Abstract
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.
Highlights
The plague pathogen, Yersinia pestis, was the reason of the death of millions of people worldwide
We provide an evidence that the I259 isoform of Pla is present exclusively in the endemic Y. pestis strains that is supported by sequencing the pla genes from 118 strains of Y. pestis belonging to seven of eight biovars of subsp. microtus [37], and circulating on the territory of the former Soviet Union and Mongolia
We found no significant difference in LD50 values and dose-dependent survival assays between the strains with the I259 and T259 Pla by using a subcutaneous route of challenge of mice and guinea pigs, or intradermal challenge of mice
Summary
The plague pathogen, Yersinia pestis, was the reason of the death of millions of people worldwide. The Pla activity is not required to initiate lethal disease by intravenous injection (i.v.), which provides immediate access to fixed macrophages lining the capillary beds of the liver and spleen [12]. This relationship was verified with isogenic pla mutants of epidemic Y. pestis strains KIM and CO92, which showed up to 106 logs of reduced virulence by the s.c. route, in comparison with the i.v. route, in a murine model of plague [11, 13]. The major role in this process has been attributed to Pla because this protease resembles mammalian plasminogen activators in function and converts plasminogen to plasmin by limited proteolysis [14], possibly leading to clarification of fibrin deposits that could hinder bacterial migration in the circulation [15]
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