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

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

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

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

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

<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

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