Functional Genomics: A program to use serial analysis of gene expression (SAGE), microarrays, RNA-Seq and proteomics for basdiomycete fungal pathogens.
We have initiated a program to use a variety of methods to characterize the transcriptome and proteome of C. neoformans. For example, we have several inter-related projects that make use of transcriptional profiling under conditions that are relevant to infection of the an animal host. Examples of these conditions include:
1. Growth at 25°C and 37°C for serotype A and D strains
2. Growth in low iron and iron-replete media, and in media containing different iron sources (e.g., ferric chloride, heme and transferrin). This work includes an analysis of the impact of defects in iron-related transcription factors (e.g., Cir1, HapX) on transcription.
3. Transcription during infection (cerebral spinal fluid, lung and macrophage infection)
4. Growth of cAMP pathway mutants (pka1 and pkr1) in comparison with wild type cells. We have completed a parallel analysis of wild type and equivalent PKA mutants in U. maydis and this will allow a comparison of the PKA-regulated gene sets between different basidiomycete species.
Structural Genomics: Physical mapping and genome sequencing.
Canada’s Michael Smith Genome Sciences Centre in Vancouver established a high through facility for the rapid construction of physical maps of genomes by BAC clone fingerprinting. We have partnered with this group to construct physical maps for six strains of C. neoformans and one strain of U. hordei. This partnership has allowed us to initiate a program of comparative genomics for C. neoformans strains of serotypes A and D, and for C. gattii strains.
The physical map for U. hordei was used to characterize the 500 kb MAT-1 locus. With funding from the NIH mouse BAC sequencing initiative, we selected five BAC clones spanning the MAT-1 locus of U. hordei and completed the sequence of this region in partnership with the University of Oklahoma’s Advanced Center for Genome Technology http://www.genome.ou.edu/cneo.html.
We are also partnering with the Michael Smith Genome Sciences Center to sequence of the genome of the C. gattii strain WM276. This project was funded by Genome Canada and Genome British Columbia (http://www.genomebc.ca), and the sequence is available in GenBank. An analysis of the genome was published along with the sequence of the C. gattii strain R265 (an outbreak isolate from Vancouver Island) generated by the Broad Institute.
Molecular genetic analysis of iron and infection-regulated genes in C. neoformans.
We are using the data generated by our transcriptional profiling and proteomics programs to identify genes with potential relevance to virulence for subsequent analysis. We are focused on genes that are regulated by iron levels in the growth medium and that encode functions involved in iron acquisition, capsule formation and transport. Some of these genes are also regulated in mutants that are defective in cAMP signaling, and this finding illustrates the comparative opportunities from the expression data from different growth conditions and strain backgrounds. We have disrupted a number of these genes and we are currently characterizing phenotypes including virulence. In parallel studies, we are also characterizing genes that are expressed during experimental meningitis. The transcripts for some of these genes have only been detected in cells from an infection and these may encode important virulence functions.
Molecular genetic analysis of cAMP signaling in Ustilago maydis.
We are taking four approaches to dissect the cAMP pathway that controls the morphological transition between budding and filamentous growth in U. maydis. First, we have constructed disruption mutants in key signaling components such as PKA (ubc1 and adr1 mutants) and we have identified suppressor mutations that influence the morphology of these strains. This approach led to the identification of the hgl1 gene that appears to encode a novel regulator of morphogenesis.
A second approach makes use of the U. maydis genome sequence to identify candidate genes for subsequent disruption. The third approach is based on a search for potential signals upstream of the cAMP pathway and this work resulted in the discovery that lipids influence morphogenesis in U. maydis. Finally, we are using the transcriptome data collected for PKA mutants of U. maydis in a genome-wide search for target genes that are regulated by cAMP signaling.