Yersinia pestis Caf1 Protein: Effect of Sequence Polymorphism on Intrinsic Disorder Propensity, Serological Cross-Reactivity and Cross-Protectivity of Isoforms.

Yersinia pestis Caf1 is a multifunctional protein responsible for antiphagocytic activity and is a key protective antigen. It is generally conserved between globally distributed Y. pestis strains, but Y. pestis subsp. microtus biovar caucasica strains circulating within populations of common voles in Georgia and Armenia were reported to carry a single substitution of alanine to serine. We investigated polymorphism of the Caf1 sequences among other Y. pestis subsp. microtus strains, which have a limited virulence in guinea pigs and in humans. Sequencing of caf1 genes from 119 Y. pestis strains belonging to different biovars within subsp. microtus showed that the Caf1 proteins exist in three isoforms, the global type Caf1NT1 (Ala48 Phe117), type Caf1NT2 (Ser48 Phe117) found in Transcaucasian-highland and Pre-Araks natural plague foci #4-7, and a novel Caf1NT3 type (Ala48 Val117) endemic in Dagestan-highland natural plague focus #39. Both minor types are the progenies of the global isoform. In this report, Caf1 polymorphism was analyzed by comparing predicted intrinsic disorder propensities and potential protein-protein interactivities of the three Caf1 isoforms. The analysis revealed that these properties of Caf1 protein are minimally affected by its polymorphism. All protein isoforms could be equally detected by an immunochromatography test for plague at the lowest protein concentration tested (1.0 ng/mL), which is the detection limit. When compared to the classic Caf1NT1 isoform, the endemic Caf1NT2 or Caf1NT3 had lower immunoreactivity in ELISA and lower indices of self- and cross-protection. Despite a visible reduction in cross-protection between all Caf1 isoforms, our data suggest that polymorphism in the caf1 gene may not allow the carriers of Caf1NT2 or Caf1NT3 variants escaping from the Caf1NT1-mediated immunity to plague in the case of a low-dose flea-borne infection

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

Evaluating intrinsic disorder propensities of different Caf1 isoforms.

<p>(A) Disorder profiles obtained for the analyzed proteins by PONDR® VSL2 (Caf1_NT1 (dashed dark yellow line), Caf1_NT2 (solid gray line), and Caf1_NT3 (dotted dark red line)) and PONDR-FIT (Caf1_NT1 (dashed yellow line), Caf1_NT2 (solid black line), and Caf1_NT3 (dotted red line)). Disorder scores above 0.5 correspond to the residues/regions predicted to be intrinsically disordered. Colored shades around the corresponding PONDR-FIT curves represent distributions of errors in evaluation of disorder propensity. (B) Comparison of the disorder profiles obtained for Caf1 isoforms by PONDR VLXT (Caf1_NT1 (dashed dark yellow line), Caf1_NT2 (solid gray line), and Caf1_NT3 (dotted dark red line)) and their intrinsic disorder-based interactability (Caf1_NT1 (dashed yellow line), Caf1_NT2 (solid black line), and Caf1_NT3 (dotted red line)) predicted using the ANCHOR algorithm [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162308#pone.0162308.ref051" target="_blank">51</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162308#pone.0162308.ref052" target="_blank">52</a>]. To simplify comparison of disorder predisposition and presence of potential disorder-based binding sites, ANCHOR data are present in the (1 –ANCHOR score form). Therefore, in PONDR® VLXT profiles, regions with scores above 0.5 are predicted to be intrinsically disordered, whereas in the ANCHOR profiles, regions with probability below 0.5 are predicted as binding regions.</p

Correlation between serum antibody titers and immunity indices.

<p>Capital case–isoforms used for immunization; lower case–genotypes of the strains used for challenge.</p

Survival of immunized mice in response to bacterial challenge.

<p>Groups of 8 BALB/c mice that were immunized with Caf1_NT1 (A, D), Caf1_NT2 (B, E), or Caf1_NT3 (C, F) isoforms were challenged with <i>Y</i>. <i>pestis</i> strains producing different Caf1 isoforms: Caf1_NT1 (circles); Caf1_NT2 (squares); or Caf1_NT3 (triangles)), at high (2000 LD₅₀, panels A-C), or low (200 LD₅₀, panels D-F) doses. Survival was monitored for 21 days after the infection. *<i>P</i><0.05; **<i>P</i><0.01 (Log-rank Mantel-Cox test). The results have been acquired with n = 8 BALB/c for each dose of subcutaneous infection.</p

Caf1 isoform cross-reactivity.

<p>Mice were immunized with NT1 (blue bars), NT2 (red bars) or NT3 (green bars) and then bled on day 29 after first (I) or day 43 after second immunization (II) and sera samples were tested in ELISA against NT1, NT2 or NT3 isoforms. Data are means ±SEM.</p

Multiple sequence alignment of the isoforms of Caf1 protein found in different <i>Y</i>. <i>pestis</i> strains.

<p>Multiple sequence alignment of the isoforms of Caf1 protein found in different <i>Y</i>. <i>pestis</i> strains.</p

Indices of immunity (II) induced by the three Caf1 isoforms.

<p>Indices of immunity (II) induced by the three Caf1 isoforms.</p

<i>Y</i>. <i>pestis</i> strains used in this study for Caf1 isolation, serologic cross-reactivity tests, and virulence experiments.

<p><i>Y</i>. <i>pestis</i> strains used in this study for Caf1 isolation, serologic cross-reactivity tests, and virulence experiments.</p