The study of regulatory networks in human pathogens such as Cryptococcus neoformans provides insights into host-pathogen interactions that may allow for correlation of gene expression patterns with clinical outcomes. In the present study, deletion of the cryptococcal copper-dependent transcription factor 1 (Cuf1) led to defects in growth and virulence factor expression in low copper conditions. In mouse models, cuf1Δ strains exhibited reduced dissemination to the brain, but no change in lung growth, suggesting copper is limiting in neurologic infections. To examine this further, a biologic probe of available copper was constructed using the cryptococcal CUF1-dependent copper transporter, CTR4. Fungal cells demonstrated high CTR4 expression levels after phagocytosis by macrophage-like J774.16 cells and during infection of mouse brains, but not lungs, consistent with limited copper availability during neurologic infection. This was extended to human brain infections by demonstrating CTR4 expression during C. neoformans infection of an AIDS patient. Moreover, high CTR4 expression by cryptococcal strains from 24 solid organ transplant patients was associated with dissemination to the CNS. Our results suggest that copper acquisition plays a central role in fungal pathogenesis during neurologic infection and that measurement of stable traits such as CTR4 expression may be useful for risk stratification of individuals with cryptococcosis.

Autophagy is a process by which cells recycle cytoplasm and defective organelles during stress situations such as nutrient starvation. It can also be used by host cells as an immune defense mechanism to eliminate infectious pathogens. Here we describe the use of autophagy as a survival mechanism and virulence-associated trait by the human fungal pathogen Cryptococcus neoformans. We report that a mutant form of C. neoformans lacking the Vps34 PI3K (vps34Δ), which is known to be involved in autophagy in ascomycete yeast, was defective in the formation of autophagy-related 8-labeled (Atg8-labeled) vesicles and showed a dramatic attenuation in virulence in mouse models of infection. In addition, autophagic vesicles were observed in WT but not vps34Δ cells after phagocytosis by a murine macrophage cell line, and Atg8 expression was exhibited in WT C. neoformans during human infection of brain. To dissect the contribution of defective autophagy in vps34Δ C. neoformans during pathogenesis, a strain of C. neoformans in which Atg8 expression was knocked down by RNA interference was constructed and these fungi also demonstrated markedly attenuated virulence in a mouse model of infection. These results demonstrated PI3K signaling and autophagy as a virulence-associated trait and survival mechanism during infection with a fungal pathogen. Moreover, the data show that molecular dissection of such pathogen stress-response pathways may identify new approaches for chemotherapeutic interventions.

Trichosporon asahii (Trichosporon beigelii) infections are rare but have been associated with a wide spectrum of clinical manifestations, ranging from superficial involvement in immunocompetent individuals to severe systemic disease in immunocompromised patients. We report on the recent recovery of T. asahii isolates with reduced susceptibility in vitro to amphotericin B (AMB), flucytosine, and azoles from six nongranulocytopenic patients who exhibited risk factors and who developed either superficial infections (four individuals) or invasive infections (two individuals) while in intensive care units. The latter two patients responded clinically and microbiologically to AMB treatment. All six isolates were closely related according to random amplified polymorphic DNA studies and showed 71% similarity by amplified fragment length polymorphism analysis, suggesting a common nosocomial origin. We also review the literature pertaining to T. asahii infections and discuss the salient characteristics of this fungus and recent taxonomic proposals for the genus.Trichosporon infections are associated with a wide spectrum of clinical manifestations, ranging from superficial cutaneous involvement in immunocompetent individuals to severe systemic disease in immunocompromised patients (9, 44). Trichosporon asahii (Trichosporon beigelii) has increasingly been described as an opportunistic pathogen involved in disseminated infections in patients with profound granulocytopenia (9,11,26). Less commonly reported risk factors associated with infections caused by this agent include treatment with immunosuppressive drugs, transplantation, AIDS, extensive burns, and the presence of implanted prosthetic devices (9,11,14,20,24,27,29).Trichosporon species were the most common non-Candida cause of fungemia at a national cancer institute (23). Disseminated Trichosporon infections in immunocompromised patients are frequently fatal, despite therapy with amphotericin B (AMB) (9,44,46). This antifungal agent has been shown to have a limited in vitro effect against Trichosporon species. In contrast, azoles have been demonstrated to have in vitro activity against members of this genus and their use has been associated with favorable responses in animal models (2,3,9,27,31,32,45,46).We report on the recent recovery of T. asahii isolates resistant in vitro to AMB and azoles from six nongranulocytopenic patients who developed either invasive or superficial infections while hospitalized in different intensive care units (ICUs). We describe the demographic and major clinical characteristics of these patients and review the literature pertaining to T. asahii infections. Results from molecular biology-based studies based on random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analyses suggest that the isolates recovered from specimens of these six patients were closely related. In addition, we discuss the salient characteristics of this fungus and recent taxonomic proposals for the genus. MATERIALS AND METHODSCase re...

