Cloning and Characterization of a Sialidase from the Opportunistic Fungal Pathogen, Aspergillus fumigatus

J. H. Yeung , M. L. Warwas, D. Indurugalla, A. O. Mooers, A. Bennet, M. M. Moore

Author address: 

Simon Fraser Univ., Burnaby, BC, CANADA.

Abstract: 

Aspergillus fumigatus is the most prevalent airborne fungal pathogen in developed countries and it is the main cause of invasive pulmonary aspergillosis in immunocompromised patients. Previous research in our laboratory identified sialic acids on A. fumigatus. Sialic acids are charged monosaccharides known to play important roles in microbial pathogenesis. Although sialic acids are present on the fungal surface, the mechanism of their biosynthesis/acquisition is unknown. Recently, we identified a gene in A. fumigatus encoding a sialidase, an enzyme that cleaves terminal sialic acids from glycoconjugates. Computational analysis and structural modeling identified key traits that were common in other microbial sialidases including Asp box and FRIP domains, and a canonical 6-bladed beta-propeller structure. The gene encoding the putative fungal sialdiase was cloned and expressed in E. coli. The recombinant A. fumigatus sialidase was able cleave the synthetic sialic acid substrate, 4-methylubelliferyl alpha-D-N-acetylneuraminic acid, released sialic acid from biological substrates such as fetuin and colominic acid, and had a pH optimum of 3.5. A. fumigatus sialidase did not exhibit any trans-sialidase activity. Kinetic characterization revealed that the purified sialidase strongly preferred alpha2,3- over alpha2,6- linked sialic acid. qPCR studies showed that exposure to human serum induced sialidase expression. However, A. fumigatus was unable to use either sialic acid or colominic acid as sole carbon sources. Finally, sialidase activity was not detected in culture filtrates. These data suggest that surface sialic acids may be acquired from release of sialic acid from glycoconjugates. A. fumigatus sialidase may represent a suitable target for deletion leading to the creation of a sialic acid-deficient mutant strain of A. fumigatus and allowing us assess the importance of sialic acids in A. fumigatus virulence in vivo.
2010

abstract No: 

F-1689

Full conference title: 

110th General Meeting American Society for Microbiology
    • ASM 110th (2010)