Aspergillus fumigatus is the leading cause of invasive mold infection and is a serious problem in immunocompromised populations worldwide. We have previously shown that survival of A. fumigatus in serum may be related to secretion of siderophores. In this study, we identified and characterized the sidA gene of A. fumigatus, which encodes L-ornithine N 5 -oxygenase, the first committed step in hydroxamate siderophore biosynthesis. A. fumigatus sidA codes for a protein of 501 amino acids with significant homology to other fungal L-ornithine N 5 -oxygenases. A stable ⌬sidA strain was created by deletion of A. fumigatus sidA. This strain was unable to synthesize the siderophores N,N؆,Nٟ-triacetylfusarinine C (TAF) and ferricrocin. Growth of the ⌬sidA strain was the same as that of the wild type in rich media; however, the ⌬sidA strain was unable to grow in low-iron defined media or media containing 10% human serum unless supplemented with TAF or ferricrocin. No significant differences in ferric reduction activities were observed between the parental strain and the ⌬sidA strain, indicating that blocking siderophore secretion did not result in upregulation of this pathway. Unlike the parental strain, the ⌬sidA strain was unable to remove iron from human transferrin. A rescued strain (⌬sidA ؉ sidA) was constructed; it produced siderophores and had the same growth as the wild type on iron-limited media. Unlike the wild-type and rescued strains, the ⌬sidA strain was avirulent in a mouse model of invasive aspergillosis, indicating that sidA is necessary for A. fumigatus virulence.

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 FeCl 3 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.

Abstract-The acute toxicity of wastewater generated during the extraction of bitumen from oil sands is believed to be due to naphthenic acids (NAs). To determine the factors that affect the rate of degradation of representative NAs in microcosms containing wastewater and the acute toxicity of treated and untreated wastewater, the effects of temperature, dissolved oxygen concentration, and phosphate addition on the rate of 14 CO 2 release from two representative naphthenic acid substrates, (linear) U-14 C-palmitic acid (PA) and (bicyclic) decahydro-2-naphthoic acid-8-14 C (DHNA), were monitored. Tailings pond water (TPW) contained microorganisms well adapted to mineralizing both PA and DHNA: PA was degraded more quickly (10-15% in 4 weeks) compared to DHNA (2-4% in 8 weeks). On addition of phosphate, the rate of NA degradation increased up to twofold in the first 4 weeks, with a concurrent increase in the rate of oxygen consumption by oil sands TPW. The degradation rate then declined to levels equivalent to those measured in flasks without phosphate. The observed plateau was not due to phosphate limitation. Decreases in either the dissolved oxygen concentration or the temperature reduced the rate. Phosphate addition also significantly decreased the acute toxicity of TPW to fathead minnows. In contrast, Microtox analyses showed no reduction in the toxicity of treated or untreated TPW after incubation for up to 8 weeks at 15ЊC.

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. Methods and Results:  Eleven wetlands, both natural and process‐affected, and one tailings settling pond in Northern Alberta were studied. The natural wetlands and process‐affected sites fell into two distinct groups based on their water chemistry. The extent of degradation of a 14C‐labelled monocyclic NA surrogate [14C‐cyclohexane carboxylic acid (CCA)] was relatively uniform in all sediments (approximately 30%) after 14 days. In contrast, degradation of a bicyclic NA surrogate [14C‐decahydronaphthoic acid (DHNA)]was significantly lower in non process‐affected sediments. Enrichment cultures, obtained from an active tailings settling pond, using commercially available NAs as the sole carbon source, resulted in the isolation of a co‐culture containing Pseudomonas putida and Pseudomonas fluorescens. Quantitative GC–MS analysis showed that the co‐culture removed >95% of the commercial NAs, and partially degraded the process NAs from OSPW with a resulting NA profile similar to that from ‘aged wetlands’. Conclusions:  Exposure to NAs induced and/or selected micro‐organisms capable of more effectively degrading bicyclic NAs. Native Pseudomonas spp. extensively degraded fresh, commercial NA. The recalcitrant NAs resembled those found in process‐affected wetlands. Significance and Impact of the Study:  These results suggest that it may be possible to manipulate the existing environmental conditions to select for a microbial community exhibiting higher rates of NA degradation. This will have significant impact on the design of artificial wetlands for water treatment.

Siderophores have been identified as virulence factors in the opportunistic fungal pathogen Aspergillus fumigatus. The 14-pass transmembrane protein MirB is postulated to function as a siderophore transporter, responsible for uptake of the hydroxamate siderophore N,N=,N؆-triacetylfusarinine C (TAFC). Our aim was to identify amino acids of A. fumigatus MirB that are crucial for uptake of TAFC. Site-directed mutagenesis was used to create MirB mutants. Expression of wild-type and mutant proteins in the Saccharomyces cerevisiae strain PHY14, which lacks endogenous siderophore transporters, was confirmed by Western blotting. TAFC transport assays using 55 Fe-labeled TAFC and growth assays with Fe-TAFC as the sole iron source identified alanine 125, tyrosine 577, loop 3, and the second half of loop 7 (Loop7Del2) as crucial for function, since their substitution or deletion abrogated uptake completely. Wild-type MirB transported ferricrocin and coprogen as well as TAFC but not ferrichrysin. MirB was localized by fluorescence microscopy using antisera raised against a MirB extracellular loop peptide. Immunofluorescence microscopy showed that in yeast, wild-type MirB had a punctate distribution under the plasma membrane, as did the A125D and Y577A strains, indicating that the defect in transport of these mutants was unlikely to be due to mislocalization or degradation. MirB immunolocalization in A. fumigatus showed that the transporter was found in vesicles which cycled between the cytoplasm and the plasma membrane and was concentrated at the hyphal tips. The location of MirB was not influenced by the presence of the siderophore TAFC but was sensitive to internal iron stores.