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 an opportunistic fungal pathogen that causes a life-threatening invasive fungal disease (invasive aspergillosis, IA) in immunocompromised individuals. The first step of pathogenesis is thought to be the attachment of conidia to proteins in lung tissue. Previous studies in our laboratory have shown that conidia adhere to basal lamina proteins via negatively charged sugars on their surface, presumably sialic acids. Sialic acids are a family of more than 50 substituted derivatives of a nine-carbon monosaccharide, neuraminic acid. The purpose of this study was 2-fold: (1) to determine the structure of sialic acids and the glycan acceptor on A. fumigatus oligosaccharides and (2) to determine the effect on the removal of sialic acids from conidia on conidial binding to the extracellular matrix protein fibronectin and phagocytosis of conidia by cultured macrophages and type 2 pneumocytes. Surface sialic acids were removed using Micromonospora viridifaciens sialidase or using acetic acid, mild acid hydrolysis. Lectin binding studies revealed that the majority of conidial sialic acids are alpha2,6-linked to a galactose residue. High-pressure liquid chromatography of derivatized sialic acids released from conidia revealed that unsubstituted N-acetylneuraminic acid is the predominant sialic acid on the surface of conidia. Enzymatic removal of sialic acid significantly decreased the binding of conidia to fibronectin by greater than 65% when compared with sham-treated controls. In addition, removal of sialic acids decreased conidial uptake by cultured murine macrophages and Type 2 pneumocytes by 33% and 53%, respectively. Hence, sialylated molecules on A. fumigatus conidia are ligands for both professional and nonprofessional phagocytes.

A gene encoding a putative sialidase was identified in the genome of the opportunistic fungal pathogen, Aspergillus fumigatus. Computational analysis showed that this protein has Asp box and FRIP domains, it was predicted to have an extracellular localization, and a mass of 42 kDa, all of which are characteristics of sialidases. Structural modeling predicted a canonical 6-bladed β-propeller structure with the model's highly conserved catalytic residues aligning well with those of an experimentally determined sialidase structure. The gene encoding the putative Af sialidase was cloned and expressed in Escherichia coli. Enzymatic characterization found that the enzyme was able to cleave the synthetic sialic acid substrate, 4-methylumbelliferyl α-D-N-acetylneuraminic acid (MUN), and had a pH optimum of 3.5. Further kinetic characterization using 4-methylumbelliferyl α-D-N-acetylneuraminylgalactopyranoside revealed that Af sialidase preferred α2-3-linked sialic acids over the α2-6 isomers. No trans-sialidase activity was detected. qPCR studies showed that exposure to MEM plus human serum induced expression. Purified Af sialidase released sialic acid from diverse substrates such as mucin, fetuin, epithelial cell glycans and colominic acid, though A.fumigatus was unable to use either sialic acid or colominic acid as a sole source of carbon. Phylogenetic analysis revealed that the fungal sialidases were more closely related to those of bacteria than to sialidases from other eukaryotes.

BackgroundDual-energy CT (DECT) has shown tremendous potential as a novel non-invasive method of urate detection in patients with gout. ObjectivesOur aim was to determine the concordance between urate volume and distribution measured on DECT with clinical presentation among patients with gout.MethodsWe conducted a retrospective descriptive study of patients with gout who were referred by a rheumatologist for gout DECT scans between January 2008 and February 2018. At our institution, routine DECT scans for gout consist of four sets of images with limbs scanned in pairs: the hands/wrists, elbows, knees and ankles/feet. We obtained volumetric measurements for all four anatomical regions, and assessed the concordance with clinical presentation as retrieved from patient electronic health record.ResultsA total of 182 patients were included in this study; 96 patients (80.2% male, age range: 27-90, mean age: 62) had urate deposits on DECT scans. Among urate-positive patients, the mean total volume of deposits was 2.45 cm3 (hands/wrists: 0.17 cm3, 7%; elbows: 0.62 cm3, 25%; knees: 0.70 cm3, 28%; ankles/feet: 0.96 cm3, 39%). The average number of urate-positive joints was 2.5, higher than that of clinically symptomatic joints (1.9). Discordance between DECT results and clinical symptoms were seen more often in elbows (46 urate-positive vs. 22 symptomatic) and knees (68 vs. 43), compared with hands/wrists (31 vs. 30) and ankles/feet (90 vs. 87). Only in 25 (26.0%) patients, the distribution of symptomatic joints fully matched the distribution of urate deposits. In 6 patients (6.3%), there was no overlap between these two distribution patterns.ConclusionOn DECT scans, most urate deposits in gout patients occur in the ankles/feet, followed by knees, elbows, and hands/wrists. DECT scans can reveal urate deposit in asymptomatic joints, especially in elbows and knees. Assessing the concordance of urate distribution with clinical presentation in all limb joints in gout patients, our results can help understand the pathophysiology of urate deposition in gout, and guide the development of DECT protocols for the screening, assessment and follow-up management of gout patients.Reference[1] Nicolaou S, Liang T, Murphy DT, Korzan JR, Ouellette H, Munk P. Dual-energy CT: a promising new technique for assessment of the musculoskeletal system. AJR Am J Roentgenol. 2012; 199:S78-86.Disclosure of InterestsBo Gong: None declared, Mark Warwas: None declared, Michael O’Keeffe: None declared, Nicole Tsao: None declared, Mary De Vera: None declared, Kamran Shojania Shareholder of: Stock options in Augurex – biotech company., Grant/research support from: Doing a vasculitis study with BMS, Faisal Khosa: None declared, Savvas Nicolaou Grant/research support from: The Department of Radiology, Vancouver General Hospital has a Master Research Agreement with Siemens Healthcare, Forcheim, Germany (non-pharmaceutical company).