15 research outputs found
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
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 Ni<sup>2+</sup> 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 His<sub>6</sub>-tag, with the HCT-8 cell surface were detected by ELISA using anti-His<sub>6</sub> 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
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
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
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 Ca<sup>2+</sup> 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
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<sub>i</sub> 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 CaCl<sub>2</sub> or 5 mM MgCl<sub>2</sub> with or without addition of 5 mM EDTA. <b>B</b>. Substrate preference of rCApy. Assays were done in presence of 5 mM CaCl<sub>2</sub>.</p
Schematic of non-specific (<i>S</i>. Typhi and CpG) and specific (<i>C</i>. <i>parvum</i> priming) mucosal exposures that modulate host immunity and protect against cryptosporidiosis during malnutrition.
<p>Strategies to enhance immune defenses against <i>Cryptosporidium</i> infection during malnutrition were investigated in a protein deficient murine model that replicates clinical features of childhood cryptosporidiosis. Whereas the well-nourished host (black) clears <i>Cryptosporidium</i> with little evidence of a secondary immune response (dark blue), mucosal vaccination with <i>Cryptosporidium</i> antigens expressed in an <i>S</i>. Typhi vector can elicit strongly boosted IFNγ-predominant immune responses to subsequent challenge (light blue). Vaccine attenuates the mild disease caused by <i>Cryptosporidium</i> in well-nourished hosts. In protein malnourished hosts (light grey) there is ongoing depletion of mucosal lymphocytes including Th1-type effectors. This results in enhanced disease after primary <i>C</i>. <i>parvum</i> challenge with a response characterized by decreased IFNγ but increased IL13 and tendency toward Th2-type cytokines (red). Unlike in nourished hosts, vaccine does not further enhance IFNγ to primary <i>C</i>. <i>parvum</i> challenge, but rather the <i>S</i>. Typhi vector alone drives increased IL17A and partially attenuates disease severity similar to the TLR9 agonist CpG (yellow). <i>C</i>. <i>parvum</i> priming, however, leverages a robust secondary Th1-type response to <i>C</i>. <i>parvum</i> during protein malnutrition, and even at low-doses in this model establishes a mucosal imprinted population of CD8<sup>+</sup> T-cells along with protective immunity to subsequent re-challenge (dark grey).</p
Protein malnutrition alters basal immune responses to primary <i>C</i>. <i>parvum</i> exposure, but secondary responses are intact.
<p>Immunologic responses to two different recombinant <i>Cryptosporidium</i> sporozoite antigens (CApy and Cp15) were performed at 13–15 days post <i>C</i>. <i>parvum</i> challenge in mice fed either control-diet (<i>C</i>. <i>parvum</i><sup>CD</sup>) or protein-deficient diet (<i>C</i>. <i>parvum</i><sup>pd</sup>) and results were compared with naïve age and diet-matched uninfected controls (PBS<sup>CD</sup> and PBS<sup>pd</sup>). Mice began respective diets 12 days prior to <i>C</i>. <i>parvum</i> challenge and remained on the same diets post-challenge. (A) Cytokine secretion in splenocytes of naïve (uninfected) CD or PD-fed mic after stimulation with <i>Cryptosporidium</i> antigens. (B) Serum antibody production as anti-CApy or anti-Cp15 IgG titer (<i>*P<</i>0.05). (C) Cytokines secreted after CApy or Cp15 antigen stimulation in (C) mesenteric lymph nodes. (D) Cytokine secretion in splenocytes expressed as fold change relative to CD-fed uninfected controls. (*<i>P</i><0.05 as indicated). Data is representative of pooled individual responses from two separate tissue harvests (n = 4-5/group).</p
<i>C</i>. <i>parvum</i> priming protects against re-challenge despite continuous protein malnutrition.
<p>Growth (A) and parasite fecal shedding (B) following challenge with 10<sup>7</sup> <i>C</i>. <i>parvum</i> oocysts in either naïve (PBS-<i>C</i>. <i>parvum</i> 10<sup>7</sup>) PD-fed mice, or mice previously exposed to either 10<sup>6</sup> (Cp 10<sup>6</sup>) or 10<sup>7</sup> (Cp 10<sup>7</sup>) <i>C</i>. <i>parvum</i> oocysts 21 days prior as indicated (n = 5/group). (A) *<i>P</i><0.05, **<i>P</i><0.01 for PBS-PBS vs PBS-<i>C</i>. <i>parvum</i> 10<sup>7</sup>, <sup>#</sup><i>P</i><0.05, <sup>###</sup> <i>P</i><0.001 <sup>####</sup> <i>P</i><0.0001 for Cp 10<sup>6-</sup><i>C</i>. <i>parvum</i> 10<sup>7</sup> vs. PBS-<i>C</i>. <i>parvum</i> 10<sup>7</sup>, <i>^</i><i>P</i><0.05, and ^^<i>P</i><0.01, ^^^^<i>P</i><0.0001 for Cp 10<sup>7-</sup><i>C</i>. <i>parvum</i> 10<sup>7</sup> vs. PBS-<i>C</i>. <i>parvum</i> 10<sup>7</sup>. (B) *<i>P</i><0.05, **<i>P</i><0.01 for Cp 10<sup>6-</sup><i>C</i>. <i>parvum</i> 10<sup>7</sup> or Cp 10<sup>7</sup>-<i>C</i>. <i>parvum</i> 10<sup>7</sup> vs. PBS-<i>C</i>. <i>parvum</i> 10<sup>7</sup>.</p