Crystal Structures of Polymorphic Prion Protein β1 Peptides Reveal Variable Steric Zipper Conformations

The pathogenesis of prion diseases is associated with the conformational conversion of normal, predominantly α-helical prion protein (PrP^C) into a pathogenic form that is enriched with β-sheets (PrP^Sc). Several PrP^C crystal structures have revealed β1-mediated intermolecular sheets, suggesting that the β1 strand may contribute to a possible initiation site for β-sheet-mediated PrP^Sc propagation. This β1 strand contains the polymorphic residue 129 that influences disease susceptibility and phenotype. To investigate the effect of the residue 129 polymorphism on the conformation of amyloid-like continuous β-sheets formed by β1, crystal structures of β1 peptides containing each of the polymorphic residues were determined. To probe the conformational influence of the peptide construct design, four different lengths of β1 peptides were studied. From the 12 peptides studied, 11 yielded crystal structures ranging in resolution from 0.9 to 1.4 Å. This ensemble of β1 crystal structures reveals conformational differences that are influenced by both the nature of the polymorphic residue and the extent of the peptide construct, indicating that comprehensive studies in which peptide constructs vary are a more rigorous approach to surveying conformational possibilities

IFN-γ-producing capability of <i>Salmonella</i>-specific CD4 T cells is reduced during peak malaria-parasite infection.

<p>Mice were vaccinated with <i>S</i>. Typhimurium BRD509::2W1S (Vac). On day 42, vaccinated mice were infected with <i>P</i>. <i>yoelii</i> (<i>P</i>.<i>y</i>.). At 14 days post-malaria parasite infection, cells were analyzed. (<b>A</b>) Spleens and livers were analyzed by flow cytometry. Using 2W1S::I-A^b tetramer staining on CD4 T cells, we evaluated both the percentage (%) and absolute number (#) of 2W1S-specific CD4 T cell populations in the liver and spleen. Data are shown as Mean+SEM (n = 3–4). Representative dot plots are shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004027#pntd.0004027.s005" target="_blank">S5 Fig</a>. To simulate CD4 T cells <i>in vivo</i>, <i>P</i>. <i>yoelii</i>-infected or control mice were treated i.v. with heat-killed <i>S</i>. Typhimurium lysates (+ HKST) or PBS. After 4 hours, splenic CD44+ CD4 T cells (<b>B</b>) and 2W1S-specific CD4 T cells (<b>C</b>) were analyzed by flow cytometry for IFN-γ production. Data represent Mean+SEM of three independent experiments (n = 9). To assess antigen-independent stimulation, splenic CD4 T cells were stimulated with PMA and Ionomycin (Iono) for 4 hours. IFN-γ production was evaluated by flow cytometry for antigen-experienced (CD44+) <b>(D)</b> or all CD4 T cells <b>(E)</b>. Bars represent Mean+SEM (n = 3–4). Significance of differences between groups was determined using a Student’s <i>t</i> test (*, <i>p</i><0.05; ns, not significant).</p

<i>P</i>. <i>yoelii</i> infection increases IL-10 expression and IL-10 blockade partially restores protection.

<p>C57BL/6 mice were vaccinated with <i>S</i>. Typhimurium BRD509::2W1S (Vac) and then given <i>P</i>. <i>yoelii</i> (<i>P</i>.<i>y</i>.) as described above. (<b>A</b>) Expression analysis of inflammatory markers by qRT-PCR at 14 d post malaria and 3 days post SL1344 challenge (D17). Transcript levels of interferon-γ (<i>Ifng</i>) and interleukin-10 (<i>Il10</i>) were determined in whole spleen tissue, normalized to beta-actin levels and shown as fold-change over mock-treated mice. Bars indicate mean+SEM (n = 4). (<b>B-C</b>) IL-10 blockade was performed as described in methods. (<b>B</b>) Parasite burden, shown as % infected red blood cells (RBCs), was determined on Giemsa-stained blood smears from tails. (<b>C</b>). Antibody-treated mice were challenged with 750 CFU of virulent <i>S</i>. Typhimurium via the i.v. route at 14 days after <i>P</i>. <i>yoelii</i> infection. At 3 d post-challenge, bacterial burden was determined in livers and normalized to tissue weight. Data represent Mean+SEM (n = 4–5 per group). Significance of differences between groups was determined using a Student’s <i>t</i> test (*, <i>p</i><0.05; ns, not significant).</p

<i>P</i>. <i>yoelii</i> infection increases PD1 expression on NTS-specific CD4 T cells.

<p>C57BL/6 mice were vaccinated with <i>S</i>. Typhimurium BRD509:2W1S (Vac) and then given <i>P</i>. <i>yoelii</i> (<i>P</i>.<i>y</i>.) as described above. (<b>A</b>) At 14 d post <i>P</i>. <i>yoelii</i> infection, splenic T cells were evaluated by flow cytometry for expression of CTLA-4 and PD1. Bar graphs show percentage of CTLA-4+, and PD1+ among 2W1S-specific CD4 T cells. Data are shown as Mean+SEM (n = 10). (<b>B-D</b>) <i>P</i>. <i>yoelii</i>-infected mice were given either an antibody cocktail consisting of αCTLA-4, αLAG3 and αPDL-1 or isotype control antibodies over 7 days as described in methods. (<b>B</b>) Parasite burden with blocking antibody cocktail (n = 3) or isotype control (n = 2), as determined by blood smears. (<b>C</b>) At 14 days post-<i>P</i>. <i>yoelii</i> infection, all the treated mice were injected i.v. with heat-killed <i>S</i>. Typhimurium lysates for 4 hours. Splenic CD4 T cells were analyzed by flow cytometry for IFN-γ production. Bar graphs show percentage of IFN-γ producing Tbet+ CD44+ 2W1S-specific CD4 T cells with blocking antibody cocktail (n = 3) or isotype control (n = 2). (<b>D</b>) Antibody-treated mice were challenged with 750 CFU of virulent <i>S</i>. Typhimurium via the i.v. route at 14 days after <i>P</i>. <i>yoelii</i> infection. At 3 d post-challenge, bacterial burden was determined in livers and normalized to tissue weight. Data represent Mean+SEM (n = 5 per group). Data are shown as Mean+SEM. Significance of differences between groups was determined using a Student’s <i>t</i> test (*, <i>p</i><0.05; ns, not significant).</p

Transient loss in protection to <i>Salmonella</i> challenge during peak malaria-parasite infection in <i>Salmonella</i>-vaccinated mice.

<p>C57BL/6 mice were vaccinated i.v. with 5x10⁵ CFU attenuated <i>S</i>. Typhimurium BRD509 (Vac). Forty-two days later, a group of vaccinated mice was inoculated i.p. with blood containing 4x10⁷<i>P</i>. <i>yoelii</i>-infected red blood cells (RBC) and the rest were given control blood. (<b>A</b>) Parasite burden, shown as % infected red blood cells (RBCs) (n = 25). Data represent Mean±SEM. (<b>B</b>) RBC concentrations (10⁶/uL) at either 14 or 28 days after malaria parasite inoculation as determined by complete blood counts (n = 4–8). Bars represent the Mean+SEM. (<b>C</b>) Mice were challenged with 750–1000 CFU of virulent <i>S</i>. Typhimurium via the i.v. route at either 14 days (<b>C</b>, left) or 28 days (<b>C</b>, right) after <i>P</i>. <i>yoelii</i> infection. At 3 d post-challenge, bacterial burden was determined in livers from mice infected with <i>S</i>. Typhimurium only (Control) or co-infected (<i>P</i>. <i>yoelii</i>). (<b>D</b>) C57BL/6 (n = 5), which are <i>Nramp1</i>/<i>Slc11a1</i>^<i>-/-</i>, or C57BL/6 expressing a functional allele of <i>Slc11a1</i> (n = 3–13), were vaccinated, then inoculated with <i>P</i>. <i>yoelii</i> and challenged i.v. with virulent <i>S</i>. Typhimurium at 14 days after <i>P</i>. <i>yoelii</i> infection. <i>S</i>. Typhimurium burden was quantified in the liver at 3 days after challenge with virulent <i>S</i>. Typhimurium. X, not performed. Data represent Mean+SEM (n = 4–8). Significance of differences between groups was determined using a Student’s <i>t</i> test (*, <i>p</i><0.05; ns, not significant).</p

