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

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

Kinetics of survival in subcutaneously inoculated mice.

<p>(A and B) The dose-dependent survival assays and (C, D) mean time to death post infection of mice (<i>n</i> = 8 in a group) inoculated with three different doses of the strains (A, C) C-267 and (B, D) 231 isogenic derivatives (Pla (-)) deficient in Pla or producing its I259 (pKP3455) or T259 (pKPEV) isoform. *, <i>P</i><0.05, **, <i>P</i><0.01, ***, <i>P</i><0.001. The planned injection dose of 100 cfu was actually equal to 98 (C-267), 115 (C-267pKP3455), 123 (C-267pKPEV), 103 (231pPst^-), 95 (231pPst^-pKP3455) or 75 (231pPst^-pKPEV) cfu.</p

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

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

Comparison of survival in mice subcutaneously or intradermally inoculated with the strain C-267.

<p>The dose-dependent survival assays 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 (squares) or producing its I259 (circles) or T259 (triangles) isoform. The planned injection dose of 100 cfu was actually equal to 53 (C-267), 58 (C-267pKP3455), 74 (C-267pKPEV) cfu.</p

Pla isoforms enzymatic activities.

<p>Pla isoforms enzymatic activities.</p

Comparative virulence of <i>Y</i>. <i>pestis</i> C-267 derivatives differing in ability to produce Pla isoforms in the case of subcutaneous or intradermal infection.

<p>Comparative virulence of <i>Y</i>. <i>pestis</i> C-267 derivatives differing in ability to produce Pla isoforms in the case of subcutaneous or intradermal infection.</p