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

Peptidoglycan-Free Bacterial Ghosts Confer Enhanced Protection against Yersinia pestis Infection

To develop a modern plague vaccine, we used hypo-endotoxic Yersinia pestis bacterial ghosts (BGs) with combinations of genes encoding the bacteriophage ɸX174 lysis-mediating protein E and/or holin-endolysin systems from λ or L-413C phages. Expression of the protein E gene resulted in the BGs that retained the shape of the original bacterium. Co-expression of this gene with genes coding for holin-endolysin system of the phage L-413C caused formation of structures resembling collapsed sacs. Such structures, which have lost their rigidity, were also formed as a result of the expression of only the L-413C holin-endolysin genes. A similar holin-endolysin system from phage λ containing mutated holin gene S and intact genes R-Rz coding for the endolysins caused generation of mixtures of BGs that had (i) practically preserved and (ii) completely lost their original rigidity. The addition of protein E to the work of this system shifted the equilibrium in the mixture towards the collapsed sacs. The collapse of the structure of BGs can be explained by endolysis of peptidoglycan sacculi. Immunizations of laboratory animals with the variants of BGs followed by infection with a wild-type Y. pestis strain showed that bacterial envelopes protected only cavies. BGs with maximally hydrolyzed peptidoglycan had a greater protectivity compared to BGs with a preserved peptidoglycan skeleton

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

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

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

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

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