Robert C. Davidson

Signal Transduction Cascades Regulating Fungal Development and Virulence

SUMMARY Cellular differentiation, mating, and filamentous growth are regulated in many fungi by environmental and nutritional signals. For example, in response to nitrogen limitation, diploid cells of the yeast Saccharomyces cerevisiae undergo a dimorphic transition to filamentous growth referred to as pseudohyphal differentiation. Yeast filamentous growth is regulated, in part, by two conserved signal transduction cascades: a mitogen-activated protein kinase cascade and a G-protein regulated cyclic AMP signaling pathway. Related signaling cascades play an analogous role in regulating mating and virulence in the plant fungal pathogen Ustilago maydis and the human fungal pathogens Cryptococcus neoformans and Candida albicans. We review here studies on the signaling cascades that regulate development of these and other fungi. This analysis illustrates both how the model yeast S. cerevisiae can serve as a paradigm for signaling in other organisms and also how studies in other fungi provide insights into conserved signaling pathways that operate in many divergent organisms.

A PCR-based strategy to generate integrative targeting alleles with large regions of homology

Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle for which genetic and molecular techniques are well developed. The entire genome sequence of one C. neoformans strain is nearing completion. The efficient use of this sequence is dependent upon the development of methods to perform more rapid genetic analysis including gene-disruption techniques. A modified PCR overlap technique to generate targeting constructs for gene disruption that contain large regions of gene homology is described. This technique was used to disrupt or delete more than a dozen genes with efficiencies comparable to those previously reported using cloning technology to generate targeting constructs. Moreover, it is shown that disruptions can be made using this technique in a variety of strain backgrounds, including the pathogenic serotype A isolate H99 and recently characterized stable diploid strains. In combination with the availability of the complete genomic sequence, this gene-disruption technique should pave the way for higher throughput genetic analysis of this important pathogenic fungus.

A MAP kinase cascade composed of cell type specific and non‐specific elements controls mating and differentiation of the fungal pathogen Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle in which the α allele of the mating type locus is linked to virulence and haploid differentiation. Here we analysed a conserved MAP kinase cascade composed of mating‐type specific (Ste11α, Ste12α) and non‐specific (Ste7, Cpk1) elements. Gene disruption experiments demonstrate that this specialized MAP kinase pathway is required for both mating and cell type‐specific differentiation but not for virulence. The Ste11α, Ste7 and Cpk1 kinases were found to act as a co‐ordinate signalling module, whereas the Ste12α transcription factor functions with a redundant partner or in a branched or parallel signalling pathway. Our studies illustrate how MAP kinase cascades can be constructed from cell type‐specific and non‐specific components, yielding pathways that contribute to cell type‐specific patterns of signalling and differentiation.

Pheromones Stimulate Mating and Differentiation via Paracrine and Autocrine Signaling in Cryptococcus neoformans

ABSTRACT Cryptococcus neoformans is a pathogenic fungus with a defined sexual cycle involving haploid MATα and MATa cells. Interestingly, MATα strains are more common, are more virulent than congenic MATa strains, and undergo haploid fruiting in response to nitrogen limitation or MATa cells. Three genes encoding the MFα pheromone were identified in the MATα mating-type locus and shown to be transcriptionally induced by limiting nutrients and coculture with MATa cells. The MFα1, MFα2, and MFα3 genes were mutated, individually and in combination. MATα strains lacking MFα pheromone failed to induce morphological changes in MATa cells. Pheromoneless MATα mutants were fusion and mating impaired but not sterile and mated at ∼1% the wild-type level. The pheromoneless MATα mutants were also partially defective in haploid fruiting, and overexpression of MFα pheromone enhanced haploid fruiting. Overexpression of MFa pheromone also enhanced haploid fruiting of MATα cells and stimulated conjugation tube formation in MATa cells. A conserved G-protein activated mitogen-activated protein kinase signaling pathway was found to be required for both induction and response to mating pheromones. The MFα pheromone was not essential for virulence of C. neoformans but does contribute to the overall virulence composite. These studies define paracrine and autocrine pheromone response pathways that signal mating and differentiation of this pathogenic fungus.

Characterization of the MFα pheromone of the human fungal pathogen Cryptococcus neoformans

Cryptococcus neoformans is an important human pathogenic fungus with a defined sexual cycle and well‐developed molecular and genetic approaches. C. neoformans is predominantly haploid and has two mating types, MATa and MATα. Mating is known to be regulated by nutritional limitation and thought also to be regulated by pheromones. Previously, a portion of the MATα locus was cloned, and a presumptive pheromone gene, MFα1, was identified by its ability to induce conjugation tube‐like filaments when introduced by transformation into MATa cells. Here, the ability of the MFα1 gene to induce these morphological changes in MATa cells was used as a phenotypic assay to perform a structure–function analysis of the gene. We show that the MFα1 open reading frame is required for the morphological response of MATa cells. We also find that the cysteine residue of the C‐terminal CAAX motif is required for activity of the MFα1 pheromone. In addition, we use a reporter system to measure the expression levels of the MFα1 pheromone gene and find that two signals, nutrient starvation and the presence of factors secreted by mating partner cells, impinge on this promoter and regulate MFα1 expression. We identify a second pheromone gene, MFα2, and show phenotypically that this gene is also expressed. Finally, we have synthesized the MFα1 pheromone and show that only the predicted mature modified form of the α‐factor peptide triggers morphological responses in MATa cells.

Retinal ischemia and cell proliferation in the rat: the role of soluble mitogens

Temporary retinal ischemia in the rat leads to a proliferation of endothelial cells and glial cells. We tested the hypothesis that this proliferation is caused by a release of soluble mitogens from the ischemic retina. Conditioned media were prepared from normal rat retina and from retina that had undergone 2 h ischemia and 48 h reperfusion, at which time it showed intense mitotic activity. The conditioned media were placed in cultures of fibroblasts, bovine adrenal capillary endothelial cells, and rat brain astrocytes. Cell proliferation in vitro was stimulated by the retinal extracts in all cell cultures. However, the cell proliferation in cultures with conditioned media from normal retina was similar to that in cultures with conditioned media from ischemic and proliferating retina. Although these data are consistent with the presence of soluble growth factors in the retina, they also indicate that release of these growth factors into the surrounding milieu after transient retinal ischemia is not altered to a degree that would explain the dramatic increase in mitosis.