Margo M. Moore

Inhibition of hepatocyte plasma membrane Ca2+-ATPase activity by menadione metabolism and its restoration by thiols

Incubation of isolated rat hepatocytes with cytotoxic concentrations of menadione resulted in inhibition of plasma membrane Ca2+‐ATPase activity. This could be restored by subsequent treatment with either dithiothreitol or reduced glutathione, suggesting that the inhibition by menadione was due to oxidation of sulfhydryl groups critical for Ca2+‐ATPase activity.

Uptake of Aspergillus fumigatus Conidia by phagocytic and nonphagocytic cells in vitro: quantitation using strains expressing green fluorescent protein.

ABSTRACT Several pathogenic fungal organisms enter eukaryotic cells and manipulate the host cell environment to favor their own growth and survival. Aspergillus fumigatus is a saprophytic fungus that causes invasive lung disease in the immunocompromised host. To determine whether A. fumigatus could enter eukaryotic cells, we studied the uptake of two different GFP-expressing A. fumigatus strains into A549 lung epithelial cells, human umbilical vein endothelial (HUVE) cells, and J774 murine macrophages in vitro. A549 cells internalized 30% of the bound conidia whereas HUVE and J774 cells internalized 50 and 90%, respectively. Conidia within A549 cells remained viable for 6 h; however, 60 to 80% of conidia within J774 cells were killed after only 4 h. Live and heat-killed conidia were internalized to the same extent by A549 cells. After 6 h, almost none of the conidia inside A549 cells had germinated, whereas extracellular conidia had developed germ tubes. Internalization of conidia by A549 cells was a temperature-dependent process and required rearrangement of the underlying host cell cytoskeleton; uptake was inhibited by 75% with 0.5 μM cytochalasin D and by 65% with 5 μM colchicine. Fluorescent labeling of infected A549 cells with rhodamine phalloidin provided visible evidence of cytoskeletal alteration as many of the intracellular conidia were contained in actin-coated phagosomes. These data provide evidence that significant numbers of A. fumigatus conidia can be internalized by nonprofessional phagocytes in vitro and these cells may serve as reservoirs for immune cell evasion and dissemination throughout the host.

Degradation of naphthenic acids by sediment micro‐organisms

Aims:  Naphthenic acids (NAs) are naturally occurring, linear and cyclic carboxylic surfactants associated with the acidic fraction of petroleum. NAs account for most of the acute aquatic toxicity of oil sands process‐affected water (OSPW). The toxicity of OSPW can be reduced by microbial degradation. The aim of this research was to determine the extent of NA degradation by sediment microbial communities exposed to varying amounts of OSPW.

Aspergillus fumigatus conidia survive and germinate in acidic organelles of A549 epithelial cells

Aspergillus fumigatus is an environmental mould that can cause invasive disease in the immunocompromised host. Previous work has shown that conidia can be internalized by lung epithelial cells (A549) and murine macrophages (J774) in vitro. Therefore, the purpose of this study was to determine the fate of A. fumigatus conidia within the endosomal network of these cells. Co-localization of conidia expressing green fluorescent protein with proteins present in the early endosomal (CD71) and lysosomal (CD63, LAMP-1) membrane was assessed using confocal microscopy. In J774 cells, 75% of internalized conidia were found in phagosomes containing LAMP-1 120 minutes post-infection. In A549 cells, 55% and 58% of internalized conidia were found to co-localize with LAMP-1 and CD63 by 24 hours. Cathepsin D also co-localized with internalized conidia in A549 cells. Phagosomes containing conidia were shown to be acidified in both cell types. Less than 1% of the initial inoculum survived in J774 cells by 12 hours post-infection. After 24 hours, 3% of internalized conidia survived in A549 cells and 34% of these had germinated. By 36 hours, the germlings were able to escape the phagosome and form extracellular hyphae without lysis of the host cell.

Survival of Aspergillus fumigatus in serum involves removal of iron from transferrin: The role of siderophores

ABSTRACT Aspergillus fumigatus is a filamentous fungus which can cause invasive disease in immunocompromised individuals. A. fumigatus can grow in medium containing up to 80% human serum, despite very low concentrations of free iron. The purpose of this study was to determine the mechanism by which A. fumigatus obtains iron from the serum iron-binding protein transferrin. In iron-depleted minimal essential medium (MEM), A. fumigatus growth was supported by the addition of holotransferrin (holoTf) or FeCl3 but not by the addition of apotransferrin (apoTf). Proteolytic degradation of transferrin by A. fumigatus occurred in MEM-serum; however, transferrin degradation did not occur until late logarithmic phase. Moreover, transferrin was not degraded by A. fumigatus incubated in MEM-holoTf. Urea polyacrylamide gel electrophoresis showed that in MEM-holoTf, holoTf was completely converted to apoTf by A. fumigatus. In human serum, all of the monoferric transferrin was converted to apoTf within 8 h. Siderophores were secreted by A. fumigatus after 8 h of growth in MEM-serum and 12 h in MEM-holoTf. The involvement of small molecules in iron acquisition was confirmed by the fact that transferrin was deferrated by A. fumigatus even when physically separated by a 12-kDa-cutoff membrane. Five siderophores were purified from A. fumigatus culture medium, and the two major siderophores were identified as triacetylfusarinine C and ferricrocin. Both triacetylfusarinine C and ferricrocin removed iron from holoTf with an affinity comparable to that of ferrichrome. These data indicate that A. fumigatus survival in human serum in vitro involves siderophore-mediated removal of iron from transferrin. Proteolytic degradation of transferrin may play a secondary role in iron acquisition.

