Immunoproteomic analyses of the protein extract from the <i>Leishmania infantum</i> amastigote-like stage.

<p>2DE gels obtained after the separation of total protein extracts (150 µg) of amastigote-like stages by 2DE (first dimension: IEF pH range 4–7, second dimension: 12% SDS-PAGE), and staining with colloidal Coomassie Brilliant Blue G-250 (A, as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001430#pntd-0001430-g001" target="_blank">Figure 1</a>). Immunoblots of reactive spots were identified after incubation of the membrane with pools of sera of asymptomatic (B) or symptomatic (C) VL dogs. Bound antibodies were detected with goat anti-dog IgG antibodies at a 1∶5.000 dilution. The x-axis represents the tentative isoeletric point (<i>pI</i>), while the y-axis represents the approximate molecular weight (kDa) as determined by a commercial 2DE gel marker (BenchMark Protein Ladder). Protein spots were numbered, and their identities are listed in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001430#pntd-0001430-g006" target="_blank">Figure 6</a>. Immunoblots are a reliable representation of three independent experiments.</p

Two-dimensional profiles of the total extracts from <i>Leishmania infantum</i> promastigote and amastigote-like stages.

<p>2DE gels were obtained after separation of promastigote (in A) and amastigote-like (in B) protein extracts (150 µg, each one) by 2DE (first dimension: IEF pH range 4–7, second dimension: 12% SDS-PAGE), and staining with colloidal Coomassie Brilliant Blue G-250. 2DE gels were derived from three independent protein preparations. One representative preparation of each parasite stage was used in this study.</p

Proteins of <i>Leishmania infantum</i> amastigotes-like identified by an immunoproteomic approach.

<p>a) Sera samples of dogs with VL. b) Name of the identified protein and species: Lmj, <i>L. major</i>; Lbr, <i>L. braziliensis</i>; Li, <i>L. infantum</i>; Ld, <i>L. donovani</i>. c) Accession numbers according to NCBI. d) Experimental expected and predicted molecular weights (<i>M</i>r, in KDa). e) Experimental expected and predicted isoeletric points (p<i>I</i>).</p

Comparison of spots identified in protein extracts from promastigote and amastigote-like stages of <i>Leishmania infantum</i>.

<p>Diagrams show the percentage of protein spots identified in either individual or combined parasite stages. In A, the percentage of total proteins identified by asymptomatic (19/18%), symptomatic (64/62%), and a combination of both sera classes (21/20%). In B, the percentage of proteins from the promastigote stage identified by asymptomatic (10/20%), symptomatic (25/49%), and a combination of both sera classes (16/31%). In C, the percentage of proteins from amastigote-like stage identified by asymptomatic (9/17%), symptomatic (39/74%), and a combination of both sera classes (5/9%).</p

Proteins of <i>Leishmania infantum</i> promastigotes identified by an immunoproteomic approach.

<p>a) Sera samples of dogs with VL. b) Name of the identified protein and species: Lmj, <i>L. major</i>; Lbr, <i>L. braziliensis</i>; Li, <i>L. infantum</i>; Ld, <i>L. donovani</i>. c) Accession numbers according to NCBI. d) Experimental expected and predicted molecular weights (<i>M</i>r, in KDa). e) Experimental expected and predicted isoeletric points (p<i>I</i>).</p

Immunoproteomic analyses of the protein extract from the <i>Leishmania infantum</i> promastigote stage.

<p>2DE gels obtained after separation of total protein extract (150 µg) of promastigote stage by 2DE (first dimension: IEF pH range 4–7, second dimension: 12% SDS-PAGE), and staining with colloidal Coomassie Brilliant Blue G-250 (A, as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001430#pntd-0001430-g001" target="_blank">Figure 1</a>). Immunoblots of reactive spots were identified after incubation of the membrane with pools of sera of asymptomatic (B) or symptomatic (C) VL dogs. Bound antibodies were detected with goat anti-dog IgG antibodies at a 1∶5.000 dilution. The x-axis represents the tentative isoeletric point (<i>pI</i>), while the y-axis represents the approximate molecular weight (kDa) as determined by a commercial 2DE gel marker (BenchMark Protein Ladder). Protein spots were numbered, and their identities are given in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001430#pntd-0001430-g005" target="_blank">Figure 5</a>. Immunoblots are a reliable representation of three independent experiments.</p

Two-dimensional profiles of cultures from <i>Leishmania amazonensis</i>.

