SUMMARY: The human fungal pathogen Cryptococcus neoformans is characterized by its ability to induce a distinct polysaccharide capsule in response to a number of host-specific environmental stimuli. The induction of capsule is a complex biological process encompassing regulation at multiple steps, including the biosynthesis, transport, and maintenance of the polysaccharide at the cell surface. By precisely regulating the composition of its cell surface and secreted polysaccharides, C. neoformans has developed intricate ways to establish chronic infection and dormancy in the human host. The plasticity of the capsule structure in response to various host conditions also underscores the complex relationship between host and parasite. Much of this precise regulation of capsule is achieved through the transcriptional responses of multiple conserved signaling pathways that have been coopted to regulate this C. neoformans -specific virulence-associated phenotype. This review focuses on specific host stimuli that trigger the activation of the signal transduction cascades and on the downstream transcriptional responses that are required for robust encapsulation around the cell.

Cryptococcus neoformans is a prevalent human fungal pathogen that must survive within various tissues in order to establish a human infection. We have identified the C. neoformans Rim101 transcription factor, a highly conserved pH-response regulator in many fungal species. The rim101Δ mutant strain displays growth defects similar to other fungal species in the presence of alkaline pH, increased salt concentrations, and iron limitation. However, the rim101Δ strain is also characterized by a striking defect in capsule, an important virulence-associated phenotype. This capsular defect is likely due to alterations in polysaccharide attachment to the cell surface, not in polysaccharide biosynthesis. In contrast to many other C. neoformans capsule-defective strains, the rim101Δ mutant is hypervirulent in animal models of cryptococcosis. Whereas Rim101 activation in other fungal species occurs through the conserved Rim pathway, we demonstrate that C. neoformans Rim101 is also activated by the cAMP/PKA pathway. We report here that C. neoformans uses PKA and the Rim pathway to regulate the localization, activation, and processing of the Rim101 transcription factor. We also demonstrate specific host-relevant activating conditions for Rim101 cleavage, showing that C. neoformans has co-opted conserved signaling pathways to respond to the specific niche within the infected host. These results establish a novel mechanism for Rim101 activation and the integration of two conserved signaling cascades in response to host environmental conditions. Cryptococcus neoformans is an environmental fungus and an opportunistic human pathogen. Survival of this fungus within a human host depends on its ability to sense the host environment and respond with protective cellular changes. It is known that the cAMP/PKA signal transduction cascade is important for sensing host-specific environments and regulating the cellular adaptations, such as capsule and increased iron uptake, that are necessary for growth inside the infected host. Here we document that, unlike what has been described in other fungal species, a C. neoformans Rim101 homologue is directly regulated by PKA. The Rim101 signaling pathway is also involved in capsule regulation and virulence. Our study demonstrates that Rim101 integrates two conserved signal transduction cascades, and it is important in regulating microbial pathogenesis.

The signaling molecule cyclic AMP (cAMP) is a ubiquitous second messenger that enables cells to detect and respond to extracellular signals. cAMP is generated by the enzyme adenylyl cyclase, which is activated or inhibited by the Gα subunits of heterotrimeric G proteins in response to ligand-activated G-protein-coupled receptors. Here we identified the unique gene ( CAC1 ) encoding adenylyl cyclase in the opportunistic fungal pathogen Cryptococcus neoformans . The CAC1 gene was disrupted by transformation and homologous recombination. In stark contrast to the situation for Saccharomyces cerevisiae , in which adenylyl cyclase is essential, C. neoformans cac1 mutant strains were viable and had no vegetative growth defect. Furthermore, cac1 mutants maintained the yeast-like morphology of wild-type cells, in contrast to the constitutively filamentous phenotype found upon the loss of adenylyl cyclase in another basidiomycete pathogen, Ustilago maydis . Like C. neoformans mutants lacking the Gα protein Gpa1, cac1 mutants were mating defective and failed to produce two inducible virulence factors: capsule and melanin. As a consequence, cac1 mutant strains were avirulent in animal models of cryptococcal meningitis. Reintroduction of the wild-type CAC1 gene or the addition of exogenous cAMP suppressed cac1 mutant phenotypes. Moreover, the overexpression of adenylyl cyclase restored mating and virulence factor production in gpa1 mutant strains. Physiological studies revealed that the Gα protein Gpa1 and adenylyl cyclase controlled cAMP production in response to glucose, and no cAMP was detectable in extracts from cac1 or gpa1 mutant strains. These findings provide direct evidence that Gpa1 and adenylyl cyclase function in a conserved signal transduction pathway controlling cAMP production, hyphal differentiation, and virulence of this human fungal pathogen.

