Abstract
BackgroundTubercidin (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 findingsAfter 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/SignificanceThis 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.
Highlights
Leishmania spp. are the causative agents of leishmaniasis, a parasitic protozoan disease that affects 12 million people worldwide with an estimated annual incidence of approximately 1 million, including both visceral and cutaneous cases [1].The leishmaniasis chemotherapy is complicated because most of drugs used are expensive, toxic, and require long periods of supervised therapy [2]
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
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
Summary
Leishmania spp. are the causative agents of leishmaniasis, a parasitic protozoan disease that affects 12 million people worldwide with an estimated annual incidence of approximately 1 million, including both visceral and cutaneous cases [1].The leishmaniasis chemotherapy is complicated because most of drugs used are expensive, toxic, and require long periods of supervised therapy [2]. Pentavalent antimonial is the WHOrecommended drug for the treatment of leishmaniasis; it has several side effects and reports of parasite resistance have been described worldwide [3]. Miltefosine is the first effective oral drug developed to treat visceral leishmaniasis. It has been used in India for more than a decade [4] and an increase in the failure rate has been reported [5, 6]. Tubercidin (TUB) is a toxic adenosine analog with potential antiparasitic activity against Leishmania, with mechanism of action and resistance that are not completely understood. TUB is toxic to the mammalian host, these findings can provide evidences for a rational drug design based on purine pathway against leishmaniasis
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