Michael S. Price

Aspergillus niger absorbs copper and zinc from swine wastewater

Wastewater from swine confined-housing operations contains elevated levels of copper and zinc due to their abundance in feed. These metals may accumulate to phytotoxic levels in some agricultural soils of North Carolina due to land application of treated swine effluent. We evaluated fungi for their ability to remove these metals from wastewater and found Aspergillus niger best suited for this purpose. A. niger was able to grow on plates amended with copper at a level five times that inhibitory to the growth of Saccharomyes cerevisiae. We also found evidence for internal absorption as the mechanism used by A. niger to detoxify its environment of copper, a property of the fungus that has not been previously exploited for metal bioremediation. In this report, we show that A. niger is capable of removing 91% of the copper and 70% of the zinc from treated swine effluent.

Transcription Factor Nrg1 Mediates Capsule Formation, Stress Response, and Pathogenesis in Cryptococcus neoformans

ABSTRACT The Cryptococcus neoformans NRG1 gene was identified using gene microarrays to define putative transcription factor genes regulated by the cyclic AMP (cAMP) signal transduction pathway. Disruption of NRG1 results in delayed capsule formation and mating, two phenotypes that are directly controlled by cAMP signaling. Putative targets of the Nrg1 transcription factor were identified using a second genome microarray to define differences in the transcriptomes of the wild-type and nrg1 mutant strains. These experiments implicate Nrg1 in the transcriptional control of multiple genes involved in carbohydrate metabolism and substrate oxidation, as well as the UGD1 gene encoding a UDP-glucose dehydrogenase required for polysaccharide capsule production and cell wall integrity. In addition to being under transcriptional control of the cAMP pathway, Nrg1 contains a putative protein kinase A phosphorylation site; mutation of this motif results in reduced Nrg1 activity. Consistent with prior studies in hypocapsular mutants, the nrg1 mutant strain is attenuated in an animal model of disseminated cryptococcal disease.

Cryptococcus neoformans Requires a Functional Glycolytic Pathway for Disease but Not Persistence in the Host

ABSTRACT Cryptococcus neoformans is an important fungal pathogen of immunocompromised individuals, with a close relative, Cryptococcus gattii, emerging as a serious threat for the immunocompetent. During initial infection, C. neoformans colonizes the airspaces of the lungs, resulting in pneumonia, and subsequently migrates to the central nervous system (CNS). We sought to understand fungal carbon utilization during colonization of these fundamentally different niches within the host, in particular the roles of gluconeogenesis and glycolysis. We created mutants at key points in the gluconeogenesis/glycolysis metabolic pathways that are restricted for growth on lactate and glucose, respectively. A phosphoenolpyruvate carboxykinase mutant (the pck1∆ mutant), blocked for entry of 2- and 3-carbon substrates into gluconeogenesis and attenuated for virulence in a murine inhalation model, showed wild-type (WT) persistence in a rabbit cerebrospinal fluid (CSF) model of cryptococcosis. Conversely, both the pyruvate kinase (pyk1∆) and the hexose kinase I and II (hxk1∆/hxk2∆) mutants, which show impaired glucose utilization, exhibited severely attenuated virulence in the murine inhalation model of cryptococcosis and decreased persistence in the CNS in both the rabbit CSF and the murine inhalation models while displaying adequate persistence in the lungs of mice. These data suggest that glucose utilization is critical for virulence of C. neoformans and persistence of the yeast in the CNS. IMPORTANCE Cryptococcus neoformans is an emerging fungal pathogen of humans and is responsible for approximately 625,000 deaths annually among those suffering from HIV infection/AIDS. The ability of this fungus to persist in the host, coupled with its propensity to colonize the CNS, makes the understanding of nutrient acquisition in the host a primary concern. In this study, we report a requirement of glucose utilization for virulence of C. neoformans that is separate from its role in ATP production in the pathogen. Furthermore, we show that inhibition of glycolysis is a viable antifungal drug target, and impaired ATP production via the PYK1 deletion may serve as a model for dormant/chronic fungal infection in the host. Taken together, these results demonstrate the critical importance of understanding basic metabolic processes of the fungus in the context of host-pathogen interactions. Cryptococcus neoformans is an emerging fungal pathogen of humans and is responsible for approximately 625,000 deaths annually among those suffering from HIV infection/AIDS. The ability of this fungus to persist in the host, coupled with its propensity to colonize the CNS, makes the understanding of nutrient acquisition in the host a primary concern. In this study, we report a requirement of glucose utilization for virulence of C. neoformans that is separate from its role in ATP production in the pathogen. Furthermore, we show that inhibition of glycolysis is a viable antifungal drug target, and impaired ATP production via the PYK1 deletion may serve as a model for dormant/chronic fungal infection in the host. Taken together, these results demonstrate the critical importance of understanding basic metabolic processes of the fungus in the context of host-pathogen interactions.