Cryptococcus neoformansis an opportunistic human fungal pathogen that elaborates several virulence attributes, including a polysaccharide capsule and melanin pigments. A conserved Gα 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 Gα 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 theLAC1gene, encoding the laccase enzyme that catalyzes a rate-limiting step in diphenol oxidation and melanin production. The second gene identified (LAC2) is adjacent to theLAC1gene and encodes a second laccase that shares 75% nucleotide identity withLAC1. Similar to theLAC1gene,LAC2is induced in response to glucose deprivation. However,LAC2basal transcript levels are much lower than those forLAC1. Accordingly, alac2mutation results in only a modest delay in melanin formation.LAC2overexpression suppresses the melanin defects ofgpa1andlac1mutants and partially restores virulence of these strains. These studies provide mechanistic insights into the regulation of capsule and melanin production by theC. neoformanscAMP pathway and demonstrate that multiple laccases contribute toC. neoformansmelanin production and pathogenesis.

Recent investigations have yielded both profound insights into the mechanisms required by pathogenic fungi for virulence within the human host, as well as novel potential targets for antifungal therapeutics. Some of these studies have resulted in the identification of novel compounds that act against these pathways and also demonstrate potent antifungal activity. However, considerable effort is required to move from pre-clinical compound testing to true clinical trials, a necessary step toward ultimately bringing new drugs to market. The rising incidence of invasive fungal infections mandates continued efforts to identify new strategies for antifungal therapy. Moreover, these life-threatening infections often occur in our most vulnerable patient populations. In addition to finding completely novel antifungal compounds, there is also a renewed effort to redirect existing drugs for use as antifungal agents. Several recent screens have identified potent antifungal activity in compounds previously indicated for other uses in humans. Together, the combined efforts of academic investigators and the pharmaceutical industry is resulting in exciting new possibilities for the treatment of invasive fungal infections.

Infectious microorganisms often play a role in modulating the immune responses of their infected hosts. We demonstrate thatCryptococcus neoformanssignals through the Rim101 transcription factor to regulate cell wall composition and the host-pathogen interface. In the absence of Rim101,C. neoformansexhibits an altered cell surface in response to host signals, generating an excessive and ineffective immune response that results in accelerated host death. This host immune response to therim101Δ mutant strain is characterized by increased neutrophil influx into the infected lungs and an altered pattern of host cytokine expression compared to the response to wild-type cryptococcal infection. To identify genes associated with the observed phenotypes, we performed whole-genome RNA sequencing experiments under capsule-inducing conditions. We defined the downstream regulon of the Rim101 transcription factor and determined potential cell wall processes involved in the capsule attachment defects and altered mechanisms of virulence in therim101Δ mutant. The cell wall generates structural stability for the cell and allows the attachment of surface molecules such as capsule polysaccharides. In turn, the capsule provides an effective mask for the immunogenic cell wall, shielding it from recognition by the host immune system.IMPORTANCECryptococcus neoformansis an opportunistic human pathogen that is a significant cause of death in immunocompromised individuals. There are two major causes of death due to this pathogen: meningitis due to uncontrolled fungal proliferation in the brain in the face of a weakened immune system and immune reconstitution inflammatory syndrome characterized by an overactive immune response to subclinical levels of the pathogen. In this study, we examined howC. neoformansuses the conserved Rim101 transcription factor to specifically remodel the host-pathogen interface, thus regulating the host immune response. These studies explored the complex ways in which successful microbial pathogens induce phenotypes that ensure their own survival while simultaneously controlling the nature and degree of the associated host response.