Laccase is a major virulence factor of the pathogenic fungus Cryptococcus neoformans, which afflicts both immunocompetent and immunocompromised individuals. In the present study, laccase was expressed in C. neoformans lac1⌬ cells as a fusion protein with an N-terminal green fluorescent protein (GFP) using C. neoformans codon usage. The fusion protein was robustly localized to the cell wall at physiological pH, but it was mislocalized at low pH. Structural analysis of the laccase identified a C-terminal region unique to C. neoformans, and expression studies showed that the region was required for efficient transport to the cell wall both in vitro and during infection of mouse lungs. During infection of mice, adherence to alveolar macrophages was also associated with a partial mislocalization of GFP-laccase within cytosolic vesicles. In addition, recovery of cryptococcal cells from lungs of two strains of mice (CBA/J and Swiss Albino) later in infection was also associated with cytosolic mislocalization, but cells from the brain showed almost exclusive localization to cell walls, suggesting that there was more efficient cell wall targeting during infection of the brain. These data suggest that host cell antifungal defenses may reduce effective cell wall targeting of laccase during infection of the lung but not during infection of the brain, which may contribute to a more predominant role for the enzyme during infection of the brain.Cryptococcus neoformans is a basidiomycete fungal pathogen that infects both immunocompetent and immunocompromised individuals (2, 34). This fungus has several characteristics that allow it to survive during pathogenesis, and it produces numerous factors that cause host cell damage. These characteristics and factors include the ability to grow at 37°C, production of a polysaccharide capsule, expression of laccase, urease, and phospholipase, and production of mannitol (4,5,10,27,32,35). The relative importance of each of the characteristics is not fully understood and may be strain dependent; however, the presence of capsule and the presence of laccase are considered major virulence factors.Laccase expression has been correlated with virulence in numerous studies using multiple strains of the fungus (15,29,32). Laccase has been proposed to contribute to virulence through production of melanin pigments and prevention of iron-dependent Fenton products, with resultant accentuation of extrapulmonary dissemination to the brain (14,19,26,36). However, laccase plays little role in pulmonary persistence (26). C. neoformans has two laccase genes in its genome, LAC1 and LAC2, but only LAC1 is expressed significantly under most conditions and deletion of LAC2 results in no reduction in virulence in mice (31, 41). Laccase from C. neoformans is a member of a class of multicopper oxidases that are primarily extracellular proteins expressed in fungi, plants, and insects (24). The main functions in these organisms are polymerization of monolignols to produce the lignin structure of the cell wall of plan...

Mannan binding lectin (MBL) is an innate immune mediator belonging to the collectin family known to bind to the surfaces of many viruses, bacteria, and fungi. However, pathogenic strains of the fungus Cryptococcus neoformans are resistant to MBL binding. To dissect the mechanism of cryptococcal resistance to MBL, we compared MBL binding to an encapsulated wild-type strain, an encapsulated ccr4⌬ mutant defective in cell integrity, and an acapsular cap60⌬ strain. No MBL binding was detected on wild-type C. neoformans. In contrast, the ccr4⌬ mutant bound MBL to the cell wall, predominantly at the ends of enlarged buds, whereas the acapsular strain bound MBL only at the bud neck and bud scars. In addition, the ccr4⌬ mutant was sensitive to the cell wall-active antifungal caspofungin and other cell wall stress inducers, and its virulence was reduced in a mouse model of cryptococcosis. Interestingly, treatment of wild-type cells with caspofungin also increased MBL binding to C. neoformans. These results suggest that both the presence of capsule and wild-type cell wall architecture preclude MBL binding to C. neoformans.

SummaryHsp70 proteins are a well-known class of chaperones that have also been described to have roles in cellular regulation. Here, we show that a Cryptococcus neoformans Hsp70 homologue Ssa1 acts as a DNAbinding transcriptional co-activator of the fungal virulence factor, laccase, via binding to a GC-rich element within the 5Ј-UAS in response to glucose starvation, iron, copper, calcium and temperature. In addition, Ssa1 forms a regulatory complex with heat shock transcription factor and TATA-binding protein during laccase induction. Furthermore, deletion of Ssa1 results in reduced laccase and attenuated virulence using a mouse model. These results indicate that Hsp70 functions as a stress-related transcriptional co-activator required for fungal virulence.

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AMB-related toxicity is associated with induction of IL-1beta, TNF-alpha and apoptosis in organs. These effects were not observed with AMB-AG conjugate, suggesting its potential as a safer formulation for therapy.