Pneumococcal lysis induced by LytA promotes GAPDH surface localization.

<p>(A) Growth profiles of the R6 strain in CY and CY + 1% Cho and of the R6 Δ<i>lytA</i> mutant in CY medium. (B) Bacterial suspensions of the R6 strain grown in CY and in CY + 1% Cho and the R6 Δ<i>lytA</i> mutant grown in CY were treated by alkaline buffer to release surface-associated proteins. Proteins present in the pellet (P) fraction corresponding to the cytoplasmic extract and in the alkaline supernatant fraction, which contains proteins detached from the cell surface, were analyzed by Western blot using appropriate polyclonal antibodies. Samples were analyzed on the same polyacrylamide gel. Left panel: detection of FtsZ (44.4 kDa) used to monitor the non-lytic effect of the alkaline treatment. Right panel: detection of GAPDH (38 kDa). Equivalent amount of loading material was determined based on OD_600nm values and gel scanning quantification and not on CFU measurements since the chaining morphology of the R6 Δ<i>lytA</i> mutant and the one induced by the presence of 1% Cho alters colony counting [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125377#pone.0125377.ref065" target="_blank">65</a>]. This procedure was also applied in experiments showed in Figs 1C, 1D and 1E. (C) Quantification of pneumococcal GAPDH associated to the bacterial surface in the R6 strain grown in CY and in CY + 1% Cho and in the R6 Δ<i>lytA</i> mutant grown in CY. GAPDH was detected by Western blot and quantification of the signal was performed. The average of three independent experiments is shown. (D) Same protocol as 1C. Amount of GAPDH associated to the cell wall fraction at different stages of growth. (E) Same protocol as 1C. Amount of GAPDH associated to the cell wall fraction analyzed by subcellular fractionation.</p

Surface GAPDH promotes binding to C1q.

<p>Bacterial culture was withdrawn at mid-exponential growth phase (OD_600nm 0.3). FITC-labeled bacteria were incubated for 1 h at 4°C on 1 μg of C1q coated on 96-wells plate. After five washes, the fluorescence of FITC was measured. A representative experiment of 3 independent experiments is shown including the standard deviation of triplicate points. Significance was determined by t-test analysis on 3 independent experiments.</p

GAPDH increases C1q deposition on pneumococcal cell wall.

<p>Pneumococcal sacculi containing only peptidoglycan were deposited on 96-wells plate and incubated with or without purified GAPDH. Normal human serum (NHS) was added and subsequent C1q and C4 deposition was measured using anti-C1q and anti-C4 antibodies. A representative experiment of 2 independent experiments is shown including the standard deviation of triplicate points.</p

Stained PEPperCHIP® array B.

<p>The permutation scans of the WT peptide (upper left) and of variant ⁶TAMFQDP<b>F</b>ER¹⁵ are highlighted by white frames. Replaced positions together with the WT amino-acid are indicated on the left of the upper left frame. Amino-acids introduced at each position are indicated on top of the frame. Duplicated spots are easily identified.</p

Comparison of the two WT permutation patterns.

<p>Intensities were normalized with respect to the mean intensity recorded for the scFv1F4—WT peptide interaction. Black and grey bars correspond to the normalized intensities in arrays A and B, respectively. White bars represent the WT sequence (normalized value = 1). Stars indicate single replacements in starting variants (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143374#pone.0143374.t001" target="_blank">Table 1</a>).</p

Context dependence of the effect on k_d of replacements.

<p>Positions 11, 12 and 13 in the peptide sequences are shown in red, blue and green, respectively. The filling is white for the WT residue (D₁₁, P₁₂, Q₁₃) and colored for the modified residue (S₁₁, V₁₂, F₁₃). The ratio k_d variant / k_d WT, as measured by SPR in HBS (black) or HBS200 (grey), is given next to each arrow, together with the nature of the replacement.</p

Correlation between SPR constants and fluorescence signals.

<p>The k_d (A, B) and ΔG (C, D) values are plotted against fluorescence signals from arrays A (A,C) and B (B,D). The k_d as measured in HBS (grey markers) and HBS-200 (white markers) is shown in log scale. Peptide names are indicated in A and B.</p

Permutation patterns for variants modified at positions 11, 12 and 13.

<p>Variant patterns are represented as red lines and superimposed with the WT pattern (blue). Fluorescence signals were normalized with respect to that recorded with each starting peptide. Patterns at the replaced positions are in green for double variants and not shown for single variants.</p

Binding parameters deduced from SPR measurements in HBS.

<p>^a Sequence replacements relative to WT are in bold and underlined.</p><p>Binding parameters deduced from SPR measurements in HBS.</p

Spot fluorescence data for the starting peptides.

<p>^a Sequence replacements relative to WT are in red.</p><p>^b Mean intensities > 10% that of the WT peptide are in bold.</p><p>^c Standard deviation calculated as <math><mrow><mi>s</mi><mo>=</mo><msqrt><mrow><mn>1</mn><mrow><mi>N</mi><mo>−</mo><mn>1</mn></mrow><mo>∑</mo><mrow><mi>i</mi><mo>=</mo><mn>1</mn></mrow><mi>N</mi><mrow><mrow><mrow><mo>(</mo><mrow><msub><mi>x</mi><mi>i</mi></msub><mo>−</mo><mi>x</mi><mo>¯</mo></mrow><mo>)</mo></mrow></mrow><mn>2</mn></mrow></mrow></msqrt></mrow></math></p><p>^d The permutation of the WT decapeptide in array A was described in Vernet <i>et al</i>., 2015 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143374#pone.0143374.ref028" target="_blank">28</a>].</p><p>Spot fluorescence data for the starting peptides.</p