Fungal Pathogenesis
We are studying basidiomycete fungi that are either economically important plant pathogens or life-threatening pathogens of humans. The plant pathogens include Ustilago maydis and Ustilago hordei. These fungi are highly amenable to molecular genetic experimentation and cause dramatic diseases on maize and barley, respectively. We are also studying the basidiomycete fungus Cryptococcus neoformans to explore aspects of fungal pathogenesis relevant to human health. This fungus is an opportunistic pathogen of immunocompromised people such as patients suffering from HIV/AIDS. The related species C. gattii can infect immunocompetent people as demonstrated by the emergence of cryptococcosis on Vancouver Island in British Columbia.
Ustilago maydis and Ustilago hordei
U. maydis and U. hordei have fascinating mating systems that are intimately associated with the ability of these fungi to infect plant hosts. Mating is controlled by genes at two genetic loci called a and b. The a locus encodes pheromones and pheromone receptors that mediate recognition between mating partners. The b locus encodes homeodomain proteins that control a switch from budding to filamentous growth. Thus mating is required to establish the filamentous cell type that is the only infectious form of these fungi. We have characterized the b genes and we are exploring the differences in the genomic organization of the mating type genes in the two species. The a and b genes are on separate chromosomes in U. maydis and they are linked together on the same chromosome to create a single MAT locus in U. hordei. Surprisingly, our physical mapping and genome sequencing efforts revealed that the MAT locus is approximately 500 kb in size in U. hordei making it one of the largest fungal mating-type loci characterized to date. In addition to mating, we found that the cAMP signal transduction pathway plays a central role in the control of morphogenesis and pathogenesis for U. maydis. Specifically, we have shown that mutants defective in the cAMP pathway are altered in cell shape and virulence. For example, mutants defective in adenylyl cyclase or in cAMP-dependent protein kinase (PKA) no longer grow by budding and instead display constitutive filamentous growth. In contrast, cells defective in the regulatory subunit of PKA display a multiple budding phenotype. In recent work, we have characterized the nutritional signals that influence filamentous growth and phenotypes associated with the biotrophic development of U. maydis in infected maize tissue.
Cryptococcus neoformans and Cryptococcus gattii
Immunocompromised people (e.g., AIDS patients) are particularly susceptible to opportunistic fungal pathogens such as C. neoformans. In fact, C. neoformans causes life-threatening CNS infections in AIDS patients and is thought to be responsible for ~15% of all AIDS-associated deaths. The fungus also infects people receiving immunosuppressive therapy. A limited arsenal of antifungal drugs (e.g., amphotericin B, fluconazole, flucytosine)is available, and these drugs generally suppress but do not cure cryptococcal meningitis. The related species C. gattii also infects immunocompetent individuals as recently demonstrated by the emergence of cryptococcosis in British Columbia. Our work on C. neoformans focused initially on building structural and functional genomic resources to support the efforts of the research community and to explore specific aspects of virulence. In particular, we have collaborated with the Michael Smith Genome Sciences Centre in Vancouver to construct BAC clone physical maps of six strains of the fungus representing the strains and serotypes being used for four different genome sequencing projects. Our group took the lead in efforts to generate reference genome sequences for two strains of C. gattii including the strain R265 that represents the main genotype causing disease on Vancouver Island. We also initiated a program of genome-wide analysis of gene expression for C. neoformans. Specifically, we employed serial analysis of gene expression (SAGE), microarrays and RNA-Seq to examine transcript levels in the context of iron deprivation, host iron sources, infection (macrophage, lung and cerebral spinal fluid), metabolic defects, and cAMP signaling. This work is generating a detailed view of gene expression during adaptation of the fungus to the host environment. Follow up experiments include the construction and analysis of knock out mutants with defects in genes identified by transcriptional profiling and proteomics. We are particularly interested in genes regulated by iron and the cAMP signaling pathway as well as genes specifically expressed during infection. Recently, we initiated a detailed examination of the role of the proteasome in the regulation of virulence factor elaboration in C. neoformans.
Funding
Work in our group is supported by the Natural Sciences and Engineering Research Council of Canada, the Canadian Institutes of Health Research, The National Institute of Allergy and Infectious Diseases (NIH), the BC Lung Association, a Scholar Award in Molecular Pathogenic Mycology to JK from the Burroughs Wellcome Fund, and fellowship funding from the Canadian Institute for Advanced Research (Fungal Kingdom: Threats & Opportunities).