PubMed Health⌕ Search

Biomedical subjects

Gary M Cox

Publications and source records attributed to Gary M Cox.

At least 37 records · Page 2Linked to original sources
PubMed2004

Cryptococcus neoformans methionine synthase: expression analysis and requirement for virulence.

This paper describes (i) the expression profile of the methionine synthase gene (MET6) in the human pathogenic fungus Cryptococcus neoformans and (ii) the phenotypes of a C. neoformans met6 mutant. In contrast to the MET3 gene, which showed no significant change in expression in any environmental condition tested, the MET6 gene showed a substantial induction in response to methionine and a dramatic transcriptional induction in response to homocysteine. Like a met3 mutant, the met6 mutant was a methionine auxotroph. However, relative to a met3 mutant, the met6 mutant grew very slowly and was less heat-shock resistant. In contrast to a met3 mutant, the met6 mutant lost viability when starved of methionine, and it was deficient in capsule formation. Like a met3 mutant, the met6 mutant was avirulent. In contrast to a met3 mutant, the met6 mutant was hypersensitive to fluconazole and to the calcineurin inhibitors FK506 and cyclosporin A. A synergistic fungicidal effect was also found between each of these drugs and met6. The phenotypic differences between the met3 and met6 mutants may be due to the accumulation in met6 mutants of homocysteine, a toxic metabolic intermediate that inhibits sterol biosynthesis.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed2004

Cyclic AMP-dependent protein kinase catalytic subunits have divergent roles in virulence factor production in two varieties of the fungal pathogen Cryptococcus neoformans.

Our earlier findings established that cyclic AMP-dependent protein kinase functions in a signaling cascade that regulates mating and virulence of Cryptococcus neoformans var. grubii (serotype A). Mutants lacking the serotype A protein kinase A (PKA) catalytic subunit Pka1 are unable to mate, fail to produce melanin or capsule, and are avirulent in animal models, whereas mutants lacking the PKA regulatory subunit Pkr1 overproduce capsule and are hypervirulent. Because other mutations have been observed to confer different phenotypes in two diverged varieties of C. neoformans (grubii variety [serotype A] and neoformans variety [serotype D]), we analyzed the functions of the PKA genes in the serotype D neoformans variety. Surprisingly, the Pka1 catalytic subunit was not required for mating, haploid fruiting, or melanin or capsule production of serotype D strains. Here we identify a second PKA catalytic subunit gene, PKA2, that is present in both serotype A and D strains of C. neoformans. The divergent Pka2 catalytic subunit was found to regulate mating, haploid fruiting, and virulence factor production in serotype D strains. In contrast, Pka2 has no role in mating, melanin production, or capsule formation in serotype A strains. Our studies illustrate how different components of signaling pathways can be co-opted and functionally specialized during the evolution of related but distinct varieties or subspecies of a human fungal pathogen.

Alleles↗
PubMed2004

Novel chimeric spermidine synthase-saccharopine dehydrogenase gene (SPE3-LYS9) in the human pathogen Cryptococcus neoformans.

The Cryptococcus neoformans LYS9 gene (encoding saccharopine dehydrogenase) was cloned and found to be part of an evolutionarily conserved chimera with SPE3 (encoding spermidine synthase). spe3-lys9, spe3-LYS9, and SPE3-lys9 mutants were constructed, and these were auxotrophic for lysine and spermidine, spermidine, and lysine, respectively. Thus, SPE3-LYS9 encodes functional spermidine synthase and saccharopine dehydrogenase gene products. In contrast to Saccharomyces cerevisiae spe3 mutants, the polyamine auxotrophy of C. neoformans spe3-LYS9 mutants was not satisfied by spermine. In vitro phenotypes of spe3-LYS9 mutants included reduced capsule and melanin production and growth rate, while SPE3-lys9 mutants grew slowly at 30 degrees C, were temperature sensitive in rich medium, and died upon lysine starvation. Consistent with the importance of saccharopine dehydrogenase and spermidine synthase in vitro, spe3-lys9 mutants were avirulent and unable to survive in vivo and both functions individually contributed to virulence. SPE3-LYS9 mRNA levels showed little evidence of being influenced by exogenous spermidine or lysine or starvation for spermidine or lysine; thus, any regulation is likely to be posttranscriptional. Expression in S. cerevisiae of the full-length C. neoformans SPE3-LYS9 cDNA complemented a lys9 mutant but not a spe3 mutant. However, expression in S. cerevisiae of a truncated gene product, consisting of only C. neoformans SPE3, complemented a spe3 mutant, suggesting possible modes of regulation. Therefore, we identified and describe a novel chimeric SPE3-LYS9 gene, which may link spermidine and lysine biosynthesis in C. neoformans.