Adhesion of Aspergillus species to extracellular matrix proteins : Evidence for involvement of negatively charged carbohydrates on the conidial surface

ABSTRACT Invasive lung disease caused by Aspergillus species is a potentially fatal infection in immunocompromised patients. The adhesion of Aspergillus fumigatus conidia to proteins in the basal lamina is thought to be an initial step in the development of invasive aspergillosis. The purpose of this study was to determine the mechanism of adhesion of A. fumigatus conidiospores to basal-lamina proteins and to determine whether conidia possess unique adhesins which allow them to colonize the host. We compared conidia from different Aspergillus species for the ability to bind to purified fibronectin and intact basal lamina. Adhesion assays using immobilized fibronectin or type II pneumocyte-derived basal lamina showed that A. fumigatus conidia bound significantly better than those of other Aspergillus species to both fibronectin and intact basal lamina. Neither desialylation nor complete deglycosylation of fibronectin decreased the binding of A. fumigatus conidia to fibronectin, suggesting that oligosaccharides on fibronectin were not involved in conidiospore binding. Further evidence for this hypothesis came from experiments using purified fragments of fibronectin; A. fumigatusconidia preferentially bound to the nonglycosylated 40-kDa fragment which contains the glycosaminoglycan (GAG) binding domain. Negatively charged carbohydrates, including dextran sulfate and heparin, as well as high-ionic-strength buffers, inhibited binding of A. fumigatus conidia to both fibronectin and intact basal lamina, suggesting that negatively charged carbohydrates on the surface of the conidium may bind to the GAG binding domain of fibronectin and other basal-lamina proteins. These data provide evidence for a novel mechanism of conidial attachment whereby adherence to fibronectin and other basal-lamina proteins is mediated via negatively charged carbohydrates on the conidial surface.

Differences in sialic acid density in pathogenic and non-pathogenic Aspergillus species

ASPERGILLUS: fumigatus is a ubiquitous soil fungus that causes invasive lung disease in the immunocompromised host. The structure of the conidial wall has not been well characterized although it is thought that adhesins present on the surface are involved in attachment of the conidia to host lung cells and proteins, which is a prerequisite for the establishment of infection. Negatively charged carbohydrates on the conidial surface have been previously identified as the molecules responsible for attachment of conidia to extracellular matrix proteins. The aim of this research was to identify carbohydrates on the conidial surface that contribute to its negative charge. Direct chemical analysis and indirect binding assays have demonstrated that A. fumigatus possesses sialic acids on the conidial surface. Pre-treatment of A. fumigatus conidia with sialidase decreased binding of a sialic acid-specific lectin, Limax flavus agglutinin (LFA), to the conidial surface and decreased adhesion of conidia to the positively charged polymer poly L-lysine. Two other sialic acid-specific lectins, Maackia amurensis agglutinin and Sambucus nigra agglutinin, exhibited negligible binding to A. fumigatus conidia indicating that 2,3-alpha- and 2,6-alpha-linked sialic acids are not the major structures found on the conidial surface. Mild acid hydrolysis and purification of conidial wall carbohydrates yielded a product that had the same R(F) as the Neu5Ac standard when analysed by high-performance thin-layer chromatography. A density of 6.7 x 10(5) sialic acid residues per conidium was estimated using a colorimetric assay. Conidia grown on a minimal medium lacking sialic acid also reacted with LFA, indicating that sialic acid biosynthesis occurs de novo. Sialic acid biosynthesis was shown to be regulated by nutrient composition: the density of sialic acids on the surface of conidia grown in minimal media was lower than that observed when conidia were grown on rich, complex media. It has previously been shown that pathogenic Aspergillus species adhere to basal lamina proteins to a greater extent than non-pathogenic Aspergillus species. To determine whether the expression of sialic acid on the conidial surface was correlated with adhesion to basal lamina, conidia from other non-pathogenic Aspergillus species were tested for their reactivity towards LFA. Flow cytometric analysis demonstrated that A. fumigatus had a significantly greater sialic acid density than three non-pathogenic Aspergillus species. Sialic acids on the conidial wall may be involved in adhesion to fibronectin, a component of the basal lamina, as binding of A. fumigatus conidia to fibronectin was strongly inhibited in the presence of a sialylated glycoprotein.