A Chitinase from Tex6 Maize Kernels Inhibits Growth of Aspergillus flavus

ABSTRACT The maize inbred Tex6 has resistance to colonization and aflatoxin accumulation by Aspergillus flavus. A protein inhibitory to growth of A. flavus has been identified from aqueous extracts of mature Tex6 seeds. This study reports the purification of a chitinase associated with this inhibitory activity to electrophoretic homogeneity and the further characterization of its properties. The inhibitory protein, which has an M(r) of 29,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is an endochitinase that is also capable of exochitinase activity. The enzyme has an optimal pH of 5.5 and a temperature optimum of 45 degrees C. Chitinase activity in maize kernels peaked approximately 36 days after pollination. The Tex6 chitinase purified in this study is capable of inhibiting the growth of A. flavus by 50% at a concentration of 20 mug/ml. Our data indicate that chitinase activity in Tex6 kernels makes a major contribution to the antifungal activity in this maize genotype. Partial peptide sequence of the chitinase showed it to differ from previously reported chitinases.

Improved protocols for functional analysis in the pathogenic fungus Aspergillus flavus

BackgroundAn available whole genome sequence for Aspergillus flavus provides the opportunity to characterize factors involved in pathogenicity and to elucidate the regulatory networks involved in aflatoxin biosynthesis. Functional analysis of genes within the genome is greatly facilitated by the ability to disrupt or mis-express target genes and then evaluate their result on the phenotype of the fungus. Large-scale functional analysis requires an efficient genetic transformation system and the ability to readily select transformants with altered expression, and usually requires generation of double (or multi) gene deletion strains or the use of prototrophic strains. However, dominant selectable markers, an efficient transformation system and an efficient screening system for transformants in A. flavus are absent.ResultsThe efficiency of the genetic transformation system for A. flavus based on uracil auxotrophy was improved. In addition, A. flavus was shown to be sensitive to the antibiotic, phleomycin. Transformation of A. flavus with the ble gene for resistance to phleomycin resulted in stable transformants when selected on 100 μg/ml phleomycin. We also compared the phleomycin system with one based on complementation for uracil auxotrophy which was confirmed by uracil and 5-fluoroorotic acid selection and via transformation with the pyr4 gene from Neurospora crassa and pyrG gene from A. nidulans in A. flavus NRRL 3357. A transformation protocol using pyr4 as a selectable marker resulted in site specific disruption of a target gene. A rapid and convenient colony PCR method for screening genetically altered transformants was also developed in this study.ConclusionWe employed phleomycin resistance as a new positive selectable marker for genetic transformation of A. flavus. The experiments outlined herein constitute the first report of the use of the antibiotic phleomycin for transformation of A. flavus. Further, we demonstrated that this transformation protocol could be used for directed gene disruption in A. flavus. The significance of this is twofold. First, it allows strains to be transformed without having to generate an auxotrophic mutation, which is time consuming and may result in undesirable mutations. Second, this protocol allows for double gene knockouts when used in conjunction with existing strains with auxotrophic mutations.To further facilitate functional analysis in this strain we developed a colony PCR-based method that is a rapid and convenient method for screening genetically altered transformants. This work will be of interest to those working on molecular biology of aflatoxin metabolism in A. flavus, especially for functional analysis using gene deletion and gene expression.

The Cryptococcus neoformans Rho-GDP Dissociation Inhibitor Mediates Intracellular Survival and Virulence

ABSTRACT Rho-GDP dissociation inhibitors (Rho-GDI) are repressors of Rho-type monomeric GTPases that control fundamental cellular processes, such as cytoskeletal arrangement, vesicle trafficking, and polarized growth. We identified and altered the expression of the gene encoding a Rho-GDI homolog in the human fungal pathogen Cryptococcus neoformans and investigated its impact on pathogenicity in animal models of cryptococcosis. Consistent with its predicted function to inhibit and sequester Rho-type GTPases, overexpression of RDI1 results in cytosolic localization of Cdc42. Likely as a result of this finding, RDI1-overexpressing strains exhibited altered morphology compared to that of the wild type, with apparent defects in maintaining proper cell polarity and cytokinesis. RDI1 deletion resulted in increased vacuole size in tissue culture medium and aberrant cell morphology at neutral pH. Maintenance of normal cell morphology is vital for C. neoformans pathogenicity. Accordingly, the rdi1Δ mutant strain also showed reduced intracellular survival in macrophages and severe attenuation of virulence in two murine models of cryptococcosis. This reduction in virulence of the rdi1Δ mutant occurs in the absence of major growth defects in rich medium and with classical virulence-associated phenotypes.

