Two Isoforms of Yersinia Pestis Plasminogen Activator Pla: Intraspecies Distribution, Intrinsic Disorder Propensity, and Contribution to Virulence

It has been shown previously that several endemic Y. pestis isolates with limited virulence contained the I259 isoform of the outer membrane protease Pla, while the epidemic highly virulent strains possessed only the T259 Pla isoform. Our sequence analysis of the pla gene from 118 Y. pestis subsp. microtus strains revealed that the I259 isoform was present exclusively in the endemic strains providing a convictive evidence of more ancestral origin of this isoform. Analysis of the effects of the I259T polymorphism on the intrinsic disorder propensity of Pla revealed that the I259T mutation slightly increases the intrinsic disorder propensity of the C-terminal tail of Pla and makes this protein slightly more prone for disorder-based protein-protein interactions, suggesting that the T259 Pla could be functionally more active than the I259 Pla. This assumption was proven experimentally by assessing the coagulase and fibrinolytic activities of the two Pla isoforms in human plasma, as well as in a direct fluorometric assay with the Pla peptide substrate. The virulence testing of Pla-negative or expressing the I259 and T259 Pla isoforms Y. pestis subsp. microtus and subsp. pestis strains did not reveal any significant difference in LD50 values and dose-dependent survival assays between them by using a subcutaneous route of challenge of mice and guinea pigs or intradermal challenge of mice. However, a significant decrease in time-to-death was observed in animals infected with the epidemic T259 Pla-producing strains as compared to the parent Pla-negative variants. Survival curves of the endemic I259 Pla+ strains fit between them, but significant difference in mean time to death post infection between the Pla−strains and their I259 Pla+ variants could be seen only in the isogenic set of subsp. pestis strains. These findings suggest an essential role for the outer membrane protease Pla evolution in Y. pestis bubonic infection exacerbation that is necessary for intensification of epidemic process from endemic natural focality with sporadic cases in men to rapidly expanding epizootics followed by human epidemic outbreaks, local epidemics or even pandemics

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

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