Joseph Heitman
Chair, Department of Molecular Genetics and Microbiology

Signal transduction cascades regulating development and virulence of microorganisms

Our research focuses on how cells sense their environment and communicate with other cells. We employ genetic and biochemical approaches to study two divergent single-celled eukaryotic organisms, the yeast Saccharomyces cerevisiae and the pathogenic fungus Cryptococcus neoformans . These organisms both grow as budding yeasts and appear quite similar, yet they have been diverging over 500 million years of evolution such that one is now a harmless saprophyte and the other a virulent human pathogen that causes life threatening infections of the central nervous system in patients with compromised immunity. We are interested in what their comparison can teach us, both about conserved cellular principles and about the unique adaptations that have occured during the evolution of a human pathogen.

All organisms sense and respond to nutrients. We study how cells sense nutrients during filamentous differentiation in S. cerevisiae and mating and virulence in C. neoformans. In response to limiting nutrients, yeast cells differentiate, undergoing a dimorphic transition called pseudohyphal growth in which the cells elongate and grow in filaments extending away from the colony and into the growth medium to forage for nutrients. We have delineated nutrient sensing signal transduction cascades that regulate filamentous growth. One is regulated by an ammonium permease that we propose functions both to transport and to sense ammonium ions. Intriguingly, this cell surface sensor is structurally, and functionally, related to the Rh blood group antigens in mammals.

The second nutrient sensing pathway is controlled by a novel G protein-coupled receptor, Gpr1, which senses sugars and activates the coupled G alpha protein Gpa2 to stimulate cAMP production by adenylyl cyclase. cAMP then activates cAMP-dependent protein kinase, and the Tpk2 catalytic subunit of this enzyme activates filamentous growth, in part by regulating transcription factors that modulate expression of cell surface proteins involved in adhesion and invasion. The divergent PKA catalytic subunits Tpk1 and Tpk3 play a distinct role and inhibit filamentation.

Importantly, the Gpr1 receptor is the first fungal G-protein coupled receptor whose ligand is not a mating pheromone. Gpr1 homologs have been identified in fission yeast and the human fungal pathogen Candida albicans. Homologs of the coupled G alpha protein are expressed in a dozen different fungi, suggesting the pathway is broadly conserved. Because this family of novel fungal G-protein coupled receptors plays a conserved role in glucose sensing, these receptors could be related to the receptors involved in sweet taste transduction in drosophila and mammals.

Our studies in yeast have served as a model to elucidate signaling cascades that control mating, filamentation, and virulence in the pathogenic fungus C. neoformans . This organism is an ideal model pathogen, and has a defined sexual cycle, exogenous DNA can be introduced by transformation, genes are readily disrupted by homologous recombination, and animal models for studies of infection have been well developed. Genome sequencing projects are in progress for three related by diverged pathogenic forms of C. neoformans . We have capitalized upon these advances to analyze the molecular determinants of virulence. Much of this work is conducted in collaboration with the Duke University Mycology Research Unit, which includes the labs of John Perfect, Gary Cox, Andy Alspaugh, Fred Dietrich, Tom Mitchell, John McCusker, Rytas Vilgalys, and our group.

Our studies have revealed a conserved nutrient sensing pathway conrols virulence of 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 induce expression of virulence factors during infection. Mutants lacking the G alpha protein Gpa1, adenylyl cyclase, or the Pka1 catalytic subunit of protein kinase A are unable to produce two specialized virulence factors, the antioxidant pigment melanin and an antiphagocytic polysaccharide capsule, in response to host cues. Importantly, these mutant strains are attenuated in animals. In contrast, mutation of the protein kinase A regulatory subunit Pkr1 enhances capsule production and results in hypervirulence. Further studies aim to identify the receptors, transcription factors and target genes of this pathway.

