Formulation of a defined V8 medium for induction of sexual development of Cryptococcus neoformans.

For over 3 decades, sexual development in the human fungal pathogen Cryptococcus neoformans and other fungi has been initiated by growing compatible mating partners on V8 juice medium. Although this medium is an efficient inducer of sexual development, the mechanism by which it promotes the process is unknown. To understand how V8 juice medium induces sexual development, we attempted to purify inducing factors from V8 juice, and we carried out a complete compositional analysis of V8 juice. We discovered that no single factor is responsible for the effects of V8 juice medium. Rather, the unique composition of V8 juice medium provides the proper nutrient composition for inducing and sustaining complete sexual development. Utilizing these findings, we developed a defined V8 (DV8) medium that mimics V8 juice medium in sexual development assays. Then, using DV8 as a tool, we explored the roles that specific molecules play in enhancing sexual development. Surprisingly, we discovered that copper is a key factor, leading to an upregulation of the mating pheromone genes MFa and MFalpha, both required for the initial steps in sexual development. The utilization of DV8 to investigate the effects of copper on sexual development presented here is an example of how defining the conditions that induce sexual development will advance the study of C. neoformans.

Plasminogen alleles influence susceptibility to invasive aspergillosis.

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.

Transcriptional network of multiple capsule and melanin genes governed by the Cryptococcus neoformans cyclic AMP cascade.

Cryptococcus neoformans is an opportunistic human fungal pathogen that elaborates several virulence attributes, including a polysaccharide capsule and melanin pigments. A conserved Galpha 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 Galpha 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.

Cytochrome c peroxidase contributes to the antioxidant defense of Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic pathogen and the leading cause of fungal meningitis. To survive within the host, this organism must be able to protect itself from oxidative stress. Cytochrome c peroxidase (Ccp1) is a mitochondrial antioxidant that catalyzes the degradation of hydrogen peroxide. In the present study, we characterized the contribution of the C. neoformans Ccp1 to antioxidant defense and for virulence. Consistent with studies of Ccp1 function in Sacchromyces cerevisiae, we found that Ccp1 contributes to resistance against exogenous oxidative stress in vitro. However, the oxidative stress phenotype does not diminish the virulence of ccp1 mutant strains in a murine model of C. neoformans disease. These results suggest that Ccp1 is involved in a complex system of protection against exogenous oxidative stress and that the elimination of this component of the antioxidant defense system does not diminish the virulence of C. neoformans.

Adenylyl cyclase-associated protein Aca1 regulates virulence and differentiation of Cryptococcus neoformans via the cyclic AMP-protein kinase A cascade.

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 Galpha 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.

Extracellular calcium and magnesium, but not iron, are needed for optimal growth of Blastomyces dermatitidis yeast form cells in vitro.

In the present study, we demonstrate that the yeast form of Blastomyces dermatitidis can proliferate for short periods of time in the absence of ferric iron but not in the absence of calcium or magnesium. The results of this study shed light on the resistance of B. dermatitidis to chelating agents, such as deferoxamine, and may explain how B. dermatitidis resists the iron-binding activity of serum transferrin.

Cerebral phaeohyphomycosis in an immunodeficient child treated medically with combination antifungal therapy.

Cerebral phaeohyphomycosis is a rare fungal infection with a poor prognosis when using conventional antifungal therapy in the absence of neurosurgical intervention. We present a case of a pediatric patient with inoperable Cladophialophora bantiana cerebral abscesses. To our knowledge, this child's case is the first reported to be treated with the combination of the newer triazole voriconazole and the new echinocandin caspofungin. Although our patient subsequently died, the natural rapid progression of the disease seemed to be altered by the antifungal combination alone, in the absence of surgery. Despite the fatal outcome for our patient, we encourage other clinicians to try unique medical approaches for this historically life-threatening infection when adjunctive surgery is impossible.