The haploid basidiomycete yeast Cryptococcus neoformans is an opportunistic human fungal pathogen that causes high morbidity and mortality among the immunocompromised population, particularly in the HIV-infected community. C. neoformans is divided into four haploid molecular types: C. neoformans var. grubii (molecular types VNI, VNII, and VNB) causes ~95% of infections and C. neoformans var. neoformans (molecular type VNIV) causes ~5% of infections. Studies have shown that while the genomes of C. neoformans var. grubii molecular type VNI strain H99 and C. neoformans var. neoformans molecular type VNIV strain JEC21 are highly syntenic overall, the presence of translocations, large-scale deletions and duplications have been identified. Previous studies have shown that extensive karyotypic alterations exist between C. neoformans strains collected not only from the natural environment versus clinical isolates, but also from amongst isolates obtained from the same patient at different time points during infection. Karyotypic change can occur early or late during infection, and likely confers a selective advantage in the human host as karyotypic variants often predominate in later stages of infection. Common evolutionary mechanisms by which adaptation occur include single nucleotide polymorphisms, chromosomal rearrangements, gene duplications and transposon movements. These events are often found in the subtelomeric regions of chromosomes, the transitional regions between chromosome-specific sequences and telomeric repeat units.
This thesis begins with the characterisation of the subtelomeres of the C. neoformans var. grubii molecular type VNI strain H99. The average length of the telomere tract in strain H99 is ~110 bp, and the number of telomere repeat units varied between 6 – 21 units. The length of the subtelomeres in C. neoformans was found to be ~40 kb, defined by the enrichment of sugar transporters in these regions. The gene density of C. neoformans subtelomeres is equivalent to the gene density in the interstitial region of chromosomes, with no discernible boundaries between the subtelomeres and the transition into the interstitial regions of the chromosomes. Short segments of the subtelomeres in H99 were found to be duplicated and subsequently undergone translocation. The majority of the predicted genes are uncharacterised; based on protein domain analyses, the genes are predicted to possess enzymatic and transport functions. Analyses of subcultures of the C. neoformans var. grubii reference strain H99 from different laboratories revealed that a ~20 kb region containing nine putative genes in the right subtelomere of chromosome 9 is absent in two H99-derived strains, H99 Eunuch and H99 CMO18. This finding supports the occurrence of small-scale rearrangements in the subtelomeres of C. neoformans. In order to determine if there are any differences in the subtelomeres between the other sequenced strains of Cryptococcus that could give an insight into why one molecular type would be more virulent than the others, and also the possible changes that could take place during infection, comparative bioinformatic analyses against strain H99 was carried out to using the sequenced genomes of C. neoformans var. neoformans strain JEC21 and the less pathogenic sister species, Cryptococcus gattii strain WM276. Comparative analyses revealed that most subtelomeres from the different genomes were generally syntenic. However, a number of subtelomeres in the three genomes had undergone rearrangements; two subtelomeres in JEC21 and WM276 respectively, correspond to interstitial regions of the H99 genome, while five subtelomeres in H99 have been rearranged based on comparison to the regions in JEC21 and WM276.
While the comparison of the three species of Cryptococcus does give insight into the differences in the subtelomeres, it is not representative of the possible changes that could take place during clinical infection. In order to gain a better understanding of the changes taking place in the subtelomeres during infection, a comparison of subtelomeric lengths between serial clinical isolates was performed using Southern hybridisation. While the majority of the subtelomeres did not show significant changes in length, we found evidence of a gene amplification event that occurred in the right subtelomere of chromosome 3. A gene amplification involving the arsenite efflux transporter-encoding gene ARR3 was found in isolates belonging to the molecular type VNI subclade A5. This is the first evidence showing that adaption through gene amplification was a mechanism that is employed by the fungus in the environment, and possibly during infection.
The work from this study provides a better understanding of the mechanisms that C. neoformans possibly employs during adaptation in the environment, and opens up new avenues of research into other adaptive mechanisms that might be at play in the fungus during infection.