<p>The 2-DE gels were obtained after the separation of stationary promastigotes extracts (R0, R10, R20, and R30 passages; 650 µg of each extract) by 2-DE (first dimension: IEF pH range 4–7; second dimension: 12% SDS-PAGE) and staining with colloidal Coomassie Brilliant Blue G-250. The gel fragments in the lower portion of the figures represent evaluated amplifications (see within the dotted lines). 2-DE gels of each passage were derived from four independent protein preparations of each passage. One representative preparation of each sample is showed in this study.</p

Immunoblotting validation of some proteins in <i>Leishmania amazonensis</i>.

<p>Representative immunoblotting of some proteins that presented a significant decrease or increase in their expression content between R0 and R30 passages, using promastigote and amastigotes-like forms of <i>L. amazonensis</i>, are shown here. For each protein [α-tubulin, in A; paraflagellar rod protein 1D, in B; glucose-regulated protein 78 (GRP78) in C, and heat shock protein 83 (HSP83), in D], this image presents one example of correspondent 2-DE spot of promastigote form obtained from R0 or R30 passages. The antibodies used to validate each spot are described in the material and methods section. Asterisks represent the comparison between the expression of the protein in the R0 condition in relation to the R30 sample in each parasite stage, applying the Student's t-test (<i>P</i><0.05), and the numbers represent the relative variation of each protein in comparison to R0 of each parasite stage. All experiments were performed in triplicate.</p

Infection of BALB/c mice.

<p>Mice (n = 8) were infected subcutaneously with 1×10⁶ stationary promastigotes of <i>Leishmania amazonensis</i>. Lesion development in the infected footpads was monitored weekly, up to 8 weeks after infection. Mean ± standard deviation (SD) are shown in (A). Parasite load in the infected footpads, spleen, and liver was analyzed in all animals (B). Other mice (n = 8, per group) were subcutaneously infected with 1×10⁶ stationary promastigotes of <i>L. amazonensis</i> obtained from R0 or R30 passages, and the lesion development was monitored up to 8 weeks after infection. Mean ± SD of the groups are shown (C). The parasite load in the infected footpads, spleen, and liver was also evaluated in these groups (D). The experiments were repeated three times, and presented similar results. *Significant difference between the R0 and R30 groups (<i>P</i><0.05).</p

Identification of proteins that presented a significant increase in their expression content.

a<p>⁾ Spots match ID number obtained from ImageMaster Platinum;</p>b<p>⁾ Name of the identified protein;</p>c<p>⁾ Uniprot identification code;</p>d<p>⁾ Experimentally predicted and expected isoelectric point (<i>pI</i>);</p>e<p>⁾ Experimentally predicted and expected molecular weight (<i>Mr</i>, in kDa);</p>f<p>⁾ Number of identified peptides by MS;</p>g<p>⁾ Percentage of the protein sequence covered by identified peptides;</p>h<p>⁾ Normalized data from R0 represented by mean values of each condition divided by R30 value;</p>i<p>⁾ Fold represents the maximum spot intensity mean value of the conditions divided by the smallest value;</p>j<p>) One-way ANOVA (<i>P</i><0.01) obtained from spot analysis;</p>k<p>⁾ Biological functions according to NCBI, UniProt, and Gene Ontology databases;</p>l<p>⁾ Biological activity and/or immunological application described in other studies: <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Joshi1" target="_blank">[53]</a> Joshi et al., 1996; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Tielens1" target="_blank">[54]</a> Tielens et al., 2010; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Soto1" target="_blank">[55]</a> Soto et al., 1996; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Lackovic1" target="_blank">[56]</a> Lackovic et al., 2010; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Scher1" target="_blank">[57]</a> Scher et al., 2012; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-SnchezCaete1" target="_blank">[58]</a> Sánchez-Cañete et al., 2009; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Peris1" target="_blank">[59]</a> Peris et al., 1994; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Steiner2" target="_blank">[60]</a> Steiner et al., 2007; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Jaramillo1" target="_blank">[61]</a> Jaramillo et al., 2011; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-DuclertSavatier1" target="_blank">[62]</a> Duclert-Savatier et al., 2009; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Celeste1" target="_blank">[63]</a> Celeste et al., 2004; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Liu1" target="_blank">[64]</a> Liu et al., 2011; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Leblanc1" target="_blank">[65]</a> Leblanc et al., 1998; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Priest1" target="_blank">[66]</a> Priest et al., 1996; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-BakkerGrunwald1" target="_blank">[67]</a> Bakker-Grunwald, 1992; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Banerjee1" target="_blank">[68]</a> Banerjee et al., 2006; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Casanova1" target="_blank">[69]</a> Casanova et al., 2008; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Jensen1" target="_blank">[70]</a> Jensen et al., 2001. The proteins were identified through the data included in the NCBI database (dated June 2012) for <i>Leishmania spp.</i></p