Signal transduction cascades regulating development and virulence of microorganisms
Our research focuses on how cells sense their environment and communicate with other cells via signal transduction cascades. We employ genetic and biochemical approaches to study two divergent single-celled eukaryotic organisms: the yeast Saccharomyces cerevisiae and the pathogenic fungus Cryptococcus neoformans, which causes life-threatening infections of the central nervous system in patients with compromised immunity. Our studies address the mechanisms of action and targets of immunosuppressive, antifungal drugs and the mechanisms by which yeast and fungal cells sense and respond to the environment and the host. Our findings reveal elements of signal transduction cascades conserved from yeast and fungi to humans over the billion years of evolution separating us from a common ancestor.

All organisms sense and respond to nutrients. Many signal transduction pathways in microorganisms are devoted to sensing the availability of nutrients, including sugars, amino acids, and nitrogen sources. We study how cells sense nutrients during filamentous differentiation in S. cerevisiae and which regulate mating, haploid fruiting, and virulence in C. neoformans. In response to adverse nutritional conditions, diploid yeast cells differentiate, undergoing a dimorphic transition called pseudohyphal growth in which the cells elongate and then grow in filaments that extend away from the colony and into the growth medium to forage for nutrients. We have delineated a signal transduction cascade that regulates filamentous growth. This cascade is initiated by a novel G protein-coupled receptor, Gpr1, which senses glucose and activates the coupled G-alpha protein Gpa2 to regulate cAMP production by adenylyl cyclase. The second messenger cAMP then activates cAMP-dependent protein kinase, and the Tpk2 catalytic subunit of this enzyme activates filamentous growth, in part by regulating the Flo8 and Sfl1 transcription factors that modulate expression of the Flo11 protein on the surface of the cell that is required for cell-cell adhesion. In contrast, the related PKA catalytic subunits Tpk1 and Tpk3 play a negative role in regulating filamentation, either by feedback inhibition of cAMP production or by competing with Tpk2 for downstream targets.

We have also discovered that the high affinity ammonium transporter Mep2 is required for filamentous differentiation in response to limiting ammonium ions. This function of Mep2 is distinct from two other related ammonium transporters, which also transport ammonium ions but do not regulate filamentous growth. Our current hypothesis is that Mep2 serves as a sensor or receptor of ammonium ions, and we are working to identify other components of this signaling pathway. Recent studies by others reveal the mammalian Rh Blood group antigens are functional homologs of the yeast Mep ammonium transporters.

Our studies in S. cerevisiae have served as a model to elucidate signal transduction cascades that regulate mating, filamentation, and virulence in the less well studied pathogenic fungus C. neoformans. This organism is an ideal model pathogen. It has a defined sexual cycle, exogenous DNA can be introduced by transformation, genes can be disrupted by homologous recombination, animal models for studies of infection have been well developed, and the genome sequence is in progress. We have capitalized upon these advances to analyze the molecular determinants of virulence in C. neoformans. In addition, we are part of a larger collaborative effort, the Duke University Mycology Research Unit, which is funded by an NIH program project grant and now encompasses eight groups at Duke (John Perfect, Gary Cox, Andy Alspaugh, Fred Dietrich, Rytas Vilgalys, John McCusker, Tom Mitchell, and our group) focusing on C. neoformans as a model fungal pathogen.

Our studies have revealed a conserved pathway is dedicated to nutrient sensing in C. neoformans. This pathway is composed of the G-alpha protein Gpa1 (the homolog of yeast Gpa2), which signals via adenylyl cyclase, cAMP, and cAMP dependent protein kinase to regulate expression of the capsule and melanin virulence factors required for infection. A second pathway is a MAP kinase signaling cascade that coordinately regulates mating, filamentation, and virulence in response to pheromones. We have identified and synthesized the lipid modified peptide pheromone that activates this cascade. We have found that haploid filamentation of MAT-alpha strains is activated in response to mating pheromones, suggesting that this differentiation pathway plays a role in early steps during mating. Analogous studies by others have recently revealed a similar role for mating pheromones in filamentous differentiation in S. cerevisiae. We have identified and characterized several conserved components, including the Gß protein Gpb1, homologs of the Ste20, Ste11, and Ste7 kinases, the MAP kinase homolog Cpk1, and the transcription factor Ste12, and have demonstrated that these proteins participate in mating, filamentation, or virulence.

