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...

Deaths caused by systemic mycoses such as paracoccidioidomycosis, cryptococcosis, histoplasmosis, candidiasis, aspergillosis, coccidioidomycosis and zygomycosis amounted to 3,583 between 1996-2006

IgM and IgA to the Cryptococcus neoformans capsular glucuronoxylomannan (GXM) promote complement-independent phagocytosis by macrophages with efficiency comparable to that of IgG1. IgM- and IgA-mediated phagocytosis of C. neoformans was proportional to CR3 expression, inhibited by Abs to CR3 (CD11b/CD18) and CR4 (CD11c/CD18), and dramatically reduced with macrophages of CD18-deficient mice. IgM and IgA promoted ingestion of yeast cells by CHO cells expressing CR3 and CR4. In contrast, IgG1-mediated phagocytosis was only partially inhibited by Abs to CR3 and CR4. Phagocytosis by IgM and IgA but not IgG1 was inhibited by soluble GXM, which binds CD18. Involvement of CR in antibody-mediated complement-independent phagocytosis indicates a new link between innate and adaptive immune systems.

Complement component 3 (C3) is the major opsonin for the pathogenic fungus Cryptococcus neoformans in the non-immune host. However, the efficiency of complement-mediated opsonization varies, depending on the strain, through mechanisms that are not understood. Analysis of complement-mediated phagocytosis for 12 strains grown in Sabouraud medium revealed that phagocytic indices were inversely correlated with capsule volume. In contrast, there was no correlation between phagocytic index and capsule volume for IgG1-opsonized cells. When capsule size was increased, the efficacy of complement-mediated phagocytosis decreased, whereas that of antibody-mediated phagocytosis increased. C3 localized inside the capsule and at the outer capsule edge for poorly phagocytozed and well-phagocytozed strains, respectively. Blocking experiments revealed that complement-mediated phagocytosis occurred through complement receptor 3 (CR3), without significant involvement of CR1 or CR4. Blocking experiments with antibodies to C3 did not completely abrogate yeast cell uptake, consistent with phagocytosis through glucuronoxylomannan-CR3 interactions. Our data explain how some large encapsulated cells avoid phagocytosis and suggest a novel strategy for immune evasion whereby a microbial capsule interferes with phagocytosis by modifying the location of C3 deposition.

IntroductionCryptococcosis affects 5-30% of patients with AIDS. It is caused by Cryptococcus neoformans, an encapsulated yeast that is acquired by inhalation and ultimately results in a chronic meningoencephalitis with a high tendency to relapse despite effective antifungal therapy (1). Several virulence factors (2-5) including a polysaccharide capsule (6, 7) are described for C. neoformans. Most virulence factors are invariably present (8) in clinical strains. Genetic inactivation of their regulatory proteins, which are part of signaling pathways, renders the yeast avirulent (9, 10). It is, however, unclear whether regulation of virulence factors occurs in vivo during chronic infection in order to enhance the virulence of an infecting C. neoformans strain. Therefore, it is often assumed that failure to eradicate the fungus is a function of a diminished cellmediated immune response (CMI). Several observations, however, indicate that pathogen-specific factors contribute to the pathogenesis of cryptococcosis. First, prolonged in vitro and in vivo passage of C. neoformans isolates can result in phenotypic changes associated with differences in virulence (11)(12)(13)(14). Second, serial isolates from chronically infected patients can exhibit differences in virulence (15). Third, C. neoformans variety gattii strains cause symptomatic infections primarily in immunocompetent hosts even in areas where AIDS is endemic (16).Reversible phenotypic switching can result in microevolution, defined as rapid changing of an organism to a stable, inherited phenotype (17). Microevolution and phenotypic switching can be achieved by several mechanisms, some of which involve changes of genes and follow a mendelian inheritance pattern whereas others (e.g., silencing) are epigenetic and inherited in a nonmendelian fashion. For certain pathogens, the emergence of phenotype variants is associated with invasion of mucosal barriers and immune evasion (18). For fungi, phenotypic switching is an in vitro phenomenon defined as the spontaneous emergence of colonies with altered colony morphology at rates higher than the somatic mutation rates (19-21). Phenotypic switching was demonstrated for C. neoformans in vitro in three different clinical strains and resulted in enhanced virulence (22,23). Despite an association with virulence, phenotypic switching in vivo has been demonstrated only in pathogens with complex life cycles that require the switch for survival in the host (24). Most infections require high doses, and because of the expected in vitro switching rates (10 -2 to 10 -5 ), these inocula are likely to be contaminated with in vitro switch variants. Therefore, it is difficult to demonstrate that the emergence of new phenotypes in the infected host are the result of in vivo switching events and do not represent in vivo selection of variants from an initially heterogeneous pathogen population (25). Phenotypic switching has been linked to the virulence of many pathogens, including fungi. However, it has not been conclusively shown to occur...

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Melanin pigments are substances produced by a broad variety of pathogenic microorganisms, including bacteria, fungi, and helminths. Microbes predominantly produce melanin pigment via tyrosinases, laccases, catecholases, and the polyketide synthase pathway. In fungi, melanin is deposited in the cell wall and cytoplasm, and melanin particles (''ghosts'') can be isolated from these fungi that have the same size and shape of the original cells. Melanin has been reported in several human pathogenic dimorphic fungi including Paracoccidioides brasiliensis, Sporothrix schenckii, Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides posadasii. Melanization appears to contribute to virulence by reducing the susceptibility of melanized fungi to host defense mechanisms and antifungal drugs.

Macrophages are key players during Paracoccidioides brasiliensis infection. However, the relative contribution of the fungal response to counteracting macrophage activity remains poorly understood. In this work, we evaluated the P. brasiliensis proteomic response to macrophage internalization. A total of 308 differentially expressed proteins were detected in P. brasiliensis during infection. The positively regulated proteins included those involved in alternative carbon metabolism, such as enzymes involved in gluconeogenesis, beta-oxidation of fatty acids and amino acids catabolism. The down-regulated proteins during P. brasiliensis internalization in macrophages included those related to glycolysis and protein synthesis. Proteins involved in the oxidative stress response in P. brasiliensis yeast cells were also up-regulated during macrophage infection, including superoxide dismutases (SOD), thioredoxins (THX) and cytochrome c peroxidase (CCP). Antisense knockdown mutants evaluated the importance of CCP during macrophage infection. The results suggested that CCP is involved in a complex system of protection against oxidative stress and that gene silencing of this component of the antioxidant system diminished the survival of P. brasiliensis in macrophages and in a murine model of infection.