Cell Proliferation↗
PubMed2004

Identification of Cryptococcus neoformans temperature-regulated genes with a genomic-DNA microarray.

The ability to survive and proliferate at 37 degrees C is an essential virulence attribute of pathogenic microorganisms. A partial-genome microarray was used to profile gene expression in the human-pathogenic fungus Cryptococcus neoformans during growth at 37 degrees C. Genes with orthologs involved in stress responses were induced during growth at 37 degrees C, suggesting that a conserved transcriptional program is used by C. neoformans to alter gene expression during stressful conditions. A gene encoding the transcription factor homolog Mga2 was induced at 37 degrees C and found to be important for high-temperature growth. Genes encoding fatty acid biosynthetic enzymes were identified as potential targets of Mga2, suggesting that membrane remodeling is an important component of adaptation to high growth temperatures. mga2Delta mutants were extremely sensitive to the ergosterol synthesis inhibitor fluconazole, indicating a coordination of the synthesis of membrane component precursors. Unexpectedly, genes involved in amino acid and pyrimidine biosynthesis were repressed at 37 degrees C, but components of these pathways were found to be required for high-temperature growth. Our findings demonstrate the utility of even partial-genome microarrays for delineating regulatory cascades that contribute to microbial pathogenesis.

Cryptococcus neoformans↗
PubMed2004

Adenylyl cyclase-associated protein Aca1 regulates virulence and differentiation of Cryptococcus neoformans via the cyclic AMP-protein kinase A cascade.

The evolutionarily conserved cyclic AMP (cAMP) signaling pathway controls cell functions in response to environmental cues in organisms as diverse as yeast and mammals. In the basidiomycetous human pathogenic fungus Cryptococcus neoformans, the cAMP pathway governs virulence and morphological differentiation. Here we identified and characterized adenylyl cyclase-associated protein, Aca1, which functions in parallel with the Galpha subunit Gpa1 to control the adenylyl cyclase (Cac1). Aca1 interacted with the C terminus of Cac1 in the yeast two-hybrid system. By molecular and genetic approaches, Aca1 was shown to play a critical role in mating by regulating cell fusion and filamentous growth in a cAMP-dependent manner. Aca1 also regulates melanin and capsule production via the Cac1-cAMP-protein kinase A pathway. Genetic epistasis studies support models in which Aca1 and Gpa1 are necessary and sufficient components that cooperate to activate adenylyl cyclase. Taken together, these studies further define the cAMP signaling cascade controlling virulence of this ubiquitous human fungal pathogen.

Activating Transcription Factors↗
PubMed2003

Enzymes that counteract nitrosative stress promote fungal virulence.

Enzymes that protect cells from reactive oxygen species (superoxide dismutase, catalase, peroxidase) have well-established roles in mammalian biology and microbial pathogenesis. Two recently identified enzymes detoxify nitric oxide (NO)-related molecules; flavohemoglobin denitrosylase consumes NO, and S-nitrosoglutathione (GSNO) reductase metabolizes GSNO. Although both enzymes protect microorganisms from nitrosative challenge in vitro, their relevance has not been established in physiological contexts. Here we studied their biological functions in Cryptococcus neoformans, an established human fungal pathogen that replicates in macrophages and whose growth in vitro and in infected animals is controlled by NO bioactivity. We show that both flavohemoglobin denitrosylase and GSNO reductase contribute to C. neoformans pathogenesis. FHB1 and GNO1 mutations abolished NO- and GSNO-consuming activity, respectively. Growth of fhb1 mutant cells was inhibited by nitrosative challenge, whereas that of gno1 mutants was not. fhb1 mutants showed attenuated virulence in a murine model, and virulence was restored in iNOS(-/-) animals. Survival of the fhb1 mutant was also reduced in activated macrophages and restored to wild-type by inhibition of NOS activity. Combining mutations in flavohemoglobin and GSNO reductase, or flavohemoglobin and superoxide dismutase, further attenuated virulence. These studies illustrate that fungal pathogens elaborate enzymatic defenses against nitrosative stress mounted by the host.

Animals↗
PubMed2003

The Cryptococcus neoformans MAP kinase Mpk1 regulates cell integrity in response to antifungal drugs and loss of calcineurin function.

Cell wall integrity is crucial for fungal growth, development and stress survival. In the model yeast Saccharomyces cerevisiae, the cell integrity Mpk1/Slt2 MAP kinase and calcineurin pathways monitor cell wall integrity and promote cell wall remodelling under stress conditions. We have identified the Cryptococcus neoformans homologue of the S. cerevisiae Mpk1/Slt2 MAP kinase and have characterized its role in the maintenance of cell integrity in response to elevated growth temperature and in the presence of cell wall synthesis inhibitors. C. neoformans Mpk1 is required for growth at 37 degrees C in vitro, and this growth defect is suppressed by osmotic stabilization. C. neoformans mutants lacking Mpk1 are attenuated for virulence in the mouse model of cryptococcosis. Phosphorylation of Mpk1 is induced in response to perturbations of cell wall biosynthesis by the antifungal drugs nikkomycin Z (a chitin synthase inhibitor), caspofungin (a beta-1,3-glucan synthase inhibitor), or FK506 (a calcineurin inhibitor), and mutants lacking Mpk1 display enhanced sensitivity to nikkomycin Z and caspofungin. Lastly, we show that calcineurin and Mpk1 play complementing roles in regulating cell integrity in C. neoformans. Our studies demonstrate that pharmacological inhibition of the cell integrity pathway would enhance the activity of antifungal drugs that target the cell wall.

Animals↗
PubMed2003

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 alpha 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 (Ste11alpha, Ste12alpha) 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 Ste11alpha, Ste7 and Cpk1 kinases were found to act as a co-ordinate signalling module, whereas the Ste12alpha 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.

Animals↗
PubMed2003

Phospholipid-binding protein Cts1 controls septation and functions coordinately with calcineurin in Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes life-threatening meningoencephalitis in immunocompromised patients. The Ca(2+)-calmodulin-activated protein phosphatase calcineurin is necessary for virulence of C. neoformans. Mutants lacking the calcineurin catalytic (Cna1) or regulatory (Cnb1) subunit fail to grow at elevated temperature and are defective in virulence and hyphal elongation. Here we isolated a multicopy suppressor gene, CTS1, which restores growth of a calcineurin mutant strain at 37 degrees C. The CTS1 gene (for calcineurin temperature suppressor 1) encodes a protein containing a C2 domain and a leucine zipper motif that may function as an effector of calcineurin. The CTS1 gene was disrupted by homologous recombination, and cts1 mutants were viable but exhibited defects in cell separation, growth, mating, and haploid fruiting. In addition, cts1 mutants were inviable when calcineurin was mutated or inhibited. Taken together, these findings suggest that calcineurin and Cts1 function in parallel pathways that regulate growth, cell separation, and hyphal elongation.

Calcineurin↗
PubMed2003

Superoxide dismutase influences the virulence of Cryptococcus neoformans by affecting growth within macrophages.

Superoxide dismutase (SOD) is an enzyme that converts superoxide radicals into hydrogen peroxide and molecular oxygen and has been shown to contribute to the virulence of many human-pathogenic bacteria through its ability to neutralize toxic levels of reactive oxygen species generated by the host. SOD has also been speculated to be important in the pathogenesis of fungal infections, but the role of this enzyme has not been rigorously investigated. To examine the contribution of SOD to the pathogenesis of fungal infections, we cloned the Cu,Zn SOD-encoding gene (SOD1) from the human-pathogenic yeast Cryptococcus neoformans and made mutants via targeted disruption. The sod1 mutant strains had marked decreases in SOD activity and were strikingly more susceptible to reactive oxygen species in vitro. A sod1 mutant was significantly less virulent than the wild-type strain and two independent reconstituted strains, as measured by cumulative survival in the mouse inhalational model. In vitro studies established that the sod1 strain had attenuated growth compared to the growth of the wild type and a reconstituted strain inside macrophages producing reduced amounts of nitric oxide. These findings demonstrate that (i) the Cu,Zn SOD contributes to virulence but is not required for pathogenicity in C. neoformans; (ii) the decreased virulence of the sod1 strain may be due to increased susceptibility to oxygen radicals within macrophages; and (iii) other antioxidant defense systems in C. neoformans can compensate for the loss of the Cu,Zn SOD in vivo.

Animals↗
PubMed2003

Role of alternative oxidase gene in pathogenesis of Cryptococcus neoformans.

We identified a homologue of the alternative oxidase gene in a screen to identify genes that are preferentially transcribed in response to a shift to 37 degrees C in the human-pathogenic yeast Cryptococcus neoformans. Alternative oxidases are nucleus-encoded mitochondrial proteins that have two putative roles: they can function in parallel with the classic cytochrome oxidative pathway to produce ATP, and they may counter oxidative stress within the mitochondria. The C. neoformans alternative oxidase gene (AOX1) was found to exist as a single copy in the genome, and it encodes a putative protein of 401 amino acids. An aox1 mutant strain was created using targeted gene disruption, and the mutant strain was reconstituted to wild type using a full-length AOX1. Compared to both the wild-type and reconstituted strains, the aox1 mutant strain was not temperature sensitive but did have significant impairment of both respiration and growth when treated with inhibitors of the classic cytochrome oxidative pathway. The aox1 mutant strain was also found to be more sensitive to the oxidative stressor tert-butyl hydroperoxide. The aox1 mutant strain was significantly less virulent than both the wild type and the reconstituted strain in the murine inhalational model, and it also had significantly impaired growth within a macrophage-like cell line. These data demonstrate that the alternative oxidase of C. neoformans can make a significant contribution to metabolism, has a role in the yeast's defense against exogenous oxidative stress, and contributes to the virulence composite of this organism, possibly by improving survival within phagocytic cells.

Animals↗
PubMed2003

Role of PLB1 in pulmonary inflammation and cryptococcal eicosanoid production.

Cryptococcal phospholipase (PLB1) is a secreted enzyme with lysophospholipase hydrolase and lysophospholipase transacylase activities. To investigate the role of PLB1 in the evasion of host immune responses, we characterized pulmonary immune responses to the parental (H99), the plb1 mutant, and the plb1(rec) reconstituted mutant strains of Cryptococcus neoformans in mice. PLB1 was required for virulence during infection acquired via the respiratory tract. Mice infected with either H99 or the plb1(rec) strain generated a nonprotective inflammatory response with subsequent eosinophilia, while mice infected with the plb1 mutant generated a protective immune response that controlled the infection. Because PLB1 is believed to facilitate virulence through host cell lysis, we examined the interaction of these strains with macrophages. The plb1(rec) mutant exhibited decreased survival during coculture with macrophages. One factor which may be involved in the survival of yeast in the presence of macrophages is fungal eicosanoid production. Host eicosanoids have been shown to down-modulate macrophage functions. plb1 exhibited a defect in eicosanoid production derived from exogenous arachidonoyl-phosphatidylcholine, suggesting that PLB1 is required for the release of arachidonic acid from phospholipids. These data suggest that cryptococcal PLB1 may act as a virulence factor by enhancing the ability to survive macrophage antifungal defenses, possibly by facilitating fungal eicosanoid production.

Animals↗
PubMed2003

Sexual cycle of Cryptococcus neoformans var. grubii and virulence of congenic a and alpha isolates.

Cryptococcus neoformans is a human-pathogenic fungus that has evolved into three distinct varieties that infect most prominently the central nervous system. A sexual cycle involving haploid cells of a and alpha mating types has been reported for two varieties (C. neoformans var. neoformans, serotype D, and C. neoformans var. gattii, serotypes B and C), yet the vast majority of infections involve a distinct variety (C. neoformans var. grubii, serotype A) that has been thought to be clonal and restricted to the alpha mating type. We recently identified the first serotype A isolate of the a mating type which had been thought to be extinct (strain 125.91). Here we report that this unusual strain can mate with a subset of pathogenic serotype A strains to produce a filamentous dikaryon with fused clamp connections, basidia, and viable recombinant basidiospores. One meiotic segregant mated poorly with the serotype A reference strain H99 but robustly with a crg1 mutant that lacks a regulator of G protein signaling and is hyperresponsive to mating pheromone. This meiotic segregant was used to create congenic a and alpha mating type serotype A strains. Virulence tests with rabbit and murine models of cryptococcal meningitis showed that the serotype A congenic a and alpha mating type strains had equivalent virulence in animal models, in contrast to previous studies linking the alpha mating type to increased virulence in congenic serotype D strains. Our studies highlight a role for sexual recombination in the evolution of a human fungal pathogen and provide a robust genetic platform to establish the molecular determinants of virulence.

Animals↗
PubMed2003

Identification of App1 as a regulator of phagocytosis and virulence of Cryptococcus neoformans.

