TUB resistance profiles of cosTUB2 and its respective deletions compared with LmjF parasites.

<p>TUB resistance profiles of cosTUB2 and its respective deletions compared with LmjF parasites.</p

Cross-resistance profiles of cosTUB2 and pTRP transfectants compared with LmjF parasites.

<p>Cross-resistance profiles of cosTUB2 and pTRP transfectants compared with LmjF parasites.</p

Evaluation of <i>trp</i> mRNA expression.

<p><i>trp</i> mRNA expression in promastigotes of LmjF, and the lines transfected with the cLHYG vector, cosTUB2, pSNBR vector and pTRP were determined by <i>trp</i>-specific RT-qPCR. Data were based on quantification of the target and were normalized by <i>gapdh</i> expression. (*) p < 0.0001, compared with the line transfected with the vector cLHYG or with LmjF. The values are the mean ± SEM of three independent biological replicates.</p

TRP expression in transfectant lines, as determined by Western blotting.

<p>Total extracts of promastigotes in the stationary phase for LmjF, cosTUB2 and pTRP transfectants were lysed and the proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane and immunoblotted with an anti-TRP polyclonal antibody. An anti-α-tubulin antibody was used as a control. The images were scanned using an Odyssey CLx imaging system (Li-COR). (A) Western blot analysis of LmjF, cosTUB2 and pTRP transfectants. (B) The bands were quantified using Image Studio2.1 Software (Li-COR) and the results for TRP were normalized against α-tubulin for blotting comparisons. Statistical analysis was performed using Mann-Whitney U test. (*) p < 0.05, compared with LmjF.</p

Structural prediction of the TRP.

<p>Protein modeling, prediction and analysis, according to Phyre2 web portal for TRP. The predicted protein contains 36% alpha helix, 6% beta strand and 3% transmembrane helix, in addition to multidrug efflux transporter, hydrolase/transport protein and transferase folds.</p

Restriction map of cosTUB2 and functional analysis.

<p>Linear representation of the cosTUB2 restriction map and the four deletions generated by partial digestion with <i>Apa</i>I (A). The restriction sites of <i>Cla</i>I (C), <i>Eco</i>RI (EI) and <i>Eco</i>RV (E) are also indicated in the Figure. TUB resistance is indicated by (+). The white arrows indicate the coding regions of the hypothetical proteins (LmjF.31.2040, LmjF.31.2050 and LmjF.31.2060), non-coding RNA (LmjF.31.ncRNA), glycoprotein-like (GP63-like) (LmjF.31.2000), succinyl-diaminopimelate-desuccinylase-like protein (SDD-like) (LmjF.31.2020) and ubiquitin-fusion protein (LmjF.31.2030). The black arrow indicates the <i>TRP</i> gene (LmjF.31.2010). The shaded boxes represent the cLHYG vector and the blank box represents the pSNBR vector.</p

Mapping and alignment of the genomic region around <i>trp</i> gene for six <i>Leishmania</i> species.

<p>Map of the genomic region of cosTUB2 from <i>L</i>. <i>major</i> Friedlin (LmjF) and localization of the <i>trp</i> gene (LmjF.31.2010) in comparison with the following <i>Leishmania</i> spp., according to TriTrypDB: <i>L</i>. <i>(V</i>.<i>) braziliensis</i> MHOM/BR/75/M2904 (LbrM), <i>L</i>. <i>(L</i>.<i>) donovani</i> BPK282A1 (LdBPK), <i>L</i>. <i>(L</i>.<i>) infantum</i> JPCM5 (LinJ), <i>L</i>. <i>(L</i>.<i>) mexicana</i> MHOM/GT/2001/U1103 (LmxM), <i>L</i>. <i>(L</i>.<i>) tarentolae</i> Parrot-TarII (LtaP). The white arrows indicate the coding regions of hypothetical proteins (LmjF.31.2040, LmjF.31.2050 and LmjF.31.2060), non-coding RNA (LmjF.31.ncRNA), glycoprotein-like 63 (GP63-like) (LmjF.31.2000), succinyl-diaminopimelate-desuccinylase-like (SDD-like) protein (LmjF.31.2020) and the ubiquitin-fusion protein (LmjF.31.2030). The black arrow indicates the <i>trp</i> gene (LmjF.31.2010).</p