Our complementary studies reveal a MAP kinase signaling cascade functions in parallel with the G protein-cAMP-PKA signaling pathway and senses pheromones during mating. We have synthesized the lipid modified peptide pheromone that activates this cascade. We developed a novel confrontation assay and find that the pheromones direct signaling between mating partners. In particular, the MFalpha pheromone produced by MAT alpha cells triggers confronting MAT alpha cells to filament and sporulate by a process known as haploid fruiting, which is thought to produce the highly infectious basidiospores that are inhaled into the alveoli of the lung. Our studies further suggest haploid fruiting is linked to the sexual cycle, and functions to enable distant mating partners to locate each other. Recent studies by others reveal mating pheromones also trigger filamentous differentiation of S. cerevisiae haploid cells. Together, these studies illustrate an ancient link between filamentation, the sexual cycle, and sporulation that has been coopted during evolution of a pathogen. Several conserved components of the MAP kinase pathway, including the Gbeta protein Gpb1, homologs of the Ste20 and Ste7 kinases, the MAP kinase homolog Cpk1, and transcription factors that function during mating and differentiation have also been identified.

Interestingly, mating type has been 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 MAT alpha strains. Our studies reveal divergent alleles of the Ste20 kinase are encoded by the MAT alpha and MATa loci. Mutants lacking the Ste20a kinase are mating imparied and attenuated for virulence, providing a molecular link between the MATa locus, the MAP kinase cascade, and virulence. Recently we identified genes encoded by the corresponding MATa mating type locus. Using these mating type specific genes we have: 1) discovered and characterized unusual self-filamentous diploid strains of C. neoformans that arise following genetic crosses, 2) shown that unusual hybrid diploid strains also occur in nature and are infectious, and 3) discovered an unusual MAT alpha isolate of the serotype A lineage that was thought to be extinct. These studies have redefined central features of the life cycle and sexual cycle of this pathogenic fungus. Studies in progress aim to define the structure, evolution, and functions of the mating type loci.

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 parallel, we employ natural toxins as molecular probes to dissect signaling. We focus on the immunosuppressants cyclosporin, FK506, and rapamycin, which suppress the immune system by blocking signaling events required for T-cell activation. These agents are widely used to treat transplant rejection, all three are natural products of soil microorganisms that likely play a role in nature as toxins to inhibit competing microorganisms. Based on this hypothesis, we analyzed the mechanisms of drug action in S. cerevisiae and discovered the signaling cascades targeted by these drugs are highly conserved.

Each of these drugs diffuses into the cell and associates with a binding protein, cyclosporin with cyclophilin and FK506 and rapamycin with FKBP. Both drug binding proteins are enzymes that catalyze a rate limiting step in protein folding following synthesis by the ribosome. The drugs bind to and inhibit the enzyme active sites, but this is not how cell function is disrupted. For example, yeast and fungal cells missing the cyclophilin or FKBP proteins are viable and resistant to the toxic effects of these drugs. Thus, these compounds do not kill the cell by inhibiting the binding proteins, because cells lacking the proteins are still viable. Instead, the protein-drug complexes are the active agents, and these complexes bind to and inhibit signaling molecules. The target of the cyclophilin-cyclosporin 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 of C. neoformans and are identifying other elements of these pathways. A calcineurin binding protein identified in C. neoformans is conserved in yeast and humans and may represent a calcineurin effector or inhibitor. The human homolog is encoded by the first gene in the Down's syndrome critical region on human chromosome 21. Both calcineurin and the the human DSCR1 protein are highly expressed in the heart and the brain, two tissues prominently effected in Down's Syndrome patients, suggesting overexpression of DCSR1 and perturbations in calcineurin signaling could underlie some of the clinical manifestations of this common syndrome.

We found that the cyclophilin A enzyme has novel nuclear functions, and implicated one target as a histone deacetylase complex. Two related cyclophilin A homologs, Cpa1 and Cpa2, are expressed in C. neoformans and mediate cyclosporin A antifungal action and share a function important for growth. These findings provide genetically tractable model systems to explore the in vivo functions of this conserved but enigmatic family of protein folding enzymes. In summary, our studies began with unusual natural product toxins and have led to the identification of conserved targets whose diverse functions in growth and signaling remain to be elucidated.

Much of experimental biology has been based on the premise that studies of model organisms, including bacteria, yeast, insects, and worms, would reveal conserved principles that govern how all organisms function. Our studies support this view and suggest further studies of model organisms will contribute much to our understanding of the molecular basis of life.

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