Phosphorylation status of α-tubulin upon adhesion of trypomastigotes to laminin, fibronectin or BSA.

<p>(<b>A</b>) Representative immunoblotting of immunoprecipitated α-tubulin phosphorylated (p-α-tubulin) during the times indicated. (<b>B</b>) Quantitation of 3 independent experiments as described in (<b>A</b>); asterisk represents a comparison between the 5 min and 120 min points by the Student's t-test with p<0.001, indicating a progressive α-tubulin dephosphorylation. (<b>C</b>) Representative immunoblotting of phosphorylated (pS, pT, pY, top) and total (bottom) soluble (S) and insoluble (INS) immunoprecipitated α-tubulin from trypomastigotes incubated for 2h with BSA, laminin (L) or fibronectin (F). (<b>D</b>) Calculation of the phosphorylation ratio relative to BSA from the experiments in (<b>C</b>); ratio: relative intensity of phosphorylated-tubulin treatment/control (BSA); on the left, 49.1 corresponds to the molecular mass standard in kDa.</p

Phosphorylation status of PAR proteins upon incubation of trypomastigotes to laminin, fibronectin or BSA.

<p>(<b>A</b>) Representative immunoblotting of phosphorylated (pS, pT, pY) and PAR proteins immunoprecipitated during the incubation time. (<b>B</b>) Quantitation of 3 independent experiments as described in (<b>A</b>); asterisk represents a comparison between the 5 min and 120 min points by the Student's t-test with p<0.05, indicating a progressive PAR dephosphorylation. (<b>C</b>) Representative immunoblotting of phosphorylated (pS, pT, pY, top) and total (bottom) soluble (S) and insoluble (INS) immunoprecipitated PAR proteins from trypomastigotes incubated for 2 h with BSA, laminin (L) or fibronectin (F). (<b>D</b>) Calculation of the phosphorylation ratio relative to BSA from the experiments in (<b>C</b>); ratio: relative intensity of phosphorylated-PAR treatment/control (BSA); on the left, 80 corresponds to the molecular mass standard in kDa.</p

Trypomastigote proteins modified by phosphorylation/dephosphorylation upon adhesion to laminin or fibronectin.

<p>Incubation was for 2 h: (<b>A</b>) Phosphorylated proteins were stained with Pro-Q Diamond. (<b>B</b>) total protein profile developed with colloidal Coomassie blue staining; molecular mass markers (kDa) are shown on the ordinates; spots with variation in phosphorylation status are circled in red. (<b>C</b>) profile of phosphorylated and dephosphorylated spots upon 2 h incubation. (<b>D</b>) functional distribution of proteins from C.</p

Phosphorylation status of ERK1/2 in trypomastigotes incubated with laminin, fibronectin or BSA.

<p>(<b>A</b>) Representative immunoblotting of phosphorylated ERK 1/2 (p-ERK 1/2) or GAPDH protein during the times indicated. (<b>B</b>) Calculation of the ERK 1/2 phosphorylation ratio for each experimental point relative to BSA from 2 experiments as in (<b>A</b>); on the left, 49.1 and 34.8 correspond to molecular mass standards in kDa. Asterisks represent a comparison between the 5 min and 120 min points by the Student's t-test with p<0.05 for fibronectin and p<0.2 for laminin.</p

Phenotype of <i>T. cruzi</i> trypomastigotes incubated with fibronectin, laminin or BSA.

<p>Incubation was for 2 h: (<b>A</b>) Localization of phosphoproteins is shown by the reactivity with anti-pS, -pT, -pY antibodies (red); nucleus and kinetoplast stained with DAPI (blue) and DIC images were also shown; white bars represent 3.2 µm. (<b>B</b>) Quantitation of parasite modifications (arrows) due to treatment with fibronectin and laminin, respectively, as compared to BSA treatment is shown; 6 fields with approximately 40 parasites each have been examined; asterisks represent a p<0.001 when experimental points were compared with the control by the Student's t-test.</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

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

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

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

Identification of proteins that presented a significant decrease 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-Tull1" target="_blank">[22]</a> Tull et al., 2010; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Oliveira1" target="_blank">[23]</a> Oliveira et al., 2006; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Daifalla1" target="_blank">[24]</a> Daifalla et al., 2011; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-IyerJ1" target="_blank">[25]</a> Iyer et al., 2008; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-HungerGlaser1" target="_blank">[26]</a> Hunger-Glaser et al., 1999; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Werbovetz1" target="_blank">[27]</a> Werbovetz et al., 1999; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Feng1" target="_blank">[28]</a> Feng et al., 2011; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Niemirowicz1" target="_blank">[29]</a> Niemirowicz et al., 2007; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-HungerGlaser2" target="_blank">[30]</a> Hunger-Glaser et al., 1997; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Alcolea1" target="_blank">[31]</a> Alcolea et al., 2009; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Bhaskar1" target="_blank">[32]</a> Bhaskar et al., 2012; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Khanra1" target="_blank">[33]</a> Khanra et al., 2012; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Berberich1" target="_blank">[34]</a> Berberich et al., 2003; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Moore1" target="_blank">[35]</a> Moore et al., 1996; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Swenerton1" target="_blank">[36]</a> Swenerton et al., 2011; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Hummadi1" target="_blank">[37]</a> Hummadi et al., 2006; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Martins1" target="_blank">[38]</a> Martins et al., 2006; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Burns1" target="_blank">[39]</a> Burns et al., 1993; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Kushawaha1" target="_blank">[40]</a> Kushawaha et al., 2011; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Misra1" target="_blank">[41]</a> Misra et al., 2005; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Bringaud1" target="_blank">[42]</a> Bringaud et al., 1995; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Eggleson1" target="_blank">[43]</a> Eggleson et al., 1999; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Mureev1" target="_blank">[44]</a> Mureev et al., 2007; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Steiner1" target="_blank">[45]</a> Steiner et al., 2007; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-MartnezRodrguez1" target="_blank">[46]</a> Martínez-Rodríguez et al., 2012; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Drummelsmith2" target="_blank">[47]</a> Drummelsmith et al., 2004; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Achour1" target="_blank">[48]</a> Achour et al., 2002; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Buda1" target="_blank">[49]</a> Buda et al., 2013; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Alcolea2" target="_blank">[50]</a> Alcolea et al., 2011; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Martn1" target="_blank">[51]</a> Martín et al., 2009; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Silva1" target="_blank">[52]</a> Silva et al., 2012. The proteins were identified through the data included in the NCBI database (dated June 2012) for <i>Leishmania spp</i>.</p