The Aspergillus fumigatus Sialidase (Kdnase) Contributes to Cell Wall Integrity and Virulence in Amphotericin B-Treated Mice.

Aspergillus fumigatus is a filamentous fungus that can cause a life-threatening invasive pulmonary aspergillosis (IPA) in immunocompromised individuals. We previously characterized an exo-sialidase from A. fumigatus that prefers the sialic acid substrate, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (Kdn); hence it is a Kdnase. Sialidases are known virulence factors in other pathogens; therefore, the goal of our study was to evaluate the importance of Kdnase in A. fumigatus. A kdnase knockout strain (Δkdnase) was unable to grow on medium containing Kdn and displayed reduced growth and abnormal morphology. Δkdnase was more sensitive than wild type to hyperosmotic conditions and the antifungal agent, amphotericin B. In contrast, Δkdnase had increased resistance to nikkomycin, Congo Red and Calcofluor White indicating activation of compensatory cell wall chitin deposition. Increased cell wall thickness and chitin content in Δkdnase were confirmed by electron and immunofluorescence microscopy. In a neutropenic mouse model of invasive aspergillosis, the Δkdnase strain had attenuated virulence and a significantly lower lung fungal burden but only in animals that received liposomal amphotericin B after spore exposure. Macrophage numbers were almost twofold higher in lung sections from mice that received the Δkdnase strain, possibly related to higher survival of macrophages that internalized the Δkdnase conidia. Thus, A. fumigatus Kdnase is important for fungal cell wall integrity and virulence, and because Kdnase is not present in the host, it may represent a potential target for the development of novel antifungal agents.

Kinetic and structural evaluation of selected active site mutants of the Aspergillus fumigatus KDNase (sialidase).

Aspergillus fumigatus is an airborne fungal pathogen. We previously cloned and characterized an exo-sialidase from A. fumigatus and showed that it preferred 2-keto-3-deoxynononic acid (KDN) as a substrate to N-acetylneuraminic acid (Neu5Ac). The purpose of this study was to investigate the structure-function relationships of critical catalytic site residues. Site-directed mutagenesis was used to create three mutant recombinant enzymes: the catalytic nucleophile (Y358H), the general acid/base catalyst (D84A), and an enlargement of the binding pocket to attempt to accommodate the N-acetyl group of Neu5Ac (R171L). Crystal structures for all enzymes were determined. The D84A mutation had an effect in decreasing the activity of AfKDNase that was stronger than that of the same mutation in the structurally similar sialidase from the bacterium Micromonospora viridifaciens. These data suggest that the catalytic acid is more important in the reaction of AfKDNase and that catalysis is less dependent on nucleophilic or electrostatic stabilization of the developing positive charge at the transition state for hydrolysis. Removal of the catalytic nucleophile (Y358H) significantly lowered the activity of the enzyme, but this mutant remained a retaining glycosidase as demonstrated by nuclear magnetic resonance spectroscopic analysis. This is a novel finding that has not been shown with other sialidases. Kinetic activity measured at pH 5.2 revealed that R171L had higher activity on a Neu5Ac-based substrate than wild-type KDNase; hence, leucine in place of arginine in the binding pocket improved catalysis toward Neu5Ac substrates. Hence, whether a sialidase is primarily a KDNase or a neuraminidase is due in part to the presence of an amino acid that creates a steric clash with the N-acetyl group.

The Aspergillus fumigatus sialidase is a 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid hydrolase (KDNase): structural and mechanistic insights.

Aspergillus fumigatus is a filamentous fungus that can cause severe respiratory disease in immunocompromised individuals. A putative sialidase from A. fumigatus was recently cloned and shown to be relatively poor in cleaving N-acetylneuraminic acid (Neu5Ac) in comparison with bacterial sialidases. Here we present the first crystal structure of a fungal sialidase. When the apo structure was compared with bacterial sialidase structures, the active site of the Aspergillus enzyme suggested that Neu5Ac would be a poor substrate because of a smaller pocket that normally accommodates the acetamido group of Neu5Ac in sialidases. A sialic acid with a hydroxyl in place of an acetamido group is 2-keto-3-deoxynononic acid (KDN). We show that KDN is the preferred substrate for the A. fumigatus sialidase and that A. fumigatus can utilize KDN as a sole carbon source. A 1.45-Å resolution crystal structure of the enzyme in complex with KDN reveals KDN in the active site in a boat conformation and nearby a second binding site occupied by KDN in a chair conformation, suggesting that polyKDN may be a natural substrate. The enzyme is not inhibited by the sialidase transition state analog 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en) but is inhibited by the related 2,3-didehydro-2,3-dideoxy-D-glycero-D-galacto-nonulosonic acid that we show bound to the enzyme in a 1.84-Å resolution crystal structure. Using a fluorinated KDN substrate, we present a 1.5-Å resolution structure of a covalently bound catalytic intermediate. The A. fumigatus sialidase is therefore a KDNase with a similar catalytic mechanism to Neu5Ac exosialidases, and this study represents the first structure of a KDNase.

Cloning and characterization of a sialidase from the filamentous fungus, Aspergillus fumigatus.

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 beta-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 alpha-D-N-acetylneuraminic acid (MUN), and had a pH optimum of 3.5. Further kinetic characterization using 4-methylumbelliferyl alpha-D-N-acetylneuraminylgalactopyranoside revealed that Af sialidase preferred alpha2-3-linked sialic acids over the alpha2-6 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.

Combined inhibition of the phosphatidylinositol 3-kinase/Akt and Ras/mitogen-activated protein kinase pathways results in synergistic effects in glioblastoma cells.

The present study uses cell-based screening assays to assess the anticancer effects of targeting phosphatidylinositol 3-kinase-regulated integrin-linked kinase (ILK) in combination with small-molecule inhibitors of Raf-1 or mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase (MEK). The objective was to determine if synergistic interactions are achievable through the use of agents targeting two key cell signaling pathways involved in regulating glioblastoma cancer. The phosphatidylinositol 3-kinase/protein kinase B (PKB)/Akt and the Ras/MAPK pathway were targeted for their involvement in cell survival and cell proliferation, respectively. The glioblastoma cell lines U87MG, SF-188, and U251MG were transiently transfected with an antisense oligonucleotide targeting ILK (ILKAS) alone or in combination with the Raf-1 inhibitor GW5074 or with the MEK inhibitor U0126. Dose and combination effects were analyzed by the Chou and Talalay median-effect method and indicated that combinations targeting ILK with either Raf-1 or MEK resulted in a synergistic interaction. Glioblastoma cells transfected with ILKAS exhibited reduced levels of ILK and phosphorylated PKB/Akt on Ser473 but not PKB/Akt on Thr308 as shown by immunoblot analysis. These results were confirmed using glioblastoma cells transfected with ILK small interfering RNA, which also suggested enhanced gene silencing when used in combination with U0126. U87MG glioblastoma cells showed a 90% (P < 0.05) reduction in colony formation in soft agar with exposure to ILKAS in combination with GW5074 compared with control colonies. A substantial increase in Annexin V-positive cells as determined by using fluorescence-activated cell sorting methods were seen in combinations that included ILKAS. Combinations targeting ILK and components of the Ras/MAPK pathway result in synergy and could potentially be more effective against glioblastoma cancer than monotherapy.