two main varieties, C. neoformans var. neoformans (serotype D) and C. neoformans var. grubii (serotype A), as well as a rare hybrid variety, C. neoformans (serotype AD). The global incidence of cryptococcosis among AIDS sufferers is approximately 30 % with 90 % of these cases being attributed to serotype A. Cryptococcus neoformans has been isolated from numerous environmental sources including guano, soil, and particularly decaying wood and tree hollows. Infection occurs when particles originating from these environments are inhaled. The ecological niche of C. neoformans was thought to be avian guano; however, recent findings indicate that the true ecological niche may rather be woody material. Representatives of this species, particularly C. neoformans var. grubii, were found to grow on agar plates containing carboxymethyl cellulose as carbon source; however, little is known about its ability to degrade hemi-cellulose. As such, the overall aim of this project was to study the interactions of C. neoformans var. grubii ATCC H99 with cellulosic and hemi-cellulosic materials. Growth studies revealed that C. neoformans var. grubii ATCC H99 was capable of utilizing carboxymethyl cellulose, glucomannan and galactomannan as sole carbon sources. This yeast also assimilated simple degradation products of lignocellulose such as L-arabinose, D-galactose, D-glucose, D-mannose, L-rhamnose and D-xylose. D-Mannose and D-glucose resulted in the highest maximum specific growth rates. Screening the genome of C. neoformans var. grubii ATCC H99 resulted in the identification of three putative cellulases, specifically an endo-glucanase (EC7) and two cellulases (CC1 and CC6). Evaluation of the deduced amino acid sequences indicated that all three enzymes belong to glycoside hydrolase family 5 (GHF5). Phylogenetic analyses revealed that the three enzymes grouped in distinct clades with other GHF5 members. Automated homology modeling of the three-dimensional structure revealed that CC1 and CC6 displayed the classical (α/β)8 TIM barrel fold associated with GHF5. Modeling of EC7 did not produce the classic GHF5 structure, suggesting that this enzyme may be classed in a separate GHF. Green fluorescent protein (GFP) tagging confirmed that CC6 forms part of the cryptococcal secretome. Real-time quantitative PCR (qPCR) analyses indicated that the three proteins responded differently on a transcriptional level in the presence of various carbohydrates. Cellulase CC6 displayed the most dynamic expression profile, indicating up-regulation in the presence of mannose, galactose and cellobiose. The use of Acacia mearnsii debris and the aqueous extract thereof also resulted in a significant up-regulation of all three enzymes. This confirms previous findings that the woody phyloplane is a natural habitat of C. neoformans var. grubii. In the presence of mucin, the transcription of CC6 was up-regulated. Similar to laccase and urease, CC6 may aid the survival of C. neoformans within the human respiratory system. Understanding the carbohydrate metabolic regulatory system and its impact on virulence would increase our overall knowledge of this pathogen‘s survival capabilities and infection strategies.