Expansion of endogenous flagellin_427–441- or SseJ_329–341-specific CD4 T cells after peptide immunization or <i>Salmonella</i> infection of mice.

<p>C57BL/6 mice were immunized sub-cutaneously with either 100 µg of flagellin_427–441 or SseJ_329–341 peptide in the presence of CFA or infected with 5x10⁵<i>Salmonella</i> (BRD509). On day 7, inguinal, axillary, and brachial lymph nodes were isolated from peptide-immunized mice (Immunized). On day 35, spleens were harvested from C57BL/6 mice infected intravenously with <i>Salmonella</i> BRD509 (Infected). Endogenous flagellin_427–441- or SseJ_329–341-specific CD4 T cells were detected using Flagellin:I-A^b or SseJ:I-A^b tetramers, enriched with anti-fluorochrome microbeads, stained with antibodies to several surface markers, and examined by flow cytometry. CD4 T cells from naive, immunized, and <i>Salmonella</i>-infected mice, were detected among CD11c⁻CD11b⁻F4/80⁻B220⁻CD3⁺ cells, and further analyzed for expression of CD44 and Flagellin:I-A^b or SseJ:I-A^b tetramer positive cells. CD4 T cells from the unbound column fraction and CD8 T cells within the bound fraction are also shown as controls. FACS plots are representative of three mice per group and three replicate experiments.</p

Mucosal flagellin-specific CD4 T cells produce IL-22 upon restimulation.

<p>C57BL/6 mice were infected three times orally with 5×10⁹ Salmonella (BRD509) at one-month intervals. On day 7 after the third infection, CD4 T cells from spleen and MLN, including Peyer's patches, were isolated. Purified CD4 T cells were restimulated with 10 µM peptide (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002499#ppat-1002499-t003" target="_blank">Table 3</a>) for 16 h in the presence of irradiated splenocytes. Culture supernatants were collected and IL-22 production measured by ELISA. Data show IL-22 production in culture supernatants as scatter plots representing individual mice. Data are pooled from three separate experiments. Numbers above each group indicate statistical significance and show the <i>p</i> value of a comparison between media alone (no stimulation) and peptide stimulation group.</p

Natural <i>Salmonella</i> class-II epitopes used in this study.

<p>Natural <i>Salmonella</i> class-II epitopes used in this study.</p

Differential regulation of bacterial Flagellin (FliC) and SseJ in vitro and in vivo.

<p>For in vitro detection of bacterial mRNA, <i>Salmonella</i> (BRD509) were cultured under SPI1-inducing conditions (LB broth, for 3hours) or SPI2-inducing conditions (modified N minimal medium, for 6hours) (<i>Top)</i>. For in vivo detection, C57BL/6 mice were infected intravenously with 5X10⁵<i>Salmonella</i> (BRD509), and spleens harvested 30 min or 5 hours later (<i>Bottom</i>). Bacterial RNA was isolated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002499#s4" target="_blank">Materials and Methods</a>. Expression of Flagellin (FliC) or SseJ mRNA was quantified by real-time qPCR. Bar graphs show the mean number ± SEM of Flagellin (FliC) or SseJ mRNA transcripts normalized to the respective amount of 16S rRNA in each sample. Data are pooled from two separate experiments (in vitro) or three separate experiments using a total of nine mice (in vivo). Numbers above indicate statistical significance and show the <i>p</i> value of a comparison between the groups.</p

Salmonella peptides screened for immunogenicity in C57BL/6 mice.

<p>Salmonella peptides screened for immunogenicity in C57BL/6 mice.</p