Sexual reproduction and the emergence and evolution of microbial pathogens
Our studies focus on the human fungal pathogen Cryptococcus, which causes life-threatening infections of the central nervous system in both immunocompromised and immunocompetent hosts. This organism is a basidiomycete and therefore divergent from other common human fungal pathogens and model fungi. We contributed to define the sexual cycle involving haploid alpha and a cells, and to apply Falkow’s molecular postulates of virulence employing gene disruption approaches and robust animal virulence models. These efforts have defined the molecular basis for antifungal drug action and synergistic combinations and elucidated roles for calcineurin in fungal virulence and drug tolerance in C. neoformans, Candida albicans, and Aspergillus fumigatus. We are currently exploring the potential of combining calcineurin or Hsp90 inhibitors with existing antifungal agents against a panel of pathogenic fungi in infectious settings including systemic, ocular, and cutaneous models. We have participated in championing and organizing the fungal kingdom genome sequencing project. These efforts are nearly completed for five strains representing three related but divergent varieties of Cryptococcus, all of which are pathogenic in humans and have unique environmental and virulence attributes. In addition, we contributed to enlist the Broad Fungal Genome initiative to sequence a group of Candida species related to Candida albicans to explore their potential for sexual reproduction, including meiosis, and detailed molecular and genetic studies are in progress for the species Candida lusitaniae on the functions of the mating type locus and conserved meiotic machinery. Finally, the fungal genome initiative of the Department of Energy Joint Genome Institute is currently sequencing the basidiomycete Tremella mesenterica, and Keisha Findley in our group serves as the community coordinator for this genome project.
Parallel studies have focused on the structure, function, and evolution of the fungal mating type locus, which is linked to differentiation and virulence potential in Cryptococcus. Signaling cascades that control virulence and mating have been defined, and the a and alpha alleles of the mating type locus have been cloned and sequenced from two varieties and the sibling species C. gattii. The MAT locus spans over 100 kb and contains more than 20 genes, several of which function in differentiation and virulence. The MAT alleles are composed of divergent sets of the same genes that evolved by extensive remodeling from a common ancestral DNA region. The only MAT allele specific genes encode two homeodomain proteins, Sxi1alpha and Sxi2a, which physically interact and are necessary and sufficient to govern post-fusion events enabling completion of the sexual cycle. A detailed model has been developed for the evolution of MAT from an ancestral tetrapolar mating system, revealing parallels with the evolution and features of sex chromosomes of plants and animals. We have contributed to define the structure of the mating type locus from the human dimorphic fungal pathogens, Histoplasma capsulatum, Coccidioides immitis and C. posadasii, revealing that all three retain both mating types, consistent with extant sexual cycles that remain to be explored. These studies also reveal how genes have been captured into the MAT locus, with implications for expansions of MAT that have occurred in other pathogenic fungi, including C. albicans and C. neoformans and C. gattii. Finally, we have defined the structure of the mating type locus in Phycomyces blakesleeanus, the first representative of the Zygomycete phylum in which MAT has been identified. This reveals that the sexM and sexP loci each contain only a single gene, and each encodes a divergent HMG domain transcription factor homolog, with implications for the origins of sex determination and the evolution of sex chromosomes.
We have defined the sexual cycles for the most common pathogenic variety of Cryptococcus (serotype A, variety grubii), recapitulated the sexual cycle for the divergent gattii variety that infects immunocompetent hosts with implications for an unusually fertile clonal alpha isolate causing an outbreak on Vancouver Island, and contributed to the discovery of a unique population of serotype A strains undergoing active recombination in sub-Saharan Africa. Recent studies have demonstrated that sexual reproduction occurs on Pigeon guano medium, and during a pathogenic association with plants, two common environmental niches in which Cryptococcus may complete its sexual cycle in nature to produce infectious spores. Our studies reveal an enhanced virulence potential of alpha strains during co-infection with a strains, and the molecular basis for this enhanced virulence is being explored involving pheromone production and sensing via cell-cell signaling analogous to quorum sensing in bacteria and other fungi. This model is being examined in detail in both murine virulence models and in heterologous hosts, including insects. Our investigations have revealed that monokaryotic fruiting represents a modified form of the sexual cycle that can occur between partners of only one mating type, and which involves a ploidy shift, meiosis, and production of recombinant haploid progeny that may represent the infectious propagules. Recent population genetic studies implicate this laboratory defined same sex mating cycle in the origin and ongoing outbreak of Cryptococcus gattii on Vancouver Island. In collaboration with Kieren Marr, we have identified the first index case for expansion of the Vancouver Island outbreak into the United States, and further studies of environmental, veterinary, and human isolates are ongoing. Our studies of unusual hybrid isolates of Cryptococcus neoformans (alphaADalpha) demonstrate that same sex mating occurs in nature and has given rise to hybrids which exhibit hybrid fitness and are pathogenic. Analysis of a different hybrid lineage (aADalpha) provides evidence that these isolates descend from a mating event in sub-Saharan Africa that gave rise to a hybrid that emigrated worldwide and is a common cause of infection. Taken together, these complementary lines of investigation illustrate the potential roles of sexual recombination in the evolution and virulence of a species cluster of human fungal pathogens with implications for other eukaryotic microbial pathogens, including fungi, parasites, and bacterial pathogens.