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Nanofiber mats hold potential in numerous applications that interface with microorganisms. However, a fundamental study that quantifies the transport of microorganisms into three-dimensional microenvironments, such as nanofiber mats, has not yet been conducted. Here, we evaluate the microbial uptake capacity of three hydrophilic cellulose sorbents, a high surface area electrospun nanofiber mat, as well as two commercial products, a macrofibrous Fisherbrand fabric and an adsorptive Sartorius membrane. The small average fiber diameter (∼1.0 μm) and large porosity of the nanofiber mats enabled a 21 times greater collection of Escherichia coli K12 per milligram of material than the macrofibrous Fisherbrand controls and 220 times more than the Sartorius controls. In most cases, the exposure time of the nanofiber mats to the microorganisms was sufficient to reach a quasi-equilibrium state of microbial uptake, allowing the calculation of an adsorption coefficient (Keq) that relates the concentration of cells in the sorbent to the concentration of cells remaining in solution. The Keq of the nanofiber mats was 420, compared to 9.2 and 0.67 for the Fisherbrand and Sartorius controls, respectively. In addition to E. coli, we studied the cellulose nanofiber mat uptake of two additional medically relevant and distinct microorganisms, Gram-negative Pseudomonas aeruginosa PA01 and Gram-positive Staphylococcus aureus MW2, to probe whether microorganism removal is bacteria-specific. The high uptake capacity of all three bacteria by the nanofiber mats indicates that microbial uptake is independent of the microorganism's adhesion mechanism. This work suggests that cellulose nanofiber mat “sponges” are a green platform technology that has the potential to remove detrimental microorganisms from wounds, trap bacteria within a protective military textile, or remediate contaminated water.

Here we describe the measurement of the inactivation rate constants for the mechanism-based inactivator 2,3-difluorosialic acid acting upon the sialidase from Micromonospora viridifaciens. Using double mixing stopped-flow experiments conducted in a 3-(N-morpholino)propanesulfonic acid buffer (100 mmol/L, pH 7.00) at 25 °C, the derived kinetic parameters are kinact/Ki= (3.9 ± 0.8) × 106(mol/L)–1s–1and Ki= 1.7 ± 0.4 μmol/L. We demonstrate that the inhibitory efficiency of the inactivation event is similar to the catalytic efficiency for this sialidase acting upon a typical substrate, 4-methylumbelliferone α-d-sialoside, kcat/Km= (7.2 ± 2.8) × 106(mol/L)–1s–1. Furthermore, we show that the catalytic efficiencies for inactivation and hydrolysis by the Kdnase from Aspergillus fumigatus are similar for the corresponding Kdn-analogues. We conclude that the deactivating effect of incorporating an axial 3-fluoro substituent onto the sialic acid scaffold is comparable to the enhanced activation that occurs when the 4-methylumbelliferone leaving group is changed to the more nucleofugal fluoride ion.

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.

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.

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.

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. [Mol Cancer Ther 2006;5(3):645–54]