Evaluating intrinsic disorder propensities of different Caf1 isoforms.

<p>(A) Disorder profiles obtained for the analyzed proteins by PONDR® VSL2 (Caf1_NT1 (dashed dark yellow line), Caf1_NT2 (solid gray line), and Caf1_NT3 (dotted dark red line)) and PONDR-FIT (Caf1_NT1 (dashed yellow line), Caf1_NT2 (solid black line), and Caf1_NT3 (dotted red line)). Disorder scores above 0.5 correspond to the residues/regions predicted to be intrinsically disordered. Colored shades around the corresponding PONDR-FIT curves represent distributions of errors in evaluation of disorder propensity. (B) Comparison of the disorder profiles obtained for Caf1 isoforms by PONDR VLXT (Caf1_NT1 (dashed dark yellow line), Caf1_NT2 (solid gray line), and Caf1_NT3 (dotted dark red line)) and their intrinsic disorder-based interactability (Caf1_NT1 (dashed yellow line), Caf1_NT2 (solid black line), and Caf1_NT3 (dotted red line)) predicted using the ANCHOR algorithm [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162308#pone.0162308.ref051" target="_blank">51</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162308#pone.0162308.ref052" target="_blank">52</a>]. To simplify comparison of disorder predisposition and presence of potential disorder-based binding sites, ANCHOR data are present in the (1 –ANCHOR score form). Therefore, in PONDR® VLXT profiles, regions with scores above 0.5 are predicted to be intrinsically disordered, whereas in the ANCHOR profiles, regions with probability below 0.5 are predicted as binding regions.</p

Caf1 isoform cross-reactivity.

<p>Mice were immunized with NT1 (blue bars), NT2 (red bars) or NT3 (green bars) and then bled on day 29 after first (I) or day 43 after second immunization (II) and sera samples were tested in ELISA against NT1, NT2 or NT3 isoforms. Data are means ±SEM.</p

Multiple sequence alignment of the isoforms of Caf1 protein found in different <i>Y</i>. <i>pestis</i> strains.

<p>Multiple sequence alignment of the isoforms of Caf1 protein found in different <i>Y</i>. <i>pestis</i> strains.</p

<i>Y</i>. <i>pestis</i> strains used in this study for Caf1 isolation, serologic cross-reactivity tests, and virulence experiments.

<p><i>Y</i>. <i>pestis</i> strains used in this study for Caf1 isolation, serologic cross-reactivity tests, and virulence experiments.</p

Indices of immunity (II) induced by the three Caf1 isoforms.

<p>Indices of immunity (II) induced by the three Caf1 isoforms.</p

Correlation between serum antibody titers and immunity indices.

<p>Capital case–isoforms used for immunization; lower case–genotypes of the strains used for challenge.</p

Survival of immunized mice in response to bacterial challenge.

<p>Groups of 8 BALB/c mice that were immunized with Caf1_NT1 (A, D), Caf1_NT2 (B, E), or Caf1_NT3 (C, F) isoforms were challenged with <i>Y</i>. <i>pestis</i> strains producing different Caf1 isoforms: Caf1_NT1 (circles); Caf1_NT2 (squares); or Caf1_NT3 (triangles)), at high (2000 LD₅₀, panels A-C), or low (200 LD₅₀, panels D-F) doses. Survival was monitored for 21 days after the infection. *<i>P</i><0.05; **<i>P</i><0.01 (Log-rank Mantel-Cox test). The results have been acquired with n = 8 BALB/c for each dose of subcutaneous infection.</p

Strains and plasmids used in this study for testing Pla activity and virulence experiments.

<p>Strains and plasmids used in this study for testing Pla activity and virulence experiments.</p

SDS-PAGE (right) and immunoblot analysis (left) of whole-cell lysates of the indicated by numbers <i>Y</i>. <i>pestis</i> strains with antibodies to Pla.

<p>Bacteria were cultured at the temperatures indicated on the right for each blot-gel pair. Molecular weight markers (Novex sharp protein standard, Life technologies) are shown in Italics on the left. Numbers and horizontal lines in the middle indicate Pla. The lower band represents the autoprocessed form of Pla. Track numbers correspond to: 1 –C-376pCD1^-pkPEV, 2 –C-376pCD1^-pkPI-3455, 3 –C-376pCD1^-, 4 –C-585pCD1^-pkPEV, 5 –C-585pCD1^-pkPI-3455, 6 –C-585pCD1^-, 7 –C-824pCD1^-pkPEV, 8 –C-824pCD1^-pkPI-3455, 9 –C-824pCD1^-, 10 – 358pCD1^-pPst^-pkPEV, 11 – 358pCD1^-pPst^-pkPI-3455, 12 – 358pCD1^-pPst^-, 13 –KM217pkPEV, 14 –KM217pkPI-3455, 15 –KM 217.</p

Kinetics of survival in subcutaneously inoculated guinea pigs.

<p>(A, B, C, D) The dose-dependent survival assays and (E and F) mean time to death post infection of guinea pigs (<i>n</i> = 6 in a group per inoculum) inoculated with four different doses of the strain 231 isogenic derivatives (Pla (-)) deficient in Pla or producing its I259 or T259 isoform. 10³ (A), 10⁴ (B), 10⁵(C, E), and 10⁶ (D, F) cfu of <i>Y</i>. <i>pestis</i> variants, respectively. The planned injection dose of 100 cfu was actually equal to 103 (231pPst^-), 95 (231pPst^-pKP3455) or 75 (231pPst^-pKPEV) cfu. Compared by ANOVA. *<i>P</i><0.05.</p