Anti-SP0845^23–350 antibodies are functional as assessed by blood bactericidal assay.

<p>Pneumococcal cells (100–150 cfu) from strains TIGR4 (A), ATCC 6303 (B), ATCC 6314 (C) and D39 (D) were pretreated with either preimmune (PI, dark bar) or mouse anti-SP0845^23–350 hyperimmune (HI, grey bar) sera and incubated with peripheral blood from CBA/N mice at 37°C for 3 h with rotation. The surviving bacteria were enumerated by plating serial dilutions on TSA plates in duplicate. The data represents the mean ± SD values. The data was analyzed using Student's unpaired t test.</p

Purification and immunoblotting of SP0845.

<p>SP0845^23–350 was expressed in <i>E</i>. <i>coli</i> and purified using Ni-NTA affinity and DEAE sepharose anion-exchange chromatography. The purified recombinant protein was resolved on 12% SDS-PAG and stained with Coomassie brilliant blue R250 (A) or transferred to nitrocellulose membrane and visualized using anti-histidine affinity tag antibody followed by horseradish peroxidase conjugated goat anti-mouse Ig as secondary antibody and 3, 3' diaminobenzidine/ H₂O₂ as substrate (B). The molecular mass marker (in kDa) is shown on the left of the panels.</p

Route of calcium entry differentially regulates PspA induced PD-L1 expression.

<p>Mouse bone marrow derived DCs were incubated with bio-pharmacological inhibitors to indicated molecules for 1h followed by stimulation with 15 μg/ml PspA for 24h. PD-L1 levels were monitored by flow cytometry. Dotted lines represent unstimulated cells. Thin lines represent cells stimulated with PspA. Bold lines represent cells treated with inhibitors to indicated molecules followed by stimulation with PspA. One of three independent experiments is shown. PD-L1 expression is represented as bar graph indicating fold increase in Relative Mean Fluorescence Intensity (MFI) for various groups. Bars represent mean ± SD of three independent experiments. For Panel B, mouse bone marrow derived DCs were incubated in the presence or absence of TMB-8 or EGTA for 1h followed by stimulations with 15 μg/ml PspA for 24h. Cells were incubated with phycoerythrin conjugated anti-mouse PD-L1 antibody. Merged images with DAPI (blue) and PD-L1 (red) staining are depicted. For Panel C total RNA was isolated from cells stimulated as indicated and PD-L1 transcript levels were measured by semi-quantitative RT-PCR. One of three independent experiments is shown.</p

Conserved Surface Accessible Nucleoside ABC Transporter Component SP0845 Is Essential for Pneumococcal Virulence and Confers Protection <i>In Vivo</i>

<div><p><i>Streptococcus pneumoniae</i> is a leading cause of bacterial pneumonia, sepsis and meningitis. Surface accessible proteins of <i>S. pneumoniae</i> are being explored for the development of a protein-based vaccine in order to overcome the limitations of existing polysaccharide-based pneumococcal vaccines. To identify a potential vaccine candidate, we resolved surface-associated proteins of <i>S. pneumoniae</i> TIGR4 strain using two-dimensional gel electrophoresis followed by immunoblotting with antisera generated against whole heat-killed TIGR4. Ten immunoreactive spots were identified by mass spectrometric analysis that included a putative lipoprotein SP0845. Analysis of the inferred amino acid sequence of <i>sp0845</i> homologues from 36 pneumococcal strains indicated that SP0845 was highly conserved (>98% identity) and showed less than 11% identity with any human protein. Our bioinformatic and functional analyses demonstrated that SP0845 is the substrate-binding protein of an ATP-binding cassette (ABC) transporter that is involved in nucleoside uptake with cytidine, uridine, guanosine and inosine as the preferred substrates. Deletion of the gene encoding SP0845 renders pneumococci avirulent suggesting that it is essential for virulence. Immunoblot analysis suggested that SP0845 is expressed in <i>in vitro</i> grown pneumococci and during mice infection. Immunofluorescence microscopy and flow cytometry data indicated that SP0845 is surface exposed in encapsulated strains and accessible to antibodies. Subcutaneous immunization with recombinant SP0845 induced high titer antibodies in mice. Hyperimmune sera raised against SP0845 promoted killing of encapsulated pneumococcal strains in a blood bactericidal assay. Immunization with SP0845 protected mice from intraperitoneal challenge with heterologous pneumococcal serotypes. Based on its surface accessibility, role in virulence and ability to elicit protective immunity, we propose that SP0845 may be a potential candidate for a protein-based pneumococcal vaccine.</p></div

Immunoreactive pneumococcal proteins identified in this study by mass spectrometry.

<p>^<i>a</i> For spot numbers 1 through 8 refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118154#pone.0118154.g001" target="_blank">Fig. 1</a>. Spot number 9 and 10 were identified from an independent experiment performed under similar experimental conditions.</p><p>^<i>b</i> Gene and protein annotations are according to the genome sequence of <i>S</i>. <i>pneumoniae</i> TIGR4 (GenBank accession no. AE005672). ORF, open reading frame.</p><p>^<i>c</i> Percentage of total protein sequence covered by the experimentally detected peptides.</p><p>^<i>d</i> Identification probability of the fragment match.</p><p>^<i>e</i> MASCOT scores greater than the cut-off value were considered statistically significant (<i>p</i> ≤ 0.05).</p><p>Immunoreactive pneumococcal proteins identified in this study by mass spectrometry.</p

SP0845-specific serum antibody endpoint titers.

<p>^<i>a</i> The SP0845-specific antibody endpoint titer was determined by ELISA after pooling serum from 12 mice immunized subcutaneously with recombinant SP0845. The endpoint titer was determined using two times the optical density obtained for the preimmune sera (diluted 1 in 100) as the cut off value.</p><p>SP0845-specific serum antibody endpoint titers.</p

SPD_0739 (homologue of SP0845) is involved in nucleoside import by pneumococci.

<p>(A) Wildtype (WT) D39, <i>spd_0739</i> deficient (KO), <i>spd_0739</i> deficient strain genetically complemented with <i>spd_0739</i> (GC) and <i>spd_0739</i> deficient strain transformed with pDC123_DS (vector control; VC) were propagated either in the absence or presence of 0.1 mM 5-fluorouridine (5FU). (B) Wildtype TIGR4 and its <i>sp0845</i> deficient derivative were propagated either in the absence or presence of 0.1 mM 5FU. (C) Wildtype D39 and a second independently generated <i>spd_0739</i> deficient mutant were grown either in the absence or presence of 0.1 mM 5FU. The absorbance of the pneumococcal cultures was recorded at 620 nm after 6 h. For panels A, B and C the statistical significance was determined using the Student's t test with the corresponding 'No 5FU' sample as the reference. (D) Wildtype D39 was grown in presence of 0.05 mM 5FU and the indicated solutes at a concentration of 0.5 mM. Six hours later the absorbance was recorded. Wildtype D39 in the absence of 5FU served as the positive control. Error bars represent mean ± SD. The data presented is a representative of three independent experiments each performed in duplicates. One-way ANOVA analysis was performed using D39 sample with 5FU in the absence of any competing solute as the reference.</p

PspA upregulates expression of PD-L1 on DCs.

<p>A, mouse bone marrow derived DCs were stimulated with indicated concentrations of PspA for 24h. B, DCs were stimulated with 15μg/ml PspA for indicated times. PD-L1 expression was monitored by flow cytometry. Bold lines represent expression levels in the presence of PspA while thin lines represent unstimulated controls. Data from one of three independent experiments are shown. The MFI values from three independent experiment (expressed as mean ± SD) are summarized as a bar graph in the right panel.</p

<i>S</i>. <i>pneumoniae</i> upregulates PD-L1 on DCs.

<p>For Panel A, mouse bone marrow derived DCs were infected with wild typ<i>e S</i>. <i>pneumoniae</i> strain D39, R6 or JY2008 (<i>see</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133601#sec002" target="_blank">Materials and Methods</a>) and PD-L1 levels were monitored by flow cytometry. Thin lines represent uninfected cells while bold lines represent cells incubated with indicated strain of <i>S</i>. <i>pneumoniae</i>. For Panel B, DCs were incubated with either TMB-8 or EGTA for 1h followed by incubation with D39 and PD-L1 levels were monitored by flow cytometry. Dotted line represents uninfected cells. Thin lines represent cells incubated with D39 while thick lines represent cells treated with indicated inhibitors followed by incubation with D39. Data from one of two independent experiments are shown. Bars in Panel C and D represent fold increase in Relative Mean Fluorescence Intensity (MFI; mean ± SD) for various groups. ns represents non-significant differences between compared groups.</p

SP0845 is accessible to antibodies.

<p>(A) Mid-logarithmic phase TIGR4 cells were incubated with mouse anti-SP0845^23–350 or mouse preimmune sera (negative control) followed by incubation with F(ab')₂ fragment phycoerythrin-conjugated goat anti-mouse IgG + IgM (H + L) antibody. The processed samples were observed under a fluorescence microscope. The fluorescent images obtained with anti-SP0845^23–350 and preimmune sera are in the upper left and right panels, respectively. The lower panels show the corresponding phase contrast images (magnification = 1000X). Surface expression of SP0845 was studied for pneumococcal strains TIGR4 (B), ATCC 6303 (C), ATCC 6314 (D), D39 (E) and R36A (F). Pneumococcal cells were incubated with either preimmune or anti-SP0845^23–350 sera for 1 hr followed by a FITC-conjugated F(ab')₂ fragment goat anti-mouse IgG + IgM (H + L) antibody as secondary antibody. The surface staining was detected by flow cytometry.</p