cAMP Signaling in C. neoformans
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cAMP Signaling in C. neoformans

C. neoformans (Cryptococcus neoformans) is a Basidiomycetous fungus and a significant human pathogen with worldwide distribution. Its importance as an opportunistic pathogen has increased in the past two decades, largely as a result of AIDS (Acquired Immune Deficiency Syndrome), cancer chemotherapy and immuno-suppression for organ transplants. Infections begin in the lung following inhalation of small, dessicated yeast cells or spores, both of which are small enough to fit into the alveoli of the lung. The organism then spreads via the blood to other organs, especially the CNS (Central Nervous System). Survival in the CNS is enhanced by the presence of abundant neurotransmitters that are scavenged as the diphenolic precursors to synthesize the virulence factor Melanin (Ref.1). Cryptococcal infection is now the leading cause of fungal Meningoencephalitis, which is fatal if untreated. C. neoformans exists predominantly as haploid yeast. It is heterothallic and has a bipolar mating system. C. neoformans strains are classified into five serotypes (A, B, C, D and AD) and three varieties: C. neoformans var. grubii, C. neoformans var. neoformans and C. neoformans var. gattii. C. neoformans var. gattii is thought to be limited in nature to regions surrounding certain eucalyptus trees and most infections with this variety occur in tropical and subtropical regions. C. neoformans var. neoformans is associated with pigeon nests and soil contaminated with bird excreta. Of the subtypes, C. neoformans var. grubii serotype A is the most common cause of human disease. C. neoformans is an excellent model organism for the study of biological processes in pathogenic fungi (Ref.2).

Several different signaling pathways that regulates morphogenesis and virulence in C. neoformans, includes cAMP (Cyclic Adenosine 3,5-monophosphate)-PKA (cAMP-Dependent Protein Kinase) pathway, a conserved MAPK (Mitogen-Activated Protein Kinase) pathway and a Calcineurin-regulated pathway (Ref.3). The GPA1 (Guanine Nucleotide-Binding Protein-Alpha Subunit) protein of a cAMP-PKA pathway is important for morphogenesis and virulence. GPA1 is a component of a signaling cascade that transduces changes in extracellular concentrations of nutrients via the cAMP pathway. Additionally, Ras1 a member of small GTP-binding proteins is implicated in control of both the cAMP pathway and MAPK cascade and may mediate cross talk between MAPK and cAMP-dependent signaling pathways. GPB1 (G-Protein-Beta Subunit-GPB1) and GPA3 (G-Protein-Alpha Subunit-GPA3) function in response to and activates a pheromone (MF-Alpha and MFa) induced MAPK cascade during mating; GPB1 does not function in the nutrient-regulated GPA1 pathway. Ras1 protein regulates filamentous and invasive growth via both MAPK and cAMP pathways (Ref.4).

The cAMP pathway regulates several important cellular processes in C. neoformans, including Capsule production, Melanin formation, Mating, Filamentation and Virulence. In the nutrient-sensing cAMP-PKA signaling the GPR1 (G protein-Coupled Receptor) is coupled to a highly conserved G-Alpha protein, GPA1. Binding of ligand to the GPR1 receptor stimulates Guanine nucleotide exchange on GPA1, resulting in its activation. The GPA1 protein is coupled to AC (Adenylate Cyclase)/Cac1. GPA1 activates AC to produce cAMP which in turn regulates PKA. Targets of PKA largely remain to be identified but most likely will include regulation of a variety of transcription factors through its R/Regulatory (PKR1) and C/Catalytic (PKA1 and PKA2) subunits (Ref.5). Binding of cAMP to the R-subunits causes them to undergo a conformational change and to dissociate from the C-subunits, releasing the latter from inhibition. Once active, PKA can cause changes in gene expression and physiology by phosphorylation of effector proteins. One candidate target of PKA, which is involved in positive regulation of capsule production, is the transcription factor Ste12Alpha. The Ras-cAMP pathway plays a vital role in detecting and responding to nutrients, although the connection between pathway activation and nutrient availability has remained obscure. GPA1 also functions in a signaling pathway that acts parallel to Ras1. Ras1 sense changes in the extracellular environment and regulate cAMP synthesis and cell cycle progression (Ref.6).

C. neoformans MAPK cascade functions in a pheromone response pathway during mating. In this organism, GPB1 plays an active role in mediating responses to pheromones in the early steps of mating via a MAPK cascade (Ste20a/Ste20Alpha, Ste11Alpha, Ste7, Cpk1 (Mitogen-Activated Protein Kinase-Cpk1/Stress-Activated Protein Kinase-Cpk1), Ste12). Pheromones, Pheromone Receptors (CPR-Alpha, CPRa1 (Pheromone Receptor-CPRa1) and Ste3) and the coupled G-protein GPB1 regulates haploid fruiting in C. neoformans. Ras1 functions upstream of GPB1 in the mating process. Ras1 and PKA acts as candidates for transmitting a signal between MAPK and cAMP signaling pathways (Ref.7). C. neoformans Ste12Alpha and Znf1 transcription factors play a specialized function in haploid fruiting, but Ste12Alpha is dispensable or redundant for mating and virulence. Ste12Alpha is required to regulate responses to nitrogen starvation and desiccation that trigger haploid filamentation and sporulation. The Ras1 protein and the MAPK cascade are depicted as signaling upstream of Ste12Alpha. Mating is regulated by both pheromone and nutrients via pathways involving the GPB1 and GPA1. MAPK cascade and PKA regulate Ste12Alpha and Znf1, which play only a modest role in regulating mating (Ref.6 & 8).

The molecular basis of capsule regulation is controlled by cAMP-signaling and Genetics has been used to define many of the members of this pathway. However many unanswered questions remain. What is the nature of the exact signal that activates the pathway? What are the identities of the transmembrane receptor and of downstream effectors? Capsule structure depends on environment and may vary among cells in a population, but the mechanisms controlling these differences are yet to be defined. The capsule polysaccharides that are shed into the external milieu enable C. neoformans to resist the host immune response (Ref.9). Little is known, however, about how this occurs. Are the old parts of the capsule sloughed off as new material is added, or is the release of capsular material a regulated process under specific cellular control? Also, how does degradation of the capsule take place? The molecular mechanisms of these processes remain obscure. Finally, although the structures of the main components of the capsule are now known, how do these components combine to form the three-dimensional structure of the capsule? A better understanding of the biochemical and biophysical properties of the capsule is needed to answer these questions. However by adaptation of new technologies to identify the cause of Cryptococcal infection, from genome sequencing and RNA interference to structural studies, the coming years may prove to be an exciting time for studies of Cryptococcal biology (Ref.10).