Browsing by Author "Botes, Angela"

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    Diversity and ontogeny of Cryptococcus neoformans var. grubii originating from South Africa
    (Stellenbosch : Stellenbosch University, 2007-12) Botes, Angela; Botha, Alfred; Boekhout, T. E.; Stellenbosch University. Faculty of Sciences. Dept. of Microbiology.
    ENGLISH ABSTRACT: Cryptococcus neoformans (Sanfelice) Vuillemin is an opportunistic fungal pathogen responsible for causing meningitis predominantly in immuno-compromised individuals, particularly in those suffering from human immuno virus (HIV) acquired immuno-deficiency syndrome (AIDS). Two main varieties are known, C. neoformans var. neoformans (serotype D) and C. neoformans var. grubii (serotype A), as well as a hybrid variety, C. neoformans (serotype AD). These serotypes may belong to one of two mating types, namely mating type a (MATa), or mating type alpha (MATα). Several molecular typing methods were previously developed to classify C. neoformans into four major genotypic groups, namely VNI, VNII, VNIII and VNIV. In addition to clinical samples, these yeasts are known to occur in a wide diversity of environmental habitats including soil, avian guano, trees and decaying wood. The study had two main objectives, firstly to obtain an indication of the prevailing C. neoformans genotypes occurring within the HIV positive and AIDS populations of South Africa and to obtain an indication of its distribution within the environment, particularly within the Western Cape Province, South Africa. Secondly, to examine whether C. neoformans is able to grow and interact with selected microbes when cultured on woody debris and to determine if C. neoformans is capable of producing its ontogenic stages on this woody debris. Despite attempts to isolate C. neoformans from 256 environmental samples originating from a variety of habitats in South Africa, a total of only four isolates were obtained from the environment. None were isolated from environmental sources in the Western Cape Province, South Africa. The four environmental C. neoformans var. grubii strains isolated from soil in the North West province of South Africa, and 32 clinical C. neoformans strains originating from the Gauteng and Western Cape provinces of South Africa were subsequently identified and characterized. Strains were identified by sequencing the internal transcribed spacer (ITS) region of the ribosomal gene cluster, while serotypes and mating types were confirmed using polymerase chain reaction (PCR) primers. The genotype of each strain was determined by employing three PCR-based typing techniques, namely PCR fingerprinting using the mini-satellite M13, microsatellite (GACA)4 and random amplified polymorphic DNA (RAPD) analysis, as well as restriction fragment length polymorphism (RFLP) analysis of the phopholipase B1 gene. A total of 97 % of the strains were identified as C. neoformans var. grubii (serotype A), while only one strain was identified as C. neoformans var. neoformans (serotype D). All strains were found to be MATα and haploid. The majority of strains grouped into genotype VNI (75.6 %), seven strains represented genotype VNII (21.2 %), while only one strain represented genotype VNIV (3 %). These results are in accordance with previous and current literature stating that C. neoformans var. grubii (serotype A, MATα, VNI) is responsible for the majority of cryptococcal infections. Using plate assays, all the C. neoformans strains were screened for wood degrading enzymes. All strains tested positive for cellulase activity, 6 % of strains tested positive for laccase production at 22 ºC, but no strains were able to degrade xylan. Subsequently, three C. neoformans var. grubii strains, originating from clinical and environmental samples, all representing the same genotype (VNI) and mating type (MATα), were evaluated for growth on Acacia mearnsii and Eucalyptus camaldulensis debris. While minimal differences were noted between strains, those cultured on A. mearnsii yielded significantly higher cell counts. Finally, all strains were mated on Acacia mearnsii and Eucalyptus camaldulensis debris, as well as V8 juice and yeast carbon base (YCB) agar to determine whether C. neoformans strains were capable of both dikaryotic and monokaryotic fruiting when cultured on woody debris. A total of 19 %, 6 %, 42 % and 72 % of the C. neoformans strains were able to mate when crossed on A. mearnsii and E. camaldulensis debris, V8 juice and YCB agar, respectively. Monokaryotic fruiting was observed in 3 %, 3 % and 3 % of strains when C. neoformans was cultured on Acacia mearnsii, Eucalyptus camaldulensis debris and YCB, respectively. This may be the first observation of C. neoformans in a hyphal phase when cultured on medium comprised solely of woody debris, the perceived natural habitat of this yeast.
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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.