Binding of FHbp to immobilized human FH by surface plasmon resonance.

<p>Purified human FH (1500 response units) was coupled to the biosensor chip. Different concentrations (3 to 316 nM) of purified, recombinant FHbp were used to determine equilibrium dissociation constants (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128185#pone.0128185.t001" target="_blank">Table 1</a></b>). <b>A.</b> Data for injection of 100 nM of wild-type (WT) FHbp (grey line) or Q38R mutant (black line) are shown. <b>B-E.</b> Data for E92K, R130G, S223R and H248L mutants are shown with WT FHbp (same data as in <b>Panel A</b>). Binding equivalent to two response units was considered as the limit of detection.</p

Thermal stability of FHbp ID 1 mutants measured by differential scanning calorimetry.

<p><b>A.</b> Wild-type (WT) FHbp ID 1 (dashed grey line) and Q38R mutant (solid black line). <b>B-E.</b> E92K, R130G, S223R and H248L mutants (solid black lines), each shown compared to the same data for the WT ID 1 as in Panel A. FHbp (0.5 mg/ml in PBS) was heated at a scan rate of 60°C/h and the excess heat capacity (ΔCp) was measured. Reference buffer data were subtracted and the data were normalized based on the molecular weight of FHbp (27.7 kDa). The lower and higher temperature transitions corresponded with the unfolding of the N- and C-terminal domains [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128185#pone.0128185.ref044" target="_blank">44</a>].</p

Detection of meningococcal FHbp on the surface of <i>E</i>. <i>coli</i>.

<p><b>A.</b> Binding of anti-FHbp mAbs JAR 41 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128185#pone.0128185.ref029" target="_blank">29</a>] and mAb502 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128185#pone.0128185.ref030" target="_blank">30</a>] to <i>E</i>. <i>coli</i> transformed with plasmid expressing wild-type FHbp (solid blue line), R41S mutant (dashed green line) or empty plasmid (shaded grey histogram). <b>B.</b> Binding of purified human FH to same strains. <b>C.</b> Binding of <i>E</i>. <i>coli</i> expressing FHbp mutant library by the same mAbs as in Panel A (solid blue line) or human FH (dashed green line). <b>D.</b> Two-dimensional plot of FHbp mutant library stained simultaneously for binding of FH (Alexa Fluor 488) and anti-FHbp mAbs (Alexa Fluor 647). The FHbp R41S mutant (dashed green lines in Panels A and B) was used to define gates for the sub-population with high mAb binding and low FH binding (quadrant Q5 in upper left of Panel D; 0.3% of events).</p

Location of mutations in FHbp in relation to FH.

<p><b>A.</b> Plot of distance between each FHbp residue and closest atom in FH in the crystal structure of the FHbp-FH complex (PDB ID 2W80) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128185#pone.0128185.ref023" target="_blank">23</a>]. Distances greater than 6 Å are depicted as 12 Å. The lipidated Cys residue in the mature protein is designated as residue number 1. <b>B.</b> Histogram showing number of mutations at each residue position in FHbp. Above the histogram are the known FH interaction residues (filled circles) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128185#pone.0128185.ref023" target="_blank">23</a>], residues mutated in previous studies (open circles) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128185#pone.0128185.ref022" target="_blank">22</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128185#pone.0128185.ref023" target="_blank">23</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128185#pone.0128185.ref027" target="_blank">27</a>] and residues mutated in the present study (filled stars). <b>C.</b> Location of residues with mutations identified to decrease binding of FH to FHbp (magenta). The location of residue R41, which previously was identified as an important FH binding residue is shown in yellow. <b>D.</b> Location of FH fragment (grey) relative to residues with mutations identified to decrease binding of FH to FHbp.</p

Serum bactericidal antibody responses of mice to mutant FHbp vaccines.

<p><b>A.</b> Control mice immunized with FHbp mutant vaccines. Each symbol represents the serum bactericidal antibody titer of an individual mouse and the horizontal lines represent the geometric mean titer (GMT) of each vaccine group. There were no statistically significant pairwise differences between the WT FHbp and either of the FHbp mutants (P≥ 0.62). <b>B.</b> Human FH transgenic mice immunized with FHbp mutant vaccines. The GMT for the R41S mutant was 13-fold higher than for the wild-type FHbp, 20-fold higher for S223R, and 19-fold higher for H248L. The P values for Mann-Whitney tests comparing each mutant group with the WT group are shown.</p

Primers used for site-specific mutagenesis.

<p>^a Name of primer for each mutant based on numbering of mature FHbp ID 1 sequence, beginning with lipidated cysteine residue; F, forward; R, reverse</p><p>^b Sequence shown from 5’ to 3’; nucleotide changes relative to wild-type sequence in forward primer are underlined</p><p>^c Annealing temperature used to amplify mutant FHbp gene</p><p>Primers used for site-specific mutagenesis.</p

Morphology of ESDC and MHC-II expression was observed by confocal microscopy.

<p>Stimulated ESDCs were imaged using a Confocal Zeiss LSM510; (A), phase contrast, (B) cell stimulated with TNFα and stained with anti-MHC Class II FITC conjugated antibody. Scale bar 10 µm.</p

Primers for semi-quantitative Real Time-PCR.

<p>Primers for semi-quantitative Real Time-PCR.</p

The invasion assay was also evaluated by Flow Cytometry.

<p>The ability of <i>S.</i> Typhimurium SL1344(p1C/1), able to express GFP once inside the host cell, to invade ESDCs was measured by monitoring the GFP expression using FACS analysis. Cells were analyzed for GFP expressing bacteria at time 2 and 4 hours post-infection. The horizontal bar in each histogram represents the percentage of fluorescent positive cells on 10.000 events. FITC subset in non infected cells is <1%. These data are representative of at least 3 independent experiments.</p

Concentration of IL-2 in the culture supernatant of naïve MF2.2d9 T cells mixed with either BMDCs or ESDCs incubated for 24 with ovalbumin, TNFα or alone with Concavallin A (ConA).

<p>These data are representative of at least 2 independent experiments. Statistical analysis performed with one-way ANOVA.</p><p>Ovalbumin 10 µg/ml, TNFα 5000 WHOSU/ml, Concavallin A 5 µM/ml.</p