Interestingly, mating type is linked to physiology and virulence of C. neoformans. Strains of the MAT-alpha mating type are more common in nature, most clinical isolates are MAT-alpha, and MAT-alpha strains are more virulent than congenic MATa strains. We have constructed bacterial artificial chromosome libraries and isolated clones spanning the mating type loci. Mapping and sequencing of these regions reveals they encode divergent homologs of many components of the MAP kinase signaling pathway. Using primers designed to these regions, we have identified the first MATa isolate of the pathogenic variety grubii strains of C. neoformans, demonstrated that unusual serotype AD strains are diploid and heterozygous at the mating type loci, and characterized congenic MAT-alpha/MATa diploids we have isolated. We are now analyzing the functions of the mating type loci by gene disruption. For example, mutants lacking the Ste20alpha kinase have defects in cytokinesis and capsule prodution, are inviable at 39°C, and are attenuated for virulence in animal models of cryptococcal meningitis. These studies provide a molecular link between the MAT-alpha locus, the MAP kinase cascade, and virulence in this fungal pathogen. In summary, these complementary studies in yeast and pathogenic fungi reveal two distinct signaling pathways that function coordinately to sense different environmental signals and give rise to appropriate developmental fates.

In complementary studies, we employ natural toxins as molecular probes to dissect signaling. We have focused on the immunosuppressive drugs, cyclosporin (CsA), FK506, and rapamycin, which suppress the immune system by blocking activation of T-lymphocytes. CsA, FK506, and rapamycin are widely used to treat and prevent graft rejection in organ transplant recipients. These compounds are all natural products of soil microorganisms and play a role in nature distinct from immunosuppression, likely as toxins to inhibit growth of competing microorganisms. Based on this hypothesis, we have analyzed in detail the mechanisms of drug action in S. cerevisiae. These studies reveal signaling cascades targeted by these drugs are conserved from yeast and pathogenic fungi to humans.

Each of these inhibitory molecules diffuses into the cell and associates with a binding protein, CsA with cyclophilin and FK506 and rapamycin with FKBP. The cyclophilin and FKBP proteins are enzymes that catalyze a rate limiting step in protein folding. The drugs bind to and inhibit the enzyme active sites, but this is not how cell function is disrupted. Yeast and fungal cells missing the cyclophilin or FKBP proteins are viable and completely resistant to these drugs. Thus, these compounds do not kill the cell by inhibiting the binding proteins. Instead, the protein-drug complexes are the active agents, and these complexes bind and inhibit signaling molecules. The target of the cyclophilin-CsA and FKBP-FK506 complexes is calcineurin, a conserved calcium sensing protein phosphatase.

Our studies now address the normal cellular functions of these drug targets. We discovered that calcineurin is required for mating, filamentation, and virulence in C. neoformans and are identifying the calcineurin substrates regulating these processes. A calcineurin binding protein has been identified in C. neoformans that is conserved in yeast and humans and may represent a calcineurin effector. This calcineurin binding protein is the first gene in the Down's syndrome critical region on human chromosome 21. Both calcineurin and the calcineurin binding protein DSCR1 are highly expressed in the heart and the brain, two tissues prominently effected in Down's Syndrome patients, suggesting DSCR1 overexpression and perturbations in calcineurin signaling could underlie some of the clinical manifestations of this disorder.

In collaboration with Steve Hanes in Albany, we have found the functions of the cyclophilin A prolyl isomerase overlap with a second prolyl isomerase, the parvulin homolog Ess1/Pin1. We have identified targets of the Ess1 and cyclophilin A proteins as the CTD domain of RNA polymerase II and a histone deacetylase complex. We have found that two closely related cyclophilin A homologs, Cpa1 and Cpa2, are expressed in C. neoformans. The Cpa1 and Cpa2 cyclophilin A proteins mediate cyclosporin A antifungal action and have a shared function that is important for cell growth. These findings provide a second genetically tractable model system in which to explore the in vivo functions of this conserved but enigmatic family of protein folding enzymes. In summary, our studies began with the unusual properties of natural product toxins and have now led to the identification of conserved target proteins whose diverse functions in cell growth and signal transduction remain to be elucidated.