Genealogy can illuminate the evolutionary path of important human pathogens. In some microbes, strict clonal reproduction predominates, as with the worldwide dissemination of Mycobacterium leprae, the cause of leprosy. In other pathogens, sexual reproduction yields clones with novel attributes, for example, enabling the efficient, oral transmission of the parasite Toxoplasma gondii. However, the roles of clonal or sexual propagation in the origins of many other microbial pathogen outbreaks remain unknown, like the recent fungal meningoencephalitis outbreak on Vancouver Island, Canada, caused by Cryptococcus gattii. Here we show that the C. gattii outbreak isolates comprise two distinct genotypes. The majority of isolates are hypervirulent and have an identical genotype that is unique to the Pacific Northwest. A minority of the isolates are significantly less virulent and share an identical genotype with fertile isolates from an Australian recombining population. Genotypic analysis reveals evidence of sexual reproduction, in which the majority genotype is the predicted offspring. However, instead of the classic a-alpha sexual cycle, the majority outbreak clone appears to have descended from two alpha mating-type parents. Analysis of nuclear content revealed a diploid environmental isolate homozygous for the major genotype, an intermediate produced during same-sex mating. These studies demonstrate how cryptic same-sex reproduction can enable expansion of a human pathogen to a new geographical niche and contribute to the ongoing production of infectious spores. This has implications for the emergence of other microbial pathogens and inbreeding in host range expansion in the fungal and other kingdoms.

Cryptococcus neoformans was first described as a human fungal pathogen more than a century ago. One aspect of the C. neoformans infectious life cycle that has been the subject of earnest debate is whether the spores are pathogenic. Despite much speculation, no direct evidence has been presented to resolve this outstanding question. We present evidence that C. neoformans spores are pathogenic in a mouse intranasal inhalation model of infection. In addition, we provide mechanistic insights into spore-host interactions. We found that C. neoformans spores were phagocytosed by alveolar macrophages via interactions between fungal ␤-(1,3)-glucan and the host receptors Dectin-1 and CD11b. Moreover, we discovered an important link between spore survival and macrophage activation state: intracellular spores were susceptible to reactive oxygen-nitrogen species. We anticipate these results will serve as the basis for a model to further investigate the pathogenic implications of infections caused by fungal spores.

Cryptococcus neoformans is an opportunistic human fungal pathogen that elaborates several virulence attributes, including a polysaccharide capsule and melanin pigments. A conserved G␣ protein/cyclic AMP (cAMP) pathway controls melanin and capsule production. To identify targets of this pathway, we used an expression profiling approach to define genes that are transcriptionally regulated by the G␣ protein Gpa1. This approach revealed that Gpa1 transcriptionally regulates multiple genes involved in capsule assembly and identified two additional genes with a marked dependence on Gpa1 for transcription. The first is the LAC1 gene, encoding the laccase enzyme that catalyzes a rate-limiting step in diphenol oxidation and melanin production. The second gene identified (LAC2) is adjacent to the LAC1 gene and encodes a second laccase that shares 75% nucleotide identity with LAC1. Similar to the LAC1 gene, LAC2 is induced in response to glucose deprivation. However, LAC2 basal transcript levels are much lower than those for LAC1. Accordingly, a lac2 mutation results in only a modest delay in melanin formation. LAC2 overexpression suppresses the melanin defects of gpa1 and lac1 mutants and partially restores virulence of these strains. These studies provide mechanistic insights into the regulation of capsule and melanin production by the C. neoformans cAMP pathway and demonstrate that multiple laccases contribute to C. neoformans melanin production and pathogenesis.

Invasive aspergillosis (IA) is a common and life-threatening infection in immunocompromised individuals. A number of environmental and epidemiologic risk factors for developing IA have been identified. However, genetic factors that affect risk for developing IA have not been clearly identified. We report that host genetic differences influence outcome following establishment of pulmonary aspergillosis in an exogenously immune suppressed mouse model. Computational haplotype-based genetic analysis indicated that genetic variation within the biologically plausible positional candidate gene plasminogen (Plg; Gene ID 18855) correlated with murine outcome. There was a single nonsynonymous coding change (Gly110Ser) where the minor allele was found in all of the susceptible strains, but not in the resistant strains. A nonsynonymous single nucleotide polymorphism (Asp472Asn) was also identified in the human homolog (PLG; Gene ID 5340). An association study within a cohort of 236 allogeneic hematopoietic stem cell transplant (HSCT) recipients revealed that alleles at this SNP significantly affected the risk of developing IA after HSCT. Furthermore, we demonstrated that plasminogen directly binds to Aspergillus fumigatus. We propose that genetic variation within the plasminogen pathway influences the pathogenesis of this invasive fungal infection.

The evolutionarily conserved cyclic AMP (cAMP) signaling pathway controls cell functions in response to environmental cues in organisms as diverse as yeast and mammals. In the basidiomycetous human pathogenic fungus Cryptococcus neoformans, the cAMP pathway governs virulence and morphological differentiation. Here we identified and characterized adenylyl cyclase-associated protein, Aca1, which functions in parallel with the G␣ subunit Gpa1 to control the adenylyl cyclase (Cac1). Aca1 interacted with the C terminus of Cac1 in the yeast two-hybrid system. By molecular and genetic approaches, Aca1 was shown to play a critical role in mating by regulating cell fusion and filamentous growth in a cAMP-dependent manner. Aca1 also regulates melanin and capsule production via the Cac1-cAMP-protein kinase A pathway. Genetic epistasis studies support models in which Aca1 and Gpa1 are necessary and sufficient components that cooperate to activate adenylyl cyclase. Taken together, these studies further define the cAMP signaling cascade controlling virulence of this ubiquitous human fungal pathogen.Cryptococcus neoformans is a heterothallic, basidiomycetous, pathogenic fungus that causes serious infections of the central nervous system in individuals immunocompromised by AIDS or undergoing organ transplantation, cytotoxic chemotherapy, or corticosteroid therapy (6, 31). The virulence of C. neoformans is influenced by several factors, including the production of an antiphagocytic polysaccharide capsule (7,20,27), the use of melanin as an antioxidant (26, 51), growth at host physiological temperature (37 to 39°C) (27, 29), and prototrophy (41). Although not directly involved in the virulence of C. neoformans, mating and filamentous growth may play a survival role in the environment and also promote dissemination of the pathogen into the host. Signaling cascades regulating virulence and differentiation of C. neoformans have been extensively studied, including a mitogen-activated protein kinase (MAPK) pathway, a G-protein-regulated cyclic AMP (cAMP) pathway, a Ras-specific pathway, and the calcineurin pathway (for reviews, see references 29 and 48).The MAPK cascade regulates mating processes involving morphological differentiation, such as the dikaryotic mycelia, basidia, and basidiospores, which are produced in response to peptide pheromones secreted by opposite mating-type cells (24,32,43). The MAPK pathway is composed of mating-typespecific (Ste3␣/a, Ste20␣/a, Ste11␣/a, and Ste12␣/a) and nonspecific (Gpb1, Ste7, and Cpk1) elements (12, 49). Gene disruption experiments revealed that the MAPK pathway is required for mating and cell type-specific differentiation but not for virulence (12). However, mating type has been associated with the virulence of serotype D (variety neoformans) strains by Kwon-Chung et al., who showed that the ␣-mating type is more virulent than the a-mating type (25). Furthermore, Del Poeta et al. demonstrated that the MF␣1 pheromone gene is induced during the late stages of central nervous system infection...