T cells down-regulate macrophage TNF production by IRAK1-mediated IL-10 expression and control innate hyperinflammation

Significance Appropriate development of inflammation is essential to protect hosts from microbial infections, but inflammation can occasionally overshoot and cause collateral damage in hosts. Such hyperinflammation happens during endotoxemia and sepsis, and is attributed to excessive production of proinflammatory cytokines by host cells, such as macrophages. In this study, we demonstrate an unconventional mechanism by which T cells regulate cytokine production of macrophages. It is our general understanding that fast-acting immune cells (such as macrophages) “instruct” how T cells should behave through the step termed antigen presentation. However, what we show here is that T cells instruct macrophages to down-regulate a key proinflammatory cytokine, TNF, within hours after the initiation of endotoxemia. Endotoxemia is caused by excessive inflammation, but the immune system has various mechanisms to avoid collateral organ damage in endotoxemia. A handful of reports have shown that innate immune responses are suppressed by the adaptive immune system. However, the molecular mechanism by which adaptive immune cells suppress innate inflammatory responses is not clear. Here, we report that T cells are shown to interact with macrophages at the early stage of enodotoxemia and to prolong survival of mice through controlling TNF and IL-10 levels by macrophage CD40 stimulation. The cross-talk between CD40 and toll-like receptor (TLR4) signaling first mediates IL-1 receptor-associated kinase 1 (IRAK1) nuclear translocation and its binding to the IL-10 gene promoter in macrophages, without interfering with the NFκB pathway. IL-10 is then detected by macrophages in an autocrine fashion to destabilize Tnfa mRNA. To induce IRAK1-mediated IL-10 expression, signals from both CD40 and TLR4 are essential. CD40 signaling induces IRAK1 sumoylation in the presence of TNF receptor-associated factor 2 (TRAF2) and intracellular isoform of osteopontin (iOPN) whereas TLR4 signaling provides IFN regulatory factor 5 (IRF5) as a chaperone for sumoylated IRAK1 nuclear translocation. Interaction of T cells with macrophages was observed in the spleen in vivo after endotoxemia induction with LPS injection. Our study demonstrates a mechanistic basis for the immunosuppressive role of macrophage CD40 in LPS endotoxemia.

Identification of Genes from the Fungal Pathogen Cryptococcus neoformans Related to Transmigration into the Central Nervous System

Background A mouse brain transmigration assessment (MBTA) was created to investigate the central nervous system (CNS) pathogenesis of cryptococcal meningoencephalitis. Methodology/Principal Findings Two cryptococcal mutants were identified from a pool of 109 pre-selected mutants that were signature-tagged with the nourseothricin acetyltransferase (NAT) resistance cassette. These two mutants displayed abnormal transmigration into the central nervous system. One mutant displaying decreased transmigration contains a null mutation in the putative FNX1 gene, whereas the other mutant possessing a null mutation in the putative RUB1 gene exhibited increased transmigration into the brain. Two macrophage adhesion-defective mutants in the pool, 12F1 and 3C9, showed reduced phagocytosis by macrophages, but displayed no defects in CNS entry suggesting that transit within macrophages (the “Trojan horse” model of CNS entry) is not the primary mechanism for C. neoformans migration into the CNS in this MBTA. Conclusions/Significance This research design provides a new strategy for genetic impact studies on how Cryptococcus passes through the blood-brain barrier (BBB), and the specific isolated mutants in this assay support a transcellular mechanism of CNS entry.

Host Defenses Against Cryptococcosis

The interaction of pathogenic Cryptococcus species with their various hosts is somewhat unique compared to other fungal pathogens such as Aspergillus fumigatus and Candida albicans. Cryptococcus shares an intimate association with host immune cells, leading to enhanced intracellular growth. Furthermore, unlike most other fungal pathogens, the signs and symptoms of cryptococcal disease are typically self-inflicted by the host during the host’s attempt to clear this invader from sensitive organ systems such as the central nervous system. In this review, we will summarize the story of host-Cryptococcus interactions to date and explore strategies to exploit the current knowledge for treatment of cryptococcal infections.

Pleiotropic effects of deubiquitinating enzyme Ubp5 on growth and pathogenesis of Cryptococcus neoformans.

Ubiquitination is a reversible protein modification that influences various cellular processes in eukaryotic cells. Deubiquitinating enzymes remove ubiquitin, maintain ubiquitin homeostasis and regulate protein degradation via the ubiquitination pathway. Cryptococcus neoformans is an important basidiomycete pathogen that causes life-threatening meningoencephalitis primarily in the immunocompromised population. In order to understand the possible influence deubiquitinases have on growth and virulence of the model pathogenic yeast Cryptococcus neoformans, we generated deletion mutants of seven putative deubiquitinase genes. Compared to other deubiquitinating enzyme mutants, a ubp5Δ mutant exhibited severely attenuated virulence and many distinct phenotypes, including decreased capsule formation, hypomelanization, defective sporulation, and elevated sensitivity to several external stressors (such as high temperature, oxidative and nitrosative stresses, high salts, and antifungal agents). Ubp5 is likely the major deubiquitinating enzyme for stress responses in C. neoformans, which further delineates the evolutionary divergence of Cryptococcus from the model yeast S. cerevisiae, and provides an important paradigm for understanding the potential role of deubiquitination in virulence by other pathogenic fungi. Other putative deubiquitinase mutants (doa4Δ and ubp13Δ) share some phenotypes with the ubp5Δ mutant, illustrating functional overlap among deubiquitinating enzymes in C. neoformans. Therefore, deubiquitinating enzymes (especially Ubp5) are essential for the virulence composite of C. neoformans and provide an additional yeast survival and propagation advantage in the host.