CApy-specific serum blocks C. parvum infection of HCT-8 cells.

<p><i>C. parvum</i> sporozoites were preincubated with mouse anti-rCApy serum or unrelated control serum (dilution 1∶10), followed by incubation with HCT-8 cells for 24 hours and infection was quantified by RT-PCR as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031030#s2" target="_blank">Materials and Methods</a>. The values are the means three independently performed experiments performed, error bars represent standard deviations.</p

Glycosylation of CApy in sporozoites.

<p><i>C. parvum</i> sporozoites were treated as recommended in the Enzymatic Carbo Release Kit™ (QAbio, San Mateo, CA) for identification of glycosylation. In brief, sporozoites were suspended in Carbo Release buffer, and denaturation buffer was added. After incubation at 100°C for 5 min, samples were chilled on ice and Triton-X was added. The enzymes PNGase, Sialidase, ß-Galactosidase, Glucosaminidase, O-Glycosidase were added alone or in combinations. Following incubation at 37°C for 16 hours, protein loading buffer was added and samples were incubated again at 100°C for 5 min. Proteins were separated by 12% SDS-PAGE, transferred to nitrocellulose and probed with mouse anti-rCApy serum.</p

Enzymatic characterization of recombinant CApy.

<p>Apyrase activity was measured in 20 mM MOPS buffer, pH 7.4, and a final nucleotide concentration of 2.5 mM. Activity units are micromoles of P_i liberated per milligram of protein per hour at 23°C. The graphs present the mean and standard deviations of triplicate experiments. <b>A</b>. Cofactor specificity of rCApy. ATPase and ADPase activity of rCApy was measured in presence of either 5 mM CaCl₂ or 5 mM MgCl₂ with or without addition of 5 mM EDTA. <b>B</b>. Substrate preference of rCApy. Assays were done in presence of 5 mM CaCl₂.</p

Expression of CApy in oocysts and sporozoites.

<p>CApy is expressed in oocysts and sporozoites. Aliquots of NP-40 soluble (S) or insoluble (I) <i>C. parvum</i> oocyst and sporozoite fractions were resolved by 12% SDS-PAGE, transferred to nitrocellulose and probed with mouse anti-rCApy serum as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031030#s2" target="_blank">Materials and Methods</a>.</p

Expression, Purification and Refolding of recombinant CApy.

<p>The electrophoretic analysis (SDS-PAGE, 12% PAA under reducing conditions) shows extracts of non-induced and induced <i>E. coli</i> cultures (lane 1 and 2) bearing the CApy gene in the pTriEx-4 expression vector from samples taken at different steps of protein purification (lanes 3–10). Following bacterial cell lysis the soluble (supernatant, lane 3) and insoluble (pellet, lane 4) fractions show that CApy was mostly found in the insoluble fraction in form of inclusion bodies (IBs). The solubilized IBs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031030#s2" target="_blank">Materials and Methods</a>) were loaded onto a Ni²⁺ chelate column and purified under denaturing conditions, flow-through (lane 5), wash (lane 6), and elution (lane 7) fractions were collected. The eluate containing the purified CApy was refolded by dialysis against folding buffer (lane 8), which was subsequently dialysed against PBS, pH 7.4 (lane 9) or 20 mM MOPS, pH 7.4 (lane10).</p

Binding of recombinant CApy to intestinal epithelial cells.

<p>Increasing concentrations of protein in the range of 5–60 µg/ml, rCApy or control protein, were added to 96-well plates containing formaldehyde-fixed adherent HCT-8 cells (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031030#s2" target="_blank">Material and Methods</a>. Interactions of proteins, both carrying a His₆-tag, with the HCT-8 cell surface were detected by ELISA using anti-His₆ HRP-conjugated antibody. The bound antibody was revealed by using o-phenylenediamine as a substrate. Respective results of one of three experiments are shown, and are expressed as absorbance at 450 nm. The values are the means of one experiment performed in triplicates, error bars represent standard deviations. An unrelated bacterial protein (Nus) was used as a control (C).</p

Schematic outline of CApy and multiple sequence alignment of the apyrase domain of Cryptosporidium sp. with apyrase homologs of their respective hosts.

<p>A. Alignment of selected apyrase sequences. Sequence alignment was performed using the CLUSTAL 2W algorithm. The GenBank accession numbers of sequences are given in parentheses. CH, <i>Cryptosporidium hominis</i> (XP_666945); CP, <i>Cryptosporidium parvum</i> (XP_627524); CM, <i>Cryptosporidium muris</i> (XP_002140694); HS, <i>Homo sapiens</i> (NP_620148); MM, <i>Mus musculus</i> (EDL34666), BT, <i>Bos Taurus</i> (XP_596269). The enzymatic activity of the human apyrase has been established by biochemical analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031030#pone.0031030-Dai1" target="_blank">[56]</a>, therefore residues important for nucleotide and Ca²⁺ binding in the human apyrase and the predicted counterparts in corresponding apyrases of other species are indicated by gray shadows and pluses. Residues that differ from the human sequence at positions with potential impact on enzymatic activity are colored red. Cysteine residues are shadowed light blue. B. CApy is a protein comprising 345 aa, including a 22-aa signal peptide (SP), and an apyrase domain spanning residues 26–345.</p

Schematic overview of genes exhibiting a change of 2-fold or more in response to melatonin associated with stress response and signaling.

<p>Fold change in transcript levels is represented by the color scale with darkest blue indicating genes with the greatest increase in transcript levels (4-fold or greater) and darkest red indicating genes with the greatest decrease in transcript levels (at least 4-fold). Each gene involved in stress responses is represented once.</p

Receptors, kinases, and genes involved in calcium signaling whose expression levels were affected by 1 mM melatonin by at least 2 fold.

<p>Receptors, kinases, and genes involved in calcium signaling whose expression levels were affected by 1 mM melatonin by at least 2 fold.</p

Venn Diagram representing the overlap of the molecular responses to low (100 pM) and high (1 mM) melatonin treatments.

<p>The number in the parenthesis indicates the number of genes altered by melatonin for each treatment.</p