Characterization of a Novel Endoplasmic Reticulum Protein Involved in Tubercidin Resistance in Leishmania major

Abstract

Background: Tubercidin (TUB) is a toxic adenosine analog with potential antiparasitic activity against Leishmania, with mechanism of action and resistance that are not completely understood. For understanding the mechanisms of action and identifying the potential metabolic pathways affected by this drug, we employed in this study an overexpression/selection approach using TUB for the identification of potential targets, as well as, drug resistance genes in L. major. Although, TUB is toxic to the mammalian host, these findings can provide evidences for a rational drug design based on purine pathway against leishmaniasis.

Methodology/principal findings: After transfection of a cosmid genomic library into L. major Friedlin (LmjF) parasites and application of the overexpression/selection method, we identified two cosmids (cosTUB1 and cosTU2) containing two different loci capable of conferring significant levels of TUB resistance. In the cosTUB1 contained a gene encoding NUPM1-like protein, which has been previously described as associated with TUB resistance in L. amazonensis. In the cosTUB2 we identified and characterized a gene encoding a 63 kDa protein that we denoted as tubercidin-resistance protein (TRP). Functional analysis revealed that the transfectants were less susceptible to TUB than LmjF parasites or those transfected with the control vector. In addition, the trp mRNA and protein levels in cosTUB2 transfectants were higher than LmjF. TRP immunolocalization revealed that it was co-localized to the endoplasmic reticulum (ER), a cellular compartment with many functions. In silico predictions indicated that TRP contains only a hypothetical transmembrane domain. Thus, it is likely that TRP is a lumen protein involved in multidrug efflux transport that may be involved in the purine metabolic pathway.

Conclusions/significance: This study demonstrated for the first time that TRP is associated with TUB resistance in Leishmania. The next challenge is to determine how TRP mediates TUB resistance and whether purine metabolism is affected by this protein in the parasite. Finally, these findings may be helpful for the development of alternative anti-leishmanial drugs that target purine pathway.

Fig 1. Restriction map of cosTUB2 and functional analysis.

Linear representation of the cosTUB2 restriction map and the four deletions generated by partial digestion with ApaI (A). The restriction sites of ClaI (C), EcoRI (EI) and EcoRV (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 TRP gene (LmjF.31.2010). The shaded boxes represent the cLHYG vector and the blank box represents the pSNBR vector.

Fig 2. Evaluation of trp mRNA expression.

trp mRNA expression in promastigotes of LmjF, and the lines transfected with the cLHYG vector, cosTUB2, pSNBR vector and pTRP were determined by trp-specific RT-qPCR. Data were based on quantification of the target and were normalized by gapdh 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.

Fig 3. TRP expression in transfectant lines, as determined by Western blotting.

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.

Fig 4. Mapping and alignment of the genomic region around trp gene for six Leishmania species.

Map of the genomic region of cosTUB2 from L. major Friedlin (LmjF) and localization of the trp gene (LmjF.31.2010) in comparison with the following Leishmania spp., according to TriTrypDB: L. (V.) braziliensis MHOM/BR/75/M2904 (LbrM), L. (L.) donovani BPK282A1 (LdBPK), L. (L.) infantum JPCM5 (LinJ), L. (L.) mexicana MHOM/GT/2001/U1103 (LmxM), L. (L.) tarentolae 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 trp gene (LmjF.31.2010).

Fig 5. Structural prediction of the TRP.

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.

Fig 6. Cellular localization of TRP in L. major.

Promastigotes in the stationary phase of LmjF and transfected line with pTRP showing phase-contrast image, DNA staining using DAPI (blue), anti-TRP polyclonal antibody visualized with an anti-rabbit secondary antibody conjugated to Alexa488 (green), anti-BiP/GRP78 visualized with an anti-mouse secondary antibody conjugated to Alexa594 (red) and merged image. All images were acquired using a Zeiss LSM confocal microscope. Bar 10μm.