Biomedical subjects

Gary M Cox

Publications and source records attributed to Gary M Cox.

42 records · Page 3Linked to original sources

PubMed2002

Molecular and genetic analysis of the Cryptococcus neoformans MET3 gene and a met3 mutant.

The Cryptococcus neoformans MET3 cDNA (encoding ATP sulfurylase) was cloned by complementation of the corresponding met3 mutation in Saccharomyces cerevisiae. Sequence analysis showed high similarity between the deduced amino acid sequence of the C. neoformans Met3p and other fungal ATP sulfurylases. A C. neoformans met3 mutant was made by targeted insertional mutagenesis, which had the expected auxotrophic phenotype, and reconstituted the met3 mutant to Met(+). In vitro, the C. neoformans met3 mutant had a substantial defect in melanin formation, significantly reduced growth rate, and greatly increased thermotolerance. In the murine inhalation infection model, the met3 mutant was avirulent and was deficient in its ability to survive in mice. It is concluded that, in contrast to the yeast form of Histoplasma capsulatum, in C. neoformans the sulfate-assimilation arm of the methionine biosynthetic pathway plays an important role in vitro, even in the presence of abundant exogenous methionine, and is critical for virulence, and indeed for survival, in vivo.

Animals↗

PubMed2002

Adenylyl cyclase functions downstream of the Galpha protein Gpa1 and controls mating and pathogenicity of Cryptococcus neoformans.

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 Galpha 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 Galpha protein Gpal, 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 gpal mutant strains. Physiological studies revealed that the Galpha 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 Gpal and adenylyl cyclase function in a conserved signal transduction pathway controlling cAMP production, hyphal differentiation, and virulence of this human fungal pathogen.

Adenylyl Cyclases↗

PubMed2002

Mating-type-specific and nonspecific PAK kinases play shared and divergent roles in Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle involving fusion of haploid MATalpha and MATa cells. Virulence has been linked to the mating type, and MATalpha cells are more virulent than congenic MATa cells. To study the link between the mating type and virulence, we functionally analyzed three genes encoding homologs of the p21-activated protein kinase family: STE20alpha, STE20a, and PAK1. In contrast to the STE20 genes that were previously shown to be in the mating-type locus, the PAK1 gene is unlinked to the mating type. The STE20alpha, STE20a, and PAK1 genes were disrupted in serotype A and D strains of C. neoformans, revealing central but distinct roles in mating, differentiation, cytokinesis, and virulence. ste20alpha pak1 and ste20a pak1 double mutants were synthetically lethal, indicating that these related kinases share an essential function. In summary, our studies identify an association between the STE20alpha gene, the MATalpha locus, and virulence in a serotype A clinical isolate and provide evidence that PAK kinases function in a MAP kinase signaling cascade controlling the mating, differentiation, and virulence of this fungal pathogen.

Alleles↗

PubMed2002

Pheromones stimulate mating and differentiation via paracrine and autocrine signaling in Cryptococcus neoformans.

Cryptococcus neoformans is a pathogenic fungus with a defined sexual cycle involving haploid MATalpha and MATa cells. Interestingly, MATalpha 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 MFalpha pheromone were identified in the MATalpha mating-type locus and shown to be transcriptionally induced by limiting nutrients and coculture with MATa cells. The MFalpha1, MFalpha2, and MFalpha3 genes were mutated, individually and in combination. MATalpha strains lacking MFalpha pheromone failed to induce morphological changes in MATa cells. Pheromoneless MATalpha mutants were fusion and mating impaired but not sterile and mated at approximately 1% the wild-type level. The pheromoneless MATalpha mutants were also partially defective in haploid fruiting, and overexpression of MFalpha pheromone enhanced haploid fruiting. Overexpression of MFa pheromone also enhanced haploid fruiting of MATalpha 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 MFalpha 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.

Autocrine Communication↗

PubMed2002

Relationship of the glyoxylate pathway to the pathogenesis of Cryptococcus neoformans.

Functional genomics has become a major focus in the study of microbial pathogenesis. This study used a functional genomic tool, differential display reverse transcription-PCR, to identify a transcriptional profile of Cryptococcus neoformans cells as they produced meningitis in an immunosuppressed host. This serial global gene expression during infection allowed for the identification of up- and down-regulated genes during infection. During this profiling, a single gene for the enzyme isocitrate lyase (ICL1) was found to be up regulated at 1 week of infection in a rabbit meningitis model and during a time of maximum host cellular response. The finding suggested that this enzyme and the glyoxylate shunt pathway are important to this yeast's energy production during infection. However, site-directed icl1 mutants had no apparent virulence defect in two animal models and no growth defect within macrophages. These observations suggest that although the yeast responded to a certain environmental cue(s) by an increase in ICL1 expression during infection, this gene was not necessary for progression of a C. neoformans infection. Compounds that specifically target only ICL1 are unlikely to cripple C. neoformans growth in vivo.

Amino Acid Sequence↗

PubMedDate not supplied

Evaluation of host immune responses to pulmonary cryptococcosis using a temperature-sensitive C. neoformans calcineurin A mutant strain.

Cryptococcus neoformans is an opportunistic fungal pathogen that threatens individuals with impaired cell-mediated immunity (CMI). Presently, there are no standardized vaccines available to prevent cryptococcal infections and conventional anti-fungal drug therapy does not induce host immune reactivity and thus cannot efficiently resolve C. neoformans infections in immunocompromised individuals. The present study was designed to characterize pulmonary immune responses following infection with an avirulent temperature-sensitive (ts) mutant, calcineurin A1 (cna1) compared to the pathogenic C. neoformans strain H99 and its potential to induce protective anti-cryptococcal immunity. Host CMI responses in cna1-inoculated mice were observed to be dose-dependent, and comprise increases in pulmonary macrophages and CD4(+) T lymphocytes. However, cytokine analysis demonstrated a mixed pulmonary cytokine response (increases in IL-4, and MCP-1) with no induction of IFN-gamma. Also, pre-immunization with the ts cna1 mutant did not result in protection from a subsequent secondary pulmonary infection with the pathogenic C. neoformans strain H99. Taken together, these results suggest that host pulmonary CMI responses to the ts cna1 mutant that is eventually eliminated from the host without the induction of IFN-gamma appear to be dose-dependent, diverse, and require further stimulation to induce C. neoformans-specific Th1-type cytokine responses to resolve subsequent experimental pulmonary cryptococcal infections.

Animals↗