Polymorphism of the Cysteine Protease YopT from Yersinia Pestis

Antibiotic therapy of plague is hampered by the recent isolation of Yersinia pestis strain resistant to all of antibiotics recommended for cure. This has constrained a quest for new antimicrobials taking aim at alternative targets. Recently Y. pestis cysteine protease YopT has been explored as a potential drug target. Targets conserved in the pathogen populations should be more efficacious; therefore, we evaluated intraspecies variability in yopT genes and their products. 114 Y. pestis isolates were screened. Only two YopT full-size isoforms were found among them. The endemic allele (N149) was present in biovar caucasica from Dagestan-highland natural plague focus # 39. The biovar caucasica strains from Transcaucasian highland (# 4-6) and Pre-Araks (# 7) plague foci also contained the N149 allele. These strains from foci # 4 7 possessed a truncated version of YopT that was a consequence of a frame-shift due to the deletion of a single nucleotide at position 71 bp. Computational analyses showed that although the SNP at the position 149 has a very minimal effect of the intrinsic disorder propensity of YopT proteins, whereas the N-terminal truncations of the YopT detected in bv. caucasica strains Pestoides F_YopT1 and F_YopT2, and Pestoides G generated isoforms with the significantly modified intrinsic disorder propensities and with reduced capability to interact with lost ability to utilize their N-terminal tail for the disorder-based interactions with biological partners. Considering that representatives of biovar caucasica were reported to be the reason of sporadic cases of human plague, this study supports the necessity of additional testing of globally disseminated YopT (S149) isoform as a potential target for treatment of plague caused by the strains producing different YopT isoforms

Polymorphism of the Cysteine Protease YopT from Yersinia Pestis

Antibiotic therapy of plague is hampered by the recent isolation of Yersinia pestis strain resistant to all of antibiotics recommended for cure. This has constrained a quest for new antimicrobials taking aim at alternative targets. Recently Y. pestis cysteine protease YopT has been explored as a potential drug target. Targets conserved in the pathogen populations should be more efficacious; therefore, we evaluated intraspecies variability in yopT genes and their products. 114 Y. pestis isolates were screened. Only two YopT full-size isoforms were found among them. The endemic allele (N149) was present in biovar caucasica from Dagestan-highland natural plague focus # 39. The biovar caucasica strains from Transcaucasian highland (# 4-6) and Pre-Araks (# 7) plague foci also contained the N149 allele. These strains from foci # 4 7 possessed a truncated version of YopT that was a consequence of a frame-shift due to the deletion of a single nucleotide at position 71 bp. Computational analyses showed that although the SNP at the position 149 has a very minimal effect of the intrinsic disorder propensity of YopT proteins, whereas the N-terminal truncations of the YopT detected in bv. caucasica strains Pestoides F_YopT1 and F_YopT2, and Pestoides G generated isoforms with the significantly modified intrinsic disorder propensities and with reduced capability to interact with lost ability to utilize their N-terminal tail for the disorder-based interactions with biological partners. Considering that representatives of biovar caucasica were reported to be the reason of sporadic cases of human plague, this study supports the necessity of additional testing of globally disseminated YopT (S149) isoform as a potential target for treatment of plague caused by the strains producing different YopT isoforms

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

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 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

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

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 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