Diversity and ontogeny of Cryptococcus neoformans var. grubii originating from South Africa

Botes, Angela (2007-12)

Thesis (MSc) -- Stellenbosch University, 2007.

Thesis

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.

AFRIKAANSE OPSOMMING: Cryptococcus neoformans (Sanfelice) Vuillemin is 'n opportunistiese fungus patogeen wat verantwoordelik is vir breinvliesontsteking hoofsaaklik in immuno-gekompromiteerde individue, veral in diegene met menslike immunovirus (MIV) verworwe immuniteitsgebreksindroom (VIGS). Twee hoof variëteite is bekend, C. neoformans var. neoformans (serotipe D) en C. neoformans var. grubii (serotipe A), asook 'n hibried, C. neoformans (serotipe AD). Hierdie serotipes mag aan een van twee paringstipes behoort, naamlik paringstipe a (MATa), of paringstipe alfa (MATα). Verskeie molekulêre tiperingsmetodes is voorheen ontwikkel om C. neoformans in vier hoof genotipiese groepe, naamlik VNI, VNII, VNIII en VNIV te klassifiseer. Benewens kliniese monsters, is hierdie giste bekend vir hul voorkoms in 'n wye verskeidenheid omgewingshabitatte, insluitende grond, voëlmis, bome en verrottende hout. Die studie het twee hoof doelwitte, eerstens om 'n aanduiding te kry van die algemene C. neoformans genotipes wat in die MIV positiewe en VIGS populasies van Suid-Afrika voorkom asook die verspreiding daarvan in die omgewing, veral in die Wes-Kaap Provinsie. Tweedens, om te bepaal of C. neoformans in staat is om te groei en 'n interaksie met geselekteerde mikrobes te hê wanneer dit op houtagtige plantafval gekweek word en of C. neoformans in staat is om sy ontogeniese stadia op die plantafval te produseer. Ten spyte van pogings om C. neoformans uit 256 omgewingsmonsters vanuit 'n verskeidenheid habitatte in Suid-Afrika te isoleer, is 'n totaal van slegs vier isolate uit die omgewing verkry. Nie een is uit omgewingsbronne in die Wes-Kaap van Suid-Afrika geïsoleer nie. Die vier omgewings C. neoformans var. grubii stamme, geïsoleer uit grond van die Noordwes Provinsie van Suid-Afrika, en 32 kliniese C. neoformans stamme afkomstig van die Gauteng en Wes-Kaap provinsies van Suid-Afrika, is vervolgens geïdentifiseer en gekarakteriseer. Stamme is geïdentifiseer deur die volgordebepaling van die interne getranskribeerde spasie (ITS) area van die ribosomale geengroep, terwyl serotipes en paringstipes is deur polimerase kettingreaksie (PCR) peilers bevestig. Die genotipe van elke stam is bepaal deur gebruik te maak van drie PCR-gebasseerde tiperingstegnieke, naamlik PCR-tipering met behulp van die mini-satelliet M13, mikrosatelliet (GACA)4 en lukraak ge-amplifiseerde polimorfiese DNA (RAPD) analise, asook beperkings-fragment-lengte-polimorfisme (RFLP) analise van die fosfolipase B1 geen. 'n Totaal van 97 % van die stamme is geïdentifiseer as C. neoformans var. grubii (serotipe A), terwyl slegs een stam as C. neoformans var. neoformans (serotipe D) geïdentifiseer is. Alle stamme was MATα en haploïed. Die meerderheid van die stamme is in genotipe VNI (75.6 %) gegroepeer, sewe stamme behoort tot genotipe VNII (21.2 %), terwyl net een stam genotipe VNIV (3 %) verteenwoordig. Hierdie resultate is in ooreenstemming met vorige en huidige literatuur wat aandui dat C. neoformans var. grubii (serotipe A, MATα, VNI) vir die meerderheid van cryptococcus-infeksies verantwoordelik is. Al die C. neoformans stamme is vir houtdegraderende ensieme getoets deur middel van plaat-toetse. Alle stamme het positief getoets vir sellulase aktiwiteit, 6 % van die stamme het positief getoets vir lakkaseproduksie by 22 ºC, maar geen stamme was in staat om xilaan af te breek nie. Gevolglik is drie C. neoformans var. grubii stamme afkomstig van kliniese en omgewingsmonsters, almal verteenwoordigend van dieselfde genotipe (VNI) en paringstipe (MATα), geëvalueer vir groei op Acacia mearnsii en Eucalyptus camaldulensis afval. Terwyl minimale verskille tussen die twee stamme opgemerk is, het dié wat op A. mearnsii gekweek is, beduidend hoër selgetalle gelewer. Laastens is alle stamme op A. mearnsii en E. camaldulensis afval afgepaar, asook op V8 sap en gis-koolstofbasis (YCB) agar om te bepaal of C. neoformans stamme in staat is tot beide dikariotiese en monokariotiese vrugvorming wanneer dit op houtagtige afval gekweek word. 'n Totaal van 19 %, 6 %, 42 % en 72 % van die C. neoformans stamme was in staat om op onderskeidelik A. mearnsii en E. camaldulensis afval, V8 sap en YCB agar te paar. Monokariotiese vrugvorming is opgemerk in 3 %, 3 % en 3 % van die stamme wanneer C. neoformans op onderskeidelik A. mearnsii, E. camaldulensis afval en YCB gekweek is. Hierdie mag die eerste waarneming wees van C. neoformans in 'n hifeuse fase wanneer gekweek op 'n medium wat slegs uit houtagtige afval bestaan, die veronderstelde natuurlike habitat van hierdie gis.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/79315
This item appears in the following collections: