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.Invasive fungal infections in humans are increasing in prevalence in parallel with the growing population of immunocompromised patients. There is a need for new antifungal drugs to treat these infections since the drugs currently available are either excessively toxic or lack broad fungicidal properties. Studies on the pathogenesis of fungal infections should provide insights that can help with the diagnosis and treatment of these important human diseases. Cryptococcus neoformans is a basidiomycetous yeast that has been used successfully as a model pathogenic fungus in a variety of molecular pathogenesis studies. We used C. neoformans to evaluate the contribution of superoxide dismutase (SOD) to the pathogenesis of fungal infections.SODs are metalloenzymes that detoxify oxygen radicals through the conversion of superoxide to hydrogen peroxide and oxygen (20). These enzymes are present in virtually all cells, and this very high degree of conservation is testament to their importance in cellular homeostasis. The primary role of SODs is to protect cells from endogenously generated superoxide anion, which is a by-product of normal aerobic respiration. SODs can be complexed with iron, manganese, and copper plus zinc. The iron and manganese SODs are genetically similar to each other, whereas the Cu,Zn SOD exhibits no significant homology with the other two enzymes (16,20,21,32). Eukaryotic cells generally contain an Mn SOD in the mitochondrial matrix and a Cu,Zn SOD which is located pre...

OBJECTIVE-Liver-specific inactivation of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) by a dominant-negative transgene (L-SACC1 mice) impaired insulin clearance, caused insulin resistance, and increased hepatic lipogenesis. To discern whether this phenotype reflects a physiological function of CEACAM1 rather than the effect of the dominant-negative transgene, we characterized the metabolic phenotype of mice with null mutation of the Ceacam1 gene (Cc1RESEARCH DESIGN AND METHODS-Mice were originally generated on a mixed C57BL/6x129sv genetic background and then backcrossed 12 times onto the C57BL/6 background. More than 70 male mice of each of the Cc1 Ϫ/Ϫ and wild-type Cc1 ϩ/ϩ groups were subjected to metabolic analyses, including insulin tolerance, hyperinsulinemic-euglycemic clamp studies, insulin secretion in response to glucose, and determination of fasting serum insulin, C-peptide, triglyceride, and free fatty acid levels. RESULTS-Like L-SACC1, Cc1Ϫ/Ϫ mice exhibited impairment of insulin clearance and hyperinsulinemia, which caused insulin resistance beginning at 2 months of age, when the mutation was maintained on a mixed C57BL/6x129sv background, but not until 5-6 months of age on a homogeneous inbred C57BL/6 genetic background. Hyperinsulinemic-euglycemic clamp studies revealed that the inbred Cc1 Ϫ/Ϫ mice developed insulin resistance primarily in liver. Despite substantial expression of CEACAM1 in pancreatic ␤-cells, insulin secretion in response to glucose in vivo and in isolated islets was normal in Cc1 Ϫ/Ϫ mice (inbred and outbred strains). CONCLUSIONS-Intact insulin secretion in response to glucose and impairment of insulin clearance in L-SACC1 and Cc1Ϫ/Ϫ mice suggest that the principal role of CEACAM1 in insulin action is to mediate insulin clearance in liver.

Glucocorticoid hormones control diverse physiological processes, including metabolism and immunity, by activating the major glucocorticoid receptor (GR) isoform, GRalpha. However, humans express an alternative isoform, human (h)GRbeta, that acts as an inhibitor of hGRalpha to produce a state of glucocorticoid resistance. Indeed, evidence exists that hGRbeta contributes to many diseases and resistance to glucocorticoid hormone therapy. However, rigorous testing of the GRbeta contribution has not been possible, because rodents, especially mice, are not thought to express the beta-isoform. Here, we report expression of GRbeta mRNA and protein in the mouse. The mGRbeta isoform arises from a distinct alternative splicing mechanism utilizing intron 8, rather than exon 9 as in humans. The splicing event produces a form of beta that is similar in structure and functionality to hGRbeta. Mouse (m)GRbeta has a degenerate C-terminal region that is the same size as hGRbeta. Using a variety of newly developed tools, such as a mGRbeta-specific antibody and constructs for overexpression and short hairpin RNA knockdown, we demonstrate that mGRbeta cannot bind dexamethasone agonist, is inhibitory of mGRalpha, and is up-regulated by inflammatory signals. These properties are the same as reported for hGRbeta. Additionally, novel data is presented that mGRbeta is involved in metabolism. When murine tissue culture cells are treated with insulin, no effect on mGRalpha expression was observed, but GRbeta was elevated. In mice subjected to fasting-refeeding, a large increase of GRbeta was seen in the liver, whereas mGRalpha was unchanged. This work uncovers the much-needed rodent model of GRbeta for investigations of physiology and disease.

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Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) regulates insulin sensitivity by promoting hepatic insulin clearance. Liver-specific inactivation or global null-mutation of Ceacam1 impairs hepatic insulin extraction to cause chronic hyperinsulinemia, resulting in insulin resistance and visceral obesity. In this study we investigated whether diet-induced insulin resistance implicates changes in hepatic CEACAM1. We report that feeding C57/BL6J mice a high-fat diet reduced hepatic CEACAM1 levels by >50% beginning at 21 days, causing hyperinsulinemia, insulin resistance, and elevation in hepatic triacylglycerol content. Conversely, liver-specific inducible CEACAM1 expression prevented hyperinsulinemia and markedly limited insulin resistance and hepatic lipid accumulation that were induced by prolonged high-fat intake. This was partly mediated by increased hepatic β-fatty acid oxidation and energy expenditure. The data demonstrate that the high-fat diet reduced hepatic CEACAM1 expression and that overexpressing CEACAM1 in liver curtailed diet-induced metabolic abnormalities by protecting hepatic insulin clearance.

The ability of Cryptococcus neoformans to grow at the mammalian body temperature (37°C to 39°C) is a well-established virulence factor. Growth of C. neoformans at this physiological temperature requires calcineurin, a Ca 2؉ /calmodulin-dependent protein phosphatase. When cytosolic calcium concentrations are low (ϳ50 to 100 nM), calcineurin is inactive and becomes active only when cytosolic calcium concentrations rise (ϳ1 to 10 M) through the activation of calcium channels. In this study we analyzed the function of Cch1 in C. neoformans and found that Cch1 is a Ca 2؉ -permeable channel that mediates calcium entry in C. neoformans. Analysis of the Cch1 protein sequence revealed differences in the voltage sensor (S4 regions), suggesting that Cch1 may have diminished voltage sensitivity or possibly an alternative gating mechanism. The inability of the cch1 mutant to grow under conditions of limited extracellular calcium concentrations ([Ca 2؉ ] extracellular , ϳ100 nM) suggested that Cch1 was required for calcium uptake in low-calcium environments. These results are consistent with the role of ScCch1 in mediating high-affinity calcium uptake in Saccharomyces cerevisiae. Although the growth defect of the cch1 mutant under conditions of limited [Ca 2؉ ] extracellular (ϳ100 nM) became more severe with increasing temperature (25°C to 38.5°), this temperature sensitivity was not observed when the cch1 mutant was grown on rich medium ([Ca 2؉ ] extracellular , ϳ0.140 mM). Accordingly, the cch1 mutant strain displayed only attenuated virulence when tested in the mouse inhalation model of cryptococcosis, further suggesting that C. neoformans may have a limited requirement for Cch1 and that this requirement appears to include ion stress tolerance.

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°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.Cryptococcus neoformans is a basidiomycetous fungus and a major human pathogen. This pathogenic fungus has a wide human host range by producing infections in both immunocompromised and immunocompetent hosts (6). Understanding the mechanisms of how this encapsulated yeast can so effectively attack a susceptible host has received renewed attention as the infrastructure for molecular biology of this yeast has matured. Several phenotypic factors in the virulence composite, such as formation of a polysaccharide capsule (7), melanin production (45), urease synthesis (11), phospholipase secretion (10), mannose production (46), high-temperature growth (28), and several signaling molecules and pathways for these factors (44,28

Cryptococcus neoformans is an encapsulated fungal pathogen that primarily infects the central nervous system of immunocompromised individuals, causing life-threatening meningoencephalitis. The capacity of C. neoformans to subvert host defenses and disseminate by intracellular parasitism of alveolar macrophages in the immune-compromised host has led to studies to evaluate genes associated with C. neoformans resistance to oxidative stress. In the present study, we identify and characterize a C. neoformans homologue to SKN7, a transcription factor in Saccharomyces cerevisiae that regulates the oxidative stress response, cell cycle, and cell wall biosynthesis. To examine the contribution of SKN7 in the pathogenesis of fungal infections, we created skn7 mutants via targeted disruption. The skn7 mutants were observed to be more susceptible to reactive oxygen species in vitro and were significantly less virulent than the wild-type strain and a reconstituted strain as measured by cumulative survival in the mouse inhalational model. The Skn7 protein was observed to be important for expression of thioredoxin reductase in response to oxidative challenge. Interestingly, skn7 mutants were also observed to flocculate following in vitro culture, a novel phenotype not observed in skn7 mutants derived from other fungi. These findings demonstrate that SKN7 contributes to the virulence composite but is not required for pathogenicity in C. neoformans. In addition, flocculation of C. neoformans skn7 mutants suggests a potentially unique function of SKN7 not previously observed in other cryptococcal strains or skn7 mutants.Cryptococcus neoformans, the etiological agent of cryptococcosis, is an encapsulated fungal pathogen that infects the central nervous system of immunocompromised individuals, causing life-threatening meningoencephalitis (31). Cryptococcosis occurs in approximately 5 to 25% of AIDS patients worldwide (37), and studies have shown that 2.8% of organ transplant recipients can develop C. neoformans infections, resulting in an overall death rate of 42% (16). Although highly active antiretroviral therapy has contributed to a significant decrease in the incidence of cryptococcosis in AIDS patients in developed countries (2), increases in organ transplant recipients and patients undergoing extensive corticosteroid therapy forecast a rise of cryptococcosis in other high-risk populations. In medically advanced countries the acute mortality rate is between 10 and 25% (37), and conventional antifungal agents are often excessively toxic, lack potent fungicidal properties, or are being rendered less effective by the emergence of drug-resistant strains. Therefore, continued studies are needed to identify novel targets for the development of drugs or vaccines to combat cryptococcal infections.Obligate aerobic microorganisms, such as C. neoformans, are under persistent exposure to endogenous oxidative stress caused by the incomplete reduction of oxygen to water, which yields reactive oxygen species (ROS) such as H 2 O 2 , the hydrox...