Antibody titres of pooled sera from immunised mice before challenge with D39.

<p>Antibody titres were measured via ELISA. Titre was defined as the dilution of mouse serum giving half-maximal absorbance, and was rounded to two significant figures.</p

Coils prediction server output for PhtD.

<p>The Coils server (<a href="http://embnet.vital-it.ch/software/COILS_form.html" target="_blank">http://embnet.vital-it.ch/software/COILS_form.html</a>) was used to compare the amino acid sequence of PhtD to a database of proteins known to form coiled-coils to yield a prediction of which regions (if any) will fold in this conformation. The x-axis represents the position in the protein by amino acid number (starting at the N-terminus) and the y-axis shows how strongly that region is predicted to form a coiled coil. ‘Window’ refers to the width of the amino acid ‘window’ that is scanned at one time.</p

Antibody binding capacity of truncated derivatives of PhtD.

<p>ELISA was used to examine binding of anti-PhtD serum to PhtD and truncated derivatives. PdB is an unrelated negative control protein (genetic toxoid derivative of pneumolysin [34]) to which no binding is expected. A: proteins truncated from the C terminus. B: proteins truncated from the N terminus. C: PhtD adjunct protein.</p

Presence and exposure of Pht proteins in strain P9 compared to D39.

<p>A: Western blotting using pneumococcal cell lysates with anti-PhtD (left) or anti-PhtABDE (right) polyclonal sera. Mobilities of molecular size markers are indicated. B and C: detection of PhtD on the surface of D39 (B) and P9 (C) by flow cytometry using antisera generated against PhtD truncated derivatives. NMS, normal mouse serum (negative control). Median fluorescence intensities for 10,000 events are displayed.</p

Circular dichroism spectroscopy analysis of PhtD and truncated derivatives.

<p>Results are shown as proportions of each protein predicted to be folded in each of the indicated secondary structural conformations, or predicted to be unordered.</p

Survival times of mice challenged with D39 after immunisation with the indicated antigens.

<p>Median survival times are indicated by horizontal lines. Significant differences from the alum only control group are indicated where present (*, <i>P</i><0.05; ****, <i>P</i><0.0001).</p

SDS-PAGE analysis of purified truncated derivatives of PhtD.

<p>Truncated derivatives of PhtD were analysed by SDS-PAGE using a 12% acrylamide gel and subsequently stained using Coomassie R250. 1 µg of each protein was used. The identity of the protein is indicated above each lane (FL; full length).</p

Truncated forms of PhtD used in this study.

<p>Proteins are shown with known or predicted domains annotated. The N termini are to the left.</p

Antibody titres of pooled sera from immunised mice before challenge with P9.

<p>Antibody titres were measured via ELISA. Titre was defined as the dilution of mouse serum giving half-maximal absorbance, and was rounded to two significant figures.</p

Survival times of mice challenged with P9 after immunisation with the indicated antigens.

<p>Median survival times are indicated by horizontal lines. Significant differences from the alum only control group are indicated where present (*, <i>P</i><0.05; **, <i>P</i><0.01).</p