The structure of the manganese oxide on the sheath of the bacterium Leptothrix discophora : An XAFS study

Abstract In natural waters, manganese oxides (MnOx) are important in mediating the bioavailability of trace metals such as Ni, Cu, Zn, Cd, and Pb, as these metals readily adsorb to the MnOx surface. Manganese from a variety of anthropogenic sources usually enters the aquatic environment in dissolved form as Mn2+. It is subsequently oxidized under oxic and neutral (pH = 6-7) conditions. Often this oxidation is catalyzed by bacteria, such as Leptothrix discophora, as part of their natural metabolic process. Mn K-edge X-ray Absorption Fine Structure Spectroscopy (XAFS) was used to investigate the local structure of manganese oxide on the sheath produced by the bacterium Leptothrix discophora SP-6. The features observed in the near edge region of the Mn K-edge spectrum indicate the presence of three oxidation states of manganese: Mn2+, Mn3+, and Mn4+. Fitting the experimental XAFS data identifies the bacterial MnOx as being composed of single-layer microcrystals with layers similar to those occurring in Na-birnessite. Some MnO6 octahedra might lie outside the layer plane, sharing corners with those in the layer plane. X-ray diffraction results for the same samples are consistent with the single-layer structure.

The role of carotenoids in preventing oxidative damage in the pigmented yeast, Rhodotorula mucilaginosa.

Rhodotorula mucilaginosa is an obligate aerobic yeast which contains a high concentration of carotenoid pigment. To test whether carotenoids are able to protect R. mucilaginosa against oxidative injury, yeast cells in liquid culture were incubated with duroquinone (DQ) (100 microM), a redox-cycling quinone known to generate intracellular O2-. or were grown in a hyperoxic atmosphere (80% O2) under conditions where carotenoid concentrations were altered either intracellularly or extracellularly. Neither of these oxidative challenges affected cell growth unless carotenogenesis was blocked by the addition of diphenylamine (50 microM). In the diphenylamine-treated nonpigmented cells, growth was completely inhibited by DQ and by hyperoxia. In normoxia, however, diphenylamine alone reduced growth by only 30%. The growth inhibition observed in diphenylamine-treated cells exposed to hyperoxia was primarily mycocidal rather than mycostatic since plating of these cells onto solid media revealed that only 25% of the cells were viable after 50 h of incubation when compared to plated control cells. Addition of 10 microM beta-carotene to diphenylamine-treated cells completely prevented the growth inhibition caused by either hyperoxia or DQ. Carotenoids, therefore, are able to prevent oxidant-induced cytotoxicity in R. mucilaginosa. Analysis of the absorption spectra of chloroform extracts of beta-carotene-supplemented cells showed that beta-carotene, not the endogenous carotenoid, torularhodin, was the major carotenoid present in these cells. Superoxide dismutase (SOD) activity in R. mucilaginosa was compared with that of another yeast, Saccharomyces cerevisiae by two methods: (i) activity staining of proteins separated by gel electrophoresis and (ii) measurement of inhibition of ferricytochrome c reduction. By these techniques, the R. mucilaginosa SOD activity had the characteristics of Mn-SOD. No Cu/ZnSOD activity was detected. Thus, the apparent absence of Cu/ZnSOD may make the antioxidant capability of endogenous carotenoids even more critical in preventing oxidative damage in R. mucilaginosa.

Serum Stimulates Growth of and Proteinase Secretion by Aspergillus fumigatus

ABSTRACT Serum contains iron-binding proteins, which inhibit the growth of most pathogenic microorganisms, including fungi. The purpose of this research was to investigate the effect of serum on growth of the opportunistic fungal pathogen Aspergillus fumigatus. Supplementing minimal essential medium (MEM) with up to 80% human serum or up to 80% fetal bovine serum (FBS) stimulated growth and increased the amount of A. fumigatus dry biomass approximately fourfold. In addition, a 100-fold increase in proteinase secretion, as measured by azocasein hydrolysis, was observed when 10% human serum or 10% FBS was added to MEM. The fungal proteinases secreted in serum-containing media were shown to degrade 3H-labeled basal lamina proteins. The factor in serum that stimulated proteinase secretion was larger than 10 kDa and was 85% inactivated when the serum was heated for 30 min at 66°C. The proportions of proteinases of each catalytic class secreted by A. fumigatus in the presence of serum were different from the proportions secreted in media containing single proteins. Proteinase secretion did not result from increased protein concentration in the medium per se because bovine serum albumin (BSA) at a concentration equivalent to the concentration of serum produced only 20% of the proteinase activity per milligram (dry weight) that was produced by FBS. Addition of BSA plus 100 μM FeCl3 to MEM resulted in the same level of growth as addition of serum, indicating that a combination of nutritional factors in serum may stimulate growth. However, the level of proteinase secretion was still only 30% of the level observed with FBS. These data indicate that serum does not inhibit the growth of A. fumigatus and that the nutrients in serum result in high levels of proteinase secretion, potentially increasing the invasiveness of this species.