Genomic approaches to explore virulence in the fungal pathogen Cryptococcus neoformans

The fungal pathogen Cryptococcus neoformans is the leading cause of encephalitis in people with the acquired immunodeficiency syndrome (AIDS). The range of drugs available to treat C. neoformans is limited in number and efficacy; therefore, there is a dire need for new, more powerful therapeutics. To achieve this we need to better understand how the fungus can cause disease, which will lead to the identification of factors relating to virulence and ultimately may provide new drug targets. The plethora of emerging genomic resources has allowed for targeted biology to focus on key genes in important cellular processes that relate to virulence. The work described in this thesis contributes to the development of genomic resources for biological investigations in Cryptococcus neoformans . This work has three specific components: (1) physical mapping of the genomes for strains serotypes A and D; (2) analysis of the low iron transcriptome in a serotype A strain; and (3) characterization of a putative siderophore (iron) transporter gene, SIT1, that was identified by transcriptome analysis. The first component involved the hybridization of 125 markers to a set of 9,216 BAC clones from the strain JEC21 (serotype D) and to 6,528 clones from the strain H99 (serotype A). These data provided the first genome-wide comparison of gene synteny between two strains of the fungus, and linked contigs to specific karyotype bands. The second component of the work involved the analysis of the low iron transcriptome for the serotype A strain, H99 using serial analysis of gene expression (SAGE). A number of interesting genes were identified in the low iron transcriptome including those involved with the response to stress and mechanisms of iron uptake. A key finding was that the low iron transcriptome was remarkably similar to the in vivo library from cells grown in rabbit cerebral spinal fluid (CSF) and significantly distinct from the libraries grown in yeast nutrient broth (YNB). The third component of the work focused on the gene SIT1, which encodes a putative siderophore transporter. The gene was characterized in three strain backgrounds of varying virulence. This work showed that SIT1 was important for iron utilization in conditions of low iron or when a siderophore was provided as the sole iron source. Further, there were pleiotrophic phenotypes for a number of virulence-related attributes including melanization, cAMP signaling and cell wall integrity. Finally, throughout the entire body of work, multiple differences were identified between strains of the same or different serotypes on a genomic and biological level, and this variation may lend insight into differences in virulence between strains.