Comparative genome hybridization reveals widespread genome variation in pathogenic cryptococcus species

Genome variability can influence the virulence of pathogenic microbes. The availability of genome sequences for strains of the AIDS-associated fungal pathogens Cryptococcus neoformans and C. gattii presented an opportunity to use Comparative Genome Hybridization (CGH) to examine genome variability between strains of different molecular subtypes and ploidy. CGH analysis of 15 strains revealed extensive genomic variation including regions of difference (deletions and amplifications) and chromosome copy number variability. Although no common genomic change was observed for these 15 strains, three key observations came out of these studies. First, CGH identified putative recombination sites and the origins of specific segments of the genome for the common laboratory strain, JEC21. Second, CGH and subsequent PCR-RFLP (PCR-Restriction Fragment Length Polymorphism) analysis on 33 clinical, environmental and laboratory-generated AD hybrid strains revealed that chromosome 1 from the serotype A genome is preferentially retained in clinical strains. Third, CGH and subsequent qRT-PCR (quantitative real-time PCR) analysis revealed disomy for chromosome 13 in two clinical strains: CBS7779 and WM626. Further qRT-PCR and phenotypic studies on CBS7779 revealed a correlation between variable melanin production and disomy. Specifically, highly melanized strains were monosomic for chromosome 13 and less melanized strains were disomic for this chromosome. This correlation, however, only held for the initial CBS7779 isolates. That is, subsequent screens of highly-melanized and less-melanized isolates derived from the initial CBS7779 strain no longer followed this pattern. These subsequent screens, however, did reveal that 1) disomy, once established, was a relatively stable trait and 2) having disomy at chromosome 13 seemed to increase the probability of developing disomy at chromosome 4. Finally, qRT-PCR of 13 additional strains from AIDS patients revealed that disomy of both chromosome 13 and chromosome 4 is common in freshly isolated, clinical strains. Overall, the data presented in this thesis reveal novel aspects of genome variability and lay the foundation for future studies on the relevance of variation in the virulence of C. neoformans.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat