PI3K signaling of autophagy is required for starvation tolerance and virulenceof Cryptococcus neoformans.

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 (vps34Delta), 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 vps34Delta 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 vps34Delta 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.

Role of a CUF1/CTR4 copper regulatory axis in the virulence of Cryptococcus neoformans.

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, cuf1Delta 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.

Interleukin-6 and interleukin-10 are expressed in organs of normal young and old mice.

Interleukin-6 (IL-6) is a pleiotropic inflammatory cytokine, also endowed with inflammation-inhibiting properties. The status of interleukin-10 (IL-10) as an anti-inflammatory cytokine is more solidly established. The roles of IL-6 and IL-10 in the context of organ physiology, and their possible modulation by the aging process, are not satisfactorily understood. The purpose of this work was to characterize organ IL-6 and IL-10 expression in different cellular compartments in mice, under steady-state and stress conditions. The former was evaluated by immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA) analyses of organ lysates (LYS) (addressing the intracellular compartment), while the latter was assessed by ELISA analyses of organ-conditioned media (CM), obtained after 48 hrs of organ culturing (addressing the potential of cytokine secretion/diffusion). Under steady-state conditions, the overall level of IL-6 and IL-10 expression was relatively low in both age groups (exceptionally, IHC staining demonstrated an enhanced expression of these cytokines in the heart, skeletal muscle and brain of young mice). Much more elevated levels of IL-6 and IL-10 expression were demonstrated in organ CM, possibly emphasizing the role of these cytokines in the context of organ stress. This was most characteristically shown in the highly specialized organs (heart, skeletal muscle and kidney) and liver of old mice, as compared with the other lymphoreticular organs (lungs, spleen, small intestine) tested. Thus, IL-10 was markedly upregulated in the highly specialized organs, while IL-6 was considerably reduced in the lymphoreticular organs. In addition, aging appears to be associated with altered patterns of intracellular expression and secretion/diffusion potentials of IL-6 and IL-10 in the heart and skeletal muscle, as demonstrated by reduced IHC staining on one hand, and an increased detection in organ CM, on the other. These findings may contribute to a better understanding of the unique functions of organ IL-6 and IL-10 in various age groups, and suggest an important role in organ response to stress in old age.

Different patterns of interleukin-1alpha and interleukin-1beta expression in organs of normal young and old mice.

The different physiological roles of interleukin-1alpha (IL-1alpha) and interleukin-1beta (IL-1beta) are not well understood, especially when considering the apparent overlap and redundancy of the two IL-1 molecules. Characterization of IL-1alpha and IL-1beta expression was performed in this study in organs from young and old mice, using immunohistochemistry and ELISA (enzyme-linked immunosorbent assay). The results indicate that organ IL-1alpha and IL-1beta display different patterns of expression: IL-1alpha is manifested more prominently in lymphoreticular organs (lungs, small intestine, spleen, liver), while IL-1beta is more evident in highly specialized and more vulnerable organs, which do not play a leading role in defense against infections and intoxication (heart, brain, skeletal muscle, kidney). This differential expression is more accentuated in old mice, possibly pointing to the special relevance of these cytokines to organ homeostasis in old age. These findings may shed new light on the physiological functions of IL-1alpha and IL-1beta, and may also lead to the development of improved therapeutic approaches, based on the specific manipulation of these cytokines.

Binding of serum mannan binding lectin to a cell integrity-defective Cryptococcus neoformans ccr4Delta mutant.

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 ccr4Delta mutant defective in cell integrity, and an acapsular cap60Delta strain. No MBL binding was detected on wild-type C. neoformans. In contrast, the ccr4Delta 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 ccr4Delta 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.

Cell wall targeting of laccase of Cryptococcus neoformans during infection of mice.

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 lac1Delta 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.

The Hsp70 member, Ssa1, acts as a DNA-binding transcriptional co-activator of laccase in Cryptococcus neoformans.

Hsp70 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 DNA-binding transcriptional co-activator of the fungal virulence factor, laccase, via binding to a GC-rich element within the 5'-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.

Induction of interleukin-1beta, tumour necrosis factor-alpha and apoptosis in mouse organs by amphotericin B is neutralized by conjugation with arabinogalactan.

OBJECTIVES: To investigate the possibilities that: (i) organ toxicity of amphotericin B-deoxycholate (AMB-DOC) is related to induction of interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha) and apoptosis in target organs; and (ii) the reduced toxicity resulting from the conjugation of AMB with water-soluble arabinogalactan (AMB-AG), is related to modulation of these parameters. METHODS: Organ expression of IL-1beta and TNF-alpha was evaluated by enzyme-linked immunosorbent assay (ELISA) in mouse organ biological fluids and in situ by immunohistochemistry. Tissue damage was evaluated histologically, and apoptosis was demonstrated by terminal dUTP nick end-labelling (TUNEL) staining. AMB-AG conjugate was compared with the micellar (AMB-DOC) and liposomal (AmBisome) AMB formulations. RESULTS: Treatment with AMB-AG or AmBisome caused no observable histopathological damage in the kidneys. In contrast, treatment with AMB-DOC resulted in disruptive changes and apoptosis in renal tubular cells. These effects were found to correlate with induction of high levels of IL-1beta and TNF-alpha in kidney lysates. Unlike AMB-AG, AMB-DOC also induced enhanced IL-1beta and TNF-alpha expression in lysates of lungs, brain, liver and spleen. The marked elevation of these inflammation-apoptosis-promoting cytokines after treatment with AMB-DOC may mediate its systemic and local renal damage. Treatment with AMB-AG (but not AmBisome) appears to uniquely modulate the in situ expression of IL-1beta and enhance secretion of TNF-alpha in kidneys, effects possibly involved in prevention of apoptosis. CONCLUSIONS: 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.