Comparison of mean time to death post infection in mice subcutaneously or intradermally inoculated with the C-267.

<p>The mean time to death post infection of mice (<i>n</i> = 8 in a group per inoculum) inoculated either subcutaneously (upper panels), or intradermally (lower panels) using three different doses of 10 (A, D), 100 (B, E), or 1000 (C, F) cfu of the strain C-267 isogenic derivatives (Pla (-)) deficient in Pla (circles) or producing its I259 (triangles) or T259 (squares) isoform. The planned injection dose of 100 cfu was actually equal to 53 (C-267), 58 (C-267pKP3455), 74 (C-267pKPEV) cfu.</p

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

Comparison of different routes of infection of mice with the same isogenic strains, C-267 derivatives.

<p>*, <i>P</i><0.05; **, <i>P</i><0.01. Mann-Whitney test. 10 sc/10 id, 10 cfu via subcutaneous/intradermal route. The actual injection doses were equal to 5 (C-267), 6 (C-267pKP3455), 7 (C-267pKPEV) cfu per mouse.</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

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

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

Predicted intrinsic disorder propensities of the two <i>Y</i>. <i>pestis</i> Pla isoforms.

<p>A. Evaluating predicted intrinsic disorder propensities of the two Pla isoforms [<i>Y</i>. <i>pestis</i> subsp. <i>microtus</i> 91001 (solid black and gray lines) and <i>Y</i>. <i>pestis</i> subsp. <i>pestis</i> CO92 (dashed red and dark red lines)] by PONDR^® VSL2 (solid gray and dashed dark red lines) and POND-FIT (solid black and dashed red lines). Disorder scores above 0.5 correspond to the residues/regions predicted to be intrinsically disordered. Colored (light pink and light gray) shades around the corresponding PONDR-FIT curves represent distributions of errors in evaluation of disorder propensity. B. Comparison of the disorder propensities of the two forms of Pla (UniProt ID: P17811), with those of its homologues, outer membrane protease E (PgtE) from <i>S</i>. <i>enterica</i> (UniProt ID: P06185) and OmpT protein from <i>E</i>. <i>coli</i> O157:H7 (UniProt ID: P58603). The corresponding disorder profiles were obtained by PONDR^® VSL2. Sequences were aligned using the ClustalW2 multiple sequence alignment tool and the results of the alignment are shown in panel C.</p

Functional activity of two isoforms of Pla protease determined by the fluorometric assay with the Pla substrate DABCYL-Arg-Arg-Ile-Asn-Arg-Glu (EDANS)-NH₂.

<p>The kinetics of the substrate cleavage was measured in quadruplicates for <i>Y</i>. <i>pestis</i> strains KIM D47, KIM D47/pPCP1Km, and KIM D47/pPCP1KmCh grown at either 26°C (A) or 37°C (B). The graphs were plotted as arithmetic means ± standard deviations. Statistical analysis of Pla activity data was done by one-way ANOVA with a Bonferroni <i>post hoc</i> test. The differences between the groups expressing the Pla (T259) and (I259) isoforms for both growth temperatures were statistically significant (<i>P</i> < 0.01).</p

Subcutaneous virulence of <i>Y</i>. <i>pestis</i> strains differing in ability to produce Pla isoforms.

<p>Subcutaneous virulence of <i>Y</i>. <i>pestis</i> strains differing in ability to produce Pla isoforms.</p