Identification of differentially expressed proteins from Leishmania amazonensis associated with the loss of virulence of the parasites.

BACKGROUND: The present study analyzed whether or not the in vitro cultivation for long periods of time of pre-isolated Leishmania amazonensis from lesions of chronically infected BALB/c mice was able to interfere in the parasites' infectivity using in vivo and in vitro experiments. In addition, the proteins that presented a significant decrease or increase in their protein expression content were identified applying a proteomic approach. METHODOLOGY/PRINCIPAL FINDINGS: Parasites were cultured in vitro for 150 days. Aliquots were collected on the day 0 of culture (R0), as well as after ten (R10; 50 days of culture), twenty (R20; 100 days of culture), and thirty (R30; 150 days of culture) passages, and were used to analyze the parasites' in vitro and in vivo infectivity, as well as to perform the proteomic approach. Approximately 837, 967, 935, and 872 spots were found in 2-DE gels prepared from R0, R10, R20, and R30 samples, respectively. A total of 37 spots presented a significant decrease in their intensity of expression, whereas a significant increase in protein content during cultivation could be observed for 19 proteins (both cases >2.0 folds). Some of these identified proteins can be described, such as diagnosis and/or vaccine candidates, while others are involved in the infectivity of Leishmania. It is interesting to note that six proteins, considered hypothetical in Leishmania, showed a significant decrease in their expression and were also identified. CONCLUSIONS/SIGNIFICANCE: The present study contributes to the understanding that the cultivation of parasites over long periods of time may well be related to the possible loss of infectivity of L. amazonensis. The identified proteins that presented a significant decrease in their expression during cultivation, including the hypothetical, may also be related to this loss of parasites' infectivity, and applied in future studies, including vaccine candidates and/or immunotherapeutic targets against leishmaniasis

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; 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<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; 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<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