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

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.
Author Summary The identification of genes associated with drug resistance has contributed for understanding of the mechanisms of action of compounds against Leishmania, as well as, in the identification of the resistance mechanisms mediated by the proteins encoded by these genes. Differently from the mammalian host, Leishmania is unable to synthetize purine nucleotides de novo and must rescue preformed purines from its host. Due to this metabolic difference between host and parasite, the purine metabolism can be considered as a potential target for drug targeting. TUB is a toxic adenosine analog that was already demonstrated as effective against Leishmania. Using a strategy of gene overexpression after cosmid genomic library transfection, we isolated, mapped, sequenced and identified two genes involved in TUB resistance in L. major. In one of the cosmids, we identified NUPM1-like protein, an atypical multidrug resistance protein previously described in L. amazonensis involved in TUB resistance. The other cosmid contains a novel resistance marker involved in TUB resistance, described here as the TRP. Co-localization of TRP in the ER of LmjF and in silico structural predictions indicated that TRP might be an ER lumen protein. Our findings may be useful to elucidate the purine pathway in the parasite and to understand the role of TRP in the mechanism of TUB resistance.