Cryptococcus neoformans is a fungal pathogen that, after inhalation, can disseminate to the brain. Host alveolar macrophages (AMs) represent the first defense against the fungus. Once phagocytosed by AMs, fungal cells are killed by a concerted mechanism, involving the host-cellular response. If the cellular response is impaired, phagocytosis of the fungus may be detrimental for the host, since C. neoformans can grow within macrophages. Here, we identified a novel cryptococcal gene encoding antiphagocytic protein 1 (App1). App1 is a cryptococcal cytoplasmic protein that is secreted extracellularly and found in the serum of infected patients. App1 does not affect melanin production, capsule formation, or growth of C. neoformans. Treatment with recombinant App1 inhibited phagocytosis of fungal cells through a complement-mediated mechanism, and Deltaapp1 mutant is readily phagocytosed by AMs. Interestingly, the Deltaapp1 mutant strain showed a decreased virulence in mice deficient for complement C5 (A/Jcr), but it was hypervirulent in mice deficient for T and NK cells (Tgepsilon26). This study identifies App1 as a novel regulator of virulence for C. neoformans, and it highlights that internalization of fungal cells by AMs increases the dissemination of C. neoformans when the host cellular response is impaired.

Animals↗
PubMed2002

Strain-dependent effects of environmental signals on the production of extracellular phospholipase by Cryptococcus neoformans.

Extracellular phospholipase (PL) activities comprising phospholipase B, lysophospholipase and lysophospholipase transacylase have been identified in culture supernatants of Cryptococcus neoformans and contribute to virulence. We found that PL production was optimal after fungal growth at 30 degrees C and secretion at 37 degrees C for all six C. neoformans isolates studied (four C. neoformans var. neoformans and two C. neoformans var. gattii). No increase in PL activity was found in one strain, NU-2, in low iron or tissue culture media, conditions where upregulation of other virulence factors has been reported. The most virulent strains in an intravenous mouse model of infection were best able to produce PL at growth and secretion temperatures of 37 degrees C, in tissue culture media and under assay conditions of pH 7.0.

Buffers↗
PubMed2002

Ras1 and Ras2 contribute shared and unique roles in physiology and virulence of Cryptococcus neoformans.

The Ras1 signal transduction pathway controls the ability of the pathogenic fungus Cryptococcus neoformans to grow at high temperatures and to mate. A second RAS gene was identified in this organism. RAS2 is expressed at a very low level compared to RAS1, and a ras2 mutation caused no alterations in vegetative growth rate, differentiation or virulence factor expression. The ras2 mutant strain was equally virulent to the wild-type strain in the murine inhalational model of cryptococcosis. Although a ras1 ras2 double mutant strain is viable, mutation of both RAS genes results in a decreased growth rate at all temperatures compared to strains with either single mutation. Overexpression of the RAS2 gene completely suppressed the ras1 mutant mating defect and partially suppressed its high temperature growth defect. After prolonged incubation at a restrictive temperature, the ras1 mutant demonstrated actin polarity defects that were also partially suppressed by RAS2 overexpression. These studies indicate that the C. neoformans Ras1 and Ras2 proteins share overlapping functions, but also play distinct signalling roles. Our findings also suggest a mechanism by which Ras1 controls growth of this pathogenic fungus at 37 degrees C, supporting a conserved role for Ras homologues in microbial cellular differentiation, morphogenesis and virulence.

Actins↗
PubMed2002

Antisense repression in Cryptococcus neoformans as a laboratory tool and potential antifungal strategy.

Antisense repression was used as a method to alter gene function in the human-pathogenic fungus Cryptococcus neoformans. The calcineurin A gene (CNA1) and the laccase gene (LAC1) were targeted since disruption of these loci results in phenotypes that are easy to screen (temperature sensitivity and lack of melanin, respectively). Serotype D yeasts were transformed with a plasmid containing the CNA1 cDNA in an antisense orientation under the control of the inducible GAL7 promoter, and serotype A yeasts were transformed with a plasmid containing the LAC1 cDNA in an antisense orientation under the control of the constitutive actin promoter. The calcineurin transformants demonstrated a temperature-sensitive phenotype only when grown on galactose, and the laccase transformants had decreased melanin production. Northern blot analysis of the calcineurin antisense transformants confirmed that the inducible phenotype was associated with a decrease in the native CNA1 transcript levels. Furthermore, it was possible to modestly impair growth of C. neoformans at 37 degrees C by using a 30 bp antisense oligonucleotide targeting CNA1. Antisense repression is now available as a tool for molecular studies in this organism, and may be applicable to other human-pathogenic fungi that have less amenable genetic systems.

Antifungal Agents↗